Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation

The expansion, or mucification, of the mouse cumulus oophorus in vitro requires the presence of an enabling factor secreted by the oocyte as well as stimulation with follicle-stimulating hormone (FSH). This study focuses on (1) the ability of mouse oocytes to secrete the enabling factor at various times during oocyte growth and maturation, (2) the temporal relationships between the development of the capacity of the oocyte to undergo germinal vesicle breakdown, the ability of the oocyte to secrete cumulus expansion-enabling factor, and the capacity of the cumulus oophorus to undergo expansion, and (3) the role of the oocyte in the differentiation of granulosa cells as functional cumulus cells. Growing, meiotically incompetent oocytes did not produce detectable amounts of cumulus expansion-enabling factor, but fully grown meiosis-arrested oocytes, maturing oocytes, and metaphase II oocytes did. Detectable quantities of enabling factor were produced by zygotes, but not by two-cell stage to morula embryos. The ability of oocytes to secrete cumulus expansion enabling factor and the capacity of cumulus cells to respond to FSH and the enabling factor are temporally correlated with the acquisition of oocyte competence to undergo germinal vesicle breakdown. Mural granulosa cells of antral follicles do not expand in response to FSH even in the presence of cumulus expansion-enabling factor, showing that mural granulosa cells and cumulus cells are functionally distinct cell types. The perioocytic granulosa cells of preantral follicles isolated from 12-day-old mice differentiate into functional cumulus cells during a 7-day period in culture. Oocytectomized granulosa cell complexes grown in medium conditioned by either growing or fully grown oocytes were comparable in size to intact complexes and maintained their 3-dimensional integrity to a greater degree than oocytectomized complexes grown in unconditioned medium. After 7 days, the oocytectomized complexes were stimulated with FSH in the presence of enabling factor, but no expansion was observed whether or not the oocytectomized complexes grew in the presence of oocyte-conditioned medium. These results suggest that a factor(s) secreted by the oocyte affects granulosa cell proliferation and the structural organization of the follicle, but continual close association with the oocyte appears necessary for the differentiation of granulosa cells into functional cumulus cells, insofar as they are capable of undergoing expansion.     

Lack of effect of oocytectomy on expansion of the porcine cumulus.

Oocyte-cumulus cell complexes (OCC) and complexes with an attached piece of membrana granulosa (C + P), isolated from prepubertal or cyclic gilts stimulated with pregnant mares' serum gonadotrophin, were cultured in media supplemented with follicle-stimulating hormone (FSH; 0.01-1.0 micrograms/ml) or forskolin (50-100 mumol/l) for 24 and 32 h. FSH and forskolin each induced dose-dependent cumulus and membrana granulosa expansion. After 2 h of culture, FSH (0.1 microgram/ml) or forskolin (100 mumol/l) increased the contents of intracellular adenosine 3',5'-phosphate (cAMP) in OCC from prepubertal gilts to almost 10 times that in unstimulated complexes. After 24 h of culture in media supplemented with FSH (0.1 microgram/ml) or forskolin (100 mumol/l), the oocytectomized OCC and C + P showed similar expansion to that of the control groups. The intracellular cAMP contents in intact and oocytectomized OCCs were similar in all groups except those treated with FSH, in which the intact OCCs had significantly higher contents than their oocytectomized counterparts (P less than 0.01). After hyaluronidase treatment, cumulus and membrana granulosa cells of intact and oocytectomized OCC and C + P were suspended, except for those of the innermost layers of the corona radiata. The results suggest that increases in cAMP contents and synthesis of an extracellular, hyaluronidase-sensitive mucus by pig OCC and C + P induced by FSH or forskolin are not dependent on the oocyte.     

