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Department of Cellular and Molecular Medicine (F.H.T., J.-F.E., B.C.V.), University of Ottawa, Ottawa Regional Cancer Centre, Ottawa, Ontario, Canada K1H 1C4; and Department of Reproductive Medicine (S.S.), University of California, San Diego, School of Medicine, La Jolla, California 92093-0633
Address all correspondence and requests for reprints to: Fiona H. Thomas, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa Regional Cancer Centre, Ottawa, Ontario, Canada K1H 1C4. E-mail: fthomas{at}uottawa.ca.
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Abstract |
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Introduction |
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Kit ligand (KL) is expressed in granulosa cells as either membrane-bound or soluble proteins arising from alternatively spliced mRNAs (7). Soluble KL (KL-1) can be cleaved due to the presence of an 84-bp exon (exon 6), which encodes a proteolytic cleavage site, allowing the extracellular domain to be released as a soluble product. Membrane-bound KL (KL-2) lacks this exon, is not efficiently cleaved, and thus remains more stably on the membrane (7). The ratio of KL-1/KL-2 mRNA differs between tissues (7), between ovaries of mice of different ages (8), and between granulosa cells of preovulatory and ovulatory rat follicles (9), which suggests that these transcripts are differentially regulated. Kit, the receptor for KL, is expressed in oocytes at all stages of follicular development in the mouse ovary (10), and several studies have demonstrated that female mice with naturally occurring mutations in KL or Kit are infertile due to developmental abnormalities (10, 11, 12, 13). Sl mutations, which have either no KL-2 (Sld) or no KL-1 (SlKl–2) have very different phenotypes (14, 15). For example, mice homozygous for the Sld allele, which only produce KL-1, are sterile due to a deficiency in germ cells (14). However, mice that exclusively produce KL-2 are fertile (15), suggesting that KL-2 may be the principal isoform required for oocyte development.
Growth/differentiation factor (GDF)-9 is localized exclusively to oocytes at all stages of follicular growth, except primordial follicles, in neonatal and adult mice (16). The pattern of GDF-9 expression and results from GDF-9 gene knockout studies suggest that this factor may play an autocrine role in the regulation of oocyte development and maturation and/or a paracrine role in the regulation of granulosa cell proliferation and differentiation (17, 18). These studies have also led to the hypothesis that GDF-9 regulates KL expression, because KL mRNA levels are elevated in the absence of GDF-9 in vivo (18).
Bone morphogenetic protein (BMP)-15 is an oocyte-specific homologue of GDF-9 and has also been cloned in mice (19). In sheep, where this factor has been well studied, the Inverdale fecundity gene (FecX) carries an inactivating mutation in BMP-15 (20), implicating this factor in the control of ovulation rate. In rodents, both GDF-9 and BMP-15 promote proliferation of granulosa cells from small antral follicles (21, 22, 23). BMP-15 has also been reported to inhibit FSH-stimulated progesterone production by rat granulosa cells (22). Evidence of interactions among GDF-9, BMP-15, and KL in vitro has been recently reported (24, 25). For example, recombinant GDF-9 inhibits KL mRNA expression in mouse preantral granulosa cells (24), whereas BMP-15 promotes KL expression in monolayers of granulosa cells from rat early antral follicles (25). GDF-9 has also been shown to induce expression of the BMP antagonist, gremlin (26). To date, there is no information on the role of BMP-15 on murine oocyte or follicular development in vitro. In addition, the effect of BMP-15 on the development of preantral granulosa cells that have maintained their in vivo architecture remains to be established.
Endocrine control of follicular development by FSH rests on a network of intrafollicular interactions (27). For example, FSH promotes proliferation and differentiation of preantral follicles via paracrine factors such as IGF-1 and activin (28, 29). In addition, FSH regulates KL expression in granulosa cells from murine preantral follicles (30). There have been no reports, however, of a role for FSH in regulation of KL and oocyte factors such as GDF-9 and BMP-15 during the development of intact preantral OGCs in vitro. Moreover, because FSH and paracrine growth factors are known to be regulators of follicle development in vivo, it is important to investigate whether these factors play a role in oocyte growth in vitro. FSH treatment has been shown to stimulate oocyte growth in bovine preantral follicles in vitro (31), but a specific role for FSH or the possibility of a dose-dependent role of FSH in promoting oocyte growth in rodent culture systems have not been confirmed. As for KL, a role in promoting early oocyte growth in vitro has previously been demonstrated in mice (32). However, the specific roles of membrane-bound and soluble KL during oocyte development have not been investigated.
