Expression of connexin 43 mRNA and protein in developing follicles of prepubertal porcine ovaries

Ž .Comparative Biochemistry and Physiology Part B 130 2001 43�55

Expression of connexin 43 mRNA and protein indeveloping follicles of prepubertal porcine ovaries �

Crystal M. Meltona, Gretchen M. Zaunbrecher b, Goro Yoshizakic,Reynaldo Patinod, Scott Whisnant e, Alexia Rendonf, Vaughan H. Leea,f,�˜

aDepartment of Animal Science and Food Technology, Texas Tech Uni�ersity, Lubbock, TX 79406, USAbDepartment of Veterinary Anatomy and Public Health, Texas A and M Uni�ersity, College Station, TX 77843, USA

cDepartment of Aquatic Biosciences, Tokyo Uni�ersity of Fisheries, Minato-ku, Tokyo 108-8477, JapandU.S. Geological Sur�ey Texas Cooperati�e Fish and Wildlife Research Unit, Texas Tech Uni�ersity, Lubbock, TX 79409-2120,

USAeDepartment of Animal Science, North Carolina State Uni�ersity, Raleigh, NC 27695-7621, USA

fDepartment of Cell Biology and Biochemistry, Texas Tech Uni�ersity Health Sciences Center, 3601 4th Street, Lubbock,TX 79430, USA

Received 2 February 2001; received in revised form 29 March 2001; accepted 3 April 2001

Abstract

A major form of cell�cell communication is mediated by gap junctions, aggregations of intercellular channelsŽ . Ž .composed of connexins Cxs , which are responsible for exchange of low molecular weight �1200 Da cytosolic

materials. These channels are a growing family of related proteins. This study was designed to determine the ontogenyŽ . Žof connexin 43 Cx43 during early stages of follicular development in prepubertal porcine ovaries. A partial-length 412

.base cDNA clone was obtained from mature porcine ovaries and determined to have 98% identity with publishedporcine Cx43. Northern blot analysis demonstrated a 4.3-kb mRNA in total RNA isolated from prepubertal and adultporcine ovaries. In-situ hybridization revealed that Cx43 mRNA was detectable in granulosa cells of primary follicles butundetectable in dormant primordial follicles. The intensity of the signal increased with follicular growth and wasgreatest in the large antral follicles. Immunohistochemical evaluation indicated that Cx43 protein expression correlatedwith the presence of Cx43 mRNA. These results indicate that substantial amounts of Cx43 are first expressed ingranulosa cells following activation of follicular development and that this expression increases throughout folliculargrowth and maturation. These findings suggest an association between the enhancement of intercellular gap-junctionalcommunication and onset of follicular growth. � 2001 Elsevier Science Inc. All rights reserved.

Keywords: Follicular development; Connexin 43; Ovary; Gap junctions; Granulosa cells; Prepubertal ovarian development; Porcineovary

� Ž .This work was supported in part by Texas Higher Education Coordinating Board ATP grant �010674-0059b-1997 VHL , USDAŽ .grant �96-35203-3465 RP , Texas State Line Item for Efficient Pork Production, and a Howard Hughes Medical Institute grant

through the Undergraduate Biological Sciences Education Program to Texas Tech University.� Corresponding author. Tel.: �1-806-743-2714; fax: �1-806-743-2990.

Ž .E-mail address: [email protected] V.H. Lee .

1096-4959�01�$ - see front matter � 2001 Elsevier Science Inc. All rights reserved.Ž .PII: S 1 0 9 6 - 4 9 5 9 0 1 0 0 4 0 3 - 1

( )C.M. Melton et al. � Comparati�e Biochemistry and Physiology Part B 130 2001 43�5544

1. Introduction

Gap junctions are described as aggregates ofintercellular channels, composed of the protein

Ž .Cx, between adjacent cells Paul, 1995 . Cx pro-teins oligomerize as hexamers within the plasmamembrane of one cell to form ‘hemichannels’ or‘connexons.’ The channel itself is formed whenextracellular domains of Cxs in adjacent cellsinteract to form a complete intercellular channelŽ .Paul, 1995 . These channels act as conduits al-lowing diffusional movement of ions, metabolites

Žand other potential low molecular weight �1200.Da signaling molecules from one cell to another

ŽSimpson et al., 1977; Simon et al., 1997; White.and Paul, 1999 .

