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Ovary |
Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23507
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicle, granulosa cells, growth factors, ovulation, ovum
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GDF-9 is also present in the cells of the primate follicle. Immunocytochemistry has been used to localize GDF-9 to the oocytes of human [8] and macaque [9] ovaries, and GDF-9 mRNA has been detected in both human oocytes and granulosa cells [10] obtained from women undergoing in vitro fertilization (IVF). Culture of human ovarian biopsies with GDF-9 enhanced the progression of follicular maturation to the secondary follicle stage [11], indicating a role for GDF-9 in follicular recruitment and early development, as was seen in rodent follicles. However, little is known regarding GDF-9 expression and the role of GDF-9 in the development of the primate follicle during the periovulatory interval, the time between the ovulatory gonadotropin surge and follicle rupture. To address this question, rhesus monkey oocytes, granulosa cells, and follicular fluid were obtained at specific times during the periovulatory interval to determine when GDF-9 is expressed by the primate ovulatory follicle. In addition, granulosa cells were cultured with GDF-9 to determine whether GDF-9 acts directly at primate granulosa cells to regulate specific periovulatory processes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The general care and housing of rhesus monkeys (Macaca mulatta) at the Oregon National Primate Research Center (ONPRC) were described previously [12]. Animal protocols and experiments were approved by the ONPRC Animal Care and Use Committee, and studies were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Adult females with regular menstrual cycles were checked daily for menses, and blood samples were obtained daily from unanesthetized monkeys by saphenous venipuncture beginning on the first day of treatment. Serum was stored at -20°C. Serum estradiol [13] and progesterone [14] concentrations were measured by RIA; intra- and interassay coefficients of variation were <10%. LH concentrations were determined by the mouse Leydig cell bioassay [15] using monkey LH RP-1 as the standard (supplied by the NIH Hormone Distribution Program); intra- and interassay coefficients of variation for the LH bioassay were <15%. These assays were performed by the Endocrine Services Laboratory at ONPRC.
A controlled ovarian stimulation model developed for the collection of multiple oocytes for IVF [16] was used to obtain oocytes, granulosa cells, and follicular fluid. Beginning within 3 days of initiation of menstruation, rhesus monkeys received 60 IU of recombinant human (r-h) FSH (Serono Reproductive Biology Institute, Rockland, MA) for 6 days, followed by 60 IU of r-hFSH plus 60 IU r-hLH (Serono) for 3 days to stimulate the growth of multiple follicles. Animals also received the GnRH antagonist Antide (Serono; 0.5 mg/kg body weight) daily to prevent an endogenous ovulatory LH surge. Adequate follicular development was monitored by serum estradiol levels and by ultrasonography [17]. Follicular aspiration was performed on anesthetized animals during aseptic surgery before (0 h) and 12, 24, 27, 33, or 36 h after administration of 1000 IU r-hCG (Serono). During spontaneous menstrual cycles, follicle rupture in rhesus monkeys occurs approximately 40 h after the ovulatory gonadotropin surge [18], so these times span the periovulatory interval. Previous studies verified ovulation sites on ovaries and oocytes in the oviducts following this protocol [16, 19]. To obtain undiluted follicular fluid, oocytes, and granulosa cells, each follicle was pierced with a 22-ga needle, and the aspirated contents of all follicles >4 mm in diameter were pooled.
Whole ovaries (n = 2–4/time point) were also obtained from monkeys undergoing controlled ovarian stimulation. Additional whole ovaries (n = 5) were collected from monkeys experiencing spontaneous menstrual cycles around the expected time of the endogenous LH surge [18]. These ovaries were obtained before the LH surge (n = 2), during the LH surge (n = 2), and after the LH surge (n = 1) based on serum estradiol, LH, and progesterone levels before and at the time of organ removal. In all experiments, n = 1 reflects follicular fluid, cells, or tissue obtained from an individual animal.