The aims of this study were: 1) to determine the effect of FSH on regulation of expression of KL-1, KL-2, Kit, GDF-9, and BMP-15 mRNA during development of OGCs in vitro; 2) to investigate the role of BMP-15 in regulation of KL expression; and 3) to correlate mRNA expression patterns with oocyte growth in vitro.
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Materials and Methods |
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Treatment groups
A preliminary set of experiments (n = 3) was set up to establish patterns of expression of KL, BMP-15, GDF-9, and Kit mRNA in OGCs on d 0, 3, and 7 of culture under basal culture conditions. OGCs isolated from 12-, 15-, and 19-d-old mice were also obtained to determine expression of KL-1 and KL-2 mRNA in vivo. Second, the role of FSH in the regulation of expression of these factors during a 3-d culture period was examined. Human recombinant FSH was added to the medium at a concentration of 0.05 ng/ml or 5 ng/ml (provided by the National Hormone and Pituitary Program). Isolated OGCs were assigned randomly to the treatment groups. OGCs were incubated for 3 d in a sterile atmosphere of humidified air with 5% CO2 at 37 C. This experiment was performed five times under identical conditions. In the next experiment, Gleevec [an inhibitor of Kit tyrosine kinase activity (34); Novartis, Basel, Switzerland] was added to the culture medium to assess the role of KL in oocyte growth and modulation of expression of specific paracrine factors. The Gleevec stock solution was dissolved in dimethylsulfoxide (DMSO), and this stock was added to culture medium at a concentration of 5 µM. For this experiment, the groups without Gleevec contained the same volume of DMSO as a control. In a separate experiment, soluble mouse KL-1 (Genzyme, Missisauga, Ontario, Canada), at a concentration previously determined to optimally stimulate oocyte growth (10 ng/ml; results not shown), alone and in combination with FSH, was used to determine the effect of an alteration in KL-1/KL-2 ratio on oocyte growth. Finally, to investigate the role of oocyte-specific BMP-15 on modulation of KL, as well as its effect on oocyte and granulosa cell development, OGCs were treated with recombinant human BMP-15 (22) at concentrations of 10 or 100 ng/ml (25). The Gleevec, KL-1, and BMP-15 cultures were performed three times under identical conditions.
Assessment of oocyte growth and granulosa cell proliferation
Preantral OGCs (77.2 ± 1.4 µm at the time of collection) were randomly assigned to the culture groups described above and incubated for 3 d. At the end of the culture period, OGC diameters were measured (as an indicator of granulosa cell proliferation) using an inverted microscope (Leitz Diavert, Wetzlar, Germany). For measurement of oocyte diameter (including the zona pellucida), granulosa cells were first removed from the oocytes by incubating for 5 min in bovine pancreatic Dnase 1 (0.01% in PBS; Sigma-Aldrich, Oakville, Ontario, Canada), with repeated pipetting to completely dissociate the granulosa cells. Mean oocyte and OGC diameters were obtained from a minimum of 10 OGCs per treatment per experiment, and each experiment was performed three times.
Quantification of KL-1, KL-2, Kit, BMP-15, and GDF-9 mRNA expression using real-time RT-PCR
Total RNA from approximately 100 OGCs per treatment per experiment was extracted using the RNeasy Mini Kit (Qiagen, Missisauga, Ontario, Canada), following the manufacturer’s instructions. To avoid contamination by genomic DNA, each total RNA sample was treated with deoxyribonuclease, using the DNA-free kit (Ambion). The concentration of RNA in the samples was assessed using an Eppendorf BioPhotometer (Eppendorf AG, Hamburg, Germany). RNA was reverse-transcribed using Superscript III (Invitrogen) according to the manufacturer’s instructions. Genomic DNA contamination was excluded in each PCR using a control for each sample that was generated in the absence of reverse transcriptase.
