REGULATION OF CELL DEATH DURING MURINE PLACENTAL

DEVELOPMENT AND ITS DYSREGULATION DUE TO XENOBIOTIC EXPOSURE

By

Jacqueline Detmar

A thesis submitted in conformity with the requirements for the

Degree of Doctor of Philosophy

Institute of Medical Sciences

University of Toronto

© Copyright by Jacqueline Detmar 2007

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Title: Regulation of cell death during murine placental development and its dysregulation due to xenobiotic

exposure. Degree: Doctor of Philosophy Name: Jacqueline Detmar Department: Institute of Medical Science

University of Toronto, 2007

THESIS ABSTRACT:

It is well established that during human placentation, cell death is a tightly

controlled process regulating normal trophoblast turnover, differentiation and

invasion. As such, we hypothesize that cell death profiles during murine

placentation will exhibit similar features to those observed in human placenta.

Furthermore, we hypothesize that dysregulation of murine trophoblast cell death due

to polycyclic aromatic hydrocarbon (PAH) exposure will result in altered placental

cell death, having consequent deleterious effects on the fetus. Examination of

murine placental cell death over gestation revealed patterns and rates similar to

those observed in human placenta. In addition, genetic background exerted a

profound effect on cell death rates and patterns, altering developmental outcome of

both normal and PAH-induced models of placental insufficiencies. Deficiency in the

proapoptotic gene Bax resulted in intrauterine growth restriction (IUGR),

accompanied by a placental phenotype characterized by accumulation of trophoblast

giant cells and alterations in labyrinthine architecture. This gene also contributed to

embryonic lethality as in its absence, embryos were protected from PAH-induced

resorptions. In addition, we have determined that PAHs are directly responsible for

alterations in the placental vasculature, associated with deficient cell death within the

labyrinthine regiont. This event is mediated by aryl hydrocarbon receptor (AhR), as

ii

Ahf-deficient conceptuses were protected from aberrant placental cell death and

IUGR. The results of these studies indicate that murine placental death rates and

patterns parallel those observed in human placentation during normal development

and after exposure to xenobiotics such as PAHs. Thus, murine placentation

provides a useful model to investigate the molecular pathways involved in cell death

signaling during human placental development.

iil

ACKNOWLEDGEMENTS

This work could not have been achieved without the efforts of many people to

whom | will always be grateful. First and foremost, | would like to thank my

supervisors, Drs. Robert Casper and Andrea Jurisicova, from whom | not only

received academic guidance, but also moral and emotional support. Drs. S. Lee

Adamson, Isabella Caniggia and Janet Rossant deserve many thanks for their

infinite patience and careful attention to my work. | feel honoured to be indebted to

such kind and intelligent women.

Also deserving of recognition are the many members of the Casper-Brown

laboratory, for their assistance and encouragement throughout the years. A special

thank you goes to summer students, Tatiania Rabaglino, Natalie DiTomasso, Raquel

DeSouza and Roxanne Fernandes. | am very proud of their accomplishments and

am forever grateful for their help. All of my colleagues in the laboratory deserve

many thanks, not only for their technical assistance, but also for their willingness to

laugh at my bad jokes.

| wish to thank my parents, Jack and Dini Detmar, the members of my

extended family and my long-time friends for their staunch confidence in my

capabilities, for tolerating my many absences and — to me, the most important — for

opening their arms to me whenever | was in need of comfort.

Lastly, | would like to extend appreciation to the Natural Sciences and

Engineering Research Council, the Richard Venn and Carol Mitchell Fellowship, and

the Al and Hannah Perly Scholarship Fund for providing me with scholarships over

the past six years.

iv

ATTRIBUTIONS

The following people have contributed to the generation of data reported in

the present thesis:

In chapter 2, N. DiTomasso assisted with dissection and genotyping of Bax

tissues, which were originally obtained from G. Perez, University of Michigan, USA.

In chapter 3, T. Rabaglino and Y. Taniuchi assisted with genotyping Bax and

Hrk tissues. TUNEL staining and cell counts of preimplantation embryos were done

by T. Rabaglino and A. Jurisicova. Beth Acton assisted with deconvolution

microscopy of preimplantation embryos after in situ caspase-3,-7 enzyme assay and

mitochondrial membrane potential assay. AhR and Bax immunocytochemistry of

preimplantation embryos was done by A. Jurisicova, who also assisted with in vitro

treatments of blastocysts. Jaymin Oh performed dot blots of exposed embryos. The

technicians of the transgenic facility did post-treatment embryo transfers. Hrk mice

were obtained from G. Nunez and A. Benito, University of Michigan, USA. The data

presented in chapter 3 has been published in the following form: Jacqui Detmar,

Tatiana Rabaglino, Yoshinari Taniuchi, Jaymin Oh, Beth M. Acton, Adalberto Benito,

Gabriel Nunez, Andrea Jurisicova. “Embryonic loss due to polycyclic aromatic

hydrocarbon exposure is mediated by Bax.” Apoptosis 2006 11; 1413-1425.

In chapter 4, Y. Taniuchi and X. Shang assisted with genotyping AhR tissues.

M. Rennie in collaboration with John Sled and K. Whiteley did placental vasculature

casting; M. Rennie also did microCT analyses and K. Whiteley prepared casts for

SEMs and assisted with electron microscopy. Computer software programs for

microCT analyses were designed by John Sled. Ultrasound biomicroscopy was

performed by D. Qu. AhR mice were obtained from J. Tilly, Harvard University,

Boston, Massachusetts, USA.

TABLE OF CONTENTS

TABLE OF CONTENTS ........:::::ecesescesseoessssrescessccnennnsenseneessecessnneesssaeensensansesseeseness vi

CHAPTER 1: Introduction ..........ccscssssssersssssssseessssssscessssonssssensenssesesenessensenesessoseens 1

1.10 Cell death oo. eee eeccseeeeeeesneeeeeescaeeeneeeeressenaeeeeesessaneesesssneeeresses 1

1.2 Human placental development ...............cccccecccccssesseeceeseeeeesseeeesseessenes 15

1.3. Cell death in human placental pathologies................ccccccssssseeeeseseeees 18

1.4 Murine placental developMent..............sccccceceessssssssseseceesssseneesessseeeeeess 21

1.5 The mouse placenta as a model for the human placenta................... 26

1.6 The placenta and fetal programmMing...................cccccccecseseseeeceeceeeeeeneees 28

1.7 Cell death during human placental development...............c:sccccesseeeeees 29

1.8 Cell death during murine placental development..............c:cceeseseeeeees 32

1.9 Maternal exposure to cigarette smoke, polycyclic aromatic

hydrocarbons and placentation .............ccccececeeseeessesenssneseseeeeseesssseeees 40

1.10 The aryl hydrocarbon reCeptor .0......... cc ceeeeeeeseeeeeeceeeeeeeeseeeseesaseeeeeeeees 44

CHAPTER 2: Murine placental cell death exhibits an organized pattern over

gestation and placental deficiency of pro-apoptotic Bax leads to altered labyrinthine architecture and IUGR........ccsssssssssssssssessssseseeessensaconsnerssesnsessennenssszes 51

2.1 ADSEraCt ..ecccesssssssssssnecsseeeeeeceeneesesenssusensnnneeeeessensnencauneesgennecanuauenauanenssssessnssenansens 51

2.2 INTFOCUCTION...........c::eeeeceeeeeeeneeeeeseseneanennneneseesessssssonsnnanscansenensnssauseseseseessnsneneneses 52

2.3 Materials and Methods ...........:ccccsssssssnssssssssessssscscesssseessssnesssonessesnsnnnenesensenseess 55

2.3.1 Animal housing, Mating And tiSSUC COIECTION............cccccccccceeceeeseeeeeees 55

2.3.2 Terminal deoxynucleotidyl transferase dUTP nick-end labeling......... 56

2.3.3 Giant cell counts in Bax-deficient PIACENtAEC............ccccccccccceseeesneeeeeees 60

2.3.4 Immunohistochemistry and lectin histOChEMIStry ...........ccccccccccceeecees 60

2.3.5 CASPASE-3 ENZYME ASSAY .....eccceeseceeeessteceeeeeeeeetsansaeeceteenaneceesteneees 62

Vi

2.3.6 WeStern DIOTHAG 2.0... ceececeesseeceeeeseeeeeseeeecseeesecssessecnessecenecsnseaseneaes 62

2.3.7 Statistical ANALYSIS 02... ecceceeceesceeeseeceseceseeeessseessaeeesseeenseeesssusenasessaaess 64

2.4 RESUIRS........ceccsencessreessesnnnenuenesseceunesnseneeeesasenseeeeesseneaeeesessenneeseesenssneaeaenenenaaeneess 65

2.4.1. ICR and C57BI/6 conceptuses and decidua display similar numbers and patterns of TUNEL positivity at 7.5 .......cccccscsscscccesssssssceeesssssneeees 65

2.4.2 Murine placentae exhibit organized cell death patterns over gestation; however, differences in the number of TUNEL-positive cells exist Detween the twWO SUFAINS......ccccccccccccscsesssessescsesseessssnssssssnsenecseneeeeseseseess 68

2.4.3 Caspase-3 expression and localization are similar for ICR and C57BI/6 PIACONNAC. eee ee ccc ccccccccccecceeeeeeceeeeeeeeeeceesencessssecsaaaaaaaaauauaassaesseaessseesees 84

2.4.4 Bax localizes to TGCs and the labyrinth of murine placenta and Bax deficiency leads to reduced TGC death and intrauterine growth

FOSUIICTION 00. .eceeseceeceeeeseceeeeeennaeeeeeesnaneeeeetenaaeaeeeeeseccsaauasessnsaneesesesssnseees 87

2.5 DISCUSSION. ..ccccccccsssessnnsensseessssssssseesesnsenencnesecersesesnenneneseesaeeneneseessesesseeseesensesenes 100

3.1 ADStract «2.2... cceeseeeeeeeeeesseneeeeeeesaneneseanssnensssaceessoeesseneensceeessseaeneseessenseennensensagenes 110

3.2 INtrOCUCTION..........::ccceesseeeeeeenssnseeeeensnnneeeeenssenneeeseessnnaneaeeeesssneuseerasenneeerenseneoes 110

3.3 Materials and Methods ..............::ccccssseeceeesssesneeeeesssnseeesesessnsecoeseessneeneesensnssees 113

3.3.1 In vivo BaP and DMBA treatment............cccccccccessseeeesneeeesseeeesessteseens 113

3.3.2 Mating and tiSSu@ COMNOCtION ..........cccececeessesstsnneeneeeeeeeeessenssnneanenseseess 113

3.3.3 PCR GONOty Ping ............ccscccccccceeesseessssennnaceneansneeeeeeeeesssssssaaeaneneeeeess 114

3.3.4 Collection of in vivo PAH-treated preimplantation eMbry06.............. 115

3.3.5 Collection and transfer of in vittro DMBA-treated eEMbryo6..............+. 116

3.3.6 Analysis of cell number and Cell Meath .........ccccccccccceeeeeeeessttseeeeenenees 117

3.3.7 Mitochondrial membrane potential ANALYSIS...........ccccccccccssssesteeeeeees 118

3.3.8 Single EMbryO CASPASE ACTIVILY ASSAY .......cccccsssssessssscceceeccesesssnseneaes 118

3.3.9 Expression of Bax and Hrk transcripts in exposed embryos. ............ 119

3.3.10 Immunocytochemical localization of Bax And ANR.............:cccccsesseeee 120

3.3.11 Statistical ANALYSIS ........cceceeseeeessneeensaceseeensaeeeeeaeeessaeeeessaaeesnenaeess 120

Vii

3.4 RESUITS.....cccccssccscssnscecsccsesssnscesccccescccsesenensccnsssacecccnsusonsesceuesconasssuassaneesenseneceens 121

3.4.1 Effect of DMBA on murine preimplantation embryos in vitro ............ 121

3.4.2 Cellular and molecular pathways activated by DMBA...............:10000 122

3.4.3 Maternal exposure to PAHs results in decreased pregnancy and INCTEASEM FESOMPTION FALCS..........ccccccccscccececseeeeeeesssesaceececeseeeeeeensnennes 129

3.5 DISCUSSION........ccssseeeesssneressseeesseneensnneeecsaneesesseneesssannesessneneeseeeeeeseneeensensenenseeas 138

CHAPTER 4: Maternal exposure to polycyclic aromatic hydrocarbons leads to altered placental vasculature and IUGR in C57BI/6 mice, which is rescued by

ADR eFICIONCY. ............0enseassnssesesssssssececesnnecccesssesnneesensensaceaoooooosessessenssanoeouanansonnees 146

4.1 ADSI oo censnetesennenssnssnssssssenccseesneneecnecsssnanesseseeeneasooesonssaseeuseeeseeeesersconanaees 146

4.2 INTFOCUCTION.............:cecceeeesessenseseeeeeeeeeesnnssnssnsneeeeonaeceaeeusneeeesenenseanensersssereesnnes 147

4.3 Materials and Methods ............::cccsssssessessssseneesssnsseansnesonsnnsseeserssnseesesesessenenes 149

4.3.1: In vivo BaP and DMBA treatment.............cccccccccscsssececceeeeeeeeseesenennes 149

4.3.2: Mating And tisSue CONOCtION ...........cccccccceccccsecseseeceeeeeecscuanasseesesessaaaes 150

4.3.3: Vascular casting and ultraSOUNA DIOMICFOSCOPY ......cccccccccceseseeeneeeees 151

4.3.4: Terminal deoxynucleotidyl transferase dUTP nick-end labeling...... 153

4.3.5: Histological Staining...........cccccccccccccccccccceeceeceeeescsuenaneseseeeeesssseuenseanea 154

4.3.6: Immunohistochemistry and Lectin Histochemistry .............ccccceeeeees 154

4.3.7: CASPASO-3 CNZYIME ASSAY ........cccccceeecee eee e ee teeseeeeeeenenneeeeenttaneeeeeennas 155

4.3.8; WeStOr DIOTLING .2......cccceeeseennstecececeeeeeseeneeeeeeeeesnsnananeeeeeceeeesseesnenensaea 155

4.3.9: Statistical ANAlYSIS...........ccccccsceeeccceceeeceeeeeeeeeceececunnsaeeeeeseesesssensesnensea 155

4.4 RESUIES.........cccsscessssesssesssssceessnessecessnsessneeesnsessneneesnseesennenssnsnenonesnenessesssconeessnes 156

4.4.1: Maternal exposure to PAHs prior to conception leads to |UGR and altered labyrinthine vasculature in C57BYV/6 MICE... eee 156

4.4.2 Reduced umbilical vessel diameter and total fetoplacental vascular surface area and volume in d15.5 placentae from PAH-exposed dams

aneeeeeeeeeeeeeeeeeeeeeneesnseaaaeeeeeessesanaaaneaeeeeseecesesereeeeeseusecenuaneseseseseuenseenenes 159

Vill

4.4.3 Both fetal and maternal compartments of d15.5 PAH-exposed placentae exhibit altered cell death rates and changes in cell death

ITIALKOLS ooo. cece eee cceneeeeecesnneeeenenessnneeenensnsaeesensessaneesessnsaaseeeeeeseenaaaeess 164

4.4.4. AhR-deficient fetuses are protected from IUGR due to chronic maternal EOXPOSUIC 10 PAHS oo. eeccescssccseccesessnssecccssssaceeeesenssssecessssnneseesesseneneeess 182

4.5 DISCUSSION...........::::cccccesssessneseeeneeeeeneneausetessnansessasasnsanuessesnsnseneososaensoeneesesssaanes 191

CHAPTER 5: SUMMARY AND GLOBAL CONCLUSIONS ............:::sssseseserseeeees 201

-CHAPTER 6: FUTURE DIRECTIONG........sssssssssssssssssesssensessssssessseesseeenssesesense 208

1x

List of Original Publications in this Thesis

Detmar J., Rabaglino T., Taniuchi Y., Oh J., Acton B.M., Benito A., Nunez G. and

Jurisicova A. (2006) Embryonic loss due to polycyclic aromatic hydrocarbons is

mediated by Bax. Apoptosis 11: 1413-1425.

Jurisicova A., Detmar J., and Caniggia |. (2005) Molecular mechanisms of

trophoblast survival: From implantation to birth. Birth Defects Research (Part C) 75:

262-280.

LIST OF FIGURES

Figure 1.1 Schematic representation of the cell death pathway. ............cescseseceeseees 4

Figure 1.2 Schematic representations of analogous cell and tissue types between NuMAN ANd MUTLiNe PlACENtAE. .............cceeessecccceeceeeesssneneeececseeeeceeeseseceseesseessssseeeeteess 17

Figure 1.3 Schematic representations of murine placental development and the Murine placental Darricr. 20... cceseeeessseecsssneccesseccssnnesssssseeeessseeeecesensessssaueeeseneesesnnegs 22

Figure 1.4 Schematic representation of the aryl hydrocarbon receptor pathway. ... 45

Figure 2.1 Low-magnification placental section of d15.5 ICR placenta, demonstrating various regions of placenta used for histomorphometry. .................. 59

Figure 2.2 Cell death patterns in d7.5 ICR and C57BI/6 conceptuses. ...............06 66

Figure 2.3 Trophoblast giant cell death patterns in ICR and C57BL/6 placentae over

GESTATION. o.oo. eee eee eesneeeeeeeeenenaeeeeescecneeneeseeeeeeeeeseneeeeseeseesesnauseseeeceneaueeeeescnsteaeeses 70

Figure 2.4 Percentage of trophoblast giant cells containing TUNEL-positive corpses in ICR and C57BL/6 placentae over gestation. .........:ccsssscsccsssscessssseessssseessseeeesnnes 72

Figure 2.5 TUNEL patterns in ICR and C57BI/6 chorionic plate and labyrinth over GESTATION. 0... ee eeeeeensenceceeeeeeeeeeceeeeeessssenneneseeeeeessessaneeasaeasaasseceeeeaeeeeeeceseneguaeaeeas 75

Figure 2.6 TUNEL patterns in ICR and C57BI/6 junctional zone at specified tIMEPOINTS OVEF GESTATION. ..........ccccccessecceessseessssseesssseessseeeeecessseseeseueeusseeeresesseaeacsaeers 79

Figure 2.7 Maternal decidual cell death patterns in ICR and C57BL/6 placentae at specified timepoints Over Gestation. ............cccssssccccssssssceeeceeessenseteecessessneeeesessssteeeeess 82

Figure 2.8 Caspase-3 expression and activity in |CR and C57BI/6 placentae are SiMilar Over GeStatiOn. ...........: cc eeseeessceeeesessceceeeeesaeeecesenaneeaeeeeessssasseeeeeesssseeesesssnaaeess 85

Figure 2.9 Bax deficiency in murine placentae leads to decreased TGC death over GESTATION. 20... eee ceessseeeeesesensenaeeeseessesseeecessssueesesssssseseeeseceegenseeesesesssnaeeeessenetese 88

Figure 2.10 Bax deficiency in murine placentae leads to altered expression levels of placental hormones and cell death Marke’. .............cccccccseessssssseeneececeseceecececeesesenens 91

X1

Figure 2.11 Cleavage levels of active caspase-3 cellular substrates are not altered In Bax-deficient PlaCentae. ...........cecccssneccssseesssneesssseecesessseeecsssseecesssseeeesenseseseeeeesaas 93

Figure 2.12 Bax is expressed in the murine labyrinth. .............cccssssecssssesssteeesserees 96

Figure 2.13 Bax deficiency leads to abnormal labyrinthine structure and intrauterine QFOWEN FeStrICTION. .......... cece ceeeeeeeeeeeeeeeeeeeseeeeeeesssaaeeeeeeeeeeten seeeeeneeeeeteeessgaananeaeeteseeeaaaa 98

Figure 2.14 Schematic representation of cell death patterns over development in the MOUSE PIACOMMA. oo... eecceeeeseeeeeesesssssenccersceceeeeeesessussesuessseseeeeesseueesaaaasesssnssssesseseaseea 102

Figure 3.1 Exposure of murine preimplantation embryos to DMBA increases the cell death index and AhR antagonist (ANF) precedes this effect. ...............::::csceceeeeeees 123

Figure 3.2 Cell death regulatory proteins are increased in DMBA-treated murine DIASTOCYSIS. .........cccccsseseseeeccceneccseeeeecceneaceeseceesesesseessaeassseeessaaseecescsenaaseeceseesenenaeagasens 125

Figure 3.3 In vitro exposure of ICR preimplantation embryos to DMBA had no effect ON PN1 and PN21 WeiGhtS. ........ eee ee eeeceeneeeeeeeteeeeeeeeeneeeeeespeeseesseseeneneseeeeeseee 130

Figure 3.4 Chronic maternal exposure to PAHs prior to conception results in reduced cell number per blastocyst in d3.5, ICR preimplantation embryos. .......... 132

Figure 3.5 Chronic maternal exposure to PAHs prior to conception results in an increased number of resorptions and a decreased number of viable embryos, with a

greater proportion of male embryos represented in the live offspring. ................5 134

Figure 3.6 Bax-deficient, but not Hrk-deficient, embryos are rescued from resorption after chronic maternal exposure to PAHs, prior to Conception. .............ccccceeesseees 136

Figure 4.1 Maternal exposure to PAHs prior to conception leads to IUGR and altered labyrinthine architecture in C57BI/6 mice at d15.5 gestation. ................06 157

Figure 4.2 Maternal exposure to PAHs prior to conception in C57BI/6 mice results in aberrant placental microvasculature in the fetal compartment.................:::::scccceres 160

Figure 4.3 Maternal exposure to PAHs prior to conception in C57BI/6 mice results in aberrant morphology of blood spaces in maternal compartment. .............::::ccccceeee 162

Figure 4.4 Two-dimensional renderings of d15.5 fetal placental vessels from vehicle and PAH-exposed dams, after micro-computed tomography. ..............sssessssreeeeees 165

Xli

Figure 4.5 Micro-computed tomography of fetal placental casts reveals decreased

arterial surface area and umbilical vessel diameter in placenta from PAH-exposed C57BYV6 Cams. ..........cceesessecceeeeceseeeeeesaeecssateccesaeessesueecenaeseesssseeecssesesessnaeecssaeesesseas 167

Figure 4.6 Chronic exposure to PAHs prior to conception does not alter fetal heart rate, abdominal cross-sectional area, nor umbilical artery pulse velocity, as determined by ultrasound DIOMICFOSCOP)................cssssssssessssssssseeeceeeeeseesssssssesenaeee 169

Figure 4.7 Chronic exposure to PAHs prior to pregnancy leads to altered cell death patterns in d15.5 placentae of C57BI/6 Aas. ..........cccssccccssssseeessseeeesseeceessteessaees 172

Figure 4.8 Chronic exposure to PAHs prior to pregnancy leads to increased chorionic plate cell death, but TGC death is unaffected... eeccccceeseeeeee eens 174

Figure 4.9 Chronic exposure to PAHs prior to conception disrupts the expression levels of executioner caspases in d15.5 C57BI/6 placentae. ...........ccccsesseresseeeees 176

Figure 4.10 Cleavage levels of active caspase-3 cellular substrates are reduced in d15.5 PAH-exposed placentae. .0..........cccceccccseeesesceseeeeeeeeesssneeeeeescsnseeeessssaseeeeenses 178

Figure 4.11 Chronic exposure to PAHs prior to conception disrupts the balance of apoptotic and anti-apoptotic proteins in d15.5 C57BI/6 placentae. .................00 180

Figure 4.12 Chronic exposure to PAHs prior to conception up-regulated AhR expression and known target genes of AhR in C57BI/6 placentae. ...............:c0 183

Figure 4.13. Aryl hydrocarbon receptor is expressed in the fetal endothelium of the mouse placenta and AhR deficiency rescues the IUGR phenotype in dams chronically exposed to PAHs prior to CONCEPTION. ...........ccccccecessssceeeessssseceeessssneeeees 185

Figure 4.14 Maternal exposure to PAHs does not alter proliferation in d15.5 C57BI/6 placentae, as evidenced by Ki-67 immunohistochemistry.............ccccscccccessssteeeeesees 188

Figure 4.15 Maternal exposure to PAHs results in altered cell death rates in different regions of C57BI/6 placentae. ...00........eeeeesesccceeeeseseceeeeeeeeesseanereesessnseeeeesssnnseseesenes 189

Figure 4.16 Maternal exposure to PAHs prior to conception results in increased resorption rates in ICR dams and IUGR in C57BI/6 dams. ...........ccccceccessssesseereeees 196

Figure 6.1 Cell death markers in trophoblast stem cells cultured under differentiating CONCITIONS OVEF TIME. 2.0... eeeetenteeeeceeeeeeeeeeeeeeceeseaeasaeaeasessseceeeseaccesececenseeeeneeneeess 210

Xili

Figure 6.2 Polycyclic aromatic hydrocarbon treatment in ICR dams alters levels of UNK cells in early placental GECIGUA. ..........ceseeeeteeeeesesesseeeeeeeeenateserecseeeteetesnananeas 213

Figure 6.3 AhR-deficient placentae exhibit defective labyrinthine architecture and altered expression of cell death and vascular Markels. ...........sssssssesessssseeeesseeeeesses 217

XIV

2D 3D

AhR

AHRE AIF

ANF

ANOVA AP Apaf-1

Arnt

Bad

BaP Bak

Bax Bcl-2 Bcl-x,

Bcl-xs

BH3

Bid

Bik

Bmf bp:

BSA

CDI cDNA

c-FLIP CP CTB

d7.5

°C DAB DAPI

DD

DED DMBA

DMSO DNA DNase |

dNTP DRE DTT

E7.0

EndoG ECF

List of Abbreviations

two-dimensional three-dimensional aryl hydrocarbon receptor aryl hydrocarbon response element apoptosis inducing factor

a-napthoflavone analysis of variance alkaline phosphatase apoptotic protease activating factor 1

aryl hydrocarbon receptor nuclear translocator

Bcl-associated death promoter benzo(a)pyrene Bcl-1-associated killer Bcl-2-associated X protein B-cell lymphoma 2 Bcl-2-like X protein (long isoform) Bcl-2-like X protein (short isoform) Bcl-2 homology domain 3

BH interacting domain death agonist

Bcl-2-interacting killer Bcl-2-modifying factor base pairs

bovine serum albumin cell death index complementary DNA

cellular FADD-like ICE inhibitory protein chorionic plate cytotrophoblast 7.5 days post coitum

degrees Celsius diaminobenzidine

4,6-diamidino-2-phenylindole death domain death effector domain dimethylbenz(a)anthracene

dimethyl! sulfoxide deoxyribonucleic acid deoxyribonuclease | deoxynucleotide dioxin response element dithiothreitol embryonic day 7.0

endonuclease G enhanced chemifluorescence

XV

EPC

ER

FADD

Fak

FasL

FITC

GlyT Het

Hrk

IAP ICE ICM IHC

1U

JC-1

JZ kb kDa KO M

mg mL

mRNA PAH Parp-1

PBS PBST PCD PCR PECAM-1 PL-l PL-Il PN1 pNA PUMA PTEN RITC RNA sc

SDS-PAGE SE SEM

Smac SpT ST

ectoplacental cone endoplasmic reticulum

Fas-associated death domain protein focal adhesion kinase Fas ligand fluorescein isothiocyanate trophoblast glycogen cell heterozygous Harakiri inhibitor of apoptosis interleukin-converting enzyme inner cell mass immunohistochemistry

international units

5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazoyl carbocyanine iodide junctional zone

kilobase pairs kilodalton

knockout molar

milligram milliliter

messenger ribonucleic acid polycyclic aromatic hydrocarbon poly (ADP-ribose) polymerase 1 phospate-buffered saline phosphate-buffered saline with 0.1% Tween 20 programmed cell death polymerase chain reaction platelet and endothelial cell adhesion molecule 1 placental lactogen | placental lactogen II post-natal day 1

para-nitroaniline p53 upregulated mediator of apoptosis phosphatase and tensin homolog

rhodamine isothiocyanate ribonucleic acid subcutaneous

sodium dodecyl sulfate polyacrylamide gel electrophoresis standard error of the mean scanning electron microscopy second mitochondrial activator of caspases spongiotrophoblast syncytiotrophoblast

XVi

TBS TBST Tdt TE TGC TUNEL WT Xiap Xist XRE Zfy1

Ug we

um

Tris-buffered saline

Tris-buffered saline with 0.1% Tween 20 terminal deoxynucleotidyl transferase trophectoderm

trophoblast giant cell terminal deoxynucleotidyl transferase dUTP nick-end labelling wildtype X-linked inhibitor of apoptosis inactive X-specific transcript xenobiotic response element

zinc finger protein 1, Y-linked

microgram microliter

micrometer micromolar

XVii

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CHAPTER 1

INTRODUCTION

Versions of sections 1.4-1.6 and 1.8 were published in Birth Defects Research (Part C), December, 2005, titled: Molecular mechanisms of trophoblast survival: From implantation to birth (Jurisicova A., Detmar J. and Caniggia I.). Reprinted with permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc. © Wiley-Liss, 2005.

CHAPTER 1: Introduction

1.1 Cell death

Programmed cell death (PCD) is an evolutionarily conserved process that has

been documented to play a vital role during development in many diverse

multicellular organisms, including mammals, amphibians and insects. It is well

established that cell death is required for adjustment of cell number or removal of

unnecessary cellular structures such as the tadpole tail during amphibian

metamorphosis, disintegration of larval organs during insect metamorphosis,

elimination of sex specific organs such Mullerian duct in male or Wolffian duct in

females as well as formation of digits in mammals (reviewed in [1, 2]). Cell death is

also driving force behind cellular sculpturing of embryonic structures such as

creation of proamniotic cavity and formation of tubular structures [3] including neural

tube closure [4] and development of ear canal, tooth remodeling, selection of

immune cells [5, 6] as well as elimination of misplaced, injured or otherwise

dangerous cells such as ectopic primordial germ cells [7]. While this programmed

cell death is not always apoptotic in nature, as both necrotic-like and autophagic like

death have been observed to occur in some organs [8, 9], it is always tightly

regulated by conserved molecular pathways, guarding the decision and execution of

cell death machinery [10, 11].

Bcl-2 family members

Proteins of the Bcl-2 (B-cell lymphoma 2) family are crucial regulators of the

cell death pathway (reviewed in [12, 13]). This family is composed of multi-domain,

anti-apoptotic (Bcl-2, Bcl-X,_, Bcl-w, Mcl-1) proteins which associate with several

cellular organelles, including the outer membrane of the mitochondria, the

endoplasmic reticulum (ER) or nuclear membranes and contain four characteristic,

homologous regions, called Bcl-2 homology domains (BH1-4) [14]. Their

counterparts are the multi-domain, pro-apoptotic (Bax, Bak, Mtd, Bcl-xs) proteins

that also bear BH domains — typically three or four — and act in an antagonistic

fashion to the pro-survival molecules. Additional family members include the BH3-

only proteins, which are pro-apoptotic proteins containing only the BH3 domain

(Bad, Bik, Blk, Bim, Bid, Hrk/DP5, Noxa, PUMA). The BH-3 only proteins act as

sentinels, charged with activating the cell death pathway in response to a variety of

different death stimuli. The BH3 domain is critical for engaging the anti-apoptotic

Bcl-2 proteins and rendering them inactive, thus triggering the cell death cascade

[15]. The mitochondria are the primary sites of actions of the Bcl-2 family members,

with functionally opposing proteins heterodimerizing via the BH domains. These

actions are mediated by the interactions of the BH3 a-helix on the pro-apoptotic

protein cleft formed by the a-helices of the BH1, BH2 and BH3 regions of the anti-

apoptotic proteins [16]. Therefore, both multimeric and BH3-only family members

can induce cell death by binding to pro-survival proteins such as Bel-2 and Bcl-x;

however, the BH3-only proteins cannot kill in the absence of their multi-domain

cousins, such as Bax and Bak [17].

The extrinsic cell death pathway

Death receptors and their cognate ligands belong to the tumour necrosis

factor (TNF) gene superfamily. The best characterized of these are the Fas (CD95,

Apo-1)/FasL (Fas ligand; CD95L, Apo-1L) and the TNF/TNF receptor pathways.

While the molecular mechanisms involving death receptor ligation have largely been

elucidated through studies involving cells of the immune system [18], Fas and TNFR

are expressed in a number of different cell types, including trophoblasts [19-21].

Death receptor ligands can exist in soluble form, exemplified by TNF-a or in

membrane-bound form, such as FasL or TNF-related apoptosis-inducing ligand

(TRAIL). Additionally, FasL has been shown to exist in soluble form (sFasL),

generated by proteolytic cleavage of membrane-bound FasL by matrix

metalloproteinases [22, 23], or sequestered in secretory vesicles and released into

the circulation or extracellular space [24]. Membrane-bound death receptor ligands

are largely involved in immune cell homeostasis [18] and maintaining sites of

immune privilege [21, 25]; however, the exact role of secreted ligands is

controversial, as they have been shown to have either cytoprotective or cytotoxic

capacities [24, 26].

External death cues are transmitted through death receptors located at the

cell membrane, such as the Fas receptor (CD95, Apo-1), the tumour necrosis factor

receptor (TNFR) or death receptors 3-6 (DR3-6). Ligand-bound receptors require a

set of signaling proteins bearing a distinct set of modular domains to facilitate

homotypic interactions (Figure 1.1). The Fas molecules associate to form a trimeric

Death Signal

(FasL; TNF, etc.)

Death Adaptor ae

<< Protein Te : “ (FADD, TRADD) ° ON DW

a, Death ae Si,

Receptor. v8 Procaspase-8/10

Active

caspase-8/10

@ ATP “**-@ Smac _

&

\ Be

%, @ Apat-t _-” Cytochrome ¢ & i \ ae “en

© . J Posse me Active y mG

Procaspase-3 ¢ caspase-3. @ vie ay

mea ar

pe

ON APOPTOSOME &

Procaspasé-3 lL .

Procaspase-6 ¢ | |

: . Cleavage of cytoplasmic Procaspasé-7 Active effector , and nuclear proteins Chromatin

a caspases condensation DNA YS CELL fragmentation

DEATH Nucleus

Figure 1.1 Schematic representation of the cell death pathway.

complex, with each receptor bearing a cytoplasmic tail containing the death domain

(DD), which will ultimately trigger the formation of the death-inducing signalling

complex (DISC; [27]). A cytoplasmic adaptor molecule, Fas-associated death

domain protein (FADD), also bears a DD at its C-terminus and homotypically

associates with the DD of the Fas receptor [27]. At the N-terminus of the FADD

protein is a death effector domain (DED) which binds with the DED of initiator

caspases, such as procaspase-8 or -10, through homophilic interactions. The

sequestration and clustering of these proenzymes allows autoproteolytic processing,

thereby forming active caspases [18, 27]. Inhibition of death receptor-induced

activation can be effected at this point, by cellular FADD-like interleukin-converting

enzyme inhibitory protein (c-FLIP). This protein bears two DED domains and a

caspase-like domain similar to caspase-8 but lacks proteolytic capability, thus

competitively blocking autocatalysis of the procaspases [28, 29]. Once the initiator

caspases have been activated, they cleave downstream effector pro-caspases

(caspase-3, -6, -7), producing active proteases with a variety of specific cellular

targets [30], resulting in the morphological and biochemical hallmarks of cell death

[31]. The extrinsinc death pathway can also lead to activation of mitochondrially-

mediated cell death, as caspase-8 can cleave the pro-apoptotic, BH3-only family

member, Bid, forming truncated Bid (tBid), which translocates to the mitochondria,

activating Bax and releasing cytochrome c [32, 33].