Tissue Preparation
Oocytes, granulosa cells, and follicular fluid were obtained from follicular aspirates as described previously [20]. Aspirates from an individual animal were pooled and subjected to centrifugation to pellet the oocytes and granulosa cells, and the resulting supernatant (i.e., follicular fluid) was removed and stored at -80°C. Oocytes were mechanically removed and treated with 2% hyaluronidase followed by 0.5% pronase to ensure removal of any adhering granulosa cells [21]. A granulosa cell-enriched population of the remaining cells was obtained by Percoll gradient centrifugation. Granulosa cells obtained likely represent a mixture of cumulus and mural cells. Total RNA was obtained from oocytes and granulosa cells using Trizol reagent (Invitrogen, Rockville, MD) and was stored at -20°C. Whole ovaries were fixed in 4% paraformaldehyde and embedded in paraffin.
GDF-9 mRNA Analysis
For analysis of granulosa cell GDF-9 mRNA expression, total RNA (1 µg) was treated with DNase I (Invitrogen) prior to reverse transcription (RT), which was performed using Moloney murine leukemia virus reverse transcriptase (Invitrogen) to produce cDNA as previously reported [22]. Semiquantitative RT polymerase chain reaction (PCR)-based assays similar to those previously described [22] were developed to assess GDF-9 mRNA levels. Sequences for the oligonucleotides (Invitrogen) used for PCR are shown in Table 1. The MgCl2 concentration, amount of cDNA included in each PCR, number of PCR cycles, and primer concentrations were determined empirically for each primer set. The amount of coamplified product for the amplicon under study and the internal standard cyclophilin were linear and parallel with increasing amount of cDNA. The products of both primer sets were in the exponentially increasing phase relative to the number of PCR cycles. Data are expressed as the ratio of GDF-9 to cyclophilin for each sample assayed. GDF-9 primers were designed based on the human GDF-9 sequence (accession NM 005360) in the N-terminal region. Sequence analysis was used to confirm the identity of PCR products; the nucleic acid sequence of the amplified region of monkey GDF-9 was 95% identical to the corresponding human sequence. PCR products were separated on 2% agarose gels and photographed using Polaroid 667 film (Polaroid Corp., Cambridge, MA). All samples were assayed in duplicate; a pool of granulosa cell cDNA was assayed in triplicate in each experiment to allow normalization between assays. Intra- and interassay coefficients of variation were <15%.
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To determine whether oocytes expressed GDF-9 mRNA, a very sensitive radioactive RT-PCR technique was used. Total RNA from individual germinal vesicle-intact oocytes (n = 3, each oocyte from a different female) was prepared using Trizol reagent with the addition of 20 µg of glycogen (Boehringer Mannheim, Indianapolis, IN) to aid in recovery. RT of oocyte total RNA was performed as described above. A fraction (1/20th) of the cDNA obtained from each oocyte was included in each PCR amplification. PCRs were performed as described above, except that 200 µCi 
32P-dCTP was added to each reaction. PCR primer sequences and conditions are listed in Table 1. PCR products were separated on 6% polyacrylamide gels, which were dried and exposed to X-OMAT film (Eastman Kodak, Rochester, NY) for 30–60 min.
Cyclooxygenase 2 (COX-2) mRNA could not be consistently detected in cultured monkey granulosa cell samples using the semiquantitative RT-PCR technique for analysis of GDF-9 expression by granulosa cells. For this reason, COX-2 mRNA levels were analyzed by real-time PCR using a Lightcycler (Roche, Indianapolis, IN) with primers based on the human COX-2 sequence (accession NM 000963). RT was performed as described above, and PCR was performed using the FastStart DNA Master SYBR Green I kit (Roche). COX-2 and cyclophilin content of each sample were determined in separate assays. For each assay, a standard curve consisting of at least 4 log dilutions of reverse-transcribed monkey granulosa cell RNA was included in each assay and used to generate a standard curve. All data were expressed as a ratio of COX-2 to cyclophilin for each sample. Intra- and interassay coefficients of variation were <10%.
