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.

Hypoxanthine and adenosine are present in preparations of mouse ovarian follicular fluid, and these purines maintain mouse oocytes in meiotic arrest in vitro (Eppig et al.: Biology of Reproduction 33:1041-1049. 1985). The first hypothesis tested in this study is that purines which maintain meiotic arrest act by maintaining meiosis-arresting levels of cyclic adenosine monophosphate (cAMP) in the oocyte. Oocyte-cumulus cell complexes were incubated in control medium (no added purines), or medium containing 0.75 mM adenosine, 4 mM hypoxanthine, or both for 3 hr and the percentage of the oocytes that underwent germinal vesicle breakdown (GVB) and the cAMP content of the intact complexes and the oocytes were determined. Adenosine alone had little inhibitory effect on GVB at this time point but sustained higher levels of cAMP in the oocytes. Hypoxanthine maintained 80% of cumulus cell-enclosed oocytes in meiotic arrest and also sustained higher cAMP levels in the oocytes. The addition of adenosine to hypoxanthine-containing medium increased the percentage of oocytes maintained in meiotic arrest, and increased the amount of cAMP in the oocytes above that maintained by either hypoxanthine or adenosine alone. Neither hypoxanthine, adenosine, nor hypoxanthine plus adenosine altered the cAMP content of intact complexes when assayed after 3 hr culture. Microinjection of an inhibitor of the catalytic subunit of cAMP-dependent protein kinase induced GVB in denuded oocytes cultured in medium containing hypoxanthine. This purine, therefore, maintained meiotic arrest by sustaining elevated cAMP levels within the oocytes. The second hypothesis tested in this study is that purines maintain meiosis-arresting levels of cAMP, at least in part, by inhibiting cAMP phosphodiesterase activity. In descending order of potency, 3-isobutyl-1-methylxanthine (IBMX), guanosine, hypoxanthine, adenosine, and xanthosine inhibited cAMP phosphodiesterase in oocyte lysates. Moreover, like the potent phosphodiesterase inhibitor IBMX, hypoxanthine augmented the meiotic arrest and cAMP accumulation mediated by follicle-stimulating hormone (FSH) in intact complexes. Therefore, inhibition of oocyte phosphodiesterase appears to be one mechanism by which the purines could maintain meiosis-arresting levels of cAMP.

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)

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