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Paracrine regulation of theca androgen production by granulosa cells in the ovary
Paracrine regulation of theca androgen production by granulosa cells in the ovary Yvonne D. Hoang, Ph.D., Kirsten J. McTavish, Ph.D., R. Jeffrey Chang, M.D., and Shunichi Shimasaki, Ph.D. Department of Reproductive Medicine, University of California, San Diego, School of Medicine, La Jolla, California
Objective: To test whether and to what extent inhibin mediates Cyp17 messenger RNA (mRNA) expression in theca cells (TCs) in response to FSH stimulation of granulosa cells (GCs). Design: Ex vivo and in vitro experimental study. Setting: University. Animal(s): Immature female Sprague Dawley rats. Intervention(s): Ovarian tissue explants and isolated theca cell preparations with or without GCs were treated with FSH, inhibin, inhibin antibody, or b-glycan antibody. Main Outcome Measure(s): As a key enzyme in androgen production, Cyp17 mRNA levels were measured by real-time reverse transcription–polymerase chain reaction. Result(s): After 24 hours, Cyp17 mRNA expression was dose-dependently increased by FSH in ovarian tissue explants and theca cells, suggesting that paracrine factor(s) secreted from GCs in response to FSH mediates Cyp17 mRNA expression in TCs. Antibodies against inhibin and inhibin coreceptor, b-glycan, blocked the stimulatory effect of FSH on Cyp17 mRNA expression. However, inhibin alone did not increase Cyp17 mRNA level to the same extent. Conclusion(s): These findings suggest a role for inhibin in the paracrine regulation of TC Cyp17 mRNA expression by GCs influenced by FSH; however, other paracrine factors produced by GCs Use your smartphone by virtue of FSH seem to be required. (Fertil SterilÒ 2013;100:561–7. Ó2013 by American Soto scan this QR code ciety for Reproductive Medicine.) and connect to the Key Words: Theca cell, granulosa cell, inhibin, Cyp17, polycystic ovary syndrome Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/hoangyd-paracrine-regulation-theca-granulosa-cell-ovary/
T
he production of ovarian androgen is driven by increased pituitary LH secretion, which activates CYP17. In vitro studies have shown that LH-induced increments in theca cell (TC) androgen production are dose-related (1). These findings correspond to the clinical observation that in women with polycystic ovary syndrome (PCOS), elevated serum LH
levels positively correlate with circulating T concentrations (2). In addition, in women with PCOS treated with GnRH agonists, elevated LH levels are reduced or eliminated, with a corresponding reduction in circulating androgen levels (3). Thus, increased secretion of LH may be pivotal in amplifying the production of excess androgen. However, another study
Received February 1, 2013; revised March 22, 2013; accepted April 10, 2013; published online May 23, 2013. Y.D.H. has nothing to disclose. K.J.M. has nothing to disclose. R.J.C. has nothing to disclose. S.S. has nothing to disclose. This work was supported in part by National Institutes of Health (NIH) grant R01 HD41494 and by the National Institute of Child Health and Human Development/NIH through a cooperative agreement (U54 HD012303) as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research. Reprint requests: Shunichi Shimasaki, Ph.D., Department of Reproductive Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0633 (E-mail: sshimasaki@ucsd. edu). Fertility and Sterility® Vol. 100, No. 2, August 2013 0015-0282/$36.00 Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2013.04.016 VOL. 100 NO. 2 / AUGUST 2013
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looking at LH responsiveness of TCs found increased conversion of P to androgen in women with PCOS despite normal steroid responses to LH (4). Nevertheless, the action of LH on TC androgen production has not been well quantified in women with PCOS or normal women. We recently reported that in women with PCOS, IV administration of FSH resulted in significant increases in serum 17-hydroxyprogesterone, androstenedione (A), and dehydroepiandrosterone levels (5). By comparison, androgen levels in normal women remained unchanged after FSH stimulation. Interestingly, the serum androgen increase was accompanied by fivefold-elevated inhibin B levels in women with PCOS, markedly greater than the threefold change observed in 561
ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE normal women. Moreover, in women with PCOS, serum estrogen responses were also greater than those of the normal group. These findings suggested that granulosa cells (GCs) might be partially responsible for regulating TC androgen production in women with PCOS. Both in vitro and in vivo studies conducted in animals have indicated that FSH may amplify LH-induced ovarian androgen production (6–9). In cultured human TCs, A responses to LH in the presence of inhibin were clearly increased compared with those without inhibin (10, 11). In addition, inhibin was able to negate the inhibitory effect of activin on human TC androgen production. Accordingly, in our study the significant increases in ovarian androgens exhibited by women with PCOS were accompanied by similar significant increments in FSH-stimulated inhibin B levels compared with those of normal women. A growing body of evidence supports the view that GC-derived factors are potent regulators of TC androgen secretion. For instance, members of the transforming growth factor-b superfamily of proteins are well-established regulators of ovarian steroidogenesis and follicle development (12, 13). In regard to androgen production, bone morphogenetic protein-6 potently suppressed basal and LH-induced secretion in ovine (14), bovine (15), porcine (16), and human TCs (17). Transforming growth factor-b, which is expressed by GCs and TCs (16), suppressed androgen secretion from rat (18) and human TCs (19). Activin decreased whereas inhibin increased androgen secretion from human (17, 20), rat (11), and bovine (21) TCs. Futhermore, insulin-like growth factor-1 enhances the stimulatory effect of LH on CYP17 expression (22) and androgen synthesis (10, 20, 23). When combined with stem cell factor/kit ligand, insulin-like growth factor-1 increased messenger RNAs (mRNAs) of steroid acute regulatory protein, CYP11A1, CYP17, 3b-hydroxysteroid dehydrogenase, and LH receptor (24). Although expressed by both GCs and TCs, hepatocyte growth factor reduced CYP17 expression and androgen secretion from rat TCs in the presence of LH (25). Overall it seems that there is a complex network of signals between GCs and TCs that act to finetune LH and FSH regulation of ovarian steroid production. In the course of our long-term project identifying the GCderived factor(s) that mediate(s) FSH stimulation of androgen synthesis by TCs, we have focused here on the role of inhibin in the mechanism, on the basis of our clinical data described above (5). With emphasis on Cyp17 as the pivotal enzyme in TC androgen synthesis, we studied FSH effects in isolated rat ovary explants and primary theca from immature rats.