Inhibition of oocyte maturation in the mouse: Participation of cAMP,steroid hormones,and a putative maturation-inhibitory factor

The hypothesis that cumulus cells inhibit oocyte maturation by a cAMP-dependent process was tested (R. M. Schultz, R. Montgomery, P. F. Ward-Bailey, and J. J. Eppig (1983). Dev. Biol.95, 294–304.). Treatment of isolated cumulus cell-oocyte complexes with follicle-stimulating hormone (FSH) resulted in a dose-dependent increase in both cumulus cell cAMP levels and in the extent of inhibition of germinal vesicle breakdown (GVBD), the first morphological manifestation of oocyte maturation. Furthermore, it was found that concentrations of a membrane-permeable analog of cAMP, dibutyryl cAMP (dbcAMP), that were below those required for complete meiotic inhibition had a greater inhibitory effect on cumulus cell-enclosed oocytes than on denuded oocytes. Cumulus cell-enclosed and denuded oocytes matured at the same time in the absence of dbcAMP. Ablation of the gap junctions that couple cumulus cells to the oocyte abolished the maturation-inhibitory action of cumulus cells that was promoted either by FSH or low concentrations of dbcAMP. These results are consistent with the hypothesis that inhibition of oocyte maturation is mediated by a factor of granulosa/cumulus cell origin, other than cAMP, which requires cAMP for its activity and/or generation, and an intact intercellular coupling pathway between cumulus cells and the oocyte. A variety of steroid hormones potentiated the FSH-induced inhibition of maturation in cumulus cell-enclosed oocytes. In addition, steroid hormones inhibited maturation in denuded oocytes, but only when oocyte cAMP levels were elevated by cAMP analogs or forskolin. Steroids alone did not inhibit maturation of either cumulus cell-enclosed or denuded oocytes. Moreover, the steroids alone or in combination with FSH did not affect metabolic coupling between the cumulus cells and oocytes, nor did testosterone affect the forskolin-induced level of cAMP in denuded oocytes. Therefore, it is proposed that the oocyte is a site for the synergistic activity of steroid hormones with a cAMP-dependent process in inhibiting maturation. Results of these studies are discussed in terms of the roles of intercellular communication, cAMP, a putative maturation-inhibiting factor, and steroid hormones in the inhibition of maturation of mouse oocytes.     

Regulation of oocyte maturation in the mouse: possible roles of intercellular communication, cAMP, and testosterone

Experiments were performed to determine if elevation of cumulus cell cAMP results in an increase in mouse oocyte cAMP while the heterologous gap junctions were intact. Both follicle-stimulating hormone (FSH) and cholera toxin induced a marked increase (>20-fold) in intracellular cAMP in isolated mouse cumulus cell-oocyte complexes in the presence of 3-isobutyl-1-methyl xanthine (IBMX). Concomitantly, both FSH and cholera toxin transiently inhibited resumption of meiosis of cumulus cell-enclosed but not denuded oocytes. The transient nature of the inhibitory effect produced by either FSH or cholera toxin was correlated with the cAMP level in the cumulus cell-oocyte complex. The inhibitory effect, however, was apparently not due to movement of cumulus cell cAMP to the oocyte via the functional heterologous gap junctions between cumulus cells and the oocyte. Radioimmunoassay of cAMP in oocytes free of attached cumulus cells or cumulus cell-enclosed oocytes exposed to either FSH or cholera toxin revealed that both groups of oocytes contained similar amounts of cAMP (about 0.14 fmole/oocyte). Metabolic labeling of cumulus cell-oocyte complexes with [³H]adenosine followed by incubation with either FSH or cholera toxin resulted in a marked increase in the amount of radiolabeled cAMP compared to that in unstimulated complexes. However, similar amounts of radiolabeled cAMP were found in oocytes derived from either stimulated or unstimulated complexes. Thus, we have not detected, using two methods of assay, that increasing the cAMP content of the cumulus cells results in any increase in the cAMP content of the oocyte. The apparent compartmentalization of cumulus cell cAMP elevated in response to either FSH or cholera toxin was not due to disruption of intercellular communication between the two cell types during the incubation; metabolic cooperativity was present between the two cell types and molecules of similar molecular weight and charge relative to that of cAMP were rapidly equilibrated between the two cell types. Testosterone potentiated the FSH/cholera toxin-induced transient inhibition of maturation of cumulus cell-enclosed oocytes. However, testosterone did not increase cAMP accumulation produced by either FSH or cholera toxin, decrease the rate of cAMP degradation, or promote movement of cumulus cell cAMP to the oocyte. Since cAMP elevated in response to FSH or cholera toxin appeared to be compartmentalized to cumulus cells and since neither FSH, cholera toxin, nor testosterone inhibited resumption of meiosis in denuded oocytes, it appears that the inhibitory effect promoted by FSH or cholera toxin is directly mediated by an agent other than cAMP, although cAMP generation is required for its action and that cumulus cells mediate the inhibition. These results are discussed in terms of a possible role of cAMP and steroids in regulating maturation in the mouse.     

