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  1. 1. Original ArticleModulation of Cx43 and Gap Junctional Intercellular Communicationby Androstenedione in Rat Polycystic Ovary and Granulosa Cellsin vitroRabih Talhouk 1*, Charbel Tarraf 1, Laila Kobrossy 1, Abdallah Shaito 1, Samer Bazzi 1, Dana Bazzoun 1,Marwan El-Sabban 2*1-Department of Biology, Faculty of Arts and Sciences, American University of Beirut (AUB), Beirut, Lebanon2-Department of Anatomy, Cell Biology and Physiology, Faculty of Medicine, American University of Beirut (AUB), Beirut, Lebanon Abstract Background: Gap-junctional intercellular communication (GJIC) is implicated in physicological processes and it is vitally important for granulosa cell (GC) differenti- ation and oocyte growth. We investigated the expression of connexin 43 (Cx43), a gap junctional protein, in normal and androstenedione-induced polycystic ovary (PCO), the effects of androstenedione on Cx43 expression, GJIC and progesterone production in granulosa cells in vitro.* Corresponding Authors: Methods: Isolated GCs from rat ovary were supplemented with FSH and drippedRabih Talhouk, Biology with EHS-matrix (EHS-drip) in culture media, were treated with physiological (10-7Department, Faculty of M) or pathological (10-5 M) androstenedione concentrations to induce differentiation.Arts and Sciences, Cx43 protein levels were assessed by Western blotting. Immunohistochemistry wasAmerican University of also used to determine the localization of Cx43 in GCs and corpus luteum (CL) ofBeirut, P.O. Box: 11-0236,Beirut, Lebanon controls and PCOs. Differentiation of GCs was determined by progesterone assayE-mail: and Lucifer yellow dye transfer for GJIC status. The degree of significance of vari-rtalhouk@aub.edu.lb ations between the results was analyzed by ANOVA using SPSS (version 11.5;Marwan El-Sabban, 2002).Department of Anatomy, Results: Cx43 localized in the GC layer of both the control and PCOs. Its proteinCell Biology & levels were upregulated in PCO rat ovaries. GCs in culture with or without andro-Physiology, Faculty of stenedione had a punctate membranous distribution of Cx43. However, androstene-Medicine, AmericanUniversity of Beirut, P.O. dione increased GJIC and upregulated progesterone and Cx43 protein levels. Inhibit-Box: 11-0236, Beirut, ing GJIC by 18-α GA in androstenedione-treated GCs caused partial inhibition ofLebanon progesterone production, suggesting a possible role of GJIC in mediating the actionE-mail: of androstenedione on GC differentiation.me00@aub.edu.lb Conclusion: This study presented a suitable culture model for polycystic ovary syn-Received: Aug. 9, 2011 drome and showed that Cx43 and GJIC might contribute to the pathogenesis of poly-Accepted: Dec. 3, 2011 cystic ovary syndrome. Keywords: Androstenedione, Connexins, Extracellular matrix, Gap junction intercellular communication, Granulosa cell, Ovary, Polycystic ovary. To cite this article: Talhouk R, Tarraf C, Kobrossy L, Shaito A, Bazzi S, Bazzoun D, El- Sabban M. Modulation of Cx43 and Gap Junctional Intercellular Communication by Androstenedione in Rat Polycystic Ovary and Granulosa Cells in vitro. J Reprod Infertil. 2012;13(1):21-32. Introduction he Polycystic Ovary Syndrome (PCOS) first disorders and particularly a major endocrinopathy identified in 1935 (1), accounts for 75% of in women of reproductive age (2). PCOS is a anovulatory infertility cases in women complex and heterogeneous genetic disorder char-which makes it one of the most common human acterized by androgen excess and ovulatory dys- J Reprod Infertil. 2012;13(1):21-32
  2. 2. JRI Modulation of Cx43 and Gap Junctionalfunction (1). Follicular growth is disrupted as a re- In ovaries, connexin 43 (Cx43), encoded by Gja1,sult of ovarian hyperandrogenism, hyperinsulin- has been found to be the main connexin expressedemia due to insulin resistance and distorted intra- in developing follicles forming the gap junctionsovarian paracrine signaling (3). In PCOS, fol- coupling granulosa cells (20−23). In agreementliculogensis occurs normally throughout the pre- with the role of gap junctions in folliculogenesis,antral stage; beyond which the progression of the Cx43 knockout mice have had folliculogenesisfollicle into a preovulatory one will be disturbed. arrest in their primary stage and developed incom-In addition