GDF-9 and BMP-15 Protein Analysis
Follicular fluids were diluted in PBS, heated to 95°C for 5 min, and loaded onto 4%–15% gradient polyacrylamide Tris-HCl gels (BioRad, Hercules, CA). Western blotting proceeded as reported previously for the monkey progesterone receptor [24], except that the urea denaturation step was omitted. The GDF-9 primary antibody was a goat polyclonal antibody generated against a synthetic peptide based on the mouse GDF-9 sequence (Santa Cruz Biotechnology, Santa Cruz, CA); the 20-amino acid synthetic peptide shares 100% sequence identity with human GDF-9 but only 80% sequence identity with human BMP-15 in the C-terminal region of these proteins (Santa Cruz Biotechnology, personal communication). The anti-GDF-9 antibody was used at a concentration of 0.8 µg/ml, and a bovine anti-goat IgG-horseradish peroxidase conjugate (Santa Cruz Biotechnology) was used as a secondary antibody at a 1:10 000 dilution. The BMP-15 primary antibody was a rabbit polyclonal antibody generated against r-hBMP-15 generously provided by Dr. Shunichi Shimasaki [25]; this antibody was used at a dilution of 1:10 000 followed by incubation with an anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:10 000 dilution; Amersham, Piscataway, NJ).
Primary antibody incubations took place overnight at 4°C, and secondary antibody incubations were performed for 2 h at room temperature. Bands were detected by chemiluminescence (Amersham) and exposure to X-OMAT film for up to 30 min. To remove N-linked oligosaccharides, follicular fluid was incubated for 24 h with recombinant N-glycanase (Glyco, Novato, CA) according to the manufacturer's instructions prior to Western blotting. For sham digestion, the follicular fluid was treated as for the digestion, but the enzyme was omitted.
Immunocytochemical Detection of GDF-9
Immunocytochemical detection of GDF-9 in ovarian tissues was performed with 5-µm sections of paraffin-embedded tissues [26] using the anti-GDF-9 antibody (Santa Cruz Biotechnology) described above for Western blotting. Antigen was retrieved using Antigen Retrieval Citra treatment (BioGenex Labs, San Ramon, CA), and endogenous peroxidase was quenched with 2% hydrogen peroxide in methanol. After blocking with 3% horse serum, sections were incubated with the primary antibody (4 µg/ml) at room temperature for 1 h followed by overnight incubation at 4°C. After rinsing with PBS, sections were incubated with biotinylated bovine anti-goat IgG secondary antibody and then with peroxide-conjugated avidin solution (Vector Laboratories, Burlingame, CA). All nonimmune serum and antibody incubations were performed in PBS with 0.1% Triton X-100 (Sigma Chemical Co., St. Louis, MO). Peroxidase was visualized using nickel-diaminobenzidine chromagen (Vector). Specificity of GDF-9 staining was demonstrated by preabsorbing the antibody overnight at 4°C with the GDF-9 peptide used to generate this antibody at 50-fold excess (Santa Cruz Biotechnology).
Cell Culture
Granulosa cells obtained from monkeys undergoing controlled ovarian stimulation before the administration of an ovulatory dose of gonadotropin were plated on fibronectin-coated tissue culture plates and maintained in serum-free conditions for 24 or 48 h as previously described [27]. Cultures were treated with hFSH (100 ng/ml; NIH Hormone Distribution Program), hLH (100 ng/ml; NIH), and/or recombinant rat GDF-9 (10 and 100 ng/ml; courtesy of Drs. Aaron Hsueh and Masaru Hayashi) [4]. Medium from untransfected 293T cells (used to produce recombinant rat GDF-9) was included in cultures not receiving GDF-9 as a control. Medium was then collected and stored at -20°C for analysis of progesterone and vascular endothelial growth factor (VEGF) levels. Cells were lysed in situ with Trizol reagent (Invitrogen), and total RNA was prepared with the addition of glycogen (10 µg) to improve recovery. Progesterone levels in the medium were determined by RIA as for serum steroids; total VEGF concentrations were determined using an enzyme immunoassay (R&D Systems, Minneapolis, MN) as previously described [28].