Oligonucleotide primers for PCR were designed to amplify KL-1, KL-2, Kit, BMP-15, and GDF-9 and custom ordered from Sigma Genosys. The housekeeping gene ß-actin was amplified as a reference for mRNA quantification. For the KL splice variants, it was necessary to generate primers that either amplified the region of cDNA that encompassed exon 6 (for detection of KL-1) or flanked the region encompassing the deleted exon 6 (for detection of KL-2). The primer sequences were as follows: KL-1 (5'-GAT TCC AGA GTC AGT GTC AC-3' and 3'-CCA GTA TAA GGC TCC AAA AGC AA-5'); KL-2 (5'-CTT GTC AAA ACC AAG GAG ATC TGC G-3' and 3'-CTT TGC GGC TTT CCC TTT CTC-5'); Kit (5'-AGG AGA TAA ATG GAA ACA ATT ATG T-3' and 3'-TTG AGC ATC TTT ACA GCG ACA GTC A-5'); BMP-15 (5'-GAG CGA AAA TGG TGA GGC TG-3' and 3'-GGC GAA GAA CAC TCC GTC C-5'), GDF-9 (5'-TCC TTC AAC CTC AGC GAA TA-3' and 3'-GCC CCC ATG CTA ACG AC-5'); and ß-actin (5'-GTG GGC CGC CCT AGG CAC CAG-3' and 3'-CTC TTT GAT GTC ACG CAC GAT TTC-5').
For the PCR, 2 µl cDNA was amplified in a reaction mixture (total vol, 20 µl) containing 2 µl 5x reaction buffer, 0.6 µl MgCl2 (1.5 mM), 0.4 µl deoxynucleotide triphosphate mix (0.2 mM), 0.4 µl each of forward and reverse primers (0.5 µM each primer), 0.4 µl BSA (from 25 mg/ml stock), 0.33 µl SYBR Green 1 (1/60,000; Molecular Probes, Invitrogen), 0.2 µl temperature-release Taq DNA polymerase (Platinum Taq Polymerase, Invitrogen) and 13.27 µl double distilled H2O. All reagents were obtained from Invitrogen unless stated otherwise. Real-time PCR was carried out using LightCycler technology (Roche Diagnostics, Laval, Quebec, Canada). The reaction conditions were as follows: predenaturation (95 C for 30 sec), 35 PCR cycles consisting of denaturation (95 C for 30 sec), annealing (58 C for 10 sec for KL-1, KL-2, ß-actin, BMP-15, and GDF-9; 52 C for 10 sec for Kit), extension [72 C for 22 sec (KL-2 and ß-actin), 72 C for 15 sec (BMP-15 and GDF-9), 72 C for 8 sec (KL-1 and Kit)]. Fluorescence data were acquired after the extension phase of each cycle, during an additional step at approximately 3 C below the melting temperature of each product. This excluded quantification of any nonspecific products. To quantify mRNA levels, a standard curve was generated for each transcript by amplifying serial dilutions (1, 1/5, 1/10, 1/20, 1/30) of control cDNA known to express the gene of interest.
To quantify specific gene expression in OGCs, the levels of expression of specific oocyte and granulosa cell mRNAs in each sample were calculated relative to ß-actin. To ensure the integrity of these results, an additional housekeeping gene, 18S rRNA (Ambion Inc., Austin, TX), was used as an internal standard to ensure that ß-actin mRNA was not regulated under any of the culture conditions tested. This gene has been identified as an appropriate housekeeping gene for use in quantitative PCR studies (35, 36). Expression of ß-actin and 18S rRNA mRNA was measured in cDNA samples from freshly isolated OGCs, and from OGCs cultured for 3 or 7 d. In the OGCs cultured for 3 d, the effects of FSH, BMP-15, Gleevec, and Roscovitine on ß-actin mRNA levels were determined (n = 3 experiments). Under all conditions, the expression of ß-actin did not vary when normalized against 18S rRNA (results not shown).
Inhibition of FSH-induced proliferation with Roscovitine
To quantify gene expression in OGCs, the levels of expression of specific oocyte and granulosa cell mRNAs in each sample were calculated relative to ß-actin. Because this housekeeping gene is expressed in both cell types of the complex, it became apparent that in OGCs with increased numbers of granulosa cells, expression of oocyte-specific mRNAs could be underestimated. Therefore, we performed an experiment to inhibit FSH-induced granulosa cell proliferation to validate data that showed decreased expression of oocyte-specific genes. Specifically, OGCs were incubated for 3 d with Roscovitine (0.7 or 20 µM in DMSO), a potent selective inhibitor of cyclin-dependent kinases (Sigma). For this experiment, the groups without Roscovitine contained DMSO (equivalent volume as that for 20 µM Roscovitine) as a control. At the end of the culture period, OGC diameters were measured to confirm an inhibition in proliferation, and RNA was extracted for use in real-time RT-PCR.
