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Placenta xxx (2014) 1e9

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Placenta

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Trophoblast specialisations during pregnancy in the tammar wallaby,Macropus eugenii: A morphological and lectin histochemical study

C.J.P. Jones a,*, J.N. Skepper b, M.B. Renfree c, J.D. Aplin a

aMaternal and Fetal Health Research Group, University of Manchester, Manchester Academic Health Sciences Centre, St Mary’s Hospital, Oxford Road,Manchester M13 9WL, UKbCambridge Advanced Imaging Centre, University of Cambridge, Cambridge CB2 3DY, UKcDepartment of Zoology, University of Melbourne, Victoria 3010, Australia

a r t i c l e i n f o

Article history:Accepted 24 March 2014

Keywords:TrophoblastLectinsUltrastructureWallabyMarsupial

* Corresponding author. Maternal and Fetal HealthHuman Development, University of Manchester, St MManchester M13 9WL, UK.

E-mail addresses: carolyn.jones@manchester.(C.J.P. Jones).

http://dx.doi.org/10.1016/j.placenta.2014.03.0180143-4004/� 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Jones CJPmorphological and lectin histochemical stud

a b s t r a c t

Introduction: The tammar wallaby has a short gestation (26.5 days) and vascular modifications toexpedite transport during that brief pregnancy. Here we examine trophoblast structural attributes thatwould facilitate materno-fetal exchange.Materials and methods: Four specimens of Macropus eugenii between days 23 and 26 gestation wereexamined using electron microscopy and 24 lectins to characterise glycosylated secretions and theirinternalisation.Results: Two trophoblast phenotypes were found, flattened cells generally in contact with the underlyinguterine epithelium and giant cells associated with histiotrophe. The latter appeared to penetrate uterineclefts, occasionally detach and become necrotic. Lectin histochemistry and ultrastructure indicated thepresence of many lysosomes and residual bodies especially in trophoblast giant cells; these containedglycans, mainly apically, which were also detected in secretions and cell debris. Trophoblast basalmembranes bore extensive filopodia. Giant cells were less common in vascular trilaminar areas and herethe trophoblast barrier became thinner near term.Discussion: Loss of Maackia amurensis agglutinin binding suggested cleavage of terminal sialic acidresidues as an early post-internalisation event in the trophoblast. Lectin staining indicated degradationoccurred in an apicalebasal direction, and the heavily glycosylated basal membrane appeared specialisedfor transport out of the cell.Conclusion: Granules seen ultrastructurally and histochemically, particularly in giant trophoblast cells ofthe bilaminar area, suggest that internalised histiotrophe is broken down here and nutrients transferredto the embryo via the specialised basal plasma membrane. The trilaminar vascular area contained mostlyflattened trophoblast cells, supporting the suggestion that gaseous exchange is its primary function.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The omphalopleure or choriovitelline placenta of the tammarwallaby Macropus eugenii is short-lived, with a gestation of only26.5 days [1]; after birth development of the altricial young iscontinued in the mother’s pouch. This placenta develops from thefusion of the chorionwith the yolk sac rather than the allantois as ineutherians, and remains cellular rather than developing a syncytialstructure as in many eutherian mammals. The thin, membranous

Research Group, Institute ofary’s Hospital, Oxford Road,

ac.uk, cjpj_43@yahoo.co.uk

, et al., Trophoblast specialisay, Placenta (2014), http://dx

yolk-sac placenta forms an intimate association with the endome-trium and comprises non-vascular bilaminar and vascular trilami-nar areas of trophoblast, both covered by endoderm cells that linethe yolk sac. It has been proposed that the bilaminar area is con-cerned primarily with histiotrophe uptake while the trilaminararea is mainly involved in gas exchange [2e4]. The microvilloussurface of the trophoblast lies over e and may interdigitate with e

the microvilli of the underlying uterine epitheliumwhich covers anendometrium supplied with blood vessels and furnished withuterine glands. The fetal and maternal vasculature closest to theinterface of the uterine epithelium and placental omphalopleureshows a remarkably thin glycocalyx, probably to expedite transportof nutrients and gases given the short gestation in this species [4].Vessels deeper in the endometrium display a thicker glycocalyxsimilar to those found in species with epitheliochorial placentae

tions during pregnancy in the tammar wallaby, Macropus eugenii: A.doi.org/10.1016/j.placenta.2014.03.018

C.J.P. Jones et al. / Placenta xxx (2014) 1e92

(e.g. skink, shark and camel) and endotheliochorial placentation(e.g. mink, cat, lion, dog and elephant [4]).

Here we examine the omphalopleure trophoblast to determinewhether there are any special features that would facilitatematerno-fetal nutrient or gas exchange as well as changes in itsstructure and glycosylation over the latter part of gestation in boththe bi- and trilaminar layers.

2. Materials and methods

The animals were obtained from our breeding colony in Melbourne and werethe same as those used previously; fixation and tissue preparationwere as describedbefore [4] using two animals at early day 23, one at day 24 and one full-term day 26of pregnancy after removal of pouch young (RPY) to initiate reactivation of thediapausing blastocyst. All procedures were approved by the University of Melbourne

Table 1Lectins, their major specificities and binding to cuboidal trophoblast on day 23 RPY.