Although Cxs are expressed in many organs,within each tissue individual members of this

Žfamily are differentially regulated Bruzzone et.al., 1996 . In ovarian tissue of mammals, investi-

gators have established the presence of: Cx26;Cx30.3; Cx32; Cx37; Cx40; Cx43; and Cx45ŽOkuma et al., 1996; Itahana et al., 1996; Hae-

.fliger et al., 1992 . Cx43 is generally co-expressedwith at least one other Cx, and appears to be animportant Cx associated with ovarian functionŽ .Munari-Silem and Rousset, 1996 . In the ratovary, the expression of Cx40, Cx43 and Cx45 hasbeen detected by both Western and Northern

Žblot analysis of whole ovarian tissue Okuma et.al., 1996 . Cx43, as well as other Cxs, is expressed

in developing follicles in sheep and cowsŽGrazul-Bilska et al., 1998; Johnson et al., 1999;

.Nuttinck et al., 2000 . A critical role of Cx43during activation of folliculogenesis was also sug-gested in studies with Cx43 deficient transgenicmice, where early follicular development was ar-

Žrested in the absence of this gene Juneja et al.,.1999 .

Ž .Granot and Dekel 1997 reported a directcorrelation between Cx43 mRNA and protein lev-els in ovarian follicles of immature rats. Granotand Dekel concluded that during early folliculo-genesis, the expression of Cx43 is developmen-tally regulated, whereas, after antrum formationgonadotropins regulate transcription, translationand post-translational modifications of Cx43Ž .Granot and Dekel, 1997 . Similarly, Chang et al.Ž .1999 showed increasing ovarian Cx RNA levelsduring the period of rapid follicular growth in

Ž .teleost fishes. Wiesen and Midgley 1993 foundthat the preovulatory LH surge caused a decrease

in Cx43 mRNA in granulosa cells of rat ovarianŽ .follicles. Additionally, Wiesen and Midgley 1994

demonstrated a dramatic decrease in the levels ofCx43 mRNA and protein during atresia of ratfollicles. Overall, the available information sug-gests that gap-junction coupling plays a role infollicular development and function among verte-

Žbrates Wiesen and Midgley, 1994; Zamboni, 1974;.Eppig, 1991; Patino et al., 2001 .˜

Although a homologue of Cx43 has been iden-tified in pigs, there have been no detailed studieson Cx43 mRNA expression during preantral fol-

Žlicular development in this species Itahana et al.,.1996; Lenhart et al., 1998 . The objective of the

present study was to determine whether Cx43Ž .mRNA and protein is expressed in ovarian folli-cles during early stages of follicular developmentin pigs. Northern blot analysis, in-situ hybridiza-tion, and immunohistochemistry were used toevaluate the prepubertal expression of porcineCx43. Prepubertal pigs were chosen because theirovaries contain a concentrated population of pre-

Ž .antral follicles Lee et al., 1996 . One of ouroverall goals is to determine the mechanisms ofactivation of follicular development in vertebrateovaries. The results reported here indicate thatincreased expression of Cx43 temporally corre-lates with activation of follicular development andearly differentiation of granulosa cells in prepu-bertal pig ovaries.

2. Materials and methods

2.1. Materials

Ž .Sows and gilts Sus scrofus were obtained fromthe Texas Tech swine farm in New Deal, TX. Allstudies were conducted in accord with the NIHGuidelines for the Care and Use of LaboratoryAnimals, as reviewed and approved by the AnimalCare and Use Committee at Texas Tech Univer-sity and Texas Tech University Health SciencesCenter. Restriction enzymes, T7 and Sp6 RNApolymerases, and RNase inhibitor were pur-

Ž . � 32 �chased from Promega Madison, WI . �- P -Ž . � 35 � ŽUTP �3000 Ci�mmol , �- S -UTP �1000

.Ci�mmol , and X-ray film were purchased fromŽ .Amersham Arlington Heights, IL . Proteinase K

and RNase-free DNase I were purchased fromŽ .Boehringer�Mannheim Indianapolis, IN . The

RNase A, salmon sperm DNA and yeast tRNA

( )C.M. Melton et al. � Comparati�e Biochemistry and Physiology Part B 130 2001 43�55 45

Ž .were purchased from Sigma St. Louis, MO .NTB-2 photographic emulsion and D19 developer

Žwere purchased from Eastman Kodak Co. New.Haven, CT . Other general and molecular biology

Žgrade chemicals were purchased from Fisher Pit-.tsburgh, PA .

2.2. Extraction of total RNA from o�aries

Ovaries were collected and RNA isolated fromŽovarian tissue as previously described Yoshizaki

.et al., 1994; Lee and Dunbar, 1993 . Briefly, theovarian tissue was homogenized with a polytronin a solution of guanidinium isothyocyanate andphenol. RNA was then isolated, precipitated withisopropanol, washed with 70% ethanol, and resus-

Ž .pended in diethylpyrocarbonate DEPC treatedwater. The integrity and quantity of the totalRNA was checked with a Pharmacia LKB Ultro-

Ž .spec Plus Spectrophotometer Piscataway, NJ bydetermining absorbency at 260 and 280 nm. Forprocedures involving RNA, solutions and glass-ware were treated with 0.1% DEPC and�or auto-claved when possible.