The intrinsic cell death pathway

The intrinsic cell death pathway is triggered by cellular stress signals such as

DNA damage, oxidative stress, toxin exposure or growth factor deprivation. The

common link in achieving cellular demise via these triggers is the engagement of cell

organelles, particularly the mitochondria (Figure 1.1). Proapoptotic Bcl-2 family

members such as Bax and Bak remain cytosolic until activation by apoptotic signals.

Upon stimulation, these proteins translocate to the mitochondria and form

membrane-spanning, oligomeric pores, in an as-yet, unresolved mechanism [34],

facilitating the release of several apoptotic factors, including cytochrome c,

Smac/Diablo [85, 36], endonuclease G (EndoG) [37, 38], apoptosis-inducing factor

(AIF) [39, 40] and Omi/HtrA2 [41]. In the presence of ATP/dATP, released

cytochrome c and apoptotic protease activating factor-1 (Apaf-1) recruit initiator

caspase-9, which undergoes autoproteolytic cleavage when bound to Apaf-1,

forming the oligomeric complex, the apoptosome [42]. This macromolecular

complex can then facilitate activation of downstream effector caspases, including

caspase-3, -6 and -7, which destroy or inactivate specific cellular substrates,

resulting in the demise of the cell.

Triggering cell death via the mitochondrial pathway is reliant upon several

Bcl-2 family members, including membrane-associated proteins such as Bax, which

mediate the release of apoptogenic factors from the mitochondria.

Bax — a multidomain, pro-apoptotic Bcl-2 family member

In healthy cells, Bax is a cytosolic, monomeric protein that functions in cell

death through the mitochondrial pathway [43, 44] recent evidence suggest that this

protein can also localize to the ER, functioning in an unknown role [45]. Both Bax

and Bak are widely distributed, with expression reported in many cell and tissue

types, functionally substituting for each other [45]. During apoptosis, Bax changes

conformation and integrates into the outer mitochondrial membrane and

oligomerizes, forming the mitochondrial apoptosis-induced channel (MAC) [46]. This

permeabilizes the mitochondrial membrane — via controversial mechanisms [47] —

allowing the efflux of apoptogenic proteins into the cytoplasm [48]. Recent studies

using Hela cell lines have demonstrated that Bax and Bak are functionally redundant

in the formation of the MAC complex [46].

Targetted knockout of Bax in mice revealed a series of defects including

lymphocyte hyperplasia, accumulation of neurons, abnormal ovarian follicle

morphology and accumulation of germ cells in the testes that renders Bax-deficient

males infertile [49]. Furthermore, while Bak knockout mice were developmentally

normal and fertile, Bax/Bak double knockouts typically died in the perinatal period

and exhibited persistence of the interdigital webbing, imperforate vaginal canal and

accumulation of cells in both the hematopoietic and nervous systems [50]. The

results of this study revealed that Bax and Bak have overlapping functions with

respect to apoptosis. These proteins execute crucial cell death functions during

mammalian development and tissue homeostasis, regulating the release of

apoptogenic factors from the mitochondria.

Mitochondrial mediators of apoptosis

While members of the Bcl-2 family control the mitochondrial response to

death signals, various factors released from this organelle have a critical function in

the apoptotic process (Figure 1.1) [51]. The first identified mitochondrially-derived

apoptotic factor was cytochrome c, which, until then, had been a known player in the

oxidative phosphorylation pathway, having a role as an electron shuttle protein [52].

The release of cytochrome c contributes to the formation of the apoptosome and

subsequent cleavage of downstream effector caspases. It is currently believed that

only a portion of cytochrome cis released, functioning in the apoptotic pathway and

the fraction of cytochrome c remaining in the mitochondria will continue to sustain

ATP production [53, 54]. Over time, the progressive damage to the mitochondria

becomes irreversible, ensuring cell death. The primary role of second mitochondria-

derived activator of caspase (Smac) in this pathway is to act as a molecular brake,

antagonizing the inhibitors of apoptosis proteins (IAPs), cytosolic proteins which

block the activity of various caspases, including caspase-3, -6 and -9. The

apoptosome complex was shown to contain the X-linked IAP (Xiap), which binds and

sequesters active caspase-3 and -9, thus blocking the proteolytic cascade [55]. If

the apoptotic stimulus is persistent or great in magnitude, the [AP-binding

capabilities of mitochondrially-derived proteins such as Smac, will competitively bind

cytoprotective proteins such as Xiap.

The three remaining mitochondrially-sequestered, death-inducing proteins

have been studied only in a limited context. Omi/HtrA2 is a protein with a dual

nature, as it can mediate caspase-dependent death through its ability to inhibit IAPs,

in addition to its function as a serine protease [41]. Overexpression of this protein

revealed the essential function of the catalytic domain for inducing caspase-

independent death [56]. Endonuclease G is another mitochondrially-derived protein,

shown to be localized to the nucleus under the influence of certain death stimuli, in

the context of both caspase-dependent and independent death [37]. While EndoG

has been implicated in the degradation of DNA [88], the exact role and relevance of

this molecule is uncertain. Lastly, AIF is a flavoprotein that normally executes an

oxido-reductase function in healthy cells [57]. In addition to this role, AIF is released

from the mitochondria during apoptosis and translocates to the nucleus to act as

mediator of chromatin condensation and DNA fragmentation (greater than 50 kb)

[39, 40]. The mitochondrial mediators of apoptosis are crucial for cell viability, as

cytochrome c [58], AIF [59] and Omi/HtrA2 [60] knockout mouse models exhibit

either embryonic or postnatal lethality; however, some redundancy in function

amongst these molecules and other cellular proteins does appear to exist, since

Smac- and EndoG-deficient mice are viable and have limited phenotypes linked to

the apoptotic mechanism [61, 62].

Caspases

Caspases are cysteine-dependent aspartate-directed proteases that are

present as inactive zymogens and are cleaved to an active form either

autocatalytically or by up-stream caspases. In Caenorhabditis elegans, CED-3 was

identified as a protease critically involved in the developmental cell death process.

The first mammalian caspase was originally discovered as a cytokine processing

10

enzyme and was known as interleukin-1-B-converting enzyme (ICE); since then,

fourteen different homologues have been identified [63]. Based on their prodomain

length and composition, caspases can be subdivided into upstream initiator

caspases (such as caspase-2, -8, -9 and -10) and short domain effector caspases

(caspase-3, -6 and -7). Initiator caspases possess long prodomains containing

either a DED or a caspase activation and recruitment domain (CARD), or both.

These regions facilitate interactions with adaptor molecules and promote the

process of autoproteolysis, a characteristic unique to the initiator caspases. Upon

activation, the initiator caspases cleave downstream effector caspases which act as

executioners of the cell death pathway. Active caspases are capable of cleaving

various intracellular proteins, including, but not limited to, structural elements of the

cytoplasm and nucleus, signaling molecules and proteins involved in DNA damage

repair. This leads to the disruption of survival pathways and the disassembly of

important architectural components of the cell, contributing to the morphological and

biochemical changes that characterize apoptotic cell death [30, 31].

One of the downstream targets of active caspase-3 is poly(ADP-ribose)

polymerase-1 (Parp-1), a crucial enzyme capable of binding DNA and mediating

base-excision repair, thus maintaining genomic integrity [64, 65]. Parp-1 responds

immediately to cellular stresses (radiation, ischemia, genotoxins) that result in DNA

damage, in a process that is energetically expensive. Therefore, high levels of DNA

repair lead to depletion of cellular energy stores by Parp-1, resulting in necrosis.

Thus, it is believed that the cell’s response to substantial genomic damage is to

induce apoptosis, thereby protecting neighbouring cells from the harmful,

11

proinflammatory effects of necrotic cell death [65]. A consequence of triggering the

cell death cascade is the cleavage of Parp-1 into canonical 24-kDa and 89-kDa

fragments [66, 67]. As such, the cleavage profile of Parp-1 afer Western blotting has

provided an end-point assay for the detection of cell death signaling within a

eukaryotic system [68].

There are a number of different caspases, some of which are only

peripherally involved in the regulation of cell death. The primary targets of such

caspases are proteins required for other non-apoptotic cellular responses, such as

cytokine maturation, cellular differentiation and cell cycle progression (reviewed by

[69, 70]. Furthermore, different caspase family members appear to have distinct

subcellular localization and cleave different subsets of proteins, dependent upon the

apoptotic trigger or cell type [71]. Investigations of cell death pathways in several

physiological models revealed that cells lacking certain kinds of caspases will

compensate by upregulating another caspase with similar substrate specificity [72].

Nonetheless, physiological animal studies have revealed requirements of certain

caspases in distinct cell types and along different stages of development [69].

Alternate modes of cell death

Recent evidence suggests a greater diversity in the cell death programme, as

an apoptotic-like death can occur without the activation of effector caspases. Signals

originating from established apoptosis-related factors such as death receptors,

caspases and certain Bcl-2 family members, may result in morphological and

biochemical features consistent with non-classical cell death. In addition to

12

apoptosis (Type | death), other cell death pathways include necrosis, autophagy

(Type II death), mitotic catastrophe, and senescence, which has been described as

a “living cell death” [73].

Autophagy is a non-necrotic, caspase-independent death, hallmarked by cell

membrane blebbing, partial chromatin condensation, autophagic vesicles within the

cytoplasm and increased lysosomal activity [74]. The genetic basis of autophagy is

conserved across evolution, with homologous genes identified in organisms from

yeast to humans [75]. Two types of autophagy exist, microautophagy and

macroautophagy. Microautophagy is characterized by engulfment of cellular

materials directly adjacent to the lysosomal membrane, whereas macroautophagy

consists of surrounding portions of the cytosol with double-membrane vesicles,

sequestering the contents to be degraded from the rest of the cytoplasm. Fusion of

the vesicle with lysosomes - forming an autophagosome — allows the cargo to be

degraded in a closed system, thereby protecting the cell from potentially cytotoxic

constituents [11]. The autophagic process allows recycling of old proteins and

organelles, while also providing the cells with basic building blocks for renewing

cellular machinery and structure. Triggering of the autophagic mechanism occurs

during cellular response to nutrient starvation, growth factor withdrawal, high

temperatures, hypoxia and invasion of pathogens [11, 74, 75]. However, autophagy

has also been reported to have requisite functions during development, allowing

dauer development in C. elegans [76], remodeling of the salivary glands in

Drosophila [9] and regression of the Mullerian ducts in mammals [77]. Dysregulation

of the autophagic process can result in a number of disease states, including cancer

13

[78] and the degenerative disorders, Huntington’s, Alzheimer’s and Parkinson’s

diseases [79]. Lastly, a number of Bcl-2 family members have been implicated as

having a role in autophagic cell death, including Bax, Bak, Bcl-2 and Bcl-x, [74].

Necrosis, in contrast to both apoptosis and autophagy, is not a self-contained,

regulated process, resulting in traumatic cell destruction, followed by the release of

the intracellular contents in the immediate vicinity [80]. Induction of necrosis is

typically triggered by events that have a radical and immediate effect on the cell

metabolism and machinery; these types of exposures often affect large numbers of

cells simultaneously. Examples are respiratory poisons, irradiation, physical

disruption of cellular membranes caused by altered extracellular osmolarity, extreme

pH, extreme temperatures and physical trauma [81]. In the presence of such

stressors, the cell will have difficulty producing ATP, thereby compromising ion flux

and water balance. This creates leaks in the cell membrane, causing the cell to

typically gain water and sodium ions and leading to cellular swelling. In turn, calcium

ions and other cellular constituents can leak out of the cell, causing damage to the

surrounding tissue. At this point, cellular metabolism stops and the DNA begins to

degrade in a completely nonspecific fashion. Internal membranes from organelles

such as lysosomes and peroxisomes can also be compromised, releasing their

contents into the cell itself and eventually, into the neighbouring environment [82].

Necrosis is generally deemed to be a passive process; however, there appears to be

a modicum of regulation involved and while some players of the necrotic pathway

have been identified, the underlying signaling pathways remain enigmatic [73, 82].

Finally, a number of studies have recently reported the presence of cell death with

14

necrotic features under normal physiologic conditions, as seen during renewal of

enterocytes in the small intestine [83] and during follicular maturation in the ovary

[84] . Moreover, necrotic cell death has been observed to compensate for the lack

of apoptotic proteins in mice deficient for classical cell death genes [8, 85].

Apoptosis-related molecules have functions outside of cell death

Recent evidence suggests that proteins having a well-established role in cell

death pathways have alternate functions in other cellular processes such as

proliferation, differentiation and survival. In fact, the first identified mammalian

caspase, caspase-1, was originally described by two separate groups [86, 87] as a

protease involved in cytokine maturation and was given the moniker, interleukin-

converting enzyme (ICE). Upon the cloning of the C. elegans cell death gene, ced-

3, ICE was found to be homologous in sequence and structure, and mammalian

ICE-like proteases were later discovered to have key roles in the apoptotic pathway

[88]. Both initiator and effector caspases have been linked to having roles outside of

cell death. Caspase-8 has been linked to not only lymphocyte proliferation, but has

also been shown to serve a role in trophoblast and macrophage differentiation in

both humans [89, 90] and mice [91, 92]. Similar functions have been indicated for

caspase-3, with reported involvement in B-lymphocyte [93] and neuronal [94]

proliferation and also in erythroblast [90], myoblast [95] and osteoblast [96]

differentiation. Thus far, it appears that caspases mediate their effects through

cleavage of various cellular substrates, and it is speculated that whether the

15

outcome is apoptotic or non-apoptotic depends on the extent of caspase activation

[97].

In addition to caspases, members of the Bcl-2 family have also been

implicated in having roles outside of cell death. Brady et al. [98] reported that mice

transgenic for Bax overexpression in the T-cell lineage results in an increased

number of cycling thymocytes. Furthermore, upon IL-2-induced stimulation, these T-

cells were capable of entering the S phase faster than control cells. More recently, it

was shown that mice deficient in p53 and transgenic for constitutively-expressed, T-

cell-specific Bax, exhibited a greater incidence of T-cell lymphomas compared with

p53-deficient mice [99]. In addition to regulating cell cycle events, both Bax and

Bak were demonstrated to be required in healthy cells for normal fusion and fission

of mitochondria [100]. Interestingly, while pro-apoptotic Bcl-2 family members

appear to accelerate entry into the cycle, anti-apoptotic members seem to have an

anti-proliferative effect. Transgenic mice over-expressing Bcl-2 in cells of B- and T-

cell lineage exhibited decreased lymphocyte turnover and slower entry into the cell

cycle after exposure to mitogens [101, 102]. Moreover, similar anti-proliferative

effects were observed in anti-apoptotic members, such as Bcl-xL, Mcl-1 and Bcl-2

(reviewed in [103}).

1.2 Human placental development

The placenta is a life-sustaining organ, which mediates the physiological

exchange of oxygen, nutrients and waste between mother and fetus. During human

placentation, highly proliferative cytotrophoblast (CTB) cells reside in chorionic villi of

16

two types, floating and anchoring villi. Floating villi are bathed in maternal blood and

allow gas and nutrient exchange for the developing embryo; the CTB of such villi

proliferate extensively during the first trimester and differentiate by fusing to form the

multinucleate syncytiotrophoblast (ST) layer (Figure 1.2). This layer is subject to

continuous renewal, whereby aged nuclei, together with cytoplasm, are released into

the maternal circulation as syncytial knots, to be replaced by the fusion of new CTB

cells [104, 105]. Cytotrophoblast cells in anchoring villi have two fates: either to fuse

and form the ST or, at selected sites, break through the basement membrane and

form columns of extravillous trophoblast cells (EVT). These columns physically

connect the embryo to the uterine wall and provide individual EVT cells that migrate

and invade the maternal spiral arteries of the placental bed [106]. This results in the

conversion of narrow arteries into distended uteroplacental arteries, which increases

blood flow to the placenta, providing oxygen and nutrients to the growing fetus.

Dysregulation of the cell death programme has been implicated in a number

of human gestational diseases, including preeclampsia and intrauterine growth

restriction (IUGR). Investigations into these pathologies have helped to elucidate

the cellular mechanisms of trophoblast cell death in both normal and diseased

states.

17

Human Placenta Mouse Placenta

Syncytiotrophoblast a Labyrinthine region with proliferative

Proliferative cytotrophoblast cells stem cells

Proximal extravillous trophoblast cells [|] Junctional zone with spongiotrophoblast

Distal extravillous trophoblast cells a Trophoblast giant cells

Syncytial knots @8 Trophoblast glycogen cells

Maternal decidua [|] Maternal decidua

td Maternal spiral artery ? Maiernal spiral artery

Figure 1.2 Schematic representations of analogous cell and tissue types between human and murine placentae.

18

1.3. Cell death in human placental pathologies

Preeclampsia

The majority of studies have focussed on pre-eclampsia, a placental disorder

complicating 5-7% of of pregnancies and is a major cause of maternal and perinatal

morbidity and mortality. This disease is associated with excessive shedding and

deportation of placental debris into the maternal circulation, due to unscheduled ST

cell death [107-109]. Syncytiotrophoblast renewal is facilitated by a balance of CTB

proliferation and syncytial knot release. It has been postulated that the elevated

levels of CTB proliferation observed in preeclamptic villi [110] perturb this balance,

thus forcing the ST to maintain integrity by increasing syncytial knot shedding [111].

This is believed to overwhelm the apoptotic machinery in ST cells, leading to a

truncated form of apoptosis, followed by necrosis, in a process termed “aponecrosis”

[112]. The release of non-apoptotic placental debris into the maternal circulation

results in the wide-spread, intravascular inflammatory responses noted in

preeclamptic women [107, 113]. Moreover, the Fas/FasL pathway has been

implicated in the pathophysiology of preeclampsia, as increased levels of Fas and

decreased levels of FasL have been detected in villous trophoblast [114]. Lastly,

sera from preeclamptic women have been shown to induce trophoblast cell death via

Fas-mediated sensitization [20].

Trophoblast differentiation and invasion are also impaired in preeclampsia,

leading to insufficient remodelling of the maternal spiral arteries, which retain their

vasocontrictive abilities, thus reducing placental perfusion [115-117]. In addition,

19

elevated apoptotic indices have been observed in EVT of preeclamptic placental

beds [118, 119], contributing to the arterial defects. Inadequate placental perfusion

results in a number of deleterious consequences, including placental oxidative

stress, hypoxia, ischemia/reperfusion injuries and infarcts [120, 121]. Thus, a large

number of symptoms of preeclampsia are directly and indirectly associated with

aberrations in the placental cell death programme.

Intrauterine growth restriction

Optimal embryonic and fetal growth depend upon the interaction of genetic

and environmental factors, including embryonic and maternal genetic and hormonal

profiles, adequate nutrient and oxygen supply and the appropriate development and

regulation of the maternal-placental-fetal unit. Approximately 7-9% of live infants

exhibit birth weights below the 10" percentile [122], a diagnostic feature of IUGR.

Intrauterine growth restriction is considered to be a pathological reduction in

expected fetal growth, due to intrauterine factors. This is distinct from babies that

are small for gestational age, which is based on standard values of infant weight and

is not reflective of fetal and neonatal growth dynamics [123]. There are a number of

established causes for IUGR including preeclampsia, fetal infection, malnutrition,

placental damage and exposure to cigarette smoke [123]. IUGR is further classified

as either “symmetric” or “asymmetric”. Symmetric IUGR is a proportional decrease

in length, weight and head size, while in asymmetric IUGR, the length and weight

are decreased, but there is a head sparing effect such that fetal head circumference

is appropriate for gestational age [124]. Determination of intrauterine growth is

20

assessed by the following ultrasonographic measurements: biparietal diameter,

abdominal circumference, femur length and crown-rump length [125]. While IUGR is

linked to increased rates of perinatal morbidity and mortality, it has also been

associated with the onset of childhood and adulthood cardiovascular and metabolic

diseases [123].

Placental insufficiency is a significant contributor to |UGR [123]. Known

placental pathologies of IUGR include decreased placental growth, chronic villitis,

increased fibrin deposition, umbilical cord anomalies, infarct, CTB hyperplasia and

basement membrane thickening [126-128]. In addition, overall size and surface

area are decreased in IUGR placentae, and they exhibit smaller terminal villi and

abnormal villous vasculature [129, 130]. Reduction in placental size has been

attributed to increased rates of cell death [131-134]. A number of different placental

cell subtypes appear to be affected, with unscheduled death seen in chorionic

trophoblast [135], villous stroma and trophoblast [132] and ST [126]. On the

contrary, ultrastructural studies of IUGR placentae have revealed no difference in

cell death rates [128]. Additionally, while serum from preeclamptic women

demonstrated higher levels of ST microparticle shedding, this was not observed in

women with normotensive IUGR [109]. Some of the discrepancies in apoptotic rates

have been attributed to differences in experimental technique and tissue sampling.

Moreover, the underlying molecular mechanisms involved in the pathogenesis of

IUGR are largely unresolved and might confound trophoblast cell death analyses.

21

1.4 Murine placental development

The murine embryo at implantation (d4.5) has a simple trophectoderm

surrounding the blastocyst, which will eventually give rise to all trophoblast cells in

the placenta (reviewed in [136, 137]). Trophoblast stem (TS) cells emerge from a

population of cells known as the polar trophectoderm, which overlies the inner cell

mass of the embryo. Contact with the inner cell mass causes the TS cells to

proliferate, in a process that is mediated by the growth factor, fibroblast growth factor

4 (FGF-4) [136]. Trophoblast stem cells are multi-potent, can proliferate in culture

for many generations [138] and can differentiate both in vitro and in vivo, into a

number of different trophoblast cell subtypes [139]. At day 6 of mouse development,

two diploid populations of trophoblast derivatives are established: the extra-

embryonic ectoderm and the ectoplacental cone (EPC; see Figure 1.3a).

The labyrinthine region

Between days 7.5-9.5 the placenta undergoes major morphological

remodeling, resulting in the formation of three distinct cellular regions, each with

unique morphology and function (reviewed in [136, 137]). Chorioallantoic (CA)

attachment transpires at approximately d8.5, a process whereby the allantois

attaches and interdigitates with the chorion [142]. Thereafter, nascent villous

branches become evident and are lined by very thin and elongated trophoblast cells

[142]. Further branching establishes a network of villi perfused with fetal blood

22

Figure 1.3 Schematic representations of murine placental development and

the murine placental barrier. A. Depicts the different regions or cell types with the

mouse placenta from early implantation to midgestation to late gestation. B.

Depicts the murine placental barrier, or interhemal distance. Panel B was based on

transmission electron micrographs published in Georgiades et al., 2002 [140] and

Coan et al., 2005 [141].

["] . maternal decidua

[-].. junctional zone

labyrinth

[| chorionic plate

GP trophoblast giant cells

@3 trophoblast glycogen cells

? maternal spiral artery

fetal trophoblast layer |

"| syncytiotrophoblast layer II

syncytiotrophoblast layer III

fetal capillary endothelium

23

d7.5

Uterine cavity

———__— Secondary TGC i —————-— Ectoplacental cone

Allantois

Primary. TGC

Proximal and distal endoderm

d10.5

Fetal capillary lumen

Figure 1.3

24

embedded in numerous small canals perfused by maternal blood. This region is

collectively referred to as the labyrinth (see Figure 1.3a) and is the site of fetal-

maternal exchange. During this process, the chorionic trophoblasts differentiate,

giving rise to several different cell types [143], that form the interhemal placental

barrier (Figure 1.3b). This barrier is comprised of four layers of cells that separate

the fetal and maternal circulatory systems. Lining the maternal blood spaces and

forming the first layer of the placental barrier are large, mononucleate, post-mitotic

cells, now considered to be a subtype of trophoblast giant cell (TGC), with a role in

hormone production [143]. Lying underneath, are two layers of ST cells, known as

syncytiotrophoblast layer II (proximal to maternal blood spaces) and

syncytiotrophoblast layer III (distal to maternal blood spaces). Formation of the ST

layers is a result of cell-cell fusion, which is mediated in part, by the apoptotic

pathway [104, 144]. At the time of CA attachment, the blood vessels and

mesenchyme — originating from the allantoic mesoderm — begin appearing within the

labyrinth [143] . The fetal endothelium is the fourth layer of the placental barrier.

The labyrinth of the mouse placenta is responsible for gas and nutrient exchange.

Studies involving gene knockout mice have underscored the crucial role the labyrinth

plays in maintaining viability of the fetus [145, 146].

The junctional zone

The junctional zone lies between the labyrinth and the TGC border and is

composed of spongiotrophoblast (SpT) and glycogen cells (GlyT; Figure 1.3a).

25

Spongiotrophoblast cells likely arise from the EPC, based on their strong similarities

in gene expression [137]. The SpT provides structure and support to the labyrinth

and consists of variable-sized, hormone-producing cells that appear to have limited

proliferative capabilities [147]. While the precise function of the SpT remains

enigmatic, targeted gene knockout studies have shown that disrupting this layer can

lead to embryonic lethality [148, 149]. Large, maternal blood channels pass through

the SpT and are lined by fetal trophoblast cells [147, 150], forming the maternal

blood spaces. These canals undergo extensive branching within the labyrinthine

layer, forming the maternal blood spaces, which are lined by trophoblast cells, as

described earlier. Although not completely established, it appears likely that SpT

cells can differentiate into GlyT [140, 143], which begin appearing at approximately

d12.5 [150]. These cells have a characteristic morphology, containing glycogen-rich

vacuoles and appear clustered together, forming islets within the junctional zone.

Later in gestation, GlyT invade the maternal decidua in an interstitial fashion, where

they enter a lytic phase at approximately d17.5, possibly supplying the surrounding

cells with an energy source for impending parturition [151].

Trophoblast giant cells

The trophectoderm of the blastocyst that is not in direct contact with the inner

cell mass is called the mural trophectoderm. From this population of cells arises the

primary TGCs (Figure 1.3a), which exit the cell cycle and undergo

endoreduplication, a process involving DNA replication without intervening karyo-

and cytokinesis [152, 153]. These cells then migrate to the antimesometrial pole of

26

the embryo and surround the future parietal yolk sac [143]. Secondary TGCs arise

from the cells of the EPC and later, from cells of the SpT [154]. These celis are

morphologically distinct, undergoing many rounds of endoreduplication, forming

large, polyploid nuclei. A clear border of secondary TGCs is evident in murine

placenta, separating the fetal junctional zone and the maternal decidua (Figure

1.3a). While not all the functions of TGCs have been fully elucidated, these cells

clearly have a role in implantation and invasion [137]. In addition, secondary TGCs

have been shown to produce luteotropic and lactogenic hormones, as well as

angiogenic factors [147], promoting maternal physiological adaptations to

pregnancy, parturition and future nourishment of the neonate [155]. While the

secondary TGCs comprising the giant cell border migrate only short distances, a

specialized subtype known as endovascular TGCs (EndoTs), are capable of

invasion and remodeling maternal spiral arteries [147]. These cells are

morphologically distinct, appearing smailer and more spindle-like in shape [156].

Moreover, EndoTs have a different gene expression profile compared with that seen

in TGCs comprising the giant cell border [143], supporting the idea that these cells

are a specialized subtype.

1.5. The mouse placenta as a model for the human placenta

Recent comparative studies between mouse and human placentae [140, 157]

have revealed striking similarities in cellular mechanisms and tissue framework,

providing justification for using the mouse placenta as an animal model for the

27

human placenta. The labyrinthine region of the mouse placenta is the site of

maternal-fetal exchange and exhibits a similar mechanism of syncytialization to

properly maintain the placental barrier [142]; thus, the labyrinth bears a similarity to

floating chorionic villi in humans (Figure 1.2). The junctional zone is considered

analogous to human column CTB cells, based on: the expression of several gene

markers; its role in structural support; its spatial distribution; and, its ability to give

rise to terminally differentiated GlyT cells [157, 158]. Trophoblast glycogen cells and

TGCs are capable of invading and directly contacting the maternal decidua and

therefore, are considered to be the murine equivalent of human EVT cells [159]. To

further support the use of the mouse as a model organism for human placentation,

recent studies have reported that during murine placentation, the maternal spiral

arteries are remodeled by specialized, invasive trophoblast cells [147, 150]. This is

identical to those events occurring during human placentation, where the spiral

arteries of the placental bed are invaded by EVT cells.

There are, of course, a number of important differences between the mouse

and human placenta. The majority of mammals, including non-human primates

exhibit superficial implantation and limited invasion of trophoblast cells into the

decidua, compared to that seen during human placentation [157]. Rodent

pregnancies are relatively brief and give birth to a litter of young that are

comparatively immature to human neonates. Additionally, the placental barrier

includes three trophoblast layers in the mouse, whereas in the human placenta, this

structure is comprised of a single layer of trophoblast and a discontinuous second

layer of CTB [140]. Nonetheless, the presence of homologous cell types and cellular

28

behaviours highlight the use of the mouse as a suitable model for the study of

normal placentation. Such a model is highly attractive, as it allows for genetic

manipulation and induction of various pathological conditions.

1.6 The placenta and fetal programming

In the last decade, substantial evidence has supported the idea that a

compromised in utero environment can influence health during postnatal life. Barker

[160] introduced the concept of fetal programming (heretofore referred to as the

Barker Hypothesis), which proposes that a number of organ structures and functions

undergo programming during embryonic and fetal life. This developmental

programming determines the physiologic and metabolic set points, which will

ultimately cue responses to the environment in the adult. Adverse in utero

conditions, such as placental insufficiency, inadequate maternal nutrition, and

altered maternal stress hormone profiles lead to developmental adaptations by the

embryo/fetus that readjust these set points (reviewed by [161, 162]). These

adaptive measures ostensibly have short-term benefits to the embryo and fetus, but

these changes to the genetically-determined body plan may confer a discordant

physiology on the adult, leading to increased risk of disease. It has been repeatedly

shown in human populations that IUGR fetuses resulting from placental insufficiency

are at increased risk for adverse short- and long-term outcomes such as

hypertension, obesity and type II diabetes, that can extend into adult life [160, 163-

165]. There is a rapidly-growing body of evidence suggesting that the placenta is

involved in fetal programming (reviewed by [166, 167]), with particular emphasis on

29

the impact of placental insufficiency on fetal cardiac development [168], fetal neural

development [169-171] and fetal endocrinology [172]. Proper establishment of the

placenta at these times allows the fetus to grow and attain its developmental

potential in the third trimester, in preparation for postnatal life. Investigations into

how normal placental cell death assists in shaping placental architecture and

function will provide us with information to better understand how aberrant or

insufficient cell death in this transient organ contributes to overall fetal, infant and

adult health.

1.7 Cell death during human placental development

During normal placentation, a balance between proliferation, differentiation

and apoptosis is required to regulate cellular homeostasis and is essential for

maintaining proper placental function. While proliferation and differentiation of

trophoblast cells have been extensively studied, only recently has work begun to

address the importance of apoptosis during placental development [132, 173]. Cell

death in normal human placentation has been implicated in villous trophoblast

turnover and syncytialization, maternal immune tolerance and EVT invasion [174].

Cell death in villous trophoblast turnover and syncytialization

In the first trimester placenta, there is a low level of cell death, with the

primary site of apoptosis within the CTB cells [175]. In the second and third

trimesters, a shift in cell death susceptibility occurs, with higher levels of apoptosis

30

observed in ST cells [132]. It has been proposed that since these cells are

transcriptionally less active, they require constant replenishment with the cellular

machinery from overlying “stem” cytotrophoblast cells [144]. Based on several

markers of cell death, it was postulated that apoptosis is initiated in villus

cytotrophoblast cells at the time of fusion, then delayed during syncytialization.

Apoptosis is finalized just prior to the extrusion of apoptotic nuclei, which is observed

as placental shedding in the form of syncytial knots. Several Bcl-2 family members

and other apoptosis-associated molecules have been proposed to be involved in

regulating this process [104]. Among these, caspase-8 was recently shown to play a

crucial role in this process [89]. In addition, c-FLIP, a negative regulator of caspase-

8 activation, is expressed in human placentae [176] and has been postulated to

have a regulatory role in syncytial fusion [177]. It is further proposed that high

expression levels of Bcl-2 and Mcl-1 are maintained in both CTB [104] and ST [178,

179] cells in order to balance this unique pathway of cellular differentiation.

Cell death in maternal immune tolerance

Regulation of cell death in human EVT is not as comprehensively studied,

due to the difficulty of obtaining the appropriate tissue and cell types by placental

bed biopsies. Moreover, isolation of pure, primary EVT has been unsuccessful and

cell lines, although well characterized, only partially resemble the EVT phenotype

[177]. Perhaps the greatest advancements have surrounded the issue of maternal

immune tolerance, in an attempt to elucidate the mechanisms by which the maternal

immune cells do not reject the allo-antigenic fetal tissue. Extravillous trophoblast

31

cells are situated in a potentially precarious environment, as these cells invade the

decidua or the maternal spiral arteries and thus, are exposed to cytotoxic, maternal

immune cells. First-trimester EVT express high levels of FasL [180, 181], in what is

believed to be a protective mechanism against activated, Fas-expressing maternal

leukocytes. These data are supported by murine studies, where examination of

implantation sites in FasL-deficient mice revealed extensive infiltration and necrosis

of maternal neutrophils and macrophages [182]. Interestingly, early trophoblasts

have also been shown to express Fas [183]; however, these cells consistently

exhibit resistance to Fas-mediated death, in a mechanism that has only been

partially resolved, and may involve the caspase-8 inhibitor, c-FLIP [19].

Cell death during trophoblast invasion

During implantation, the blastocyst must appose and adhere to the uterine

wall, after which embryonic-derived cells begin invading the endometrium in a

mechanism which involves death of maternal cells [174]. Using both rodent and

human in vitro models, it has been demonstrated that endometrial cells are

susceptible to trophoblast-induced death during the invasion phase of implantation

[184, 185]. A possible mechanism involves the Fas/FasL system, as maternal

endometrial cells express Fas on their apical surface and human trophectoderm and

early trophoblasts have been shown to express FasL [185]. A similar mechanism

has been elucidated in mice; however, this system of implantation appears to utilize

the TNFR1/TNF-a pathway. This would allow the embryo to erode the uterine

32

epithelium, advance into the decidua and gain access to the maternal blood supply

[186].

Another important event during human and murine placentation is maternal

artery remodelling by the fetal-derived trophoblast cells. In a process that has been

studied both in vitro [187] and in vivo [188], maternal endothelial and smooth muscle

cells of the spiral arteries are replaced by endovascular trophoblast. This alters the

architecture of the vascular wall, increasing maternal blood flow to the placenta

[189]. In the absence of spiral artery transformation, nutrient and oxygen transport

to the fetus is compromised, leading to pathological conditions such as preeclampsia

and IUGR [189, 190]. During invasion of the human spiral arteries, maternal smooth

muscle and endothelial cells are replaced by EVT cells in a mechanism that has yet

to be resolved. Recent reports suggest that fetal-derived trophoblasts induce death

in these cell types via the Fas/FasL pathway [191, 192], in a process that is

mediated by maternal uterine natural killer cells [193].

1.8 Cell death during murine placental development

While it is now clear that apoptosis plays a functional role in placental tissue

morphogenesis, the underlying mechanisms coordinating cell death have not been

studied in this context. The localization and extent of cell death in the rodent

placenta and how this correlates to cell death patterns in human placenta remain

largely unknown. While some sporadic in vivo [194, 195] and in vitro [196]

observations have surfaced during investigations into other placental events, the

33

majority of what is known about cell death during murine placentation has largely

been derived through comparative gene knockout mouse studies.