Acronym Source Major specificity

GNA Galanthus nivalisSnowdrop

Non-reducing terminal a-D-mannose,especially the mannosyl a1,3 mannose linkag

CON A Canavalia ensiformisJack bean

a-D-glucosyl and a-D-mannosyl residuesin high mannose, intermediate andsmall complex linked sequences

PSA Pisum sativumGarden pea

a-D-mannose in non-bisectedbi/tri-antennary, complex N-linked sequence

e-PHA Phaseolus vulgaris(erythroagglutinin)Kidney Bean

Bi/tri-antennary bisected complex N-linked s

l-PHA Phaseolus vulgaris(leukoagglutinin)Kidney Bean

Tri/tetra-antennary, non-bisected complexN-linked sequences

LTA Tetragonolobus purpureusLotus

L-fucosyl terminals (especially where clustereFuca1,6GlcNAc > Fuca1,2Galb1,4(Fuca1,3)-Gl

UEA-1 Ulex europaeus-1Gorse

H type 2 antigen (aL-Fuc(1,2)Galb1,4-GlcNAc

ALA Aleuria aurantiaMushroom

L-fucose linked a1,6 to GlcNAc

DBA Dolichos biflorusHorse Gram

GalNAca1,3(LFuca1,2)Galb1,3/4GlcNAcb1-

VVA Vicia villosaHairy vetch

GalNAca1-Ser/Thr and GalNAca1,3Galb1-

MPA Maclura pomiferaOsage orange

Galb1,3GalNAca1- > GalNAca1-

BSA-1B4 Bandeiraea simplicifoliaGriffonia

Gala1,3Galb1,4GlcNAcb1-

DSA Datura stramoniumJimson Weed

b1,4GlcNAc, N-acetyllactosamine > chitotrios

STA Solanum tuberosumPotato

b1,4GlcNAc oligomers

LEA Lycopersicon esculentumTomato

b1,4GlcNAc oligomers

HPA Helix pomatiaRoman Snail

Terminal GalNAca1-

AHA Arachis hypogaeaPeanut

Galb1,3GalNAcb1- > Galb1,4GlcNAcb1-

AHA þ NECA Erythrina cristagalli

Coral TreeGalb1,4GlcNAcb1-

ECA þ NSBA Glycine max

SoybeanTerminal GalNAca1- > Gala1

SBA þ NWFA Wisteria floribunda

WisteriaGalNAca1,6Galb1- > GalNAca1,3Galb1-

SNA-1 Sambucus nigraElderberry Bark

NeuNAca2,6Gal/GalNAc-

MAA Maackia amurensis NeuNAca2,3Galb1-PAA Phytolacca americana

PokeweedPoly-N-acetyllactosamine, GlcNAc oligomers

WGA Triticum vulgarisWheat germ

Di-N-acetylchitobiose, N-acetyllactosamineand some sialyl residues

0: Negative, 1: weak, 2: moderate. 3: strong, 4: intense. Granule density from sparse (þBSA-1B4 brackets: staining values on day 24.

Please cite this article in press as: Jones CJP, et al., Trophoblast specialisamorphological and lectin histochemical study, Placenta (2014), http://dx

Animal Ethics committees, and followed the National Health and Medical ResearchCouncil [5] guidelines for research on native fauna. Approximately 2 ml of yolk sacfluid was removed from tammarwallaby uteri via a 26G needle inserted directly intothe yolk sac cavity, and replaced with Superfix to fix the placenta in situ. The wholeuterus was placed into cacodylate buffer and stored at 4 �C before transport to theUK. Full-depth pieces of bilaminar and trilaminar omphalopleure from the fourspecimens were dehydrated through a graded alcohol series followed by propyleneoxide and embedded in Taab epoxy resin (Taab Laboratories Equipment, Alder-maston, UK). Some pieces of tissue were osmicated for examination at the ultra-structural level. Sections 0.75 mm thick were stained with a panel of 24 biotinylatedlectins and an avidineperoxidase revealing system, with neuraminidase pretreat-ment of some sections, as previously described [6] [except that SNA-1 was used at50 mg/ml] to determine the nature of the glycans present. Details of the sources ofthe lectins and their binding specificities can be found in Table 1. Sections wereassessed using a semi-quantitative ranking system of analysis where staining in-tensity was allocated a grade from 0 (negative) to 4 (intense staining) and also

Mv membrane Granules Basal membrane

Bi Tri Bi Tri Bi Tri

e1e2 1 3þþþ 3þþþ 1 1

4 3 2e3þþþ 3þþþ 4 3

s2e4 1e4 2e3þþ 1e3þþþ 2 4

equences 4 4 3þþþ 3þþþ 4 3

3e4 3e4 1e2þþ 1e2þþ 3 2e4

d),cNAcb

2e3 2e3 1þþ 2þþ 1 2

b1-) and Ley 1 1 1þ 1e2þ 0 0

4 4 3þþþ 4þþþ 4 4

0 0 0 0 0 0

4 4 3þ 3þ 0 1

4 4 2e3þþþ 3e4þþþ 3 3

1 (3e4) 0 (4) 0 (2e3þþ) 0 (3þþ) 0 (3e4) 0 (4)

e 4 4 3þþþ 3þþþ 4 4

4 4 3þþþ 2e3þþþ 4 3e4

4 4 2e3þþþ 2e3þþþ 4 2

3 2e3 2þþ 2þþþ 2e3 0

3e4 3e4 2e3þþ 3þþ 2 0

4 4 2e3þþþ 3e4þþþ 3 44 4 2e3þþ 3þþþ 2e3 2

4 4 2e3þþþ 3þþþ 4 42e3 2 3þ 2e3þþ 1 0

3 3 3þ 3þþ 1 04 4 3þþþ 3e4þþþ 4 4

3e4 4 2e3þþ 2e3þþþ 2e3 2e3

4 4 2e3þ 2þþþ 1e2 34 4 3þþþ 2e4þþþ 4 3

4 4 2e3þþþ 2e4þþþ 4 3

) to dense (þþþ).

tions during pregnancy in the tammar wallaby, Macropus eugenii: A.doi.org/10.1016/j.placenta.2014.03.018

C.J.P. Jones et al. / Placenta xxx (2014) 1e9 3

photographed with an �25 objective on a Leitz Dialux microscope using Image-ProPlus (Media Cybernetics) software, with a 25 mm scale bar pasted onto eachimage. For ultrastructural studies, osmicated tissues were embedded in Taab epoxyresin and thin sections were contrasted with uranyl acetate and lead citrate. Day 23RPY specimens were examined in a Philips EM 10 electron microscope and imageswere captured using a Deben camera and stored as 4 MB TIFF files. Those from 24and 26 days were examined in Cambridge on a Tecnai T20 G2 electronmicroscope at120 kV. Images were captured using a Deben camera and stored as 10 MB TIFF files.

