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Regular Article |
a The Reproductive Medicine Unit, Department of Obstetrics and Gynaecology, University of Adelaide, The Queen Elizabeth Hospital, Woodville, 5011, Adelaide, Australia
b Department of Obstetrics and Gynaecology, First Affiliated Hospital of Sun Yet Sen University of Medical Sciences, Guang Zhou, China
ABSTRACT
Cumulus cells and mural granulosa cells (MGC) are phenotypically different and there is now evidence suggesting that the oocyte plays an active role in determining the fate of follicular somatic cells. This study investigates the role of oocyte-secreted factor(s) in the regulation of the growth and differentiation of cumulus and MGC. Bovine cumulus-oocyte complexes (COC) and MGC were cultured with various hormones for 18 h followed by a further 6-h pulse of [3H]thymidine as an indicator of follicular cell DNA synthesis. The COC incorporated 11 to 14 times more [3H]thymidine than MGC in either the absence or presence of 50 ng/ml insulin-like growth factor (IGF)-I. Purified porcine FSH (450 ng/ml) added together with IGF-I marginally increased 3H incorporation in MGC relative to IGF-I alone but dramatically decreased incorporation in COC sixfold. Conversely, mean progesterone production in the presence of IGF-I + FSH was 13-fold higher from MGC than from COC, confirming a distinctive phenotype of cumulus cells. However, this phenotype was found to be dependent on the presence of the oocyte, as microsurgical removal of the oocyte (oocytectomy) resulted in an 11-fold decrease in [3H]thymidine incorporation in cumulus cells treated with IGF-I, elimination of the inhibitory effect of FSH on IGF-I-stimulated DNA synthesis, and led to a 2-fold increase in progesterone production in medium with IGF-I and FSH. All of these markers were completely restored to COC levels when oocytectomized complexes were cocultured with denuded oocytes (DO) at a concentration of 0.5 oocytes/µl, demonstrating that oocytes secrete a soluble factor(s) that promotes growth and attenuates cumulus cell progesterone secretion. In the presence of IGF-I, [3H]thymidine incorporation in MGC increased ninefold above control levels with the addition of DO. The addition of FSH to IGF-I-increased 3H counts in MGC, however, led to a decrease in counts in MGC + DO as is also observed in COC. Furthermore, progesterone production was halved when DO were added to MGC cultures, most notably in the presence of IGF-I and/or FSH. These results provide further evidence that MGC and cumulus cells have distinctive phenotypes and that the oocyte is responsible for some of the characteristic features of cumulus cells. Bovine oocytes secrete a soluble factor(s) that simultaneously promotes growth and attenuates steroidogenesis in follicular somatic cells.
cumulus cells, follicle, follicular development, FSH, granulosa cells, growth factors, ovum
INTRODUCTION
Mammalian ovarian follicles consist of layers of somatic cells surrounding the germ cell or the oocyte. As follicles grow and an antrum is formed, the somatic cells separate into two distinct subtypes: the cumulus cells, those surrounding and in intimate metabolic contact with the oocyte; and the mural granulosa cells (MGC), the cells lining the follicle forming, in the cow, a stratified epithelium with the basal lamina. Apart from anatomical differences, cumulus cells and MGC are functionally distinct (reviewed by Eppig et al. [1]). Cumulus cells produce hyularonic acid and undergo cumulus cell expansion in response to FSH, while MGC do not. Mural granulosa cells are more steroidogenically active than cumulus cells, as indicated by higher levels of mRNA expression for steroidogenic enzymes such as cholesterol side-chain cleavage cytochrome P450 [2] and cytochrome P450 aromatase [3] in rat cumulus cells. Levels of mRNA expression for a variety of growth factors and hormone receptors also differ between the two cell types; for example, rodent cumulus cells express lower mRNA levels for the LH receptor [4], kit ligand [5], and plasminogen activator [6] and higher levels for insulin-like growth factor I (IGF-I) [7] than do MGC. The two distinct cell phenotypes serve differing functions throughout folliculogenesis: cumulus cells play an essential role in the normal growth and development of the oocyte, while MGC serve primarily an endocrine function and support growth of the follicle. Furthermore, following ovulation, granulosa cells go on to an endocrine role by undergoing terminal differentiation to luteal cells, while cumulus cells mucify and are ovulated with the oocyte aiding in ovum pickup and the sperm acrosome reaction. Although some of the functional differences between the two classes of follicular somatic cells are well characterized, which factors regulate the developmental pathway of these cells is less well understood.