Tumour suppressor genes and placental cell death

Tumour suppressor genes encode for proteins that are involved in cell cycle

regulation and cellular differentiation; such proteins are often referred to as

“gatekeepers”, inhibiting or permitting cell cycle progression, depending on the

internal and external status of the cell. A number of these genes — such as p53 —

have been implicated in promoting apoptosis in various cell types, as this outcome,

or cell senescence, is usually considered preferable to the propagation of a cell with

uncontrolled proliferative potential. The mouse placenta expresses a number of

these tumour suppressors, including p53, retinoblastoma (Rb), and phosphatase

and tensin homolog (PTEN).

The p53 tumour suppressor is a ubiquitously-expressed, multi-functional

transcription factor that is activated by DNA damage [197, 198] and other cellular

stressors, such as hypoxia [199, 200]. The primary function of this protein is to

induce G, arrest [201, 202] or apoptosis by activating the appropriate target genes.

In addition to its transcriptional activity, a novel p53-mediated apoptotic pathway was

recently described, suggesting that p53 translocates to the mitochondria, inducing

mitochondrial permeabilization via interaction with members of the Bcl-2 and BH3-

only family [203, 204]. The transcriptional and apoptotic potential of p53 in murine

placental celis have been demonstrated both in vivo and in vitro. p53 has exhibited

involvement in triggering the apoptotic pathway in early, proliferating trophoblast

34

cells with compromised genomic stability [205, 206]. In vivo studies of the effects of

DNA-damage-inducing agents on rat placentation revealed that cells within the

labyrinth appear most susceptible to p53-mediated apoptosis, as evidenced by

increased levels of active caspase-3 [207, 208]. Lastly, a recent report by Soloveva

and Linzer [155], demonstrates that the loss of p53 in differentiating murine

trophoblast stem (TS) cells renders them not only resistant to DNA-damage-induced

apoptosis, but also allows them to bypass the critical G; checkpoint. This is

supported by the report that extraembryonic cells, but not embryonic cells, were

resistant to p53-dependent cell death after DNA damage [209]. p53-deficient mice

are viable, but cells derived from these animals demonstrate higher proliferation

rates in culture [210]; however, the growth dynamics of trophoblast-derived cells is

currently unknown.

Retinoblastoma (Rb) protein is also a tumour suppressor, with a prominent

role in cell cycle regulation. The phosphorylation state of Rb correlates with its

functional capacity: phosphorylation is maximal at the start of S phase (i.e. in

proliferating cells) and lowest after mitosis and entry in G, (i.e. in quiescent cells);

hence, the hypophosphorylated form of Rb suppresses cell proliferation. It was

recently demonstrated that hypophosphorylated Rb predominates in murine TS cells

deprived of Fgf-4 (a required growth factor for TS cells) and hyperphosphorylated Rb

is found in actively growing TS cells [155]; however, all forms of Rb protein were

observed to decrease in differentiating TGC in vitro [155]. Initial studies producing

Rb-null mice revealed that these mice die between embryonic days 13.5-15.5,

displaying abnormalities in erythropoiesis and central nervous system, due to

35

excessive apoptosis, failed differentiation and disruption of the cell cycle [211-213].

Additionally, it was later reported that Rb mutant placentae exhibit increased

numbers of trophoblast cells in the labyrinth, leading to disrupted architecture of this

placental region and resulting in decreased placental vascularization and transport

[146]. These data indicate that Rb mutant placentae appear to have an

unprogrammed proliferation defect within the labyrinth, offsetting the balance

between the cell death and proliferation pathways. In order to determine whether

the embryonic phenotype was secondary to a placental defect, tetraploid

aggregation and conditional knockout approaches were employed to provide Rb-

deficient embryos with wildtype placentae. Under these conditions, the knockout

embryos were carried to term, but died shortly after birth [146], suggesting that the

majority of embryonic phenotypes in Rb-deficient embryos are caused by a

dysfunctional placenta and are therefore, not due to a cell-autonomous Rb

requirement by the embryo. Rb family members bridge the cell death and cycle

pathways through the direct repression of E2F/Dp transcription factors [214]. Free

E2F/Dp heterodimers stimulate entry into the S phase of the cell cycle and either

subsequent proliferation or apoptosis, depending on the cellular context [215].

Recently, it was reported that Dp1-deficient embryos die by embryonic day 12.5 and

demonstrate increased rates of apoptosis in early extraembryonic cells [216].

Moreover, embryonic lethality caused by Dp1 deficiency was shown to be largely

due to placental defects, as late-gestation Dp1-null fetuses were obtained after

placental rescue [216].

36

PTEN is a tumour suppressor that has been shown to inhibit cell migration,

cell spreading and focal adhesion formation through interactions with focal adhesion

kinase [217, 218]. Additionally, PTEN has been shown to negatively regulate

phosphoinositide-dependent kinase 1 (PDK1), which phosphorylates and activates

protein kinase B (PKB/Akt1), triggering a well-established survival pathway [219,

220]. PTEN is ubiquitously expressed in embryonic day 7.5 mouse embryos and its

inactivation in the mouse causes embryonic lethality at d9.5 [221, 222]. Knockout

embryos demonstrate regions of increased proliferation, including the allantois, the

expansion of which is hypothesized to inhibit chorioallantoic fusion and therefore,

cause embryonic death [221]. Interestingly, murine trophoblast and endothelial cells

have been demonstrated to express PKBo/Akt1, and the consequences of

PKB/Akt1-deficiency include reductions in the decidual basalis, glycogen-containing

SpT and vascularization [223]. These defects indicate a deficiency in placentation,

leading to the observed fetal growth reduction, neonatal lethality and diminished life

span after exposure to genotoxic stress [223]. Lastly, it has also been shown that

PKB/Akt1 signalling is involved in differentiation of murine TS cells to TGC in vitro

[224]. Since TGCs in the mouse placenta are involved in the production of

numerous protein and steroid hormones, it is possible that reduced placental and

fetal growth may be due to the impaired differentiation of this cell type.

Cell death-associated genes and placentation

While many cell death regulatory molecules are expressed in the mouse

placenta, their roles in this organ have not been functionally characterized. Bruce

37

(baculovirus inhibitor of apoptosis repeat-containing [BIR] ubiquitin-conjugating

enzyme) is a large (528 kDa) protein that has both an N-terminal BIR domain and a

C-terminal ubiquitin-conjugating domain (UBC); these regions provide Bruce with

both antiapoptotic [225-227] and ubiquitylation capabilities [227, 228]. There are

several inhibitor of apoptosis (IAP) proteins, including Bruce, which function as cell

death antagonists by suppressing pro-apoptotic proteins such as Smac and active

caspase-9 [227, 228]. The murine Bruce gene is highly conserved, as human

APOLLON shares 92% identity with Bruce and has been demonstrated to confer

chemotherapeutic resistance to certain cancer cells [226]. Lotz et al. [229] reported

that Bruce is highly expressed in the labyrinth and SpT of the placenta; lower

expression levels were observed in TGCs. Analysis of earlier developmental stages

revealed that Bruce can also be detected in the chorion, within the cells of the EPC

and in early TGCs as well as in the late gastrula stage embryo [230, 231].

Three separate groups of investigators have produced Bruce-deficient mice,

with two groups observing a trophoblast proliferation-related phenotype and no

alterations in cell death [229, 230]. The third group also observed proliferation

defects but additionally reported increased rates of apoptosis in Bruce-deficient

placentae [231]. Loss of Bruce leads to embryonic and/or perinatal growth reduction

and lethality, which can likely be attributed to the observed placental defects.

Proliferation was severely reduced in the SpT [229, 230], and was diminished in the

labyrinth [229]. In contrast, Ren et al. [231] reported defects in trophoblast cell

death, with elevated levels of Bax, Bak and caspase-2, and activation of the

mitochondrial cell death cascade in embryonic fibroblasts obtained from mutant

38

embryos. Moreover, p53 expression was also shown to be elevated in Bruce-

deficient placentae, particularly in SpT cells, and silencing of p53 and Bruce

expression in cell lines resulted in improved cell viability [231]. Thus, p53 appears to

acts as a downstream effector of Bruce, and in the absence of Bruce,

mitochondrially-mediated apoptosis ensues [231]. The conflicting results of these

studies is perhaps, not so unexpected in hindsight. Bruce is a chimaeric molecule,

with both ubiquitylation and apoptois-inhibiting capabilities, underscoring the

multifunctional nature of this protein. Yeast homologs of Bruce have likewise been

shown to be involved in cell division [232, 233]. On the other hand, as previously

stated, Bruce is also clearly associated with regulation of the apoptotic pathway and

further molecular analysis into the nature of this enigmatic — yet exciting — molecule

in the right cellular context is required in order to further elucidate the function of

Bruce during mammalian placentation.

Daxx (Fas death domain-associated protein) was initially reported as a highly

conserved protein, interacting with the intracellular domain of Fas and enhancing

Fas-mediated apoptosis in overexpression studies [234]. As is the case with Bruce,

Daxx is a multifunctional protein with seemingly contradictory functions. It has been

shown to be involved in both extrinsic (TGF-B -mediated) and intrinsic (p53-

dependent DNA damage) apoptosis pathways [235, 236]. In addition to these cell

death functions, Daxx is also capable of transcriptionally repressing CRE, E2F1,

Sp1, NF-«B and the androgen receptor [237, 238], demonstrating further potential in

modulating cellular behavior. Daxx is expressed in a number of murine [239] and

human tissues [240], including the placenta. Daxx deficiency in mice leads to

39

embryonic lethality by day 9.5 and both embryonic and extraembryonic lineages are

diminished in comparison to wildtype littermates, marked by increased rates of

apoptosis by day 7.5 and day 8.5 [241]. This enhanced apoptosis was unexpected,

since Daxx had until then, only been associated with promoting apoptotic events in

the cell; however, further investigation revealed that Daxx mutant embryonic stem

(ES) cell lines also had elevated rates of apoptosis. It was later revealed that a

similar Daxx deletion triggered cell death by stimulating the JNK/p38-Bim-Bax

pathway, leading to the activation of caspase-9 and caspase-3 [242]. Lastly, Daxx

has recently been reported to play a role in viral protection, as Daxx-null fibroblast

cell lines demonstrated enhanced viral gene expression compared to Daxx-

complemented cells [243]. Therefore, it is intriguing to speculate that in addition to

the role of Daxx as a regulator of cell death in the placenta, it may also be involved

in protecting the trophoblast and hence, the fetus, from viral invasion.

Prostaglandin Fz (PGF2a) receptor (FP) is a G-protein-coupled receptor that

has been shown to induce the apoptotic cascade by activating caspase-8 in luteal

cells [244]. Interestingly, FP lacks an intracytoplasmic region possessing any of the

traditional death or caspase-recruiting domains that characterize other, typical death

associated receptors. Prostaglandin Fz, receptor is highly expressed in the uterus

[245], but it has also been shown to be expressed in the mouse [246] and human

[239] placenta. Homozygous deletion of FP resulted in developmentally normal and

viable mice, but FP-deficient females failed to deliver their fetuses at term, leading to

in utero death, followed by resorption [247]. No changes were detected in mutant

placental and decidual weights, nor were there any disparities in placental cell death

40

patterns; however, elevated decidual cell death over gestation was noted [194].

Further characterization of phenotype revealed alterations of decidual cell death

patterns once post-term fetuses were categorized as either live or dead. Elevated

decidual cell death in dead fetuses was associated with alterations in the Bax:Bcl-2

ratio and increased active caspase-3 levels [248]. Given these observations, it was

hypothesized that decidual cell death was necessary for normal term delivery of the

conceptus and that a Bax:Bcl-2 “rheostat” is involved in regulating apoptosis in the

postterm placenta [248]. On the contrary, upregulation of Bcl-2 was reported in the

decidua of abortion-prone mice, perhaps serving as a compensatory or protective

mechanism [249], especially considering the premature “delivery” of these preterm

placentae.

It is evident that cell death plays a key role in the appropriate maturation of

the placenta and that pro- and anti-apoptotic expression patterns can be

manipulated in the fetal-placental-maternal unit in order to optimize the uterine

environment for healthy gestation.

1.9 Maternal exposure to cigarette smoke, polycyclic aromatic hydrocarbons and placentation

Polycyclic aromatic hydrocarbons (PAH) such as 7,12-

dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (BaP) are released into the

environment as a result of incomplete combustion of fossil fuels; however, the

primary route of human exposure to these compounds is cigarette smoke [250].

Epidemiological studies have revealed that exposure to pollution and smoking during

41

pregnancy is associated with many adverse outcomes, including intrauterine fetal

growth restriction, preterm delivery and increased perinatal mortality [251-253].

However, the association between exposure to tobacco products and miscarriage

has yet to be firmly established. While some studies have reported an increased

risk of spontaneous abortion due to smoking during natural or assisted conception

[254-256], others have reported that no significant association exists [257]. These

inconsistencies may be due to inaccurate self-reporting on the part of the subjects,

the varying numbers of cigarettes smoked from subject to subject and the time at

which the miscarriage occurred, as earlier abortions may go undetected.

Another smoking-related pathology that has been observed in human

populations is delayed conception and infertility. Several epidemiological studies

have reported reduced pregnancy rates in women who were currently smoking,

compared to women who were never smokers and ex-smokers (i.e. those who had

ceased smoking for one year before attempting conception) [258-260]. One study

further demonstrated that women exposed to environmental tobacco smoke were

more likely to fail to conceive within six months, compared to non-exposed women

[261].

Cigarette smoking during pregnancy also influences the placental

vasculature, resulting in reduced dimensions of the fetal capillaries [262, 263] and

increased uterine artery resistance [264]. This is consistent with a reduction of

surface area and length of villous capillaries in placenta of smokers at term [265], as

well as the observed decrease in ST apoptosis [175, 266], likely a consequence of

abnormal trophoblast turnover. The observed reduction in fetal capillary dimensions,

42

possibly due to insufficient remodelling of maternal spiral arteries into vessels of low

resistance, affects placental blood flow [267]. Reduced placental perfusion has

been implicated in poor exchange of gases and nutrients between fetus and mother

[268], possibly leading to IUGR.

Several reports have indicated cellular and trophoblast differentiation defects

in placentae of heavy smokers accompanied by alterations in the cell death rates

[175, 269]. Cytotrophoblast cells, isolated from first trimester placenta of smoking

mothers, have reduced invasive potential and poor differentiation capabilities in vitro

[269]. This is accompanied by an increase in the number of columns of

cytotrophoblast origin that failed to reach the uterus [270]. Gruslin et al. [175],

reported an increased rate of apoptosis in smokers during first trimester, but

decreased rates at term. These findings suggest that trophoblast cells from first and

third trimester placenta may differ in cell death susceptibility, which may be due to

changes in the expression of different sets of cell death genes.

Cigarette smoke contains many different chemical compounds, most of which

have unknown biological activity. While some studies determined nicotine as the

cause of uteroplacental insufficiency [269, 270], these predictions were recently

questioned since no adverse maternal or fetal outcomes were observed in patients

undergoing nicotine replacement therapy during pregnancy [271]. Thus, it was

concluded that the myriad of cellular and molecular abnormalities observed in

placentae and newborns of smoking mothers could be caused by exposure to one or

more of the other thousands of chemicals found in cigarette smoke or by their

metabolic by-products. This is consistent with observations of significant

43

accumulation of PAHs in placenta [272], leading to DNA adduct formation and

altered activity of harmful downstream enzymes induced by PAHs in placental

samples of either smokers [273], or women living in areas of significant

environmental pollution [252, 274].

Animal studies using various types and sources of PAHs have also shown

that exposure to these toxins can lead to unfavourable reproductive outcomes. It

was recently shown in rats, that mid-gestation inhalation of aerosolized BaP resulted

in decreased fetal survival in a dose-dependent manner [275]; similar results have

also been reported in pregnant rats [276] and mice [277] exposed to BaP.

Resorption of the entire litter was observed in pregnant mice orally exposed to

carbon black oil, which is a petroleum refinery by-product, containing several classes

of hydrocarbons, including PAHs [278].

While there is a plethora of animal studies involving the exposure of pregnant

animals to PAHs, there is virtually no data on the reproductive outcome after

chronic, maternal exposure to PAHs. Previous studies in rats have determined that

after intravenous BaP exposure, this chemical demonstrated a long half-life in a

number of different tissues [279], and that chronic, oral exposure of BaP (50-100

mg/kg) leads to persistence of DNA adducts in liver and lung tissue [280].

Therefore, it would appear that PAH exposure prior to pregnancy has long-lasting

effects, likely due to accumulation of these compounds and their metabolites in

various tissues.

44

1.10 The aryl hydrocarbon receptor

There are several possible ways that PAHs can alter cell function at the

molecular level. These compounds are capable of directly altering DNA structure via

formation of DNA adducts [281] resulting in DNA mutations in affected cells. In

addition, PAHs interact with aryl hydrocarbon receptor (AhR), a member of the basic

helix-loop-helix family of transcription factors, having a broad range of tissue

expression [282]. AhR is an intracellular receptor that binds aryl hydrocarbons and

plays a major role as a xenobiotic sensor, inducing expression of metabolic enzymes

to rid the cell of these potentially harmful compounds. In the absence of ligand, AnR

exists in the cytosol associated with heat shock protein 90 (HSP90) and XAP2, an

immunophilin-related protein (Figure 1.4). Extracellular ligands, such as PAHs,

diffuse through the plasma membrane and bind to AhR, causing a conformational

change that exposes a nuclear localization sequence. The entire complex

subsequently translocates to the nucleus, whereupon the AhR-ligand complex

dissociates from the associated proteins and binds to aryl hydrocarbon nuclear

translocator (Arnt). At this point, the AnR-Arnt complex is transcriptionally active and

binds to specific DNA sequence called dioxin response, or AnR response elements

(DREs/AHREs), resulting in the expression of Phase | and Phase I! detoxifying

enzymes [283]. AhR appears incapable of homodimerizing and partners only with

Arnt. This is supported by in situ hybridization studies of murine embryos over

gestational timepoints, where Arnt was revealed to have overlapping expression in

those tissues expressing AhR [284].

45

a .

LO ligand “ binding

Lo OS NS

.

\ |

\ eo ° j

‘Transcription of PhaseJ’and Phase Ii \ f detoxification enzymes; \ other transcribed genes include those - 3 \ ‘ involved in cell cycle, differentiation and growth. control, apoptosis

~N 4

Nucleus

Figure 1.4 Schematic representation of the aryl hydrocarbon receptor pathway.

46

A great deal of cross-talk exists between AhR and other cellular pathways

signalling proliferation [285], cell cycle arrest [286], hypoxia [287] and response to

hormones [288], leading to a diverse range of biological outcomes. While the

majority of studies investigating ANR function have revolved around its ligands, it has

become increasingly clear that AnR does not need an exogenous ligand to activate

its signaling pathway. This was initially observed during studies in murine embryonic

palate development [289] and later supported by targetted gene knockout of AHR.

Homozygous mutant mice are viable and fertile, but are smaller, exhibit reduced

fecundity and have hepatic, hematopoietic and immune defects [290, 291]. In

addition, AhR-deficient mice are resistant to BaP and dioxin exposure. Currently,

there is no information as to a possible placental phenotype due to AMR deficiency;

however, both Arnt and AhR mRNA transcripts exist in a number of different female

reproductive tissues in both humans and rodents [292-295], with high expression

levels being reported in the placenta [292, 293, 296].

In contrast to AhR, Arnt has multiple binding partners and is involved in

various signalling pathways, regulating cellular responses to hypoxia, angiogenesis

[297] [298] and embryonic development [299]. Targetted gene knockout of Arnt in

mice resulted in embryonic lethality at d10.5, exhibiting defects in vascularization

and embryonic development [298, 300]. Moreover, Arnt-deficient placentae were

reported to have greatly reduced labyrinth and SpT, and increased numbers of

TGCs [300], suggesting that Arnt may play a role in trophoblast cell fate. In light of

the results reported after gene knockout studies, an additional role for AnR during

development of both embryonic and adult tissues was proposed. Robles et al. [301]

47

demonstrated that AnR was required for normal ovarian germ cell endowment, as

AhR-deficient mice had twice the follicular reserve as their wildtype counterparts. In

addition, cardiac hypertrophy [302] and patent hepatic ductus venosus [303] were

reported in adult mice deficient in ANR. Moreover, mice transgenic for a

constitutively active AnR have a reduced lifespan and are prone to gastric tumours

[304]. However, while the existence of an AnR-regulated pathway during normal

development appears likely, the identities of possible endogenous ligands and

developmental cues remain enigmatic.

The aryl hydrocarbon receptor has been postulated to be highly tolerant to

evolutionary adaptations. A number of studies have reported the existence of one

copy of AhR in invertebrates such as C. elegans [305], D. melanogaster [306] and

several mollusks (reviewed in [307]); however, the AhR found in invertebrates does

not bind the typical ligands seen in vertebrates. Interestingly, non-mammalian

vertebrates (e.g. birds, reptiles, fish, amphibians) possess two copies of AhRs that

mediate xenobiotic sensing and activate transcriptional machinery resulting in the

profound phenotypic consensequences due to dioxin exposure that are observed in

these animals (reviewed in [308]). The mammalian genome has one copy of AhR,

which has also exhibited diversity at both the gene and protein level. Human AhR,

in addition to some of its target genes such as CYP 1A1 and GST M1 [809], has

been shown to be highly polymorphic (reviewed in [283, 310]). The consequence of

such genetic polymorphisms in AhR results in inter-individual variations in PAH

metabolism [309] and unpredictable susceptibilities to diseases such as cigarette-

smoking-related lung cancer [311] and hypertension [312]. Both mouse and rat AhR

48

exhibit polymorphic gene sequences (reviewed in [283, 310]), yielding the highly

variable phenotypes that are observed after dioxin or PAH exposure in these

animals. In fact, four murine AhR alleles exist, originally discovered in “dioxin-

resistant” DBA/2 and “dioxin-sensitive” C57BI/6 mice [313]. Examination of the AhR

gene in 13 mouse lines confirmed the identification of these four alleles and

evolutionary analyses yielded a high tolerance to nucleotide substitution in murine

AhR [814]. Thus, AhR is subject to a high degree of evolutionary pressure within a

number of evolutionarily divergent organisms.

1.

49

HYPOTHESES

Murine placental cell death is a regulated event, required for optimal

placentation and dysregulation of this process affects embryonic and fetal

development.

Chronic maternal exposure to polycyclic aromatic hydrocarbons prior to

conception will interfere with normal cell death, perturbing normal placental

development, which will have deleterious consequences on the offspring.

The aryl hydrocarbon receptor and its downstream, pro-apoptotic target, Bax,

are involved in regulating murine placental cell death and deficiencies in

these genes will rescue the observed PAH-induced embryonic and fetal

phenotypes.

50

CHAPTER 2

MURINE PLACENTAL CELL DEATH EXHIBITS AN ORGANIZED PATTERN

OVER GESTATION AND PLACENTAL DEFICIENCY OF PRO-APOPTOTIC BAX

LEADS TO ALTERED LABYRINTHINE ARCHITECTURE AND IUGR

51

CHAPTER 2: Murine placental cell death exhibits an organized pattern over gestation and placental deficiency of pro-apoptotic Bax leads to altered labyrinthine architecture and IUGR

2.1 Abstract

While cell death in the human placenta has been recognized as a physiological

event, virtually no studies have been done on the mouse placenta. Herein, we

report the results of a systematic and quantitative examination into cell death

patterns in both ICR and C57BV/6 placentae, over gestation. Furthermore, we

investigated the effects of Bax deficiency on murine placentation and the

consequent outcome of this deletion on the fetus. Over gestation, ICR and C57BI/6

placentae exhibited TUNEL-positive patterns similar to those observed in human

placentae, with sporadic, infrequent death observed in early placentae, which

increased towards term. The most striking observation was the organized pattern of

TUNEL-positive cells surrounding the vessels of the labyrinth at mid-gestation, with

an apparent role in remodeling the vasculature of this region. While cell death

patterns were similar for both strains of placentae examined, C57BI/6 placentae

demonstrated greater numbers of dead cells in almost all placental regions, starting

at d13.5. Bax, a pro-apoptotic, Bcl-2 family member, immunolocalized to a subset of

trophoblast giant cells (TGCs) and to cells within the labyrinth. Bax deficiency in the

mouse placenta resulted in an altered labyrinthine architecture, leading to fetal

intrauterine growth restriction and additionally, revealed a role for Bax in the

programmed cell death (PCD) of trophoblast giant cells (TGCs).

52

2.2 Introduction

Programmed cell death has been shown to be an important part of a large

number of biological processes, including embryogenesis, organogenesis, tissue

remodeling and normal cellular homeostasis [315]. Hallmark features of apoptosis

include cell/nuclear shrinkage, chromatin condensation, DNA fragmentation and

membrane blebbing, leading to the production of apoptotic bodies, which are

efficiently eliminated by phagocytosis [316]. However, cells do not always die along

the apoptotic pathway and may exhibit signs of autophagy (characterized by

autophagic vacuolization of cytoplasmic contents and lack of chromatin

condensation), necrosis (cytoplasmic swelling, plasma membrane rupture, moderate

chromatin condensation, leaky nuclear envelope), or may display combined features

of several types of cell death, such as aponecrosis or apoptosis accompanied with

autophagy (for reviews, see [73, 317]. Alterations in cell death rates are known to

have pathological consequences, with a deficiency in cell death resulting in cancer

or autoimmune disorders and an augmentation or acceleration of cell death leading

to degenerative diseases. Thus, a fine balance in the loss and production of new

cells is essential for all organs, particularly for those that depend on tissue

remodeling.

During normal placentation, proliferation, differentiation and apoptosis

contribute in a balanced fashion to maintain homeostasis and proper placental

function. While proliferation and differentiation of trophoblast cells have been

extensively investigated, only recently have studies begun to address the

importance of cell death during normal and abnormal placentation in human [131,

53

132]. In a healthy placenta, cell death has been implicated in regulation of

trophoblast turnover. Syncytiotrophoblast cells require constant replenishment by

overlying “stem” cytotrophoblast cells [144] and this renewal is driven by molecules

regulating the cell death cascade. It has been postulated that apoptosis is initiated

in villus cytotrophoblast cells at the time of fusion, delayed during syncytialization,

then subsequently finalized just prior to the extrusion of apoptotic nuclei [104], which

are termed syncytial knots. This process of cell fusion uniquely provides the

transcriptionally quiescent ST with new materials to maintain the maternal-fetal

exchange unit. Only recently has a functional link between cell death and placental

vasculogenesis been established in humans [318]; however, the localization and

extent of cell death in the rodent placenta, and how this compares with cell death

patterns in the human placenta remain largely unknown.

During invasion of the spiral arteries, maternal smooth muscle and endothelial

cells are replaced by EVT cells in a mechanism that has still yet to be resolved.

Recent reports suggest that fetal-derived trophoblasts induce death in these cell

types via the Fas/FasL pathway [191, 192], in a process that is mediated by

maternal uterine natural killer cells [193]. Extravillous trophoblast cells are situated

in a potentially precarious environment, as these cells invade the decidua or the

maternal spiral arteries and thus, are exposed to cytotoxic, maternal immune cells.

In what is believed to be a cytoprotective mechanism against Fas-expressing

maternal leukocytes, first-trimester EVT express high levels of FasL [180, 181].

Interestingly, early trophoblasts have also been shown to express Fas [183];

however, these cells consistently exhibit resistance to Fas-mediated death. On the

54

contrary, a recent study reports the susceptbility of EVT to interferon-y-induced

death — a cytokine produced by maternal uNK cells — impeding EVT invasion and

maternal spiral artery remodelling [319]. This is supported by studies of tubal

pregnancy, where decreased rates of EVT apoptosis have been attributed to the

altered immune microenvironment [320].

Comparative studies between mouse and human placentae have

demonstrated striking similarities in the molecular and cellular framework during

development of this tissue. Between 7.5-9.5 days post coitum, the developing EPC

undergoes major morphological remodeling, resulting in the formation of three

distinct cellular regions, each with unique morphology and function (for review [136]).

The fusion of the allantois with the chorion and subsequent branching, establishes a

network of villi surrounded by small canals collectively referred to as the labyrinth.

This region is responsible for gas and nutrient exchange, and thus bears a similarity

to floating chorionic villi in human. The junctional zone consists of variable-sized

SpT cells with the capability of differentiating into secondary TGCs and GlyTs. Due

to the expression of several gene markers, as well as to their spatial distribution, the

cells of this region are considered to be analogous to human cytotrophoblast cells

found in columns [158]. The two invasive trophoblast cell types in the mouse

placenta are GlyTs, a cell having distinct morphology but unknown function, and

TGCs. These latter cells invade the endometrium and are positioned in direct

contact with the maternal decidua; thus, TGCs are analogous to human extravillous

trophoblast cells. Trophoblast giant cells have distinctively large, polyploid nuclei

and are efficient in producing several key regulatory, luteotropic and lactogenic

55

hormones, as well as angiogenic factors [159]. In addition, it was recently reported

that a subset of specialized murine TGCs are capable of invasion and remodeling

maternal spiral arteries [147], a physiologically important event that also occurs

during human placentation.

Here, we report the results of a systematic analysis of the temporal and

spatial distribution of dying cells throughout gestation in the placentae of two

different strains of mice. In addition, we have established the expression profile of

several cell death-related markers in the placentae of both mouse strains and we

describe a placental phenotype in mice deficient in Bax, a pivotal, pro-apoptotic Bcl-

2-related gene.

2.3 Materials and Methods

2.3.1 Animal housing, mating and tissue collection

Six-week-old ICR, (Harlan, Indianapolis, IN, USA), C57BI/6 (National Cancer

Institute, Frederick, Maryland, USA) and Bax heterozygous (C57BI/6 [49]) virgin

females were mated with the appropriate male stud (i.e. ICR, C57BI/6 and Bax

heterozygous males) of proven fertility. Gestational age was determined based on

the presence of a vaginal plug, with the morning of detection being day 0.5 (d0.5)

post coitum (pc). Animals were maintained in a controlled room with a 12h light: 12

h dark cycle and allowed ad libitum access to rodent chow and water.

Pregnant ICR and C57BI/6 females (from d7.5-d18.5) were euthanized by

cervical dislocation and the uterine horns were removed. Approximately 3-5

56

conceptuses were collected with the uterine tissue still intact and immersion-fixed in

ice-cold 10% phosphate-buffered formalin for 24 hours. Fixed tissue were

subsequently washed 2 x 1 hour in PBS and stored in 70% ethanol at 4°C. The

remaining uterine horn was placed in phosphate-buffered saline (PBS) and the

uterine tissue was torn open with forceps and conceptuses teased out. Decidual

tissue was removed from d7.5 and d8.5 EPC and discarded, while the decidua was

left intact on d9.5-d18.5 placentae; after determining normal development by

morphological assessment, embryos/fetuses were discarded. Ectoplacental cones

from the same dam were pooled in order to have enough tissue for immunoblotting

analyses. Tissues for immunoblotting and caspase-3 assays were stored at -80°C.

Bax heterozygous females were euthanized at d15.5 and d18.5; uterine horns

were collected in PBS, and conceptuses were freed from uterine tissue as described

above. Wet weights of fetuses and placentae (with attached decidua) were recorded

and placentae were either frozen on dry ice or fixed in ice-cold, 10% phosphate-

buffered formalin for 24 hours. A piece of fetal forelimb tissue was removed,

washed in PBS, placed on dry ice and stored at -20°C for genotyping, as previously

described [321]. All animal experiments were conducted using protocols approved

by the Animal Care Committee at the Samuel Lunenfeld Research Institute, Mount

Sinai Hospital.

2.3.2. Terminal deoxynucleotidyl transferase dUTP nick-end labeling

Three healthy conceptuses (d7.5) or placentae (d9.5-d11.5, d13.5, d15.5 and

d18.5) from three different ICR or C57BI/6 dams were embedded in paraffin using

57

routine histological techniques; d7.5 conceptuses were embedded whole, while

later-gestation placentae were cut slightly lateral to the midline and the larger half

was embedded to obtain transverse sections. Tissue blocks containing d7.5

conceptuses were serially sectioned at 5 um thick and examined under the

microscope before staining. Those sections containing the EPC at the approximate

midline and sections corresponding to 50 and 100 um distant to one side of the

midline, were chosen for each sample to be used for TUNEL staining. Tissue blocks

containing placentae were trimmed until just before the midline (estimated by the

presence of the umbilical cord) and 5 um serial sections were cut until approximately

150-200 ym of tissue had been sectioned. Those sections at midline, and at 50 ym

and 100 um from midline were used for TUNEL staining.

Sections were deparaffinized and treated with 10 g/mL proteinase K

(Invitrogen, Burlington, ON, Canada) in PBS for 13 minutes, followed by brief

washes with MilliQ water and PBS. Endogenous peroxidase was quenched in 3%

H2O2 in methanol for 30 minutes, slides were washed in PBS and pre-equilibriated

for 10 minutes at room temperature in a solution of 1x One-Phor-All PLUS buffer

(GE Healthcare, Baie d'Urfe, QC, Canada) supplemented with 0.1% Triton X-100

(Sigma, Oakville, Ontario, Canada). TUNEL reaction mixture was prepared with the

following final concentrations, diluted in 0.1% Triton X-100: 1 x One-Phor-All PLUS

buffer, 10 uM biotin-16-dUTP (Enzo Life Sciences, Farmingdale, NY, USA), 1 uM

dATP (Fermentas, Burlington, ON, Canada) and 20 IU of terminal deoxynucleotidyl

transferase, FPLC™ pure enzyme (GE Healthcare). Sections were incubated with

the TUNEL reaction mixture in a humidified chamber for 90 minutes at 37°C and

58

then washed with PBS. Streptavidin-horseradish peroxidase reagent (Vector

Laboratories, Burlingame, CA, USA) was used for detection of incorporated,

biotinylated nucleotides and the colour reaction was developed using

diaminobenzidine (DAB) substrate (Sigma). Sections were counter-stained with

methyl green, dehydrated and mounted. Histomorphometric analyses were done on

each placental section, with the researcher blind to the strain of mouse and

gestational timepoint, using a Zeiss 9901 microscope, a Retiga 1300 camera and

BioQuant® Software. For d7.5, the conceptus was divided into regions of

mesometrial decidua, anti-mesometrial decidua and EPC; all TGCs and allantoic

cells were counted in each section. For all other timepoints, placental regions were

divided into chorionic plate (CP) and labyrinthine regions, the junctional zone (area

between the labyrinth and giant cell border) and the maternal compartment (area

between the giant cell border and the myometrium; see Figure 2.1). All TGC ells

were counted for each section, except for those that were contained within the

extreme lateral edges of the placenta, adjacent to the decidua parietalis. Tissue

areas were determined at 25x, 100x or 500x magnification, depending on the size of

the region; TUNEL-positive cells were counted at 500x magnification, scanning each

section in its entirety. If a group of dead cells in the maternal compartment

contained greater than nine TUNEL-positive cells, these were considered to be focal

points of death and the area of these foci was determined at 500x magnification.

The rate of this type of death was expressed as a percent of the area exhibiting

TUNEL staining, over the total area of the tissue in the maternal compartment. The

different data sets for the three sections were averaged to obtain final areas and cell

59

Maternal compartment

TGC border

Junctional zone

Labyrinth

Chorionic plate

i oe : 1mm f sANS.

d15.5 ICR placenta outlining approximate regions of measurement for d10.5 - d18.5 placenta

Figure 2.1 Low-magnification placental section of d15.5 ICR placenta,

demonstrating various regions of placenta used for histomorphometry.

Masson trichrome-stained section was used for this diagram as methyl green-

stained sections were too faint at low magnification for visualization.

