Growth Differentiation Factor-9 Stimulates Inhibin Production and Activates Smad2 in Cultured Rat Granulosa Cells
Abstractd!p, http://www.100md.com
Ovarian inhibin production is stimulated by FSH and several TGFß family ligands including activins and bone morphogenetic proteins. Growth differentiation factor-9 (GDF-9) derived by the oocyte is a member of the TGFß/activin family, and we have previously shown that GDF-9 treatment stimulates ovarian inhibin-{alpha} content in explants of neonatal ovaries. However, little is known about GDF-9 regulation of inhibin production in granulosa cells and downstream signaling proteins activated by GDF-9. Here, we used cultured rat granulosa cells to examine the influence of GDF-9 on basal and FSH-stimulated inhibin production, expression of inhibin subunit transcripts, and the GDF-9 activation of Smad phosphorylation. Granulosa cells from small antral follicles of diethylstilbestrol-primed immature rats were cultured with FSH in the presence or absence of increasing concentrations of GDF-9. Secreted dimeric inhibin A and inhibin B were quantified using specific ELISAs, whereas inhibin subunit RNAs were analyzed by Northern blotting using 32P-labeled inhibin subunit cDNA probes. Similar to FSH, treatment with GDF-9 stimulated dose- and time-dependent increases of both inhibin A and inhibin B production. Furthermore, coincubation of cells with GDF-9 and FSH led to a synergistic stimulation of both inhibin A and inhibin B production. GDF-9 treatment also increased mRNA expression for inhibin-{alpha} and inhibin-ß subunits. To investigate Smad activation, granulosa cell lysates were analyzed in immunoblots using antiphosphoSmad1 and antiphosphoSmad2 antibodies. GDF-9 treatment increased Smad2, but not Smad1, phosphorylation with increasing doses of GDF-9 leading to a dose-dependent increase in phosphoSmad2 levels. To further investigate inhibin-{alpha} gene promoter activation by GDF-9, granulosa cells were transiently transfected with an inhibin-{alpha} promoter-luciferase reporter construct and cultured with different hormones before assaying for luciferase activity. Treatment with FSH or GDF-9 resulted in increased inhibin-{alpha} gene promoter activity, and combined treatment with both led to synergistic increases. The present data demonstrate that oocyte-derived GDF-9, alone or together with pituitary-derived FSH, stimulates inhibin production, inhibin subunit mRNA expression, and inhibin-{alpha} promoter activity by rat granulosa cells. The synergistic stimulation of inhibin secretion by the paracrine hormone GDF-9 and the endocrine hormone FSH could play an important role in the feedback regulation of FSH release, thus leading to the modulation of follicle maturation and ovulation.
Introductionmm7:n, 百拇医药
DEVELOPMENT OF THE mammalian ovary is characterized by the initial endowment of a fixed number of primordial follicles. The pool of primordial follicles is gradually depleted during reproductive life. It is well accepted that follicular growth and differentiation are controlled by pituitary gonadotropins as well as follicular paracrine factors of granulosa and theca cell origin (1, 2, 3); many studies have indicated that the oocyte contributes to follicular development as well.mm7:n, 百拇医药
Growth differentiation factor-9 (GDF-9) belongs to the TGFß superfamily and is expressed exclusively in the oocyte (4). The essential role of GDF-9 in folliculogenesis was demonstrated following targeted deletion of the GDF-9 gene in mice (5). Our previous results demonstrated that treatment with GDF-9 induces preantral follicle growth (6) and granulosa cell proliferation but inhibits gonadotropin-induced granulosa cell differentiation in vitro (7).
TGFß and bone morphogenetic proteins (BMPs) signal through type I and type II serine/threonine kinase receptors by activating intracellular Smad family proteins. Based on our studies using the ectodomains of putative receptors for GDF-9, it appears that GDF-9 also interacts with receptors of the TGFß and BMP receptor family, and BMPRII is one of the receptors for GDF-9 (8). Inhibins produced by granulosa cells form a negative feedback loop between the anterior pituitary and the ovary and are important in the regulation of FSH secretion (9). Because our earlier data indicated that GDF-9 treatment increases the inhibin-{alpha} content in ovarian explant cultures (6), we tested whether treatment with recombinant GDF-9 can promote inhibin production in cultured granulosa cells. We further assessed if Smad1 or Smad2 could be phosphorylated by treatment with GDF-9 in granulosa cells to elucidate the signaling pathway of GDF-9 on inhibin production. Based on the inhibin-{alpha} promoter reporter gene system (10), we also tested whether GDF-9 directly stimulates inhibin-{alpha} promoter activity in transfected granulosa cells.
Materials and Methodskdy, 百拇医药
Animalskdy, 百拇医药
Immature female Sprague Dawley rats were obtained from Simonsen Laboratories (Gilroy, CA). Animals (25 d old, body weight from 55–65 g) were anesthesized and killed using CO2 at 72 h after insertion of diethylstilbestrol implants (11). All animals were housed under controlled humidity, temperature, and light regimen, and fed ad libitum on standard rat chow. Animal care was consistent with institutional and NIH guidelines.kdy, 百拇医药
Reagents and hormoneskdy, 百拇医药
McCoy’s 5a medium (modified) and L-15 Leibovitz medium were obtained from Life Technologies, Inc. (Santa Clara, CA). Recombinant human FSH (Org 32489E) was from NV Organon (Oss, The Netherlands). L-glutamine, penicillin, and streptomycin were purchased from BioWhittaker, Inc. (Wakersville, MD). Recombinant activin A and BMP-2 were from R&D Systems (Minneapolis, MN).kdy, 百拇医药
Recombinant GDF-9 was generated in transfected mammalian cells and characterized as previously described (6). Briefly, expression vectors for wild-type and epitope-tagged GDF-9 were constructed using pcDNA3.1 Zeo (Invitrogen, San Diego, CA). N-terminal-tagged GDF-9, encoding a Flag epitope for the M1 antibody followed by six histidine residues fused to the amino-terminus of mature GDF-9, showed no bioactivity and served as a negative control. Human embryonic kidney 293T cells were transfected with the expression vector, and clonal cell lines stably expressing wild-type and tagged GDF-9 were selected under 1 mg/ml of Zeocin (Invitrogen). Conditioned media were harvested after 4 d of serum-free culture. Quantitation of N-tagged GDF-9 was done after purification with nickel column and measurement of protein content using the Micro BCA protein assay kit (Perstorp Life Science, Rockford, IL). Purified N-tagged GDF-9 was then used as a standard for the quantitation of wild-type GDF-9 by immunoblots using specific GDF-9 antibodies.