to the role of the endocrine control in petent oocytes (24, 25). Furthermore; GCs lackingthe development of the preantral follicle, there are Cx43 have displayed lower proliferation rate andvarious paracrine and autocrine factors identified reduced response to oocyte-derived mitogens re-in small follicles, playing roles during early fol- sulting in an impaired function of these cells (26,licular growth not to mention a critical role of the 27).oocyte in this process (4−7). Studies have shown The regulation of GJIC and connexin proteinthat granulosa cells (GCs) in antral follicles in expression by steroids has been documented inPCOs exhibit decreased aromatase activity, alter- several cell types (28−30), including ovarian cellsed response to growth factors, hormones and early (31); causing either a downregulation or an eleva-leutinization (8−10). tion in the expression of gap junctional proteins. Normally, inactive primordial follicles undergo An in vitro study revealed that GJIC up-regulationarrest at prophase I and contain nongrowing oo- in granulosa-oocyte complex is independent ofcytes and squamous pregranulosa cells supported gonadotropins (32). However, its breakdown isby a basal lamina. As these follicles get activated gonadotropin dependent and occurs by the cluster-the squamous pregranulosa cells will develop to ing of Cx43 in lipid raft microdomains of theform a single layer of cuboidal granulosa cells that cells.will later divide to form a multilayered follicle Taking into account the importance of GJIC for(11). It is clear that tissue remodeling is essential GC differentiation and oocyte growth, in additionfor follicular maturation; as such, the communica- to the important role of steroids in controllingtion between GCs and the extracellular matrix GJIC, the current study was designed to study(ECM) is inevitable for normal folliculogenesis. GJIC in GCs of androstenedione-induced PCOSThe role of ECM in GC function has been investi- in rats using in vivo and in vitro models. Specific-gated since early 1980s and continues with the ally, we investigated the expression of Cx43, aimprovement in the in vitro culture systems (12). gap junctional protein, in normal and androstene-Attempts to study GC differentiation in vitro has dione-induced PCO, the effects of androstenedi-focused on the effects of soluble mediators such one on Cx43 expression, GJIC, and progesteroneas hormones, growth factors, cytokines and neuro- production in vitro.transmitters (13−15); however, the crucial role ofthe microenvironment in GC differentiation is Methodspoorly understood. It has been shown that anti- Materials: Androstenedione, Diethylstilbestrolbodies which block cell-ECM interactions have (DES), equine chorionic gonadotrophic (eCG) andresulted in the loss of progesterone synthesis in human chorionic gonadotrophic (hCG) hormones,cultured rat GCs in vitro (16). In addition, studies follicle stimulating hormone (FSH), Hanks Bal-have revealed that ECM rescues primary GCs anced Salt Solution (HBSS), Dulbecco’s Minimalfrom apoptosis (17). Consistent with this, it has Essential Medium (DMEM-F12), penicillin/ strep-been shown that gonadotrophins and ECM syn- tomycin, trypsin-EDTA, poly-L-lysine and 18αergize to regulate GC differentiation, as measured glycyrrhetinic acid (18αGA) were purchased fromby progesterone production, and induction of gap Sigma Chemical Co. (St Louis, Missouri, USA).junction formation (18). Protease inhibitors, CompleteTM, were obtained Gap-junctional intercellular communication from Boehringer (Mannheim, Germany). Fetal bo-(GJIC) is implicated in many normal physiologic- vine serum (FBS) was purchased from Gibcoal and pathological processes. GYIC has been (Paisely, Scotland). The source of growth factordocumented to play a crucial role in the physi- reduced EHS (Engelberth-Holm-Swarm tumor)-ology of the ovarian follicle. GJIC has been also matrix, MatrigelTM, was from Collaborative Bio-implicated in the control of steroidogenesis (19). medical Products (Bedford, MA, USA). PVDF22 J Reprod Infertil, Vol 13, No 1, Jan/Mar 2012
