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Regular Article |
a The Jackson Laboratory, Bar Harbor, Maine 04609
b Department of Pathology, Baylor College of Medicine, Houston, Texas 77030
ABSTRACT
Oocytes secrete factors that regulate the development of the surrounding granulosa cells in ovarian follicles. KIT ligand (KL) mRNA expression in granulosa cells is thought to be regulated by oocytes; however, the factor(s) that mediate this effect are not known. One candidate is the oocyte-specific gene product growth differentiation factor-9 (GDF-9). This study examined the effect of recombinant GDF-9 (rGDF-9) on steady-state KL mRNA expression levels in preantral and mural granulosa cells in vitro. Furthermore, the study compared the effect of rGDF-9 with that of coculture with oocytes at different developmental stages. As determined by RNase protection assay, both KL-1 and KL-2 mRNA levels in preantral and mural granulosa cells were suppressed by 25–250 ng/ml rGDF-9. Fully grown oocytes also suppressed both KL-1 and KL-2 mRNA expression levels. Partly grown oocytes isolated from 7-, 10-, or 12-day-old mice either had no effect on KL mRNA levels or promoted KL-1 mRNA steady-state expression. It is concluded that GDF-9 is likely to mediate the action of fully grown, but not partly grown, oocytes on granulosa cell KL mRNA expression.
cumulus cells, developmental biology, gametogenesis, granulosa cells, growth factors, oocyte development, ovary, ovum
INTRODUCTION
There is accumulating evidence that mammalian oocytes secrete factors that regulate the development of the surrounding granulosa cells in ovarian follicles. Circumstantial evidence in support of this assertion is provided by studies showing that in antral follicles there are differences in the level of expression of a number of genes in those cells closest to the oocyte, the cumulus cells, compared to the more distant mural granulosa cells. For example, mRNAs encoding LH receptor, KIT ligand (KL), and urokinase plasminogen activator are expressed at higher levels in mural granulosa compared to cumulus cells [1, 2]. On the other hand, mRNAs encoding insulin-like growth factor-1, cyclooxygenase-2, and the prostaglandin EP2 receptor are expressed at higher levels in cumulus than mural granulosa cells [3–5].
Additional evidence that oocytes secrete factors that regulate the development of the surrounding granulosa cells in ovarian follicles is provided by studies showing that oocytes regulate granulosa cell function in vitro. For example, oocytes secrete paracrine signals that enable cumulus cells to undergo cumulus expansion in response to FSH stimulation [6–9] and suppress LH receptor mRNA expression by granulosa cells [10]. Furthermore, oocytes secrete signals that regulate expression by granulosa cells of both KL-1 and KL-2 mRNA, the alternate mRNA splice-variants of KL [11].
The paracrine signaling factors produced by oocytes that regulate follicle development are not well characterized. An advance in our understanding of this area was provided by the observation that growth differentiation factor-9 (GDF-9) is an oocyte-specific gene product in the ovary [12]. Mice not expressing GDF-9 were subsequently shown to exhibit a block in follicle development at the primary stage [13, 14]. In addition, recombinant GDF-9 (rGDF-9) treatment regulates aspects of granulosa cell function in vitro that have previously been shown to be regulated by oocytes [15, 16]. Growth differentiation factor-9 is therefore likely to be an important mediator of the actions of oocytes on granulosa cells. However, it is not known whether GDF-9 regulates KL mRNA expression by granulosa cells. In addition, it is not clear from previous studies whether GDF-9 mediates the actions of oocytes at all stages of their development. Of particular interest, GDF-9 protein has been localized within oocytes of early primary follicles onward [15]. Despite this, partly grown and fully grown oocytes have different effects on a number of aspects of granulosa cell function [9, 11]. The current study was undertaken to 1) examine the action of rGDF-9 on KL mRNA expression by granulosa cells and 2) compare this action with that of oocytes at different stages of development.
MATERIALS AND METHODS
Granulosa Cell and Oocyte Isolation and Culture
Oocytes at five stages of development were used in granulosa cell/oocyte coculture experiments. Fully grown, meiotically competent oocytes were isolated by gentle pipetting of cumulus-oocyte complexes collected from the preovulatory follicles of 22-day-old (C57BL/6J x SJL/J)F1 mice that had been primed with 5 IU eCG 44 to 48 h earlier. Partly grown (PG), meiotically incompetent oocytes were collected from the preantral follicles of 7-day-old mice (PG7), 10-day-old mice (PG10), and 12-day-old mice (PG12) by collagenase digestion as described previously [10]. PG oocytes were also isolated by gentle pipetting of granulosa cell-oocyte complexes collected from the antral follicles of 15-day-old mice (PG15).