2.3. Re�erse transcriptase�polymerase chain reaction( )RT�PCR

Ž .Total RNA 2 �g from ovaries of cycling pigswas heated for 5 min at 80�C, and immediatelyused for first-strand cDNA synthesis. The reac-tion mixture included: 20 units of RNasin ribonu-clease inhibitor; 20 nmole of each dNTP; 100pmole of random hexamer; 50 mM of Tris�HClŽ .pH 8.3 ; 40 mM of KCl, 10 mM of DTT; 7 mM ofMgCl ; and 100 units of M-MLV reverse tran-2

Ž .scriptase RNase H Minus in 20 �l. The reactionwas carried out at room temperature for 10 minand at 37�C for 1 h. Two highly conserved regionsof Xenopus Cx30, 38 and 43; rat 43; and human32 were used to construct mixed primers for PCR

Žamplification of Cx43 cDNA Yoshizaki et al.,. �1994 . The forward primer was TTCCC-

Ž . Ž . Ž . Ž .Ž . Ž . �CT AT ACT TC ACT CA CT AG T CGT CG� Ž . Ž .and the reverse primer was GT CT TT CT TC-

Ž .Ž . Ž . Ž .Ž . �ACGT AG TGGG ACGT C GT AGT GA .PCR was performed using 10 �l of cDNA reac-tion product as template. Five �l of each PCRproduct were electrophoresed in 5% polyacryl-

Žamide gels using a 1-kb DNA ladder Gibco BRL,.Gaithersburg, MD as a reference to estimate the

molecular size of the amplified fragments. The

remainder of the PCR products were elec-trophoresed in 2% low-melting point agarose gels

Žand the fragments were isolated Sambrook et al.,. Ž .1989 , cloned into pGEM-T vector Promega ,

and sequenced using a Sequenase Version 2 DNAŽ .sequencing kit Amersham .

2.4. Synthesis of radiolabeled riboprobes

A 412 base pig Cx43 cDNA fragment inpGEM-T was used to synthesize cRNA probesŽ .Lee, 2000 . The Cx43 cDNA was between theSacII and SalI restriction sites, and linearizedplasmids were used to generate riboprobes. Tran-scripts synthesized with T7 RNA polymerase wereantisense for Cx43, and transcripts synthesizedwith Sp6 polymerase were the sense strand.Labeled riboprobes were separated from unincor-

� 32 � � 35 �porated �- P -UTP or �- S -UTP on BioRadP30 spin columns, and then stored at �80�C.

2.5. Northern blot analysis

Ž . ŽTotal RNA 20 �g from ovaries 1-, 70-, 84-.and 140-day-old gilts was separated on 1.5%

agarose�formaldehyde gels and transferred toŽ .Biotrans Membrane ICN Biochemicals using es-Žtablished protocols Lee and Dunbar, 1993; Lee

.et al., 1998 . Membranes were then hybridized�32 �overnight at 50�C with Cx43 P -riboprobes

Ž .specific activity 2�3 cpm�ng at a concentrationof 1�106 cpm�ml. The membranes were washedonce in formamide wash containing: 50% for-mamide, 1X SSC and 10-mM dithiothreitol at50�C for 30 min; followed by a 30-min roomtemperature wash with 0.5X SSC; and a finalwash in 0.1X SSC at 65�C for 2 h. 1X SSC is 150mM NaCl and 15 mM NaCitrate. Membraneswere wrapped in Saran Wrap and exposed onX-ray film for 5�7 days before being developedthrough an Xomatic automatic film processor.

2.6. Tissue collection and preparation for in-situhybridization and immunohistochemistry

Ovaries were collected from prepubertal pigsŽ .three or more animals for each age at 1, 21, 56,70, 84 and 140 days of age, and from adult cyclingsows. Prepubertal pigs were chosen at these agesbecause their ovaries contain a population of

Žprogressively more mature preantral follicles Lee.et al., 1996 . Ovaries were then fixed in 4%


Ž .paraformaldehyde in PBS pH 7.0�7.4 overnight.Tissues were washed with 50% ethanol and 70%ethanol, three times each for 30 min at 4�C,dehydrated, and embedded in Paraplast � PlusŽ .Sherwood Medical Labs, St. Louis, MO . Paraffinwax blocks were cut at 5 �m in serial sections andplaced on Superfrost�Plus slides. Slides weredried overnight at 37�C, placed in desiccatedboxes, and stored at 4�C until used for in-situhybridization of Cx43 mRNA.