60

death numbers for each placenta. Photomicrographs were taken using a Leitz

DMRXE microscope, a Sony DXC-970MD camera and Northern Eclipse® software.

2.3.3 Giant cell counts in Bax-deficient placentae

Formalin-fixed Bax WT and KO littermate d15.5 and d18.5 placentae were

embedded in paraffin using routine histological procedures; an approximately

midline section was labeled using the TUNEL procedure described above. Each

section was examined blind using a Zeiss 872 E compound microscope, at 400x

magnification and the number of TUNEL-positive and -negative cells per placental

section was counted.

2.3.4 Immunohistochemistry and lectin histochemistry

Sections were deparaffinized in xylene, rehydrated and underwent microwave

antigen retrieval in 10 mM citrate buffer, pH 6.0. Sections were allowed to cool

down to room temperature, washed, then blocked with 10% horse serum + 10%

BSA in PBS with 0.1% Tween 20 (PBST). Anti-active-caspase-3 antibody (Cell

Signalling, Danvers, MA, USA) was diluted 1:500 in 5% horse serum + 5% BSA in

PBS. Slides were then held at 4°C overnight, washed in PBS, followed by a 2-hour

incubation at room temperature with biotinylated anti-rabbit antibody (1:200 dilution

in 5% horse serum + 5% BSA) from a VectaStain ABC kit (Vector Labs). The ABC

solution was prepared according to the manufacturer’s instructions and detection

61

using DAB substrate was done as described above, followed by counterstaining with

hematoxylin.

Immunofluorescence using anti-Bax NT antibody (Upstate, Lake Placid, NY,

USA) was similar to the method described above, except an autofluorescence

quenching step was added before blocking (slides held in 0.1% Sudan Black in 70%

ethanol for 30 minutes, followed by washes in PBS) and the step to quench

endogenous peroxidase was omitted. In addition, streptavidin, conjugated to

fluorescein isothiocyanate (FITC; Chemicon, Temecula, CA, USA) was used for

detection, nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI;

Sigma) and slides were mounted with anti-fade medium (Vector Labs). Sections

were examined and imaged on a deconvolution microscope (Olympus IX70, Applied

Precision Inc., lssaquah, WA, USA), using FITC and DAPI filters. Images were

acquired using DeltaVision Software (Applied Precision Inc., Issaquah, WA, USA)

and the DAPI channel was assigned a red wavelength to allow for greater resolution

of photomicrographs.

Lectin histochemistry was used to identify the fetal endothelium. The

methodology used was similar to that described for immunohistochemistry, except

antigen retrieval was omitted and secondary antibody was not needed. Instead,

sections were blocked and probed with 50 ug/mL biotinylated Bandeiraea

simplicifolia (Sigma) for 1 hour at room temperature. This was followed by

peroxidase quenching using 1% H2O2 in PBS, then washes in PBS. Detection of

reaction using the ABC complex and DAB substrate was done as described above.

62

Images of sections after immunohistochemistry and lectin histochemistry were taken

using the same instrumentation as that described for TUNEL photomicrography.

2.3.5 Caspase-3 enzyme assay

Enzymatic activity of caspase-3 in murine placental tissues was assessed

using the Caspase-3 Cellular Activity Assay Kit PLUS (Biomol, Plymouth Meeting,

PA, USA). Briefly, d15.5 placentae from both ICR and C57BI/6 mice (1 placenta

from each of n=4 dams) were dissected into either maternal-enriched or fetal-

enriched fractions (Figure 2.10a), weighed and homogenized in cell lysis buffer at a

ratio of 1 mg tissue: 5 wL lysis buffer. Protein concentration was determined using

the Bradford assay (BioRad, Mississauga, ON, Canada) and 25 ug of total protein

_lysate was used for each sample in the assay. Thereafter, the assay was performed

according to the manufacturer’s protocol, using the colorimetric, pNA substrate

provided. Absorbance readings were obtained using a pQuant microtitre plate

reader (Molecular Devices, Sunnyvale CA, USA), with readings taken every 10

minutes for a total of 120 minutes; the assay plate was held at 37°C between

readings. Calculation of enzymatic activity was done using the slope of the linear

portion of the time course.

2.3.6 Western blotting

To assess protein expression in ICR and C57BI/6 placentae, two to three

placentae from each dam were placed in PBS containing a Complete™ protease

63

inhibitor tablet (Roche, Laval, QC, Canada), dissolved according to manufacturer's

instructions. Pooled placentae were weighed, placed on ice and 1 x SDS sample

buffer (62.5 mM Tris-HCl, pH 6.8; 2% w/v SDS; 10% glycerol; 50 mM DTT; and

0.01% w/v bromophenol blue) was added at a ratio of 1 mg tissue: 9 uL sample

buffer. The tissue was vigorously homogenized over ice, followed by several

passages through an 18 % gauge needle and a 23 % gauge needle fitted to a

syringe, in order to more thoroughly homogenize the tissue and shear the DNA. If

not used immediately, samples were stored at -80°C. Individual d15.5 Bax WT

(n=5) or KO placentae (n=5) were similarly treated, except decidual tissue was

removed (Figure 2.10a) prior to weighing and homogenization. Lastly, samples of

labyrinthine tissue from WT, KO or heterozygous (Het) d14.5 and d18.5 placentae

were “punched out” (Figure 2.12b) using a 2-mm Keyes tissue punch (Roboz

Surgical Instruments, Gaithersburg, MD, USA).

Standard denaturing acrylamide gels of varying concentrations and

electrophoresis buffer were prepared according to instructions provided by the

manufacturer of the electrophoresis apparatus (Novex). For each lane, 8.5 UL of

sample was mixed with 8.5 pL of 1 x SDS buffer supplemented with B-

mercaptoethanol (Sigma) at a ratio of 49:1. Samples were then held at 100°C for 3

minutes, allowed to cool and centrifuged briefly; 15 pL of sample was loaded per

lane. Proteins were transferred to 0.2 um Biodyne nitrocellulose (VWR) and blots

were stained with Ponceau S (Sigma) and then blocked for 1 hour with 5% skim milk

in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBST) for 1 hour. The following

primary antibodies were used: anti-caspase-3 (1:500; Cell Signalling); anti-Bax NT

64

(1:500); anti-placental lactogen | (1:100; Chemicon); anti-placental lactogen II

(1:100; Chemicon); anti-cleaved Parp-1 (1:500, Cell Signalling); and anti-PECAM-1

(1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Blots were stripped and

reprobed with anti-B-actin antibody (1:400, Santa Cruz) to correct for protein loading.

Each antibody was diluted in 3% skim milk in TBS and membranes were held

overnight at 4°C, with gentle shaking. Blots were then washed 4 x 5 minutes with

TBST and probed with a solution of 1:1000 of appropriate secondary antibody

conjugated with alkaline phosphatase (AP; BioRad) in 3% skim milk in TBS. After 4

X 5-minute washes with TBST, membranes were incubated with ECF substrate (GE

Healthcare) and scanned on a STORM imager (Molecular Devices). Densitometric

analyses were done using ImageQuant® software.

2.3.7 Statistical analysis

ICR and C57BI/6 placental cell death rates and protein expression levels over

gestation within one strain and between the two strains were analyzed by one-way

and two-way ANOVAs, respectively. Cell death rates and protein expression levels

for Bax WT versus KO placentae were analyzed by two-way ANOVA. Post-hoc

comparisons of means were analyzed using the Tukey-Kramer test. All other data

were analyzed by Student’s t-test. Statistical software used was SPSS® (Version

13) and data were considered statistically significant if p < 0.05.

65

2.4 Results

2.4.1 ICR and C57BV/6 conceptuses and decidua display similar numbers and patterns of TUNEL positivity at d7.5

At d7.5, the regions demonstrating the greatest number of TUNEL-positive

cells were the mesometrial and anti-mesometrial decidual cells directly adjacent to

the EPC and distal endoderm, respectively. The anti-mesometrial decidua in both

strains studied exhibited significantly higher rates of cell death compared with that

seen in the mesometrial decidua (Figure 2.2a). Additionally, the uterine cavity often

contained TUNEL-positive, detached cells and debris, including free, fragmented

DNA (Figure 2.2b). The EPC exhibited infrequent cell death, with no discernible

evidence of an organized pattern. The allantois was likewise observed to have a low

number of dead cells; however, the majority of TUNEL-positive cells were located at

the tip of this structure. A low percentage of TUNEL-positive TGCs (Figure 2.3a)

appeared sporadically around the EPC and the extraembryonic endoderm

(surrounding the embryo), with no evident pattern. However, a greater number of

primary TGCs demonstrated condensing nuclei and morphologically appeared to be

dying, but were TUNEL-negative (Figure 2.3b). Moreover, at d7.5 for both mouse

strains, there was a greater percentage of TUNEL-negative, condensing primary

TGC, compared with the characteristic, TUNEL-positive cells typically observed in

mid- to late-gestation. Lastly, several primary TGCs contained TUNEL-positive cell

corpses that had been phagocytosed (Figure 2.4). Comparisons between the data

obtained from ICR and C57B//6 placentae at d7.5 yielded no significant differences

in TUNEL positivity in the various cell types and regions studied (i.e. mesometrial

66

Figure 2.2 Cell death patterns in d7.5 ICR and C57BI/6 conceptuses. A. Graph

depicts the number of TUNEL-positive cells per 100 um? of tissue. B.

Photomicrograph demonstrating the uterine cavity of a d7.5 ICR conceptus,

containing TUNEL-positive debris and nuclei. Mesometrial and anti-mesometrial

poles of conceptus are indicated. C. Left panel consists of low- and high-

magnification photomicrographs of d7.5 ICR EPC, with arrow indicating TUNEL-

positive trophoblast cell within EPC. Right panel consists of low- and high-

magnification photomicrographs of d7.5 embryo, surrounding primary TGCs and

anti-mesometrial decidua. Filled arrowheads indicate TUNEL-positive primary TGC

healthy primary TGC nuclei. Bars represent average values + SE, with white and

black bars denoting TUNEL-positivity in ICR placentate (n=8) and C57B//6 placentae

(n=6), respectively. Values of significant statistical difference between different

tissue regions are shown with corresponding p value (Tukey-Kramer post-hoc test).

67

A. TUNEL-positive cells in d7.5 conceptuses

p=0.0013

0.9; p=0.0019 | @ 0.8 4

g 0.7 J O-Icr ot 06 4 @ c57BI6

Bs 05 YO 10 7 °

aS o4. in 6 > 2 0.3 -

E 0.2 4 0.1 4

0 n=8 n=8 n=8

Mesometrial . Anti-mesometrial EPC

decidua decidua

B.

TUNEL-positive debris

and nuclei

‘| anti-mesometrial

pole

mesometrial

d7.5 ICR ectoplacental cone d7.5. ICR primary TGCs

Figure 2.2

68

and anti-mesometrial decidua, EPC, allantois and primary TGCs). Note that positive

(sections pre-incubated with DNase | enzyme) and negative (sections incubated

without Tdt enzyme) controls for TUNEL-staining at varying gestational timepoints

yielded high levels and absent TUNEL-positivity, respectively

2.4.2 Murine placentae exhibit organized cell death patterns over gestation; however, differences in the number of TUNEL-positive cells exist between the two strains

By d10.5, all major regions of the mouse placenta are evident. Herein, the

data will be reported for each region at the gestational timepoints examined.

Trophoblast giant cells: d10.5 — d18.5

Several different morphological features of TGC death emerged over

gestation: (1) TGC with condensing, TUNEL-positive nuclei; (2) TGC with swollen,

TUNEL-positive nuclei and TUNEL-positive cytoplasm; (3) TGC with TUNEL-

negative nuclei, but strong chromatin condensation and cell shrinkage; and, (4)

healthy TGC containing phagocytosed, TUNEL-positive cell debris and/or nuclei. At

all timepoints examined, for both ICR and C57BI/6 placentae, these features of

TUNEL-positivity in TGCs were evident. Towards the end of gestation, there were

increased rates of both types (1 and 2) of TUNEL-positive TGC in both strains

examined; however, from d15.5 — d18.5, this rate was significantly higher in C57BI/6,

compared with ICR placentae (Figure 2.3a). At d10.5, the percent cell death within

the four different groups of TGC appeared approximately equal (Figures 2.3, 2.4);

69

however, this pattern changed at d13.5 and continued until term, with the majority of

TGC in both strains exhibiting TUNEL-positive TGC nuclei (type 1 and 2). Lastly,

the sites of dead/dying TGCs — whether TUNEL-positive or condensing — were

largely confined to the lateral aspects of the placentae, with TGCs in and around the

midline of both ICR and C57B\/6 placentae typically appearing healthy. The percent

of TGCs containing TUNEL-positive cell corpses remained relatively consistent over

time, with a small, but significant decrease observed over gestation (Figure 2.4). In

addition, these cells were intermittently scattered throughout the TGC border, with

no discernible pattern evident, except for a tendency towards being at the maternal

edge of the maternal-fetal interface.

Chorionic Plate: d10.5 —d18.5

TUNEL-positive cells were observed in the chorionic plate at all timepoints,

with increasing numbers of labeled cells as gestation progressed (Figure 2.5a).

Labelled CP cells were examined at high magnification and the pattern of staining

appeared confined to the nuclei, with little to no free, labeled DNA within the

cytoplasm. The majority of cell death was located sporadically throughout the CP,

with some clustering of TUNEL-positive cells around smaller blood vessels. The

cells around the umbilical veins and arteries typically appeared unlabelled and did

not exhibit morphological signs of apoptosis at all gestational timepoints examined.

The CP of C57BI/6 placentae appeared to contain greater numbers of TUNEL-

positive cells compared with the CP of ICR placentae; however, this trend was

statistically significant only at mid-gestation (Figure 2.5a).

70

Figure 2.3 Trophoblast giant cell death patterns in ICR and C57BL/6 placentae

over gestation. Graphs depict the percentage of (A) TUNEL-positive or (B)

TUNEL-negative, condensing TGCs per placental section in ICR and C57BI/6

placentae over gestation. Accompanying photomicrographs in panel below

exemplify characteristic, TUNEL-positive (solid arrowhead) and condensing (arrows)

TGC nuclei in d15.5 ICR placentae; open arrowheads indicate healthy TGC nuclei

and asterisks (*) indicate maternal blood sinuses. Bars represent average values +

SE, with white and black bars denoting ICR placentate (ng7.5=8, nato.5=8; for all other

timepoints, n=9) and C57BI/6 placentae (nqg7.5=6, Na1o.5=8; for all other timepoints,

n=9), respectively. Within the same strain over gestation, means with the same

letter are not significantly different from each other (Tukey-Kramer test, p<0.05).

Values of significant statistical difference between ICR and C57BI/6 placentae are

shown with corresponding p value (Tukey-Kramer test).

71

A. TUNEL-positive Trophoblast Giant Cells

10 O ICR

8 @ C57BI/6

6 b,c b’

a,b a’ , 4 ; a a

: (ice 0 d7.5 d10.5 d13.5 d15.5 d17.5 d18.5

Percentage

of TUNEL-positive

TGC

per

sect

ion

(%)

TUNEL-negative, Condensing Trophoblast Giant Cells

10 C] ICR

mM C57BI/6

laaaa d10.5 d13.5 d15.5 d17.5 d18.5

Percentage

of condensing

TGC

per

sect

ion

(%)

d15.5 ICR placentae - trophoblast giant cells lining maternal venous sinuses

Figure 2.3

72

Figure 2.4 Percentage of trophoblast giant cells containing TUNEL-positive

corpses in ICR and C57BL/6 placentae over gestation. Graph depicts the

percentage of TGC containing TUNEL-positive corpses in ICR and C57BI/6

placentae over gestation. Accompanying photomicrograph below graph depicts a

TGC (cell border indicated by dashed line) containing two TUNEL-positive corpses

(arrowheads) within the cytoplasm. An adjacent, healthy TGC, without cell corpses

is indicated by the open arrowhead. Bars represent average values + SE, with white

and black bars denoting ICR placentate (ng7.5=8, Nuio.5=8; for all other timepoints,

n=9) and C57BI/6 placentae (Nng7.5s=6, Ng10.5=8; for all other timepoints, n=9),

respectively. Within the same strain over gestation, means with the same letter are

not significantly different from each other (Tukey-Kramer test, p<0.05).

Perc

enta

ge

of TGC

cont

aini

ng TUNEL-

positive co

rpse

s per

sect

ion

(%)

Trophoblast Giant Cells containing TUNEL- positive Corpses

73

9] a 8 74 a

74 ClIcr 6 - ™@ C57BI/6

3 b ob b

4) b b 3 5 Cc c

c Cc 2) d

‘ i: 0 2 0 ro 0 10 0 ™ Oo oO LO ™ eo

° o 5 o oO o

d15.5 ICR placenta: trophoblast giant cell containing cell corpses

Figure 2.4

74

Labyrinth: d10.5 — d18.5

For both ICR and C57BI/6 placentae, the cells of the labyrinth exhibited a

sporadic pattern of death until d13.5, as TUNEL-positive cells were randomly

scattered throughout this region. The number of labeled cells per 100 um? at d10.5

was similar for both strains examined (Figure 2.5b); however, at d13.5, the number

of dead cells in C57BI/6 labyrinth was elevated. At d15.5, in addition to the

previously observed sporadic cell death, a pattern of TUNEL-positive cells clustered

around the vasculature (Figure 2.5b) became evident in both ICR and C57BI/6

placentae; however, there were a greater number of these clusters in C57BI/6,

compared with ICR placentae, resulting in a significantly greater number of TUNEL-

positive cells per 100 ym? labyrinth in C57BI/6 placentae (Figure 2.5b). The majority

of these dead and/or dying cells appeared to be fetal endothelial cells and

trophoblast cells lining the maternal sinusoids; however, occasional ST nuclei were

also observed to be TUNEL-positive. As gestation progressed toward term, the

number of TUNEL-positive cells located sporadically within the labyrinth diminished

for both ICR and C57BI/6 placentae. Conversely, the number of cells dying in

groups around the vessels increased for ICR labyrinth towards d18.5, while this

pattern appeared unchanged in C57BI/6 placentae. The net result of these changing

patterns in TUNEL positivity was that there was no change with gestation in the

number of dead cells within the labyrinth in ICR placentae and a trend towards

decreased numbers of dead cells in the labyrinth in C57BI/6 placentae; however,

C57B\/6 labyrinth still demonstrated significantly greater numbers of TUNEL-

75

Figure 2.5 TUNEL patterns in ICR and C57BI/6 chorionic plate and labyrinth

over gestation. A. Graph depicts the number of TUNEL-positive nuclei per 100

um? of CP over gestation. Photomicrograph in right panel is a representative picture

of typical TUNEL-positive nuclei in d15.5 CP; arrowheads indicate TUNEL-positive

CP nuclei. B. Graph depicts the number of TUNEL-positive nuclei per 100 ym? of

labyrinth over gestation. Photomicrograph in right panel represents sporadic TUNEL

positivity seen in d15.5 labyrinth; photomicrograph in lower right panel depicts typical

cell death patterns seen along the labyrinthine vessels. Arrowheads indicate

TUNEL-positive labyrinth nuclei and arrows indicating nucleated fetal red blood cells

within dying/dead vessel. C. Graph depicts the number of TUNEL-positive nuclei

per 100 pm? of spongiotrophoblast within the labyrinth layer; accompanying

photomicrograph depicts characteristic TUNEL patterns observed in trophoblast

cells of this tissue. Dotted lines indicate the border between the labyrinth and the

spongiotrophoblast surrounding the maternal blood canal, indicated by asterisk (*);

empty arrowheads indicate TUNEL-positive nuclei in trophoblast cells encircling the

maternal blood. White bars represent average values for ICR placentae (n = 9 for all

timepoints except ng1o.s = 8) and black bars represent average values for C57BI/6

placentae (n = 9 for all timepoints except ngio.s =8) + standard error (SE). Within the

same strain over gestation, means with the same letter are not significantly different

from each other (Tukey-Kramer test, p<0.05). Values of significant statistical

difference between ICR and C57BI//6 placentae are shown with corresponding p

value (Tukey-Kramer test).

TUNEL-positive cells in Chorionic Plate

=0.04 . 1.6; nc b’

gE 144 =0.002 ; b’ — cick = § b b 5 & 1) mcs7Bve Se 1.04 85 os $3 a’ ae 06: 75 94] Zo Ee “— 4

d10.5 d13.5 d15.5 017.5 d18.5

TUNEL-positive cetls in Labyrinth

1.2 p=0.033 ~ TI ICR SIN

3 E 1.0 @ C57BI/6 by P=0.0032 2

82 08 » .p=0.0002

23 b b’ 82396 p=0.006 ge b’ ge 04 F ,

=

Woz) ag ae P = med

aa 0 dio05 d13.5 d15.5 d17.5 d18.5

TUNEL-positive Spongiotrophoblast Cells in Labyrinth

he Qa

a —

oe 1.8 Olicr p=0.019 1.6 Mi C57BI/6 p=0.007

#TUN

EL-p

osit

ive

cells

per

100 um

within La

byri

nth

(#cells/100

um

p=0.02

'

di3.5 d15.5 d17.5 di8.5

76

d15.5 C57BV/6 labyrinth - sporadic and

clustered death in upper and lower

panels, respectively

traversing through labyrinth, with encircling spongiotrophoblast cells

Figure 2.5

77

-positive cells at term, when compared with age-matched ICR labyrinth (Figure

2.5b). TUNEL-positive labyrinthine cells from both strains of placentae were

examined under high magnification for labeling patterns within the cell. Typically,

TUNEL staining was confined to the nuclei for those cells dying sporadically within

the labyrinth of both ICR and C57B\/6 placentae; little to no labeling of free DNA

within the cytoplasm was observed. For those cells clustered around the

vasculature of the labyrinth and exhibiting positive TUNEL positivity, labeled DNA

was observed both in the nuclei and cytoplasm of dying cells (Figure 2.5b).

Periodically, labeled cellular debris and free DNA were identified in both fetal vessels

and maternal blood spaces within the labyrinth.

As gestation progressed from d12.5 to term, SpT tissue appeared insinuated

within the labyrinth region of the mouse placenta, often completely differentiating to

GlyT by d18.5. At d13.5, both ICR and C57BI/6 placentae demonstrated similar,

comparatively high numbers of TUNEL-positive cells within these SpT pockets.

From d15.5 — d18.5, there was a significantly greater number of dead cells in these

regions in C57BI/6, compared with ICR labyrinth (Figure 2.5c). Although occasional

SpT/GlyT cells within the labyrinth were TUNEL-positive and sporadically placed, the

majority of labeled trophoblasts were directly adjacent to large, maternal blood

canals traversing through the labyrinth (Figure 2.5c).

Junctional zone: d10.5 — d18.5

The cells of the junctional zone (JZ) consist of SoT and GlyT. Cells of the JZ

in the placenta of both ICR and C57BI/6 mice exhibited a trend towards increased

numbers of TUNEL-positive cells over gestation (Figure 2.6). At earlier

78

developmental timepoints, cell death within the JZ was sporadic; however, at d15.5

in ICR placentae, SpT cells within the JZ, bordering on the labyrinth, began to exhibit

small clusters of TUNEL-positive cells (less than ten positive cells per group) which

was was apparent through to d18.5. Furthermore, GlyT cells of the JZ in d15.5 ICR

placentae similarly demonstrated focal regions of collective cell elimination at the

maternal-fetal interface (Figure 2.6); however, these focal regions were much

smaller in size (typically less than ten TUNEL-positive nuclei). These patterns of

grouped, TUNEL-positive cells at the maternal-fetal interface and the labyrinth-JZ

border were not evident in C57BV/6 placentae until d17.5 and d18.5,

respectively; indeed, the pattern of cell death in these regions appeared sporadic at

these timepoints. At high magnification, the labeling in trophoblast cells of the JZ

was typically confined to the nuclei; however, occasional TUNEL-positive cells were

observed to have staining within the cytoplasm, especially in regions of clustered cell

death. At d10.5 and d18.5, the JZ of both ICR and C57BI/6 placentae exhibited

similar numbers and patterns of dead cells; however, at d13.5 — d17.5, cells of the

JZ in ICR placentae demonstrated significantly higher numbers of TUNEL-positive

cells, compared with C57BI/6 placentae.

The maternal compartment: d10.5 ~ d18.5

At mid-gestation, all TUNEL-positive cells were scattered within the maternal

compartment, and largely appeared to be cells of maternal origin (i.e.

decidual cells, uterine natural killer cells, glandular epithelium); the number of

TUNEL-positive cells in the maternal compartment increased over gestation, for both

79

Figure 2.6 TUNEL patterns in ICR and C57BV/6 junctional zone at specified

timepoints over gestation. Graph depicts the number of TUNEL-positive nuclei

per 100 um of junctional zone tissue over gestation. Accompanying

photomicrograph is a representative picture of typical TUNEL-positive nuclei in d15.5

junctional zone. Dotted line delineates the border between the labyrinth and the

spongiotrophoblast layer of the junctional zone; arrowheads indicate TUNEL-positive

spongiotrophoblast nuclei. White bars represent average values for ICR placentae

(n = 9 for all timepoints except ngio.s5 = 8) and black bars represent average values

for C57BI/6 placentae (n = 9 for all timepoints except ngios =8) + standard error

(SE). Within the same strain over gestation, means with the same letter are not

significantly different from each other (Tukey-Kramer test, p<0.05). Values of

significant statistical difference between ICR and C57BI/6 placentae are shown with

corresponding p value (Tukey-Kramer test).

#TUN

EL-p

osit

ive

cells

per

190

um2

of JZ (#

cell

s/10

0 um

*)

02 92

090

90 oO

bh

Oo bk

UT OD

i L

i L

}

80

TUNEL-positive cells in Junctional Zone

p=0.011

p=0.033 b

b

p=0.0008 a o) ©: a

~~»

ied)

b’

o

PP

wh

I

d105 d13.5 di5.5 d17.5 d18.5

d15.51C

Figure 2.6

81

mouse strains studied (Figure 2.7a). In addition, there was elevated sporadic cell

death within the maternal compartment of C57BI/6 placentae, compared with ICR

placentae. At d15.5, focal regions of TUNEL positivity were observed in the

maternal compartment, located closely to the fetal-maternal interface (Figure 2.7b,c).

Additionally, clusters of TUNEL-positive cells often appeared to encase the large,

maternal arteries entering the JZ of the placenta and occasionally surrounded

maternal venous sinuses as well. The pattern and timing of this death varied

between the two mouse strains examined. For ICR placentae, the peak percent

area of clustered cell death within the maternal compartment was at d17.5, followed

by a dramatic decrease at d18.5 (Figure 2.7b). At this timepoint, these foci were

largely TUNEL-negative (only a faint brown stain was evident), and instead

appeared as masses of acellular tissue. In C57BI/6 placentae, a similar clustering of

dead/dying cells around the maternal blood canals was observed; however, the

percentage of TUNEL-positive lesions in the maternal compartment of C57BI/6

placentae was significantly lower than those in ICR placentae at all timepoints

examined (Figure 2.7b). Additionally, there was little to no evidence of TUNEL-

negative, acellular patches of tissue at the maternal-fetal interface — as seen in ICR

placentae — in C57BI/6 placentae at d18.5.

82

Figure 2.7 Maternal decidual cell death patterns in ICR and C57BL/6 placentae

at specified timepoints over gestation. A. Graph depicts the number of sporadic,

TUNEL-positive nuclei within maternal decidua of ICR and C57B//6 placentae over

gestation. Accompanying photomicrograph illustrates a region of maternal decidua

with sporadic, TUNEL-positive nuclei (arrowheads). B. Graph represents the

percentage of area in the maternal compartment that exhibits focal regions of

TUNEL-positivity for ICR and C57BV/6 placentae over gestation. Accompanying

photomicrograph depicts a focal region of TUNEL-positive nuclei within the maternal

compartment, with open arrowheads indicating healthy TGC in the TGC border. C.

Photomicrograph of maternal vessel (demarcated by dotted line) in decidua at

maternal-fetal interface, surrounded by foci of TUNEL-positive nuclei and acellular

tissue. Solid line represents one of the focal areas of TUNEL positivity. Asterisk (*)

represents a patch of acellular material. Bars represent average values + SE, with

white and black bars denoting TUNEL-positivity in ICR placentate (ng7.5=8, ngi9.5=8;

for all other timepoints, n=9) and C57Bl/6 placentae (ng7.5=6, Ngio.5=8; for all other

timepoints, n=9), respectively. Within the same strain over gestation, means with

the same letter are not significantly different from each other (Tukey-Kramer test,

p<0.05). Values of significant statistical difference between ICR and C57BI/6

placentae are shown with corresponding p value (Tukey-Kramer test).

#TUNEL-positive

cell

s pe

r 2

(#cells/100

ym

o

) of maternal_tissue

Oo

100

ym

83

Sporadic TUNEL-positive cells in Maternal

compartment

CIcR p=0.028

6 5 m CS57BI/6 A d’

2 4 ype

p=0.031 cd’ 1 p=0.017 = b

8 7 b’,c’ : b’

| aa’ oa a

44

0 d10.5 di3.5 d155. di7.5 d185

Focal regions of TUNEL-positivity

in Maternal Compartment

RBS os

d15.5 ICR placenta - sporadic cell death in maternal decidua

i.

focal cell d15.5 ICR placenta - death in maternal decidua

wn

s p<0.0001 8 2 oo 164 b

85 12 a = p=0.0033 z 8 p=0.0005 - ‘ere 8 4 a - "SC a

SoS £—

o 2

= i ke ae J

§ 0 o d15.5 d17.5 d18.5

C.

d17.5:ICR placenta - foci of TUNEL- positive nuclei and acellular

tissue surrounding maternal vessel at maternal-fetal interface

84

2.4.3 Caspase-3 expression and localization are similar for ICR and C57BI/6 placentae

Activation of caspase-3 is considered to be a hallmark feature of cell death by

apoptosis. Both ICR and C57B//6 placental lysates exhibited similar levels of

cleaved caspase-3, with peak expression at early- to mid-gestation, followed by a

gradual decrease over the remaining developmental timepoints (Figure 2.8a). Note

that sections incubated with secondary antibody only, exhibited no staining. In

addition, caspase-3 enzyme assays revealed significantly higher activity in fetal-

enriched, compared with maternal-enriched fractions of d15.5 placentae, for both

strains examined (Figure 2.8b). While both fetal-enriched and maternal-enriched

placental lysates of C57BI/6 origin displayed higher levels of caspase-3 activity than

ICR placentae, this difference did not reach statistical significance.

Immunohistochemical analysis using cleaved-caspase-3 antibody on d15.5 placental

sections resulted in patterns of reactivity that were comparable to those seen after

TUNEL (Figure 2.8d). Evaluation of cleaved Parp-1 by immunoblotting revealed a

gradual decline of this protein in ICR placentae over gestation; however, in C57BI/6

placentae, cleaved Parp-1 peaked at d11.5, followed by decreased levels until term

(Figure 2.8c). Moreover, the cleavage profile of Parp-1 consistently demonstrated a

lack of the classical 89-kDa fragment and instead, exhibited Parp-1 cleavage

products of approximately 75 and 52 kDa.

85

Figure 2.8 Caspase-3 expression and activity in ICR and C57BI/6 placentae are

similar over gestation. A. Graph demonstrates procaspase-3 cleavage in

placental lysates as a densitometric ratio of cleaved caspase-3:total caspase-3 over

gestation, after Western blotting. Accompanying picture below graph is a

representative immunoblot of placental lysates from the indicated timepoints, against

anti-caspase-3 and anti-B-actin antibodies. B. Graph demonstrates the differences

in caspase-3 activity between the fetal and maternal compartments for ICR and

C57BI/6 placentae at d15.5. C. Graph depicts production of cleaved Parp-1

fragment at 75 kDa over gestation and accompanying picture below is a

representative immunoblot of placental lysates against anti-cleaved-Parp-1 and anti-

B-actin antibodies. Parp-1 cleavage fragments at 75 and 52 kDa are indicated and

arrow represents the putative position of the 89-kDa fragment. D. Representative

photomicrographs of fetal labyrinth (left panel) and maternal compartmental

placental tissue (right panel) in d15.5 C57BV/6 placenta, after cleaved caspase-3

immunohistochemistry. Arrowheads indicate cells positive for cleaved caspase-3,

arrows indicate nucleated fetal red blood cells within positively stained labyrinthine

vessels and asterisks (*) demarcate maternal blood spaces. Bars represent average

values + SE and n=3 for both ICR and C57BI/6 placentae, except where indicated in

panel C. Values of significant statistical difference are shown with corresponding p

value (Tukey-Kramer test).

Acti

vity

(p

mol/

min)

fo

r 25

pg

> Cleaved Caspase-3 Profile

0.8

0.6

3 0.4

Dénsitometric

ratio

of cleaved

caspase-3:total

caspase-3

3 r 0 “we

86

O Cleaved Parp-1 (75 kDa) Profile

0.7 p=0.0017 O IcR

Gi ier mM C57BI6 Mm. cé7aVe

Dens

itom

etri

c ra

tio

of cl

eave

d Parp-1

(75

kDa)

-act

in

09.5. d105 11.5: 0135 d16.5 17.5 18.5 9.6 di15.° di55 = 18.5

dood B-actin

oS 5.6 2.949 BGO Qh” yor ge

w Cagpase-3 Enzyme Assay

=0.006 : =0,016 =

np

o 5

aN

placental

tysate

NS

net re

32-35 kDa Procaspase-3

17-19 kDa Cleaved caspase-3

a a

t+ 75 kDa Parp-1 cleavage wb 52 kDa fragments

aii a FS] P-actin

~ 9.5 = —-d11.5— -d15.5- +d18.5~

d15.5 Fetal Labyrinth 15.5 Maternal Compartment ICR

CS7BI/6

Ei Sa EEEaamme md

towards towards decidua SpT and

Oo

Fetal-enriched Maternal-enriched maternal chorionic plate GlyTcells

compartment

Figure 2.8

87

2.4.4 Bax localizes to TGCs and the labyrinth of murine placenta and Bax deficiency leads to reduced TGC death and intrauterine growth restriction

Bax is a pro-apoptotic, channel-forming protein that has been implicated in

the regulation of cell death in the human placenta [322, 323]. Western blot

assessment of Bax expression in placental lysates revealed that in the ICR strain,

Bax levels significantly peak at mid-gestation and decrease towards term (Figure

| 2.9a). Interestingly, Bax expression in C57BI/6 placental lysates appears to

precipitously dip at mid-gestation. At all timepoints examined, Bax expression was

significantly greater in ICR placentae compared with C57BI/6 placentae (Figure

2.9a). Localization of Bax protein was determined by immunofluorescence in d15.5

placenta and revealed positive staining maternal decidua (data not shown), labyrinth

and a subset of TGC; other placental cell subtypes did not exhibit Bax staining.

Since Bax expression was detected in a subset of TGCs (Figure 2.9b),

patterns of death in this cell type were assessed in Bax WT and KO placental

sections using TUNEL. This assay demonstrated a significantl decrease in the

number of TUNEL-positive TGCs in Bax KO, compared with WT placentae, at both

d15.5 and d18.5 (Figure 2.9c). In addition, a greater proportion of TUNEL-positive

Bax KO TGCs exhibited the morphological features of necrosis and/or autophagy, as

opposed to the largely apoptotic or aponecrotic death that was observed in the

majority of TUNEL-positive Bax WT TGCs. Moreover, Bax KO placentae at d15.5

and d18.5 displayed significantly greater numbers of TGC per section, compared

with WT placentae at the same timepoints (Figure 2.9d, e). Further to this, while

Bax WT placentae yielded a significant decrease in the total number of TGCs per

88

Figure 2.9 Bax deficiency in murine placentae leads to decreased TGC death

over gestation. A. Graphs depict Bax expression levels in ICR (n=3 for all

timepoints) and C57BV/6 (n=3 for all timepoints) placental lysates over gestation. B.