While the critical role follicular somatic cells play in oocyte development has been well demonstrated, it is now increasingly evident that oocytes play an active role in the development of follicular cells. Oocytes were first implicated in the regulation of follicular cells when el-Fouly et al. [8] removed oocytes from preovulatory rabbit follicles resulting in spontaneous luteinization. It has since been demonstrated that oocyte-derived factors modulate a broad range of follicular cell functions, including cumulus cell mucification and hyaluronic acid production [9–11], cellular proliferation [12], steroid synthesis [13], urokinase plasminogen activator (uPA) [6] and LH receptor (LHR) [14] mRNA expression, kit-ligand (KL) mRNA expression [15], and synthesis of inhibins [16]. Although none of these oocyte-derived factor(s) has been characterized, many of the effects of oocytes on follicular cells can be mimicked by members of the transforming growth factor (TGF) superfamily, in particular by TGFß1 and growth differentiation factor 9 (GDF-9) [17, 18]. Indeed, oocyte-derived GDF-9 is essential for normal development as GDF-9-deficient female mice are infertile due to a complete block in folliculogenesis at the type 3b stage [19]. These mice exhibit downregulated ovarian mRNA expression of activin ßB, follistatin, and cyclooxygenase 2 (COX-2), and substantial upregulation of KL expression [20]. Increased granulosa cell KL production in GDF-9 knockout mice may account for the abnormally large oocytes found in these mice [21]. Addition of recombinant GDF-9 to cultured rat follicular cells increases follicle growth and inhibin 
production [22] and induces COX-2, hyaluronan synthase 2 (HAS2), steroidogenic acute regulatory protein (StAR) mRNA synthesis, but suppresses uPA and LHR mRNA synthesis in mouse granulosa cells [18]. These findings indicate that oocyte-secreted factor(s) play a critical and multifaceted role not only in the normal processes of folliculogenesis but also in directing the somatic cells to regulate its own development.
To date, very little is known of the requirement of oocyte-derived factors for normal follicular development in nonrodent species. What little is known indicates that there may be important differences between rodent and nonrodent species. For example, rodent cumulus cells are unable to undergo mucification in response to FSH in the absence of the cumulus expansion enabling factor (CEEF) secreted by oocytes. Although bovine and porcine oocytes produce CEEF, cumulus expansion may be independent of this factor [23–26]. However, like murine oocytes, porcine oocytes are able to regulate cumulus cell progesterone and estradiol synthesis [27]. Bovine oocytes have also been shown to promote rat granulosa cell proliferation [28] and dimeric inhibin A and B secretion [16]. As there is a relatively poor understanding of bovine oocyte-secreted factor(s), the objectives of this study were to investigate the role of bovine oocytes in modulating cumulus and MGC functions in terms of proliferative and steroidogenic capacities.
MATERIALS AND METHODS
Collection of Follicular Cells and Culture Conditions
Bovine ovaries were collected from a local abattoir and transported to the laboratory in warm saline. Follicular cells were collected from antral follicles (~2–6 mm), the stage of development and atresia status of which were unknown. Follicles were mechanically aspirated using an 18-gauge needle and a 10-ml syringe into 25 mM Hepes-buffered tissue culture medium-199 (H-TCM; ICN, Costa Mesa, CA) supplemented with 2 mM Na pyruvate (Sigma, St. Louis, MO), 100 U/ml penicillin G (Sigma), and 100 mg/ml streptomycin sulfate (Sigma) and either 5% fetal calf serum (FCS; Trace Scientific, Clayton, Australia; experiments 1 and 2) or 0.2 mg/ml polyvinyl alcohol (PVA; Sigma; experiment 3). With the aid of a dissecting microscope, completely enclosed cumulus-oocyte complexes (COC) with an evenly pigmented cytoplasm were selected from the aspirate. Complexes were washed twice in each of H-TCM and bicarbonate-buffered TCM (B-TCM) with PVA, Na pyruvate, and antibiotics, before groups of 14–16 COC were transferred to individual wells of 96-well flat-bottomed plates (Falcon, Franklin Lakes, NJ). Cells were treated with or without 50 ng/ml recombinant human insulin-like growth factor I (IGF-I; Gro-pep, Adelaide, Australia) and/or 450 ng/ml (equivalent to 2 µg/ml NIH-pFSH-P1) purified porcine pituitary FSH (Folltropin V; Vetrepharm, London, Canada). Depending on treatments of individual experiments, hormones and B-TCM were added to wells to make up the final volume of 250 µl (experiment 1) or 125 ml (experiments 2 and 3). Cells were cultured in an atmosphere of 39°C, 96% humidity in 5% CO2 in air for 18 h, followed by a further 6-h pulse of 0.8 µCi tritiated thymidine ([3H]thymidine, ICN). At the completion of culture a fraction of the culture media was collected and frozen (-20°C) for steroid analysis.