FSH,EGF,胰岛素促进小鼠卵母细胞体外减数分裂恢复机制的研究

FSH、EGF和胰岛素均对体外培养的小鼠卵母细胞的减数分裂的恢复起促进作用,而FSH的促进作用滞后,但作用后使卵丘细胞扩散。三者的促进作用似受卵巢颗粒细胞内游离钙和cAMP的调节。EGF和胰岛素可使培养的颗粒细胞内的cAMP水平降低;同时FSH使单个卵丘细胞内的游离Ca~(2 )水平降低,而胰岛素无影响。所以FSH、EGF和胰岛素诱发卵母细胞成熟的机制不同:EGF通过细胞内Ca~(2 )的升高和cAMP水平的下降促使卵母细胞的减数分裂恢复;FSH降低卵丘细胞内Ca~(2 )的水平,但由于卵丘细胞与卵母细胞之间的联系被打断,最终使GVBD发生;而胰岛素的作用只涉及胞内cAMP的变化。     

Induction of maturation in cumulus cell-enclosed mouse oocytes by follicle-stimulating hormone and epidermal growth factor: evidence for a positive stimulus of somatic cell origin

The efficacy of follicle-stimulating hormone (FSH), epidermal growth factor (EGF), and dibutyryl cGMP (dbcGMP) as inducers of germinal vesicle breakdown (GVBD) in cumulus cell-enclosed mouse oocytes was examined when meiotic arrest was maintained in vitro with purines, dibutyryl cAMP (dbcAMP), or the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). When FSH was added to hypoxanthine (HX)-containing medium, the effect on oocyte maturation was at first inhibitory and later stimulatory. EGF stimulated GVBD at all time points tested. FSH and EGF also induced GVBD when oocytes were arrested with dbcAMP, IBMX, or guanosine. Dibutyryl cGMP stimulated GVBD when meiotic arrest was maintained with HX, but not when oocytes were meiotically arrested with guanosine, and was inhibitory in dbcAMP-supplemented medium. FSH and dbcGMP produced a transient delay of oocyte maturation in control medium, but the FSH effect was much more pronounced. EGF had no effect on maturation kinetics. The actions of FSH and EGF required the presence of cumulus cells. Both agents significantly stimulated cAMP production in oocyte-cumulus cell complexes. A brief exposure of complexes to a high concentration of dbcAMP induced GVBD, suggesting that FSH and EGF may act via a cAMP-dependent process. The frequency of FSH- and EGF-induced GVBD in cumulus cell-enclosed oocytes was significantly higher than the frequency of GVBD when oocytes were cultured while denuded of cumulus cells. of maturation is apparently not mediated solely by oocyte-cumulus cell uncoupling and termination of the transfer of an inhibitory meiotic signal from cumulus cells to the oocyte. The data suggest the generation of a positive signal within cumulus cells in response to hormone treatment that acts upon the oocyte to stimulate GVBD in the continued presence of inhibitory factors.     