MATERIALS AND METHODS Reagents and Supplies Female Sprague Dawley rats were purchased from Charles River Laboratories. McCoy's 5a and M199 medium were purchased from Gibco (Life Technologies). Diethylstilbestrol, collagenase type IA, hCG, penicillin/streptomycin, Triton X-100, and DNase were purchased from Sigma Chemical. Silastic tubing was purchased from Dow Corning. Thin-wall polyallomer tubes were obtained from Beckman-Coulter. Ovine FSH was obtained from Dr. A. F. Parlow at the National 562
Hormone and Peptide Program of the National Institute of Diabetes and Digestive and Kidney Diseases, Harbor-UCLA Medical Center. Inhibin antiserum was kindly provided by Dr. Nicholas Ling of the Whittier Institute (La Jolla, CA), which was derived from rabbits that were immunized with an amino terminal 30-amino acid chain of porcine inhibin a-subunit coupled to bovine serum albumin (BSA) (26). Goat anti-human b-glycan/TGFBRIII sc-6199 antibody was obtained from Santa Cruz Biotechnology. Recombinant inhibin A was gifted by Drs. Wolfgang H. Fischer and Wylie Vale of the Salk Institute in La Jolla, CA. Nucleospin RNA II extraction kits were purchased from Clontech Laboratories. iQ SYBR Green Supermix qPCR reagent and iScript reverse transcriptase kits were purchased from BioRad. LabTek chamber slides were obtained from Thermo Fisher Scientific. Donkey anti-rabbit fluorescein isothiocyanate conjugate– conjugated secondary antibodies were purchased from R&D Systems. ProLong Gold Antifade 6-diamino-2-phenylindole reagent was obtained from Invitrogen (Life Technologies). ThinCert hanging tissue culture inserts and plates were purchased from Greiner Bio-One North America.
Isolation and Culture of TCs All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of California, San Diego. Female Sprague Dawley rats (24 days old) were implanted SC with silastic implants containing 10 mg diethylstilbestrol (DES) to stimulate follicle growth, without luteinizing the follicles. Four days after DES implantation, ovarian follicles were punctured with needles to release GCs and oocytes. Oocytes were then removed from the GCs by passing the cell suspension through nylon mesh having a 40-mm pore size (27). The remaining ovary tissue was finely minced with a scalpel and digested in 100 mL per ovary of M199 medium with 0.35 mg/mL collagenase type IA, 10 ug/mL DNase, and 10 mg/mL BSA at 37 C. The tissue was digested for a total of 30 minutes and gently agitated with a Pasteur pipette every 5 minutes then centrifuged for 4 minutes at 1,000 rpm at room temperature. Media containing collagenase was aspirated, and cells were resuspended in 5 mL fresh M199. Large debris and oocytes were subsequently removed using 100- and 40-mm cell strainers, respectively. Discontinuous Percoll gradients were created according to the methods of Magoffin and Erickson (28). This method yields an enriched population of theca-interstitial cells which, in this report, are referred to as TC. Briefly, gradients were formed in 5-mL thin-wall polyallomer tubes. First, 750 mL of a 50% Percoll solution was added to the bottom of the tube, then 1.5 mL of a 36% Percoll solution followed by 2.25 mL of the cell suspension carefully layered on top. Theca cells were separated by centrifuging the gradients at 2,000 rpm for 25 minutes at 4 C in a swinging bucket rotor. Theca cells migrated to the interface between the 36% and 50% Percoll layers and were collected from the tube using a 20-gauge needle and syringe. Theca cells were then washed twice in M199 and resuspended in McCoy's 5a medium with penicillin/streptomycin. Cells were plated in serum-coated 24-well plates at 0.25 106 cells per well. For antibody VOL. 100 NO. 2 / AUGUST 2013
Fertility and Sterility® neutralization studies, TCs were plated and immediately pretreated with 0–2 mg of inhibin or b-glycan antibodies for 1 hour at 37 C. Follicle-stimulating hormone (10 ng/mL) was added, cells were cultured for 48 hours, and total RNA was analyzed by real-time reverse transcription–polymerase chain reaction (real-time RT-PCR).
Isolation of Total RNA and Real Time RT-PCR Theca cell preparations (0.25 106 cells) were cultured with increasing concentrations of FSH or hCG (1, 3, or 10 ng/mL) in serum-coated 24-well plates with 500 mL serum-free McCoy's 5a medium for 48 hours. In separate experiments, cells were cultured with either inhibin alone or FSH (10 ng/mL) and increasing concentrations of b-glycan or inhibin antibodies (0.02, 0.2, or 2 mg/mL) as mentioned. After 48 hours cell culture medium was aspirated, and total RNA was isolated from cells using the Clontech Nucleospin RNA II kit according to the manufacturer's instructions. Total RNA and purity was quantified by measuring sample absorbance at 260 and 280 nm. Samples were stored at 80 C until assayed. Primers were designed to span exon boundaries to exclude possible genomic DNA amplification on the basis of National Center for Biotechnology Information nucleotide sequences as follows: rat Cyp17 (NM_012753.1) and rat L19 (BC058135.1). Primer sequences used were as follows: Cyp17 (forward: 50 -ACAAGGCTAACGTTGACTCC-30 ) and (reverse, 50 -TGGTCAATCTCCTTTTGGATCTTC-30 ) and L19 (forward, 50 -ATGTAAAGAGCAGCCGGA AC-30 ) and (reverse, 50 -ATTGGTCTGATCCACCACAC-30 ). Relative abundance of Cyp17 mRNA was calculated by the DDCt method and normalized to L19 expression. Briefly, the DCt was calculated by subtracting the cycle threshold (Ct) of the housekeeping gene from that of the target gene. The Ct of the sample with the lowest Cyp17 gene expression was subtracted from every sample (DDCt). Last, the relative expression level was calculated using the 2-DDCt method.
Immunofluorescence Polyclonal antibodies against human cytochrome P450c17 (29) and porcine follistatin (30, 31) were generously gifted by Drs. Alan Conley and Nicholas Ling, respectively. Theca cell preparations (0.25 105) were cultured in eight-well chamber slides in McCoy's 5a medium without treatment overnight. The next morning, culture media were removed and cells were washed twice with phosphate-buffered saline (PBS). Cells were fixed for 10 minutes at 4 C in 4% paraformaldehyde, washed twice with PBS, and permeabilized in PBS with 0.1% Triton X-100 at room temperature for 30 minutes. Nonspecific binding was blocked with 2% BSA and 0.1% triton X-100 in PBS (blocking buffer) for 1 hour. Primary antibody was diluted to 1:250 in blocking buffer and applied to cells for 1 hour, followed by three washes in PBS for 10 minutes each. Cells were incubated with donkey antirabbit fluorescein isothiocyanate conjugate–conjugated antibodies (1:200) for 30 minutes at room temperature, protected from light. Three PBS washes were performed, and nuclei were stained with ProLong Gold Antifade Reagent (Invitrogen) with 6-diamino-2-phenylindole reagent (Invitrogen). VOL. 100 NO. 2 / AUGUST 2013
Evaluation of TC-specific Cyp17 and GC-specific follistatin immunostaining confirmed that these TCs preparations were predominantly TCs, with only 5% GC contamination.