Data Analysis
All gels were scanned, and specific bands were analyzed densitometrically using the GelDoc system (BioRad). Linear relationships were analyzed using Origin (Microcal Software Inc., Northhampton, MA). All data were assessed for heterogeneity of variance using the Bartlett test and were log transformed when necessary. One-way ANOVA was used to analyze GDF-9 mRNA and follicular fluid GDF-9 and BMP-15 protein concentrations. VEGF, progesterone, and COX-2 mRNA levels in cultured cells were assessed by two-way ANOVA with two repeated measures to examine the effects of time and treatment in vitro. Differences were never identified with respect to time in culture, and there was no interaction between time and treatment. For this reason, data for each time point were analyzed by one-way ANOVA with one repeated measure to examine only the effects of treatment. This analysis was followed by a Newman-Keuls test when indicated. Data are presented as mean ± SEM, and differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine whether GDF-9 is expressed by the primate oocyte, total RNA prepared from individual oocytes at the germinal vesicle-intact stage was subjected to RT-PCR using a radioactive nucleotide to enhance detection of amplified cDNA. An amplified fragment of the expected size (239 base pairs [bp]) was detected in all oocyte cDNA samples examined (Fig. 1). Each oocyte cDNA showed no detectable amplification of aromatase cDNA, indicating that GDF-9 mRNA detection was not the result of contamination of the oocyte by adhering granulosa cells. Granulosa cell cDNA showed strong amplification for this steroidogenic enzyme (Fig. 1).
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GDF-9 mRNA was detected by semiquantitative RT-PCR in granulosa cells obtained at different times during the periovulatory interval. GDF-9 cDNA levels did not differ among the time points examined (Fig. 2).
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The presence of GDF-9 protein in the periovulatory follicle was confirmed by Western blotting of follicular fluid. Bands migrating at 54 and 22 molecular weight (MW) were detected in each follicular fluid sample examined (Fig. 3A). GDF-9 is a glycosylated protein [4], and N-glycosidase treatment of follicular fluid yielded a shift in the apparent size of the 54-MW band when compared with undigested follicular fluid or follicular fluid subjected to sham digestion (Fig. 3C). N-glycosidase treatment also achieved digestion of recombinant rat propeptide form of GDF-9 (Fig. 3D), as indicated by the presence of a protein of smaller apparent size. Data obtained from N-glycosidase digestion of monkey follicular fluid proteins are consistent with deglycosylation of GDF-9 and support the identification of these proteins as monkey GDF-9. To confirm that GDF-9, and not the closely related protein BMP-15, was detected by Western blotting, monkey follicular fluid was also subjected to Western blotting for BMP-15. A single protein with an apparent size of 45 MW was detected in every follicular fluid sample examined, distinct from the 54- and 22-MW bands identified as GDF-9 in the present study (Fig. 3E).
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Western blotting was used to compare levels of propeptide and mature GDF-9 in monkey follicular fluid samples obtained throughout the periovulatory interval. When increasing volumes of follicular fluid were used for Western blotting, increasing signal was measured by densitometry for both the 54- and 22-MW bands (Fig. 3, A and B). These data indicate that Western blotting can be used for semiquantitative analysis of GDF-9 in monkey follicular fluid samples. Follicular fluid obtained at different times during the periovulatory interval was subjected to Western blotting for GDF-9. Both forms of GDF-9 were detected in every follicular fluid sample examined, and the optical density resulting from the propeptide GDF-9 band was higher than that resulting from the mature GDF-9 band in every sample examined. There was no change in GDF-9 concentrations in follicular fluid across the periovulatory interval (Fig. 4). This method was also used to compare BMP-15 concentrations in monkey follicular fluid samples. BMP-15 was detected in every follicular fluid sample examined, and concentrations also did not change across the periovulatory interval (data not shown).