2.7. In-situ hybridization

Tissue sections were prepared for in-situ hy-Ž . �35 �bridization as described by Lee 2000 . S -

labeled riboprobes were placed on each slideŽ 7 .1 � 10 cpm�ml and incubated at 50�Covernight. After RNase treatment to remove un-bound riboprobes, slides were: dehydrated; dried;

Ž .dipped in emulsion NTB-2�distilled water 1:1for 3 s; and dried for 2 h. Slides were developedwith D19 developer, counterstained with Harrishematoxylin, and coverslipped. Results were ana-lyzed with brightfield and darkfield microscopy onan Olympus BX50 microscope equipped with a35-mm camera. Controls consisted of substitutionof the sense riboprobe for the antisense ri-boprobe. The level of expression for Cx43 mRNAin specific cell types was designated as unde-tectable if the density of silver grains over thatcell was equal to or less than that observed in thesame area of adjacent sections probed with sensecRNAs as negative controls. At least three dif-ferent sections from each tissue sample wereevaluated.

2.8. Western blot analysis

Proteins from ovaries of 100-day-old gilts wereŽsolubilized for SDS-PAGE Lee and Dunbar,

.1994 . Solubilized proteins were fractionated by15% PAGE, transferred to Immobilon-P PVDF

Ž .membrane Millipore Corp., Bedford, MA , andprobed with rabbit anti-rat Cx43 antibodiesŽ .Zymed, San Francisco, CA . Phosphorylation ofCx43 in pig ovarian samples was identified using

Žalkaline phosphatase agarose beads Sigma, St..Louis, MO following instructions provided by the

manufacture. Briefly, 100 �g of solubilized pro-tein was incubated with 2.5 units of phosphataseovernight at 37�C. Samples were reduced with 4%2-mercaptoethanol and 30 �g fractionated, and

transferred for analysis. Non-phosphatased sam-ples received the same treatment except that thephosphatase enzyme was omitted. Membraneswere probed with rabbit anti-rat Cx43 antibodiesŽ .0.05 �g�ml in TBS containing 0.05% Tween 20and visualized with the SuperSignal West Pico

ŽChemiluminescent Substrate Pierce, Rockford,.IL .

2.9. Immunohistochemistry

Paraffin wax sections of the ovaries were probedwith rabbit anti-Cx43 using the Vectastain Elite

Ž .ABC Kit Vector Laboratories, Burlingame, CA ,and counterstained with hematoxylin to verify his-

Žtology as previously described Lee and Dunbar,.1993 . Sections of ovaries were deparaffinized,

rehydrated and treated with 0.3% peroxide inmethanol for 30 min. After three PBS washes,slides were: boiled for 10 min in 0.01 M citratebuffer; cooled to room temperature for 10 min;and washed in PBS buffer for 5 min. Sectionswere then incubated with normal goat serum for30 min, followed by an incubation with anti-Cx43Ž .2.5 �g�ml in PBS buffer containing 10% BSAovernight at 4�C. After three PBS washes, tissueswere incubated with biotinylated goat anti-rabbitIgG for 30 min and washed with PBS. Slides werethen incubated with the Avidin�Biotin reagentfor 30 min, washed three times with PBS, andreacted with 0.05% diaminobenzidine hydrochlo-ride and 0.01% hydrogen peroxide for 5 min. Thereaction was stopped by rinsing in water, andslides were examined on an Olympus BX50 mi-croscope. Controls consisted of substituting nor-mal rabbit IgG for rabbit anti-Cx43 IgG. At leastthree different sections from each tissue samplewere evaluated.

3. Results

3.1. Analysis of the porcine Cx43 cDNA fragment

A major fragment of 412 bp was amplified fromRNA isolated from adult ovarian tissue, purified,and subcloned into the pGEM-T vector. Twoclones were identical and were identified as ho-mologues of Cx43. Homology analysis was con-ducted using the GCG program and two separate

Žcomparisons were used: FASTA nucleotide iden-. Ž .tity ; and TFASTA amino acid identity . These


clones demonstrated 98% amino acid identity toŽthe published sequence for pig Cx43 Itahana et

.al., 1996 , and showed 94.2% amino acid identityŽ .with the rat Cx43 sequence Beyer et al., 1987 .

The pig Cx43 fragment showed the characteristiccysteine distribution found in the putative second

Ž .extracellular loop of Cxs Fig. 1 . The pig Cx43fragment contained: part of the first extracellularregion; the third and fourth transmembrane re-gions; the intracellular loop; and part of the sec-ond extracellular loop. Hydropathicity analysis ofthe two fragments demonstrated that each had

Žthe expected two hydrophobic regions transmem-. Žbrane and one hydrophilic region intracellular

. Žloop found in typical Cx proteins Yoshizaki et.al., 1994 .