Ovary Organ Cultures Ovaries from 4-day DES-treated Sprague Dawley rats were harvested and dissected to remove fat and connective tissue. Each ovary was dissected into 1-mm3 fragments using a sterile scalpel. Ovary pieces were cultured on 3-mm polyethylene terephthalate (PET) ThinCert hanging 12-well culture inserts for well plates in 1 mL McCoy's 5a medium for 24 hours with 0–10 ng/ mL FSH or hCG. Total RNA was extracted and gene expression measured by quantitative RT-PCR as mentioned above.
GC and TCs Cocultures Granulosa cells (1.5 106) and TCs (0.25 106) were cocultured in the upper and lower compartments, respectively, of the above-mentioned culture plates. Both the inserts and culture plates were prepared and serum coated according to the manufacturer's instructions before adding cells, which were cultured in 1 mL McCoy's 5a serum free medium with 0 or 10 ng/mL FSH.
Statistical Analysis Data from two or three replicate experiments were pooled for statistical analyses. Comparisons among multiple groups were analyzed by analysis of variance (ANOVA) followed by Tukey's post hoc analysis. Statistical analyses were carried out using StatPlus for Excel (AnalystSoft).
RESULTS FSH Stimulates Cyp17 mRNA Expression in Whole Follicles In order to elucidate the paracrine regulation of Cyp17 by FSH, ovary organ cultures were used to recapitulate the in vivo microenvironment of whole follicles. Fragments (1 mm3) of rat ovarian tissue were cultured for 24 hours with FSH (Fig. 1A) or hCG (Fig. 1B). Cyp17 mRNA expression was dose-dependently increased with increasing concentrations of FSH or hCG (0, 1, 3, or 10 ng/mL) for 24 hours. Cyp17 induction by hCG was approximately 40-fold compared with controls whereas induction by FSH was approximately 30-fold compared with controls at 10 ng/mL treatment. The effect of hCG, the canonical regulator of Cyp17 expression, was slightly stronger than that of FSH at inducing Cyp17 mRNA levels.
FSH Stimulates Cyp17 and Cyp19 mRNA Expression in GC and TC Cocultures To study the paracrine stimulation of Cyp17 mRNA expression, 1.5106 GCs (insert) and 0.25x106 TCs (well) were co-cultured in hanging well insert plates with FSH for up to 72 hours. In the presence of FSH, CYP19 mRNA expression in the GC compartment was increased as expected, reaching 16-fold control levels by 48 hours (Fig. 2A). Interestingly, Cyp17 mRNA expression in the TC compartment was also increased approximately 38-fold compared with untreated controls by 24 hours and more than 90-fold by 48 hours (Fig. 2B). 563
ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE
FIGURE 1
Follicle-stimulating hormone indirectly stimulates Cyp17 mRNA expression in whole follicles. Fragments of ovarian tissue were treated with increasing concentrations (0, 1, 3, or 10 ng/mL) of FSH (A) or hCG (B) for 24 hours. Cyp17 mRNA levels were measured by quantitative real-time RT-PCR and normalized to L19 mRNA levels. Results were evaluated by a one-way ANOVA followed by Tukey's post hoc analysis. Different letters indicate a significant difference (P<.05) between groups within each treatment. Values shown are mean SEM. n ¼ 6 per treatment group. Hoang. Paracrine regulation of theca Cyp17 mRNA. Fertil Steril 2013.
FIGURE 2
Follicle-stimulating hormone stimulates Cyp19 and Cyp17 mRNA expression in GC and TC cocultures. Granulosa cells (1.5 106) and TCs (0.25 106) were cocultured in the upper and lower compartments, respectively, of serum-coated 12-well hanging well insert plates for up to 72 hours with 0 or 10 ng/mL FSH. Total RNA was extracted from each compartment separately, then Cyp19 mRNA levels in GCs (A) and Cyp17 mRNA levels in TCs (B) were measured by quantitative real time RT-PCR and normalized to L19 mRNA levels. Results were evaluated by a one-way ANOVA followed by Tukey's post hoc analysis. Different letters indicate a significant difference (P<.05) between groups within each treatment. Values shown are mean SEM. n ¼ 6 per treatment group. Hoang. Paracrine regulation of theca Cyp17 mRNA. Fertil Steril 2013.
Further examination of the paracrine stimulation by GCs in TC preparations yielded intriguing results. Treatment with the FSH led to a significant increase in Cyp17 mRNA levels (Fig. 3) despite the presence of only a very small number (5%) of contaminating GCs in the TC preparations. This finding was surprising and suggests that a potent stimulator of Cyp17 mRNA expression is released from GCs only upon FSH stimulation. Together, these data show that FSH regulates Cyp17 mRNA expression in both dispersed TCs preparations and intact follicles.
Inhibin Antibody Blocks FSH Stimulation of Cyp17 mRNA Expression in TCs Elevated circulating inhibin B and androgen levels were observed in PCOS women compared with controls when treated with FSH (4). Therefore, we sought to determine 564
whether inhibin plays a role in the indirect stimulation of ovarian androgen production by FSH. Specifically, we examined the effects of an inhibin antibody on TCs in the presence of FSH. TC preparations were pre-treated with 0, 0.02, 0.2, or 2 mg of antibody for 1 hour before stimulation with 10 ng/mL FSH. After 24 hours, FSH clearly stimulated Cyp17 mRNA expression (Fig. 4A). The presence of the inhibin antibody resulted in a dose dependent suppression of FSH stimulatory actions with an 80% reduction in the presence of 2 mg of antibody. The inhibin antibody alone had a very slight stimulatory effect on Cyp17 expression at the highest concentration. Normal rabbit IgG did not affect the stimulation of Cyp17 mRNA expression by FSH, indicating a specific effect of the inhibin antibody. These results directly indicate that inhibin is involved in the FSH upregulation of Cyp17 mRNA expression in TCs. VOL. 100 NO. 2 / AUGUST 2013
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FIGURE 3
FIGURE 4
Follicle-stimulating hormone indirectly stimulates Cyp17 mRNA expression in TC cultures. Theca cells were treated with increasing concentrations (0, 1, 3, or 10 ng/mL) of FSH for 24 hours. Cyp17 mRNA levels were measured by quantitative real time RT-PCR and normalized to L19 mRNA levels. Results were evaluated by a oneway ANOVA followed by Tukey's post hoc analysis. Different letters indicate a significant difference (P<.05) between groups within each treatment. Values shown are mean SEM. n ¼ 6 per treatment group. Hoang. Paracrine regulation of theca Cyp17 mRNA. Fertil Steril 2013.
Inhibin Co-Receptor, b-glycan, Blocks FSH Stimulation of Cyp17 mRNA Expression in TCs TCs were pre-treated with 0, 0.02, 0.2, or 2 mg of b-glycan antibody for 1 hour before stimulation with 10 ng/mL FSH. After 24 hours, we observed a dose dependent effect of FSH on Cyp17 mRNA expression with maximal immunoneutralization in the presence of 2 mg of b-glycan antibody (Fig. 4B). The same amount of b-glycan antibody on its own did not affect Cyp17 mRNA expression. Furthermore, normal goat IgG did not affect the stimulation of Cyp17 mRNA expression by FSH, indicating a specific effect of the b-glycan antibody. These results suggest that inhibin acting via its co-receptor, bglycan, may be involved in the FSH upregulation of Cyp17 mRNA expression.