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The GDF-9 antibody used for Western blotting was also used for immunocytochemical detection of GDF-9 in monkey ovaries (Fig. 5). Dark cytoplasmic staining was present in oocytes and granulosa cells near the oocyte; nuclear staining was not observed in oocytes or granulosa cells (Fig. 5, E and F). Staining was not seen when the primary antibody was omitted or when the primary antibody was preabsorbed with the peptide used to generate the antibody (Fig. 5, A–C), indicating specific staining for GDF-9. Stromal staining (Fig. 5D) was present when the primary antibody was preabsorbed, indicating that this staining does not represent detection of GDF-9. This nonspecific stromal staining was primarily observed in red blood cells and interstitial cells thought to be remnants of corpora lutea from previous menstrual cycles.
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Oocytes and granulosa cells of antral follicles 
1 mm in diameter showed cytoplasmic immunostaining for GDF-9 (Fig. 5). The pattern of GDF-9 immunostaining within the oocyte varied among oocytes, with some oocytes showing staining dispersed throughout the cytoplasm and others with staining restricted to portions of the cytoplasm (Fig. 5, E and F). Similar asymmetry of GDF-9 staining within mouse oocytes has been previously reported [7]. Oocyte staining patterns did not appear to be related to follicle size or exposure to ovulatory gonadotropin. Cumulus and mural granulosa cells near the oocyte showed immunostaining, but mural granulosa cells not adjacent to the cumulus were consistently devoid of staining (Fig. 5D). GDF-9 staining of granulosa cells was observed in periovulatory follicles within ovaries removed during both natural menstrual cycles and at specific times during controlled ovarian stimulation. No change in the localization of GDF-9 immunostaining across the periovulatory interval or in response to gonadotropin administration was observed.
GDF-9 Regulation of Granulosa Cell Function In Vitro
Granulosa cells were maintained in culture for 48 h in the absence and presence of GDF-9, LH, and FSH to determine whether GDF-9 modulates granulosa cell functions known to be induced by the ovulatory gonadotropin surge in vivo (Fig. 6). After 24 h of culture, GDF-9 (100 ng/ml) stimulated VEGF concentrations above those measured in control cultures, as did LH and FSH both alone and with GDF-9 (P < 0.05). No differences in VEGF concentrations were observed in control and GDF-9-treated cultures after 48 h of treatment (data not shown). Medium from cell cultures was also assayed for progesterone. Progesterone concentrations in cultures treated with either dose of GDF-9 were not different from control concentrations at 24 or 48 h of culture (data not shown). Treatment with LH and FSH increased progesterone concentrations above control levels at both time points examined (P < 0.05); addition of GDF-9 to gonadotropin-stimulated cultures did not alter progesterone concentrations compared with cultures receiving LH or FSH only. Exposure of granulosa cells to GDF-9, LH, and/or FSH did not alter COX-2 mRNA levels after 24 or 48 h of culture (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The sites of GDF-9 production differ between primate and rodent follicles. In rodent follicles, the oocyte is thought to be the sole site of ovarian GDF-9 synthesis, with GDF-9 expression localized to follicles of the primary to preovulatory stages [4]. Previous studies in monkeys [9] and women [29, 30] confirmed GDF-9 expression by primate oocytes located in primary, secondary, and antral follicles. However, GDF-9 mRNA has also been amplified from oocytes and granulosa cells obtained from women undergoing treatment for infertility [10], and possible detection of GDF-9 expression in cumulus granulosa cells of human antral follicles has also been reported [29]. In the present study, we have extended the findings of Gougeon and Busso [9] to show that granulosa cells and oocytes of monkey antral follicles express GDF-9. GDF-9 expression was identified in primate oocytes from large preovulatory follicles and by granulosa cells obtained during the interval between the ovulatory gonadotropin surge and follicle rupture. Granulosa cells near the oocyte showed the strongest immunostaining, suggesting that cumulus cells may produce the majority of GDF-9 mRNA detected in the mixed population of granulosa cells assayed. Because immunostaining of the ovarian stroma was not eliminated by preabsorption of the GDF-9 primary antibody, it is unclear whether theca or other stromal cells produce significant amounts of GDF-9 in periovulatory follicles. GDF-9 concentrations and expression patterns do not appear to be regulated by the ovulatory gonadotropin surge. GDF-9 action may be most important during early follicular development [11]. However, regulation of components of the GDF-9 response pathway, including GDF-9 receptors [31], intracellular messengers [32], and possible binding proteins [33–35], may modulate the activity of GDF-9 within the follicle during the periovulatory interval.
