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.     

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.     

Effect of FSH and HCG on progesterone secretion by cultured oocyte-cumulus complexes and granulosa cells from porcine antral follicles: Influence of follicular development

Oocyte-cumulus complexes and granulosa cells were harvested from small (1–2 mm), medium (3–5 mm), and large (6–12 mm) porcine antral follicles and cultured for 2 and 3 days. The effects of various doses of purified hCG and human FSH on progesterone secretion and monolayer formation were examined. After a 2-day culture period it was found that FSH was more effective in stimulation of progesterone secretion by cultured oocyte-cumulus complexes than in granulosa cells harvested from small follicles (P < 0.01), whereas hCG was more effective in stimulating progesterone secretion in granulosa cells than in oocytecumulus complexes harvested from large follicles. In contrast, after a 3-day culture period, granulosa cells secreted more progesterone compared to oocytecumulus complexes under control conditions or in the presence of hCG or FSH. After 3 days both FSH and hCG stimulated progesterone secretion by oocytecumulus complexes and granulosa cells; however, the hormone effect was greater upon granulosa cells than oocyte-cumulus complexes. After 3 days of culture in the case of both follicular cell types, there was a greater response to FSH in the case of cells harvested from small compared to large follicles. The reverse was true in the case of hCG responsiveness. Monolayer formation ability of oocyte-cumulus complexes was greater in the case of complexes harvested from small and medium than complexes harvested from large follicles. Addition of hCG to the cultures led to a dose-dependent decrease in monolayer formation by oocyte-cumulus complexes harvested from all sizes of follicles.     

FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles

The hypothesis was tested that bovine preantral follicles can be stimulated to grow in vitro by FSH and by the mitogens, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but not by transforming growth factor-beta (TGFbeta), which generally inhibits EGF and bFGF action. Preantral follicles, 60 to 179 mum in diameter, were isolated from fetal ovaries by treatment with collagenase and DNase and cultured for 6 d in serum-free medium, with or without FSH and growth factors. Basic FGF (50 ng/ml), and to a lesser extent FSH (100 ng/ml) and EGF (50 ng/ml), stimulated thymidine incorporation by granulosa cells in bovine preantral follicles compared to control cultures (8-, 4- and 2.5-fold the labeling index of the controls; P < 0.05). Alone TGFbeta (10 ng/ml) had no effect on (3)H-thymidine incorporation, but it completely inhibited the bFGF- but not the FSH-stimulated increase in the labeling index and mean follicular diameter of preantral follicles (P < 0.05). By the end of the culture period oocytes in most treatments had degenerated, and the few surviving oocytes were in preantral follicles cultured with FSH or bFGF. Progesterone accumulation was greater (P < 0.05) in the presence of FSH (100 ng/ml) or EGF (50 ng/ml) than with bFGF, TGFbeta or control medium. Basic FGF strongly inhibited the effect of FSH on progesterone secretion (P < 0.05). Only FSH stimulated the conversion of exogenous testosterone to estradiol and both bFGF and TGFbeta markedly inhibited FSH-stimulated estradiol accumulation. These results indicate that proliferation of granulosa cells of bovine preantral follicles can be stimulated by bFGF, FSH and EGF, whereas TGFbeta inhibits growth, and that they are steroidogenically active in culture. Basic FGF and TGFbeta antagonize FSH-stimulated steroid production by granulosa cells of cultured bovine preantral follicles.     

Effect of insulin-like growth factor-I during preantral follicular culture on steroidogenesis, in vitro oocyte maturation, and embryo development in mice

Insulin-like growth factor-I (IGF-I) is involved in the regulation of ovarian follicular development and has been shown to potentiate the FSH responsiveness of granulosa cells from preantral follicles. The aim of the present study was to investigate the effect of IGF-I during preantral follicular culture on steroidogenesis, subsequent oocyte maturation, fertilization, and embryo development in mice. Preantral follicles were isolated mechanically and cultured for 12 days in a simplified culture medium supplemented with 1% fetal calf serum, recombinant human FSH, transferrin, and selenium. In these conditions, follicles were able to grow and produce oocytes that could be matured and fertilized. The first experiment analyzed the effect of different concentrations of IGF-I (0, 10, 50, or 100 ng/ml) added to the culture medium on the follicular survival, steroidogenesis, and the oocyte maturation process. The presence of IGF-I during follicular growth increased the secretion of estradiol but had no effect on the subsequent oocyte survival and maturation rates. In the second experiment, IGF-I (0 or 50 ng/ml) was added to the culture medium during follicular growth, oocyte maturation, or both, and subsequent oocyte fertilization and embryo development rates were evaluated. Oocyte fertilization rates were comparable in the presence or absence of IGF-I. However, the blastocyst development rate was enhanced after follicular culture in the presence of IGF-I. Moreover, the total cell number of the blastocysts observed after differential labeling staining was also higher when follicles were cultured or matured in the presence of IGF-I.     