To measure the thickness of the trophoblast, lectin-stained images representingapproximately 300 mm (such as that shown in Fig. 4a) were enlarged on the com-puter screen till the 25 mm scale bar measured 25 mm (1 mmh 1 mm) and an imageextended 260 mm across the computer screen. Twenty orthogonal measurementswere made at 12e15 mm intervals across the linear trophoblast from the basallamina to the microvillous surface on each of three stained sections (CON A, DSA,LEA) of both bilaminar and trilaminar regions, giving 60 measurements in each area.Only images of cuboidal cells were analysed tomake a valid comparison between bi-and trilaminar areas. The arithmetic means � SD were obtained and data analysedusing the KruskaleWallis test (Prism software) to identify any significant reductionin trophoblast thickness in bilaminar and trilaminar areas over the gestationalperiod studied (days 23e26 RPY).

3. Results

3.1. General structure

The trophoblast cells of the omphalopleure exhibited two phe-notypes: cells that formed interdigitations with, or had becomedetached from, the underlying uterine epithelium, were cuboidal orflattened but elsewhere, especially where there were secretionsbetween the uterine epithelium and trophoblast cells, they weremuch larger, rounded or tongue shaped (Fig. 1a); we refer to theseas giant cells (GC) since these are somewhat analogous to the giantcells of some epitheliochorial eutherian placenta. In some instancesit appeared as if GC dipped into histiotrophe-filled clefts or grooveswithin the endometrium, some becoming detached; these becamedegenerate and formed cell debris (vide infra). The nuclei in bothphenotypes were greatly increased in size e up to 30 mm long e

compared to maternal cells which rarely exceeded 9 mm in theirlongest diameter. Sometimes binucleate profiles could be seen introphoblast as well as in endodermal cells. Both cuboidal and GCcontained granules which, at the light microscope level, were moreplentiful apically in cuboidal cells, while in some GC they tended tobe ubiquitous. In the trilaminar area (Fig. 1b), trophoblast wascovered by connective tissue and capillaries; the upper surface wascovered by endoderm cells as in the bilaminar layer. During tissuepreparation the connective tissue often separated from the un-derlying trophoblast cells, as seen in the figure, and secretions oftencontracted away from uterine epithelial cells.

Fig. 1. General structure. Toluidine blue-stained 0.5 mm sections of a day 26 omphalopleuEndoderm cells overlie the trophoblast cells which interdigitate with the underlying uterinearea can just be detected (arrowheads). Giant cells overlie an area rich in secretions (*). Beneaendoderm cells overlie connective tissue in which lie fetal capillaries. This structure has becepithelium via a microvillous membrane (arrowheads). Maternal capillaries can be seen undecells, Mat cap: maternal capillaries, Fetal cap: fetal capillary, Tr: trophoblast.

Please cite this article in press as: Jones CJP, et al., Trophoblast specialisamorphological and lectin histochemical study, Placenta (2014), http://dx

3.2. Electron microscopy

3.2.1. Cuboidal cellsApical microvilli on cuboidal cells (Fig. 2a) were closely packed

with a central core of microfilaments extending into the apicalcytoplasm (Fig. 2b, arrow). In the day 23 specimen, these microvilliwere separated from the sparser uterine epithelial microvilli and inmany cases appeared to be embedded in secretions (Fig. 2b, *). Atlater stages, the trophoblast and uterine microvilli sometimesloosely interdigitated with a touching of apposing surfaces (Fig. 2d,arrow); secretions were more filamentous and seemed to emanatefrom uterine epithelial microvilli (Fig. 2c and d). Here, trophoblastmicrovilli were shorter and more stubby.

Subapically, the numerous electron-lucent vacuoles and pino-cytotic vesicles sometimes contained dark secretory material(Fig. 2b) and there were various granules, some with heteroge-neous contents and others more homogeneous, suggestive of het-erolysosomes and/or residual bodies (Fig. 2a and b). Later inpregnancy, granules were often less apparent in cuboidal cells(Fig. 2c). The basal areas generally contained parallel cisternae ofrough endoplasmic reticulum and clusters of fat droplets (Fig. 2a).Nuclei were large with dispersed chromatin.

3.2.2. Giant cells (GC)Ultrastructurally, these cells were similar to cuboidal cells

except that they were much larger and often packed with a varietyof pleomorphic granules (Fig. 2e), many suggestive of hetero-lysosomes or residual bodies, leaving little room for other organ-elles (Fig. 2f). At the bases of microvilli, pinocytotic vesicles andendocytic systems were often present containing secretory mate-rial (Fig. 3a, arrows) together with vacuoles with electron-lucentcontents and a variety of granules were present. Golgi bodies, oftenwith dilated saccules, were dotted around the cytoplasm (Fig. 3b).Rod-shaped mitochondria were present while cisternae of roughendoplasmic reticulum, often in parallel arrays, and fat dropletsoccupied the more basal areas (Fig. 3c). By day 24 of the 26.5-daygestation, although cuboidal cells showed fewer granules, theywere still present in giant cells (Fig. 3d).