3.2. Northern blot analysis of Cx43 in porcine o�aries

To determine whether Cx43 mRNA was ex-pressed in prepubertal pig ovaries, Northern blotanalysis was conducted using total porcine ovar-

�32 �ian RNA and P -labeled cRNA probes for Cx43.A 4.3-kb mRNA for Cx43 was detectable through-out prepubertal ovarian development at 1, 70, 84

Ž .and 140 days of age Fig. 2 . This Northern blot isrepresentative of experiments with three different

samples of RNA for each age. Cx43 mRNA wasalso detected in RNA isolated from adult ovarian

Ž .tissue data not shown .

3.3. In-situ hybridization of Cx43 mRNA in porcineo�aries

To determine cellular localization of Cx43mRNA, prepubertal and adult ovaries were col-lected for in-situ hybridization. In porcine ovariesfrom 1-day-old animals, Cx43 mRNA was local-ized in the interstitial cells of the ovarian cortex

Ž .between clusters of germ cells Fig. 3a,b . Cx43mRNA was undetectable in: germ cells; the stro-mal tissue in the medullar and hilar regions; andthe ovarian surface epithelium. Adjacent sectionswere probed with sense cRNA probes as negativecontrols to demonstrate background levels oflabeling and specificity of the antisense cRNAsŽ .Fig. 3c,d . Cx43 mRNA was also localized ininterstitial cells between the newly formed pri-

Ž .mordial follicles Fig. 3e,f . However, Cx43 mRNAwas undetected in granulosa cells or oocytes ofthe primordial follicles.

In ovaries collected from 56-day-old pigs Cx43mRNA was localized in the granulosa cells of

Ž .growing primary follicles Fig. 4a,b and sec-

Ž . Ž .Fig. 1. Nucleotide cDNA and deduced amino acid sequence prot of the cloned 412 base cDNA fragment of porcine connexin 43Ž .Cx43 . The conserved cysteine residues characteristic of the second extracellular loop of Cx43 are indicated by asterisks.


Ž .Fig. 2. Connexin 43 Cx43 mRNA expression in prepubertalŽ .porcine ovaries. a autoradiographs of Northern blot analysis

�32 �of porcine ovarian RNA probed with a P -labeled cRNAprobe for Cx43. Expression of Cx43 mRNA was detectablethroughout prepubertal ovarian development. Shown are 1,

Ž .70, 84 and 140 days of age. b The integrity of total RNAŽ .28S was confirmed in ethidium bromide-stained agarose gelsbefore transfer and Northern blot analysis.

Ž .ondary follicles Fig. 4c,d . Follicle diametersobserved in these experiments ranged from 55�80�m for primary follicles and 120�400 �m forsecondary follicles. Cx43 mRNA was also de-tectable in presumptive theca cells associated with

Ž .growing preantral follicles Fig. 4c,d . However,labeling for Cx43 mRNA was undetectable in

Žgranulosa cells of all primordial follicles diame-.ter�25�32 �m and in all oocytes from pre-

antral stage follicles observed in prepubertal pigs.No specific labeling was detectable in adjacentsections probed with sense cRNA probes as nega-tive controls.

Ž .In ovaries from late prepubertal 140-day-oldand adult pigs, the granulosa cells of healthy,developing preantral and antral follicles con-tained abundant amounts of the Cx43 mRNAŽ .Fig. 5a,b . In addition, the theca cells surround-ing these growing antral follicles demonstratedspecific labeling for Cx43 mRNA. Cx43 gap-junc-tion mRNA was undetectable in oocytes of folli-cles at all stages of development. Cx43 mRNA

Ž .was undetectable in granulosa cells Fig. 5c,d ofmore than 12 atretic follicles evaluated in sec-tions from 140-day-old and adult animals.

3.4. Expression and localization of Cx43 protein inporcine o�aries

To verify that expression of Cx43 mRNA wasaccompanied by synthesis into protein, Westernblot analysis and immunohistochemistry were usedto analyze porcine ovarian tissues. Western blotanalysis using rabbit anti-rat Cx43 immunoglobin

Ždemonstrated a characteristic doublet 43 and 45. ŽkDa of two immunoreactive protein species Fig.

.6 . When treated with phosphatase, the doubletwas converted to a single band that migrated at

Ž .the same mobility 43 kDa indicating that theŽ45-kDa band is the phosphorylated form Lenhart

.et al., 1998 . This experiment was repeated threetimes with similar results.