  3. 3. Talhouk R, et al. JRImembranes and α-32PdCTP were supplied by were excised and surrounding tissues were re-Amersham Pharmacia Biotech (Uppsala, Swe- moved.den), and rat-specific progesterone RIA kits were Cell culture: For GC culture, ovaries were wash-obtained from Immunotech (France). Rabbit anti- ed in cell culture medium (DMEM-F 12) contain-connexin 43 was obtained from Zymed (San ing 5% FBS. GCs were harvested in DMEM-F 12Francisco, CA, USA). Anti-rabbit and anti-goat containing 5% FBS by follicle puncture using 25-IgG HRP conjugated antibodies, β actin anti- gauge needle fitted to a 1 ml insulin syringe. Thebodies, Enhanced Chemiluminescence (ECL) kit GCs were isolated from the excised ovaries usingand ECL markers were obtained from Santa Cruz gentle pressure with a blunt microspatula. TheBiotechnology (Santa Cruz, CA, USA). Lucifer culture medium was collected and the cells wereYellow CH (LY), Secondary goat anti-rabbit IgG sedimented by centrifugation at 150 g for 5 min atFITC-conjugate, propidium iodide, and Prolong 4 °C. The pellet was then washed with DMEM-AntifadeTM kit were purchased from Molecular F12 and resuspended in l ml of DMEM-F 12. TheProbes (Leiden, Netherlands). Biorad DC protein cells were counted using a hemocytometer andassay reagent was obtained from Biorad (Her- plated in a 24-well cell culture plate at a seedingcules, CA, USA). All other material used was of density of l.5x105 cells/ml. Cells were maintainedmolecular biology grade. in a humidified incubator (95% air, 5% C02) at 37 Animals, hormone treatments and tissue sections: °C. GCs were plated on serum precoated tissueImmature 21−24 day old female Sprague Dawley culture plates in serum supplemented (5% FBS)rats were obtained from the animal care facility at DMEM-F12 media in the presence of 20 ng/mlthe American University of Beirut. All animal FSH and 1% penicillin/streptomycin. The mediumcare requirements were fulfilled, and animals was changed on the first day after plating and onwere given food and water ad libitum. Rats were subsequent days as needed. Serum was removedsacrificed by cervical dislocation. For in vivo on day 1 after plating and diluted MatrigelTMstudies, rats were divided into five groups. The (1.5% vol/vol) was dripped (hereafter referred tocontrol group received injections of corn oil (100 as EHS-drip), onto cells (40). After washing, cellsµl/day for 3 days, subcutaneously [s.c.]) and were grown on plastic without matrigel.ovaries were subsequently removed one day after GCs were cultured with or without androstenedi-the last injection. Another group of rats received one at 10-7 or 10-5 M (dissolved in 100% ethanol)injections of DES (1 mg/day, for 3 days, s.c.); the as of day 1 after plating. Cells were trypsinizedrats were then either sacrificed (DES group; and counted (Trypan blue viable counts) using aovaries enriched in preantral follicle-undifferenti- hemocytometer, and the conditioned medium wasated GCs) or subsequently received injections of collected for progesterone quantification using aeCG (15 IU, intraperitoneal injection [i.p.]). radioimmunoassay kit according to the manufac-Forty-eight hours later, the latter group of animals turer’s instructions.were either sacrificed (eCG group; ovaries enrich-ed in antral follicle-differentiated GCs) or admin- In experiments where 18αGA was added to GCs,istered hCG (15 IU; i.p.) and sacrificed eight 50 µM 18αGA (dissolved in DMSO) was added tohours, thereafter (hCG group; ovaries enriched in the cultured GCs on day l of culture. Media wereluteal cells). This protocol was used to mimic the changed on a daily basis and accompanied withdifferent follicular developmental stages in the rat repeated androstenedione treatments.ovary (33). The PCOS group received daily inject- Progesterone assay: Cells were cultured ontions of androstenedione at a concentration of plastic, EHS-drip in 24-well plates. Media condi-6 mg/100 g body weight dissolved in 200 µl corn tioned beta cells were collected on daily basisoil for 21 days. It is established that this treatment until day 6 of culture. Upon collection, 40 µl ofis sufficient to induce PCOS in 24 day-old female diluted protease inhibitors cocktail was added torats (34−39). The ovaries were removed, fixed in the media. The sampled media were stored at -704% buffered formaldehyde, embedded in paraffin, °C for subsequent analysis. Progesterone concen-and sectioned at 5 µm and mounted onto Vecta- tration in the media was quantified using a solid-bond coated slides. Sections were used for im- phase radioimmunoassay (RIA) kit. The assaymuunofluorescence as described later. For GC was performed according to the manufacturer’sculture, rats received daily injections of DES (l instructions. Samples from each treatment weremg/day, s.c.) for three consecutive days. Ovaries run in duplicates and the values were converted to J Reprod Infertil, Vol 13, No 1, Jan/Mar 2012 23