Unless otherwise stated, oocyte coculture experiments were undertaken at a concentration of 2 oocytes/µl culture medium for fully grown oocytes; 3 oocytes/µl culture medium for PG15 oocytes; 4 oocytes/µl culture medium for PG12 oocytes; 6 oocytes/µl culture medium for PG10 oocytes; and 9 oocytes/µl culture medium for PG7 oocytes. These concentrations were chosen to be comparable on the basis of oocyte volume [17]. Previous experiments in this laboratory have used oocytes at similar concentrations in coculture with granulosa cells. In such experiments functional responses of granulosa cells have been maintained: for example, fully grown oocytes have been shown to promote cumulus expansion in the presence of FSH [8]. In addition, two-dimensional PAGE analysis of proteins newly synthesized by granulosa cells cultured in the presence of 2 oocytes/µl culture medium have shown that granulosa cells treated in this way exhibit similar expression patterns to control granulosa cells cultured without oocytes [10]. Both of these observations suggest that the oocyte/granulosa cell coculture system used in the current experiments does not negatively affect granulosa cell health. Culture experiments in which oocytes were present were supplemented with 100 µM 3-isobutyl-1-methylxanthine (IBMX; Aldrich, Milwaukee, WI) to maintain fully grown oocytes at the germinal vesicle stage and to maintain common experimental treatment conditions when partly grown oocytes were used. Initial studies found no detectable effect of IBMX on KL-1 and KL-2 mRNA expression (data not shown). Depending on the individual experiment, oocytes were cocultured with either preantral granulosa cells or highly differentiated granulosa cells expressing markers of the preovulatory mural granulosa cell phenotype.
Granulosa cells from preantral follicles were cultured as described previously [11]. Briefly, preantral granulosa cell-oocyte complexes were isolated from 12-day-old mice by collagenase digestion [18] and then cultured following removal of the oocyte (oocytectomy), in 48-well tissue culture dishes (Corning Inc., Corning, NY) for 48 h. Approximately 250 oocytectomized preantral granulosa cell-oocyte complexes were cultured in 150 µl Waymouth (MB752/1) culture medium supplemented with 0.23 mM pyruvic acid, 50 mg/L streptomycin sulfate, 75 mg/L penicillin G (Sigma Chemical Co., St. Louis, MO), 3 mg/ml BSA (ICN Biochemicals, Aurora, OH), and ITS (insulin: 5 µg/ml; transferrin 5 µg/ml; selenium 5 ng/ml; Collaborative Research, Inc., Bedford, MA). Either denuded oocytes or rGDF-9 were added to the 150-µl drops at the same time as the target granulosa cells.
Granulosa cells of a more highly differentiated phenotype from antral follicles were also cultured as described previously [10, 19]. In this culture system, granulosa cells were isolated from the antral follicles of 20-day-old mice and cultured in a total volume of 150 µl M199 culture medium plus BSA using 48-well tissue culture dishes. Cells were treated with recombinant human follicle stimulating hormone (FSH; 0.025 IU/ml; Organon, Oss, Netherlands) and testosterone (1 ng/ml; Sigma). This treatment has previously been shown to result in high levels of KL and LH receptor mRNA expression, both markers of a mural granulosa cell phenotype, after 48 h of culture [10, 11].
Recombinant GDF-9: Preparation and Use
Mouse rGDF-9 conditioned medium was prepared as described previously [15]. Briefly, a full-length mouse rGDF-9 cDNA was cloned into the expression plasmid pHTop containing the cleavage enzyme PACE before transfection into Chinese hamster ovary (CHO) cells under standard conditions. Cells expressing rGDF-9 were selected in 0.02 µM methotrexate before clonal expansion in 0.05 µM methotrexate. Once CHO cells had reached confluency, the cells were washed twice with PBS and incubated with Opti-MEM (Gibco BRL, Gaithersburg, MD) containing 1 mg/ml heparin (Sigma) for 48 h. Following collection of the conditioned media, the concentration of mouse rGDF-9 was estimated as previously described [15]. For experiments examining the effect of rGDF-9, appropriate volumes of rGDF-9 conditioned medium were added to cultures, replacing an identical volume of Waymouth/M199 culture medium. Control conditioned medium (CCM) was produced using CHO cells transfected with pHTop-PACE expression vector alone (no GDF-9). The CCM was included in treatment groups that received less or no rGDF-9 conditioned medium, so that treatment groups contained equal volumes of conditioned medium.