Mouse oocytes regulate hyaluronic acid synthesis and mucification by FSH-stimulated cumulus cells

Mucification (or expansion) of the cumulus cells surrounding the oocyte is thought to depend on the direct action of gonadotropins in stimulating production and deposition of hyaluronic acid (HA) in the extracellular matrix. We now report that the oocyte is essential for this process. Either follicle-stimulating hormone (FSH) at 1 micrograms/ml or dibutyryl cAMP at 2 mM induces mucification of intact cumulus cell-oocyte complexes (COCs) in vitro, but fails to stimulate mucification of isolated cumulus cells. HA synthesis by FSH-stimulated cumulus cells is only approximately 3.5% of the value achieved by FSH-stimulated COCs. Isolated oocytes cultured with or without FSH do not synthesize detectable amounts of HA but induce isolated cumulus cells to increase HA synthesis approximately 13-fold in cocultures with FSH. Medium conditioned by isolated oocytes for 5 hr induces nearly the same level of HA synthesis by cumulus cells under the same culture conditions. FSH also stimulates cumulus cells to increase synthesis of dermatan sulfate proteoglycans (DS-PGs) approximately 3-fold, but this stimulation does not depend upon the presence of oocytes. The results indicate that oocytes produce a soluble factor(s) essential in combination with FSH to stimulate HA, but not DS-PG, synthesis by cumulus cells in vitro and that this factor(s) acts independently or downstream from the FSH-induced formation of cAMP.     

Meiotic state of bovine oocytes is regulated by interactions between cAMP,cumulus, and granulosa

Bovine oocytes are arrested at the prophase of first meiotic cell cycle. Meiosis resumes in oocytes of pre-ovulatory follicles upon LH surge. However, oocytes from secondary follicles spontaneously resume meiosis in the absence of hormones if removed from the follicle and cultured in vitro. The nature of meiotic arrestor in bovine follicles is poorly understood. In this study we investigated the role of cell-cell interactions between granulosa and cumulus cells and the oocyte in mediating maintenance of meiotic arrest by cAMP. We sorted oocytes as granulosa-cumulus oocyte complexes (GCOC) if surrounded with cumulus cells attached to a large granulosa investment or cumulus oocytes complexes (COC) if surrounded with cumulus cells only and investigated the role cAMP in maintenance of meiotic arrest in these oocytes under various conditions. In hormone- and serum-free medium both GCOC and COC enclosed oocytes resumed meiosis. When [cAMP](i) was elevated with addition of invasive adenylate cyclase (iAC) GCOC enclosed oocytes were maintained in the prophase with intact germinal vesicle (GV) while COC enclosed oocytes underwent GV breakdown (GVBD). iAC elevated [cAMP](i) in both types of oocytes to the same level. If oocytes were liberated from the cumulus and granulosa cells, they re-initiated meiosis in serum and hormone free medium, but remained in the GV stage if iAC was added to the medium. Untreated GCOC and COC enclosed oocytes extruded first polar body at the same frequency in hormone-supplemented media. GCOC and COC enclosed oocytes but not denuded oocytes (DO) cultured without somatic cells acquired developmental competence if cultured in hormone-containing medium. It is concluded that maintenance of meiotic arrest is regulated by the interplay of [cAMP](i), and cumulus and granulosa cells.     