Inhibin Alone Does Not Stimulate Cyp17 mRNA Expression in TCs We further tested whether addition of inhibin, instead of FSH, to the TC culture is sufficient to increase Cyp17 mRNA expression. As inhibin B was not available for this study, TCs were treated with increasing concentrations of inhibin A (0, 3, 100, 300 ng/mL) for 24 hours (Fig. 4C). Cyp17 mRNA expression was stimulated by 4-fold only at a super-high concentration (300 ng/ml) of inhibin compared with untreated controls.
Involvement of inhibin in the stimulatory effect of FSH on Cyp17 mRNA expression in TC cultures. Theca cells were pretreated with increasing concentrations (0, 0.02, 0.2, or 2 mg/mL) of inhibin antibody (Inh) (A) or b-glycan antibody (BG) (B), then stimulated with FSH (0 or 10 ng/mL) for 24 hours. Theca cells were also treated with increasing concentrations of inhibin A (0, 30, 100, or 300 ng/mL) for 24 hours. Cyp17 mRNA levels were measured by quantitative real time RT-PCR and normalized to L19 mRNA levels (C). Results were evaluated by a one-way ANOVA followed by Tukey's post hoc analysis. Different letters indicate a significant difference (P<.05) between groups within each treatment. Values shown are mean SEM. n ¼ 6 per treatment group. Hoang. Paracrine regulation of theca Cyp17 mRNA. Fertil Steril 2013.
DISCUSSION According to the two-cell/two-gonadotropin theory, ovarian steroid synthesis is governed by a specific pattern of enzyme expression whereby androgens are produced by Cyp17 in TCs VOL. 100 NO. 2 / AUGUST 2013
and then subsequently aromatized in GCs. Based on findings that GCs can regulate TC androgenic pathways in a paracrine fashion in cell culture models (6, 7, 31), we sought to confirm 565
ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE that this phenomenon exists in our rat follicle model. The rationale of the use of this model is attributed to our previous in vivo finding that FSH stimulates androgen production in PCOS women (4). In this system, an active network between GCs and TCs with oocytes allows for extensive crosstalk and intercellular signaling. In this regard, a role for oocyte-secreted factors in regulating androgen production has been reported. For instance, in immortalized rat TCs, oocyte-derived GDF-9 directly upregulated basal and forskolin-induced Cyp17 mRNA expression in addition to androstenedione and progesterone secretion (32). Furthermore, in vivo GDF-9 treatment increased Cyp17 protein levels in rat ovaries (33). Because of the intact signaling networks present in whole follicles, paracrine feedback loops between the cell types may also enhance steroid production. It is likely that other oocytefactors, which have not been identified, are involved in regulating follicular androgen production. Surprisingly, the paracrine effect of GC-derived factors was present in our TC coculture model which only contained 5% GC. These data suggested the release of potent regulators of androgen synthetic enzymes from GCs upon FSH stimulation. Several members of the TGF-beta superfamily have been implicated in regulating ovarian steroidogenesis, particularly inhibin, as observed in our clinical studies as mentioned (4). Inhibin is a heterodimeric protein that has no known receptor and is thought to function as an inhibitor of activin actions. Inhibin binds to membrane bound co-receptor protein, b-glycan, to form a complex that has high affinity for activin type II receptors (34) resulting in their sequestration and inhibition of activin signaling. Expression of b-glycan has been localized to the GCs, TCs, and oocytes of rat ovaries (34, 35). Therefore, it was hypothesized that GC-derived inhibin functions as an antagonist of activin signaling in TCs by sequestering activin type II receptors, resulting in eventual stimulation of Cyp17 expression within the TC. The stimulation of Cyp17 by FSH was blocked when TCs were pre-treated with either inhibin or betaglycan antibody implicating this pathway in the paracrine regulation of Cyp17 by GCs. The lack of Cyp17 induction in the presence of inhibin alone was surprising to us since we thought the direct effect of inhibin was much more potent than FSH, which exerts an indirect effect on TC Cyp17 mRNA expression. There is little information available about the activity of inhibin B vs. inhibin A, with one study showing that inhibin A was 2fold less potent than inhibin B in suppressing FSH production in pituitary cells (37). However, the authors stated that the difference was attributed to the purity of inhibin A being slightly less than inhibin B. Therefore, it is highly unlikely that inhibin B would exhibit much greater activity than inhibin A in our culture system. Collectively, we conclude that inhibin is essential but insufficient to stimulate Cyp17 mRNA expression in TCs, thus there must be other GC factors synergistically acting with inhibin to stimulate TC Cyp17 mRNA expression. In summary, in line with our recent clinical findings that IV FSH treatment increased serum androgen levels in women with PCOS but not normal women, we show that FSH treat566
ment of rat ovarian explants and TCs increased Cyp17 mRNA levels similar to hCG treatment. Further, because increases in ovarian androgens paralleled increases in inhibin B levels in FSH-treated women with PCOS (5), we also evaluated the contribution of GC-derived inhibin on the FSHinduced stimulation of Cyp17 mRNA. Although inhibin and b-glycan antibodies dose-dependently blocked the stimulatory effect of FSH, inhibin alone only stimulated a partial increase in Cyp17 mRNA. Overall our study shows that FSH-stimulated GC-derived factors, including but not limited to inhibin, act in a paracrine manner to stimulate theca cell Cyp17 mRNA expression. Acknowledgments: The authors thank Dr. Alan Conley and Drs. Wolfgang Fischer and Wylie Vale for their gifts of the Cyp17 antibody and inhibin A; and Dr. Nicholas Ling for gifts of inhibin and follistatin antibodies.