Preparation and culture of granulosa cells\};, http://www.100md.com
Granulosa cells were obtained from small antral follicles of estrogen-treated immature rats. Ovaries were punctured in L-15 Leibovitz medium. Ovarian debris was removed, and the remaining medium containing granulosa cells was collected after low-speed centrifugation at 500 x g for 10 min. Granulosa cells were dispersed by repeated washing and suspended into the culture medium (McCoy’s 5a supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin).\};, http://www.100md.com
Assessment of inhibin A and inhibin B production\};, http://www.100md.com
Granulosa cells (1 x 105 viable cells/ml) were cultured in 24-well plates (Corning, Inc., Corning, NY) in the presence or absence of increasing concentrations of GDF-9, or the inactive N-tagged GDF-9, with or without FSH. Conditioned media were harvested after 24, 48, and 72 h and stored at -80 C until measurement of inhibin content. The amounts of dimeric inhibin A and inhibin B were quantified using specific ELISAs (Serotec, Oxford, UK). To achieve concentrations within the assay range, the spent culture media from each experiment were diluted and serial dilutions of spent culture media were found to be linear with inhibin A and inhibin B standards.
Northern blot hybridization:?4g, http://www.100md.com
For RNA blot analysis, granulosa cells were harvested 48 h after treatment with FSH or GDF-9, and total RNA was isolated using an RNeasy extraction kit (QIAGEN Inc., Valencia, CA). RNA was quantitated by absorbance measurement at 260 nm. Eight micrograms of total RNA were size-fractionated in 1.5% agarose gels before transfer to Hybond-N nylon membranes (Amersham Pharmacia Biotech, Little Chalfont, UK). For the detection of inhibin-{alpha} , ßA-, and ßB-subunit mRNAs by Northern blot hybridization, rat cDNA probes of the respective inhibin subunits were prepared as previously described (12, 13). Levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA were used as a loading control. Northern blot hybridizations were performed overnight at 42 C, and the filters were washed three times for 20 min with 0.1–1x saline sodium citrate/1% sodium dodecyl sulfate at 50 C. Hybridized filters were exposed at 23 C overnight to Fujifilm phosphoimaging plates before analysis using a Fujifilm IP-Reader Bio-Imaging Analyzer BAS 1500 (Fuji Photo Film Co., Ltd., Tokyo, Japan). The signals of the three different inhibin subunits were quantified and normalized to the expression levels of GAPDH.
Western blotting analysis of Smad proteinso, 百拇医药
To investigate Smad activation by GDF-9, granulosa cells (2 x 106 viable cells/ml) were cultured for 2 h. Media were changed to remove nonattached cells and replaced by fresh media containing different hormones. Granulosa cells were harvested at 30, 60, and 90 min after culture and washed once on ice with cold PBS before lysis in the Laemmli buffer containing ß-mercaptoethanol. Cells were gently sonicated on ice for 15 sec with a MSE sonicator (Sanyo Corp., Osaka, Japan) and boiled for 3 min. Proteins were separated on 8% SDS-PAGE gels and electroblotted onto Amersham Hybond-electrochemiluminescence (for Smad2 experiments) and Hybond-P membranes (for Smad1 experiments) (Amersham Pharmacia Biotech, Arlington Heights, IL). For the detection of Smad1 and Smad2 phosphorylated at the carboxyl terminus, membranes were blocked for 1 h at room temperature in Tris-buffered saline-0.1% Tween containing 5% fat-free dry milk, after which membranes were incubated with an antiphosphoSmad2 antibody diluted at 1:8000 at 4 C overnight or with an antiphosphoSmad1 antibody diluted at 1:8000 at 4 C overnight (14). The secondary antibodies were used following manufacturer’s instructions (Roche Molecular Biochemicals, Indianapolis, IN). Immunoreactive proteins were detected using enhanced chemiluminescence (ECL kit, Amersham Pharmacia Biotech).
Transfection of inhibin-{alpha} promoter-luciferase reporter construct into granulosa cells\, 百拇医药
Granulosa cells (5 x 105 viable cells/well) were cultured in culture medium supplemented with 10% FBS for 2 h. Before transfection, medium was changed to serum-free medium and cells were cotransfected with 2.5 µg of inhibin-{alpha} promoter luciferase plasmid and 0.1 µg of p-Rous sarcoma virus (RSV)-ß-galactosidase (gal) for 4 h at 37 C using lipofectamine (Life Technologies, Inc., Gaithersburg, MD). The pRSV-ß-gal vector containing the lacZ gene encoding ß-gal driven by the RSV long terminal repeat was used to monitor transfection efficiency (15). Cells were then cultured for 24 h in the presence or absence of GDF-9 with or without FSH in media containing 1% FBS.\, 百拇医药
To harvest cells, lysis buffer (200 µl) (Promega Corp., Madison, WI) was added into each well and 30 µl of the supernatant was used for luciferase determination using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). Fifty microliters of the cell lysate were also used to measure ß-gal activity (16). The activity of the inhibin-{alpha} promoter is expressed as the ratio of relative light unit/ß-gal activity.
Data analysis!^ogp, 百拇医药
All experimental data are presented as the mean ± SEM of duplicate measurements of triplicate cultures and each experiment was repeated at least three times. Statistical significance was determined by Student’s paired t test or ANOVA for multiple group comparisons and by Duncan’s multiple range test for effects of multiple doses and treatments. Significance was accepted at P < 0.05.!^ogp, 百拇医药
Results!^ogp, 百拇医药
Effect of GDF-9 treatment on basal and FSH-stimulated inhibin A and inhibin B production!^ogp, 百拇医药
Using a serum-free culture of granulosa cells, we tested the effect of recombinant GDF-9 on inhibin production. As shown in Fig. 1, treatment with recombinant GDF-9 (3–150 ng/ml) for 2 d increased inhibin A and inhibin B production in a dose-dependent manner as compared with the control (Ct) group (P < 0.01). The maximum increases in inhibin A (3.2-fold) and inhibin B (10-fold) production were achieved at 150 ng/ml of GDF-9. FSH at a dose of 3 ng/ml also increased inhibin A and inhibin B production by 5- and 2-fold, respectively. Furthermore, cotreatment with 3 ng/ml FSH and increasing doses of GDF-9 led to synergistic increases in inhibin production as compared with the group treated with FSH alone. Compared with FSH alone, concentrations of GDF-9 at 3–150 ng/ml augmented FSH action by 6-fold for inhibin A and by 20-fold for inhibin B (P < 0.05). Consistent with earlier studies (6), treatment with N-tagged GDF-9 did not augment FSH action, demonstrating the specific action of GDF-9.
fig.ommitteedw!, 百拇医药
Figure 1. Effect of treatment with GDF-9 on basal and FSH-stimulated inhibin A (A) and inhibin B (B) production by cultured granulosa cells. Granulosa cells (2 x 105 cells) were cultured for 48 h in serum-free McCoy’s 5a medium in the presence or absence of increasing doses of GDF-9 with or without 3 ng/ml FSH. Some cells were treated with FSH and 150 ng/ml of N-tagged GDF-9 (NG). Medium inhibin concentration was measured by two-site enzyme-linked immunoassay. Similar data were obtained from two additional experiments. Each pointrepresents mean ± SEM of triplicate wells. Ct, Control.w!, 百拇医药
To study the time course of the GDF-9 stimulation of inhibin production, granulosa cells were cultured for up to 3 d with or without FSH, GDF-9, or a combination of both. Treatment with FSH and GDF-9 stimulated inhibin production with synergistic increases observed at 48 h after cotreatment. At 72 h after treatment with FSH and GDF-9, 5.2- and 11.5-fold increases above FSH treatment alone were found for inhibin A and inhibin B, respectively. In contrast, treatment with N-tagged GDF-9 did not increase basal or FSH-stimulated inhibin A or inhibin B production at any time point tested (Fig. 2).