  4. 4. JRI Modulation of Cx43 and Gap Junctionalconcentrations by extrapolation from a standard before proceeding in the immunostaining. All in-curve. cubations and washings were done at room tem- Western blot analysis: Total proteins extracted perature.either from ovaries or GCs in culture were ana- Lucjfer yellow dye transfer assay: GCs were grownlyzed by western blotting for Cx43 as described on EHS-drip in 8-chamber polystyrene tissue cul-by El-Sabban et al. Briefly, ovaries were homo- ture treated glass slides at 3.0x105 cells/ chamber.genized for 2 min, into RIPA buffer supplemented Scrape-loading technique was performed usingwith protease inhibitors (40 µl/l ml of RIPA). The Lucifer Yellow (LY) dye (41, 42).homogenate was then centrifuged at 10,000 g for Statistics: The degree of significance of vari-30 min where the supernatant was removed and ations between average levels of progesteronestored at -70 °C. For GCs in culture, protein ex- from triplicate wells was determined after appro-traction was performed on day 4 of culture by priate Analysis of Variance (ANOVA) usingscraping the cells in RIPA buffer supplemented Least Significant Difference (LSD) test. All ana-with protease inhibitors. The scraped cells were lyses were performed using SPSS version; 11.5then sheared, centrifuged at 10,000 g for 30 min for windows SPSS Inc., Chicago, IL, USA).and the supernatant was removed and stored inaliquots at -70 °C. Biorad assay was used for Resultsquantification of protein content of samples To reveal a potential role of Cx43 in GC func-extracted from both cells and ovaries. Twenty µg tion, we aimed to characterize its distribution inof protein were run on a 12% SDS-PAGE and rat ovaries from hormone-treated animals withblotted. Membranes were then blocked in 3% different follicular developmental stages and inskimmed milk for 1 hr, incubated with primary those from rats with PCOs after androstenedionerabbit anti-connexin 43 according to the supplier’s induction. Sections were by stained H&E to revealsuggestion in 5% skimmed milk for 1 hr at room follicular development in each of control, treatedtemperature then for 1 hr at room temperature in and PCOs groups. As figure 1 (A-E) indicates,3% skimmed milk solution containing goat anti- control ovaries had mostly primordial and primaryrabbit IgG conjugated to horse raddish peroxidase follicles (Figure 1A). The DES-treated group had(1:5000). Immunoreactivity was detected using their ovaries filled with preantral follicles (Figureenhanced chemiluminescence. 1B). Follicles were at the early antral to late antral Immunohistochemistry: GCs were cultured on stage in ovaries of the eCG group (Figure 1C).poly-L-lysine coated EHS-dripped coverslips in The hCG ovaries were filled with follicles at the24-well plates as described previously. On day 4 preovulatory stage having the oocyte in an eccen-of culture, cells were washed with warm HBSS tric position (Figure 1D). Polycystic ovaries wereand fixed in cold (-20 °C) 70% ethanol. Fixed enlarged and occupied with follicular cysts thatcells were rinsed twice with phosphate-buffered were either devoid from oocytes or had theirsaline (PBS), blocked for 1 hr with 3% normal oocytes surrounded with a maximum of twogoat serum and incubated for 2 hr at room tem- layers of GCs. Immunolocalization of Cx43 in ratperature with rabbit anti-connexin 43. The cells ovaries of DES, eCG, hCG, and PCO groupswere then incubated for 1 hr with secondary goat stained with H&E stains (Figure 1, A-E) revealedanti-rabbit IgG (H+L) FITC-conjugate. Concen- that Cx43 localized to the GCs of all animaltrations of the primary and secondary antibodies groups (Figure 1, F-J). Cx43 was not detected atwere used as recommended by the supplier. the thecal cell layer or at the border between GCsNuclei were counterstained with propidium iodide and oocytes where Cx26 and Cx32 were found to(5 µg/ml). Washing with PBS was performed be predominant, respectively (data not shown).twice between incubations. Cells were then