Immunohistochemical localization using rabbitanti-rat Cx43 immunoglobin revealed the patternof Cx43 gap-junction protein expression mirroredthe pattern of mRNA expression. The expressionpatterns for Cx43 proteins were similar for ovariesat different ages, so examples from 140-day-oldovaries are presented here, since this tissue con-tained follicles at all stages of development. Somenon-specific staining was observed with the rabbitIgG used as a negative control, therefore, con-trols are shown for comparison. Cx43 protein was

Ž .detectable dark brown reaction product in gran-ulosa cells of primary follicles but undetectable in

Ž .primordial follicles Fig. 7a,b . Interstitial cellssurrounding primordial follicles in the ovariancortex demonstrated a punctate pattern of label-ing for Cx43 protein in neonatal and prepubertal

Ž .porcine ovaries Fig. 7a,b . Cx43 gap-junction pro-tein was localized in granulosa cells of the growing

Ž .preantral follicles Fig. 7c,d , however, primordialfollicles did not contain detectable levels of ex-pression. Labeling for the Cx43 gap-junction pro-tein was abundant in mural granulosa cells anddetectable in theca cells surrounding antral folli-

Ž .cles Fig. 7e,f . Cx43 gap-junction protein wasundetectable in all oocytes and granulosa cells of

Ž .all atretic follicles observed Fig. 7g,h .

4. Discussion

A fragment of Cx43 RNA was isolated byRT�PCR from cycling porcine ovaries. The de-duced amino acid sequence of this fragment is98% identical to the porcine Cx43 sequence re-


Ž . Ž . ŽFig. 3. In-situ hybridization of connexin 43 Cx43 mRNA in 1-day-old ovarian sections probed with antisense A, B, E, F or sense C,. Ž .D Cx43 riboprobes and detected by autoradiography. A, B Expression of Cx43 mRNA in interstitial cells surrounding clusters of

Ž . Ž . Ž .oogonia in the ovarian cortex arrows . C, D Adjacent section as negative control. E, F Expression of Cx43 mRNA in interstitial cellsŽ . Ž . Ž .between primordial follicles arrows but undetectable in granulosa cells of the primordial follicles p . Bar in A �36 �m and applies

Ž .to B�F .

Ž .ported by Itahana et al. 1996 . The present studyis the first to demonstrate that Cx43 mRNA isexpressed in porcine granulosa cells of preantralfollicles during prepubertal ovarian maturation.The pattern of Cx43 protein expression correlateswith that of Cx43 mRNA. The enhanced expres-sion of Cx43 in granulosa cells of primary, ascompared to primordial, follicles suggest that itspresence is associated with activation of folliculardevelopment and the early steps of differentiationin granulosa cells. Furthermore, these results areconsistent with the concept that gap junctions areinvolved in follicular development and function.

Analysis of neonatal porcine ovarian tissue de-monstrates Cx43 is expressed in the interstitial

cells surrounding clusters of oogonia and newlyassembled primordial follicles. The spatial associ-ation of Cx43 with clusters of oocytes where pri-mordial follicles are forming suggests gap junc-tions have a significant function during germ celldevelopment and early follicular assembly. Gapjunctions were previously shown among rete ovariicells and between rete cells and oocytes in fetalmouse ovaries, but the specific Cxs involved were

Ž .not determined Mitchell and Burghardt, 1986 . Arecent study suggested a critical role for Cx43 inthe prenatal development of mouse gonadsŽ .Juneja et al., 1999 . In transgenic mice lackingCx43, neonatal ovaries were small due to a defi-

Žciency in primordial germ cells Juneja et al.,


Ž . Ž . ŽFig. 4. In-situ hybridization of connexin 43 Cx43 mRNA in 56-day-old porcine ovaries. A, C Brightfield photomicrographs and B,. Ž .D darkfield photomicrographs of ovarian sections probed with antisense Cx43 riboprobes and detected by autoradiography. A, B

Ž . Ž . ŽExpression of Cx43 mRNA is detectable in granulosa cells of primary follicles arrows , but undetectable in primordial follicles p . C,. Ž . Ž .D Expression of Cx43 mRNA is detectable in a secondary follicle arrows and some theca cells arrowheads , but undetectable in

Ž . Ž . Ž .primordial follicles p . Bar in A �36 �m and applies to B�D .

Ž . Ž . Ž .Fig. 5. In-situ hybridization of connexin 43 Cx43 mRNA in adult porcine ovaries. A, C Brightfield photomicrographs and B, DŽ .darkfield photomicrographs of ovarian sections probed with antisense Cx43 riboprobes and detected by autoradiography. A, B

Ž .Expression of Cx43 mRNA is abundant in mural granulosa cells of antral follicles arrows and detectable in adjacent theca cellsŽ . Ž . Ž . Ž .arrowheads . C, D Expression of Cx43 mRNA is undetectable in atretic follicles. Bar in A �36 �m and applies to B�D .