Anti-Bax immunofluorescence in d15.5 Bax WT placenta, demonstrating positive

reactivity in TGCs and lack of staining in Bax KO TGC. C. Graph indicating the

percentage of TUNEL-positive cells per section in Bax WT and KO placentae, at

d15.5 and d18.5. D. Graph representing the total number of giant cells per section

in Bax WT and KO placentae, at d15.5 and d18.5. E. Hematoxylin-stained placental

sections of d15.5 Bax WT and KO placentae. The TGC border between the

maternal decidua and the fetal junctional zone has been demarcated in orange,

indicating the greater number of TGCs in Bax-deficient placentae. Bars represent

average values + SE. Values of significant statistical difference between ICR and

C57B\/6 placentae, or between Bax WT and KO placentae are shown with the

corresponding p value (Tukey-Kramer test). Within the same strain over gestation,

means with the same letter are not significantly different from each other (Tukey-

Kramer test, p<0.05). Nuclei in immunofluorescence photomicrographs are red and

Bax reactivity is green.

89

A. - Bax Expression in Placental Lysates over Gestation

8 2 p=0.001 C0 cr, ne3 3 1.8 b o 1.6 M@ C5786, n=3 6 14 242 p=0.012

c o's pr0.005 — p<0.001 p=0.02 BY p=0.002 3 06 a, ie

gS 04 ; ; bod % 02 a’d’ a’, a a‘. | ; B 0 d O d9.5 d10.5 d115 d13.5 15.5 d17.5 d13.5

B.

d15.5 Bax WT d15.5 Bax KO

C. Giant cell counts after TUNEL D. Total giant cell count

Bax WT = YD p=0.002

3 °° 7=0,008 mBaxkO 200 2 25 2 g p=0.04 5 160

25 20 ° 2% g 120 g x 15 7)

© 8 410 8 80 Wl < 5 5 & 40 te tH

ES 0 0

d18.5 d15.5

d15.5 Bax WT d15.5 Bax KO 7 mate

Figure 2.9

90

section from d15.5 to d18.5, Bax KO placentae did not exhibit such a decline over

the latter part of gestation (Figure 2.9d). Lastly, immunoblotting and densitometric

analysis of fetal-enriched placental lysates revealed increased levels of placental

lactogen | (PL-I) and placental lactogen II (PL-II) — both of which are TGC secretory

products — in Bax-deficient tissue (Figure 2.10b).

While total caspase-3 levels were significantly up-regulated in Bax KO

compared with Bax WT placentae, the ratio of cleaved caspase-3:total caspase-3

did not differ between the genotypes (Figure 2.10c). To ascertain whether caspase-

3 activity was affected, the same immunoblots were stripped and probed with

antibodies against PTEN, FAK and p21, which are known caspase-3 substrates [93,

324, 325]. This yielded no difference in cleaved protein:total protein (Figure 2.11),

verifying that indeed, caspase-3 activity is unchanged in Bax-deficient placental

lysates. Interestingly, levels of the 75 kDa fragment of cleaved Parp-1 was

significantly higher in Bax KO, compared with WT placentae (Figure 2.10d), while

the level of the 52 kDa fragment of Parp-1 was not significantly different between the

two genotypes (data not shown).

Bax expression was also identified in the labyrinth, with immunoreactivity

localized localized to the fetal endothelium and either one or both of the ST layers.

This was absent in Bax-deficient cells, confirming the specificity of the antibody

(Figure 2.12a). Confirmation of Bax expression in the labyrinth was determined by

Western blotting of labyrinthine-enriched lysates. As shown in Figure 2.12b, Bax

protein was detected in the placental labyrinth of heterozygous fetuses from Bax-

deficient dams. This supports the idea that detectable Bax in the labyrinth was

91

Figure 2.10 Bax deficiency in murine placentae leads to altered expression

levels of placental hormones and cell death markers. A. Photomicrographs of

dissection of d15.5 placental tissue into fetal-enriched and maternal-enriched

fractions; tissue was subsequently used for Western blotting (Bax placentae) or

caspase-3 enzyme assays (ICR and C57BI/6 placentae). B. Expression patterns for

PL-I and PL-II in d15.5 fetal-enriched, Bax WT and KO placental lysates after

immunoblotting and densitometric analysis. C. Caspase-3 expression patterns in

d15.5 fetal-enriched, Bax WT and KO placental lysates, after immunoblotting and

densitometric analysis. D. Parp-1 cleavage profile of 75-kDa fragment in d15.5

fetal-enriched, Bax WT and KO placental lysates after immunoblotting and

densitometric analysis. Representative immunoblot bands for Bax WT and KO

placental lysates are depicted alongside corresponding graphs, with B-actin used as

a control for protein loading. Bars represent average values + SE. Values of

significant statistical difference between Bax WT and KO placentae are shown with

the corresponding p value (Tukey-Kramer test).

Transverse slice of d15.5 placenta ~1-2 mm thick

Dens

itom

etri

c ratio

of

cleaved

casp

ase-

3:to

tal

caspase-3

Densitometric

ratio

of PL

~1

or total

caspase-3:actin

92

Se Discarded or used for caspase-3 assays

Maternal-enriched

fraction

Fetal-enriched fraction

Western blotting and Densitometry

Placental Lactogen Expression

in-d15.5 Placentae

p=0.007

12 Bax WT

40 | @ Bax KO s

3 WT KO WT KO - SS mee

2 dn. . ‘Guanes come . . 5 Lt R-actin R-actin

PL-I PL-Il

Caspase-3 Expression in d15.5

Placentae

p=0.03 0.45

0.35 @ Bax KO 0.3 Pro-caspase-3

0.25 0.2 Cleaved

0.15 caspase-3 0.1

0,05 = ee 0 oo : fS-actin

Cleaved caspase-3: — Total caspase-3:

total caspase-3 actin

D. Cleaved Parp-1 (75 kDa) in d15.5 Placentae

3 G © 16 "p=0.04 os BE 12 WT_KO #0 " i ss SS a aa cleaved Parp-1 2n™ 0.8 5 —

ao

—Ea04

a Bax WT Bax KO

Figure 2.10

93

Figure 2.11 Cleavage levels of active caspase-3 cellular substrates are not

altered in Bax-deficient placentae. Graph depicts densitometric ratios of

cleaved:total protein in d15.5 placental lysates, for known cellular targets of active

caspase-3. Representative immunoblot bands for Bax WT and KO placental lysates

are depicted alongside corresponding graphs, with B-actin used as a control for

protein loading. Bars represent average values + SE.

Dens

itom

etri

c ra

tio

of cl

eave

d

protein:total

prot

ein

Cleaved:total

94

Cleavage Profiles of Caspase-3 Substrates in d15:5, fetal-enriched Placentae

Cleaved:total Cleaved:total

FAK p21 PTEN

FAK: WT KO 125 kDa Full-length

75 kDa i Cleaved

p21:

21 kDa Full-length

14kDa —=} Cleaved

PTEN:

60 kDa Full-length

30 kDa Cleaved

Figure 2.11

95

contributed by the fetus and not the mother. Lastly, expression levels of PECAM-1

(an endothelial cell marker) were decreased in fetal-enriched placental lysates from

Bax-deficient placentae, compared with wildtype placental lysates (Figure 2.13a).

To more closely examine the labyrinthine architecture, lectin histochemistry was

used to distinguish the fetal endothelium [326]. The labyrinthine vasculature of Bax-

deficient placentae (Figure 2.13b) appeared slightly disorganized, with a looseness

and dilatation of both the endothelium-lined fetal vessels and the cytotrophoblast-

lined maternal blood channels at the same gestational timepoint. Moreover, this

altered labyrinthine architecture appeared more profound at later gestational stages.

To determine whether the morphological defects seen in the labyrinth had an impact

on the fetus, placental and fetal weights were obtained after heterozygous matings.

This revealed that while no difference was seen in fetal or placental weight at d15.5,

Bax KO fetuses at d18.5 demonstrated a small, but significant decrease in weight,

compared with d18.5 Bax WT fetuses (Figure 2.13c); no difference was observed for

placental wet weights.

96

Figure 2.12 Bax is expressed in the murine labyrinth. A. Anti-Bax

immunofiuorescence in d15.5 Bax WT placenta, demonstrating positive reactivity in

labyrinthine cells and lack of reactivity in the labyrinth of Bax KO placenta. B.

Photomicrograph in the left panel displays the results of microdissection, after

“punching” out portions of labyrinth and trimming off SpT from a slice of d15.5

placenta. Immunoblot to the right demonstrates the banding pattern obtained after

probing labyrinth-enriched lysates with anti-Bax and anti-B-actin antibodies. The

gestational timepoints and the fetal and maternal genotypes of the labyrinthine

lysates in each lane are indicated above and below the blots, respectively. Nuclei in

immunofluorescence photomicrographs are red and Bax reactivity is green.

Asterisks (*) represent maternal blood spaces and (tf) represent fetal vessels.

97

d15.5 Bax WT Labyrinth

d15.5 Bax KO Labyrinth

B. Confirmation of Bax expression in the labyrinth by microdissection

and immunoblotting:

Bax expression due to fetal contribution

to labyrinth

[oT d14.5 di85 di4.5 d18.5 d14.5 d185

Labyrinty Spongiotrophoblast &

ee ee -~— Bax

Labyrinth (.

— {$-actin

Fetal genotype: WT WT Het Het KO KO Maternal genotype: Het WT KO KO KO KO

Figure 2.12

98

Figure 2.13 Bax deficiency leads to abnormal labyrinthine structure and

intrauterine growth restriction. A. Graph depicts PECAM-1 expression in fetal-

enriched, d15.5 Bax WT and KO placental lysates, relative to the levels of B-actin.

B. Photomicrographs depict d15.5 and d18.5 Bax WT and KO labyrinth, after

probing with Bandeiraea simplicifolia and counter-staining with hematoxylin. C.

Graph depicts the wet placental and fetal weights obtained at d15.5 and d18.5, after

heterozygous matings. Bars represent average values + SE and values of

significant statistical difference are shown with the corresponding p value (Student's

t-test).

Weight

(9)

99

A. PECAN-1 Expression in Fetal-enriched

Placentae

= (0.07 a vO 2s 0.06 a E 8 0.05 Su 0.04 * p=0.04 gg i = A A B-actin

By 0.02 o. & 0.01 L n=5

d15.5 Bax WT d15.5 Bax KO

Low magnification: 15.5 labyrinth d18.5 labyrinth To err Se name

Bax WT |

Bax KO

d15.5 Placental and Fetal Weights d18.5 Placental and Fetal Weights

Bax WT p=0.05

m Bax KO

3S = 2 a

=

Piacenta Fetus Placenta Fetus

From.n=8 dams From n=8 dams

Figure 2.13

100

2.5 Discussion

Several pathological conditions complicating human pregnancies such as

preeclampsia , intrauterine growth restriction (for reviews see [20, 177]) and

exposure to cigarette smoke, have been shown to have aberrant placental cell death

profiles [327]. Although normal, human placental cell death over gestation has been

well studied, a systematic search into the extent and patterns of cell death in the

mouse placenta has been unavailable until now. The existence of homologous cell

types and cellular behaviour between these two species highlights the use of the

mouse as a suitable model for study of human placentation. Thus, analysis of

murine placental cell death patterns and molecular pathways is crucial, as it can aid

the study and interpretation of various genetic models and pathological conditions.

As has been previously shown in the human placenta [328, 329], cell death

appears comparatively infrequently during early placental development in the

mouse, giving way to a greater number of, and more organized patterns of dying

cells towards the end of gestation (Figure 2.14). In addition, different modes of cell

death (i.e. apoptosis, autophagy, aponecrosis, necrosis) were observed in several

placental cell types at varying gestational timepoints. Thus, sporadic, as well as

organized patterns of cell death are evident in the mouse placenta over gestation.

Strikingly apparent were the pockets of cell death that emerged at d15.5 in

the maternal compartment, closely adjacent to the M-F interface. A number of these

TUNEL-positive foci clustered around the large, maternal blood canals, creating the

impression that such massive, systematic death may provide a clean, efficient

separation of the placenta from the uterine wall upon parturition. Concomitant with

101

this expansion of dying cells in the maternal compartment, was an increased number

of TUNEL-positive cells in the junctional zone, particularly in the SpT and GlyT cells

within the fetal compartment. We propose that these clustered regions of death

within the junctional zone provide a similar function as that death which is seen in

the maternal compartment: specifically, to permit a clean separation from the

uterine wall, thereby protecting the mother and fetus from blood loss during delivery.

The observed trend of decreased cell death within the maternal and fetal

compartments adjacent to the M-F interface in inbred, C57BI/6 placenta are

suggestive of developmental delay, when compared with the outbred ICR placenta.

As fetuses of C57BI/6 mice exhibit growth curves indicative of IUGR (personal

communications, S.L. Adamson; and [330)), it is therefore possible, that abnormal

rates of cell death in these two regions surrounding the M-F interface have not yet

received the appropriate cell death cues. However, investigations into the precise

timing of both copulation and parturition in these two strains, or direct quantification

of placental markers over gestation, would be needed to clarify whether the

observed patterns in cell death reflect issues with developmental timing in the

C57BI/6 placenta.

Programmed cell death has been shown to have an important role in

embryonic development, with an impact on shaping such organs as the heart, digits

and brain (reviewed in [331-333]); it has also been implicated as having a role in

human placental morphogenesis [111]. Throughout life, PCD also functions to

102

Figure 2.14 Schematic representation of cell death patterns over development

in the mouse placenta. Pictures reflect typical cell death patterns as revealed by

TUNEL, that were observed in transverse sections of ICR conceptuses and

placentae at the timepoints examined in this study.

103

Dead cells, debris and free, fragmented DNA in uterine cavity

TUNEL-positive cells in maternal decidua - mainly

|

_*——-—~ Occasional TUNEL-positive EPC at mesometrial and anti- So a

!

i |

Severai_.in distal tip of allantois mesometrial poles

Occasional dead primary TGC

Occasional in proximal and distal endoderm

Corpses within primary TGC

d10.5

Sporadic cell death in maternal decidua

Occasional TUNEL-positive TGC

—\-— Corpses within TGC

maternal decidua and

fetal glycogen cells =~ Sporadic cell death in labyrinth and

\ chorionic plate

spongiotrophoblast

labyrinth

Sporadic cell death in maternal decidua

TUNEL-positive secondary TGC (| @ OO

chorionic plate

GB trophoblast giant cells Sporadic spongiotrophoblast ceil death

GE | dead or dying —w cells

TUNEL-positive labyrinthine ceils along

vasculature Focal regions of TUNEL-positive maternal decidual and fetal spongiotrophablast/glycogen

cells starting to form - mainly around large maternal vessels

Spongiotrophoblast surrounding large

vessels within labyrinth starting to die

Greater number of TUNEL-positive chorionic plate cells

d18.5 Regions of acellular tissue embedded with small, sporadic, TUNEL-

positive nuclei

Focal regions of TUNEL-positive maternal decidual and fetal

spongiotrophoblast/glycagen cells

Clustered spongiotrophoblast cell

death now evident

Figure 2.14

104

promote neovascularization in nascent or newly-functioning tissues, or to allow for

vessel regression and occlusion, as seen in the postpartum uterus (reviewed in [334,

335]). During early- to mid-gestation, cell death in the murine labyrinth occurred

sporadically and was infrequently observed; however, as development proceeded

from d13.5, TUNEL-positive cells exhibited typical clustering along the labyrinthine

vessels, with a trend towards an increased number of foci at d15.5, which was

slightly reduced by d18.5. It is likely that this “peak and decline” of labyrinthine cell

death, which was observed in both strains of mice studied, is a reflection of the

massive amount of remodeling required to serve increasing fetal demand. Thus, at

day 15.5, labyrinthine cell death along the vasculature is elevated — when fetal

demand is still comparatively low — to prepare for the impending vascular volume

and surface area [336] that is required at d18.5, when fetal demand is at a

maximum. While specific cell types of the labyrinth undergoing death were at times

difficult to identify, the fact that they were cells lining the vasculature was suggested

by the proximity of the dying cells to the fetal or maternal red blood cells within the

regressing vessels. Moreover, immunohistochemistry using anti-active-caspase-3

antibody revealed similar staining patterns along the vasculature as those seen in

TUNEL. It is possible that the cells involved may be endothelial cells (ECs) directly

involved in angiogenesis, as these have been shown to be more susceptible to

apoptosis compared to quiescent ECs [337]. In fact, it has been proposed that

priming angiogenic endothelial cells towards the cell death pathway allows for a

certain degree of “vascular pruning” during neovascularization [338].

105

Strain differences in labyrinthine cell death and in SpT pockets within the

labyrinth were readily apparent at mid-gestation, with C57BI/6 placentae displaying

significantly greater numbers of TUNEL-positive cells. A possible explanation for

this may be a placental response to reduced fetal growth by upregulating vascular

remodeling, leading to increased cell death; however, an equally compelling

argument is that the enhanced labyrinthine death in C57BI/6 mice results in IUGR.

In addition, dysregulated cell death has been proposed to be a mitigating factor in

gestational diseases such as preeclampsia and IUGR. Recently, two independent

studies have demonstrated higher rates of apoptosis in placental villi complicated by

IUGR [839, 340]; Levy et al. [339] reported that this increase was not associated

with upregulation of Bax protein [339]. These data support the hypothesis of

putative IUGR in C57BI/6 fetuses, as placentae from these dams exhibited elevated

cell death rates and decreased Bax expression, compared with ICR placentae.

Examinations of Parp-1 cleavage profile provided a platform for speculation

into possible alternative pathways involved in cell death execution during

placentation. Immunoblotting of placental lysates obtained from both ICR and

C57BI/6 mice at different gestational timepoints yielded Parp-1 cleavage fragments

at ~75 and 52 kDa, but a complete absence of the canonical, 89-kDa fragment,

known to be a product resulting from caspase-3 cleavage in other somatic cells [66,

341]. Various lysis buffers, two different anti-cleaved-Parp-1 antibodies and

modifications in sample preparation for gel loading were used to test the veracity of

this result, but all immunoblots demonstrated identical Parp-1 cleavage profiles.

Cells undergoing necrotic death have been reported to exhibit a different Parp-1

106

cleavage profile, yielding two major bands at 89 and 50 kDa [342], with lysosomal

cathepsins implicated in the production of this altered Parp-1 cleavage profile [343].

However, cell death triggered by the lysosomal pathway is not always necrotic in

nature; emerging evidence has shown that certain signals induce cathepsin release

from lysosomes, leading to the activation of the apoptotic pathway (for review see

[344]). Additionally, calpain, which is a Ca**-dependent cysteine protease, has

likewise been shown to produce 70-kDa [345] and 40-kDa [346] Parp-1 cleavage

fragments. Moreover, calpain has been associated with apoptotic events in

numerous cell types, but particularly in neurons [345, 347] and leukocytes [348,

349]. Interestingly, while the Parp-1 cleavage profile in ICR placentae followed a

similar pattern to cleaved caspase-3, C57BI/6 placental lysates did not exhibit such

uniformity, suggesting that the increases observed in the latter tissue may be due to

the activation of different caspases or alternate cell death pathways. In contrast,

different Parp-1 cleavage profiles may indicate roles for calpain and/or cathepsins in

trophoblast differentiation, as both these proteases have been associated with

differentiation events in other tissues [350-352]. Finally, both cathepsins and

calpains have been implicated during the development of both human and murine

placentae [353-356].

While the impact of disrupting various Bcl-2 family members has been

previously studied in many tissues and cell types, the placenta was not included in

these analyses [321, 357]. As a role for Bax in human placentation was also

proposed [322, 323], we decided to explore the potential of a placental phenotype

caused by a deficiency in this proapoptotic Bcl-2 family member. Both Bax and Bak

107

remain cytosolic until activation by apoptotic signals, upon which these proteins

translocate to the mitochondria and form membrane-spanning, oligomeric pores, in

an as-yet, unresolved mechanism [34], facilitating the release of several apoptotic

factors. The final consequence of this action is the formation of the apoptosome

[42], which leads to activation of downstream caspases, having a variety of specific

cellular targets [30]. In this study, we report Bax expression profiles in ICR

placentae, that peak at midgestation and gradually decrease towards the end of

gestation; however, Bax expression in C57BI/6 placentae was greatly diminished

compared with that seen in ICR placentae. Immunohistochemical studies revealed

positive Bax staining in a subset of TGCs and in a subset of cells within the

labyrinth. Moreover, the study of Bax-deficient placentae revealed the functional

significance of Bax in these two cellular layers. Bax KO placentae obtained from

heterozygous crosses exhibited increased TGC numbers compared with placentae

from wildtype littermates, attributable to diminished death rates of this cell type.

Additionally, immunoblotting studies revealed higher levels of PL-I and PL-Il in Bax-

deficient placentae, likely reflecting elevated TGC numbers. Moreover, TGC in Bax

KO placentae demonstrated a greater tendency towards autophagic- and/or

necrotic-like death, possibly caused by activating alternate cell death pathways due

to the absence of Bax and/or caspase activation [74]; however, the exact nature of

this death will require further investigation. Interestingly, while total caspase-3

expression and the 75-kDa Parp-1 cleavage fragment were upregulated in d15.5

Bax KO placentae, levels of cleaved caspase-3 were similar between WT and KO

placentae. The significance behind these seemingly contradictory data remain

108

uncertain; however, future studies will involve elucidating the different molecular

pathways involved in murine placental cell death, and hopefully, the meaning of such

enigmatic results will be made more apparent. Examination of the Bax KO labyrinth

region revealed an altered architecture, with apparent dilatation of both the maternal

blood spaces and fetal vessels. This was accompanied by an overall loose

organization of the labyrinthine vasculature, which also exhibited reduced

expression levels of PECAM-1. Further to this, d18.5 Bax KO fetuses exhibited a

small but significant degree of IUGR compared with Bax WT littermates, which is

likely attributable to the observed labyrinthine defects.

Conclusions

In conclusion, cell death patterns during murine placental development

appear similar to those seen in human placentae, with an overall increase towards

the end of gestation. While it is evident that cell death is involved in shaping the

vasculature of the murine labyrinth, the molecular pathways functioning in this region

and in other regions of the mouse placenta, remain to be elucidated. Lastly, murine

placentae deficient in Bax exhibit reduced TGC death and an altered labyrinthine

architecture that likely contributes to the observed IUGR; however, further studies

involving tetraploid rescue experiments will be required to determine whether this

growth restriction can be improved by correcting placental insufficiency.

109

CHAPTER 3

EMBRYONIC LOSS DUE TO EXPOSURE TO POLYCYCLIC AROMATIC

HYDROCARBONS IS MEDIATED BY BAX

A version of this chapter is published in Apoptosis, 2006, Volume 11, Issue 8, pp. 1413-1425, (Detmar J., Rabaglino T., Taniuchi Y., Oh J., Acton B., Benito A., Nunez

G. and Jurisicova A.). Reprinted with kind permission from Springer Science and Business Media. © Springer Science and Business Media, 2006.CHAPTER 3: Embryonic loss due to exposure to polycyclic aromatic hydrocarbons is mediated by Bax

REFERENCE: Detmar J., Rabaglino T., Taniuchi Y., Oh J., Acton BM., Benito A., Nunez G. and Jurisicova A. (2006) Embryonic loss due to exposure to polycyclic aromatic hydrocarbons is mediated by Bax. Apoptosis 11:1413-1425.

110

3.1 Abstract

The high miscarriage rates observed in women smokers raises the possibility

that chemicals in cigarette smoke could be detrimental to embryo development.

Previous studies have established that polycyclic aromatic hydrocarbons (PAHs),

transactivate the aryl hydrocarbon receptor (AhR), leading to cell death. Herein we

show that PAH exposure results in murine embryo cell death, acting as a potential

mechanism underlying cigarette-smoking-induced pregnancy loss. Cell death was

preceded by increases in Bax levels, activation of caspase-3 and decreased litter

size. Chronic exposure of females to PAHs prior to conception impaired

development, resulting in a higher number of resorptions. This embryonic loss could

not be prevented by the disruption of Harakiri, but was diminished in embryos

lacking Bax. We conclude that exposure of early embryos to PAHs reduces the

allocation of cells to the embryonic and placental lineages by inducing apoptosis in a

Bax-dependent manner, thus compromising the developmental potential of exposed

embryos.

3.2 Introduction

Cigarette smoking has been associated with a number of female reproductive

problems, including compromised oocyte maturation, ovarian failure, sub-fecundity,

and infertility (reviewed in [358, 359]). Such negative effects have been documented

for both active and passive (i.e. second-hand) smoking, using both human

epidemiological and animal model studies (reviewed in [250, 251, 253, 360]). The

111

most serious outcomes of tested PAHs include impaired preimplantation embryo

development, early embryo mortality, stillbirths, intrauterine growth retardation and

reduced neonatal survival [252, 253, 361]. Several mechanisms have been

proposed to explain the mechanisms surrounding female sub-fertility and exposure

to tobacco smoke as it relates to ovarian [362, 363] or trophoblast [364, 365]

function, but there is still a dearth of information regarding the molecular

mechanisms involved.

Polycyclic aromatic hydrocarbons (PAHs), such as 7,12-

dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (BaP) are released into the

environment in high quantities as a result of the incomplete combustion of fossil

fuels (wood and coal). PAHs may also be found in furnace gases and automobile

exhaust fumes; however, the primary route of human exposure to these compounds

is cigarette smoke. A recent study reported variable PAH levels amongst the

different brands of cigarettes, with the total amount of PAHs in mainstream smoke

ranging from 1 to 1.6 wg per cigarette [366].

There are several possible mechanisms by which PAHs can alter cell function

at the molecular level. These compounds are capable of directly altering DNA

structure by forming DNA adducts [281], resulting in DNA mutations in affected cells.

In addition, these compounds interact with aryl hydrocarbon receptor (AhR), a

member of the basic helix-loop-helix family of transcription factors. Both BaP and

DMBA elicit a biological response, at least in part, by activation of this receptor,

which is present in its unbound form in the cytoplasm [367]. Upon ligand binding,

the AhR translocates to the nucleus and heterodimerizes with the aryl hydrocarbon

112

nuclear translocator (ARNT). This heterodimeric transcription factor recognizes

specific DNA sequences called aryl hydrocarbon-, dioxin- or xenobiotic-response

elements (AHRE, DRE or XRE) and thus modulates the expression of target genes.

The expression of a variety of genes has been reported to be altered by ligand-

bound AhR, including xenobiotic metabolizing enzymes, growth factors and other

transcription factors [367, 368]. We have recently reported that both human and

murine ovaries respond to DMBA exposure by activating cell death within oocytes of

resting primordial follicles in an AnR-dependent manner [369]. While a large volume

of information exists regarding the toxic effects of PAHs, comparatively little is

known about the mechanisms by which these compounds exert their apoptotic

effect. The PAH/AhR complex activates the pro-apoptotic Bcl-2 family member Bax,

expression of which appears to be necessary for the induction of cell death in

oocytes, since animals lacking functional Bax are almost completely resistant to

PAH driven follicular atresia [8369, 370]. Additionally, Harakiri (Hrk), a pro-apoptotic,

BH3-only family member [371] was demonstrated to be up-regulated in Jurkat T

cells, after exposure to dioxin, a halogenated aromatic hydrocarbon with known AhR

agonist activity [372]. The purpose of the current study was to determine the

biological effects of PAHs and elucidate the mechanisms involved after in vitro and

in vivo exposure of murine preimplantation embryos to PAHs. The in vivo animal

model used (i.e. chronic, slow-release exposure of female mice to PAHs prior to

conception) was implemented to mimic the conditions that are frequently seen in

human populations, where many women stop smoking either before or during early

pregnancy, generally due to fetal health concerns [373, 374].

113

3.3 Materials and Methods

3.3.1 In vivo BaP and DMBA treatment

Hrk wildtype (WT), Hrk knockout (KO) or Bax heterozygous (Het) mice were

backcrossed for two generations onto an ICR background. Six-week-old ICR,

(Harlan, Indianapolis, IN, USA), Fz Hrk WT, Fe Hrk KO and Fe Bax Het virgin, female

mice were randomly separated into PAH-treated or vehicle-treated groups and

group-housed in separate cages. Animals were maintained in a controlled room with

a 12h light: 12 h dark cycle and allowed ad libitum access to rodent chow and water.

Separate preparations of 2 mg/mL BaP (Sigma, Oakville, ON, Canada) or DMBA

(Sigma) were dissolved in corn oil and subjected to waterbath sonication, to allow

full dissolution of the PAHs. These solutions were then mixed 1:1 for a final

concentration of 1 mg/mL DMBA and 1 mg/mL BaP, or 2 mg/mL PAHs. Animals

were given subcutaneous injections under the scruff of the neck, using a 26% gauge

needle. The following regimen was employed: one dose (2 mg/kg) per week for 3

weeks, then three weeks’ rest, followed by one dose per week for three weeks. The

final, cumulative dose for PAH-treated mice was 12 mg/kg; vehicle-treated animals

were given proportional injections of corn oil, according to body weight.

3.3.2 Mating and tissue collection

Four days after the last injection, female mice were mated with the

appropriate male stud (i.e. F2 Hrk WT males, Fo Hrk KO males, F2 Bax Het males, or

114

ICR males) of proven fertility. Gestational age was determined based on the

presence of a vaginal plug, with the morning of detection being embryonic day 0.5

(d0.5) of gestation. Plugged females were removed from the male and group-

housed in separate cages; it was ensured at all times, that PAH-treated and vehicle-

treated females were placed in different cages. Pregnant dams were euthanized by

cervical dislocation; ICR conceptuses were collected at various gestational

timepoints (d7.5, d9.5, d10.5 or d12.5), while Hrk WT, Hrk KO and Bax Het

conceptuses were collected only at d12.5. The pregnant uteri were placed in

phosphate-buffered saline (PBS) and the number of conceptuses and resorptions

was recorded. Uterine horns were torn open with forceps and conceptuses teased

out. Decidual tissue was removed from the embryonic membranes, which were

subsequently torn open to expose the embryo. Early embryos (d7.5) were assessed

for viability by morphology; later embryos (d9.5, d10.5 or d12.5) were assessed for

viability by morphology and the presence of a beating heart. Where required,

embryos were washed well with PBS and stored at -20°C for PCR genotyping. ICR

placentae with attached decidua were collected at d9.5, d10.5 and d12.5, and

placed in 10% phosphate-buffered formalin for 24 hours at 4°C for 24 hours, after

which the tissue was washed well with PBS and stored in 70% (v/v) ethanol until

paraffin embedding.

3.3.3 PCR Genotyping

Hrk Fz mice, Bax Fz mice and Bax embryos were genotyped by PCR as

previously described [357, 375] . ICR embryos collected at d9.5 or 10.5 were

115

digested overnight at 55°C in lysis buffer (60 mM KCI, 10 mM Tris-HCl, pH 8.3, 2

mM MgCl, 0.1 mg/mL gelatin, 0.45% Nonidet P40, 0.45% Tween 20) with

proteinase K (0.5 mg/mL final concentration). Proteinase K was heat-inactivated at

95°C for 10 minutes. PCR genotyping for the sex chromosomes was done in

separate reaction tubes, using primers for the Xist gene (X chromosome; forward

primer: 5° TTG CGG GAT TCG CCT TGA TT 3’; reverse primer: 5’ TGA GCA GCC

CTT AAA GCC AC 3’) and the Zfy gene (Y chromosome; forward primer: 5’ GAC

TAG ACA TGT CTT AAC ATC TGT CC 3’; reverse primer: 5’ CCT ATT GCA TGG

ACA GCA GCT TAT G 3’). X-chromosome PCR conditions were as follows: 2 mM

MgCle, 400 nM each primer and 200 nM dNTPs. Y-chromosome PCR conditions

were as follows: 2 mM MgCl, 800 nM each primer and 200 nM dNTPs. Cycling

conditions for both X and Y PCR were identical: 3 cycles of 95°C for 4 min., 52°C

for 1 min., 72°C for 1 min., followed by 32 cycles of 95°C for 1 min., 52°C for 1 min.

and 72°C for 1 min. Known male and female DNA obtained from ear punches were

used as controls. PCR reactions were run on a 2% gel and evaluated for the

presence of the 220 bp X band and the 200 bp Y band.

3.3.4 Collection of in vivo PAH-treated preimplantation embryos

PAH-treated and vehicle-treated ICR female mice were superovulated using 5

IU of pregnant mare serum gonadotropin (Sigma), followed 48 hours later by 5 IU of

human chorionic gonadotropin (Wyeth, St-Laurent, QC, Canada). The mice were

subsequently mated and the gestational timepoint was established as described

above. Embryos were retrieved at d3.5, by flushing the uterus with modified human

116

tubal fluid medium (Irvine Scientific, CA) then fixed in 4% paraformaldehyde, diluted

in PBS on microscope slides, air-dried and stored at 4°C until further use.

3.3.5 Collection and transfer of in vitro DMBA-treated embryos

Eight- to twelve-week old ICR female mice (Harlan) were superovulated using

the previously described protocol. Embryos were retrieved at the zygote stage

(d0.5) and placed in double-well dishes containing KSOM with amino acids

(Specialty Media, Phillipsburg, NJ, USA). In the afternoon of day 2.5, 8-cell embryos

were selected from a pool of cultured embryos and were randomly divided into

treatment groups (20-30 per group/per experiment). Embryos were cultured in 50 pL

microdrops under mineral oil (Specialty Media) supplemented with vehicle (DMSO)

diluted in the culture medium (0.002%) or DMBA (1 uM). DMBA stock was prepared

in DMSO as a 50 mM solution and diluted in medium prior to addition to a culture.

The synthetic AhR antagonist, o-naphthoflavone (ANF) was diluted in DMSO as

stock of 10 mM. The final dose used as an inhibitor of ANR was 2 uM, as previously

determined [369]. All compounds were purchased from Sigma. Embryos were

cultured in a humidified incubator at 37°C, 5% COs for forty-eight hours to assess

embryo development and evaluate other parameters associated with the activation

of cell death. Embryos that were not used for transfer were washed and fixed in 4%

paraformaldehyde, as described above.

To determine the developmental potential of treated embryos, blastocysts

exposed for 24 or 48 hours to vehicle or DMBA, were transferred into the uterine

117

horns of pseudopregnant females, mated with vasectomized males 2.5 days prior

transfer. Each female received 10 embryos (5 per uterine horn) in three

independent experiments with three females per group/experiment. Females were

allowed to deliver and the number of pups at birth was determined by visual

examination the morning after delivery, which was considered posinatal day 1

(PN1). Each pup was weighed on PN1 and PN21.

3.3.6 Analysis of cell number and cell death

Nuclear staining with the fluorochrome, 4,6-diamidino-2-phenylindole (DAPI,

Sigma) was used to analyze chromatin status. Embryos that had been treated with

vehicle or DMBA in vitro, then fixed and stored at 4°C, were stained in a 50 wg/mL

solution of DAPI in PBS for five minutes, washed with PBS, mounted with 30%

glycerol and viewed under a Zeiss Axioplan microscope. Assessment of mitosis and

cell death was based on DNA condensation, fragmentation and nuclear morphology

as previously described [376]. The cell death index (CDI) was calculated as the

percent of total cells that exhibited intense DAPI staining due to condensation of

chromatin, which in mammalian blastocysts is known to precede DNA fragmentation

[377]

Embryos that had been exposed to vehicle or BaP-DMBA mixture in vivo

were treated as above, but underwent further analysis using TUNEL assays; these

assays were performed as previously described [377].