Follicle stimulating hormone-induced DNA synthesis in the granulosa cells of hamster preantral follicles involves activation of cyclin-dependent kinase-4 rather than cyclin d2 synthesis

Although cyclin D2 mRNA synthesis precedes gonadotropin-induced DNA synthesis in quiescent granulosa cells in culture, it is unclear whether a similar mechanism exists for the granulosa cells of growing preantral follicles in cyclic animals. The objective was to evaluate whether the synthesis of cyclin D2 protein was a prerequisite for FSH-induced DNA synthesis in the granulosa cells of intact preantral follicles of cyclic hamsters. Preantral follicles from cyclic hamsters were cultured in the presence or absence of FSH, and cell cycle parameters were examined. FSH stimulated cyclin-dependent kinase (CDK)-4 activity by 2 h and DNA synthesis by 4 h without altering the levels of cyclin D2 in the granulosa cells. The FSH effect was mimicked by epidermal growth factor administered in vivo. Although FSH increased the levels of cyclin D2 mRNA, it also stimulated the degradation of cyclin D2 as well as p27(Kip1) and p19(INK4) proteins. FSH activation of CDK4 was mediated by cAMP and ERK-1/2. In contrast to granulosa cells in intact follicles, FSH or cAMP significantly increased cyclin D2 protein levels in cultured granulosa cells but failed to induce DNA synthesis. Collectively, these data suggest that granulosa cells of preantral follicles, which are destined to enter the S phase during the estrous cycle, contain necessary amounts of cyclin D2 and other G1 phase components. FSH stimulation results in the formation and activation of the cyclin D2/CDK4 complex leading to DNA synthesis. This mechanism may be necessary for rapid movement of follicles from preantral to antral stages during the short duration of the murine estrous cycle.     

In vitro development of sheep preantral follicles

Preantral ovarian follicles isolated from prepubertal sheep ovaries were individually cultured for 6 days in the presence of increasing doses of FSH (ranging from 0.01 to 1 microg/ml) and under two different oxygen concentrations, 20% and 5% O2. Follicle development was evaluated on the basis of antral cavity formation as well as the presence of healthy cumulus oocyte complexes. Follicle growth was enhanced by FSH addition to culture medium, while the use of a low oxygen concentration slightly stimulated this process. However, when follicles were cultured in the presence of high doses of FSH (1 microgram/ml) and under low oxygen concentration, a high proportion of them showed the presence of an antral cavity and of a healthy cumulus-oocyte complex. In addition, under this specific culture condition sheep preantral follicles released higher levels of estradiol as compared to those secreted at lower FSH concentrations or under 20% O2. When the meiotic competence of oocytes derived from follicles cultured at 1 microgram/ml FSH was assessed, no significant difference was recorded between the two oxygen groups. These results show that the culture conditions here identified are beneficial to in vitro growth and differentiation of sheep preantral follicles.     

Developmental and hormonal regulation of cumulus expansion and secretion of cumulus expansion-enabling factor (CEEF) in goat follicles