Lateral membranes of both trophoblast cell types were con-nected by apical junctional complexes and periodic desmosomes orlong tight junctions (Fig. 3e, arrows, arrowhead) while basal plasmamembranes generally exhibited filopodia which made sporadicdesmosomal contact (Figs. 2a and 3f, arrows) with overlyingendoderm cells. In the trilaminar area, heterotypic contact via

re. a) Bilaminar area showing both cuboidal (left) and giant trophoblast cells (right).epithelium on the left hand side of the image; a faint line indicating the microvillousth the uterine epithelium, maternal capillaries can be seen. b) Trilaminar vascular area;ome detached from the underlying trophoblast which is closely attached to the uterinerneath the uterine epithelium. Scale bars: 25 mm. Cu Tr: cuboidal trophoblast, GC: giant

tions during pregnancy in the tammar wallaby, Macropus eugenii: A.doi.org/10.1016/j.placenta.2014.03.018

Fig. 2. Electron microscopy of trophoblast. a) Electron microscopy of cuboidal cells covered by endoderm cells at 23 days showing apical microvilli, some subapical vacuoles and avariety of granules. Fat droplets and cisternae of rough endoplasmic reticulum are present basally and the basal plasma membrane is highly convoluted (*). Cells are joined bydesmosomes on their lateral membranes as are endodermal cells to trophoblast (arrows). b) The day 23 cuboidal trophoblast microvilli contain microfilaments that extend downinto the apical cytoplasm (arrow); they are surrounded by secretions and debris (*) some of which appear to contain residual membranous structures. c) At 26 days there are fewgranules in the cuboidal trophoblast; basal convolutions can be seen surrounded by a tenuous basal lamina. d) Filamentous material, possibly part of the uterine glycocalyx, can beseen between the cuboidal trophoblast and uterine epithelial microvilli on day 26. Contact between the two layers is tenuous though occasional membrane-to-membrane contactcan be seen (arrow). e) These giant cells have numerous pleomorphic granules and their microvilli are embedded in secretions. Fat and cisternae of RER can be seen basally. f) Agiant cell packed full of large, homogeneous electron dense granules at 23 days. Part of the nucleus can be seen. Mv: microvilli, RER: rough endoplasmic reticulum.

C.J.P. Jones et al. / Placenta xxx (2014) 1e94

electron dense adhesion plaques between trophoblast and con-nective tissue cells was less robust, accounting for the frequentseparation of this layer from the trophoblast during processing. Avery tenuous, wispy basal lamina could be detected in some places.

Evidence of cell debris and degenerate cells was sometimes seenin the lumen between trophoblast and uterine epithelium, oftenadherent to cell apical surface of cells (Fig. 3g, *).

3.3. Lectin histochemistry

3.3.1. Cuboidal cellsBinding of e-PHA in bi- and trilaminar areas (Fig. 4ael) gave

results similar to that of several other lectins recognising N-linkedstructures (see Table 1) over the time course studied. The presenceof O-linked structures was also indicated by binding of HPA, WFA,

Please cite this article in press as: Jones CJP, et al., Trophoblast specialisamorphological and lectin histochemical study, Placenta (2014), http://dx

AHA and SBA. The affinity of the cuboidal trophoblast for lectinswas often very variable, depending on whether the cells were incontact with the underlying epithelium or separated by a layer ofsecretions; in the latter case, staining was generally increased(Fig. 4i, double arrowheads). Variability was also seen in microvilliwhen parted from the underlying cells (Fig. 4e and i), especially ondays 24 and 26 in areas of attachment (Fig. 4e, short arrow). Day 23trophoblast was generally separated from the uterine epithelium(Fig. 4aed) and microvilli bound most lectins strongly except forUEA-1, DBA and BSA-1B4. At later stages, such microvillous stainingwas generally only seen where there was separation between thetwo layers.

A distinct polarity was evident in glycosylated granule distri-bution within the trophoblast cells with most located apically. Thiswas clearly seen with GNA (Fig. 5a) but a gradient was seen even

tions during pregnancy in the tammar wallaby, Macropus eugenii: A.doi.org/10.1016/j.placenta.2014.03.018

Fig. 3. Electron microscopy of trophoblast. a) Day 24 bilaminar giant cell showing pinocytosis (arrows) and many different types of vacuole and granule suggesting different stagesof breakdown of ingested material. b) Day 23 giant cell with Golgi bodies associated with the various granules. c) An image of the basal area of a giant cell showing the roughendoplasmic reticulum and fat droplets with a convoluted basal plasma membrane (*) adjoining an endoderm cell. d) At 26 days this giant cell is embedded in secretion and is full ofpleomorphic granules. e) An apical tight junction (arrowhead) and desmosomes (arrows) unite two adjoining giant cells which have dark secretions around the microvillous surface(*) against a background of electron-lucent material. f) Desmosomal attachments between a day 23 convoluted bilaminar cuboidal trophoblast basal plasma membrane and anendodermal cell. g) Part of a degenerate cell (*) is attached to a giant cell in the trilaminar area at day 23. Mv: microvilli, G: Golgi body, RER: rough endoplasmic reticulum, End:endoderm cell.

C.J.P. Jones et al. / Placenta xxx (2014) 1e9 5

with lectins that bound throughout the cell, with heavier stainingapically. The trophoblast bound most lectins on day 23, with l-PHA,LTA, UEA-1 and DBA staining only weakly. There was no detectablebinding of BSA-1B4 on day 23 (Fig. 5b), but by day 24, microvilloussurfaces of both the trophoblast cells and uterine epithelium andsome intracellular granules (Fig. 5c) were stained as well as necroticmaterial or debris. With most other lectins, stained granulesdiminished sharply in number by day 24 (Fig. 4eeg) andmore so byday 26 in both bi- and trilaminar regions (Fig. 4i and k). This was,however, variable especially on day 26, where some regions werealmost completely devoid of granules while in others they wereplentiful. Generally, areas with few granules showed trophoblastclosely apposed to the uterine epithelium, while separated areastended to have stronger staining (Fig. 4i, double arrowheads) sug-gesting that uptake was dependent on the presence of histiotropheand did not occur via uterine epithelium.