Ž .Fig. 6. Western blot analysis of porcine connexin 43 Cx43 .Each lane contains 30 �g of solubilized porcine ovarian pro-

Ž .teins, and blots were probed with rabbit anti-rat Cx43. pOvarian proteins treated overnight with 2.5 units of alkaline

Ž .phosphatase. np Ovarian proteins incubated overnight with-out alkaline phosphatase as a mock control. In non-alkalinephosphatase treated samples, porcine Cx43 appears as twobands at 43 and 45 kDa. In alkaline phosphatase treatedsamples, porcine Cx43 appears as a single band at 43 kDa.

.1999 . This deficit in germ cell proliferation ispresumably due to a loss of gap-junctional com-munication among the somatic cells associated

Žwith the primordial germ cells Juneja et al.,.1999 . Overall, the previous and present results

are consistent with the concept that Cx43 is re-quired for optimal development of germ cells andassembly of primordial follicles.

Despite the presence of Cx43 in interstitialcells surrounding the forming follicles, Cx43 wasundetectable in primordial follicles. This observa-tion is in agreement with studies in rodents, cowsand teleost fish suggesting that gap junctions first

Žappear at the onset of follicular growth Burghartand Anderson, 1981; Johnson et al., 1999; Iwa-

.matsu et al., 1988 . A recent study with bovineovaries suggested that Cx43 is expressed in granu-

Žlosa cells of primordial follicles Nuttinck et al.,.2000 . The dissimilar findings with bovine follicles

could be a result of species variation or the use ofmethods with different sensitivities of detection.

Expression of porcine Cx43 mRNA is enhancedwhen follicular development resumes and thesquamous granulosa cells of primordial folliclesdifferentiate and become cuboidal. As folliculardevelopment continues, primary and secondaryfollicles express Cx43 mRNA and protein in thegrowing granulosa cell layers. This observationindicates that gap junctions containing Cx43 areformed among granulosa cells of the developingpreantral follicles. This pattern of Cx43 expres-sion is similar to that reported in sheep and cowsŽ .Grazul-Bilska et al., 1998; Johnson et al., 1999 .Although additional Cxs may be expressed ingranulosa cells, Cx43 appears to have a specificand critical role in early follicular development

since the absence of this gene blocks the earlystages of follicular development in mouse ovariesŽ .Juneja et al., 1999 . The increased expression ofporcine Cx43 in granulosa cells during the transi-tion of primordial to primary follicles suggeststhat Cx43 may have a similar role in the activa-tion of follicular development in pig ovaries. Thisrole would appear to be specific to gap-junctionalcommunication among granulosa cells, sincemouse follicles lacking the oocyte connexin, Cx37,progress through preantral development normallyŽ .Simon et al., 1997 . Alternatively, the loss ofCx37 in oocytes could be compensated by otherconnexins.

Activation of follicular development remainspoorly understood, partly because of the lack ofquantifiable indicators for initiation of folliculardevelopment. The process of follicular activationhas been commonly monitored by morphologicalchanges, such as enlargement of the oocyte andtransition of the squamous granulosa cells to

Žcuboidal cells Hirshfield, 1992; Parrott and Skin-ner, 1999; Braw-Tal and Yossefi, 1997; Oktay et

.al., 1997 . Additionally, proliferation of granulosacells has been associated with initiation of follicu-lar development, and has been measured by triti-

Žated thymidine incorporation Zeleznik et al.,.1980; Roy and Greenwald, 1986; Hirshfield, 1991

and the expression of proliferating cell nuclearŽ . Žantigen PCNA Oktay et al., 1995; Lundy et al.,

.1999 . While morphological classification andmeasurement of proliferation in granulosa cellsare still useful indicators for the onset of follicu-logenesis, evaluation of early follicle developmentshould include other aspects of granulosa celldifferentiation. The augmented expression ofCx43 in granulosa cells of primary follicles, ascompared to primordial follicles, indicates thatenhanced transcription of this gene is associatedwith and can be used as a marker of activation offollicular development.

Large antral follicles are present at 140 days ofage, and the mural granulosa cells of these folli-cles also express abundant levels of Cx43 mRNAand protein. This spatio-temporal distributionpattern is similar to that previously reported for

ŽCx43 in antral follicles of pigs and rats Itahanaet al., 1996; Lenhart et al., 1998; Wiesen and

.Midgley, 1993 . Furthermore, Cx43 mRNA andprotein are also found in the theca cell layer. Thisobservation suggests that communication amongtheca cells and among granulosa cells in antral


Ž . Ž .Fig. 7. Immunohistochemical localization of connexin 43 Cx43 protein in prepubertal porcine ovaries. A, C, E, G Sections probedŽ . Ž .with Cx43 antibodies and B, D, F, H probed with non-specific rabbit IgG as negative controls. A Expression of Cx43 protein in

Ž . Ž .granulosa cells of primary follicles arrows and interstitial cells in the ovarian cortex arrowheads but undetectable in granulosa cellsŽ . Ž . Ž .of the primordial follicles p . C Expression of Cx43 protein in granulosa cells of growing secondary follicles arrows but undetectable