Statistical analyses
All results are expressed as mean ± SEM of at least three independent experiments. Mean oocyte and OGC diameters, from a minimum of 10 OGCs per treatment per experiment, were compared between experimental groups using a one-way ANOVA for multiple comparisons, followed by Student’s t tests. For each transcript, all treatment groups were quantified simultaneously in a single LightCycler run. To correct for differences in both RNA quality and quantity between samples, data were normalized by dividing the quantity of target gene by the quantity of ß-actin. Mean mRNA levels from a minimum of 100 OGCs per treatment per experiment were compared using ANOVA and Student’s t tests. P values < 0.05 were accepted as statistically significant.
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). However, after d 3, the KL-1/KL-2 ratio increased significantly, due to a 5-fold decrease in KL-2 mRNA expression (P < 0.05) (Fig. 1B). KL-1 mRNA expression decreased between d 0 and d 3, then increased again between d 3 and d 7 (P < 0.05) (Fig. 1B). The ratio of KL-1/KL-2 mRNA increased during follicular development in vivo (between postnatal d 15 and 19) (Fig. 1C) (P < 0.05), and this mirrored the pattern observed during culture of OGCs in vitro. Oocyte diameters measured on d 0, 3, and 7 of culture revealed that the majority of oocyte growth occurred during the first 3 d of culture in control medium (P < 0.05), with no further significant growth between d 3 and d 7 (Fig. 1D). Both BMP-15 and GDF-9 steady-state mRNA levels increased during the first 3 d of culture (Fig. 2A), and BMP-15 mRNA increased further from d 3 to d 7 (P < 0.05), whereas GDF-9 expression remained constant. Kit mRNA expression did not change during the 7-d culture period (Fig. 2B).
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). The decrease in KL-1/KL-2 ratio in the presence of 0.05 ng/ml FSH was due to a 2-fold increase in KL-2 mRNA expression (Fig. 3B). There was no increase in KL-2 expression in OGCs treated with 5 ng/ml FSH. There was no significant effect of 0.05 ng/ml FSH on BMP-15 expression, whereas BMP-15 mRNA was significantly decreased in OGCs treated with 5 ng/ml FSH (P < 0.05), compared with controls (Fig. 3C). There was no significant effect of FSH on KL-1, GDF-9, or Kit expression (Fig. 3, B, C, and D, respectively). To validate the finding that FSH inhibited oocyte-specific BMP-15 expression, FSH-induced granulosa cell proliferation was inhibited with Roscovitine, a potent selective inhibitor of cyclin-dependent kinases (Fig. 4A). It was found that FSH-induced inhibition of BMP-15 mRNA expression still occurred in OGCs treated with Roscovitine (Fig. 4B), confirming an FSH-specific effect rather than a consequence of increased GC/oocyte mRNA ratio in these samples.
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), the possibility of a regulatory feedback loop between KL and BMP-15 was investigated. It was found that when recombinant BMP-15 was added to OGCs in culture, there was a significant increase in both KL-1 and KL-2 expression, compared with controls (P < 0.05) (Fig. 6, A and B). Unlike FSH, however, there was no significant alteration of KL-1/KL-2 mRNA ratio by BMP-15 (Fig. 6C).
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). However, oocyte diameter was significantly increased over control levels only in the presence of 0.05 ng/ml FSH (Fig. 7B). In addition, oocyte growth, induced in the presence of 0.05 ng/ml FSH, was suppressed by Gleevec (P < 0.05) (Fig. 7C). Lastly, KL-1 alone stimulated oocyte growth, but this effect was not as pronounced as with low FSH (Fig. 7D). In addition, when the KL-1/KL-2 ratio was increased by addition of soluble KL-1 to cultures in the presence of 0.05 ng/ml FSH, FSH-induced oocyte growth was suppressed (P < 0.05) (Fig. 7D). BMP-15 had no significant effect on granulosa cell proliferation or oocyte diameter (Fig. 7, E and F).