Secretion of cumulus expansion-enabling factor (CEEF) in porcine follicles

The objective of this study was to find out whether porcine cumulus and mural granulosa cells can secrete cumulus expansion-enabling factor (CEEF). Culture drops of M-199 medium were conditioned with denuded porcine oocytes (1 oocyte/μl), cumulus cells from oocytectomized complexes (1 OOX/μl), pieces of mural granulosa isolated from preantral to preovulatory follicles (1000 cells/μl), or oviductal cells (1000 cells/μl) for 24 hr. The production of CEEF was assessed by the addition of mouse OOX and follicle-stimulating hormone (FSH) (1 μg/ml) to microdrops of the conditioned medium. After 16–18 hr, expansion of the mouse OOX was scored on a scale of 0 to 4 by morphologic criteria. Mouse OOX did not expand in nonconditioned FSH-supplemented medium. Immature porcine oocytes produced +3 to +4 expansion of the mouse OOX. Granulosa cells isolated from preantral and early antral follicles and cumulus cells isolated from all stages of follicle development constitutively secreted CEEF under in vitro conditions. Mural granulosa cells of small, medium, and preovulatory (PMSG) follicles also secreted CEEF in vitro; however, FSH or leutenizing hormone (LH) stimulation was essential for this secretion. Hormonally induced secretion of CEEF was accompanied by expansion of the mural granulosa itself. Granulosa cells isolated from follicles of gilts 20 hr after PMSG and human chorionic gonadotropin (hCG) administration did not produce CEEF and did not expand in response to FSH and LH in vitro. CEEF activity also was found in the follicular fluid of small antral follicles, was reduced in medium follicles, and was not detectable in PMSG-stimulated follicles. However, CEEF activity was reestablished in the follicular fluid of preovulatory follicles by hCG injection, conceivably due to increased production of CEEF by cumulus cells. We conclude that (1) porcine cumulus and mural granulosa cells are capable of CEEF production in vitro and (2) autocrine secretion of CEEF by cumulus cells is involved in regulation of porcine cumulus expansion both in vitro and in vivo. Mol. Reprod. Dev. 49:141–149, 1998. © 1998 Wiley-Liss, Inc.     

Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific growth factors that appear to play key roles in granulosa cell development and fertility in most mammalian species. We have evaluated the role(s) of these paracrine factors in the development and function of both the cumulus cells and oocytes by assessing cumulus expansion, oocyte maturation, fertilization, and preimplantation embryogenesis in Gdf9+/-Bmp15-/- [hereafter, double mutant (DM)] mice. We found that cumulus expansion, as well as the expression of hyaluronon synthase 2 (Has2) mRNA was impaired in DM oocyte-cumulus cell complexes. This aberrant cumulus expansion was not remedied by coculture with normal wild-type (WT) oocytes, indicating that the development and/or differentiation of cumulus cells in the DM, up to the stage of the preovulatory luteinizing hormone (LH) surge, is impaired. In addition, DM oocytes failed to enable FSH to induce cumulus expansion in WT oocytectomized (OOX) cumulus. Moreover, LH-induced oocyte meiotic resumption was significantly delayed in vivo, and this delayed resumption of meiosis was correlated with the reduced activation of mitogen-activated protein kinase (MAPK) in the cumulus cells, thus suggesting that GDF9 and BMP15 also regulate the function of cumulus cells after the preovulatory LH surge. Although spontaneous in vitro oocyte maturation occurred normally, oocyte fertilization and preimplantation embryogenesis were significantly altered in the DM, suggesting that the full complement of both GDF9 and BMP15 are essential for the development and function of oocytes. Because receptors for GDF9 and BMP15 have not yet been identified in mouse oocytes, the effects of the mutations in the Bmp15 and Gdf9 genes on oocyte development and functions must be produced indirectly by first affecting the granulosa cells and then the oocyte. Therefore, this study provides further evidence for the existence and functioning of an oocyte-granulosa cell regulatory loop.     

Regulation of murine oocyte meiosis: evidence for a gonadotropin- induced, cAMP-dependent reduction in a maturation inhibitor