Experiment 1—The Effect of Oocytectomy on Cumulus Cell Function
To examine if the oocyte is required for normal cumulus cell proliferative and steroidogenic activities, the contents of each oocyte were microsurgically removed from the COC (oocytectomy) as previously described [9]. The resulting oocytectomized complexes (OOX) consist of a hollow zona pellucida surrounded by several layers of cumulus cells. Groups of 14–16 COC or OOX were assigned to the following treatments: 1) control, 2) FSH, 3) IGF-I, or 4) FSH + IGF-I, and each treatment was carried out in duplicate wells of 250 µl. Seven replicates of this experiment were conducted using a total of 845 and 841 COC and OOX complexes, respectively.
Experiment 2—The Effect of Coculturing Denuded Oocytes with Oocytectomized Complexes
To determine if a soluble oocyte-secreted factor(s) is responsible for the high capacity of cumulus cells to incorporate [3H]thymidine, OOX complexes were cocultured with denuded oocytes (DO) and compared to COC. Oocytes were mechanically denuded of cumulus cells by repeated passage through a fine-bore fire-polished glass pipette in H-TCM + 5% FCS. After two washes in B-TCM + PVA, 62 DO were added to wells containing OOX complexes in a total of 125 µl (0.5 DO/µl). A 3 x 2 factorial experiment was conducted whereby either OOX, OOX + DO, or COC were treated with either IGF-I or IGF-I + FSH. Treatments were carried out in single wells per experiment and the experiment was replicated five times.
Experiment 3—Comparison of MGC to COC and the Effect of Denuded Oocytes on MGC
This experiment was conducted to allow a direct comparison between COC and MGC in terms of their relative abilities to incorporate [3H]thymidine and synthesize progesterone. After removal of any large pieces of stromal and thecal tissue from follicular aspirates, MGC were collected by centrifugation and were washed twice in B-TCM + PVA. A fraction of the MGC was dissociated (see below) for cell counts and then 50 000 undissociated MGC were added to wells containing hormones ± 62 DO to make up a final volume of 125 µl (0.4 x 106 MGC/ml and 0.5 DO/µl). The MGC, MGC + DO, and COC were then cultured in the following treatments: 1) control, 2) FSH, 3) IGF-I, or 4) IGF-1 and FSH and the experiment replicated five times.
Determination of Cell Numbers
Mural granulosa cells and COC (groups of 30) were dissociated by incubating for 5 min in H-TCM supplemented with 9.1 mM EGTA (Sigma), pelleted, resuspended, incubated for a further 10 min in H-TCM with 2.1 mM EGTA and 0.5 mM sucrose (BDH Chemicals, Kilsyth, Australia), centrifuged, resuspended in a known volume of H-TCM 199 + 0.2% (w:v) BSA (Sigma), and finally counted using a hemacytometer [29]. Mural granulosa cells and COC 3H counts and progesterone results are expressed per 1000 cells.
Assessment of Cell Cultures
Culture supernatants were assayed for progesterone using an RIA kit (Diagnostic System Laboratories, Webster, TX) in accordance with the manufacturer's instructions. This kit that utilizes an 125I-labeled progesterone tracer has a sensitivity of 0.25 pmol/ml and an intraassay coefficient of variability of 5.4%. Following culture COC and MGC were harvested using a Tomtec Harvester 96 onto a filtermat and beta particle emission by incorporated [3H]thymidine was assessed using a Wallac microbeta counter after exposure to scintillation fluid (Fisions, Leies, UK). The 3H counts were used as a measure of incorporation of [3H]thymidine into follicular cell DNA and is indicative of the proportion of cells in S-phase, thereby providing an indicator of DNA synthesis.