The objective of this article was to study the developmental and hormonal regulation of cumulus expansion and secretion of cumulus expansion-enabling factor (CEEF) in goat follicles. M-199 medium was conditioned for 24 hr with cumulus-denuded oocytes (DOs), oocytectomized complexes (OOXs), or mural granulosa cells (MGCs) from goat follicles of different sizes. Mouse OOXs and eCG were added to culture drops of the conditioned medium and cumulus expansion was scored at 18 hr of culture to assess CEEF production. While mouse OOXs did not expand, goat OOXs underwent full cumulus expansion when cultured in nonconditioned eCG-supplemented M-199 medium. When cultured in nonconditioned medium containing 10% follicular fluid (FF) from goat medium (2-4 mm) and small (0.8-1.5 mm) follicles, 71-83% mouse OOXs expanded; but expansion rates decreased (P < 0.05) at either lower or higher FF concentrations. FF from large (5-6 mm) follicles did not support mouse OOX expansion at any concentrations. While medium conditioned with DOs from follicles of all the three sizes supported expansion of 80-90% mouse OOXs, medium conditioned with mature DOs had no effect. While cumulus cells from follicles of all the three sizes secreted CEEF in the absence of gonadotropins, MGCs from large follicles became gonadotropin dependent for CEEF production. Both FSH and LH stimulated CEEF production by large follicle MGCs, but FSH had a shorter half-life than LH to expand mouse OOXs. Few meiosis-incompetent goat oocytes from small follicles underwent cumulus expansion when cultured in medium conditioned with goat DOs or cocultured with goat COCs from medium follicles. It is concluded that (1) goat cumulus expansion is independent of the oocyte; (2) the limited CEEF activity in FF from large follicles was due mainly to the inability of MGCs in these follicles to secret the factor in absence or short supply of gonadotropins; (3) the cumulus expansion inability of the meiosis incompetent goat oocytes was due to the inability of their cumulus cells to respond to rather than to produce CEEF.     

Oocyte regulation of anti-Müllerian hormone expression in granulosa cells during ovarian follicle development in mice

In the ovarian follicle, anti-Müllerian hormone (Amh) mRNA is expressed in granulosa cells from primary to preovulatory stages but becomes restricted to cumulus cells following antrum formation. Anti-Müllerian hormone regulates follicle development by attenuating the effects of follicle stimulating hormone on follicle growth and inhibiting primordial follicle recruitment. To examine the role of the oocyte in regulating granulosa cell Amh expression in the mouse, isolated oocytes and granulosa cells were co-cultured and Amh mRNA levels were analysed by real-time RT-PCR. Expression in freshly isolated granulosa cells increased with preantral follicle development but was low in the cumulus and virtually absent in the mural granulosa cells of preovulatory follicles. When preantral granulosa cells were co-cultured with oocytes from early preantral, late preantral or preovulatory follicles, and when oocytes from preovulatory follicles were co-cultured with cumulus granulosa cells, Amh expression was increased at least 2-fold compared with granulosa cells cultured alone. With oocytes from preantral but not preovulatory follicles, this was a short-range effect only observed with granulosa cells in close apposition to oocytes. We conclude that stage-specific oocyte regulation of Amh expression may play a role in intra- and inter-follicular coordination of follicle development.     

Production of buffalo embryos using oocytes from in vitro grown preantral follicles

The present study examines the use of buffalo preantral follicles as a source of oocytes for in vitro embryo production. Preantral follicles were isolated from abattoir-derived buffalo ovaries and were grown for 100 days in five different culture systems: (1) minimum essential medium (MEM); (2) coconut water; (3) MEM + ovarian mesenchymal cell (OMC) co-culture; (4) MEM + granulosa cell (GC) co-culture; or (5) MEM + cumulus cell (CC) co-culture. Low growth rates for the preantral follicles were observed when follicles were cultured in MEM or coconut water medium. Moderate growth rates were seen for OMC and GC co-cultures, and high rates of growth were observed when follicles were grown in CC co-culture. The survival of preantral follicles was low in the MEM culture (<25%), but was over 75% in the other culture systems. Oocytes were not recovered from the MEM group, while an oocyte recovery rate of 80-100% was observed when the follicles were cultured with coconut water/somatic cells. Transferable embryos could be produced only with the oocytes obtained from preantral follicles grown in the OMC and CC co-culture systems. This study demonstrates, for the first time, that it is possible to produce buffalo embryos by in vitro fertilization of oocytes derived from in vitro grown preantral follicles.     

Role of follicle stimulating hormone and epidermal growth factor in the development of porcine preantral follicle in vitro

The aim of the present study was to assess the role of follicle stimulating hormone (FSH), epidermal growth factor (EGF) or a combination of EGF and FSH on the in vitro growth of porcine preantral follicles, estradiol secretion, antrum formation, oocyte maturation and subsequent embryonic development. Porcine preantral follicles were cultured for 3 days in the absence or in the presence of FSH or EGF. Oocytes from these follicles were then matured, fertilized in vitro and embryos were cultured. Estradiol secretion and histological analysis of cultured follicles were also carried out. The results showed that when FSH, or a combination of EGF and FSH, was added to the culture medium, most of preantral follicles grew to antral follicles with high estradiol secretion and the oocytes from these antral follicles could mature, fertilize and develop to the blastocyst stage. Without FSH, or a combination of EGF and FSH, preantral follicles were unable to develop to the antral stage. Histology demonstrated that the resulting follicles were nonantral, estradiol production was reduced and none of their oocytes matured after in vitro maturation. The results indicate the essential role of FSH in promoting in vitro growth of porcine preantral follicle, estradiol secretion, antrum formation, oocyte maturation and subsequent embryonic development. EGF with FSH treatment of porcine preantral follicles improves the quality of oocytes, shown by a higher frequency of embryonic development.     