We have developed an assay that can detect relative changes in the amount of a non-cAMP inhibitor of maturation present in cumulus cells (Eppig et al., 1983, Dev. Biol., 100:39-49). Using this assay in which accelerated maturation of a group of treated cumulus cell-oocyte complexes relative to untreated complexes indicates a decrease in the amount of inhibitor, results of the experiments described here suggest a possible relationship between elevation of cAMP levels and subsequent decreased amounts of a non-cAMP inhibitor. Mouse oocytes obtained from cumulus cell-oocyte complexes treated with luteinizing hormone (LH) resumed meiosis prior to oocytes obtained from untreated complexes; the degree of acceleration of maturation was dependent on LH concentration. A similar result was obtained with follicle-stimulating hormone (FSH). Correlated with LH- or FSH-acceleration of maturation was an LH- or FSH-induced elevation of cumulus cell cAMP levels. Inhibiting LH-induced elevation of cumulus cell cAMP levels inhibited LH-induced acceleration of maturation. An initial incubation of complexes in medium containing dibutyryl cAMP (dbcAMP) also promoted acceleration of maturation. In contrast, maturation of denuded oocytes was not altered by treatment with either LH, FSH, or dbcAMP. Complexes initially incubated in dbcAMP-containing medium still demonstrated acceleration of maturation after a subsequent 2 h incubation in dbcAMP-free medium. Relative to untreated complexes, none of these treatments disrupted intercellular communication between cumulus cells and the oocyte. Elevating follicle cAMP levels with cholera toxin induced maturation of follicle-enclosed oocytes when cumulus cell-oocyte coupling was still fully maintained. These results are interpreted to indicate that gonadotropin-mediated acceleration of maturation is via a cAMP-dependent reduction in the level of a maturation inhibitor present in granulosa/cumulus cells.     

Maintenance of meiotic arrest and the induction of oocyte maturation in mouse oocyte-granulosa cell complexes developed in vitro from preantral follicles.

During the development of oocyte-granulosa cell complexes from preantral follicles in vitro, oocytes grow and acquire competence to undergo germinal vesicle breakdown (GVB). In the culture system used here, GVB-competent oocytes were maintained in meiotic arrest solely by endogenous physiological mechanisms of the granulosa cells without supplementation with meiosis-arresting substances. Addition of mycophenolic acid, an inhibitor of inosine monophosphate (IMP) dehydrogenase, induced GVB in about 70% of the GVB-competent oocytes grown in vitro. The mechanism for meiotic arrest in this system is, therefore, similar to that for arrest in vivo insofar as it requires the participation of the IMP dehydrogenase pathway. Rp-cyclic adenosine monophosphothioate, a membrane-permeable antagonist to cAMP, induced GVB by about 30% of the competent oocytes. Cyclic AMP-dependent pathways, therefore, participate in the physiological mechanism by which mouse granulosa cells maintain meiotic arrest. Complexes were grown for 10 days in medium containing 0, 1, 5, or 10 ng/ml FSH, were stimulated with either 1 microgram/ml FSH or LH, and were assessed for GVB and cumulus expansion. GVB was stimulated by FSH whether or not the complexes were grown in medium containing FSH, but LH or hCG induced GVB only when the complexes were grown in medium containing FSH. Cumulus expansion occurred in response to either FSH or LH only when complexes were grown in medium containing FSH. FSH, therefore, promotes the differentiation of granulosa cells from preantral follicles in vitro so that LH can stimulate GVB and cumulus expansion.     

Bovine cumulus cell expansion does not depend on the presence of an oocyte secreted factor

Communication between the oocyte and its somatic cells has been shown to be important in oocyte development. Here we examined how the oocyte may be involved in bovine cumulus cell expansion. Intact bovine cumulus oocyte complexes (COC) were obtained by puncturing antral follicles. From the intact COC, oocytectomised complexes (OOX) were produced by micro surgical removal of the oocyte. Clumps of cumulus cells (CC) were obtained by micro-dissection. Intact or OOX complexes or CC were matured in the presence of fetal calf serum and hFSH (6 mlU/ml) for 24 hr and the degree of expansion measured. The presence of the oocyte is not essential to allow bovine cumulus expansion to occur as expansion occurred in all groups. Murine OOX complexes from eCG primed 35–40-day-old C57BL6/CBA F₁ hybrids (known to require the presence of an oocyte secreted factor for cumulus expansion) were cultured with or without denuded bovine oocytes (1 oocyte/μl). Murine OOX complexes expanded only in the presence of denuded bovine oocytes. Thus some factor produced by bovine oocytes enabled expansion of murine OOX complexes. To determine whether the factor is secreted by bovine oocytes, murine OOX were cultured with or without media conditioned by bovine oocytes (1 oocyte/μl for 4 hr). Significant expansion of murine OOX occurred in media conditioned by bovine oocytes. This shows that the cumulus expansion enabling effect of bovine oocytes is released into the surrounding media. Media conditioned by bovine oocytes and then frozen for up to 1 month showed that the activity by the factor can withstand freezing. © 1995 wiley-Liss, Inc.     