Data Analysis
A Student's t-test was performed to determine differences in 3H incorporation and progesterone secretion by COC and MGC (Fig. 1). Differences between treatments in the amount of [3H]thymidine incorporated and progesterone produced were analyzed by generalized linear models analysis (SPSS package) and Tukey-Kramer tests. Where variances were found to be heterogenous between variables (Levene test), analyses were performed on log-transformed data. A P value of <0.05 was considered statistically significant.
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RESULTS
A direct comparison of COC to MGC revealed contrasting functional characteristics, with COC exhibiting a much higher DNA synthetic capacity and a lower capacity to produce progesterone (Fig. 1). The COC incorporated 17 times as much [3H]thymidine as MGC but secreted 10 times less progesterone.
Experiment 1—The Effect of Oocytectomy on Cumulus Cell Function
To determine if the characteristic cumulus cell phenotype is dependent on the presence of the oocyte, the contents of the oocyte were microsurgically removed from the COC (oocytectomy). Oocytectomy resulted in loss of the phenotype typical of cumulus cells with the cells exhibiting characteristics more typical of MGC, as demonstrated by a dramatic loss in proliferative capacity and an increase in the secretion of progesterone (Fig. 2). In the absence of hormones, oocytectomy resulted in a 5-fold (P < 0.001) reduction in the incorporation of [3H]thymidine compared to COC, and an 11-fold (P < 0.001) reduction in the presence of IGF-I. Follicle-stimulating hormone inhibited IGF-I-stimulated DNA synthesis in COC, as is characteristically observed in bovine COC [30], although this was not evident in OOX complexes. Synthesis of DNA in OOX complexes was not significantly different when they were cultured with IGF-I alone versus with IGF-I in combination with FSH, which is more typically observed for MGC. Progesterone production was also higher in OOX complexes compared to COC (P < 0.05), most notably in the presence of IGF-I + FSH where levels were approximately double those of COC.
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Experiment 2—The Effect of Coculturing Denuded Oocytes with Oocytectomized Complexes
To ensure that the marked decrease in the capacity of cumulus cells to incorporate [3H]thymidine after oocytectomy can be attributed to the absence of the oocyte and was not due to the trauma associated with the procedure, OOX complexes were cocultured with DO and compared to COC. The diminished capacity of OOX complexes to synthesize DNA and increased capacity to secrete progesterone was completely restored to COC levels by coculturing OOX complexes with DO at a concentration of 0.5 oocytes/µl (Fig. 3). Coculture with DO led to a 10-fold increase in [3H]thymidine incorporation in OOX complexes (P < 0.001) to levels comparable to that of COC. Furthermore, the typical FSH inhibition of IGF-I-stimulated DNA synthesis in COC was also observed in OOX + DO (i.e., the addition of FSH to IGF-I cultures led to a 10-fold decrease in 3H counts in both OOX + DO complexes and in COC, compared to in the presence of IGF-I alone). Coculturing DO with OOX complexes also decreased (P < 0.001) progesterone secretion by OOX complexes to COC levels (Fig. 3B). These results indicate that the characteristic nature of cumulus cells is dependent to a large degree on the presence of the oocyte, specifically on a soluble factor(s) secreted by oocytes.
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Experiment 3—Comparison of MGC to COC and the Effect of Denuded Oocytes on MGC
In this experiment, COC and MGC were compared in terms of their proliferative and steroidogenic capacities, and the response of MGC to cocultured DO was examined. Regardless of treatment, the functions of COC were found to contrast that of MGC with a much higher capacity to incorporate [3H]thymidine and a lower capacity to produce progesterone (Fig. 4). Incorporation of [3H]thymidine in COC was on the order of 2-fold (IGF-I + FSH) to 17-fold (IGF-I) higher than in MGC (Fig. 4A). Thymidine incorporation in COC was unaffected by treatment with FSH but stimulated 7.5-fold by IGF-I (P < 0.001), and this enhanced IGF-I effect was significantly (P < 0.001) inhibited by addition of FSH. Insulin-like growth factor I also significantly (P < 0.001) increased DNA synthesis in MGC above control levels; however, in contrast to COC, FSH in combination with IGF-I did not lower 3H counts in MGC but instead marginally increased them. The pattern of progesterone secretion by the two cell types approximates a mirror image to the incorporation of [3H]thymidine (Fig. 4B). Progesterone secretion by MGC was significantly (P < 0.001) higher than by COC in all treatments, most notably in the presence of FSH + IGF-I where levels were 10-fold above COC levels. In MGC but not in COC, progesterone secretion was marginally increased (P > 0.05) above control levels by either FSH or IGF-I, and in combination the two hormones had an additive effect.