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Otsuka F, Shimasaki S. A negative feedback system between oocyte bone morphogenetic protein 15 and granulosa cell kit ligand: its role in regulating granulosa cell mitosis. Proc Natl Acad Sci U S A 2002;99: 8060–5. Magoffin DA, Erickson GF. Purification of ovarian theca-interstitial cells by density gradient centrifugation. Endocrinology 1988;122:2345–7. Dharia S, Slane A, Jian M, Conner M, Conley AJ, Parker CRJ. Colocalization of P450c17 and cytochrome b5 in androgen-synthesizing tissues of the human. Biol Reprod 2004;71:83–8. Sugawara M, DePaolo L, Nakatani A, DiMarzo SJ, Ling N. Radioimmunoassay of follistatin: application for in vitro fertilization procedures. J Clin Endocrinol Metab 1990;71:1672–4. Nakatani A, Shimasaki S, DePaolo LV, Erickson GF, Ling N. Cyclic changes in follistatin messenger ribonucleic acid and its protein in the rat ovary during the estrous cycle. Endocrinology 1991;129:603–11. Smyth CD, Miro F, Whitelaw PF, Howles CM, Hillier SG. Ovarian thecal/interstitial androgen synthesis is enhanced by a follicle-stimulating hormone-stimulated paracrine mechanism. Endocrinology 1993;133: 1532–8. Solovyeva EV, Hayashi M, Margi K, Barkats C, Klein C, Amsterdam A, et al. Growth differentiation factor-9 stimulates rat theca-interstitial cell androgen biosynthesis. Biol Reprod 2000;63:1214–8. Vitt UA, McGee EA, Hayashi M, Hsueh AJW. In vivo treatment with GDF-9 stimulates primordial and primary follicle progression and theca cell marker CYP17 in ovaries of immature rats. Endocrinology 2000;141: 3814–20. Lewis KA, Gray PC, Blount AL, MacConell LA, Wiater E, Bilezikjian LM, et al. Betaglycan binds inhibin and can mediate functional antagonism of activin signalling. Nature 2000;404:411–4. Metzker ML. Sequencing technologies—the next generation. Nat Rev Genet 2010;11:31–46. Ling N, Ying SY, Ueno N, Esch F, Denoroy L, Guillemin R. Isolation and partial characterization of a Mr 32,000 protein with inhibin activity from porcine follicular fluid. Proc Natl Acad Sci U S A 1985;82:7217–21.
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Objective: To test whether and to what extent inhibin mediates Cyp17 messenger RNA (mRNA) expression in theca cells (TCs) in response to FSH stimulation of granulosa cells (GCs). Design: Ex vivo and in vitro experimental study. Setting: University. Animal(s): Immature female Sprague Dawley rats. Intervention(s): Ovarian tissue explants and isolated theca cell preparations with or without GCs were treated with FSH, inhibin, inhibin antibody, or b-glycan antibody. Main Outcome Measure(s): As a key enzyme in androgen production, Cyp17 mRNA levels were measured by real-time reverse transcription–polymerase chain reaction. Result(s): After 24 hours, Cyp17 mRNA expression was dose-dependently increased by FSH in ovarian tissue explants and theca cells, suggesting that paracrine factor(s) secreted from GCs in response to FSH mediates Cyp17 mRNA expression in TCs. Antibodies against inhibin and inhibin coreceptor, b-glycan, blocked the stimulatory effect of FSH on Cyp17 mRNA expression. However, inhibin alone did not increase Cyp17 mRNA level to the same extent. Conclusion(s): These findings suggest a role for inhibin in the paracrine regulation of TC Cyp17 mRNA expression by GCs influenced by FSH; however, other paracrine factors produced by GCs Use your smartphone by virtue of FSH seem to be required. (Fertil SterilÒ 2013;100:561–7. Ó2013 by American Soto scan this QR code ciety for Reproductive Medicine.) and connect to the Key Words: Theca cell, granulosa cell, inhibin, Cyp17, polycystic ovary syndrome Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/hoangyd-paracrine-regulation-theca-granulosa-cell-ovary/
T
he production of ovarian androgen is driven by increased pituitary LH secretion, which activates CYP17. In vitro studies have shown that LH-induced increments in theca cell (TC) androgen production are dose-related (1). These findings correspond to the clinical observation that in women with polycystic ovary syndrome (PCOS), elevated serum LH
levels positively correlate with circulating T concentrations (2). In addition, in women with PCOS treated with GnRH agonists, elevated LH levels are reduced or eliminated, with a corresponding reduction in circulating androgen levels (3). Thus, increased secretion of LH may be pivotal in amplifying the production of excess androgen. However, another study
Received February 1, 2013; revised March 22, 2013; accepted April 10, 2013; published online May 23, 2013. Y.D.H. has nothing to disclose. K.J.M. has nothing to disclose. R.J.C. has nothing to disclose. S.S. has nothing to disclose. This work was supported in part by National Institutes of Health (NIH) grant R01 HD41494 and by the National Institute of Child Health and Human Development/NIH through a cooperative agreement (U54 HD012303) as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research. Reprint requests: Shunichi Shimasaki, Ph.D., Department of Reproductive Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0633 (E-mail: sshimasaki@ucsd. edu). Fertility and Sterility® Vol. 100, No. 2, August 2013 0015-0282/$36.00 Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2013.04.016 VOL. 100 NO. 2 / AUGUST 2013
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looking at LH responsiveness of TCs found increased conversion of P to androgen in women with PCOS despite normal steroid responses to LH (4). Nevertheless, the action of LH on TC androgen production has not been well quantified in women with PCOS or normal women. We recently reported that in women with PCOS, IV administration of FSH resulted in significant increases in serum 17-hydroxyprogesterone, androstenedione (A), and dehydroepiandrosterone levels (5). By comparison, androgen levels in normal women remained unchanged after FSH stimulation. Interestingly, the serum androgen increase was accompanied by fivefold-elevated inhibin B levels in women with PCOS, markedly greater than the threefold change observed in 561
ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE normal women. Moreover, in women with PCOS, serum estrogen responses were also greater than those of the normal group. These findings suggested that granulosa cells (GCs) might be partially responsible for regulating TC androgen production in women with PCOS. Both in vitro and in vivo studies conducted in animals have indicated that FSH may amplify LH-induced ovarian androgen production (6–9). In cultured human TCs, A responses to LH in the presence of inhibin were clearly increased compared with those without inhibin (10, 11). In addition, inhibin was able to negate the inhibitory effect of activin on human TC androgen production. Accordingly, in our study the significant increases in ovarian androgens exhibited by women with PCOS were accompanied by similar significant increments in FSH-stimulated inhibin B levels compared with those of normal women. A growing body of evidence supports the view that GC-derived factors are potent regulators of TC androgen secretion. For instance, members of the transforming growth factor-b superfamily of proteins are well-established regulators of ovarian steroidogenesis and follicle development (12, 13). In regard to androgen production, bone morphogenetic protein-6 potently suppressed basal and LH-induced secretion in ovine (14), bovine (15), porcine (16), and human TCs (17). Transforming growth factor-b, which is expressed by GCs and TCs (16), suppressed androgen secretion from rat (18) and human TCs (19). Activin decreased whereas inhibin increased androgen secretion from human (17, 20), rat (11), and bovine (21) TCs. Futhermore, insulin-like growth factor-1 enhances the stimulatory effect of LH on CYP17 expression (22) and androgen synthesis (10, 20, 23). When combined with stem cell factor/kit ligand, insulin-like growth factor-1 increased messenger RNAs (mRNAs) of steroid acute regulatory protein, CYP11A1, CYP17, 3b-hydroxysteroid dehydrogenase, and LH receptor (24). Although expressed by both GCs and TCs, hepatocyte growth factor reduced CYP17 expression and androgen secretion from rat TCs in the presence of LH (25). Overall it seems that there is a complex network of signals between GCs and TCs that act to finetune LH and FSH regulation of ovarian steroid production. In the course of our long-term project identifying the GCderived factor(s) that mediate(s) FSH stimulation of androgen synthesis by TCs, we have focused here on the role of inhibin in the mechanism, on the basis of our clinical data described above (5). With emphasis on Cyp17 as the pivotal enzyme in TC androgen synthesis, we studied FSH effects in isolated rat ovary explants and primary theca from immature rats.