Ž . Ž . Ž . Ž .in granulosa cells of the primordial follicles p . E Expression of Cx43 protein in granulosa cells arrows and theca cells arrowheadsŽ . Ž .of an antral follicle. B An atretic follicle where expression of Cx43 protein is undetectable in granulosa cells arrows and theca cells

Ž . Ž . Ž .arrowheads . Bar in A �28 �m and applies to B�H .

follicles is mediated, at least in part, by Cx43 gapjunctions. However, other Cxs are also present in

Ž .pig ovaries Itahana et al., 1996, 1998 , and itcannot be determined from these studies which

specific ones are functional and necessary forfollicular development. It is possible that the ex-pression of multiple Cxs with different biochemi-cal properties provides a mechanism for the inte-


gration of proper signals necessary for folliculardevelopment.

Other studies have demonstrated a dramaticdecrease of Cx43 mRNA and protein in maturepreovulatory follicles after the LH surge�pro-gesterone rise. That observation implies a loss ofcommunication between granulosa cells and theoocyte during the periovulatory period. One hy-pothesis suggests that the interruption�loss ofcommunication between granulosa cells and theoocyte is solely responsible for the re-initiation of

Žoocyte meiotic maturation Munari-Silem and.Rousset, 1996 . Previous reports suggest a similar

role for Cx43 in preovulatory porcine folliclesŽ .Lenhart et al., 1998 . An alternative hypothesis isthat a positive signal of follicle cell origin isgenerated in response to gonadotropin stimula-tion, and that this signal stimulates resumption of

Ž .meiosis rodent oocytes Schultz, 1991 , similar tothe scenario proposed in other species such as

Žstarfish, teleost fishes and amphibians Kishimoto,.1999; Patino et al., 2001 .˜

Ž .The factor s or hormonal signals responsiblefor the regulation of the Cx43 gap junction genein the mammalian ovary during the folliculargrowth period, have not been determined. Granot

Ž .and Dekel 1997 concluded that during earlyfolliculogenesis, rat ovarian Cx43 gap junctionand its gene are developmentally regulated. How-ever, recent studies demonstrated that FSH up-regulates the expression of Cx43 and gap-junc-tional communication in a rat granulosa cell lineŽ .Sommersberg et al., 2000 . Also, FSH enhancesCx43 gap-junction conductance in cultured

Ž .porcine granulosa cells Godwin et al., 1993 . Thesubstantial increase in Cx43 mRNA in growingand antral follicles observed in the present studyis consistent with FSH regulation. However, afterantrum formation, modifications of Cx43 were

Žfound to be regulated by LH�hCG Granot and.Dekel, 1997 . Cx43 phosphorylation is differen-

tially regulated in ovaries of prepubertal giltsŽ .treated with eCG�hCG Lenhart et al., 1998 .

Overall, the available information is consistentwith the concept that FSH increases Cx43 gap-junction communication in granulosa cells of de-veloping follicles.

Cell�cell communication via gap junctions mayalso play a part in coordinating the process ofatresia. In the present study, expression of Cx43mRNA was undetectable in atretic follicles. Thisfinding is in agreement with the observation of

reduced amounts of Cx43 mRNA in rat ovarianŽfollicles undergoing atresia Wiesen and Midgley,

. Ž .1993 . Wiesen and Midgley 1993 also reportedthat the amount of Cx43 gap-junction mRNA wasreduced as early as 6 h after withdrawal of estra-diol in follicles undergoing atresia. It is still un-clear whether changes in gap-junction mRNA andprotein are the cause or the result of atresia.

In summary, pig Cx43 is expressed in apprecia-ble levels in granulosa cells following activation offollicular development and this expression gradu-ally increases during follicle growth. Since expres-sion of Cx43 occurs simultaneously with the mor-phological differentiation of granulosa cells dur-ing initiation of follicular development, the regu-lation of this gene may be intimately associatedwith this biological process. Furthermore, thechanging patterns of Cx43 expression in laterstages may be linked to different regulatorymechanisms that develop as follicles mature. Fu-ture studies will address the factors and mecha-nisms that are involved in regulating the expres-sion of Cx43 at these critical steps during earlyfollicular development and determine whetherthis association is coincidental or causal.

Acknowledgements

The Texas Cooperative Fish and Wildlife Re-search Unit is jointly sponsored by the U.S. Geo-logical Survey, Texas Tech University, Texas Parksand Wildlife Department and The Wildlife Man-agement Institute. The authors thank MaryCatherine Hastert for technical assistance.

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Expression of connexin 43 mRNA and protein in developing follicles of prepubertal porcine ovaries

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