To assess the effect of androstenedione on Cx43mounted on slides and staining was preserved by expression and localization and GJIC function-adding AntifadeTM to the stained cells. Cells were ality, GCs’ expression and localization of Cx43observed using a fluorescence microscope (Zeiss were examined. When GCs were plated on EHS-LSM 410, Germany). Same procedure was fol- drip with or without androstenedione, they ex-lowed for the immunohistochemistry of the pressed Cx43 as indicated in figure 2 (A-C). Toovarian sections, except that deparafinization and further analyze the effect of androstenedione onrehydration in an ethanol gradient were needed Cx43 cellular distribution in GCs culture immune-24 J Reprod Infertil, Vol 13, No 1, Jan/Mar 2012
  5. 5. Talhouk R, et al. JRI Figure 2. Immunolocalization of Cx43 (A-C) and lucifer yellow dye transfer (D-F) in GCs on day 4 of culture. GCs were plated on EHS-drip without androstenedione (A & D), with l0-7 M (B & E) or l0-5 M (C & F) androstenedione. Although Cx43 distribution was not altered in either of the cultured conditions, GJIC was markedly enhanced when cells were treated with l0-5 M (F) androstenedione. Fluores- cent microscopy images were obtained at 10X (A-C) and 20X magnifications (D-F) control untreated cultures, however, appreciable increase in LY transfer to cell layers away from the scrape sites was noted when GCs were cul- Figure 1. Hematoxylin and Eosin stains and Cx43 immuno- tured on EHS-drip with l0-5 M androstenedione localization for rat ovarian sections of control (A & F), (Figure 2F), suggesting enhanced GJIC in PCO- DES (B & G), eCG (C & H), hCG (D & I) and androstene- like conditions (34). dione (PCO; E & J) treated rats where section in the squares are magnified to reveal the clear structure of the follicle. Since Cx43 has been shown to be the major Cx Image (E) shows a section of polycystic ovary with a fol- expressed in GCs in vivo in both control and licular cyst (FC) in addition to small follicles (SF). Middle PCOs, we examined the localization and levels of panel represents enlarged areas of similar sizes representing expression of Cx43 in GCs in response to andro- the follicle features in every condition. Cx43 localizes to stenedione dosages. Western blot analysis showed the GCs in all stained sections. Micrographs are taken at 5X magnification that the treatment of rats with l0-5 M concentration of androstenedione upregulated Cx43 expressionlocalization studies were performed. Cx43 expres- in their PCOs (Figure 3A). Similarly, and parallelsion was evident at 4 days in culture and had a to enhanced GJIC noted in culture, androstene-punctate membranous appearance in the absence dione also enhanced Cx43 expression at l0-7 andor presence of androstenedione at both l0-7 M and l0-5 M by cultured GC in a dose-dependent man-l0-5 M concentrations (Figure 2, A-C). Neverthe- ner. Enhanced phosphorylation of Cx43 was alsoless, the functionality of GJIC between GCs on noted in androstenedione treated cells (Figureday 4 of culture was enhanced upon androstenedi- 3B).one treatment. Confluent cultures of GCs plated Progesterone production was used as an indica-on EHS-drip alone displayed limited dye transfer tor of the extent of differentiation of GCs. Weto neighboring cell layers (Figure 2D), although showed that the enhanced GJIC and Cx43 expres-these cells showed increased dye transfer when sion in androstenedione-treated cells was parallel-compared to cells cultured on plastic alone (data ed by an increase in progesterone production. Tonot shown). Similar transfer of the fluorescent dye assess the effect of androstenedione on progester-was observed with GCs cultured on EHS-drip one levels produced by GCs on day 4 of culture,with l0-7 M androstenedione when compared to RIA analysis was performed. Whereas GCs on p J Reprod Infertil, Vol 13, No 1, Jan/Mar 2012 25