Ribonuclease Protection Assay
The steady-state levels of KL-1 and KL-2 mRNA expression were assessed by RNase protection assay as described previously [11]. The KL probe was generated from a KL-1 cDNA (generously provided by Dr. Peter Besmer) linearized with Spe1 (Roche, Indianapolis, IN). This enzyme cut 62 base pairs past an exon boundary that is present in KL-1 and absent in KL-2, thereby enabling the two transcripts to be identified by RNase protection assay [20]. Antisense Rpl-19 RNA probe was included in all assays in order to allow differences in the quantity of mRNA between samples to be ameliorated mathematically [10, 21].
Statistical Analysis
Experiments were repeated independently between three and five times. The effect of treatment on KL-1 and KL-2 mRNA levels was assessed by ANOVA, with data for the two transcripts analyzed separately. Differences in background density between assays as quantified by the phosphor imaging system were large, thereby generating a high degree of interassay variation in average phosphor imaging units. For this reason, the data for individual replicates were first normalized so that the mean KL mRNA levels for each replicate was equal to one. The effect of treatment on the differential steady-state expression of KL-1 and KL-2 mRNA was subjected to ANOVA using the ratio of KL-1 to KL-2 before normalization. When a significant F-ratio was defined by ANOVA, groups were compared using Fisher's protected least significant difference post-hoc test.
RESULTS
Effect of rGDF-9 and Oocytes on Steady-State KL mRNA Expression in Preantral Granulosa Cells
Steady-state KL mRNA expression levels in preantral granulosa cells have previously been shown to be responsive to the effects of oocytes in coculture. Therefore, in the first experiment the effect of rGDF-9 conditioned medium on KL mRNA expression in preantral granulosa cells was examined. As described in the Materials and Methods, in this and subsequent experiments using rGDF-9 conditioned medium identical volumes of conditioned medium were used for all treatment groups, with CCM replacing rGDF-9 conditioned medium in those groups receiving less or no rGDF-9 conditioned medium. The results from the first experiment show that 25–250 ng/ml rGDF-9 suppressed steady-state KL-1 and KL-2 mRNA expression levels in the preantral granulosa cell culture system (Fig. 1). Maximal suppression of KL-1 mRNA occurred at concentrations of 100–250 ng/ml rGDF-9, whereas maximal suppression of KL-2 mRNA was found at 250 ng/ml rGDF-9. At 250 ng/ml, rGDF-9 significantly increased the ratio of KL-1:KL-2 mRNA.
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In the next experiment, the effect of 250 ng/ml rGDF-9 was compared with that of oocytes at two developmental stages (Fig. 2). We have previously shown that PG12 oocytes promote KL-1 and KL-2 mRNA expression in coculture with preantral granulosa cells, while fully grown oocytes suppress KL-1 and KL-2 mRNA expression and significantly increase the ratio of KL-1:KL-2 mRNA. However, in the current experiment, oocyte cocultures also contained CCM. Under these conditions, PG12 oocytes had no effect on KL expression, while fully grown oocytes suppressed both KL-1 and KL-2 mRNA expression but had no effect on the ratio of KL-1:KL-2 mRNA expression. As in the first experiment, 250 ng/ml rGDF-9 suppressed both KL-1 and KL-2 mRNA expression and increased the ratio of KL-1:KL-2 mRNA. The magnitude of the suppression of KL mRNA expression by 250 ng/ml rGDF-9 was similar to that of two fully grown oocytes/µl.
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To examine whether the lack of effect of fully grown oocytes on the ratio of KL-1:KL-2 mRNA was due to inclusion of CCM in the cultures, a study was made of the effect of fully grown oocytes at different concentrations in the presence or absence of CCM. The results show that, in the absence of CCM, a concentration of fully grown oocytes of 0.5 oocytes/µl is sufficient to increase significantly the ratio of KL-1:KL-2 mRNA (Fig. 3). However, in the presence of CCM, fully grown oocyte concentrations as high as 4.5/µl failed to alter the ratio of KL-1:KL-2 mRNA expression.