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In the same experiment, MGC were also cocultured with DO to determine if the proliferative effects of DO on cumulus cells could also be replicated in MGC. The addition of 0.5 DO/µl to MGC resulted in a significant (P < 0.001) enhancement of MGC DNA synthesis across all treatments, ranging from a 1.6-fold (FSH treatment) to a 9-fold increase (IGF-I), although these levels were still substantially lower than COC levels (Fig. 4A). Notably, the marked IGF-I-stimulated increase in DNA synthesis in MGC with DO was inhibited with the addition of FSH. This pattern of 3H incorporation is a characteristic feature of bovine COC (also observed in OOX + DO; experiment 2) but not of MGC, providing further evidence that exposing MGC to DO bestows cumulus cell characteristics on MGC. Progesterone production by MGC was also significantly (P < 0.001) attenuated by coculture with DO, with levels being approximately halved in all treatments (Fig. 4B).
DISCUSSION
This study has demonstrated that bovine cumulus cells and MGC have distinct and opposing capacities in terms of their abilities to synthesize DNA and secrete progesterone, and we have shown for the first time that the distinct bovine cumulus cell phenotype is dependent to a large extent on a soluble oocyte-secreted factor(s). In the absence of the oocyte-secreted factor(s), cumulus cells acquire some of the characteristics of the MGC phenotype. On the other hand, MGC respond to oocyte-secreted factor(s) by becoming more cumulus cell-like, indicating that the factor(s) is able to at least attenuate, if not partially reverse, the pathway of development to the MGC phenotype. These results support the hypothesis originally investigated in the mouse (reviewed by Eppig et al. [1]) that the default pathway of follicular cell development is toward the granulosa cell phenotype, and that oocytes obstruct the pathway of MGC differentiation and actively promote development of the cumulus cell phenotype.
Two features dramatically distinguish the function of cumulus cells from mural granulosa cells: their higher proliferative capacity and their lower steroidogenic capacity. We have shown that removal of the oocyte from the COC by oocytectomy results in cumulus cells spontaneously undergoing a rapid conversion to the MGC phenotype, markedly decreasing their proliferative capacity and becoming distinctly more steroidogenic. Most notable in cumulus cells following oocytectomy is the loss of capacity to synthesize DNA in response to IGF-I stimulation and an increase in the production of progesterone in the combined presence of IGF-I and FSH. Both these responses are features more characteristic of MGC. In restoring these values to COC levels by coculturing oocytectomized complexes with DO, we have demonstrated that the characteristic bovine cumulus cell phenotype is dependent to a large degree on a soluble factor(s) secreted by the oocyte. This confirms previous work in the mouse using different markers of the cumulus cell phenotype [6, 14].
In this study MGC responded to oocyte-secreted factor(s) by producing a phenotype intermediate between COC and MGC. This was evident in terms of both DNA synthesis and progesterone production, and this response was consistent regardless of treatment in vitro. Because oocyte-secreted factor(s) function in a dose-dependent manner [9, 13, 14, 27, 28], it is likely that culturing MGC with a higher concentration of DO would have shifted the MGC phenotype further toward the COC phenotype. It is noteworthy that not only did the addition of DO to MGC substantially enhance IGF-I-stimulated MGC DNA synthesis, but also the further addition of FSH reduced this response to COC levels. Follicle-stimulating hormone inhibition of IGF-I-stimulated DNA synthesis is a feature characteristic of bovine COC and not MGC and is associated with FSH-induced changes in extracellular matrix that occur with expansion and mucification [31]. Hence oocyte-secreted factor(s) have the effect of bestowing cumulus cell characteristics on MGC. These results demonstrate that bovine MGC have receptors for the oocyte-secreted factor(s) and that the developmental progression of MGC to a differentiated state can be slowed by these factors. Presumably in vivo, the highly differentiated MGC at the periphery of large antral follicles are either not exposed to oocyte factor(s), or have some mechanism to counter their effects. It is conceivable that a gradient of oocyte-secreted factor(s) exists across a follicle, and that generally the factor(s) act locally on cumulus cells only. This hypothesis is supported by observations by Hirshfield et al. [32] that radiolabeling in antral follicles of rats infused with [3H]thymidine is highest in the discus proligerus (the cumulus cells and the MGC supporting the COC) and is lowest in the MGC on the opposing side of the follicle. This gradient across an antral follicle could be established as a result of cumulus cells sequestering the limited secretion of oocyte factor(s), the oocyte factor(s) having a very short half life, or alternatively there may be some neutralizing activity in follicular fluid.