MATERIALS AND METHODS Reagents and Supplies Female Sprague Dawley rats were purchased from Charles River Laboratories. McCoy's 5a and M199 medium were purchased from Gibco (Life Technologies). Diethylstilbestrol, collagenase type IA, hCG, penicillin/streptomycin, Triton X-100, and DNase were purchased from Sigma Chemical. Silastic tubing was purchased from Dow Corning. Thin-wall polyallomer tubes were obtained from Beckman-Coulter. Ovine FSH was obtained from Dr. A. F. Parlow at the National 562
Hormone and Peptide Program of the National Institute of Diabetes and Digestive and Kidney Diseases, Harbor-UCLA Medical Center. Inhibin antiserum was kindly provided by Dr. Nicholas Ling of the Whittier Institute (La Jolla, CA), which was derived from rabbits that were immunized with an amino terminal 30-amino acid chain of porcine inhibin a-subunit coupled to bovine serum albumin (BSA) (26). Goat anti-human b-glycan/TGFBRIII sc-6199 antibody was obtained from Santa Cruz Biotechnology. Recombinant inhibin A was gifted by Drs. Wolfgang H. Fischer and Wylie Vale of the Salk Institute in La Jolla, CA. Nucleospin RNA II extraction kits were purchased from Clontech Laboratories. iQ SYBR Green Supermix qPCR reagent and iScript reverse transcriptase kits were purchased from BioRad. LabTek chamber slides were obtained from Thermo Fisher Scientific. Donkey anti-rabbit fluorescein isothiocyanate conjugate– conjugated secondary antibodies were purchased from R&D Systems. ProLong Gold Antifade 6-diamino-2-phenylindole reagent was obtained from Invitrogen (Life Technologies). ThinCert hanging tissue culture inserts and plates were purchased from Greiner Bio-One North America.
Isolation and Culture of TCs All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of California, San Diego. Female Sprague Dawley rats (24 days old) were implanted SC with silastic implants containing 10 mg diethylstilbestrol (DES) to stimulate follicle growth, without luteinizing the follicles. Four days after DES implantation, ovarian follicles were punctured with needles to release GCs and oocytes. Oocytes were then removed from the GCs by passing the cell suspension through nylon mesh having a 40-mm pore size (27). The remaining ovary tissue was finely minced with a scalpel and digested in 100 mL per ovary of M199 medium with 0.35 mg/mL collagenase type IA, 10 ug/mL DNase, and 10 mg/mL BSA at 37 C. The tissue was digested for a total of 30 minutes and gently agitated with a Pasteur pipette every 5 minutes then centrifuged for 4 minutes at 1,000 rpm at room temperature. Media containing collagenase was aspirated, and cells were resuspended in 5 mL fresh M199. Large debris and oocytes were subsequently removed using 100- and 40-mm cell strainers, respectively. Discontinuous Percoll gradients were created according to the methods of Magoffin and Erickson (28). This method yields an enriched population of theca-interstitial cells which, in this report, are referred to as TC. Briefly, gradients were formed in 5-mL thin-wall polyallomer tubes. First, 750 mL of a 50% Percoll solution was added to the bottom of the tube, then 1.5 mL of a 36% Percoll solution followed by 2.25 mL of the cell suspension carefully layered on top. Theca cells were separated by centrifuging the gradients at 2,000 rpm for 25 minutes at 4 C in a swinging bucket rotor. Theca cells migrated to the interface between the 36% and 50% Percoll layers and were collected from the tube using a 20-gauge needle and syringe. Theca cells were then washed twice in M199 and resuspended in McCoy's 5a medium with penicillin/streptomycin. Cells were plated in serum-coated 24-well plates at 0.25 106 cells per well. For antibody VOL. 100 NO. 2 / AUGUST 2013
Fertility and Sterility® neutralization studies, TCs were plated and immediately pretreated with 0–2 mg of inhibin or b-glycan antibodies for 1 hour at 37 C. Follicle-stimulating hormone (10 ng/mL) was added, cells were cultured for 48 hours, and total RNA was analyzed by real-time reverse transcription–polymerase chain reaction (real-time RT-PCR).
Isolation of Total RNA and Real Time RT-PCR Theca cell preparations (0.25 106 cells) were cultured with increasing concentrations of FSH or hCG (1, 3, or 10 ng/mL) in serum-coated 24-well plates with 500 mL serum-free McCoy's 5a medium for 48 hours. In separate experiments, cells were cultured with either inhibin alone or FSH (10 ng/mL) and increasing concentrations of b-glycan or inhibin antibodies (0.02, 0.2, or 2 mg/mL) as mentioned. After 48 hours cell culture medium was aspirated, and total RNA was isolated from cells using the Clontech Nucleospin RNA II kit according to the manufacturer's instructions. Total RNA and purity was quantified by measuring sample absorbance at 260 and 280 nm. Samples were stored at 80 C until assayed. Primers were designed to span exon boundaries to exclude possible genomic DNA amplification on the basis of National Center for Biotechnology Information nucleotide sequences as follows: rat Cyp17 (NM_012753.1) and rat L19 (BC058135.1). Primer sequences used were as follows: Cyp17 (forward: 50 -ACAAGGCTAACGTTGACTCC-30 ) and (reverse, 50 -TGGTCAATCTCCTTTTGGATCTTC-30 ) and L19 (forward, 50 -ATGTAAAGAGCAGCCGGA AC-30 ) and (reverse, 50 -ATTGGTCTGATCCACCACAC-30 ). Relative abundance of Cyp17 mRNA was calculated by the DDCt method and normalized to L19 expression. Briefly, the DCt was calculated by subtracting the cycle threshold (Ct) of the housekeeping gene from that of the target gene. The Ct of the sample with the lowest Cyp17 gene expression was subtracted from every sample (DDCt). Last, the relative expression level was calculated using the 2-DDCt method.