fig.ommitteedh7/tv's, 百拇医药
Figure 2. Time course of GDF-9 action on inhibin A (A) and inhibin B (B) production by cultured granulosa cells. Granulosa cells (2 x 105 cells/well) were incubated for the times indicated with medium alone (control), FSH (3 ng/ml), or FSH plus different concentrations of GDF-9 (3–150 ng/ml). At the end of the incubation, inhibin concentration in the media was assayed by two-site ELISA. Some cells were treated with 150 ng/ml of N-tagged GDF-9 (NG) with or without FSH (3 ng/ml). Similar data were obtained from two additional experiments. The results are expressed as mean ± SEM of triplicate wells.h7/tv's, 百拇医药
Effect of GDF-9 treatment on basal and FSH-stimulated inhibin subunit transcript levelsh7/tv's, 百拇医药
Our previous data showed that maximal stimulation of inhibin subunit transcript levels occurred at 48 h after FSH treatment (17). Total RNA from granulosa cells cultured for 48 h in the presence of GDF-9 (150 ng/ml) with or without FSH (3 ng/ml) was fractionated on denaturing agarose gels, and Northern blot analysis was performed with inhibin -, ßA-, and ßB-subunit cDNA probes. As shown in Fig. 3 (left panel), hybridization with the -subunit probe resulted in a major hybridization signal at about 1.5 kb, consistent with previous reports (18). Hybridization with the inhibin ßA-subunit probe resulted in a predominant hybridization signal of 6.9 kb (Fig. 3, middle panel). In contrast, two ßB-subunit mRNA species (~ 4.4 kb and ~ 3.3 kb) were detected (Fig. 3, right panel). In cultures treated with GDF-9 alone, levels of inhibin {alpha} , ßA-, and ßB-subunit mRNA, normalized based on GAPDH message levels, were elevated approximately 5.7-, 4-, and 2.3-fold, respectively. Combined treatment with FSH and GDF-9 led to an additive effect on the level of inhibin subunit mRNAs. These results demonstrated a stimulatory effect of GDF-9 in regulating inhibin subunit gene expression.
fig.ommitteedp, 百拇医药
Figure 3. Northern blot analyses of the effects of GDF-9 and/or FSH on inhibin subunit mRNA levels in cultured granulosa cells. Cells were cultured for 48 h with 150 ng/ml GDF-9 and/or 3 ng/ml FSH. Total RNA was extracted and analyzed by Northern blotting using 32P-labeled inhibin subunit cDNA probes. Arrowheads indicate the migration position of specific bands. Results were normalized for GAPDH mRNA levels. Ct, Control.p, 百拇医药
GDF-9 stimulation of Smad2 phosphorylationp, 百拇医药
Lysates from cultured granulosa cells were analyzed in immunoblots using antiphosphoSmad1 and antiphosphoSmad2 antibodies. An immunoreactive band (52 kDa) corresponding to phosphorylated Smad2 was evident (Fig. 4A). In addition, a nonspecific band around 60 kDa was found to be invariant for all treatment groups and served as a loading control. As shown in Fig. 4A, treatment with GDF-9 led to a dose-dependent increase in Smad2 phosphorylation. In contrast, treatment with FSH alone was ineffective. Also, combined treatment of FSH and GDF-9 did not have any influence on the level of Smad2 phosphorylation stimulated by GDF-9. In contrast, treatment with activin increased Smad2 phosphorylation. The stimulatory effect of GDF-9 on Smad2 phosphorylation was time dependent and evident from 30 min post stimulation with the highest level at 90 min (Fig. 4B). Smad2 phosphorylation by activin was also time dependent. Phosphorylation of Smad1 was analyzed in the same experiments using an antibody that recognizes phosphorylated Smad1, 5, and 8. As shown in Fig. 4C, treatment with BMP-2, but not with GDF-9, increased signals at 52 kDa, consistent with the molecular mass of phosphoSmad1.
fig.ommitteed&7, http://www.100md.com
Figure 4. Treatment with GDF-9 activates Smad2, but not BMP-activated Smad1, in cultured granulosa cells. A, Dose-dependent stimulation of Smad2 by GDF-9. Immunoblot analysis of Smad2 phosphorylation was performed using cell extracts following treatment with GDF-9 (30, 100, 150 ng/ml), FSH (3 ng/ml), and activin (50 ng/ml). B, Time-dependent stimulation of Smad2 by GDF-9 (150 ng/ml). C, Lack of induction of Smad1 phosphorylation by GDF-9 in cultured granulosa cells. BMP2 (50 ng/ml), GDF-9 (150 ng/ml). Arrowheads indicate the immunoreactive band of phosphorylated Smad. Ct, Control.&7, http://www.100md.com
GDF-9 stimulation of inhibin-{alpha} promoter activity&7, http://www.100md.com
To elucidate the mechanisms by which GDF-9 regulates inhibin-{alpha} gene expression, we constructed a fusion gene that contained 679 bp of the mouse inhibin-{alpha} gene promoter region linked to a luciferase reporter gene. This construct includes a single potential CRE at –122 bp and three AP2 sites at –202 bp, –176 bp, and –149 bp, respectively (15, 19). The promoter construct was transiently transfected into primary cultures of granulosa cells. To estimate the inhibin-{alpha} promoter regulation by hormones, we measured luciferase activity after the addition of GDF-9 and/or FSH to the culture medium. As shown in Fig. 5, treatment with GDF-9 and FSH increased inhibin-{alpha} gene promoter activity by 1.9- and 2.1-fold, respectively. Furthermore, combined treatment with GDF-9 and FSH increased reporter gene expression significantly greater than that seen with GDF-9 or FSH treatment alone (3.4-fold of the control level).
fig.ommitteed@lq, 百拇医药
Figure 5. GDF-9 stimulation of the inhibin-{alpha} promoter activity in transfected granulosa cells. Cells were transiently transfected with the inhibin-{alpha} promoter luciferase reporter construct and cultured for 24 h after the addition of GDF-9 (150 ng/ml), FSH (3 ng/ml), or both. Cell lysates were assayed for the activity of the luciferase reporter gene. Luciferase activity is presented as relative light units (RLU) and normalized based on ß-gal activity in cotransfected cells. Mean ± SEM. Ct, Control.@lq, 百拇医药
Discussion@lq, 百拇医药
The present data demonstrate that oocyte-derived GDF-9, alone and together with pituitary-derived FSH, stimulates dimeric inhibin production, inhibin subunit mRNA expression, and inhibin-{alpha} promoter activity in rat granulosa cells. We also present evidence that GDF-9 stimulates the phosphorylation of endogenous Smad2 but not Smad1 proteins in these cells, suggesting that GDF-9 mediates its cellular effects through a pathway similar to that used by activins and TGFß but not the one employed by BMPs.