Please cite this article in press as: Jones CJP, et al., Trophoblast specialisamorphological and lectin histochemical study, Placenta (2014), http://dx

3.3.2. Giant cells (GC)These large, rounded cells were often pressed closely together

on day 23 (Fig. 4b) while on later days they were more spread out.They were sparse in the trilaminar area on day 23 (Fig. 4d) andvery infrequent in the trilaminar 26-day (Fig. 4l) specimen butcould occasionally be seen over histiotrophe in endometrialgrooves. In contrast, numerous GC were seen in bilaminar areas,some within lumina of narrow endometrial clefts, parallel to theuterine surface. The numerous granules bound most lectins, andstaining was generally heavier at the apical side; however, theintensity was extremely variable especially at later stages (Fig. 5d).On day 23, a subpopulation of round granules in the bilaminararea, just under the microvilli, stained particularly intensely withVVA and MAA like a string of beads (inset, Fig. 4b). Lectinsincluding PSA, e-PHA, l-PHA, DSA, ECA, SBA, WFA and WGA alsobound but in these cases the staining was not selective and other

tions during pregnancy in the tammar wallaby, Macropus eugenii: A.doi.org/10.1016/j.placenta.2014.03.018

Fig. 4. Lectin histochemistry. Phaseolus vulgaris erythroagglutinin (e-PHA) staining to show changes in trophoblast thickness and granule density over time. aed) 23 days, eeh) 24days, iel) 26 days. a) At 23 days’ gestation, cuboidal trophoblast cells are seen in the bilaminar area packed with granules and with a darkly staining microvillous membrane andbasal plasma membrane (arrowheads). The cells are separated from the underlying uterine epithelium whose glycocalyx is bound by this lectin. Endoderm cells overlie thetrophoblast. b) Giant cells with prominent subapical granules that also bind MAA (inset) strongly. The apical microvillous and basal plasma membranes (arrowheads) again bindstrongly. c) Trilaminar trophoblast layer has flattened cells with darkly staining microvillous and basal plasma membranes (arrowheads) and overlying capillaries and endoderm.This layer has detached from the uterine epithelium, the glycocalyx of which can be seen. d) Occasional giant cells can be seen at a cleft or groove in the uterine epithelium filledwith dark secretions. The basal plasma membrane is intensely stained (arrowheads). The darkly staining glycocalyx of the uterine epithelium can be seen. e) By 24 days there arefew fewer granules that bind this lectin and the trophoblast is much thinner, though the basal plasma membrane still binds strongly (arrowheads). Where trophoblast is joined tothe uterine epithelium there is little detectable binding of the lectin (short arrow). f) Giant cells have fewer lectin-binding granules compared to day 23 and are surrounded bysecretions. The basal plasma membrane is well stained (arrowheads). There is a space (*) between the edge of the secretions and the uterine epitheliumwhich may be artefactual. g)The trilaminar trophoblast is also thin at this stage, with few granules, and the basal plasma membrane is not so prominent (arrowheads). h) Giant cells are surrounded by secretionbut have few lectin-binding granules. Again, they appear to dip into uterine clefts or grooves. The basal plasma membrane is well stained (arrowheads) and there is a split betweenthe secretions and uterine epithelium (*). i) At 26 days there is little lectin binding in the bilaminar area except where the trophoblast is detached from the underlying uterineepithelium. At those points, microvillous staining can be seen (double arrowheads); the underlying uterine microvillous membrane also binds in these areas. The basal plasmamembrane still stains strongly along the length of the trophoblast (single arrowheads). j) Giant cells are once more full of granules that bind this lectin, with secretion around them,and a strongly staining basal plasma membrane adjoining the endoderm (arrowheads). k) In the trilaminar area, the trophoblast layer is very thin and can just be faintly seen closelyadherent to the uterine epithelium in this area (double arrowheads). The basal plasma membrane is well stained (single arrowheads) while the capillary layer and endoderm havebecome detached. l) Giant cells in the trilaminar region are hard to detect and are not well-developed but one can be seen with a prominent basal plasma membrane (arrowheads).Profiles of uterine glands have intraluminal secretions similar to those seen extra-cellularly between the trophoblast and uterine epithelium, with a split between the secretions anduterine cells (*). Scale bars: 25 mm. Tr: trophoblast, GC: giant cell, End: endoderm, UE: uterine epithelium, Cap: capillary, Sec: secretions, MAA: Maackia amurensis agglutinin.

C.J.P. Jones et al. / Placenta xxx (2014) 1e96

granules were also stained. At day 24, the distribution of deeply-stained granules was much more haphazard in all areas while byday 26 a more uniform population was present, again, moreheavily distributed apically.

In the trilaminar region, despite cutting several blocks, exam-ples of fully developed giant cells at day 26 were extremely rare.

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Occasionally, one was found over an endometrial groove and againthe granules were most heavily distributed apically, in contact withsecretions (Fig. 4l).

Where giant cells had penetrated endometrial clefts, there wereclumps of cell debris, deeply stained with lectins (Fig. 5e). Withtoluidine blue, this debris was barely visible in contrast to darkly

tions during pregnancy in the tammar wallaby, Macropus eugenii: A.doi.org/10.1016/j.placenta.2014.03.018

Fig. 5. Lectin histochemistry of cells and secretions. a) At 23 days, the apical area of bilaminar trophoblast is full of granules that bind GNA, indicative of lysosomes. b) At 23 daysthere is virtually no detectable staining with BSA-1B4. c) By 24 days, the endoderm, trophoblast basal membrane and microvilli bind BSA-1B4 together with intracellular granules,but larger cells in the lower part of the image have unstained microvilli delineated by arrowheads. Debris stains intensely and may be coating the microvilli of the cells in the upperpart of the image. d) At 26 days the binding of STA by bilaminar giant cells is very variable, fromweak to intense. e) A bilaminar giant cell (*) stained with ECA can be seen deep in acleft; debris binds this lectin very strongly. f) A toluidine blue-stained semithin section of the trilaminar area at 24 days shows secretions to be darkly stained while cell debris stainsvery lightly. g) With WGA þ Neuraminidase the secretions are pale and cell debris binds the lectin intensely. h) ALA binds to a degenerate cell (*) and other debris (double arrows)between the trophoblast and uterine epithelium in the trilaminar area at 23 days. Within the gland lumen there is densely stained material as well as lighter components. Uterinecells appear to show a glycocalyx or adherent material bound by ALA and both light and dark secretions are present in the lower right part of the image. i) With MAA at 23 days, thegland profiles are full of secretions of different staining intensity (*) as they move through the gland. Some dense, lamellar material can be seen adhering to the uterine epitheliumand part of the trophoblast on the right. j) The gland profiles show very little binding of MAA at 24 days, though Golgi bodies are well stained; this was found with the majority oflectins. k) At 26 days there is more intense staining with MAA and this was found with most lectins at this stage. Tr: trophoblast, End: endoderm, GC: giant cell, UE: uterineepithelium, Sec ¼ secretion, Gl: gland. GNA: Galanthus nivalis agglutinin, BSA-1B4: Bandeiraea simplicifolia isolectin B4, STA: Solanum tuberosum agglutinin, ECA: Erythrina cristagalliagglutinin, WGA þ N: Wheat germ agglutinin after neuraminidase pretreatment, ALA: Aleuria aurantia agglutinin, MAA: Maackia amurensis agglutinin. Scale bars: aed, hek: 25 mm,e: 100 mm, feg: 50 mm.