Immunofluorescence Polyclonal antibodies against human cytochrome P450c17 (29) and porcine follistatin (30, 31) were generously gifted by Drs. Alan Conley and Nicholas Ling, respectively. Theca cell preparations (0.25 105) were cultured in eight-well chamber slides in McCoy's 5a medium without treatment overnight. The next morning, culture media were removed and cells were washed twice with phosphate-buffered saline (PBS). Cells were fixed for 10 minutes at 4 C in 4% paraformaldehyde, washed twice with PBS, and permeabilized in PBS with 0.1% Triton X-100 at room temperature for 30 minutes. Nonspecific binding was blocked with 2% BSA and 0.1% triton X-100 in PBS (blocking buffer) for 1 hour. Primary antibody was diluted to 1:250 in blocking buffer and applied to cells for 1 hour, followed by three washes in PBS for 10 minutes each. Cells were incubated with donkey antirabbit fluorescein isothiocyanate conjugate–conjugated antibodies (1:200) for 30 minutes at room temperature, protected from light. Three PBS washes were performed, and nuclei were stained with ProLong Gold Antifade Reagent (Invitrogen) with 6-diamino-2-phenylindole reagent (Invitrogen). VOL. 100 NO. 2 / AUGUST 2013
Evaluation of TC-specific Cyp17 and GC-specific follistatin immunostaining confirmed that these TCs preparations were predominantly TCs, with only 5% GC contamination.
Ovary Organ Cultures Ovaries from 4-day DES-treated Sprague Dawley rats were harvested and dissected to remove fat and connective tissue. Each ovary was dissected into 1-mm3 fragments using a sterile scalpel. Ovary pieces were cultured on 3-mm polyethylene terephthalate (PET) ThinCert hanging 12-well culture inserts for well plates in 1 mL McCoy's 5a medium for 24 hours with 0–10 ng/ mL FSH or hCG. Total RNA was extracted and gene expression measured by quantitative RT-PCR as mentioned above.
GC and TCs Cocultures Granulosa cells (1.5 106) and TCs (0.25 106) were cocultured in the upper and lower compartments, respectively, of the above-mentioned culture plates. Both the inserts and culture plates were prepared and serum coated according to the manufacturer's instructions before adding cells, which were cultured in 1 mL McCoy's 5a serum free medium with 0 or 10 ng/mL FSH.
Statistical Analysis Data from two or three replicate experiments were pooled for statistical analyses. Comparisons among multiple groups were analyzed by analysis of variance (ANOVA) followed by Tukey's post hoc analysis. Statistical analyses were carried out using StatPlus for Excel (AnalystSoft).
RESULTS FSH Stimulates Cyp17 mRNA Expression in Whole Follicles In order to elucidate the paracrine regulation of Cyp17 by FSH, ovary organ cultures were used to recapitulate the in vivo microenvironment of whole follicles. Fragments (1 mm3) of rat ovarian tissue were cultured for 24 hours with FSH (Fig. 1A) or hCG (Fig. 1B). Cyp17 mRNA expression was dose-dependently increased with increasing concentrations of FSH or hCG (0, 1, 3, or 10 ng/mL) for 24 hours. Cyp17 induction by hCG was approximately 40-fold compared with controls whereas induction by FSH was approximately 30-fold compared with controls at 10 ng/mL treatment. The effect of hCG, the canonical regulator of Cyp17 expression, was slightly stronger than that of FSH at inducing Cyp17 mRNA levels.
FSH Stimulates Cyp17 and Cyp19 mRNA Expression in GC and TC Cocultures To study the paracrine stimulation of Cyp17 mRNA expression, 1.5106 GCs (insert) and 0.25x106 TCs (well) were co-cultured in hanging well insert plates with FSH for up to 72 hours. In the presence of FSH, CYP19 mRNA expression in the GC compartment was increased as expected, reaching 16-fold control levels by 48 hours (Fig. 2A). Interestingly, Cyp17 mRNA expression in the TC compartment was also increased approximately 38-fold compared with untreated controls by 24 hours and more than 90-fold by 48 hours (Fig. 2B). 563
ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE
FIGURE 1
Follicle-stimulating hormone indirectly stimulates Cyp17 mRNA expression in whole follicles. Fragments of ovarian tissue were treated with increasing concentrations (0, 1, 3, or 10 ng/mL) of FSH (A) or hCG (B) for 24 hours. Cyp17 mRNA levels were measured by quantitative real-time RT-PCR and normalized to L19 mRNA levels. Results were evaluated by a one-way ANOVA followed by Tukey's post hoc analysis. Different letters indicate a significant difference (P<.05) between groups within each treatment. Values shown are mean SEM. n ¼ 6 per treatment group. Hoang. Paracrine regulation of theca Cyp17 mRNA. Fertil Steril 2013.
FIGURE 2
Follicle-stimulating hormone stimulates Cyp19 and Cyp17 mRNA expression in GC and TC cocultures. Granulosa cells (1.5 106) and TCs (0.25 106) were cocultured in the upper and lower compartments, respectively, of serum-coated 12-well hanging well insert plates for up to 72 hours with 0 or 10 ng/mL FSH. Total RNA was extracted from each compartment separately, then Cyp19 mRNA levels in GCs (A) and Cyp17 mRNA levels in TCs (B) were measured by quantitative real time RT-PCR and normalized to L19 mRNA levels. Results were evaluated by a one-way ANOVA followed by Tukey's post hoc analysis. Different letters indicate a significant difference (P<.05) between groups within each treatment. Values shown are mean SEM. n ¼ 6 per treatment group. Hoang. Paracrine regulation of theca Cyp17 mRNA. Fertil Steril 2013.
Further examination of the paracrine stimulation by GCs in TC preparations yielded intriguing results. Treatment with the FSH led to a significant increase in Cyp17 mRNA levels (Fig. 3) despite the presence of only a very small number (5%) of contaminating GCs in the TC preparations. This finding was surprising and suggests that a potent stimulator of Cyp17 mRNA expression is released from GCs only upon FSH stimulation. Together, these data show that FSH regulates Cyp17 mRNA expression in both dispersed TCs preparations and intact follicles.
Inhibin Antibody Blocks FSH Stimulation of Cyp17 mRNA Expression in TCs Elevated circulating inhibin B and androgen levels were observed in PCOS women compared with controls when treated with FSH (4). Therefore, we sought to determine 564
whether inhibin plays a role in the indirect stimulation of ovarian androgen production by FSH. Specifically, we examined the effects of an inhibin antibody on TCs in the presence of FSH. TC preparations were pre-treated with 0, 0.02, 0.2, or 2 mg of antibody for 1 hour before stimulation with 10 ng/mL FSH. After 24 hours, FSH clearly stimulated Cyp17 mRNA expression (Fig. 4A). The presence of the inhibin antibody resulted in a dose dependent suppression of FSH stimulatory actions with an 80% reduction in the presence of 2 mg of antibody. The inhibin antibody alone had a very slight stimulatory effect on Cyp17 expression at the highest concentration. Normal rabbit IgG did not affect the stimulation of Cyp17 mRNA expression by FSH, indicating a specific effect of the inhibin antibody. These results directly indicate that inhibin is involved in the FSH upregulation of Cyp17 mRNA expression in TCs. VOL. 100 NO. 2 / AUGUST 2013
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FIGURE 3
FIGURE 4
Follicle-stimulating hormone indirectly stimulates Cyp17 mRNA expression in TC cultures. Theca cells were treated with increasing concentrations (0, 1, 3, or 10 ng/mL) of FSH for 24 hours. Cyp17 mRNA levels were measured by quantitative real time RT-PCR and normalized to L19 mRNA levels. Results were evaluated by a oneway ANOVA followed by Tukey's post hoc analysis. Different letters indicate a significant difference (P<.05) between groups within each treatment. Values shown are mean SEM. n ¼ 6 per treatment group. Hoang. Paracrine regulation of theca Cyp17 mRNA. Fertil Steril 2013.