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Discussion |
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In vivo, overall KL expression is low during early preantral development (in 7- and 8-d-old mice), and it increases during later preantral stages (12- and 13-d old mice), before declining in early antral follicles of 15-d-old mice (8, 30). In mice younger than 10 d old, there is an equal distribution of both KL isoforms; whereas between 10 and 20 d of age, the KL-1/KL-2 ratio decreases (8). Expression of both KL and Kit in the ovary is consistent with a role for this ligand-receptor pair in oocyte growth (8). Although a role for soluble KL-1 in promoting early oocyte growth (in 8-d-old mice) in vitro has previously been demonstrated (32), oocyte growth was limited, and the relative roles of membrane-bound and soluble KL during oocyte development were not elucidated. In the present study, KL-1, added exogenously to the culture medium, stimulated oocyte growth, but the effect was not so pronounced as with a low concentration of FSH. From expression analysis in vivo, KL-2 is thought to be the principal isoform expressed in granulosa cells associated with growing oocytes, with a switch to KL-1 expression in granulosa cells associated with fully grown oocytes (37). Here, we have presented data to support an increase in KL-1/KL-2 ratio in OGCs isolated from 19-d-old mice, compared with 15-d-old mice. This pattern of expression is in agreement with the in vitro results, where a low steady-state KL-1/KL-2 mRNA ratio was associated with growing oocytes on d 3 of culture, with a subsequent increase in KL-1/KL-2 ratio between d 3 and 7, when no further increase in oocyte diameter is observed. Previous data has shown that, in response to hCG, there is a shift in steady-state mRNA from KL-2 to KL-1 in rat mural granulosa cells (9), and a rapid depletion of both isoforms in cumulus cells, which suggests differential functions of the KL isoforms during oocyte development and meiotic progression.
Although FSH is known to be a critical regulator of oocyte maturation (38), little is known of the role of FSH during the oocyte growth phase. Because oocytes in hypogonadal mice grow to normal size and can acquire developmental competence (39), it has been suggested that gonadotropins may not be necessary for oocyte development per se but may play a general role in supporting overall follicular development and endocrine function and preventing follicular degeneration (40). From our results, we can hypothesize a role for FSH in the modulation of KL expression to promote oocyte growth in vitro. However, the concentration of FSH is crucial for appropriate regulation of paracrine factors to promote oocyte development. Repeated ovarian stimulation of mice with gonadotropins in vivo has been reported to reduce subsequent in vitro meiotic competence of oocytes (41). Further evidence is provided by Eppig et al. (42), who reported that a high concentration of FSH, in the presence of insulin, promoted precocious differentiation of granulosa cells within preantral OGCs in culture. These follicles were also found to contain oocytes with reduced competence to undergo fertilization and preimplantation development (42). A subsequent study by the same authors used a much lower concentration of FSH during follicular growth in vitro, and the rate of oocyte fertilization and blastocyst development was significantly improved (2). Therefore, the correct concentration of FSH is important for the acquisition of oocyte developmental competence. Whether this effect is mediated through Kit signaling requires further investigation.
In GDF-9-deficient mouse ovaries, follicular development does not progress beyond the primary stage, but the oocytes within these follicles grow larger than normal (17). Interestingly, these mutant mice also have elevated levels of KL-1 and KL-2 mRNA (18). Differential regulation of the two KL transcripts is likely to be a vital component of regulation of KL expression during oocyte and follicular development. Because down-regulation of KL-2 expression coincides with the cessation of oocyte growth, these oocytes may exceed the normal maximum diameter due to continued elevation of KL-2 expression. In addition, although oocytes in GDF-9-deficient mice grow, they do not attain meiotic competence (43). The data presented here, taken together with findings from previous studies (18, 43), suggest that a premature increase in KL-1/KL-2 ratio and/or a failure to down-regulate KL-2 at the appropriate stage of development may impair oocyte growth and acquisition of developmental competence.