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The lack of effect of PG12 oocytes on KL mRNA expression in the second experiment was examined by directly comparing the effect of PG12 oocytes in the presence or absence of CCM. Surprisingly, there was no effect of PG12 oocytes either with or without the inclusion of CCM (data not shown). Because this result contradicts previous findings from our group and others [11, 22], we hypothesized that this difference may be due to subtle differences in the developmental stage of oocytes used in the different studies. We therefore compared the effect of oocytes at various developmental stages before the attainment of full size. No CCM was used in this experiment. The PG7 and PG12 oocytes had no effect on KL mRNA expression by cultured preantral granulosa cells, and PG15 oocytes suppressed KL mRNA expression (Fig. 4). However, PG10 oocytes promoted KL-1 mRNA expression, supporting the hypothesis that differences between studies in terms of the effect of PG oocytes on KL mRNA expression levels may be due to subtle differences in the developmental stage of the oocytes used.
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Effect of rGDF-9 and Oocytes on Steady-State KL mRNA Expression in Mural Granulosa Cells
The effect of rGDF-9 was next tested on mural granulosa cells. Concentrations of 25–250 ng/ml rGDF-9 suppressed both KL-1 and KL-2 mRNA expression. Maximal suppression of both KL-1 and KL-2 mRNA expression occurred at concentrations of 100–250 ng/ml rGDF-9. Unlike in the preantral granulosa cell culture system, there was no effect of rGDF-9 on the KL-1:KL-2 ratio within these concentration ranges. In the final experiment, the effects of rGDF-9 and fully grown oocytes on KL mRNA expression in the mural granulosa cell culture system were compared (Fig. 5). The results show that, at a concentration of 2/µl, fully grown oocytes had a similar effect to 250 ng/ml rGDF-9, and that control-conditioned medium had no effect on KL mRNA expression levels in this system. Consistent with our previous study [11], the suppressive action of fully grown oocytes was less in the mural granulosa cell culture system than in the preantral granulosa cell culture system (mean reduction [±SEM] in KL mRNA levels as a result of fully grown oocyte coculture: mural granulosa cells—51.4 ± 0.1%; preantral granulosa cells—72.6 ± 0.1%).
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DISCUSSION
Results from the current study show that rGDF-9 suppresses steady-state KL-1 and KL-2 mRNA expression levels in both preantral and mural granulosa cells in vitro. Fully grown oocytes also suppressed KL-1 and KL-2 mRNA expression in these culture systems. However, partly grown oocytes isolated from 7- to 12-day-old mice either had no effect on KL mRNA expression or promoted KL-1 expression. Given the similarity between the action of rGDF-9 and fully grown oocytes, the results suggest that the actions of fully grown oocytes on KL mRNA expression are mediated by GDF-9. In line with this conclusion, ongoing studies in our laboratories using medium conditioned by fully grown oocytes have confirmed that oocytes at this stage of development secrete GDF-9 in vitro. Despite this, it is possible that other factors secreted by fully grown oocytes may also participate in the regulation of KL mRNA expression by granulosa cells.
The observation that GDF-9 may mediate the actions of fully grown but not PG oocytes is also supported by studies that show that both rGDF-9 and fully grown oocytes, but not PG12 oocytes, promote cumulus expansion in vitro in the presence of FSH [9, 15]. Similarly, fully grown, but not PG12, oocytes suppress urokinase plasminogen activator production by granulosa cells, and rGDF-9 suppresses urokinase plasminogen activator mRNA expression levels in granulosa cells [15, 23].
The current study also provides new information that helps to explain aspects of the granulosa cell KL expression patterns observed in vivo. Previously, it has been shown that KL expression is low in the preantral (early secondary) follicles of 7- to 8-day-old mice. Then expression increases dramatically in the preantral (late secondary) follicles found in 12- to 13-day-old mice before declining in the early antral follicles of 15-day-old mice [11, 24]. The results presented here indicate that KL levels in early secondary preantral follicles are unlikely to be low due to a suppressive activity of PG7 oocytes. However, because PG15 oocytes suppressed KL mRNA expression in the preantral granulosa cell culture system, these results suggest that at the early antral stage of follicle development, oocytes may play an important role in suppressing steady-state KL mRNA expression levels in granulosa cells.
In the current study, coculture with fully grown oocytes in the absence of CCM, as well as treatment with rGDF-9, increased the ratio of KL-1:KL-2 mRNA expressed by preantral granulosa cells. This finding is interesting given that Kitl mutations that have either no membrane-bound forms of KL (Kitld), or no KL-1 (KitlKL-2), show very different phenotypes [1, 2], suggesting that KL-1 and KL-2 have different roles in vivo. However, because fully grown oocytes do not come into contact with preantral granulosa cells in vivo the significance of this finding in terms of follicle development is open to question. Unlike when CCM was absent from the coculture, in the presence of CCM fully grown oocyte concentrations as high as 4.5/µl failed to increase the ratio of KL-1:KL-2 mRNA. This finding may indicate that the actions of GDF-9 and of fully grown oocytes in differentially regulating KL-1 and KL-2 mRNA expression are mediated differently. However, the confounding effects of the CCM prevent firm conclusions being drawn in this regard.