The synthesis and secretion of inhibin and activin dimers in the ovary are dependent on the regulation of three known inhibin/activin subunits that are controlled not only by endocrine hormones, but also by local factors. The role of pituitary gonadotropins in the transcriptional regulation of the inhibin and activin subunit genes in granulosa cells has been well established both in vitro and in vivo (20), and the cAMP-protein kinase A pathway is mediating the gonadotropin regulation of the inhibin and activin subunit genes (20). A role for oocyte-derived factors in regulating granulosa cell inhibin production was recently proposed (21). Coculture of granulosa cells with meiotically immature oocytes was associated with a predominant stimulation of inhibin B (21), resembling the effects of GDF-9 in the present study (Fig. 1). Oocyte-derived GDF-9 acts synergistically with FSH to stimulate inhibin production, despite the inhibitory effects of GDF-9 on FSH-induced estrogen production and LH receptor formation (22, 23). Because GDF-9 null mice showed a high expression of inhibin-{alpha} message in follicles with one layer of granulosa cells (24), the GDF-9 regulation of inhibin production could be follicle stage dependent.
Although GDF-9 and FSH synergistically stimulated inhibin dimer production, no synergistic effects were seen at the mRNA level. It is possible that this discrepancy is due to additional posttranscriptional actions of GDF-9 on inhibin biosynthesis and/or release, mediated by cross-talk between the cAMP and Smad signaling pathways. The present results suggest that GDF-9 increases the sensitivity of granulosa cells to FSH, in addition to stimulating inhibin production in its own right. Because inhibin is important in the regulation of FSH secretion by the anterior pituitary, changes in granulosa cell inhibin production regulated by GDF-9 could alter antral follicle recruitment during reproductive cycles, leading to changes in ovulatory efficiency.9t-a{7z, 百拇医药
TGFß family of growth factors signal by binding to cell-surface serine kinase receptors which then activate cytoplasmic Smads and MAPKs in response to ligand binding (25, 26). Several different TGFß family members, activin A, TGFß, and BMP are capable of eliciting biological responses in cultured granulosa cells (17, 18, 27, 28, 29, 30, 31, 32), and the presence of all seven type I and five type II TGFß and BMP receptors have previously been reported (29, 30, 31, 32, 33). Also, the expression of several Smad signaling proteins in the mammalian ovary has recently been shown (32, 34, 35, 36). Although our recent data suggest that GDF-9 binds directly to BMPRII (8), the putative type I receptors mediating GDF-9 action have not been described.
For TGFß/BMP signaling, the formation of tetrameric receptor complexes containing two type I and two type II receptors allows the phosphorylation of the type I receptor by the type II receptor on the GS domain resulting in activation of the type I receptor kinase. Type I receptors specifically recognize and phosphorylate R-Smads (receptor-activated Smads). Following activation, R-Smads associate with a common Co-Smad, Smad-4. Both the R-Smad and Co-Smad in the complex may participate in DNA binding on Smad binding elements (SBE) of target genes and allow the recruitment of transcriptional cofactors. Previously, it has been shown that two distinct Smad signaling pathways, the activin/TGFß-activated pathway and the BMP-activated pathway, are important to mediate the cellular effects of most TGFß family members (25). The R-Smads, Smad2 and Smad3, are activated by TGFß or activin type I receptors, whereas Smad1, Smad5, and Smad8 are activated by BMP type I receptors. We have previously identified BMPRII as a type II receptor for GDF-9 (8). Due to the sharing of type II and possibly type I receptors between BMP and GDF-9 ligands, GDF-9 could activate the BMP receptor-specific Smad1, 5, and 8 proteins because BMPRII previously had been shown to couple only to type I BMP receptors. However, somewhat surprisingly, the present study shows that GDF-9 activated Smad2, but not Smad1, 5 or 8, suggesting that GDF-9 resembles activins and TGFß, but not BMPS, in its action. These data further suggest that type I receptors activated by GDF-9 could confer stimulation of Smad2 phosphorylation using a pathway different from BMPs. It is unclear whether the known type I receptors previously shown to activate the Smad2 pathway are one of the GDF-9 receptors in granulosa cells.
Inhibin gene expression in the ovary is stimulated by FSH, which uses cAMP as an intracellular second messenger. FSH markedly stimulates inhibin mRNA levels in cultured rat granulosa cells (34, 37). The effects of FSH are mimicked by forskolin, a pharmacological agent that activates adenylyl cyclase (35, 38). In contrast, inhibin production augmented by GDF-9 was not associated with a change in intracellular or extracellular cAMP accumulation (22). We have shown previously that cotreatment of granulosa cells with FSH plus activin resulted in a 2-fold increase in cAMP levels, demonstrating a potentiation of FSH-induced cAMP production by activin, even though treatment with activin alone had no effect (17). The inhibin-{alpha} promoter region contains several potential cAMP response elements and transcription factor AP2-binding sites that might mediate cAMP regulation. We determined here whether GDF-9 could activate the transcription of an inhibin-{alpha} promoter reporter construct known to be activated by FSH and cAMP (39). We observed that GDF-9 on its own modestly activates transcription of this construct, and treatment with FSH and GDF-9 have an additive effect. Interestingly, a potential SBE sequence, CAGACA (19, 40), was identified upstream of the AP1 site of the inhibin-{alpha} promoter. Therefore, the observed GDF-9 stimulation of Smad2 phosphorylation and inhibin {alpha} -promoter activity suggests that this GDF-9 effect might be mediated by the SBE found in the inhibin {alpha} -promoter.
It is becoming clear that the GDF-9 and GDF-9B signaling pathways play an essential role in early follicle development. BMP-15/GDF-9B, similar to GDF-9, is expressed in the oocyte (41, 42, 43) and stimulates granulosa cell proliferation and modulates granulosa cell differentiation (44). The present study on the GDF-9 stimulation of inhibin production by granulosa cells could provide clues on the twinning phenotype associated with the heterozygous GDF-9B gene mutation in Inverdale sheep (45). In sheep with the homozygous GDF-9B gene mutation (XI/XI), defective secondary follicle development was detected. Paradoxically, an increased ovulation rate was found in heterozygotes (XI/X+) (45). Although the original paper did not discuss the mechanism underlying these diametrically opposing phenotypes, our data on GDF-9 stimulation of inhibin A and inhibin B production provides a likely explanation. Release of FSH is controlled by the feedback of inhibins from the ovary. It is possible that a decreased production of GDF-9/GDF-9B dimers by these antral follicles could lead to lower inhibin production with a resultant increase in FSH secretion by the anterior pituitary.