118

3.3.7 Mitochondrial membrane potential analysis

JC-1 = (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazoyl | carbocyanine

iodide, DePsipher™ Kit TA700, R&D Systems Inc, Minneapolis, MN, USA) is a

lipophilic cationic dye which reflects the activity of mitochondrial membrane potential

and thus, is frequently used as an indicator of mitochondrial depolarization

associated with apoptosis [378]. Embryos were exposed to JC-1 and analyzed as

previously described [379]. Following staining, embryos were individually washed

and subsequently imaged on a deconvolution microscope (Olympus 1X70, Applied

Precision Inc., Issaquah, WA, USA), using fluorescein isothiocyanate (FITC) and

rhodamine isothiocyanate (RITC) filters. Optically sectioned images (each section

having a thickness of 5 um) were obtained, with a total of ten sections visualized for

each mouse embryo. Acquired images were analyzed using DeltaVision Software

(Applied Precision Inc., Issaquah, WA, USA), which allows for quantification of signal

intensity in each channel. The ratio of RITC (J-aggregate) to FITC (J-monomer)

staining was determined for all sections of the embryo, from which an average ratio

of J-aggregate to J-monomer staining for the entire embryo (n=13 for vehicle and

n=19 for DMBA-treated) was determined.

3.3.8 Single embryo caspase activity assay

This single cell fluorescent assay reflects predominantly the activity of

caspase-3 and -7, by trapping fluorochrome-conjugated caspase substrate (DEVD -

Asp-Glu-Val-Asp) within a cell. Caspase activity was assessed in exposed embryos

as previously described [321]. Briefly, embryos were cultured in the presence of

119

rhodamine-conjugated substrate (PhiPhiLux; Oncolmmunin Inc., College Park, MD)

for 3 hours, after 45 hours of exposure to vehicle or DMBA. Samples were

subsequently washed 3 times with PBS, fixed in 4% paraformaldehyde, transferred

onto slides and stained with DAPI as described above. This approach permitted

simultaneous analysis of chromatin status (diffuse or condensed), the distribution of

DNA and caspase-3-like activity. The total ratio of intensity of staining between

nuclear content and caspase-3 activity in several optical sections per embryo

(n=9/vehicle and n=10/DMBA) on rhodamine (red) and DAPI (blue) channels was

analyzed as described above for JC-1.

3.3.9 Expression of Bax and Hrk transcripts in exposed embryos

Gene expression was determined by a quantitative reverse transcription-

polymerase chain reaction followed by Southern dot blot analysis (QADB) assay

detailed previously [376, 380]. Briefly, cDNAs derived from three pools of five

blastocysts per treatment group (exposed to either DMBA or vehicle for 24 hours)

were amplified, dot blotted and analyzed by hybridization of dot blots with cDNA

probe radiolabeled by random-priming. Blots were exposed to a phospho-imager

cassette and the intensity of each dot was evaluated using ImageQuant Software.

The cDNA probe used contained part of the coding region and the 3'-untranslated

region of Bax and Hrk [376].

120

3.3.10 Immunocytochemical localization of Bax and AhR

Murine blastocysts were exposed to vehicle or DMBA for 24 hours, fixed for

10 minutes in 10% phosphate-buffered formalin, transferred to slides, air-dried and

stored at -20°C. Upon defrosting, slides were washed in PBS and microwave

antigen retrieval was performed in sodium citrate buffer (pDH=6.0) as previously

described [369]. Subsequently, slides were rinsed in PBS and blocked in 10%

normal goat serum in PBS with 0.05% Triton X. Affinity-purified rabbit anti-Bax P-19

(sc-526, Santa Cruz Technologies, Santa Cruz, CA, USA) or rabbit anti-AhR

(BioMol, Plymouth Meeting, PA, USA) polyclonal antibody diluted 1:100 in 5% goat

serum in PBS were applied to samples and incubated overnight at 4°C. Upon

further washing and incubation with diluted (1:200) secondary anti-rabbit biotinylated

antibody (Vector Laboratories, Burlingame, CA, USA), final labeling was performed

with streptavidin-Texas Red (Calbiochem, San Diego, CA, USA) and counterstained

with DAPI. The ratio of intensity of nuclear content to Bax immunostaining in several

optical sections per embryo (n=10/group) on DAPI and RITC channels was analyzed

using deconvolution microscopy, as described above.

3.3.11 Statistical analysis

Analysis of the differences in cell number, cell death, or mitotic rates between

treated and control groups was assessed by Kruskal-Wallis One Way Analysis of

Variance (ANOVA). Birth rates after transfer of exposed embryos were evaluated by

Chi-square analysis. All other statistical evaluations were done using the Student’s

121

t-test. All tests were performed using the SigmaStat statistical package (Version

1.0) and a p value < 0.05 was considered significant.

3.4 Results

3.4.1 Effect of DMBA on murine preimplantation embryos in vitro

Since previous epidemiological studies linked smoking with increased rate of

spontaneous abortions, we explored the possibility that a prototypical PAH, such as

DMBA, could function as a trigger for cell death, resulting in embryonic loss. We

used 8-cell preimplantation embryos, as this developmental stage is associated with

the second wave of embryonic genome activation, which is believed to be

responsible for the allocation of blastomeres to future cell lineages: the inner cell

mass (ICM) and the trophectoderm (TE). The dose of DMBA (1 1M) was

determined based on our previous experience with ovarian organ culture [369].

Moreover, pilot embryonic studies with this dose of DMBA did not exhibit non-

specific toxic effects, even when applied at earlier embryonic stages.

Addition of DMBA to the culture medium during the 48-hour period did not

interfere with the progression of embryos from the 8-cell stage to the expanded

blastocyst stage (d4.5). While embryos appeared slightly smaller, they contained a

distinct ICM and a defined blastocoele cavity. The total cell number, as well as the

mitotic index in the treated group, was slightly lower than in vehicle-treated embryos,

but the difference did not reach statistical significance. The only parameter

significantly (p = 0.012) affected by DMBA exposure was cell death, which was

increased in the treated embryos (Figure 3.1). Dead cells were localized to both the

122

ICM and the TE lineage and exhibited classical hallmarks of apoptosis, as indicated

by nuclear condensation, blebbing and fragmentation.

We further explored whether DMBA mediates its biological effect in the

murine blastocyst via the transcription factor, AnNR. Since previous reports

suggested embryonic expression of AhR in murine blastocyst by RT-PCR, we

performed immunocytochemistry using an anti-AhR antibody. Expression of AhR

protein was observed in both the ICM and TE, with exclusive cytoplasmic and

perinuciear localization of this protein. Upon exposure to DMBA for 24 hours,

occasional nuclear staining of AnR in a small number of cells was observed (Figure

3.2). Earlier time points (2, 4 and 6 hours) resulted in only cytoplasmic localization

of AhR. This suggests that DMBA, or its metabolites, function via nuclear

translocation and activation of AhR transcription. We further confirmed this

hypothesis by co-treatment of embryos with DMBA and ANF, a synthetic antagonist

of AhR, resulting in the rescue of DMBA-induced cell death (Figure 3.1). ANF alone

had no effect on embryo development, cell number, cell death or mitotic index.

3.4.2 Cellular and molecular pathways activated by DMBA

In order to determine whether DMBA activates similar cell death pathways in

the blastocyst as observed in primordial follicles of murine ovaries [369], we

assessed the expression of the pro-apoptotic Bcl-2 family member, Bax. Exposure

to DMBA for 24 hours significantly increased the levels of Hrk and Bax transcript in

blastocysts, when compared with vehicle-treated embryos (Figure 3.2). This was

followed in the next 24 hours by a 2-fold increase in Bax protein level/per nuclear

123

Figure 3.1 Exposure of murine preimplantation embryos to DMBA increases

the cell death index and AhR antagonist (ANF) precedes this effect. (A)

Representative micrographs showing the DAPI-stained nuclear morphology of

murine blastocysts incubated in vehicle 1 uM DMBA for 48 hours. Increased

numbers of cells with condensed and/or fragmented chromatin were observed

(arrows) in DMBA-exposed blastocysts. (B) The cell death index was significantly

increased (*p=0.0012, Student’s t-test) in DMBA-treated embryos, compared to

vehicle-treated embryos, after 48 hours. Depicted are the average values obtained

for vehicle-treated (white bars), DMBA-treated (black bars) embryos, DMBA, ANF

co-treated (dark grey bars), and ANF-treated (light grey bars) embryos + standard

error.

124

Vehicle

B. Effect of acute exposure of ANR agonist and antagonist on murine

preimplantation embryos in vitro

94 * p=0.012 8 aad

7: 0) Vehicle 2 @ 1M DMBA & a N m@ 1 uM DMBA + 2 uM ANF a en oe i 2 uM ANF L N ow iD Q 4- ul wt

Average “m

itot

ic an

d %dead

cell

Mitotic Index Cell Death Index

Figure 3.1

125

Figure 3.2 Cell death regulatory proteins are increased in DMBA-treated

murine blastocysts. Panels A-C are micrographs of representative embryos, with

cell-death-associated genes in red and nuclear staining in blue. As shown, the left

panel corresponds to vehicle-treated and the right panel to DMBA-treated embryos.

(A) Immunocytochemistry of arylhydrocarbon receptor (AhR) in murine embryos

exposed to vehicle or 1 uM DMBA for 48 hours. Representative nuclei from both

vehicle- and DMBA-treated embryos have been outlined in white to depict the

increased nuclear localization in embryos exposed to 1 uM DMBA for 48 hours. (B)

Immunocytochemistry of Bax protein in murine embryos exposed to vehicle or 1 uM

DMBA for 48 hours. (C) Trapping of caspase-3/7-cleaved substrate in vehicle-

treated or DMBA-treated murine blastocysts. (D) RT-PCR and dot-blotting of

pooled, murine embryos treated with 1 uwM DMBA for 24 hours, yielded significantly

higher Bax (**p=0.006, Student’s t-test) and Hrk ( p=0.05, Student's t-test)

transcripts, compared to pooled embryos treated with vehicle. (E) Estimation of total

Bax protein in vehicle (DMSO) or DMBA-exposed murine blastocysts. The ratio of

red/blue (protein expression/nuclear content) was determined in several optical

sections per embryo for a total of 10 embryos per group. Significantly increased Bax

(‘p=0.027) was observed in embryos held in 1 «M DMBA (black bars) for 48 hours,

compared to embryos exposed to vehicle (white bars) for the same length of time.

(F) Estimation of total caspase-3-like expression in vehicle- or DMBA-exposed

murine blastocysts. Values were obtained using a red/blue ratio as described above

for several optical sections per embryo, for a total of 10 embryos per group.

Significantly increased caspase-3/7 (‘p=0.047) was observed in embryos held in 1

126

uM DMBA for 48 hours, compared to embryos exposed to vehicle for the same

length of time. Bars represent average values + standard error.

127

; Increased Bax and Hrk expression in embryos Vehicle 1 uM DMBA D. exposed in vitro to DMBA for 24 hrs

5=0.05

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Vehicle 4M DMBA

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Vehicle 1 uM DMBA

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128

content/embryo, as determined by quantitative analysis after immunostaining (Figure

3.2). These results are consistent with observations that a 48-hour exposure to

DMBA is needed for activation of cell death, as observed in neonatal murine ovaries

(Jurisicova and Matikainen, unpublished observation). Since Bax is known to

activate the mitochondrial pathway of apoptosis, we further assessed changes in the

mitochondrial membrane potential as indicated by a shift in the spectrum of

fluorescence of the cationic dye, JC-1. Due to the fact that changes of mitochondrial

membrane potential precede other hallmarks of apoptosis [381], we assessed the

ratio of JC-1 intensity after 43 hours of DMBA exposure. While we observed a

strong trend towards a decreased ratio of red/green fluorescence

(polarized/depolarized mitochondria) in the DMBA-treated group, this decrease did

not reach statistical significance (2.20 + 0.29 for vehicle and 1.63 + 0.18 for DMBA-

treated embryos). We attribute this lack of significance to the variability amongst

individual embryos caused by the transient nature of changes in mitochondrial

membrane potential.

Activation of effector caspases (caspase-3 and -7) was evaluated by

quantitative assessment of active caspases based on the cleavage of a rhodamine-

labeled substrate (DEVD), 48 hours after exposure to DMBA. We observed a

significant (~35%) increase (p = 0.0467) in the accumulation of caspase-mediated

fluorescence/per nuclear content in DMBA-treated blastocysts when compared with

vehicle-treated embryos (Figure 3.2). Thus, it appears that DMBA activates AhR,

resulting in increased levels of Bax protein, which in turn may activate cell death via

the mitochondrial pathway, resulting in the activation of caspases-3 or -7.

129

In order to determine the long-term developmental consequences of exposure

of murine embryos to DMBA, we transferred embryos exposed to vehicle or DMBA

for 24 hours (d3.5) or 48 hours (d4.5) into the uterine horns of pseudopregnant

females. As observed for cell death rates, only exposure to DMBA for 48 hours

caused a significant decrease (p = 0.021) in the number of pups born/embryos

transferred (30% [30 pups/90 embryos] in DMBA-exposed and 50% [45 pups/90

embryos] in vehicle-treated groups). Assessment of birth weight (PN1) as well as

weight at weaning (PN21) yielded no differences between vehicle- or DMBA-

exposed pups (Figure 3.3).

3.4.3 Maternal exposure to PAHs results in decreased pregnancy and increased resorption rates

Maternal cigarette smoking has been associated with increased rates of

spontaneous abortion and reduced fecundity. Additionally, female smokers are

increasingly apt to quit smoking prior to, or upon the onset of pregnancy. Our

murine model of slow-release injections of PAHs into virgin, female mice prior to

conception, mimics this human situation and our studies focused on the pregnancy

and resorption rates in PAH-exposed versus vehicle-exposed dams.

In order to determine the effects of maternal exposure to PAHs on murine

preimplantation embryo development, ICR females were exposed to BaP and DMBA

prior to conception, superovulated and mated to ICR males; embryos were retrieved

at d3.5 and assessed for cell death and mitotic indices. Six vehicle- and PAH-

130

A. PN1 Pup Weights 2.5

2

0 Vehicle

2 1.5) m PAH-exposed <=

> oO = 1 |

0.5

0 n=26 n=23

Male Female

B. PN21 Pup Weights 20

16 _ O Vehicle

2 424 m@ PAH-exposed

B oO = 8

4

0 n=23 n=23

Male Female

Figure 3.3 In vitro exposure of ICR preimplantation embryos to DMBA had no

effect on PN1 and PN21 weights. Graphs depict weights of male and female pups

at (A) PN1 and later at (B) PN21. Pups had been exposed to vehicle or DMBA in

vitro for 24 or 48 hours during the preimplantation stage and transferred to

pseudopregnant ICR females at d3.5 or d4.5. Bars reflect average values + SE.

131

treated dams were superovulated and successfully mated, however only 4 females

from each group yielded embryos useful for study. The number of obtained morula-

or blastocyst-stage embryos was decreased in the PAH-exposed females (n=74

from vehicle-exposed dams and n=54 from PAH-exposed dams). We observed a

reduction in the number of cells per embryo, resulting in ~20% loss of total number

of cells in embryos from PAH-exposed, compared with vehicle-exposed mothers.

This was accompanied by a trend towards increased cell death and mitotic indices in

embryos from PAH-exposed dams, as assessed by DNA morphology and TUNEL

staining (Figure 3.4).

Spontaneous pregnancy rates for PAH-exposed, ICR mice were reduced, as

only 60% (9 pregnant/15 plugged) of PAH-exposed, ICR females were pregnant

after excising the uterus 7, 9, 10, or 12 days after mating (determined by the

presence of a vaginal plug), compared with 100% (15 pregnant/15 plugged) of the

vehicle-exposed females. When PAH-treated females did conceive, there was a

significant increase in abnormal embryos, starting at approximately d7.5, leading to

early embryonic death determined by the greater number of resorptions and non-

viable embryos observed in d9.5 pregnant uteri of PAH-exposed females, compared

with vehicle-exposed females (Figure 3.5).

and

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Assessment of murine ICR preimplantation embryos after maternal exposure to PAHS

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132

Effect of maternal PAH exposure on ICR blastocyst cell number

Vehicle

§ PAH-exposed n=67

r ne5d

* p=0.0004

Vehicle PAH-exposed

Figure 3.4 Chronic maternal exposure to PAHs prior to conception results in

reduced cell number per blastocyst in d3.5, ICR preimplantation embryos. (A)

Trend towards decreased % mitotic and increased % dead (condensed chromatin,

blebbing, etc.) or TUNEL-positive cells in murine blastocysts obtained from dams

exposed to PAHs, prior to conception. (B) A significantly decreased number of cells

per embryo (*p=0.0004) was observed in blastocysts obtained from dams exposed

to PAHs, compared to dams exposed to vehicle. Bars from both (A) and (B) reflect

the average values + standard error for vehicle-treated dams (n = 4, white bars) and

PAH-treated dams (n = 4, black bars).

133

There are contradicting findings in human studies regarding the impact of

cigarette smoking and environmental pollution on the sex ratio of babies born at

term. Since our model of chronic PAH exposure in female ICR mice demonstrated

increased spontaneous abortion rates, we determined the sex genotype of live

embryos at d9.5-d10.5, for both vehicle- and PAH-exposed mothers. Tissue from

resorbing conceptuses was not available for PCR analysis, as the resorbing

embryos were far too damaged for extraction of good quality DNA. Seventy-four live

embryos from vehicle-treated dams yielded an almost equal proportion of both sexes

(Figure 3.5). Fifty-three live embryos from PAH-exposed dams yielded an average

proportion of 0.73 males per litter and 0.25 females per litter. Thus, while the

average proportion of either sex per litter did not differ in the vehicle-treated group,

the average proportion of males per litter was significantly different from the average

proportion of females per litter in PAH-exposed dams (Figure 3.5).

Since we observed that two cell death genes, Hrk and Bax, are upregulated in

embryos exposed to PAHs in vitro, we decided to establish if these genes were

necessary for the activation of death observed in PAH-exposed mothers. Hrk WT or

Hrk KO [375] mice — which are fertile and exhibit no overt phenotype — were

backcrossed for two generations onto an ICR background and exposed to the slow-

release PAH protocol, as described above. All PAH-treated females, regardless of

genotype, demonstrated a reduced number of embryos per litter, compared with

their vehicle-treated counterparts (Figure 3.6). Hrk KO and WT dams treated with

134

Figure 3.5 Chronic maternal exposure to PAHs prior to conception results in

an increased number of resorptions and a decreased number of viable

embryos, with a greater proportion of male embryos represented in the live

offspring. (A) A significantly decreased (*p<0.0001) number of morphologically

normal embryos and a significantly higher (**p=0.018) number of morphologically

abnormal embryos were obtained at d7.5 from ICR mothers exposed to PAHs (n=5

dams, black bars) compared to mothers exposed to vehicle (n=4 dams, white bars).

(B) A significantly decreased (‘p<0.0001) number of viable embryos and a

significantly higher (‘p=0.02) number of nonviable embryos were obtained at d9.5

from PAH-exposed mothers (n=6 dams) compared to vehicle-exposed mothers

(n=11 dams). Bars from graphs (A) and (B) reflect average values + standard error.

(C) ICR females chronically exposed to PAHs (n=8 dams, black bars) showed a

significantly different proportion of males, compared to females, per litter (“p=0.03).

ICR females exposed to vehicle (n=5 dams, white bars) had similar proportions of

males and females per litter. Bars reflect average proportions + standard error.

> Average

number

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135

Effect of maternal exposure to PAHS on

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# embryos with # embryos with normal morphology abnormal morphology

Effect of maternal exposure to PAHS on

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+ p<0.0001

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iw # viable embryos # nonviable # resorptions

Effect of PAHs on embryo sex in d9.5-d10.5

ICR mice

(1 Vehicle

i PAH-exposed n=

Males Females

Figure 3.5

136

Figure 3.6 Bax-deficient, but not Hrk-deficient, embryos are rescued from

resorption after chronic maternal exposure to PAHs, prior to conception. (A)

Hrk-deficient dams exposed to PAHs prior to conception demonstrated a similar

number of live embryos per litter, at d12.5, compared to PAH-exposed Hrk WT

dams. Bars represent average values + standard error for PAH-exposed WT (n=7)

and KO (n=9) dams and for vehicle-exposed WT (n=5) and KO (n=8) dams. (B)

Proportion of Bax wildtype plus heterozygous embryos (WT and Het) and KO

embryos, obtained by PCR genotyping, after exposure of Bax heterozygous females

to vehicle, or to PAHs, prior to conception. The proportion of Bax KO embryos per

litter in PAH-exposed mothers is significantly higher (**p=0.024) compared to the

proportion of Bax KO embryos from vehicle-exposed mothers. The proportion of

wildtype and heterozygous embryos per litter is significantly decreased (*p=0.024) in

PAH-treated mothers compared to vehicle-treated mothers. Bars represent the

average proportion of the genotypes for live, d12.5 embryos in vehicle-treated dams

(n=7, white bars) and PAH-treated dams (n=9, black bars).

137

Maternal exposure to PAHs in d12.5 Hrk WT or Hrk KO mice

p=0.007

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G 07 4

o8 05; o 2 G44

ee 034 56 a 0.2 4

> on. zg

WT dams

p<0.001 O Vehicle M PAH-exposed

KO dams

# viable embryos

Maternal exposure to PAHs in d12.5 Bax heterozygous mice

n=65 () Vehicle, n=7 dams,

n=55 82 embryos

* p=0.024. @ PAH-exposed, n=9 dams,

85 embryos n=30 oy ** p=O.024

WT and Het KO

Genotype of viable embryos

Figure 3.6

138

PAHs demonstrated no significant difference in the number of embryos at d12.5.

Thus, disruption of Hrk is not sufficient to rescue the embryos from PAH-mediated

loss.

As Bax functions downstream of Hrk [382] and is also directly activated by

PAHs [369], this gene is more likely to have an effect on embryonic lethality.

However, as Bax KO females exhibit reduced fertility (J. Detmar and G. Perez,

unpublished observation) we used heterozygous mothers and assessed the

distribution of genotypes amongst surviving embryos at gestation day 12.5. The

genotypic proportions of offspring for heterozygous Bax females exposed to PAH

treatment differed significantly from the expected Mendelian ratios observed in

vehicle-exposed mothers, with a higher proportion of KO embryos, suggesting that

KO embryos are more resistant to embryonic loss triggered by PAHs (Figure 3.6).

3.5 Discussion

Several population-based human studies indicate that maternal cigarette

smoking carries serious reproductive hazards (reviewed in [383]). These include

delayed conception or infertility [258-260] as well as increased risk of spontaneous

abortion during natural or assisted conception [254-256]. While early pregnancy

loss has been previously linked to excessive activation of cell death [384, 385], the

molecular mechanisms involved in toxicant-mediated embryo demise are unclear.

Thus, we thought to explore this concept in an animal model, mimicking exposure to

a subset of chemicals found in cigarette smoke.

139

Animal studies using various types and sources of PAHs have shown that

exposure to these toxins can lead to decreased fetal survival [275, 277] or complete

postimplantation embryo loss [278]. Since these studies focussed on the

detrimental effects of PAH exposure in pregnant animals, we decided to test whether

female mice chronically exposed to PAHs prior to conception had a different

reproductive outcome. This model would be valuable in ascertaining what

pregnancy-related effects, if any, could be observed in animals treated with PAHs

prior to conception, a condition that is seen in human populations when women quit

smoking cigarettes before attempting to get pregnant. It was observed that ICR

females chronically exposed to a final, cumulative dose of 12 mg/kg of PAHs, prior

to conception, had a significantly decreased number of live embryos and a

significantly increased number of dead embryos at d9.5 of gestation. This translates

to a loss of approximately 35% of the litter in a PAH-exposed dam, compared with a

5% loss in control animals treated with vehicle. Additionally, PAH-exposed females

had a lower pregnancy rate compared with vehicle-treated females. Moreover, both

increased resorption rates and decreased pregnancy rates were still observed in

females up to 8 weeks after the final dose of BaP and DMBA (unpublished

observations, J. Detmar). Previous studies in rats have determined that after

intravenous BaP exposure, this chemical demonstrated a long half-life in a number

of different tissues [279] that can lead to persistence of DNA adducts in liver and

lung tissue [280]. Therefore, it would appear that PAH exposure prior to pregnancy

has long-lasting effects — at least with respect to spontaneous abortion and time to

140

conception — likely due to accumulation of these compounds and their metabolites in

various tissues.

DMBA has been extensively used as a prototypical ovotoxic compound [386]

with an unknown mode of action. We recently determined that DMBA/AhR

complexes activate the pro-apoptotic Bcl-2 family member, Bax. This pathway

appears to be necessary for the induction of cell death, since animals lacking

functional AhR or Bax are almost completely resistant to DMBA-driven follicular

atresia [369, 370]; however, dioxin — another potent AhR ligand — failed to activate

Bax and induce oocyte death in the ovary. This is due to minor changes in the

flanking nucleotide sequence adjacent to the tandem AHRE in the Bax promoter

[369]. This is also consistent with the observation that dioxin failed to compromise

preimplantation development or induce cell death in the murine blastocyst [387,

388], while DMBA proved capable of doing so in both oocytes and embryos (current

study). In both ovarian and embryo cultures, DMBA-mediated cell death required 48

hours of exposure and could be prevented by ANF, a synthetic inhibitor of AhR.

Ligand-engaged AhR resulted in the induction of Bax, followed by a slight change in

mitochondrial membrane potential and the activation of caspase-3. This was

observed in both the ICM and the TE, suggesting that the response of ovarian

follicles and cells of the early developing embryo utilize similar signalling pathways in

response to DMBA exposure. Moreover, DMBA exposure resulted in reduced live

birth rates, but only when cell death was induced at 48 hours, suggesting that cell

death contributes to either failed implantation or later embryo loss.

141

AhR expression in rabbit and mouse embryos has been previously described

[388, 389]. In the rabbit, blastocyst AnR expression is first localized to the polar

trophectoderm and eventually spreads to the embryoblast at later stages [390, 391].

Additionally, in vitro AnR antisense oligodeoxynucleotide experiments resulted in a

lower incidence of murine blastocyst formation, as well as a decreased mean

embryo cell number [389]. This suggests that AhR activity may be required for the

proper regulation of cell cycle progression. Our data indicate that excessive

activation of AhR by PAHs not only interferes with cell cycle, but also initiates

apoptosis. With the clear apoptotic effects of acute exposures to DMBA in embryos

observed in vitro, we proceeded to test the biological effects of chronic exposure to

PAHs in vivo. The use of more than one surrogate PAH and the injection of slow-

release doses over a period of nine weeks was designed to more closely parallel

PAH exposure found in human cigarette smokers. For these experiments, both

preimplantation and postimplantation embryos were examined, as preliminary in

vitro data suggested that PAHs had adverse effects on both stages in gestation.

While the mitotic and cell death indices did not reach statistical significance in

embryos exposed to PAHs in vivo, there was a trend towards increased cell death

and decreased mitosis in embryos from dams treated with PAHs, prior to

conception. Considering the transient nature of markers capable of detecting cell

death, in addition to the effective phagocytosis of cell debris by TE cells [392], even

these minor changes in cell fate resulted in a significant decrease in cell number in

PAH-exposed embryos.

142

Since embryonic resorption involves triggering the cell death cascade [393,

394], we tested whether mice with null deletions of Bax and Hrk — known

downstream targets of PAHs and dioxins, respectively — could rescue the resorption

phenotype seen in our model. Hrk is a pro-apoptotic, transcriptionally regulated,

BH3-only family member [375, 382]. It functions to induce cell death by binding to

pro-survival, Bcl-2 family members, thus de-activating their anti-apoptotic signals

and priming the cell for death [15]. Bax operates downstream of Hrk, forming pores

in the outer mitochondrial membrane and allowing release of apoptotic factors, such

as Apaf-1, Smac and cytochrome c. Therefore, the BH3-only and Bax family

members act in concert to tip the balance in the cell towards the apoptotic program

(reviewed in [15]). In our model, it was observed that deletion of Hrk did not rescue

the resorption phenotype; however, Bax KO embryos were protected from PAH-

induced death, as the proportion of Bax-deficient embryos was significantly higher in

PAH-treated, compared with vehicle-treated, Bax heterozygous females. Thus, Bax

appears to be a pro-apoptotic regulator of PAH-induced spontaneous abortion. In

addition, since Bax is a strong inducer of cell death, it is possible that the lack of

rescue seen in Hrk-deficient mice is due to overwhelming stimulation of Bax, and

that knocking out Hrk in such a model is insufficient to compensate for directly

activated Bax-driven death.

The results of our experiments indicate that maternal exposure to PAHs prior

to conception alters the sex ratio, with a significantly greater proportion of males per

litter being observed in PAH-treated compared with vehicle-treated dams.

Epidemiological studies examining the influence of cigarette smoke on human sex

143

ratios have yielded conflicting results, with reports of altered sex ratios at birth,

depending on the level of exposure, and whether the father or the mother, or both,

smoke tobacco products [395-397]; however, it was determined that exposure to

cigarette smoke does not appear to effect changes in the proportion of sperm

carrying either the X or Y chromosome [3897]. While the molecular mechanisms

behind sex skewing is unclear, it was recently reported that maternal smoking and

the genetics of signal transduction had an effect on the sex ratio at birth [398].

Additionally, alterations in the maternal sex hormone profile have been reported to

be associated with changes to the offspring sex ratio in rodents [399] and exposure

to BaP has been shown to affect sex hormone levels in pregnant rats [275].

Furthermore, female embryos may be more susceptible to apoptosis induction, as

several cell death associated genes (e.g. XIAP, AIF) are located on the X

chromosome and may impact the execution of Bax-mediated death. Hyperglycemia-

induced apoptosis of preimplantation embryos is a Bax-driven process [400] anda

recent report demonstrated differential susceptibility of male and female embryos to

apoptosis [401]; however, an increased number of viable female embryos was

observed in that study. Extrapolation of data from rodent-based studies to the

human population obviously requires extreme caution and the results of these

experiments merely underscore the need to continue investigations into the exact

relationship between cell death susceptibility, genetic sex and gene expression.

144

Conclusion

While many studies clearly indicate that antismoking interventions during

pregnancy should be a high priority as a part of prenatal care, exposure to PAHs

from second-hand smoke is more difficult to prevent. Moreover, even if women stop

smoking as soon as pregnancy is detected, this would not eliminate exposure to

PAHs, which have accumulated in maternal tissues and could still pose a significant

threat to the developing fetus. It is, therefore, important to dissect the molecular

mechanisms driving the biological effects of PAHs and develop strategies to

antagonize the adverse effects of toxic AhR ligands, as a step towards the future

design of therapies to improve fetal growth, development and survival.

145

CHAPTER 4

MATERNAL EXPOSURE TO POLYCYCLIC AROMATIC HYDROCARBONS

LEADS TO ALTERED PLACENTAL VASCULATURE AND IUGR IN C57BL/6

MICE, WHICH IS RESCUED BY AHR DEFICIENCY

146

CHAPTER 4: Maternal exposure to polycyclic aromatic hydrocarbons leads to altered placental vasculature and IUGR in C57BI/6 mice, which is rescued by AhR deficiency.

4.1 Abstract

Maternal cigarette smoking is considered to be an important risk factor

associated with fetal IUGR. Polycyclic aromatic hydrocarbons (PAHs) are well-

known constituents of cigarette smoke and the effects of acute exposure to these

chemicals at different gestational stages have been well established in a variety of

laboratory animals. In addition, many PAHs are known ligands of the aryl

hydrocarbon receptor (AhR), a cellular xenobiotic sensor responsible for activating

the metabolic machinery. In this study, we have developed a chronic, low-dose

regimen of PAH exposure to C57BI/6 mice prior to conception, which ultimately

resulted in IUGR in d15.5 fetuses. Furthermore, we observed both histological and

structural adaptations in the placental vasculature of PAH-exposed dams, with

significant reductions in arterial surface area and volume of the fetal vasculature in

d15.5 placentae. This altered vascularization was accompanied by reduced

labyrinthine and increased CP cell death rates. AhR-deficient fetuses were rescued

from PAH-induced growth restriction and exhibited no changes in the labyrinthine

cell death rate. The results of this investigation demonstrate that chronic exposure

to PAHs is a contributing factor to the development of IUGR in human smokers and

that the AhR pathway is involved.

147

4.2 Introduction

Cigarette smoking during pregnancy has serious consequences to maternal,

embryonic, fetal and neonatal health, having been associated with increased risks of

spontaneous abortion, placental abruption, placenta previa, ectopic pregnancy and

premature delivery (for reviews see [253, 360, 363]). Gestational exposure to

tobacco products is now causally associated with IUGR and is considered to be the

mediating factor in smoking-related neonatal mortality [402]. Only a small portion of

human smokers have growth-restricted babies and maternal genotype associated

with metabolism of polycyclic aromatic hydrocarbons (PAHs) has been implicated in

the development of this condition [403, 404].

While the direct cellular targets of cigarette smoke causing the IUGR

phenotype are unknown, maternal smoking has been reported to influence the

placental architecture, affecting both the vascular and trophoblast compartments.

Histomorphometric studies of placentae from smoking mothers have revealed

alterations in maternal villous spaces [263] and fetal capillary volume [262, 263, 265]

and surface area [265]. Functionally these changes are reflected by increased

resistance in both the uterine and umbilical arteries as determined by Doppler

Studies [264, 405]. In addition to alterations in placental vasculature, maternal

exposure to cigarette smoked has been associated with reduced ST apoptosis [266]

and trophoblast hyperplasia [406, 407].

There are greater than 4,000 chemical components found in cigarettes;

however, the main toxicants are a group of carcinogens known as polycyclic

148

aromatic hydrocarbons [408]. Included in this group are benzo(a)pyrene (BaP) and

dimethylbenz(a)anthracence (DMBA), which are known carcinogens and whose

toxic effects include the formation of DNA- and protein adducts, in addition to

triggering the expression of xenobiotic-metabolizing enzymes through binding to the

aryl hydrocarbon receptor (AhR). While PAHs are known environmental pollutants

generated by fossil fuel combustion, car exhaust and forest fires [409], or through

the consumption of smoked and grilled foods [410], the major source of human

exposure is through use of tobacco products [411]. Polycyclic aromatic

hydrocarbons have long been known to exert toxic effects on a variety of organs,

including those of the reproductive system (for review, see [412]). Furthermore,

PAHs have been shown to cross the placenta [413, 414] and form hemoglobin

adducts in both maternal and fetal sera [415], in addition to forming DNA adducts in

both human [272] and murine [416] trophoblasts.

The aryl hydrocarbon receptor (AhR) is a basic helix-loop-helix transcription

factor, acting as a xenobiotic sensor for a number of different hydrocarbons,

including PAHs (for review see [288, 367]). Ligands, such as BaP and DMBA,

diffuse across the cell membrane and bind to AhR, causing a conformational

change, exposing a nuclear localization sequence. This allows the receptor-ligand

complex to translocate into the nucleus, where it binds with its partner, the AhR

nuclear translocator (Arnt) and binds promoter regions at AhR/dioxin/xenobiotic

response elements (AREs/DREs/XREs), activating the transcription of cellular

detoxification machinery. AhR-deficient mice are resistant to BaP-induced

carcinogenicity [417],dioxin toxicity [290, 418], exhibit cardiac hypertrophy, reduced

149

fecundity and postnatal growth retardation, independent of exogenous ligand [291,

302]. AhR expression has been previously demonstrated in the mouse uterus and

fetal endothelium of the mouse placenta [293].