C.J.P. Jones et al. / Placenta xxx (2014) 1e9 7

stained glandular secretions (Fig. 5f and g); occasional dead cellsadjacent to the trophoblast also stained intensely with lectins(Fig. 5h, *).

The trophoblast basal plasma membrane was heavily glycosy-lated, staining with most lectins apart from UEA-1, DBA andMAA in the bilaminar area; VVA, SBA (with and withoutneuraminidase) and BSA-1B4 (Fig. 5b and c) bound here only on andafter day 24. In the trilaminar area on day 23, there was weaker orno binding to AHA, HPA and SBA (with andwithout neuraminidase)though, apart from HPA, this increased again by day 26.

3.4. Uterine glands and secretions

On day 23, uterine gland secretions in different gland profilesvaried in their lectin affinity (Fig. 5i, *), suggesting molecularmodification and variation in hydration as the secretions moved up

Please cite this article in press as: Jones CJP, et al., Trophoblast specialisamorphological and lectin histochemical study, Placenta (2014), http://dx

through the coiled glands. Golgi bodies often stained intensely,especially with e-PHA, ALA, MPA, VVA, DSA, HPA, ECA after neur-aminidase,WFA,MAA andWGA, though actual secretions bound allthe lectins apart from LTA, DBA, BSA-1B4 and AHA on day 23. Onday 24, intraluminal secretion staining diminished significantlywith all the lectins (Fig. 5j), though this recovered by day 26(Fig. 5k), matching the decline and subsequent increase in bilami-nar cell staining. Glands subjacent to bi- and trilaminar areassecreted material with a similar glycan composition. Histiotrophehad two components; one homogeneous and weakly stained withlectins, the other more stringy and densely stained (except by UEA-1 and DBA), forming lamellae in places; this tended to be foundattached to cell surfaces. Some appeared to contain cell debris andcould be composed of degenerate cells.

The cuboidal trophoblast cells thinned significantly (p < 0.001)between days 23 and 24 but between days 24 and 26 little change

tions during pregnancy in the tammar wallaby, Macropus eugenii: A.doi.org/10.1016/j.placenta.2014.03.018

Fig. 6. Histograms of cuboidal trophoblast thickness in lectin-stained sections. KruskaleWallis analysis of 60 measurements of trophoblast thickness, 20 from each of three lectin-stained sections (CON A, ALA and LEA) of each specimen. a) In the bilaminar area, the cuboidal trophoblast cells in these specimens became significantly thinner (p < 0.001)between days 23 and 24 but no significant change was seen between then and day 26. b) In the trilaminar region of the specimens analysed, the cells became significantly thinner(p < 0.001) between days 23 and 24 and also, but to a lesser extent, between days 24 and 26 (p < 0.05).

C.J.P. Jones et al. / Placenta xxx (2014) 1e98

occurred (Fig. 6a and b), as confirmed by Dunn’s Multiple Com-parison test. The mean trophoblast thicknesses (�SD) were16.73 � 6.05 mm (day23), 11.80 � 4.25 mm (day 24) and10.97 � 4.69 mm (day 26), whereas in the trilaminar region, wheregas exchange occurs, the cells continued to become significantlythinner between days 23 and 24 and days 24e26 (p < 0.001). Meantrophoblast thicknesses in this region were 12.73 � 5.25 mm (23days), 8.65 � 5.14 mm (day 24) and 6.22 � 4.45 mm (day 26).

4. Discussion

In this study we have identified several previously undescribedfeatures of placentation in M. eugenii. There are two phenotypes oftrophoblast, one with a flattened morphology mainly associatedwith adherence to the uterine epithelium, and a much larger giantcell (GC) in areas where histiotrophe fills the gap between fetal andmaternal tissues, and collects within endometrial grooves or clefts.GC contain secondary lysosomes and residual bodies, sometimes tothe point of engorgement; their distribution and appearance, withevidence of uptake of glycosylated secretions into intracellularvesicles, confirms the early suggestion by Hill [2] that the bilaminararea in marsupials is primarily concerned with uptake of nutrients.The preponderance of flatter cells in the trilaminar zone iscommensuratewith a respiratory function, and although they showultrastructural evidence for internalisation of secretions, they areprobably less active in this respect, with few lectin-binding gran-ules when adherent to uterine epithelium.

The range of lectins binding to uterine glands and their secre-tions, and to trophoblast, indicates the wide variety of glycans inhistiotrophe, confirming earlier studies using the periodic acideSchiff reaction [7]. Lectin histochemistry strongly suggests a pro-gressive breakdown of histiotrophe after its ingestion by thetrophoblast cells, for transfer to the fetal compartment. The distinctapical vacuoles in the bilaminar area on day 23 contain a2,3-linkedsialic acid residues shown by intense stainingwithMAA, which alsobinds uterine gland secretions. The absence of MAA binding deeperin the trophoblast suggests that sialic acid must then be lost alongwith GalNAca1 (VVA, SBA), and N-linked complex glycans bound byl-PHA. However, biochemical heterogeneity of lysosomes is notevident ultrastructurally, there being no discrete population ofapical granules.