This study demonstrates that GDF-9 enhances FSH-stimulated inhibin production and inhibin subunit gene expression, and activates the Smad2 pathway in rat granulosa cells. These findings emphasize the ability of this oocyte factor to modulate the effectiveness of the gonadotropins on granulosa cell function. More information is needed regarding the specific type I receptors for GDF-9 and its interaction with other hormonal signaling systems operating in the ovarian follicle.xw:, 百拇医药
Acknowledgmentsxw:, 百拇医药
We thank Caren Spencer for editorial assistance and Ms. Marjo Rissanen for technical assistance. Drs. Carl-Heldin and Peter ten Dijke kindly provided the antiphosphoSmad antibodies for this work.xw:, 百拇医药
Received June 13, 2002.xw:, 百拇医药
Accepted for publication September 4, 2002.xw:, 百拇医药
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Ovarian inhibin production is stimulated by FSH and several TGFß family ligands including activins and bone morphogenetic proteins. Growth differentiation factor-9 (GDF-9) derived by the oocyte is a member of the TGFß/activin family, and we have previously shown that GDF-9 treatment stimulates ovarian inhibin-{alpha} content in explants of neonatal ovaries. However, little is known about GDF-9 regulation of inhibin production in granulosa cells and downstream signaling proteins activated by GDF-9. Here, we used cultured rat granulosa cells to examine the influence of GDF-9 on basal and FSH-stimulated inhibin production, expression of inhibin subunit transcripts, and the GDF-9 activation of Smad phosphorylation. Granulosa cells from small antral follicles of diethylstilbestrol-primed immature rats were cultured with FSH in the presence or absence of increasing concentrations of GDF-9. Secreted dimeric inhibin A and inhibin B were quantified using specific ELISAs, whereas inhibin subunit RNAs were analyzed by Northern blotting using 32P-labeled inhibin subunit cDNA probes. Similar to FSH, treatment with GDF-9 stimulated dose- and time-dependent increases of both inhibin A and inhibin B production. Furthermore, coincubation of cells with GDF-9 and FSH led to a synergistic stimulation of both inhibin A and inhibin B production. GDF-9 treatment also increased mRNA expression for inhibin-{alpha} and inhibin-ß subunits. To investigate Smad activation, granulosa cell lysates were analyzed in immunoblots using antiphosphoSmad1 and antiphosphoSmad2 antibodies. GDF-9 treatment increased Smad2, but not Smad1, phosphorylation with increasing doses of GDF-9 leading to a dose-dependent increase in phosphoSmad2 levels. To further investigate inhibin-{alpha} gene promoter activation by GDF-9, granulosa cells were transiently transfected with an inhibin-{alpha} promoter-luciferase reporter construct and cultured with different hormones before assaying for luciferase activity. Treatment with FSH or GDF-9 resulted in increased inhibin-{alpha} gene promoter activity, and combined treatment with both led to synergistic increases. The present data demonstrate that oocyte-derived GDF-9, alone or together with pituitary-derived FSH, stimulates inhibin production, inhibin subunit mRNA expression, and inhibin-{alpha} promoter activity by rat granulosa cells. The synergistic stimulation of inhibin secretion by the paracrine hormone GDF-9 and the endocrine hormone FSH could play an important role in the feedback regulation of FSH release, thus leading to the modulation of follicle maturation and ovulation.
Introductionmm7:n, 百拇医药
DEVELOPMENT OF THE mammalian ovary is characterized by the initial endowment of a fixed number of primordial follicles. The pool of primordial follicles is gradually depleted during reproductive life. It is well accepted that follicular growth and differentiation are controlled by pituitary gonadotropins as well as follicular paracrine factors of granulosa and theca cell origin (1, 2, 3); many studies have indicated that the oocyte contributes to follicular development as well.mm7:n, 百拇医药
Growth differentiation factor-9 (GDF-9) belongs to the TGFß superfamily and is expressed exclusively in the oocyte (4). The essential role of GDF-9 in folliculogenesis was demonstrated following targeted deletion of the GDF-9 gene in mice (5). Our previous results demonstrated that treatment with GDF-9 induces preantral follicle growth (6) and granulosa cell proliferation but inhibits gonadotropin-induced granulosa cell differentiation in vitro (7).
TGFß and bone morphogenetic proteins (BMPs) signal through type I and type II serine/threonine kinase receptors by activating intracellular Smad family proteins. Based on our studies using the ectodomains of putative receptors for GDF-9, it appears that GDF-9 also interacts with receptors of the TGFß and BMP receptor family, and BMPRII is one of the receptors for GDF-9 (8). Inhibins produced by granulosa cells form a negative feedback loop between the anterior pituitary and the ovary and are important in the regulation of FSH secretion (9). Because our earlier data indicated that GDF-9 treatment increases the inhibin-{alpha} content in ovarian explant cultures (6), we tested whether treatment with recombinant GDF-9 can promote inhibin production in cultured granulosa cells. We further assessed if Smad1 or Smad2 could be phosphorylated by treatment with GDF-9 in granulosa cells to elucidate the signaling pathway of GDF-9 on inhibin production. Based on the inhibin-{alpha} promoter reporter gene system (10), we also tested whether GDF-9 directly stimulates inhibin-{alpha} promoter activity in transfected granulosa cells.
Materials and Methodskdy, 百拇医药
Animalskdy, 百拇医药
Immature female Sprague Dawley rats were obtained from Simonsen Laboratories (Gilroy, CA). Animals (25 d old, body weight from 55–65 g) were anesthesized and killed using CO2 at 72 h after insertion of diethylstilbestrol implants (11). All animals were housed under controlled humidity, temperature, and light regimen, and fed ad libitum on standard rat chow. Animal care was consistent with institutional and NIH guidelines.kdy, 百拇医药
Reagents and hormoneskdy, 百拇医药
McCoy’s 5a medium (modified) and L-15 Leibovitz medium were obtained from Life Technologies, Inc. (Santa Clara, CA). Recombinant human FSH (Org 32489E) was from NV Organon (Oss, The Netherlands). L-glutamine, penicillin, and streptomycin were purchased from BioWhittaker, Inc. (Wakersville, MD). Recombinant activin A and BMP-2 were from R&D Systems (Minneapolis, MN).kdy, 百拇医药
Recombinant GDF-9 was generated in transfected mammalian cells and characterized as previously described (6). Briefly, expression vectors for wild-type and epitope-tagged GDF-9 were constructed using pcDNA3.1 Zeo (Invitrogen, San Diego, CA). N-terminal-tagged GDF-9, encoding a Flag epitope for the M1 antibody followed by six histidine residues fused to the amino-terminus of mature GDF-9, showed no bioactivity and served as a negative control. Human embryonic kidney 293T cells were transfected with the expression vector, and clonal cell lines stably expressing wild-type and tagged GDF-9 were selected under 1 mg/ml of Zeocin (Invitrogen). Conditioned media were harvested after 4 d of serum-free culture. Quantitation of N-tagged GDF-9 was done after purification with nickel column and measurement of protein content using the Micro BCA protein assay kit (Perstorp Life Science, Rockford, IL). Purified N-tagged GDF-9 was then used as a standard for the quantitation of wild-type GDF-9 by immunoblots using specific GDF-9 antibodies.
Preparation and culture of granulosa cells\};, http://www.100md.com
Granulosa cells were obtained from small antral follicles of estrogen-treated immature rats. Ovaries were punctured in L-15 Leibovitz medium. Ovarian debris was removed, and the remaining medium containing granulosa cells was collected after low-speed centrifugation at 500 x g for 10 min. Granulosa cells were dispersed by repeated washing and suspended into the culture medium (McCoy’s 5a supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin).\};, http://www.100md.com
Assessment of inhibin A and inhibin B production\};, http://www.100md.com
Granulosa cells (1 x 105 viable cells/ml) were cultured in 24-well plates (Corning, Inc., Corning, NY) in the presence or absence of increasing concentrations of GDF-9, or the inactive N-tagged GDF-9, with or without FSH. Conditioned media were harvested after 24, 48, and 72 h and stored at -80 C until measurement of inhibin content. The amounts of dimeric inhibin A and inhibin B were quantified using specific ELISAs (Serotec, Oxford, UK). To achieve concentrations within the assay range, the spent culture media from each experiment were diluted and serial dilutions of spent culture media were found to be linear with inhibin A and inhibin B standards.