  6. 6. JRI Modulation of Cx43 and Gap JunctionalFigure 3. Effect of androstenedione on Cx43 expression. A.Western blot analysis of Cx43 protein in normal rat ovaries(lane a) and in androstenedione treated rats’ PCO ovaries(lane b). Lane H: heart sample Cx43 control. B. Westernblot analysis of Cx43 protein in GCs on day 4 of culture. Figure 4. Androstenedione induces progesterone produc-Cx43 proteins expressed in GCs cultured on EHS-drip (lane tion in GC cultures in a GJIC-dependent manner. A. GCsa), in the presence of l0-7 M (lane b) or l0-5 M (lane c) on day 4 of culture on EHS-drip in the absence (control) orandrostenedione. P0, unphosphorylated form of Cx43 presence of l0-7 M or l0-5 M androstenedione. Progesteroneprotein, P1 phosphorylated active form of Cx43. β-actin production is enhanced by androstenedione in a dose-de-protein shows for equal loading pendent manner. Note: minimal levels of progesterone (<20 pg/cell) are produced by GC cultured on plastic. The amount of progesterone (pg) secreted into the media waslastic produced very low levels of progesterone normalized per one cell. B. Effect of 18αGA on progester-(<20 pg/cell), cells on EHS-drip produced signifi- one production by GCs on EHS-drip and treated with l0-5 Mcantly (p <0.05) higher progesterone levels (> 100 and l0-7 M androstenedione and 50 µM of 18αGA showpg/cell). Furthermore, EHS-dripped GCs cultured marked downregulation of progesterone production on daywith l0-7 M and l0-5 M androstenedione, signifi- 4 of culture. Statistical analysis obtained from these experi- ments reveals statistical significance at p <0.05 representedcantly increased their progesterone levels, in a by asterisk (*). In (A) all results are significantly differentdose dependent manner, as compared to the con- from each othertrols. GCs cultured with l0-5 M androstenedioneproduced more than 700 pg/cell of progesterone transport of metabolites and signal molecules(Figure 4A). between them and also with oocytes. This cellular In order to study the role of GJIC in facilitating communication is crucial for follicular develop-progesterone production in GCs in response to ment and oocyte growth and maturation (46).androstenedione 18αGA, a gap junction inhibitor, Mice with disrupted GJs were infertile and thewas used. Increased progesterone production was communication between oocytes and the neigh-at least partly a consequence of increased GJIC boring GCs was greatly reduced (47). Aberrantsince 18αGA decreased progesterone production GJIC is involved in ovulatory dysfunction, resultsby GCs in culture. Compared to control untreated in interrupted folliculogenesis and prevents for-cells, progesterone production by GCs cultured on mation of GC layers around the oocyte in Cx43EHS-drip in the presence of l0-5 M androstenedi- null mice. Ackert et al. demonstrated that the mu-one was inhibited by about 40% in the presence of tant oocytes obtained from Cx43 null mice failed18αGA with no significant difference (p <0.05) in to undergo meiotic maturation and could not beits levels in the l0-7 M androstenedione (Figure fertilized. This correlation between abnormal fol-4B). liculogenesis, Cx43 expression and aberrant GJIC prompted our laboratory to investigate the regula- Discussion tion of Cx43 in the PCO. GJIC has been shown to regulate major cellular In vivo rat PCO model has long been estab-programs such as growth, differentiation, and apo- lished, and could be experimentally inducedptosis (41, 43−45). More specifically in the ovary, through prolonged injections of androgens in im-cell-cell communication between GCs allows mature female rats (38, 39, 48). In the current26 J Reprod Infertil, Vol 13, No 1, Jan/Mar 2012
  7. 7. Talhouk R, et al. JRIstudy, androstenedione was successfully used to dione on Cx43 expression, localization and GJICinduce PCOs in rats as verified by H&E staining to further understand how GJs display PCO phe-of PCO sections. These ovaries had evident fol- notype through their effects on GC differentiation.licular cysts, and lacked corpora lutea, as most Cx43 immunolocalization showed punctate mem-follicles developed into early follicular stages but branous distribution in GCs when cultured ondid not develop to ovulatory stages in PCOs (49, EHS-drip, in the presence of both 10-7 and 10-5 M50). In the ovary of a control 41-day old rat, Cx43 androstenedione, or in androstenedione free me-was localized to GCs of small and large follicles dium. However, scrape loading of LY showed aand the corpus luteum (CL). In androstenedione- significant increase in GJIC in the presence of 10-5induced PCO rats, Cx43 was also localized in fol- M androstenedione. Increased LY dye transfer inlicular GCs. This is consistent with previous data the presence of 10-7 M androstenedione was not asindicating that Cx43 