It is interesting to note that Gleevec attenuated FSH-induced inhibition of BMP-15 expression, suggesting a Kit-mediated action of FSH, even though high FSH did not increase KL-1 or KL-2 mRNA expression levels per se. This suggests that the effect of high FSH to increase KL-1/KL-2 ratio may result in modulation of BMP-15 expression, which does not happen when a lower FSH concentration is used. It was also found that FSH-induced inhibition of BMP-15 mRNA expression still occurred in OGCs treated with Roscovitine, which inhibits granulosa cell proliferation, confirming an FSH-specific effect rather than a consequence of increased granulosa cell/oocyte mRNA ratio in these samples. BMP-15 has previously been shown to suppress FSH receptor expression (44). Thus, a feedback loop can be proposed, whereby FSH-stimulated KL expression results in suppression of BMP-15, causing an increase in FSH receptor expression and increased FSH sensitivity that enhances FSH-induced KL expression. Because BMP-15 has also been reported to be an inhibitor of luteinization (45), precocious down-regulation of expression of this factor may be responsible, at least in part, for impaired oocyte growth in the presence of high levels of FSH.
Dibutyryl cAMP (a membrane-permeable analog of the FSH second messenger) has been shown to increase overall KL mRNA levels (32). In addition, a high concentration of FSH increased KL-1/KL-2 ratio in oocytectomized granulosa cell complex cultured with fully grown oocytes (30). The same study also reported that KL-2 mRNA levels in preantral granulosa cells were decreased when the oocytes were removed from the complexes (30). These results suggest that contact-dependent oocyte-granulosa interaction is likely to be important for regulation of KL-1/KL-2 ratio in preantral follicles; thus, the system for growth of intact OGCs reported here is physiologically relevant.
In the present study, there was a trend for increased oocyte growth in the presence of exogenous BMP-15, although this effect was not statistically significant. In addition, both KL-1 and KL-2 transcripts were up-regulated by BMP-15, but the ratio of KL-1/KL-2 was not affected, supporting the idea that the correct ratio of KL-1/KL-2 expression (decreased KL-1/KL-2) is important for stimulation of oocyte development. Both soluble and membrane-bound KL have similar binding affinities for Kit (46), but because KL-2 is membrane-anchored, it may prevent subsequent down-regulation and internalization of activated Kit, as has been demonstrated in mast cells (47). Indeed, KL-2 has been reported to induce a more persistent activation of Kit receptor kinase than the soluble form of KL (48, 49) and thus is likely to be the more potent isoform for regulation of oocyte growth during early follicular development.
BMP-15 has previously been shown to stimulate mitosis of granulosa cells in vitro from DES-stimulated rats (22). In contrast, the results of our study do not suggest a role for BMP-15 in the promotion of granulosa cell proliferation within growing follicles. However, species and/or stage-dependent differences may account for any discrepancies with previous data. In addition, the previous studies used cultured granulosa cell monolayers, which are likely to behave differently in vitro to the intact OGCs used here. BMP-15 and GDF-9 are thought to play synergistic roles in oocyte survival and folliculogenesis (50). Previously, GDF-9 has been shown to inhibit KL mRNA expression, whereas BMP-15 promotes KL expression (24, 25). The role of GDF-9 in the regulation of KL expression has previously been elucidated in mice (24), whereas information on the effect of BMP-15 on KL expression was derived from studies in rats (25). This first investigation of the effect of BMP-15 on KL expression in mouse follicles is in agreement with previous data showing stimulation of KL expression by BMP-15 in oocyte/granulosa cell coculture in rats (25). That same study also showed that soluble KL inhibits BMP-15 expression in oocytes, thus proposing a negative feedback loop between these factors within the follicle (25). In the present study, when Kit activity was inhibited using Gleevec, there was no effect on GDF-9 expression in OGCs in vitro (data not shown). Interestingly, the data also indicate that FSH-induced up-regulation of KL-2 mRNA expression is not due to a down-regulation of GDF-9 expression.
The importance of gonadotropins and intraovarian paracrine factors in ovarian function has been established but is not well understood. Here, we provide insight into the relationships between FSH and intraovarian paracrine factors during murine follicular development, as well as the relevance of these interactions for oocyte growth and granulosa cell proliferation in vitro. At present, the major benefit of culture systems that support the growth and development of immature oocytes is to advance our knowledge of oocyte development and understanding of the developmental regulation of autocrine/paracrine factors controlling these processes. This knowledge should allow the development of more successful systems for in vitro growth of oocytes for clinical application.
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Acknowledgments |
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Footnotes |
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First Published Online November 11, 2004
Abbreviations: BMP, Bone morphogenetic protein; DMSO, dimethylsulfoxide; GDF, growth/differentiation factor; KL, Kit ligand; OGC, oocyte-granulosa cell complex.
Received June 30, 2004.
Accepted for publication November 3, 2004.
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