Northern blot hybridization:?4g, http://www.100md.com
For RNA blot analysis, granulosa cells were harvested 48 h after treatment with FSH or GDF-9, and total RNA was isolated using an RNeasy extraction kit (QIAGEN Inc., Valencia, CA). RNA was quantitated by absorbance measurement at 260 nm. Eight micrograms of total RNA were size-fractionated in 1.5% agarose gels before transfer to Hybond-N nylon membranes (Amersham Pharmacia Biotech, Little Chalfont, UK). For the detection of inhibin-{alpha} , ßA-, and ßB-subunit mRNAs by Northern blot hybridization, rat cDNA probes of the respective inhibin subunits were prepared as previously described (12, 13). Levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA were used as a loading control. Northern blot hybridizations were performed overnight at 42 C, and the filters were washed three times for 20 min with 0.1–1x saline sodium citrate/1% sodium dodecyl sulfate at 50 C. Hybridized filters were exposed at 23 C overnight to Fujifilm phosphoimaging plates before analysis using a Fujifilm IP-Reader Bio-Imaging Analyzer BAS 1500 (Fuji Photo Film Co., Ltd., Tokyo, Japan). The signals of the three different inhibin subunits were quantified and normalized to the expression levels of GAPDH.
Western blotting analysis of Smad proteinso, 百拇医药
To investigate Smad activation by GDF-9, granulosa cells (2 x 106 viable cells/ml) were cultured for 2 h. Media were changed to remove nonattached cells and replaced by fresh media containing different hormones. Granulosa cells were harvested at 30, 60, and 90 min after culture and washed once on ice with cold PBS before lysis in the Laemmli buffer containing ß-mercaptoethanol. Cells were gently sonicated on ice for 15 sec with a MSE sonicator (Sanyo Corp., Osaka, Japan) and boiled for 3 min. Proteins were separated on 8% SDS-PAGE gels and electroblotted onto Amersham Hybond-electrochemiluminescence (for Smad2 experiments) and Hybond-P membranes (for Smad1 experiments) (Amersham Pharmacia Biotech, Arlington Heights, IL). For the detection of Smad1 and Smad2 phosphorylated at the carboxyl terminus, membranes were blocked for 1 h at room temperature in Tris-buffered saline-0.1% Tween containing 5% fat-free dry milk, after which membranes were incubated with an antiphosphoSmad2 antibody diluted at 1:8000 at 4 C overnight or with an antiphosphoSmad1 antibody diluted at 1:8000 at 4 C overnight (14). The secondary antibodies were used following manufacturer’s instructions (Roche Molecular Biochemicals, Indianapolis, IN). Immunoreactive proteins were detected using enhanced chemiluminescence (ECL kit, Amersham Pharmacia Biotech).
Transfection of inhibin-{alpha} promoter-luciferase reporter construct into granulosa cells\, 百拇医药
Granulosa cells (5 x 105 viable cells/well) were cultured in culture medium supplemented with 10% FBS for 2 h. Before transfection, medium was changed to serum-free medium and cells were cotransfected with 2.5 µg of inhibin-{alpha} promoter luciferase plasmid and 0.1 µg of p-Rous sarcoma virus (RSV)-ß-galactosidase (gal) for 4 h at 37 C using lipofectamine (Life Technologies, Inc., Gaithersburg, MD). The pRSV-ß-gal vector containing the lacZ gene encoding ß-gal driven by the RSV long terminal repeat was used to monitor transfection efficiency (15). Cells were then cultured for 24 h in the presence or absence of GDF-9 with or without FSH in media containing 1% FBS.\, 百拇医药
To harvest cells, lysis buffer (200 µl) (Promega Corp., Madison, WI) was added into each well and 30 µl of the supernatant was used for luciferase determination using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). Fifty microliters of the cell lysate were also used to measure ß-gal activity (16). The activity of the inhibin-{alpha} promoter is expressed as the ratio of relative light unit/ß-gal activity.
Data analysis!^ogp, 百拇医药
All experimental data are presented as the mean ± SEM of duplicate measurements of triplicate cultures and each experiment was repeated at least three times. Statistical significance was determined by Student’s paired t test or ANOVA for multiple group comparisons and by Duncan’s multiple range test for effects of multiple doses and treatments. Significance was accepted at P < 0.05.!^ogp, 百拇医药
Results!^ogp, 百拇医药
Effect of GDF-9 treatment on basal and FSH-stimulated inhibin A and inhibin B production!^ogp, 百拇医药
Using a serum-free culture of granulosa cells, we tested the effect of recombinant GDF-9 on inhibin production. As shown in Fig. 1, treatment with recombinant GDF-9 (3–150 ng/ml) for 2 d increased inhibin A and inhibin B production in a dose-dependent manner as compared with the control (Ct) group (P < 0.01). The maximum increases in inhibin A (3.2-fold) and inhibin B (10-fold) production were achieved at 150 ng/ml of GDF-9. FSH at a dose of 3 ng/ml also increased inhibin A and inhibin B production by 5- and 2-fold, respectively. Furthermore, cotreatment with 3 ng/ml FSH and increasing doses of GDF-9 led to synergistic increases in inhibin production as compared with the group treated with FSH alone. Compared with FSH alone, concentrations of GDF-9 at 3–150 ng/ml augmented FSH action by 6-fold for inhibin A and by 20-fold for inhibin B (P < 0.05). Consistent with earlier studies (6), treatment with N-tagged GDF-9 did not augment FSH action, demonstrating the specific action of GDF-9.
fig.ommitteedw!, 百拇医药
Figure 1. Effect of treatment with GDF-9 on basal and FSH-stimulated inhibin A (A) and inhibin B (B) production by cultured granulosa cells. Granulosa cells (2 x 105 cells) were cultured for 48 h in serum-free McCoy’s 5a medium in the presence or absence of increasing doses of GDF-9 with or without 3 ng/ml FSH. Some cells were treated with FSH and 150 ng/ml of N-tagged GDF-9 (NG). Medium inhibin concentration was measured by two-site enzyme-linked immunoassay. Similar data were obtained from two additional experiments. Each pointrepresents mean ± SEM of triplicate wells. Ct, Control.w!, 百拇医药
To study the time course of the GDF-9 stimulation of inhibin production, granulosa cells were cultured for up to 3 d with or without FSH, GDF-9, or a combination of both. Treatment with FSH and GDF-9 stimulated inhibin production with synergistic increases observed at 48 h after cotreatment. At 72 h after treatment with FSH and GDF-9, 5.2- and 11.5-fold increases above FSH treatment alone were found for inhibin A and inhibin B, respectively. In contrast, treatment with N-tagged GDF-9 did not increase basal or FSH-stimulated inhibin A or inhibin B production at any time point tested (Fig. 2).