Inhibin Co-Receptor, b-glycan, Blocks FSH Stimulation of Cyp17 mRNA Expression in TCs TCs were pre-treated with 0, 0.02, 0.2, or 2 mg of b-glycan antibody for 1 hour before stimulation with 10 ng/mL FSH. After 24 hours, we observed a dose dependent effect of FSH on Cyp17 mRNA expression with maximal immunoneutralization in the presence of 2 mg of b-glycan antibody (Fig. 4B). The same amount of b-glycan antibody on its own did not affect Cyp17 mRNA expression. Furthermore, normal goat IgG did not affect the stimulation of Cyp17 mRNA expression by FSH, indicating a specific effect of the b-glycan antibody. These results suggest that inhibin acting via its co-receptor, bglycan, may be involved in the FSH upregulation of Cyp17 mRNA expression.
Inhibin Alone Does Not Stimulate Cyp17 mRNA Expression in TCs We further tested whether addition of inhibin, instead of FSH, to the TC culture is sufficient to increase Cyp17 mRNA expression. As inhibin B was not available for this study, TCs were treated with increasing concentrations of inhibin A (0, 3, 100, 300 ng/mL) for 24 hours (Fig. 4C). Cyp17 mRNA expression was stimulated by 4-fold only at a super-high concentration (300 ng/ml) of inhibin compared with untreated controls.
Involvement of inhibin in the stimulatory effect of FSH on Cyp17 mRNA expression in TC cultures. Theca cells were pretreated with increasing concentrations (0, 0.02, 0.2, or 2 mg/mL) of inhibin antibody (Inh) (A) or b-glycan antibody (BG) (B), then stimulated with FSH (0 or 10 ng/mL) for 24 hours. Theca cells were also treated with increasing concentrations of inhibin A (0, 30, 100, or 300 ng/mL) for 24 hours. Cyp17 mRNA levels were measured by quantitative real time RT-PCR and normalized to L19 mRNA levels (C). Results were evaluated by a one-way ANOVA followed by Tukey's post hoc analysis. Different letters indicate a significant difference (P<.05) between groups within each treatment. Values shown are mean SEM. n ¼ 6 per treatment group. Hoang. Paracrine regulation of theca Cyp17 mRNA. Fertil Steril 2013.
DISCUSSION According to the two-cell/two-gonadotropin theory, ovarian steroid synthesis is governed by a specific pattern of enzyme expression whereby androgens are produced by Cyp17 in TCs VOL. 100 NO. 2 / AUGUST 2013
and then subsequently aromatized in GCs. Based on findings that GCs can regulate TC androgenic pathways in a paracrine fashion in cell culture models (6, 7, 31), we sought to confirm 565
ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE that this phenomenon exists in our rat follicle model. The rationale of the use of this model is attributed to our previous in vivo finding that FSH stimulates androgen production in PCOS women (4). In this system, an active network between GCs and TCs with oocytes allows for extensive crosstalk and intercellular signaling. In this regard, a role for oocyte-secreted factors in regulating androgen production has been reported. For instance, in immortalized rat TCs, oocyte-derived GDF-9 directly upregulated basal and forskolin-induced Cyp17 mRNA expression in addition to androstenedione and progesterone secretion (32). Furthermore, in vivo GDF-9 treatment increased Cyp17 protein levels in rat ovaries (33). Because of the intact signaling networks present in whole follicles, paracrine feedback loops between the cell types may also enhance steroid production. It is likely that other oocytefactors, which have not been identified, are involved in regulating follicular androgen production. Surprisingly, the paracrine effect of GC-derived factors was present in our TC coculture model which only contained 5% GC. These data suggested the release of potent regulators of androgen synthetic enzymes from GCs upon FSH stimulation. Several members of the TGF-beta superfamily have been implicated in regulating ovarian steroidogenesis, particularly inhibin, as observed in our clinical studies as mentioned (4). Inhibin is a heterodimeric protein that has no known receptor and is thought to function as an inhibitor of activin actions. Inhibin binds to membrane bound co-receptor protein, b-glycan, to form a complex that has high affinity for activin type II receptors (34) resulting in their sequestration and inhibition of activin signaling. Expression of b-glycan has been localized to the GCs, TCs, and oocytes of rat ovaries (34, 35). Therefore, it was hypothesized that GC-derived inhibin functions as an antagonist of activin signaling in TCs by sequestering activin type II receptors, resulting in eventual stimulation of Cyp17 expression within the TC. The stimulation of Cyp17 by FSH was blocked when TCs were pre-treated with either inhibin or betaglycan antibody implicating this pathway in the paracrine regulation of Cyp17 by GCs. The lack of Cyp17 induction in the presence of inhibin alone was surprising to us since we thought the direct effect of inhibin was much more potent than FSH, which exerts an indirect effect on TC Cyp17 mRNA expression. There is little information available about the activity of inhibin B vs. inhibin A, with one study showing that inhibin A was 2fold less potent than inhibin B in suppressing FSH production in pituitary cells (37). However, the authors stated that the difference was attributed to the purity of inhibin A being slightly less than inhibin B. Therefore, it is highly unlikely that inhibin B would exhibit much greater activity than inhibin A in our culture system. Collectively, we conclude that inhibin is essential but insufficient to stimulate Cyp17 mRNA expression in TCs, thus there must be other GC factors synergistically acting with inhibin to stimulate TC Cyp17 mRNA expression. In summary, in line with our recent clinical findings that IV FSH treatment increased serum androgen levels in women with PCOS but not normal women, we show that FSH treat566
ment of rat ovarian explants and TCs increased Cyp17 mRNA levels similar to hCG treatment. Further, because increases in ovarian androgens paralleled increases in inhibin B levels in FSH-treated women with PCOS (5), we also evaluated the contribution of GC-derived inhibin on the FSHinduced stimulation of Cyp17 mRNA. Although inhibin and b-glycan antibodies dose-dependently blocked the stimulatory effect of FSH, inhibin alone only stimulated a partial increase in Cyp17 mRNA. Overall our study shows that FSH-stimulated GC-derived factors, including but not limited to inhibin, act in a paracrine manner to stimulate theca cell Cyp17 mRNA expression. Acknowledgments: The authors thank Dr. Alan Conley and Drs. Wolfgang Fischer and Wylie Vale for their gifts of the Cyp17 antibody and inhibin A; and Dr. Nicholas Ling for gifts of inhibin and follistatin antibodies.
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