was present in follicular GCs evident. In parallel experiments we showed thatof rat (51), mouse (24, 52), ewe (53), cow (54), no LY transfer was noted in GC cultured on plas-and pig (55, 56). Cx43 has also been previously tic compared to those on EHS-drip (not shown).detected in rat CL (57, 58). Taken together, the data suggests a possible syn- In this study, GCs were grown in the culture ergistic role of both hormones, androstenedione inmedia under differentiation-permissive conditions, this case and ECM, in promoting GJIC. A similarsupplemented with FSH and dripped with EHS- observation was reported earlier in mammarymatrix. GCs cultured on plastic in media lacking epithelial cells (41). In fact, in human GCs, hCGandrostenedione produced very low amounts of and ECM synergize to enhance gap junction for-progesterone while those cultured on EHS-drip mation and progesterone production (18). As forproduced appreciable amounts. More so, in- GJIC, androstenedione could be acting directly oncreased progesterone production by GCs on EHS- increasing Cx gene expression in GCs, since it hasdrip was noted in response to androstenedione in a been shown previously that Cx43 promoter isdose-dependent manner. This suggested that EHS- responsive to estrogens (66), and that estrogendrip and androstenedione together synergistically upregulated Cx43 expression in GCs in vivo (57).promoted progesterone production by GCs. It has Though, estradiol and progesterone have beenpreviously been shown that growing mammary shown to decrease Cx43 protein levels whenepithelial cells enhanced their differentiation phe- administered together into cultured rat endo-notype and GJIC with EHS-drip and lactogenic metrial cells (28). Alternatively, androstenedionehormones (41, 59). During the follicular phase of could be activated by several kinases, such asovarian cycle, androstenedione produced by theca MAP kinase which is also activated by ECM (67),cells reaches a level of 10-7 M in the follicular and it has been shown to regulate GJIC (68−70).fluid. In PCO syndrome, androstenedione levels Direct regulation of GJIC in GCs by either estro-rise from 10-7 M to 10-5 M, which is the androgen gens or androgens has not been previouslyconcentration that reaches the GCs (60, 61). The studied. It was found that Cx43 protein levelsmechanism of this rise in androgen levels is un- were highly upregulated in the PCOs as comparedclear. Nevertheless, this excess clearly adds up to to the controls. This trend is not noted in the im-the complexity of PCOS and is mediated by both munohistochemistry as it only reveals the distribu-intrinsic and extrinsic factors (62). The rise in tion and localization of Cx43.androstenedione levels is either due to increased Quantification of fluorescence was not attemptedactivity of theca cells, or decreased GC ability to and Western blotting for Cx43 protein was usedaromatize the accumulating androgens into estro- for quantification purposes instead.gens (63, 64). Given that, 10-7 M (moderate) and The elevation of Cx43 protein levels observed in10-5 M (high) androstenedione concentrations PCOs in this study cannot be attributed to an in-were used to study the effects of androstenedione crease in the number of ovarian blood vessels ex-on Cx43 expression, GJIC and subsequently GC pressing Cx43, since H&E staining of both PCdifferentiation. and control ovaries did not reveal any difference Cx43 is the major Cx expressed by GCs that in vascularization (not shown). GJIC is at leastcontributes to intercellular coupling; hence play- partly mediated by Cx43 post-translational modi-ing role in GC development, maturation, differen- fication through kinase activation (41, 71) becausetiation and leutinization (46, 65). As such, we an increase in Cx43 protein levels and phosphory-intended to characterize the effect of androstene- lation in the presence of 10-7 M and 10-5 M andro- J Reprod Infertil, Vol 13, No 1, Jan/Mar 2012 27