A number of murine studies have explored the consequences of acute dioxin

or PAH exposure during gestation [419-421]. In addition, laboratory studies have

revealed that rats exposed to side-stream smoke during pregnancy yielded pups

with significant decreases in weight [422, 423]. However, there is little information

regarding the effect of maternal exposure to PAHs prior to pregnancy. As these

chemicals have been shown to accumulate in the adipose and mammary tissue

[279, 424], the slow release of unaltered PAHs into the maternal blood can still

present a toxicological threat to the growing fetus. We previously reported a murine

model [425] designed to mimic the phenomenon observed in human populations,

where women will cease smoking upon attempting, or acquiring knowledge of,

conception — typically due to fetal health concerns [373, 374]. Herein, we report that

chronic exposure to PAHs prior to gestation, results in compromised maternal and

fetal placental vascularization and altered labyrinthine architecture in C57BI/6 mice,

leading to |UGR, which is rescued by AhR deficiency.

4.3 Materials and Methods

4.3.1: In vivo BaP and DMBA treatment

C57BI/6 (National Cancer Institute, Frederick, Maryland, USA) and AhR

heterozygous (C57BI/6-AhR”"*") virgin female mice were randomly separated into

PAH-treated or vehicle-treated groups and group-housed in separate cages.

150

Animals were maintained in a controlled room with a 12h light: 12 h dark cycle and

allowed ad libitum access to rodent chow and water. Subcutaneous injections of

vehicle or PAHs were administered over a nine-week period as previously described

[425]. The final, cumulative dose for PAH-treated mice was 12 mg/kg; vehicle-

treated animals were given proportional injections of corn oil, according to body

weight.

4.3.2: Mating and tissue collection

Four days after the last injection, female mice were mated with the

appropriate male stud (i.e. C57BI/6 or AhR Het males). Gestational age was

determined based on the presence of a vaginal plug, with the morning of detection

designated as day 0.5 (d0.5) post coitum. Plugged females were removed from the

male and group-housed in separate cages; it was ensured at all times, that PAH-

treated and vehicle-treated females were placed in different cages. Pregnant dams

were euthanized by cervical dislocation at d15.5 and uteri were placed in phosphate-

buffered saline (PBS; the number of conceptuses and resorptions was recorded.

Fetal and placental wet weights were taken and placentae derived from AhR

heterozygous mothers were halved at the approximate midline. The larger half

containing the umbilicus was fixed in ice-cold, 10% phosphate-buffered formalin and

the smaller half was frozen on dry ice. C57BI/6 placentae were fixed or frozen

whole. Where required, a piece of fetal forelimb tissue was removed, washed in

PBS, placed on dry ice and stored at -20°C for PCR genotyping. Frozen tissue was

151

stored at -80°C and fixed tissue was washed twice for 1 hour in PBS and stored in

70% ethanol at 4°C. AhR fetuses were genotyped by PCR as previously described

[301]. All animal experiments were conducted using protocols approved by the

Animal Care Committee at the Samuel Lunenfeld Research Institute, Mount Sinai

Hospital.

4.3.3: Vascular casting and ultrasound biomicroscopy

Vehicle- and PAH-exposed, C57BI/6 pregnant dams were euthanized by

cervical dislocation at d15.5 and vascular casts were made with methyl methacrylate

(i.e. plastic) casting compounds and processed for scanning electron microscopy

(SEM) using established methods [150]. This technique was used to qualitatively

examine the fetal and maternal placental circulations. Briefly, plastic casts of the

feto-placental circulation were obtained by dissecting d15.5 pregnant uteri and

placing tissue into ice-cold PBS. Concepituses were individually exposed and placed

in a warm saline bath to inititate cardiac function and placental blood flow. After

inserting a glass cannula into either the umbilical vein or artery, blood was flushed

from the placental circulation with warmed saline (0.9% NaCl, 2% xylocaine, 100 IU

heparin/mL). Methyl methacrylate casting compound (Polysciences Inc.,

Warrington, PA; Batson’s number 17; 5 mL monomer base, 1.5mL. catalyst, 0.1 mL

promoter) was infused into one umbilical vessel until it exited via the other, inn order

to completely fill the feto-placental vasculature. The vessels were tied off and the

casting compounds were allowed to polymerize for several hours, after which the

152

placenta was severed from the fetus. To remove surrounding tissue, placentae were

immersed in 20% KOH and casts were subsequently washed with water and air-

dried.

Plastic casts of maternal placental circulations were obtained by euthanizing

d15.5 pregnant mouse by cervical dislocation and injecting 50 wL of 100 IU/mL

heparin into the still-beating heart. The chest was opened and a catheter inserted

into the descending aorta; a cut was made into the right atrium to serve as an exit for

the perfusate. The initial perfusate consisted of chilled saline-xylocaine mixture (as

described previously), which dilated the vessels and cleared the circulation of blood.

This was followed by injection of approximately 4-5 mL of liquid plastic (prepared as

described previously), after which the inferior vena cava was ligated. Once the

plastic hardened, the uterus was removed and cut between the individual

implantation sites. Casts were exposed by digesting the surrounding tissue with

KOH, washing with water and air-dried, as described previously. Plastic casts for

both fetal (Nvenicie=4 placentae from 2 dams; nean=5 placentae from 2 dams) and

maternal (Nvehicie=3 placentae from 2 dams; npay=3 placentae from 2 dams) placental

circulations were viewed using a FEI XL30 (FEI Systems Canada Inc., Toronto, ON)

scanning electron microscope.

Microcomputed tomography (MicroCT) was employed to obtain quantitative

measurements of the feto-placental vasculature, using methods previously

described [336]. Briefly, d15.5 pregnant uteri were collected into ice-cold PBS.

Conceptuses were individually exposed and placed in a warm saline bath to inititate

cardiac function and placental blood flow. After inserting a glass cannula into either

153

the umbilical vein or artery, blood was flushed from the placental circulation with

warmed saline (0.9% NaCl, 2% xylocaine, 100 IU heparin/mL). Radio-opaque

silicone rubber (Microfil, Flow Technology, Carver, MA, USA) was infused into the

vessels until it reached the capillaries. The vessels were tied off and the casting

compounds were allowed to polymerize for several hours, after which the placenta

was severed from the fetus. The perfused placenta was fixed in 10% phosphate-

buffered formalin and mounted in 1% agar containing 10% formalin for subsequent

scanning. Three-dimensional data sets were obtained using an MS-9 micro-CT

scanner (GE Medical Systems, London, ON, Canada) and analyzed using Amira

software (TGS, Berlin, Germany). Images and data were obtained for both arterial

(Nehicile=9 placentae from 4 dams; npay=9 placentae from 4 dams) and venous

(Nvehicle=8 placentae from 4 dams; npay=8 placentae from 4 dams) fetal vessels.

Ultrasound biomicroscopy (UBM) was utilized to assess placental

hemodynamics and fetal dimensions in vivo. Vehicle-exposed and PAH-exposed

dams were anaesthetized with isofluorane at d15.5 and crown-rump length, fetal

heart rate and pulse velocity of the uterine artery were measured using 30 MHz

ultrasonography (Vevo 770, Visualsonics, Toronto, ON, Canada). Data were

obtained for 11-15 fetuses from three vehicle-exposed dams and 17-19 fetuses from

four PAH-exposed dams.

4.3.4: Terminal deoxynucleotidyl transferase dUTP nick-end labeling

C57BI/6, AhR wildtype (WT) and AhR knockout (KO) placentae from vehicle-

or PAH-exposed dams were embedded in paraffin using routine histological

154

techniques and 5 um sections were taken at the midline. Sections were

deparaffinized and were labeled, and TUNEL-labelled cells were quantified using

techniques similar to those described in section 2.3.2.

4.3.5: Histological Staining

Sections were deparaffinized in xylene and rehydrated to water. To

distinguish cytotrophoblast cells lining the maternal blood spaces, rehydrated

sections were briefly equilibriated with alkaline phosphatase (AP; 100 mM Tris, 100

mM NaCl, 5 mM CaCh, pH 8.5) buffer, then held in AP substrate solution (NBT +

BCIP in AP buffer; Promega, Madison, WI, USA) for 25 minutes at 37°C ina

humidified chamber. Slides were washed briefly with water, stained with nuclear fast

red and counterstained with 1% tartrazine yellow. To visualize collagen deposition,

Masson’s trichrome stain was applied, after post-fixing rehydrated sections in

Zenker’s fixative for 1 hour.

4.3.6: Immunohistochemistry and Lectin Histochemistry

Sections were deparaffinized in xylene, rehydrated and underwent microwave

antigen retrieval in 10 mM citrate buffer (pH 6.0). Subsequent steps were performed

in the same manner as those described in section 2.3.4, using anti-AhR antibody

(1:500; Biomol, Plymouth Meeting, PA, USA). The techniques used for lectin

histochemistry are also described in section 2.3.4.

155

4.3.7: Caspase-3 enzyme assay

Enzymatic activity of caspase-3 in vehicle- or PAH-exposed C57BI/6

placentae was assessed using the Caspase-3 Cellular Activity Assay Kit PLUS

(Biomol, USA), as described in section 2.3.5

4.3.8: Western blotting

To assess protein expression in control or treated C57BI/6 placentae, one

placenta from each of five dams was processed as described in section 2.3.6.

Individual AhR WT and KO placentae (vehicle and PAH-exposed) were similarly

treated, after maternal decidua was removed. Polyacrylamide gels and immunoblots

were prepared as previously described (Section 2.3.6) and the following primary

antibodies were used: anti-caspase-3 and -6 (1:500; Cell Signalling, Danvers, MA,

USA); anti-cleaved Parp-1 (1:500, Cell Signalling); anti-Bax NT (1:500, Upstate,

Lake Placid, NY, USA); anti-Xiap (1:500, BD Biosciences, Mississauga, ON,

Canada); anti-FasL (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA); p53

(Vector labs, Burlington, ON, Canada); anti-FAK (Santa Cruz); and, p21

(Calbiochem, Mississauga, ON, Canada). Blots were stripped and reprobed with

anti-B-actin antibody (1:400, Santa Cruz) to correct for protein loading.

4.3.9: Statistical analysis

Statistical analysis of AnR data was done using two-way ANOVAs. All other

statistical tests were done using Student's t-test. Statistical software used was

SPSS® (Version 13) and data were considered statistically significant if p < 0.05.

156

4.4 Results

4.4.1: Maternal exposure to PAHs prior to conception leads to IUGR and altered labyrinthine vasculature in C57BI/6 mice

We have previously shown that maternal exposure to PAHs prior to

pregnancy on an outbred, ICR genetic background results in increased resorption

rates due to elevated cell death during the preimplantation stage [425]. While

C57BI/6 dams exposed to PAHs exhibited a trend towards smaller litters, this

decrease was not statistically significant (data not shown). Instead, PAH treatment

of C57BI/6 females yielded fetuses with a 14% reduction in weight compared to

fetuses from dams exposed to vehicle (Figure 4.1a). These data were supported by

crown-rump length measurements by UBM, which were significantly reduced in

fetuses from PAH-exposed dams (Figure 4.1b).

Evaluation of d15.5 placental sections using various histological techniques

revealed aberrations in the labyrinthine region, particularly in the vasculature. After

staining placental sections from PAH-exposed dams with Masson’s trichrome, it was

apparent that collagen deposition along the chorionic plate and into the large

chorionic vessels was thinner than in vehicle-treated controls (Figure 4.1c). In

addition, alkaline phosphatase and lectin (BS-l) histochemistry (markers of maternal

and fetal blood spaces, respectively) revealed alterations in the labyrinthine

architecture, characterized by dilatation of both the maternal blood spaces and fetal

microvasculature (Figure 4.1c). The other placental regions were examined but did

not exhibit any obvious defect.

157

Figure 4.1 Maternal exposure to PAHs prior to conception leads to IUGR and

altered labyrinthine architecture in C57BI/6 mice at d15.5 gestation. A. Bar

graph depicts placental and fetal weight of d15.5 conceptuses from C57BI/6 dams

exposed to vehicle (n=62 conceptuses from 8 dams) or PAHs (n=55 conceptuses

from 9 dams) prior to pregnancy. B. Graph demonstrates fetal crown-rump length

measurements of d15.5 fetuses from vehicle-exposed (n=15 fetuses from 3 dams)

and PAH-exposed (n=17 fetuses from 4 dams) dams. C. Photomicrographs in

upper panel represent the basolateral edge of d15.5 placentae, exposed to vehicle

or PAHs and stained with Masson’s trichrome (nuclei are dark red-blue, cytoplasm is

red, collagen is bright blue). Photomicrographs in middle panel are of d15.5

placental labyrinth obtained from vehicle- or PAH-exposed dams, after histochemical

Staining with alkaline phosphatase (AP) and counterstaining with nuclear fast red

and tartrazine yellow. Purple-staining outlines the maternal blood spaces due to

fetal CTB reaction with AP substrate, cytoplasm is yellow or yellow-orange and

nuclei are red. Lower panel contains photomicrographs of d15.5 C57BI/6 labyrinth

exposed to vehicle or PAHs and labeled with Bandeiraea simplicifolia (BS-l) lectin (a

marker of endothelial cells), outlining the fetal blood spaces; nuclei are

counterstained with hematoxylin. Bars represent mean values + standard error (SE)

and values of significant statistical difference are shown with the corresponding p

value. Arrows indicate the line of collagen deposition at the chorionic plate and

arrowheads indicate chorionic vessels.

158

A. d15.5 Fetal and Placental Weights B. Fetal Crown-Rump Length Measurements by UBM

0.5 p<0.001 y

0.45 14

— 0.4

S 0.35 0 Vehicle = 138 £ 03 m PAHs = 13.6 g 0.25 13.4

0.2 D 13.2 “ % 13.0

0.05 _ 12.8 _

o |ins82 12.6 n=15 Placental Weight Fetal Weight Vehicle PAHs

C. Vehicle

Masson’s trichrome for

collagen

Alkaline phosphatase for CTBs lining maternal blood

spaces

BS-I for

fetal blood

spaces

Figure 4.1

159

To qualitatively evaluate the 3-D changes induced by maternal exposure to

PAHs, plastic vascular casts of the maternal and fetal placental circulations were

prepared. Consistent with those alterations observed in histological sections,

placental vascular casts revealed several changes in the vascular tree. Casts of the

fetal placental vasculature from mothers treated with PAHs, consistently exhibited

engorged, dilated capillaries that were not evident in the feto-placental casts from

dams exposed to vehicle (Figure 4.2a, b). Additionally, PAH-exposed fetal

capillaries exhibited numerous, intricate anastomoses and a greater degree of

tortuosity compared with the more linear structures observed in the fetal casts

exposed to vehicle. Moreover, this same dilatation and engorgement was apparent

in the maternal placental canals of PAH-exposed placentae (Figure 4.3a, b). Lastly,

large maternal canals were consistently observed to be projecting out of the

junctional zone, close to the chorionic surface of placentae obtained from dams

treated with PAHs (Figure 4.3b).

4.4.2 Reduced umbilical vessel diameter and total fetoplacental vascular surface area and volume in d15.5 placentae from PAH-exposed dams

Micro-computed tomography (microCT) was employed to measure all feto-

placental vessels greater than 0.03mm in diameter, with 3-D datasets and iso-

intensity surface renderings acquired as previously described [336]. The acquired

160

Figure 4.2 Maternal exposure to PAHs prior to conception in C57BI/6 mice

results in aberrant placental microvasculature in the fetal compartment.

Scanning electron photomicrographs of corrosion casts viewed from the chorionic

side from (A) vehicle and, (B) PAH-exposed d15.5 C57BV6 placentae. Boxed region

in cast of feto-placental vasculature in upper-left panel is magnified in centre panel.

Boxed regions in centre panel are magnified in panels to the right.

161

FETAL PLACENTAL VASCULATURE CASTS

Figure 4.2

162

Figure 4.3 Maternal exposure to PAHs prior to conception in C57BI/6 mice

results in aberrant morphology of blood spaces in maternal compartment.

Scanning electron photomicrographs of corrosion casts of maternal placental

labyrinthine canals from (A) vehicle and (B) PAH-exposed d15.5 C57BI/6 placentae.

Boxed region in left panel is magnified in right panel. Arrow indicates a large,

superficial maternal canal. Superficial canals were consistently observed in

maternal-side casts obtained from PAH-exposed dams.

163

MATERNAL PLACENTAL VASCULATURE CASTS

A. Vehicle

B. PAH-exposed

Figure 4.3

164

surfaces (see Figure 4.4a, c) allowed 3-D visualization of the fetoplacental

vasculature as well as calculation of vessel diameters, surface area, and overall

lumen volume. While no overt changes in the large vessels of the venous

vasculature were observed between vehicle and PAH-exposed placentae, the

arterial surface renderings of PAH-exposed placentae displayed a greater degree of

tortuousity in some large vessels of the trees (Figure 4.4b). In addition, PAH-

exposed placentae exhibited significantly reduced arterial surface area and volume

compared to placentae from vehicle-exposed dams (Figure 4.5a, b), while no

change in venous surface area or volume was observed between the two groups.

However, both the umbilical artery and vein demonstrated significant decreases in

diameter in placentae from dams treated with PAHs compared to dams treated only

with vehicle (Figure 4.5c). In spite of these alterations in fetal and placental

vasculature, fetal abdominal cross-sectional area, fetal heart rate and the peak

systolic blood velocity in the umbilical artery did not change with PAH exposure, as

determined by Doppler studies (Figure 4.6a-c).

4.4.3 Both fetal and maternal compartments of d15.5 PAH-exposed placentae exhibit altered cell death rates and changes in cell death markers

Polycyclic aromatic hydrocarbons have been implicated in altering the

balance of cell survival and death in a number of different cell types [864, 426, 427].

As precisely regulated cell death is essential for trophoblast turnover, analyses of

cell death were implemented. Morphometric analyses of d15.5 placental sections

after TUNEL, revealed significant decreases in the number of labeled cells in the

165

Figure 4.4 Two-dimensional renderings of d15.5 fetal placental vessels from

vehicle and PAH-exposed dams, after micro-computed tomography. A. Two-

dimensional rendering of fetal arterial cast obtained from d15.5 placentae of vehicle

and PAH-exposed dams. B. Higher magnification of boxed region in (A)

demonstrating the altered curvature of the larger fetal vessels. Red arrows indicate

the direction of blood flow. C. Two-dimensional rendering of fetal venous cast

obtained from d15.5 placentae of vehicle and PAH-exposed dams.

166

MICRO-COMPUTED TOMOGRAPHY OF d15.5 FETAL PLACENTAL VESSELS UP TO 13 um RESOLUTION

A. Vehicle PAH-exposed

Arterial

Cast

Venous

Cast Figure 4.4

167

Figure 4.5 Micro-computed tomography of fetal placental casts reveals

decreased arterial surface area and umbilical vessel diameter in placenta from

PAH-exposed C57BI/6 dams. A. Graph depicting the average surface area of the

fetal, arterial or venous vasculature in vehicle and PAH-exposed placentae. B.

Graph depicting the average fetal, arterial or venous volume in vehicle and PAH-

exposed placentae. C. Graph depicting the average fetal arterial or venous

umbilical vessel diameter in vehicle or PAH-exposed placentae. Bars represent

average values + SE and values of significant statistical difference are shown with

the corresponding p value (Student’s t-test).

168

A. Vascular Surface Area

_ 120, p=0.0043 N

E 100 - | D Vehicle 80 - m@ PAHs

g 60 -

8 40 -

“© 204 > YM 4 n=9 n=8

Arterial Venous

B. Vascular Volume

4 - p=0.0032

3.5 +

E 3 | | & 2.54 D Vehicle

@ 24 m@ PAHs

E 1.54 6 1- >

0.5 4 0 n=9 n=8

Arterial Venous

C. Umbilical Vessel Diameter

06- | p=0.00014 3=0.00074

—~ 0.54 | E Oo i € 0.44 Vehicle

_ @ PAHs £2 034

reby

— 02] QO

0.1 -

0 n=9 n=7

Arterial Venous

Figure 4.5

169

Figure 4.6 Chronic exposure to PAHs prior to conception does not alter fetal

heart rate, abdominal cross-sectional area, nor umbilical artery pulse velocity,

as determined by ultrasound biomicroscopy. Graphs depict unchanged (A)

abdominal cross-sectional area in d15.5 fetuses; (B) heart rate in d15.5 fetuses; and

(C) pulse velocity of the umbilical artery, after maternal exposure to PAHs. Bars

represent average values + SE; n values reflect the number of fetuses analyzed in

vehicle (n=3) and PAH-exposed (n=4) dams.

Peak velocity (m

m/s)

Ar

ea (mm?)

Rate

(b

eats

pe

r minute)

35

30

25

250

200

150

100

50

20)

15]

10}

170

Abdominal Cross-sectional Area

n=11

Vehicle PAH-exposed

Fetal Heart Rate

n=14

Vehicle PAH-exposed

Peak Systolic Blood Velocity in the Umbilical Artery

o

zr

>

ON

[SP 6 O

o N

<A Oo

oOo

oO

n=14

Vehicle PAH-exposed

Figure 4.6

171

labyrinth and junctional zone of placentae from PAH-exposed dams (Figure 4.7a).

Based on the location and the presence of fetal red blood cells, the dead/dying cells

appear to be a combination of fetal endothelium and ST cells. In addition, the

incidence of sporadic decidual cell death, and the percent area of TUNEL-positive

tissue in the maternal compartment also significantly decreased compared with

placentae exposed to vehicle only (Figure 4.7a, b). Conversely, PAH treatment

caused a significant increase in the number of TUNEL-positive cells in the CP

(Figure 4.8a), whereas TGCs remained unaffected (Figure 4.8b). Note that positive

(section pre-incubated with DNase | enzyme) and negative (sections incubated

without Tdt enzyme) controls exhibited high levels and absence of TUNEL-positivity,

respectively

Assessment of cell death markers revealed altered expression and activity in

several key proteins. Decreased rates of placental cell death in dams exposed to

PAHs were accompanied by diminished caspase-3 enzyme activity (Figure 4.9a).

These data were supported by immunoblotting, as levels of cleaved caspase-3 and -

6 also declined significantly, while total caspase-3 and -6 levels appeared

unchanged (Figure 4.9b, c). Moreover, examination of the cleavage profiles of

known, intracellular caspase-3 substrates [93, 324, 325] revealed significantly

reduced levels of the cleaved forms of Parp-1 (Figure 4.10a), p21 (Figure 4.10b),

PTEN (Figure 4.10c) and FAK (Figure 4.10d). Additionally, Bax, a pro-apoptotic

protein known to be regulated by AhR after PAH exposure [370] exhibited

significantly decreased levels of expression in placentae from PAH-exposed,

compared with vehicle-exposed dams (Figure 4.11a). Moreover, significantly higher

172

Figure 4.7 Chronic exposure to PAHs prior to pregnancy leads to altered cell

death patterns in d15.5 placentae of C57BI/6 dams. A. Graph depicts the

number of TUNEL-positive nuclei per 100 ym? of labyrinthine, junctional zone or

decidual tissue in d15.5 vehicle or PAH-exposed placentae. Data for decidual cells

reflect positive cells sporadically placed within the maternal compartment.

Accompanying photomicrographs in right panel represents TUNEL patterns

observed in vehicle or PAH-exposed placental labyrinth. B. Graph depicts the

percent area of tissue in the maternal compartment that was regions of clustered

TUNEL-positive cells. Accompanying photomicrographs in right panel demonstrates

foci — demarcated with dashed line — of TUNEL-positive cells in the maternal

compartment. Arrows indicate TUNEL-positive nuclei and empty arrowheads

specify trophoblast giant cells in the fetal compartment. Bars represent average

values + SE and values of significant statistical difference are shown with the

corresponding p value (Student's t-test).

#TUNEL-positive

cells

2 per

100

pm

Percent

area

of Maternal

# TUNEL-positive cells

18 p=0.03

1:2 © Vehicle

1.0) p=0.0046 m PAHs 0.8

0.6

0.2

0 n=8 [li Labyrinth Junctional Decidua -

Zone sporadic death

Percent Area of Maternal Compartment

that is TUNEL-postive foci

3.5

3.0

25

2.0

1.5

1.0

°° nin Vehicle PAH-treated

-positive

(%)

Compartment

that

is TUNEL

173

Vehicle PAH-exposed

ne Ss ¥ ins atk

Foci of TUNEL-positive cells in maternal compartment - d15.5 midline placenta

Figure 4.7

174

Figure 4.8 Chronic exposure to PAHs prior to pregnancy leads to increased

chorionic plate cell death, but TGC death is unaffected. A. Graph depicts the

number of TUNEL-positive nuclei per 100 wm? of CP in d15.5 vehicle or PAH-

exposed placentae. Accompanying photomicrographs in right panel demonstrate

TUNEL staining observed in vehicle or PAH-exposed CP. B. Graph depicts the

percentage of TUNEL-positive or condensing TGC, or the percentage of TGC

containing TUNEL-positive corpses. Arrows indicate TUNEL-positive nuclei. Bars

represent average values + SE and values of significant statistical difference are

shown with the corresponding p value (Student’s t-test).

e,cells

per

100

pm

#TUNEL-positiv

175

#TUNEL-positive Chorionic Plate Cells PAH-exposed

35 p=0.03 | 3.0 5 0 5

r

n=8

Vehi PAH- —— * ehicle treated Chorionic plate - d15.5 midline placenta

B. Rates of TGC death due to. PAH Exposure

16

. 14 ey GS 12

© § 40 C1 Vehicle

m= oO m PAH-exposed o DM

ao? 8 Do fe 6 a 8 Po 4 oo

a. 2

0 n=8 i % TUNEL-positive. .. % condensed %TGC with

TGC TGC TUNEL-positive

corpses

Figure 4.8

176

Figure 4.9 Chronic exposure to PAHs prior to conception disrupts the

expression levels of executioner caspases in d15.5 C57BI/6 placentae. A.

Graph demonstrates the average caspase-3 enzyme activity levels in 50 yg of

placental lysate obtained from d15.5 vehicle or PAH-exposed dams. Representative

immunoblots are shown to the right of each graph. Bars represent average values +

SE and values of significant statistical difference are shown with the corresponding p

value (Student's t-test).

Densitometric

rati

o of

total

caspase-6:actin

Densitometric

ratio

2 o

0.5

0.4

0.3

0.2

© QO

177

A. Caspase-3 Enzyme Assay - d15.5

Placental Lysates

S ° O Vehicle Ee 6 | m™ PAH exposed Es

se 8 Eo 4 £2 3 28 5 p=0.025

2 1 0 n=5

Vehicle PAH-exposed

Caspase-3 Expression

p=0.029 35 kDa Procaspase-3

0.45

0.4 038 18 kDa Cleaved Caspase-3

0.2 0.2 = Se =) actin

Vehicle PAH- 0.1 _ = 0.0 n=6 N=6 exposed

0 — | Total Caspase-3: Cleaved Caspase-3:

actin total caspase-3

Caspase-6 Expression

35 kDa Procaspase-6

p=0.041 0.025 g g

UD =

0.02 88 9 o

0.015 2 15 kDa Cleaved , go Caspase-6

» 2 0.10 os

@ 9, Bo

v 0.005 oa Vehicle PAH- c Qo S$ exposed

a

Total Caspase-6: Cleaved Caspase-6:

actin Total Caspase-6

Figure 4.9

178

Figure 4.10 Cleavage levels of active caspase-3 cellular substrates are

reduced in d15.5 PAH-exposed placentae. Graphs demonstrate levels of cleaved

(A) Parp-1; (B) p21; (C) PTEN; and, (D) FAK, in d15.5 placental lysates from

vehicle- or PAH-exposed dams. Representative immunoblot bands for vehicle and

PAH-treated placental lysates are depicted alongside corresponding graphs. Bars

represent average values + SE and values of significant statistical difference are

shown with the corresponding p value (Student’s t-test).

5 g 0.025

2 0.020 a

© 0.015 2 a.

6 ® 0.010 = > nO

53 0.005

0

3X 0:6

ok 05 oH ef 0:4

ge 0.3

on a © 0.2

gs 0.1

0

0.05 OQ QF 0.04 © £ 22 0.03 @aa

53 0.02 a>

= 8 0.01 oOo

0

0.86

0.84

0.82 0.8

0.78

0.76

0.74

0.72 Densitometric

rati

o of

cleaved

PTEN:total PTEN

Cleaved Parp-1 (70 kDa)

+

p=0.044

n=3 n=3

Vehicle PAH-exposed

Levels of Caspase-3-specific Cleaved FAK

p=0.015

n=5 Vehicle PAH-exposed

Levels of Caspase-3-specific Cleaved p21

p=0.005

n=5

125 kDa

Vehicle PAH-exposed

Levels of Caspase-3-specific Cleaved PTEN

p=0.026

n=5

Vehicle PAH-exposed

30 kDa

179

cleaved Parp-1

" &-actin

Vehicle PAH- exposed

Vehicle

21 kDa

14.kDa

Vehicle

Vehicle

yj) Full-length FAK

cleaved FAK

PAH- exposed

#| Full-length p21

| cleaved p21

PAH-

exposed

=| Full-length PTEN

kj} cleaved PTEN

PAH- exposed

Figure 4.10

180

Figure 4.11 Chronic exposure to PAHs prior to conception disrupts the

balance of apoptotic and anti-apoptotic proteins in d15.5 C57BI/6 placentae.

Graphs demonstrate the levels of (A) Bax; (B) Xiap and, (C) FasL, as assessed by

immunoblotting, in d15.5 placental lysates from vehicle or PAH-exposed dams.

Representative immunoblots are shown to the right of each graph. Bars represent

average values + SE and values of significant statistical difference are shown with

the corresponding p value (Student’s t-test).

Densitometric

ratio

Densitometric

rati

o of

Densitometric

ratio

of FasL:actin

Bax:actin

0.2

0.15

0.1

0.05

of Xi

ap:a

ctin

oOo NM

FF

DD.

a

cn &

oo

=

tn

Nb

oO

oO a

181

Bax Expression

T 21 kDa oom! Bax

p=0.044 Vehicle PAH-

exposed

n=5

Vehicle PAH-exposed

Xiap Expression

p=0.042

i) [eed] | Xiap

i (we) (wwe! §=—R-actin

Vehicle PAH-

exposed

n=5

Vehicle PAH-exposed

FasL Expression

p=0.023

H FasL R-actin

Vehicle PAH-

exposed x

n=3

Vehicle PAH-exposed

Figure 4.11

182

expression levels of Xiap, an anti-apoptotic protein, were observed in placentae from

PAH-treated dams (Figure 4.11b). Lastly expression levels of FasL, also known to

be regulated through AhR [428] and shown to be cytoprotective in endothelial cells

[429, 430], was increased in response to PAH exposure in d15.5 placental lysates

(Figure 4.11c).

4.4.4 AhR-deficient fetuses are protected from IUGR due to chronic maternal exposure to PAHs

The aryl hydrocarbon receptor acts as an intracellular sensor, triggering the

transcription of cellular machinery and initiating the appropriate response against

potentially harmful xenobiotics. Levels of AnR expression as determined by

immunoblotting and immunohistochemistry, exhibited a small, but significant,

increase in WT placentae from PAH-exposed dams (Figure 4.12a). Furthermore,

immunoblotting and densitometric analyses of PAH-exposed placental lysates

exhibited significantly increased levels of AnR-regulated p53 [431] and its

downstream target, p21 (Figure 4.12b,c). To ascertain which placental cell types

express AhR, immunochistochemistry was performed on d15.5 placental sections.

AhR immunolocalized to the maternal endothelial cells of the decidua and to the fetal

endothelium of the labyrinthine, chorionic plate and vitelline vessels. This staining

pattern was specific, as it was not evident in the placentae of AhR KO littermates

(Figure 4.13a).

AhR heterozygous dams exposed to PAHs prior to conception yielded AhR

WT and Het offspring exhibiting a significant decrease in weight compared with

vehicle-exposed fetuses; however, PAH-exposed AhR KO fetuses demonstrated no

183

Figure 4.12 Chronic exposure to PAHs prior to conception up-regulated AhR

expression and known target genes of AhR in C57BI/6 placentae. Graphs

depict expression levels of (A) AhR; (B) p53, and (C) p21 protein in d15.5 placental

lysates from vehicle or PAH-exposed dams. Representative immunoblots are

shown to the right of each graph. Bars represent average values + SE and values of

significant statistical difference are shown with the corresponding p value (Student’s

t-test).

Densitometric

rati

o of

p53:actin

Densitometric

ratio

of Ah

R:ac

tin

Dens

itom

etri

c ra

tio

of p21:actin

0.35

0.3

0.25

0.2

0.15

0.1

0.05

184

AhR Expression

p=0.022

n=5

0.5 5

0.45 -

0.4 4

0.35.4

0.3 4

0.25 +

0.2 4

0.15 +

0.1 4

0.05 ;

Vehicle PAH-exposed

p53 Expression

p=0.024

FH p53

Vehicle PAHs

n=3

0.06

0.05

0.04

0.03

0.02

0.01

Vehicle PAH-exposed

p21 Expression

p=0.021

p21

R-actin

Vehicle PAHs

Vehicle PAH-exposed

Figure 4.12

185

Figure 4.13 Aryl hydrocarbon receptor is expressed in the fetal endothelium

of the mouse placenta and AhR deficiency rescues the IUGR phenotype in

dams chronically exposed to PAHs prior to conception. A. Photomicrographs

of d15.5 AhR WT (upper left and right panels) and AhR KO (lower left and right

panels) placenta after immunohistochemica! staining with anti-AhR antibody. Fetal

endothelial cells in the labyrinth are indicated by arrows while those of the CP are

indicated by a ¢. Asterisks specify maternal blood spaces. B. Graph exhibits d15.5

weight in AnR WT, Het and KO fetuses from heterozygous dams exposed to vehicle

(n=5 dams) or PAHs (n=9 dams) prior to conception. Bars represent average values

+ SE and values of significant statistical difference are shown with the corresponding

p value (Tukey-Kramer test).

Weight

(g)

AhR KO Labyrinth

186

Fetal Weight

AhR KO

06- p=0.036 p=0.0092

0.5 | | | O Vehicle 0.4: lm PAH-exposed 0.3 4

0.2 4

o1/ |S © = i i i

0 | Cc Cc

AhR WT AhR Het AhR KO

Figure 4.13

187

further restriction in growth compared with KO offspring from dams exposed to

vehicle only (Figure 4.13b).

The aryl hydrocarbon receptor has been implicated in a number of different

cellular pathways, including cell death, survival and the cellular stress response.

While expression of the proliferation marker, Ki67, was not obviously altered

amongst vehicle- and PAH-exposed placental sections in both AhR WT and KO

placental sections (Figure 4.14), levels of several cell death markers differed in all

four groups of placental lysates. In the absence of PAHs, placental lysates from

AhR KO fetuses exhibited statistically significant increased levels of cleaved

caspase-3 compared with placental lysates from WT littermates (Figure 4.15a).

After chronic, maternal exposure to PAHs, significantly decreased levels of cleaved

caspase-3 were observed in both PAH-exposed AhR WT and KO placental lysates.