The apical trophoblast microvillous surface is heavily glycosy-lated with a-galactose residues detected by BSA-1B4 bindingappearing on day 24 on both uterine and trophoblast cells. Com-parison of the lectin staining with electron micrographs suggeststhis may be related to intensely staining debris coating the surfaceof the cells, as well as the endogenous glycocalyx. The mechanism

Please cite this article in press as: Jones CJP, et al., Trophoblast specialisamorphological and lectin histochemical study, Placenta (2014), http://dx

by which glycans are concentrated in the cell debris is not known,but earlier studies have shown glycans to accumulate in cytosol incases of cell death [8].

The day 23 GNA binding to bi- and trilaminar apical trophoblastgranules indicates non-reducing terminal a-D-mannose [9] which isa marker for resident lysosomal glycoproteins or partially digestedproducts of ingestion [10e12]. Lysosomal activity would berequired for the breakdown of histiotrophe; as significant GNAbinding sites are absent from uterine gland secretions and stainedgranules are only rarely seen in uterine surface epithelium, so GNAcannot be used to track maternal secretory products. Instead it actsas an index of the high levels of histiotrophe ingestion and break-down within the apical trophoblast cytoplasm. On days 24 and 26of pregnancy, GNA staining extends throughout the cell, suggestingthat lysosomal activity has become more widespread. Biosynthesisof enzymes needed for histiotrophe digestion in trophoblast [3], aswell as endocrine activity, is indicated by abundant rough endo-plasmic reticulum and Golgi vacuoles seen ultrastructurally.

The giant cells are reminiscent of areolar trophoblasts found inepitheliochorial placentae [13,14] e large, tongue-shaped cellsoverlying uterine gland openings e modified for histiotrophe up-take and containing many intracellular vacuoles. In the tammarwallaby, GC vacuole size does not differ greatly from that incuboidal trophoblast; however the cells themselves are muchlarger with a greater area for uptake. The presence of cells inendometrial grooves, possibly related to the position of glandnecks, is a novel finding, but some marsupials are known to havegiant cells that penetrate the endometrial epithelium e.g. Philanderopossum [15] and Monodelphis domestica [16], a feature also ofeutherian trophoblast in the haemochorial, invasive placentae ofprimates, rodents and bats [6,17,18]. These grooves contain celldebris composed of shed trophoblast cells; their breakdownproducts may later be opportunistically ingested by overlyingtrophoblast cells as such material was often found on cell surfaces.

The large trophoblast nuclei are probably polyploid, with sometrophoblast and endoderm cells being binucleate. Giant cells havepreviously been described in trophoblast of the fat-tailed dunnartSminthopsis crassicaudata [19], large nuclei illustrated in the tropho-blast ofDidelphis virginiana in late pregnancy [1] and syncytialmassesinPerameles species [20]while in eutherianmammals giant polyploidtrophoblast cells are present in rodent and multinucleate giant cellsare found in the basal plate of the human and in epitheliochorialplacentae of species such as the camel where they secrete steroidhormones [14]. Polyploidy and syncytialisation are therefore an earlyevolutionary feature of placental cells in eutherian mammals.

The basal plasma membrane of the trophoblast forms filopodiaand is richly glycosylated, features probably associated with

tions during pregnancy in the tammar wallaby, Macropus eugenii: A.doi.org/10.1016/j.placenta.2014.03.018

C.J.P. Jones et al. / Placenta xxx (2014) 1e9 9

transporter molecules and enzymes, as seen on the basal plasmamembrane of human and feline syncytiotrophoblast [21,22]. Thestronger lectin staining of the trophoblast basal plasma membranein the bilaminar compared with trilaminar areas on day 23 mayindicate a different array of transporter enzymes, perhaps speci-alised for nutrient transfer. It is unlikely that the faint basal laminacontributed significantly to the intense lectin staining observed onthe basal surface. The widely spaced desmosomal attachments andlarge intercellular spaces between endoderm cells and trophoblastmay enable rapid diffusion of nutrients into the yolk sac.

The trophoblast becomes significantly thinner during last fewdays of pregnancy, especially in the trilaminar region where, at 26days, it is 2 mm in places. Our measurements differ somewhat fromthose found in a previous study [7] where a thinning trend was lessobvious, however the measurements in that study were far fewer,ranging from 1 to 24 readings as opposed to 60 made here. Thin-ning of this area, with loss of endothelial glycocalyx [4] probablyfacilitates diffusion for gaseous exchange with fetal blood. Fetal andmaternal tissue interdigitation is not as intimate as is found inmany epitheliochorial placentas (for example, the alpaca [23]), withfilamentous material separating the two cell layers, as previouslyshown in the tammar wallaby [1,7] and the marsupial P. opossum[15]. Uterine epithelial microvilli also have a prominent glycocalyxwhich may prevent close adhesion. The failure of interdigitatingmicrovilli frequently to bind lectins when materno-fetal contactwas close, with strong staining evident when the two membranesurfaces were separated was surprising as, ultrastructurally, ma-terial was present between individual microvilli. In epitheliochorialplacentae, such a lack of staining was attributed to glycaneglycaninteractions obscuring the lectin-binding sites [24].

Our findings suggest that trophoblastmorphology in the tammarwallaby is plastic, the phenotype exhibited depending on thecloseness of attachment to the underlying uterine epithelium. Thebilaminar seems to have more GC than the trilaminar area thoughthe effect of sampling cannot be discounted; these accumulatemasses of granules, probably mainly residual bodies followinglysosomal breakdown of ingested histiotrophe. Some turnover ofthese cells is indicatedbydebris found inunderlying clefts or groovesaswell as between trophoblast anduterineepithelium. Possibly, cellsbecome so engorged with residual bodies that they can no longerfunction efficiently and are subsequently shed. The products of suchcell breakdownmay then be utilised as a formof holocrine secretion.