fig.ommitteedh7/tv's, 百拇医药
Figure 2. Time course of GDF-9 action on inhibin A (A) and inhibin B (B) production by cultured granulosa cells. Granulosa cells (2 x 105 cells/well) were incubated for the times indicated with medium alone (control), FSH (3 ng/ml), or FSH plus different concentrations of GDF-9 (3–150 ng/ml). At the end of the incubation, inhibin concentration in the media was assayed by two-site ELISA. Some cells were treated with 150 ng/ml of N-tagged GDF-9 (NG) with or without FSH (3 ng/ml). Similar data were obtained from two additional experiments. The results are expressed as mean ± SEM of triplicate wells.h7/tv's, 百拇医药
Effect of GDF-9 treatment on basal and FSH-stimulated inhibin subunit transcript levelsh7/tv's, 百拇医药
Our previous data showed that maximal stimulation of inhibin subunit transcript levels occurred at 48 h after FSH treatment (17). Total RNA from granulosa cells cultured for 48 h in the presence of GDF-9 (150 ng/ml) with or without FSH (3 ng/ml) was fractionated on denaturing agarose gels, and Northern blot analysis was performed with inhibin -, ßA-, and ßB-subunit cDNA probes. As shown in Fig. 3 (left panel), hybridization with the -subunit probe resulted in a major hybridization signal at about 1.5 kb, consistent with previous reports (18). Hybridization with the inhibin ßA-subunit probe resulted in a predominant hybridization signal of 6.9 kb (Fig. 3, middle panel). In contrast, two ßB-subunit mRNA species (~ 4.4 kb and ~ 3.3 kb) were detected (Fig. 3, right panel). In cultures treated with GDF-9 alone, levels of inhibin {alpha} , ßA-, and ßB-subunit mRNA, normalized based on GAPDH message levels, were elevated approximately 5.7-, 4-, and 2.3-fold, respectively. Combined treatment with FSH and GDF-9 led to an additive effect on the level of inhibin subunit mRNAs. These results demonstrated a stimulatory effect of GDF-9 in regulating inhibin subunit gene expression.
fig.ommitteedp, 百拇医药
Figure 3. Northern blot analyses of the effects of GDF-9 and/or FSH on inhibin subunit mRNA levels in cultured granulosa cells. Cells were cultured for 48 h with 150 ng/ml GDF-9 and/or 3 ng/ml FSH. Total RNA was extracted and analyzed by Northern blotting using 32P-labeled inhibin subunit cDNA probes. Arrowheads indicate the migration position of specific bands. Results were normalized for GAPDH mRNA levels. Ct, Control.p, 百拇医药
GDF-9 stimulation of Smad2 phosphorylationp, 百拇医药
Lysates from cultured granulosa cells were analyzed in immunoblots using antiphosphoSmad1 and antiphosphoSmad2 antibodies. An immunoreactive band (52 kDa) corresponding to phosphorylated Smad2 was evident (Fig. 4A). In addition, a nonspecific band around 60 kDa was found to be invariant for all treatment groups and served as a loading control. As shown in Fig. 4A, treatment with GDF-9 led to a dose-dependent increase in Smad2 phosphorylation. In contrast, treatment with FSH alone was ineffective. Also, combined treatment of FSH and GDF-9 did not have any influence on the level of Smad2 phosphorylation stimulated by GDF-9. In contrast, treatment with activin increased Smad2 phosphorylation. The stimulatory effect of GDF-9 on Smad2 phosphorylation was time dependent and evident from 30 min post stimulation with the highest level at 90 min (Fig. 4B). Smad2 phosphorylation by activin was also time dependent. Phosphorylation of Smad1 was analyzed in the same experiments using an antibody that recognizes phosphorylated Smad1, 5, and 8. As shown in Fig. 4C, treatment with BMP-2, but not with GDF-9, increased signals at 52 kDa, consistent with the molecular mass of phosphoSmad1.
fig.ommitteed&7, http://www.100md.com
Figure 4. Treatment with GDF-9 activates Smad2, but not BMP-activated Smad1, in cultured granulosa cells. A, Dose-dependent stimulation of Smad2 by GDF-9. Immunoblot analysis of Smad2 phosphorylation was performed using cell extracts following treatment with GDF-9 (30, 100, 150 ng/ml), FSH (3 ng/ml), and activin (50 ng/ml). B, Time-dependent stimulation of Smad2 by GDF-9 (150 ng/ml). C, Lack of induction of Smad1 phosphorylation by GDF-9 in cultured granulosa cells. BMP2 (50 ng/ml), GDF-9 (150 ng/ml). Arrowheads indicate the immunoreactive band of phosphorylated Smad. Ct, Control.&7, http://www.100md.com
GDF-9 stimulation of inhibin-{alpha} promoter activity&7, http://www.100md.com
To elucidate the mechanisms by which GDF-9 regulates inhibin-{alpha} gene expression, we constructed a fusion gene that contained 679 bp of the mouse inhibin-{alpha} gene promoter region linked to a luciferase reporter gene. This construct includes a single potential CRE at –122 bp and three AP2 sites at –202 bp, –176 bp, and –149 bp, respectively (15, 19). The promoter construct was transiently transfected into primary cultures of granulosa cells. To estimate the inhibin-{alpha} promoter regulation by hormones, we measured luciferase activity after the addition of GDF-9 and/or FSH to the culture medium. As shown in Fig. 5, treatment with GDF-9 and FSH increased inhibin-{alpha} gene promoter activity by 1.9- and 2.1-fold, respectively. Furthermore, combined treatment with GDF-9 and FSH increased reporter gene expression significantly greater than that seen with GDF-9 or FSH treatment alone (3.4-fold of the control level).
fig.ommitteed@lq, 百拇医药
Figure 5. GDF-9 stimulation of the inhibin-{alpha} promoter activity in transfected granulosa cells. Cells were transiently transfected with the inhibin-{alpha} promoter luciferase reporter construct and cultured for 24 h after the addition of GDF-9 (150 ng/ml), FSH (3 ng/ml), or both. Cell lysates were assayed for the activity of the luciferase reporter gene. Luciferase activity is presented as relative light units (RLU) and normalized based on ß-gal activity in cotransfected cells. Mean ± SEM. Ct, Control.@lq, 百拇医药
Discussion@lq, 百拇医药
The present data demonstrate that oocyte-derived GDF-9, alone and together with pituitary-derived FSH, stimulates dimeric inhibin production, inhibin subunit mRNA expression, and inhibin-{alpha} promoter activity in rat granulosa cells. We also present evidence that GDF-9 stimulates the phosphorylation of endogenous Smad2 but not Smad1 proteins in these cells, suggesting that GDF-9 mediates its cellular effects through a pathway similar to that used by activins and TGFß but not the one employed by BMPs.