  8. 8. JRI Modulation of Cx43 and Gap Junctionalstenedione were noted. It has been documented pressed by rat ovarian GCs (82). Similarly, wethat GJIC in porcine granulosa cells was enhanced showed Cx45 immunolocalization in GCs ofby protein kinase A (PKA) phosphorylation but normal rat ovaries and PCOs, and Cx45 proteindecreased by protein kinase C (PKC) (72). Other levels were upregulated in PCOs as compared tostudies indicated that FSH-stimulated Cx43 phos- normal ones (unpublished data), further suggest-phorylation was attributable to PKA (73), and that ing the involvement of Cx45 in addition to Cx43casein kinase 1 induced Cx43 gap-junction assem- in modulating GJIC in GCs following androgenbly (74). stimulation. The role of Cx45 in PCO syndrome Androstenedione treatment of GCs on EHS-drip needs further investigation.promoted increased GJIC in GCs, in addition toincreased progesterone production. Yet, under Conclusionphysiological concentrations (10-7 M) of andro- Gap junctions form a network of bonded inter-stenedione, inhibiting GJIC did not lead to a sig- connecting GCs that surround the oocyte, and thenificant decrease in progesterone production loss of these junctions may facilitate the dissoci-levels suggesting that GJIC is not the sole medi- ation of the oocyte and disrupt normal folliculo-ator but could contribute along with ECM to pro- genesis. In conclusion, this work demonstrates forgesterone production. Cx43 effect on progesterone the first time the modulation of Cx43 in andro-production is only prominent under non-physio- stenedione-induced rat PCO. Moreover, andro-logical doses of androstenedione (10-5 M). The stenedione induced luteinization of cultured GCseffect of ECM, androstenedione and FSH on GC together with enhanced GJIC, and Cx43 expres-differentiation and progesterone production is sion suggest a role for gap junctions in partiallylikely due to the collective effects of enhanced mediating the effect of androstenedione on pro-activity and expression of the steroidogenic en- gesterone production. Further studies are requiredzymes (75), deposition of some basement mem- to understand the androstenedione-induced GJICbrane components like fibronectin and proteo- dependent signaling pathways mediating a PCO-glycans, maintenance of FSH receptors under op- like phenotype in vitro.timal culture conditions (75) and/or an increase inc-AMP production (76). Treating cultured rat GCs Acknowledgementwith cAMP increases GJIC (77, and unpublished The authors are grateful to doctors. Medhatdata), since cAMP, apart from being a second Khattar and Joana Kogan for critical reading ofmessenger, has been known to enhance GJIC and the manuscript. Wissam Mehio is acknowledgedinduce differentiation in several cell types (41, for assisting in the preparation of the manuscript.78). Knowing that GJIC may play a role in in- This work is supported by the University Re-creasing progesterone and consequently mediating search Board (RST, CGT and MES). MedicalGC differentiation, the effect of inhibiting GJIC Practice Plan, Diana Tamari Sabbagh Researchon GC differentiation had to be examined. When Fund, Terry Fox Cancer Research fund (MES),treating GCs with 18αGA, an inhibitor of GJIC and Lebanese National Council for Scientific Re-(41, 79−81), progesterone production was down- search (CGT, and RST).regulated. It is worth noting that the androstene- Declaration of conflict of interest: The authorsdione-induced differentiated phenotype, as evi- declare no conflict of interest.dent by increased progesterone production, wasunlikely due to changes in cell morphology since ReferencesGCs cultured on EHS-drip with or without andro- 1. Stein IF, Leventhal ML. Amenorrhea associated with bilateral polycystic ovaries. Am J Obstetstenedione did not show marked altered morph- Gynecol. 1935;29:181-91.ology. Therefore, the effect of androstenedione onprogesterone production may be partially medi- 2. Jones MR, Chua A, Chen YD, Li X, Krauss RM,ated by increased GJIC, either through upregu- Rotter JI, et al. Harnessing expression data to iden-lating Cx43 levels of protein expression, as seen tify novel candidate genes in polycystic ovary syndrome. PLoS One. 2011;6(5):e20120.in our study, or through promoting Cx43 phos-phorylation by activating several kinases. 3. Pigny P, Merlen E, Robert Y, Cortet-Rudelli C, The involvement of other connexins in regu- Decanter C, Jonard S, et al. Elevated serum level oflating GC function cannot be ruled out (46). In anti-mullerian hormone in patients with polycystic ovary syndrome: relationship to the ovarian folliclefact, Cx45 has been shown previously to be ex-28 J Reprod Infertil, Vol 13, No 1, Jan/Mar 2012
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