Hormonal requirements for the growth and differentiation of hamster preantral follicles in long-term culture

Preantral follicles from pro-oestrous and oestrous hamsters were isolated enzymically (Stages 1-5) and by microdissection (Stage 6) and cultured for up to 168 h in the absence or presence of 100 ng ovine FSH or LH separately or combined or 1 or 10 micrograms progesterone or estradiol-17 beta in serum-free defined medium and exposed to 1 muCi [3H]thymidine for 24 h before termination. In the presence of insulin and hydrocortisone but not gonadotrophins, the morphology of follicles from pro-oestrous animals at Stages 1-4 (1-4 layers granulosa cells; no theca) were unaffected for up to 48 h whereas for Stages 5 (5-6 layers granulosa cells and developing theca) and 6 (7-8 layers granulosa cells and theca), atresia was prominent by 24 h. FSH significantly reduced the percentage of atretic follicles in Stages 1-5 throughout the culture period; but was effective only up to 96 h for Stage-6 follicles. LH was also effective, albeit to a lesser extent. FSH increased follicular labelling indexes during every 24-h labelling period and, during a pulse-chase period, follicular DNA content and granulosa cell numbers. FSH, but not LH, induced differentiation by 96 h of preantral follicles at Stage 6 into small antral stages (Stages 7-8). FSH and LH together induced almost the same effect as FSH alone. However, neither progesterone nor oestradiol had any significant long-term effects on DNA synthesis and oestradiol induced atresia beyond 24 h. Both FSH and LH induced follicular maturation in vitro as evident from increases in progesterone, androstenedione and oestradiol production. Follicles (Stages 1-4) collected from oestrous hamsters responded to FSH to a lesser extent than did those from pro-oestrous animals, possibly because of in-vivo exposure to periovulatory changes in gonadotrophins; however, an antrum formed in Stage-6 follicles by 72 h.     

Growth and antrum formation of bovine preantral follicles in long-term culture in vitro

Culture of preantral follicles has important biotechnological implications through its potential to produce large quantities of oocytes for embryo production and transfer. A long-term culture system for bovine preantral follicles is described. Bovine preantral follicles (166 +/- 2.15 micrometer), surrounded by theca cells, were isolated from ovarian cortical slices. Follicles were cultured under conditions known to maintain granulosa cell viability in vitro. The effects of epidermal growth factor (EGF), insulin-like growth factor (IGF)-I, FSH, and coculture with bovine granulosa cells on preantral follicle growth were analyzed. Follicle and oocyte diameter increased significantly (P < 0.05) with time in culture. FSH, IGF-I, and EGF stimulated (P < 0.05) follicle growth rate but had no effect on oocyte growth. Coculture with granulosa cells inhibited FSH/IGF-I-stimulated growth. Most follicles maintained their morphology throughout culture, with the presence of a thecal layer and basement membrane surrounding the granulosa cells. Antrum formation, confirmed by confocal microscopy, occurred between Days 10 and 28 of culture. The probability of follicles reaching antrum development was 0.19 for control follicles. The addition of growth factors or FSH increased (P < 0.05) the probability of antrum development to 0.55. Follicular growth appeared to be halted by slower growth of the basement membrane, as growing follicles occasionally burst the basement membrane, extruding their granulosa cells. In conclusion, a preantral follicle culture system in which follicle morphology can be maintained for up to 28 days has been developed. In this system, FSH, EGF, and IGF-I stimulated follicle growth and enhanced antrum formation. This culture system may provide a valuable approach for studying the regulation of early follicular development and for production of oocytes for nuclear/embryo transfer, but further work is required.     