The synthesis and secretion of inhibin and activin dimers in the ovary are dependent on the regulation of three known inhibin/activin subunits that are controlled not only by endocrine hormones, but also by local factors. The role of pituitary gonadotropins in the transcriptional regulation of the inhibin and activin subunit genes in granulosa cells has been well established both in vitro and in vivo (20), and the cAMP-protein kinase A pathway is mediating the gonadotropin regulation of the inhibin and activin subunit genes (20). A role for oocyte-derived factors in regulating granulosa cell inhibin production was recently proposed (21). Coculture of granulosa cells with meiotically immature oocytes was associated with a predominant stimulation of inhibin B (21), resembling the effects of GDF-9 in the present study (Fig. 1). Oocyte-derived GDF-9 acts synergistically with FSH to stimulate inhibin production, despite the inhibitory effects of GDF-9 on FSH-induced estrogen production and LH receptor formation (22, 23). Because GDF-9 null mice showed a high expression of inhibin-{alpha} message in follicles with one layer of granulosa cells (24), the GDF-9 regulation of inhibin production could be follicle stage dependent.
Although GDF-9 and FSH synergistically stimulated inhibin dimer production, no synergistic effects were seen at the mRNA level. It is possible that this discrepancy is due to additional posttranscriptional actions of GDF-9 on inhibin biosynthesis and/or release, mediated by cross-talk between the cAMP and Smad signaling pathways. The present results suggest that GDF-9 increases the sensitivity of granulosa cells to FSH, in addition to stimulating inhibin production in its own right. Because inhibin is important in the regulation of FSH secretion by the anterior pituitary, changes in granulosa cell inhibin production regulated by GDF-9 could alter antral follicle recruitment during reproductive cycles, leading to changes in ovulatory efficiency.9t-a{7z, 百拇医药
TGFß family of growth factors signal by binding to cell-surface serine kinase receptors which then activate cytoplasmic Smads and MAPKs in response to ligand binding (25, 26). Several different TGFß family members, activin A, TGFß, and BMP are capable of eliciting biological responses in cultured granulosa cells (17, 18, 27, 28, 29, 30, 31, 32), and the presence of all seven type I and five type II TGFß and BMP receptors have previously been reported (29, 30, 31, 32, 33). Also, the expression of several Smad signaling proteins in the mammalian ovary has recently been shown (32, 34, 35, 36). Although our recent data suggest that GDF-9 binds directly to BMPRII (8), the putative type I receptors mediating GDF-9 action have not been described.
For TGFß/BMP signaling, the formation of tetrameric receptor complexes containing two type I and two type II receptors allows the phosphorylation of the type I receptor by the type II receptor on the GS domain resulting in activation of the type I receptor kinase. Type I receptors specifically recognize and phosphorylate R-Smads (receptor-activated Smads). Following activation, R-Smads associate with a common Co-Smad, Smad-4. Both the R-Smad and Co-Smad in the complex may participate in DNA binding on Smad binding elements (SBE) of target genes and allow the recruitment of transcriptional cofactors. Previously, it has been shown that two distinct Smad signaling pathways, the activin/TGFß-activated pathway and the BMP-activated pathway, are important to mediate the cellular effects of most TGFß family members (25). The R-Smads, Smad2 and Smad3, are activated by TGFß or activin type I receptors, whereas Smad1, Smad5, and Smad8 are activated by BMP type I receptors. We have previously identified BMPRII as a type II receptor for GDF-9 (8). Due to the sharing of type II and possibly type I receptors between BMP and GDF-9 ligands, GDF-9 could activate the BMP receptor-specific Smad1, 5, and 8 proteins because BMPRII previously had been shown to couple only to type I BMP receptors. However, somewhat surprisingly, the present study shows that GDF-9 activated Smad2, but not Smad1, 5 or 8, suggesting that GDF-9 resembles activins and TGFß, but not BMPS, in its action. These data further suggest that type I receptors activated by GDF-9 could confer stimulation of Smad2 phosphorylation using a pathway different from BMPs. It is unclear whether the known type I receptors previously shown to activate the Smad2 pathway are one of the GDF-9 receptors in granulosa cells.
Inhibin gene expression in the ovary is stimulated by FSH, which uses cAMP as an intracellular second messenger. FSH markedly stimulates inhibin mRNA levels in cultured rat granulosa cells (34, 37). The effects of FSH are mimicked by forskolin, a pharmacological agent that activates adenylyl cyclase (35, 38). In contrast, inhibin production augmented by GDF-9 was not associated with a change in intracellular or extracellular cAMP accumulation (22). We have shown previously that cotreatment of granulosa cells with FSH plus activin resulted in a 2-fold increase in cAMP levels, demonstrating a potentiation of FSH-induced cAMP production by activin, even though treatment with activin alone had no effect (17). The inhibin-{alpha} promoter region contains several potential cAMP response elements and transcription factor AP2-binding sites that might mediate cAMP regulation. We determined here whether GDF-9 could activate the transcription of an inhibin-{alpha} promoter reporter construct known to be activated by FSH and cAMP (39). We observed that GDF-9 on its own modestly activates transcription of this construct, and treatment with FSH and GDF-9 have an additive effect. Interestingly, a potential SBE sequence, CAGACA (19, 40), was identified upstream of the AP1 site of the inhibin-{alpha} promoter. Therefore, the observed GDF-9 stimulation of Smad2 phosphorylation and inhibin {alpha} -promoter activity suggests that this GDF-9 effect might be mediated by the SBE found in the inhibin {alpha} -promoter.
It is becoming clear that the GDF-9 and GDF-9B signaling pathways play an essential role in early follicle development. BMP-15/GDF-9B, similar to GDF-9, is expressed in the oocyte (41, 42, 43) and stimulates granulosa cell proliferation and modulates granulosa cell differentiation (44). The present study on the GDF-9 stimulation of inhibin production by granulosa cells could provide clues on the twinning phenotype associated with the heterozygous GDF-9B gene mutation in Inverdale sheep (45). In sheep with the homozygous GDF-9B gene mutation (XI/XI), defective secondary follicle development was detected. Paradoxically, an increased ovulation rate was found in heterozygotes (XI/X+) (45). Although the original paper did not discuss the mechanism underlying these diametrically opposing phenotypes, our data on GDF-9 stimulation of inhibin A and inhibin B production provides a likely explanation. Release of FSH is controlled by the feedback of inhibins from the ovary. It is possible that a decreased production of GDF-9/GDF-9B dimers by these antral follicles could lead to lower inhibin production with a resultant increase in FSH secretion by the anterior pituitary.
This study demonstrates that GDF-9 enhances FSH-stimulated inhibin production and inhibin subunit gene expression, and activates the Smad2 pathway in rat granulosa cells. These findings emphasize the ability of this oocyte factor to modulate the effectiveness of the gonadotropins on granulosa cell function. More information is needed regarding the specific type I receptors for GDF-9 and its interaction with other hormonal signaling systems operating in the ovarian follicle.xw:, 百拇医药
Acknowledgmentsxw:, 百拇医药
We thank Caren Spencer for editorial assistance and Ms. Marjo Rissanen for technical assistance. Drs. Carl-Heldin and Peter ten Dijke kindly provided the antiphosphoSmad antibodies for this work.xw:, 百拇医药
Received June 13, 2002.xw:, 百拇医药
Accepted for publication September 4, 2002.xw:, 百拇医药
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