In conclusion, this study describes novel features of the tammarmaterno-fetal interface including the presence of two trophoblastphenotypes. Intracellular granule accumulation, engorgement andsubsequent degeneration of giant cells may contribute to a nutri-tional cycle of histiotrophe. We suggest the primary route formaterno-fetal transfer of glycoprotein is via secretions into spacesand clefts between uterine epithelium and trophoblast, adjacent towhich absorptive trophoblast giant cells are present at increasedfrequency. Glycoprotein ingestion by trophoblast leads to break-down in lysosomes which can be tracked initially by loss of ter-minal sugars including sialic acid, followed by a cytoplasmic routeto the yolk sac via the basal surface from where it can be deliveredto the fetus. The trilaminar vascular area contains mostly flattenedtrophoblast cells, supporting the suggestion that gaseous exchangeis its primary function.

Please cite this article in press as: Jones CJP, et al., Trophoblast specialisamorphological and lectin histochemical study, Placenta (2014), http://dx

Conflict of interest

The authors confirm there are no known conflicts of interestassociated with this publication.

References

[1] Tyndale-Biscoe H, Renfree M. Reproductive physiology of marsupials. Cam-bridge: Cambridge University Press; 1987.

[2] Hill JP. On the foetal membranes, placentation and parturition, of the nativecat (Dasyurus viverrinus). Anat Anz 1900;18:364e73.

[3] Renfree MB. The composition of fetal fluids of the marsupial,Macropus eugenii.Dev Biol 1973;33:62e79.

[4] Jones CJP, Aplin JD, Renfree MB. The fetomaternal interface shows vascularhypoglycosylation in the tammar wallaby Macropus eugenii: comparisonwith a range of non-mammalian and eutherian placentae. Placenta2013;34:879e84.

[5] National Health and Medical Research Council. Australian code of practice forthe care and use of animals for scientific purposes. 7th ed. 2004. Canberra ACT,Australian Government.

[6] Jones CJ, Carter AM, Aplin JD, Enders AC. Glycosylation at the fetomaternalinterface in hemomonochorial placentae from five widely separated species ofmammal: is there evidence for convergent evolution? Cells Tissues Organs2007;185:269e84.

[7] Freyer C, Zeller U, Renfree MB. Ultrastructure of the placenta of the tammarwallaby, Macropus eugenii: comparison with the grey short-tailed opossum,Monodelphis domestica. J Anat 2002;201:101e19.

[8] Jones CJP, Bäcklin B-M, Stoddart RW, Dantzer V. Environmental pollutants asaetiological agents in female reproductive pathology: placental glycanexpression in normal and polychlorinated biphenyl (PCB)-exposed mink(Mustela vison). Placenta 1997;18:689e99.

[9] Shibuya N, Goldstein IJ, Van Damme EJ, Peumans WJ. Binding properties of amannose-specific lectin from the snowdrop (Galanthus nivalis) bulb. J BiolChem 1988;263:728e34.

[10] Lity�nska A, Przybylo M. Does glycosylation of lysosomal proteins show age-related changes in rat liver? Mech Ageing Dev 1998;102:33e43.

[11] Calvo A, Pastor LM, Bonet S, Pinart E, Ventura M. Characterization of the gly-coconjugates of boar testis and epididymis. J Reprod Fertil 2000;120:325e35.

[12] Tribl F, Gerlach M, Marcus K, Asan E, Tatschner T, Arzberger A, et al. “Sub-cellular proteomics” of neuromelanin granules isolated from the human brain.Mol Cell Proteomics 2005;4:945e57.

[13] Jones CJP, Wooding FBP, Dantzer V, Leiser R, Stoddart RW. A lectin bindinganalysis of glycosylation patterns during development of the equine placenta.Placenta 1999;20:45e57.

[14] Wooding FBP, Burton GJ. Comparative placentation: structures, functions andevolution. Berlin, Heidelberg: Springer-Verlag; 2008.

[15] Enders AC, Enders RK. The placenta of the four-eyed opossum (Philanderopossum). Anat Rec 1969;165:431e49.

[16] Freyer C, Zeller U, Renfree MB. Placental function in two distantly relatedmarsupials. Placenta 2007;28:249e57.

[17] Georgiades P, Ferguson-Smith AC, Burton GJ. Comparative developmentalanatomyof themurine andhumandefinitiveplacentae. Placenta 2002;23:3e19.

[18] Hemberger M, Hughes M, Cross JC. Trophoblast stem cells differentiatein vitro into invasive trophoblast giant cells. Dev Biol 2004;271:362e71.

[19] Roberts CT, Breed WG. Placentation in the dasyurid marsupial, Sminthopsiscrassicaudata, the fat-tailed dunnart, and notes on placentation of the didel-phid, Monodelphis domestica. J Reprod Fertil 1994;100:105e13.

[20] Padykula HA, Taylor JM. Ultrastructural evidence for loss of the trophoblasticlayer in the chorioallantoic placenta of Australian bandicoots (Marsupialia:Peramelidae). Anat Rec 1976;186:357e85.

[21] Borke JL, Caride A, Verma AK, Kelley LK, Smith CH, Penniston JT, et al. Calciumpump epitopes in placental trophoblast basal plasma membranes. Am JPhysiol 1989;257:c341e6.

[22] Champion EE, Glazier JD, Greenwood SL, Mann SJ, Rawlings JM, Sibley CP, et al.Localization of alkaline phosphatase and Ca2þ-ATPase in the cat placenta.Placenta 2003;24:453e61.

[23] Jones CJP, Abd-Elnaeim M, Bevilacqua E, Oliveira LV, Leiser R. Comparison ofuteroplacental glycosylation in the camel (Camelus dromedarius) and alpaca(Lama pacos). Reproduction 2002;123:115e26.

[24] Jones CJP, Dantzer V, Stoddart RW. Changes in glycan distribution withinthe porcine interhaemal barrier during gestation. Cell Tissue Res 1995;279:551e64.

tions during pregnancy in the tammar wallaby, Macropus eugenii: A.doi.org/10.1016/j.placenta.2014.03.018