In vitro growth, maturation, fertilization, and embryonic development of oocytes from porcine preantral follicles

This study was conducted to identify an in vitro culture system that would support intact porcine follicle growth from preantral follicle to antral stages, oocyte maturation, fertilization, and embryonic development; and to evaluate factors that influence porcine preantral follicle growth in vitro. Preantral follicles isolated from prepubertal porcine ovaries were cultured for 4 days in the presence of different concentrations of porcine serum and FSH, and with different numbers of follicles per well. A series of experiments showed that porcine antral follicles can be grown at a high frequency in vitro from healthy preantral follicles with intact theca when cultured in North Carolina State University 23 medium supplemented with 1.5 ng/ml FSH, 7.5% serum, and when cultured with three follicles per well. After 4 days of culture, 68% healthy cumulus-enclosed oocytes from these follicles were obtained, and 51% of the oocytes completed meiotic maturation to the metaphase II stage. Fifty-three percent of the mature oocytes underwent fertilization, 43% of the fertilized oocytes cleaved, and 13% developed to the blastocyst stage. The results show 1) that porcine preantral follicles can grow efficiently to the antral stage using these culture conditions, and 2) that oocytes from in vitro-matured porcine preantral follicles can acquire meiotic competence and undergo fertilization and embryonic development.     

Follicle-stimulating hormone and estradiol regulate antrum-like reorganization of granulosa cells in rat preantral follicle cultures

Formation of a fluid-filled antrum results from the actions of FSH and estrogen on preantral ovarian follicles in most mammalian species. To investigate the novel proposal that hormone-regulated cell-cell interactions mediate antrum formation, we isolated preantral follicles from infant (10- or 11-day-old) Wistar rats and cultured them in a substratum-adherent manner in Minimum Essential Medium supplemented with 2 mM hypoxanthine, 3 mg/ml bovine serum albumin, 5 micrograms/ml insulin, 5 micrograms/ml transferrin, and 5 ng/ml selenium. Similar cultures were previously shown to support oocyte growth and acquisition of meiotic competence. In the absence of FSH, follicles attached to the plastic surface and granulosa cells spread-out uniformly around granulosa cell-enclosed oocytes. FSH treatment caused certain follicles to show an increase between culture days 3 and 7 in appearance of conspicuous antrum-like reorganization of the granulosa cells, but without forming a completely enclosed fluid-filled cavity. This response was biphasic over 10-500 ng/ml FSH, with an optimal concentration of 50 ng/ml resulting in a mean of 37.8 +/- 4.7% of follicles showing antrum-like reorganization for 3 similar experiments. Estradiol-17 beta alone at 10(-10)-10(-8) M was without effect on this response, but at 10(-10) and 10(-9) M, it significantly augmented the action of an optimal concentration of FSH by about 2-fold in 4 experiments. In these experiments, the effect of 10(-8) M estradiol was not significantly different from FSH alone, indicating that the response to estradiol was also biphasic.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of protein synthesis patterns in mouse cumulus cells and mural granulosa cells: effects of follicle-stimulating hormone and insulin on granulosa cell differentiation in vitro.

Successful development of mammalian oocytes requires correct interactions between developing oocytes and associated granulosa cells. Development of oocyte-granulosa cell complexes from preantral follicles in vitro does not produce oocytes competent to develop to blastocysts at the same frequency as for oocytes that develop in vivo. Addition of either FSH or insulin to cultures of oocyte-granulosa cell complexes does not improve the frequency of blastocyst development, and the combination of both insulin and FSH is deleterious. Here, high-resolution 2-dimensional PAGE (2D-PAGE) and computerized gel image analysis were used to compare patterns of protein synthesis in cumulus cells and mural granulosa cells of small antral follicles, and then to assess effects of FSH and insulin on the differentiation of oocyte-associated granulosa cells (OAGCs) in vitro. Culture of OAGCs without FSH or insulin resulted in failure to synthesize many proteins at rates characteristic of cumulus cells. Either hormone used alone caused many cumulus cell proteins that were decreased in control cultures to be synthesized at nearly normal cumulus cell rates, and also caused the synthesis of other proteins to be increased or decreased. The two hormones added together produced the greatest change in protein synthetic pattern, including overexpression or underexpression of many proteins not affected by either hormone alone. Addition of these hormones to culture media thus appeared insufficient to elicit a normal cumulus cell phenotype in OAGCs and could lead to complex changes in protein synthesis that may be deleterious to oocyte development. The high-resolution 2D-PAGE approach described here should be a valuable tool in studies on oocyte and granulosa cell development in vitro, since phenotype can be evaluated globally through the display of over 1000 newly synthesized proteins rather than relying upon the expression of just a few genes.