Both cumulus and mural granulosa cells may be targets for GDF-9 action. In rodents, GDF-9 has been hypothesized to promote expansion while delaying luteinization of cumulus cells [7]. In contrast, mural granulosa cells not exposed to GDF-9 would luteinize earlier in the periovulatory interval [7]. GDF-9 may have similar actions in the cumulus of primate follicles; GDF-9 appears to be produced primarily by cumulus granulosa cells. However, the presence of mature GDF-9 protein in follicular fluid may delay luteinization throughout the remainder of the follicle. Previous studies have demonstrated that some periovulatory changes occur in the monkey follicle within 12 h of administration of an ovulatory dose of gonadotropin, including increased follicular fluid progesterone levels [20] and increased expression of steroidogenic acute regulatory gene [36] and COX-2 [26] mRNAs. Similar changes have been observed in mouse granulosa cells in response to GDF-9 exposure [7]. Some periovulatory processes, including luteinization of the follicle wall [37], occur much later after the ovulatory gonadotropin surge in the primate (24–36 h) compared with the rodent. In the present study, GDF-9 did not induce expression of COX-2 by monkey granulosa cells, whereas GDF-9 increases COX-2 expression by rodent granulosa cells [7], suggesting that additional factors may be required to induce the expression of this important gene in primate follicles. In rodents, compartmentalization of GDF-9 action may coordinate events within the rodent follicle and contribute to the relatively short (14 h) interval between the ovulatory gonadotropin surge and ovulation. In primates, GDF-9 may act at granulosa cells throughout the follicle, delaying some periovulatory processes and contributing to the longer (40 h) periovulatory interval observed in monkeys and humans.
Detection of GDF-9 protein in macaque follicular fluid suggests that GDF-9 is present throughout the periovulatory follicle, so all granulosa cells are potential targets for GDF-9 in primate follicles. In the present study, GDF-9 treatment increased granulosa cell VEGF production, indicating that primate granulosa cells are GDF-9 responsive. This is the first study to demonstrate that VEGF production by granulosa cells is GDF-9 responsive and to suggest that GDF-9 may be involved in regulating the vascularization of the granulosa cell layer, which takes place during luteinization of the follicle wall. In the present study, GDF-9 did not significantly reduce progesterone production by monkey granulosa cells in vitro. However, others have reported that GDF-9 inhibited steroidogenesis in cultured human [38] and rat [39] granulosa cells. Rodent theca cells can respond to GDF-9 in vitro [40], and in a recent study, human theca cells appeared more responsive than granulosa cells to the inhibitory effects of GDF-9 on steroidogenesis [38]. Although only granulosa cell responses to GDF-9 have been extensively studied, multiple cell types within the mammalian follicle may be important targets for GDF-9 action.
Other members of the TGFß superfamily share significant homology with GDF-9. Both GDF-9 and BMP-15 (also called GDF-9B [41, 42]) lack a specific cysteine residue that leads to a covalent bond and dimer formation in other members of the TGFß superfamily [42]. BMP-15 has been localized to the oocytes of primary through preovulatory follicles in rats [25] and primary follicles in humans [8], but expression of BMP-15 by granulosa cells has not been reported in any species. Mice homozygous for mutation of GDF-9 are infertile [2], but BMP-15 knockout mice do produce offspring [43], suggesting important differences between GDF-9 and BMP-15 action in the mammalian ovary. Data presented here indicate that BMP-15 is present in follicular fluid throughout the periovulatory interval, but further experiments will be required to determine the source and function of BMP-15 in the primate follicle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Diane M. Duffy, Department of Physiological Sciences, Eastern Virginia Medical School, 700 Olney Rd., Lewis Hall, Norfolk, VA 23507. FAX: 757 624 2269; duffydm{at}evms.edu
Received: 29 January 2003.
First decision: 23 February 2003.
Accepted: 2 April 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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