Effect of follicle-stimulating hormone on cyclic adenosine monophosphate level and on meiotic maturation in mouse cumulus cell-enclosed oocytes cultured in vitro

We have reported that in vitro treatment with follicle-stimulating hormone (FSH) delays by about 3 h spontaneous meiotic resumption in cumulus cell-enclosed mouse oocytes. In the present paper we show that the temporary meiotic block is accompanied by a transient increase of cAMP concentration in the oocyte. In cumulus cell-oocyte complexes stimulated with 1 microgram/ml FSH, cAMP significantly increases within 1 h both in the whole complex (from a basal value of 1.9 +/- 0.2 to 169 +/- 13 fmol) and in the enclosed oocyte (from 0.9 +/- 0.2 to 2.4 +/- 0.2 fmol), then progressively decreases to basal values. Stimulation by FSH does not cause any cAMP increase in denuded oocytes. As the concentration of cAMP in the cells decreases, the percentage of oocytes escaping the meiotic block imposed by FSH increases. If the complexes are cultured in the presence of 1 microgram/ml FSH plus 1 mM isobutyl-1-methylxanthine (1BMX), cAMP concentration increases approximately 250-fold in the complex, and 10-fold in the enclosed oocyte; the level of cAMP in the oocyte drops very rapidly (50% degradation in less than 2 min) if the oocyte is then transferred to IBMX-free medium. The data are discussed in terms of the possible role of cAMP transfer from cumulus cells to the oocyte in the regulation of meiotic progression in mouse oocytes.     

Interactions between somatic cells and germ cells throughout mammalian oogenesis

Oocytes and their companion somatic cells maintain a close association throughout oogenesis and this association is essential for normal oocyte and follicular development. This review summarizes current concepts of the role of the somatic cells in the regulation of mammalian oocyte growth, the maintenance of meiotic arrest, the induction of oocyte maturation, and the acquisition of full embryonic developmental competence during oocyte maturation in vitro. Gap junctions appear to mediate these regulatory processes. The regulatory interaction of oocytes and somatic cells, however, is not unidirectional; the oocyte participates in the proliferation, development, and function of the follicular somatic cells. The oocyte secretes factors that enable the cumulus cells to synthesize hyaluronic acid and undergo cumulus expansion in response to hormonal stimulation. In addition, the oocyte produces factors that promote the proliferation of granulosa cells. These interactions in vitro do not appear to require the mediation of gap junctions. The oocyte also promotes the differentiation of granulosa cells into functional cumulus cells, but this function of the oocyte appears to require the continued presence and close association of the oocyte and granulosa cells. Therefore, oocytes and follicular somatic cells are interdependent for development and function.     

Oocyte-secreted factor(s) determine functional differences between bovine mural granulosa cells and cumulus cells

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 [(3)H]thymidine as an indicator of follicular cell DNA synthesis. The COC incorporated 11 to 14 times more [(3)H]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 (3)H 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 [(3)H]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/microl, demonstrating that oocytes secrete a soluble factor(s) that promotes growth and attenuates cumulus cell progesterone secretion. In the presence of IGF-I, [(3)H]thymidine incorporation in MGC increased ninefold above control levels with the addition of DO. The addition of FSH to IGF-I-increased (3)H 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.