Morphometric analyses of TUNEL-stained placentae from AhR WT and KO

littermates of dams exposed to vehicle or PAHs, revealed a reduction in labyrinthine

cell death rates of PAH-exposed, AhR WT placentae, which was not evident in AhR

KO placentae (Figure 4.15b). Lastly, both AnR WT and KO placentae from PAH-

exposed females demonstrated significantly higher numbers of TUNEL-positive cells

in the CP, compared with that seen in AhR WT and KO placentae from dams treated

with vehicle.

188

Vehicle PAH-exposed

AhR KO eee

Ki-67 immunohistochemistry on d15.5 labyrinth

Figure 4.14 Maternal exposure to PAHs does not alter proliferation in d15.5

C57BI/6 placentae, as evidenced by Ki-67 immunohistochemistry.

Photomicrographs in upper panel represent d15.5 AhR WT labyrinth after Ki67 IHC

from vehicle (left panel) and PAH-treated (right panel) dams. Photomicrographs in

lower panel represent d15.5 AhR KO labyrinth after Ki67 IHC from vehicle (left

panel) and PAH-treated (right panel) dams.

189

Figure 4.15 Maternal exposure to PAHs results in altered cell death rates in

different regions of C57BI/6 placentae. A. Graph depicts levels of total and

cleaved caspase-3 in d15.5 fetal-enriched, AR WT or KO placental lysates from

vehicle or PAH-exposed AhR heterozygous dams. Representative immunoblots are

shown below graph. B. Graph depicts the average number of TUNEL-positive cells

per 100 pm? of labyrinthine or CP tissue in d15.5 AhR WT or KO placentae from

dams exposed to vehicle or PAHs. Bars represent average values + SE and values

of significant statistical difference are shown with the corresponding p value (Tukey-

Kramer test).

> Densitometric

ratio

of cleaved

Procaspase-3 >

Cleaved caspase-3

#TUN

EL-p

osit

ive

cell

s

caspase-3:total

casp

ase-

3 2

per

100

um

190

Cleaved Caspase-3

p=0.027 p=0.026

0.30 || 0.25 4

0:20 p=0.038

0.15 | |

0.10

~ n=3 n=3

AhR WT AhR KO

Vehicle PAHs Vehicle PAHs

4.0

3.5

3.0 2.5 2.0

1.5]

1.01

0.5)

AhR WT

33 kDa

+17 kDa

AhR KO

TUNEL data for d15.5 AhR Placentae

p=0.00013

oe

p=0.045

p=0.013

AhRWT AhRKO

«<— Labyrinth —»

AhRWT AhRKO

«<— Chorionic Plate —

Figure 4.15

191

4.5 Discussion

Cigarette smoking during pregnancy is linked with a number of detrimental

outcomes, and has now been established to be causally associated with IUGR. We

recently reported the development of a new animal model of pre-pregnancy

exposure to PAHs, which on an outbred genetic background (ICR) compromises

preimplantation development, leading to embryonic resorptions [425]. The

mechanism by which PAHs exert this phenotype involves the induction of cell death

in a Bax-dependent manner, as Bax-deficient embryos are protected from early

postimplantation embryo loss triggered by maternal exposure to PAHs. Here we

report the results of our investigations into the effects of the identical toxicant

regimen on an inbred genetic background (C57BI/6). This model closely mimics

those conditions in human populations, where women are exposed to chemicals

from cigarette smoke over a long period of time, allowing accumulation of PAHs.

Upon conception, the growing fetal and placental tissues will be exposed to both

metabolized and unaltered PAHs, which have been postulated to exert a variety of

effects on different cell and tissue types (reviewed in [432]), including human

choriocarcinoma cells [433]. Moreover, exposure to PAHs during different stages of

placental development can yield different outcomes. For example, culturing first

trimester placental cells in BaP yielded decreased expression levels of EGF

receptors, whereas Culturing term placental cells resulted in EGF receptor

desensitization [434]. In addition, undifferentiated, proliferative TS cells exposed to

BaP exhibited no changes in levels of cytochrome P450 1A1, whereas expression of

this xenobiotic-metabolizing enzyme was induced in differentiating TS cells [435].

192

In the present study, maternal exposure to PAHs prior to conception caused

fetal intrauterine growth restriction in C57BI/6 dams, as evidenced by reduced fetal

weight and length. Evaluation of two-dimensional placental sections revealed

morphological changes in the labyrinth that included dilatation of the maternal and

fetal vasculature and decreased amounts of collagen at the chorionic plate and

along the large chorionic vessels. The alterations in the labyrinthine

microvasculature were confirmed by SEM of fetal and maternal corrosion casts,

which displayed larger, engorged vessels or blood spaces, irregular anastomoses

and a greater degree of vascular branching and tortuosity. Interestingly, these

changes are strikingly similar to those reported in casts of fetal capillaries from term

placental cotyledons of smoking mothers, which included increased capillary

tortuoisty, density and branching [436]. Finally, thickening of the interhemal distance

and defective labyrinthine vascular remodeling have been reported in dioxin-

exposed rat placentae [437]. Thus, the observed changes in placental vascular

architecture of women smokers may be mediated by the action of PAHs, as

maternal administration of these compounds in our murine model resulted in a

similar vascular phenotype.

The observed changes in vascular structure may be due, in part, to the

effects of PAHs on the extracellular matrix, which we have shown to be affected in

the placentae from exposed dams. Previous reports have shown that cultured

smooth muscle cells exposed to BaP or DMBA exhibited reduced secretion of newly

synthesized collagen [438]. In addition, levels of type | and III procollagen,

accompanied by increased MMP expression, have been reported in skin fibroblasts

193

cultured with tobacco smoke extract [439, 440]. It is possible that in PAH-exposed

murine placentae, expression and/or maintenance of the extracellular matrix proteins

could facilitate the observed changes in vascular architecture. The significant

decreases in arterial surface area and volume obtained from PAH-exposed

placentae, coupled with the reduced diameters of both umbilical vessels likely have

an impact on placental blood flow and/or maternal-fetal exchange. Therefore,

alterations in vascular architecture and blood flow patterns may diminish gas and

nutrient/waste exchange between the mother and the fetus, leading to IUGR.

The importance of cell death during placental development and the

consequences of disrupting this process on fetal and maternal health is highlighted

by gestational diseases such as preeclampsia and IUGR [174, 177]. A clear

difference in cell death rates was observed between the placentae from PAH-

exposed compared with vehicle-exposed dams. A similar reduction in trophoblast

cell death was recently reported in rat placentae treated with dioxin at later

gestational timepoints [437]. Significantly reduced levels of labyrinthine cell death

were observed in PAH-treated placentae, consistent with observations in placentae

from smoking mothers, where decreased apoptosis was observed at term [175,

266]. This decline in the rate of placental cell death is supported by Western

blotting, where decreased levels of cleaved caspase-3, -6, and Bax, were observed

in PAH-exposed placental lysates. Additionally, PAH treatment up-regulated levels

of the anti-apoptotic protein Xiap, a phenomenon previously reported in the placenta

of women smokers [175]. Interestingly, while Bax levels were significantly lower in

placentae from PAH-treated dams, expression levels of FasL were significantly

194

higher. Previous reports have shown that FasL is increased in endothelial cells after

exposure to cigarette smoke extract [430] or ischemia-reperfusion injury [429],

resulting in cytoprotection against Fas-expressing cells of the immune system. FasL

is known to be regulated by AhR [428] and upregulation of this molecule could

explain the decreased cell death rates observed in PAH-exposed placentae.

Furthermore, increased expression of FasL may be a link to why cigarette smokers

have some protection against preeclampsia, where FasL has been shown to be

down-regulated [114]. Lastly, the significant decrease in decidual cell death may

contribute to the observed fetal growth restriction, as apoptosis has been shown to

be involved in remodelling the maternal spiral arteries [192, 441] and perturbations

in this process have been associated with IUGR (reviewed in [189]).

Chronic or acute exposure to ligand in vivo can readily alter expression and

activity levels of ANR [442]. In our model of pre-pregnancy exposure to PAHs,

placental AhR was activated, as evidenced by increased expression levels of p53

and p21, downstream targets of ANR; however, it is presently unknown whether AhR

is activated in the placentae of women smokers. The effects of persistent ANR

stimulation, as evidenced by transgenic mice expressing a constitutively active AhR,

include a reduced life span and the presence of gastric tumours [304]. Acute PAH

exposure typically leads to downregulation of AhR protein by proteasomal

degradation in vitro and in vivo [442, 443]; however, chronic exposure appears to

send the system into a balanced steady state of AhR transcription and degradation

in vivo [442]. In addition with altered levels of transcription, ANR binding affinity is

differentially affected by either acute or chronic doses of dioxin in vivo [444]. It has

195

been proposed that AhR is degraded to regulate the amount of activated AhR and

thus maintain appropriate levels of Arnt for Arnt-dependent pathways [443, 445].

Alterations in labyrinthine cell death were not observed in PAH-exposed AhR-

deficient fetuses — which were also rescued from the IUGR phenotype — highlighting

that PAHs are responsible for this decreased apoptosis in the labyrinth, which

appears to be mediated through the AhR pathway. Moreover, it appears that cell

death has an important physiological function in the labyrinth, likely contributing to

vascular remodeling and allowing optimal cell turnover for this metabolically active

organ. On the contrary, AhR deficiency did not rescue the increased cell death rates

observed in the CP cells, suggesting that while AnR mediates death in the labyrinth,

another cell death pathway may be utilized in the CP. In addition to the observed

collagen deficiencies in PAH-exposed placentae, the increased numbers of TUNEL-

positive, CP cells may be contributing factors to the higher incidence of premature

delivery and premature rupture of the membranes in smoking mothers [446, 447].

In light of the extensive variability in reported phenotypes from both in vivo

and in vitro studies utilizing placentae obtained from smoking mothers, the results of

the present study further illustrate the necessity of considering the genetic context of

observed phenotypes. This is underscored by the fact that only a small proportion of

smokers exhibit !UGR, which has been associated with maternal genotype [126,

403]. We previously reported that ICR dams administered chronic doses of PAHs

prior to conception exhibited a higher resorption rate than those exposed to vehicle

(Figure 4.16a and [425]); however, placental and fetal weights did not differ between

the two groups (Figure 4.16b). Here, we have shown that PAH-exposed C57BI/6

Proportion

of re

sorb

ed

Weig

ht

(g)

embryos

per

litt

er

C57BI/6 d15.5 Fetal and Placental Weights

p<0.001

an

Fetal Weight

0.5

0.45 0.4 0 Vehicle

0.35 m@ PAH-exposed 0.3 0.25

0.2

0.15

0.1

0.05 |__|n=62

Placental Weight

C57BI/6 Resorption Rate (d15.5)

0.3

0.25

0.2

0.15 |

0.1

0.05

n=11 0

Vehicle

Figure 4.16 Maternal exposure to PAHs prior to conception results in

PAH-exposed

Weight

(g)

Proportion

of resorbed

embryos

per

litt

er

0.6

0.5

0.4

0.3

0.2

0.1

196

ICR d15.5 Fetal and Placental Weights

QO Vehicle

@ PAH-exposed

al

0.4

0.35

0.3

0.25

0.2

0.15

0.1

0.05

Placenta Fetus

ICR Resorption Rate (d7.5-d12.5)

I

n=15

p<0.001

Vehicle PAH-exposed

increased resorption rates in ICR dams and IUGR in C57BI/6 dams. A. Graph

depicts significantly increased resorption rates (d7.5-d12.5) in ICR dams after PAH

treatment. B. Graph demonstrates the lack of effect of PAH treatment on d15.5

placental and fetal weights in ICR dams. C. Graph depicts the significant reduction

in d15.5 fetal weight in CS7BI/6 mice after PAH treatment, while (D) depicts the

similar resorption rates seen in PAH-exposed, compared with vehicle-exposed

C57BI/6 dams. Bars represent average values + SE and values of significant

statistical difference are shown with the corresponding p value (Student’s t-test).

197

dams yielded growth-restricted fetuses (Figure 4.16c), but no difference in resorption

rate (4.16d), compared with dams exposed to oil. Furthermore, the majority of these

mice were injected at the same time and with the same formulation, in addition to

being housed in the same room, considerably reducing the number of variables

between the groups. Lastly, AhA-deficiency has been shown to rescue KO fetuses

from PAH-induced IUGR. This receptor is notoriously susceptible to genetic

background, with several polymorphisms in both the promoter and coding regions of

the AhR gene in mice and humans [442, 448]. Such genetic variability will alter

levels of ANR expression and activity, vastly altering phenotypic outcome.

While the primary route of PAH exposure in humans is through tobacco

smoke, the results of these experiments underscore the need to assess the effects

of these compounds through exposure to environmental pollution. Both

epidemiological and clinical studies human populations have reported numerous

deleterious reproductive outcomes due to compounds such as BaP, dioxins,

polychlorinated biphenyls (PCBs) and particulate matter that is found in air, soil and

water (reviewed in [251, 252, 449]). A pivotal study was conducted in the Czech

Republic, which demonstrated the association between environmental pollution and

neonatal mortality [450]. This was followed by several several studies in the USA

and China, where regions considered to have high levels of pollution were linked to

low birth weight, sudden infant death syndrome and neonatal mortality [451, 452].

In addition to chronic exposure to pollutants, disasters such as the terrorist attacks

and subsequent collapse of the buildings of the World Trade Centre (WTC) in New

York on September 11", 2001, can result in acute exposure to compounds such as

198

PAHs, PCBs and heavy metals [453, 454]. Studies of mothers living in close

proximity to the WTC at the time of the attacks revealed an increased incidence of

DNA adduct formation in maternal and fetal white blood cells [455] that was

associated with BaP exposure [456]. Lastly, a similar study revealed that neonates

born to mothers exposed to the various pollutants in the aftermath of the WTC

attacks, exhibited reduced birth weight, length and head circumference [457]. In

light of such profound effects on female reproduction and neonatal outcome, the

murine model of PAH exposure used in these studies would also prove useful in

determining the effects of acute exposure to environmental pollution.

Conclusions

The present study utilizes an animal model of PAH exposure which mimics

the levels seen in human cigarette smokers. C57BI/6 dams chronically exposed to

PAHs prior to conception resulted in aberrant vascularization of both the labyrinthine

and large vessels of the placenta at d15.5, and fetal growth restriction. Histological

sections and corrosion casting of both the maternal and fetal vessels revealed

dilatations and disorganization of the microvasculature of PAH-exposed placentae.

In addition, fetal arterial surface area and volume were significantly diminished, as

determined by microCT, and the umbilical vessels of fetuses from PAH-exposed

dams exhibited decreased diameters. Placentae from mice treated with PAHs

exhibited altered cell death rates, compared with those from control mice,

demonstrating reductions in the labyrinthine region and increases in the CP. AhR

199

deficiency rescued the IUGR phenotype and returned the labyrinthine cell death rate

to control levels; however, it did not affect cell death in the CP. It is concluded PAH

exposure prior to conception results in altered placental vascularization and cell

death patterns that ultimately lead to IUGR.

200

CHAPTER 5

SUMMARY AND GLOBAL CONCLUSIONS

201

CHAPTER 5: SUMMARY AND GLOBAL CONCLUSIONS

Apoptosis is a required event during metazoan development, required for the

development, differentiation and sculpting of organs, in addition to its role in cell and

tissue homeostasis. Moreover, cell death enables the renewal of old cells,

supporting appropriate cell turnover, while at the same time, preventing injury to

neighbouring, healthy cells. Dysregulation of the apoptotic process can lead to a

diseased state, whereby inhibition of cell death results in the accumulation of

unwanted cells and the potential for subsequent tumourigenesis. Alternatively,

acceleration of the cell death programme can lead to degenerative disorders.

Exposure to PAHs has been previously shown trigger cell death signalling, resulting

in the activation of the apoptotic pathway and leading to the demise of crucial cell

types, such as immune cells and lung epithelium. During pregnancy, exposure to

cigarette smoke — which is the primary route of PAH exposure in humans — has

been associated with reduced fertility and a high miscarriage rate. On the other

hand, PAH exposure has also been implicated in impeding the cell death process,

resulting in tumour formation. A number of different placental phenotypes have

been reported in human conceptuses from smoking mothers, including altered cell

death rates, hyperplasia and premature rupture of the membranes. In addition,

exposure to cigarette smoke during pregnancy is now causally associated with

IUGR, a fetal outcome linked to altered placental cell death rates. While cell death

in the human placenta has been recognized as a physiological event, virtually no

studies have been done on the mouse placenta. In this thesis, | have described

normal cell death patterns and expression of apoptosis-related proteins during

202

mouse placental development and have outlined the consequences of Bax-

deficiency. Furthermore, maternal treatment with PAHs was used as a trigger to

alter cell death rates in outbred, inbred and genetically modified strains of mice.

In chapter two, | reported the results of a systematic and quantitative

examination into cell death patterns in both ICR and C57BI//6 placentae, over

gestation. In addition, the effects of Bax deficiency on murine placentation and its

effects on the fetus were investigated. Over gestation, ICR and C57BI/6 placentae

exhibited TUNEL-positive patterns similar to those observed in human placentae,

with scattered, infrequent death observed in early placentae, which increased

towards term. The most striking observation was the organized pattern of cell death

surrounding the vessels of the labyrinth at mid-gestation, with an apparent role in

remodeling the vasculature of this region. Organized, classically apoptotic cell death

was also detected in TGCs, which are almost completely eliminated towards the end

of gestation. In addition, massive aponecrotic death of intermingled GlyT and

maternal decidual cells was observed in the last quarter of pregnancy. While cell

death patterns were similar for both strains of placentae examined, C57BI/6

placentae typically demonstrated greater numbers of dead cells in almost all regions,

starting at d13.5. Moreover, the unique Parp-1 cleavage profile and TUNEL-staining

patterns observed in the mouse placenta indicate that both classical and non-

classical cell death pathways are functioning within this organ. Lastly, pro-apoptotic

Bax protein immunolocalized to a subset of TGCs and to cells within the labyrinth,

revealing a role for Bax in programmed cell death of this trophoblast cell sub-type.

In addition, Bax deficiency in the mouse placenta resulted in an altered labyrinthine

203

architecture, including fetal capillary dilatation, leading to fetal IUGR. Only recently

has a functional link between cell death and placental vasculogenesis been reported

in human placenta [318] and the studies herein are the first systemic investigations

into murine placental cell death patterns, revealing its crucial function in placental

vascularization and trophoblast turnover.

The observed defects in Bax-deficient placentae — which included decreased

rates of TGC death and dilatation of the fetal capillaries in the labyrinth — and its

possible effect on the fetus have not been previously described; however, the effects

of Bax deficiency in a number of adult tissues have been rigorously studied. This is

not an uncommon occurrence in the field of gene knockout mouse characterization,

as the placenta is an often neglected organ during these investigations, especially

when viable young are born. Therefore, these studies highlight the need to re-visit

previously constructed, gene-targetted knockout mice and examine the effects of

gene deficiencies on the placenta. This opens up numerous avenues of exploration

into placental biology and can contribute to resolving troublesome issues

surrounding human placentation, ultimately leading to improved fetal and maternal

health.

Apoptosis has been shown to play a critical role during vascular remodelling

and cardiovascular development [458, 459], but the underlying cellular mechanisms

involved remain unclear [460, 461]. Recent studies have demonstrated that cell

death is essential for cardiac outflow tract development in both birds [458] and mice

[462], and contributes to pharyngeal arch artery remodelling during murine

embryogenesis [88]. In the adult, apoptosis has been shown to be essential for re-

204

vascularizing the lung in an animal model of ischemia-induced pulmonary injury

[463]. During human placentation, apoptosis has been shown to have a crucial role

in placental morphogenesis, facilitating implantation, maternal spiral artery

remodelling and cytotrophoblast differentiation [111, 174]. Thus far, only one report

has implicated cell death during normal placental vascularization in humans [318];

however, dysregulation of this process in vascular tissues has been fairly well

studied in a number of different systems. Pulmonary hypertension in congenital

heart disease has been linked to defects in the apoptotic programme [464], and the

combined effects of augmented proliferation and unscheduled cell death contribute

to cardiac and renal hypertension [465]. Additionally, disruption of death signalling

pathways in endothelial cells by Bcl-2 overexpression or caspase inhibition impaired

the formation of vascular-like structures in in vitro [466] and in vivo angiogenesis

assays [467], with both initiator caspase-8 and executioner caspase-3 implicated in

this process. Lastly, activation of the cell death pathway has been described to be a

critical event during melanoma mimicry of vasculogenesis [468].

In chapter three, it was shown that chronic, maternal exposure of ICR mice to

PAHs prior to conception resulted in murine embryonic cell death, acting as a

potential mechanism underlying cigarette-smoking-induced pregnancy loss. Cell

death was preceded by increases in Bax, activation of caspase-3 and decreased

litter size. Moreover, the post-implantation embryonic sex ratio was altered in PAH-

exposed dams, with a greater number of male embryos surviving treatment than

female embryos. The observed embryonic loss could not be prevented by the

disruption of Hrk, but was diminished in embryos lacking Bax. These studies

205

revealed that exposure of early embryos to PAHs reduced the allocation of cells to

the embryonic and placental lineages by inducing apoptosis in a Bax-dependent

manner, thus compromising the developmental potential of exposed embryos.

In chapter four of this thesis, chronic maternal exposure of C57BI/6 mice to

PAHs prior to conception resulted in IUGR in d15.5 fetuses. Furthermore, both

histological and structural adaptations were observed in the placental vasculature of

PAH-exposed dams, with significant reductions in arterial surface area and volume

of the fetal placental vasculature. This altered vascularization was accompanied by

reduced labyrinthine and increased CP cell death rates. As PAHs are known ligands

of AhR, it was hypothesized that embryos deficient in this xenobiotic sensor would

be unaffected by PAH exposure. Indeed, AhA-deficient fetuses were rescued from

PAH-induced growth restriction and exhibited no changes in the labyrinthine cell

death rate. The results of this investigation suggest that chronic exposure to PAHs

is a contributing factor to the development of IUGR in human smokers and

implicated AhR in mediating the biological actions of these compounds.

The studies described in chapters three and four of this thesis contribute to

the growing body of knowledge involving the effects of PAH exposure on pregnancy

and placentation. Spontaneous abortion, reduced fertility rates and IUGR are

manifested in human smokers [253] and were observed in these studies in dams

exposed to PAHs, chemical components found in cigarettes. Thus, these results

recaptiulate many physiological events triggered by tobacco smoke exposure in

humans and further support the usefulness of the mouse as a model for human

placentation.

206

The effects of PAH exposure on a number of different cell and tissue types

have been rigorously studied, leading to the conclusion that PAHs can exert

opposite effects on the cell death pathway [426, 469, 470]. This appears to hold true

in C57BIV/6 placentae, where labyrinthine and decidual cells appeared resistant and

CP cells appeared susceptible to PAH-induced death. Additionally, the two different

Strains of mice produced varying phenotypes upon PAH exposure, corroborating

preceding reports that the AhR gene is highly polymorphic, with distinct biological

effects attributed to genetic background [448]. While ICR dams exposed to PAHs

exhibited higher rates of resorption, this was not evident in C57BI/6 dams. On the

other hand, PAH treatment of C57BI/6 dams yielded growth-restricted fetuses, a

condition not apparent in ICR fetuses. Moreover, AnR may be responsible for

producing these diverse outcomes, as PAH-exposed ICR embryos exhibited higher

levels of ANR and its downstream target, Bax, and AhA-deficient fetuses were

protected from PAH-induced growth restriction. Lastly, the disparity in response to

PAHs between the two strains of mice offer yet another caveat regarding the effects

of genetic background when comparing in vivo murine data between strains.

207

CHAPTER 6

FUTURE DIRECTIONS

208

CHAPTER 6: FUTURE DIRECTIONS

Bax as a mediator of trophoblast cell differentiation

Herein, | have described that Bax-deficient placentae exhibit defects in TGC

death and in labyrinthine vascular architecture. Bax is a pro-apoptotic protein that

localizes to TGC in the mouse placenta and functions in mediating the demise of this

trophoblast cell sub-type. Interestingly, Bax expression was also detected in the

labyrinth region and appeared to localize to both the endothelial and syncytial layers;

however, whether Bax immunolocalizes to the ST, or to the endothelium, or both,

was difficult to ascertain. Since the cell death pathway has been shown to function

in ST differentiation [89, 104], it is possible that Bax is involved in this process.

Therefore, precise localization of this protein needs to be determined. This could be

more firmly established by immunohistochemical double- or triple-labelling studies

using anti-Bax, anti-cytokeratin (a trophoblast marker) and/or anti-CD31 (an

endothelial marker); however, this may prove difficult as the mouse placenta

contains a great deal of autofluorescence. In addition, ultra-thin tissue sections (i.e.

~50-100 nm) may be required to definitively identify Bax expression patterns in these

cells. Quantitative RT-PCR studies are currently underway, using cDNA obtained

from both Bax WT and KO labyrinthine tissue, to determine whether Bax deficiency

alters gene expression of trophoblast differentiation markers. In parallel to these

studies, in vitro trophoblast stem (TS) studies could be conducted, exposing these

cells to ST-differentiating cell culture conditions [471], followed by gene expression

analyses, including qRT-PCR and Western blotting. If these experiments yield Bax

expression in ST cells, then derivation of Bax WT and KO trophoblast stem cell lines

209

could be considered. Currently, Bax-deficient mice are on mixed C57BI/6

background and would require back-crossing to a 129 background in order to

efficiently derive TS cell lines. Production of Bax WT and KO TS cells would provide

an in vitro model of trophoblast differentiation, to be subsequently analyzed for a

variety of genetic markers of trophoblast cell differentiation.

While we were able to establish that Bax mediates death in a subset of TGC,

analysis of TUNEL patterns in the other placental regions (i.e. labyrinth, junctional

zone, CP) are ongoing and will provide a more comprehensive overview of cell

death patterns and rates in Bax-deficient placentae. In addition, since a number of

dying Bax KO TGCs appeared necrotic and the activation of this cell death pathway

has been previously demonstrated in Bax-deficient MEFs [74], this phenotype

should be further evaluated and quantified in Bax KO placentae. Again, analysis of

TS cell cultures under undifferentiating and diffierentiating conditions and subjected

to a variety of cell death stimuli would be useful in determining gene expression

patterns of cell death markers. We have previously established that expression

levels of Bax and various caspases are upregulated in TS cells under culture

conditions differentiating towards a TGC fate (Figure 6.1) [472] and it is possible that

Bax deficiency may alter the differentiation programme of this trophoblast cell

subtype. Such information would be valuable in supporting the in vivo data

described in this thesis. Lastly, transmission electron microscopy (TEM) would be

useful in evaluating the ultrastructure of Bax KO placentae, including the interhemal

distance and TGC cellular and nuclear morphology.

210

Figure 6.1 Cell death markers in trophoblast stem cells cultured under

differentiating conditions over time. A. Western blot demonstrating increased

Bax expression in TS cells cultured under differentiating conditions for 48 hours.

Blot was stripped and re-probed with anti-B-actin antibodies as a control for protein

loading. B. Graphs depicting cleavage profiles of caspase-3, -6 and -9 in TS cells

cultured under differentiating conditions over time. Bars represent average values

+SE.

Dens

itom

etri

c ratio

of

cleaved

casp

ase:

tota

l caspase

211

TS cells cultured 48 hrs:

Bax

(3-actin

Undifferentiating Differentiating conditions conditions

Expression levels of cleaved caspases in TS cells under differentiating conditions over time

Wi Cleaved:total caspase-3

@ Cleaved:total caspase-6

Bf. Cleaved:total caspase-9

do d2 d4 dé

Days of TS cell culture.under differentiating. conditions

Figure 6.1

212

immune-mediated rejection of embryonic tissues in PAH-exposed dams

Numerous reports indicate that while high levels of PAHs are

immunosuppressive by triggering apoptosis in various types of immune cells [426,

473], low-level exposure to PAHs can stimulate the immune system [474, 475],

potentially resulting in pathologies such as asthma and autoimmune disorders. The

most abundant immune cell type in both the human and rodent placenta is the

uterine natural killer (UNK) cell, residing mainly in the maternal decidua, with

functions in maternal spiral artery remodelling, cytokine release and, it is believed,

maternal recognition of the “foreign” placental cells. While the physiological roles of

uNK cells are somewhat apparent, the pathways of activation and the mechanisms

by which cytolysis is inhibited, are unclear.

An initial pilot study investigating the extent of uNK infiltration into the

maternal decidua revealed that d9.5 ICR placentae exposed to PAHs exhibit greater

numbers of uNK cells compared with placentae exposed to vehicle (Figure 6.2).

These numbers remain high until approximately d12.5, when numbers fall back to

similar values obtained from control placentae. It is uncertain whether these cells

are acting to suppress or assist the invasion of placental trophoblast cells, as it is

believed uNK cells may be involved in both processes [319, 476, 477]. Further

investigations have revealed that FasL expression — a molecule which protects

fetally-derived placental cells from the maternal immune system — is upregulated in

PAH-exposed, ICR placentae, suggesting a failed attempt to protect the fetal

trophoblast from a PAH-induced, hyper-stimulated maternal immune system.

213

Figure 6.2 Polycyclic aromatic hydrocarbon treatment in ICR dams alters

levels of uNK cells in early placental decidua. Histomicrographs depict d9.5-

d12.5 placental sections from dams exposed to vehicle and PAHs. Histochemistry

using Dolichos biflorus lectin was used as a marker of murine uNK cells and

sections were counter-stained with hematoxylin. The population of maternal uNK

cells are upregulated in d9.5 and d10.5 PAH-exposed placentae, which is

subsequently down-regulated by d12.5.

Vehicle

PAH-

exposed

Vehicle

PAH-

exposed

Vehicle

PAH-

exposed

214

Figure 6.2

215

A proposed study would involve investigating the spontaneous resorption

phenotype observed in PAH-exposed ICR females, focusing on the proliferation and

activation status of placental immune cells. It would also be advantageous to

ascertain whether maternal factors, embryonic factors, or both, are responsible for

recruiting and activating these immune cells. To answer this question, embryo

transfer experiments could be implemented. Embryos from PAH-exposed mothers

will be transferred to non-exposed pseudopregnant females and embryos from non-

exposed mothers will be transferred to pseudopregnant PAH-treated females. Both

embryonic-derived and maternal-derived tissues would be evaluated to determine

cell death rates and levels of gene expression in control and PAH-exposed tissues.

The results of such experiments could assist in establishing the role of molecular

pathways involved in maternally-mediated spontaneous abortion and would help

further the understanding of the impact of cigarette smoking on female reproductive

immunology. Moreover, the results of such a study would support the argument that

PAH-exposure, or exposure to cigarette smoke is not just associated with pregnancy

loss, but is a direct cause of pregnancy loss, which extends into a female’s

reproductive health, even after cessation of tobacco use.

Placental phenotype due to AhR deficiency

Studies of ANR- and Arni-deficient mice indicate a defect in vascularization

[298, 478]; however, AhA-deficient placentae have not been thoroughly examined

until now. Previous reports have already shown that AnR KO mice exhibit reduced

post-natal viability and are smaller than their WT counterparts [290]. In the studies

216

described in this thesis, AnR was shown to immunolocalize to the fetal endothelium

of the placenta and AhA-deficient fetuses were resistant to PAH-induced growth

restriction. In dams exposed only to vehicle, d15.5 AhA-deficient fetuses exhibited

reduced weight compared to WT fetuses, indicating a possible placental phenotype.

Examination of AhR KO placental sections revealed alterations in the labyrinthine

architecture, which exhibited dilated and disorganized fetal vessels (Figure 6.3a).

Similar defects of the placental vasculature were seen in Bax-deficient and PAH-

treated, C57BI/6 placentae, both of which also yielded growth-restricted fetuses.

While aberrations in the labyrinthine vasculature of PAH-exposed placentae appear

associated with disturbances in cell death, AnR KO placentae exposed to vehicle

alone exhibited only slightly higher cell death rates compared with AhR WT

placentae. Interestingly, levels of cleaved caspase-3 are elevated in AnR KO

placentae, as determined by Western blotting (Figure 6.3b). Thus, if a greater

number of AnR KO and WT TUNEL-stained sections were evaluated for cell death

rates, this difference might reach statistical significance. Alternatively, the observed

increase in cleaved caspase-3 expression may reflect changes in trophoblast

differentiation, as this classical cell death marker has been implicated in human ST

differentiation [177]. Impaired differentiation and turnover of ST in KO placentae

may be responsible for the observed fetal IUGR, as this trophoblast subtype is

crucial in forming the placental barrier, the site of maternal-fetal exchange.

Therefore, evaluation of trophoblast differentiation markers by (RT-PCR should be

implemented to determine whether trophoblast differentiation pathways are

dysregulated in AhR KO placentae. Again, to provide additional support to in vivo

217

Figure 6.3 AhR-deficient placentae exhibit defective labyrinthine architecture

and altered expression of cell death and vascular markers. A.

Histomicrographs depict d15.5 AhR WT and KO placental sections stained with

Bandeiraea simplicifolia lectin, demarcating fetal blood vessels. B. Graphs depict

densitometric ratios after Western blotting, demonstrating altered gene expression

levels in d15.5 AhR KO placentae compared with AhR WT placentae.

218

d15.5 AhR WT Labyrinth d15.5 AhR KO Labyrinth

BS-l lectin

histochemistry to stain fetal

endothelium

B. Cleaved caspase-3 expression p53 expression

Ba a 4 ws 0.25 8B 04 5 © 8 0.36 p=0.027 £ o2 I 6% 03 fc 2 = 0.25 £0.15 p=0.024 Pe 02 Be o. 2 % 0.15 ga” 2® 0.1 ® 0.05 E 8 0.05 =3 o n=3 aa 0 ¥ oo s 8 AhR WT AhR KO AhR WT AhR KO

PECAM-1 expression Fit-1 expression

ue oO “

0% 0.05 5 1.0 p=0.011

cs 0.04 0.028 B ¢ 08 2 p=0. = 5% 0.03 £§ 06 ES = £3 0.02 Si 0.4 x 5 Ui 0.01 2 0.2

QO 0 n=6 n=6 & 0 n=3

AhR WT AhR KO AhR WT AhR KO

Figure 6.3

219

data, AnR WT and KO trophoblast stem cell lines could be derived; however, since

the current mouse line is on a C57BI/6 background, several generations of back-

crossing to 129 mice would be required, to gain a greater efficiency in TS cell

derivation.

The fetal endothelium of the labyrinth is an important element of the placental

barrier, forming the fourth cellular layer of the interhemal distance. Initial

investigations have yielded alterations in endothelium-specific gene expression in

AhFR-deficient placentae, with decreased levels of PECAM-1 and increased levels of

Fit-1 observed (Figure 6.3b). In addition, consistent with other reports [231, 479], we

have observed p53 immunolocalization in the fetal endothelium of the placental

labyrinth and this protein is down-regulated in KO placentae (Figure 6.3b). Since

AhR has been shown to be involved in p53 regulation [469], AhA-deficiency could

perturb normal endothelial-specific p53 expression and impair placental

vascularization, leading to the observed fetal growth restriction. Finally, tetraploid

aggregation experiments could elucidate whether placental insufficiency is a

mitigating factor in the IUGR and cardiac phenotypes observed in knockout animals.

The studies described in this thesis have elucidated an important

association between cell death and placental vascular remodelling. Since a number

of human gestational pathologies such as preeclampsia and IUGR have been

associated with dysregulated cell death pathways, and the etiologies of these

diseases are still enigmatic, continued investigations into these maladaptations

should be pursued. In addition, the results described herein underscore the

usefulness of the mouse as a model of human placentation and offer a cautionary

220

note in evaluating and describing resulting placental phenotypes in mice of different

genetic backgrounds.

221

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