The ability of oocytes to promote follicular cellular proliferation was first directly demonstrated in the mouse [12]. This study showed that oocytes secrete a soluble factor(s) that promotes growth in the relatively undifferentiated granulosa cells of preantral follicles as well as the more differentiated MGC and cumulus cells. More recently, it was demonstrated that bovine oocytes produce a labile factor(s) capable of promoting DNA synthesis in rat granulosa cells in a dose-dependent fashion [28]. This growth-promoting activity of oocytes was found to be specific to granulosa cells with other somatic cell lines unaffected by coculture with oocytes. The expression pattern of this factor is also developmentally regulated, with murine oocytes first expressing mitogenic activity concomitant with the acquisition of meiotic competence [33]. This means that immature, germinal vesicle-stage oocytes first express the factor around the time of antrum formation and continue expression throughout antral development and during oocyte meiotic maturation. Although COC from mice deficient in granulocyte-macrophage colony stimulating factor have nearly twice the number of cumulus cells per COC compared to wild-type mice, the growth-promoting activity of these oocytes is not higher [34]. A soluble, heat-stable factor(s) secreted by the oocyte also regulates granulosa cell steroidogenesis in a dose-dependent manner. While estradiol production in porcine cumulus cells is inhibited by oocyte-secreted factor(s) [27], it is increased in murine cumulus cells [13, 35], and progestin secretions are inhibited by oocytes in both species. Both the steroid-regulating and the growth-promoting activity of oocytes are thought to act either independently or downstream of FSH-induced cAMP production, as oocytectomy does not prevent FSH-induced increases in intracellular cAMP levels [9, 27, 35].
The results of this study indicate that the growth-promoting and steroid-regulating activities of oocytes are closely related to one another. To date these two functions of oocytes have been investigated in isolation. In the current experiments, regardless of treatment, the pattern of DNA synthesis presented a mirror image to the pattern of progesterone secretion. In every instance where oocytes promoted DNA synthesis, progesterone secretion from these follicular cells was attenuated to a similar extent and vice versa. Despite this, it is likely the two factors are quite distinct. The oocyte-derived growth-promoting factor is highly labile while the steroid-regulating factor is not. Media conditioned by oocytes, including heat-treated conditioned media (100°C), are highly effective in regulating follicular cell steroidogenesis [13, 27]. Although oocyte conditioned medium is able to elicit marginal increases in granulosa cell DNA synthesis [12], these increases are considerably less than those during coculture with fresh oocytes [28] (unpublished data). Likewise these oocyte-secreted factor(s) are also likely to be distinct from the CEEF and the oocyte factor that inhibits plasminogen activator production. These factors differ again in that they are effective in fresh and frozen oocyte-conditioned medium, yet are heat sensitive [6, 26, 36]. Thus there are several chemically distinct oocyte factors, the secretion of which may be coordinately regulated.
A number of members of the TGFß superfamily are produced by oocytes, and some of these factors, in particular GDF-9, can mimic the effects of oocyte-secreted factor(s). For example, mouse cumulus cells are only able to produce hyaluronic acid in response to FSH if they are exposed to oocyte-secreted factors, TGFß1 [17] or GDF-9 [18]. Granulosa cell/ovarian mRNA transcripts for uPA, LHR, and KL are all suppressed by GDF-9 [18, 20] and by oocyte-secreted factors [6, 14, 15]. Likewise, coculture with either oocytes or GDF-9 increases MGC dimeric inhibin production [16, 22]. The mitogenic effect of oocytes on granulosa cells demonstrated in this and other studies can also be mimicked by other oocyte-secreted TGFß family members including activin [37], TGFß1 [28, 38], and GDF-9 [22]. The GDF-9, or homologues such as GDF-9B [39], are good candidates for the oocyte-derived growth factor because not only are these proteins produced by oocytes during the very early stages of folliculogenesis promoting follicular growth, but GDF-9 is essential for the process [19]. Further examination of the role of GDF-9 in folliculogenesis is warranted as this protein may not only be the oocyte-derived mitogenic factor but is likely to play an important role in a broad range of oocyte-somatic cell interactions.