The conclusion that GDF-9 may mediate the actions of fully grown but not partly grown oocytes is particularly intriguing in the light of evidence that oocytes from primary follicles onward produce levels of GDF-9 protein that are detectable within oocytes using immunohistochemistry [15]. Furthermore, the Gdf9null mouse exhibits a block in follicle development at the late primary follicle stage [13, 14]. There is therefore good evidence that at the primary follicle stage the actions of oocytes are mediated, at least in part, by GDF-9. One explanation for these observations and the results from the current study is that GDF-9 is produced by all oocytes, but only secreted by oocytes at the primary follicle stage and when fully grown. On the other hand, the possibility that oocytes at all developmental stages from primary follicles onward produce and secrete GDF-9 cannot be ruled out.
Indeed, support for the latter possibility comes from the observation that rGDF-9, fully grown oocytes, and PG12 oocytes all suppress granulosa cell LH receptor mRNA expression in vitro [10, 15, 16]. Although the magnitude of this effect is lower for PG12 oocytes than for fully grown oocytes, this finding does suggest that GDF-9 mediates the action of PG12 oocytes on LH receptor mRNA expression. This being the case, the possibility that GDF-9 secretion is restricted to narrow developmental stages of the oocyte is not supported. Furthermore, the evidence suggests that there may be a degree of complexity in the signaling factors produced by the oocyte. In fact, two-dimensional PAGE studies confirm that oocytes secrete a range of different proteins [25]. Under these circumstances, an additional oocyte-secreted factor that in some way antagonizes or neutralizes the action of GDF-9 can be postulated. It is also worth speculating that the lack of an effect of PG12 oocytes on KL mRNA expression in the current, but not previous, studies [11, 22] may reflect the existence of a delicate balance between oocyte-secreted factors that either promote or suppress KL mRNA expression in granulosa cells.
Given the evidence presented here that GDF-9 down-regulates KL mRNA expression in granulosa cells, it is interesting to note that in the Gdf9null mouse, ovarian KL mRNA levels are about 32-fold higher than in wild-type ovaries [4]. This finding provides in vivo support for the current observation that GDF-9 suppresses KL mRNA levels in vitro. Combined with evidence that KL is an important factor regulating oocyte development [4, 22, 24], the data provide convincing evidence of a regulatory feedback loop between the oocyte and surrounding granulosa cells involving GDF-9 and KL/KIT signaling.
A potential role for such a regulatory feedback loop may be the control of oocyte growth, because there is evidence from both in vivo and in vitro studies that KL stimulates this process [14, 15, 22, 26]. Such a feedback loop would be hypothesized to occur in this fashion: During preantral follicle development when the majority of oocyte growth occurs, the growing oocytes promote (or at the least do not suppress) KL production by granulosa cells. The KL produced by granulosa cells then stimulates continued oocyte growth. In antral follicles, following further oocyte development, GDF-9 secretion from the oocyte becomes effective at suppressing KL production by granulosa cells. As a result the stimulus for continued oocyte growth is reduced, and growth slows, and eventually halts. Although unlikely to be a complete picture of the oocyte-granulosa cell interactions regulating oocyte growth [27], this hypothesis provides a solid conceptual framework for future studies.
In conclusion, the results from the experiments reported here indicate that rGDF-9 suppresses steady-state KL mRNA expression levels in granulosa cells in vitro. Because this action was similar to that of coculture with fully grown but not PG oocytes, these results further suggest that GDF-9 is a factor that mediates the actions of fully grown, but not PG, oocytes on KL mRNA expression by granulosa cells.
ACKNOWLEDGMENTS
We thank Dr. Martin Matzuk, of Baylor College of Medicine for the gift of the recombinant GDF-9. We are grateful to Marilyn O'Brien and Jennifer Aimone for technical support; Dr. Peter Besmer for the gift of the KL-1 cDNA; Organon for FSH; and Drs. Wes Beamer, Tom Gridley, and Martin Matzuk for their helpful comments during manuscript preparation.
FOOTNOTES
1 This research was funded by the National Institute of Child Health and Human Development, National Institutes of Health, through grant HD23839. 
2 Correspondence. FAX: 207 288 6073; jje{at}jax.org
Accepted: June 28, 2000.
Received: April 11, 2000.
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