In conclusion, this study has demonstrated that a soluble factor or factors secreted by bovine oocytes have a dramatic effect on the developmental program of follicular somatic cells. The characteristic nature of cumulus cells is largely dependent on the continual presence of the oocyte and the factor(s) that it secretes. Absence of the oocyte causes the cumulus cells to convert rapidly to the MGC phenotype. The MGC also respond to oocyte-secreted factor(s) by displaying characteristics more typical of cumulus cells, but presumably this does not occur in antral follicles in vivo. Thus, bovine oocytes seem to actively establish and maintain their own microenvironment in much the same way as mouse oocytes do [1]. This first occurs around the time of antrum formation [33] when oocyte-associated granulosa cells start to differentiate into either mural granulosa or cumulus cells. Throughout the prolonged stages of bovine antral follicle development, MGC differentiate into slowly growing, highly steroidogenic cells while the oocyte maintains cumulus cells in an alternate, distinct state. Perhaps the MGC phenotype is detrimental to the development of the oocyte. Maintenance of this specialized microenvironment around the oocyte throughout antral development may be necessary to confer full oocyte cytoplasmic maturation, as it is during this stage of development that bovine oocyte meiotic and developmental competencies are gradually acquired.
ACKNOWLEDGMENTS
The authors thank Dr. Jim Wang for statistical advice, the staff of the Reproductive Endocrine Laboratories for progesterone assays, and Lesley J. Ritter for expert technical assistance.
FOOTNOTES
First decision: 24 November 1999.
1 R.L.'s stay in Adelaide was generously supported by Serono Asia-Pacific. R.B.G. is a Research Officer of the Australian National Health and Medical Research Council (NH&MRC). 
2 Correspondence. FAX: 61 8 82227521; robert.gilchrist{at}adelaide.edu.au
Accepted: April 24, 2000.
Received: October 25, 1999.
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T.E. Hickey, D.L. Marrocco, R.B. Gilchrist, R.J. Norman, and D.T. Armstrong Interactions Between Androgen and Growth Factors in Granulosa Cell Subtypes of Porcine Antral Follicles Biol Reprod, July 1, 2004; 71(1): 45 - 52. [Abstract] [Full Text] [PDF]
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Y. Hirao, T. Itoh, M. Shimizu, K. Iga, K. Aoyagi, M. Kobayashi, M. Kacchi, H. Hoshi, and N. Takenouchi In Vitro Growth and Development of Bovine Oocyte-Granulosa Cell Complexes on the Flat Substratum: Effects of High Polyvinylpyrrolidone Concentration in Culture Medium Biol Reprod, January 1, 2004; 70(1): 83 - 91. [Abstract] [Full Text] [PDF]
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C. Glister, N. P. Groome, and P. G. Knight Oocyte-Mediated Suppression of Follicle-Stimulating Hormone- and Insulin-Like Growth Factor-Induced Secretion of Steroids and Inhibin-Related Proteins by Bovine Granulosa Cells In Vitro: Possible Role of Transforming Growth Factor {alpha} Biol Reprod, March 1, 2003; 68(3): 758 - 765. [Abstract] [Full Text] [PDF]
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I. Rothchild The Yolkless Egg and the Evolution of Eutherian Viviparity Biol Reprod, February 1, 2003; 68(2): 337 - 357. [Abstract] [Full Text] [PDF]
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A.M. Luciano, S. Modina, F. Gandolfi, A. Lauria, and D.T. Armstrong Effect of Cell-to-Cell Contact on In Vitro Deoxyribonucleic Acid Synthesis and Apoptosis Responses of Bovine Granulosa Cells to Insulin-Like Growth Factor-I and Epidermal Growth Factor Biol Reprod, December 1, 2000; 63(6): 1580 - 1585. [Abstract] [Full Text]
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