Immature (germinal vesicle stage) Rana pipiens oocytes typically remain arrested in prophase I of meiosis even after extended periods of in‐vitro culture, if not stimulated with hormones. We have, however, sporadically observed “spontaneous” occurrences of oocyte maturation in vitro without the addition of hormones. This study documents some of our observations on this phenomenon and presents experimental results concerning the effects and possible involvement of estrogen and follicle wall components in regulating spontaneous oocyte maturation. Estrogen was found to inhibit spontaneous oocyte maturation (GVBD) in a dose‐dependent fashion. Follicles in which spontaneous maturation was inhibited by estrogen retained their responsiveness (GVBD) to both frog pituitary homogenate (FPH) and progesterone stimulation. Inhibitory effects of estrogen on spontaneous maturation, however, were not reversed following incubation of washed follicles in plain culture medium without added hormones. Possible involvement of progesterone synthesis in spontaneous oocyte maturation was ascertained by simultaneously monitoring endogenous progesterone synthesis and the occurrence of spontaneous GVBD over the course of the maturation process. In spontaneous maturing follicle there was a gradual increase in basal levels of progesterone synthesis that preceded GVBD. Significantly, addition of estrogen abolished both the spontaneous progesterone production and spontaneous oocyte maturation. When FPH was added to follicles exhibiting spontaneous oocyte maturation, progesterone production was augmented and the time course of oocyte maturation was greatly accelerated. Involvement of ovarian components in the maturation process was investigated by selective removal of various follicle layers by microdissection. Removal of follicle epithelium and theca layer (defolliculation) markedly decreased spontaneous and FPH‐induced maturation, whereas removal of the entire follicle wall (denudation) completely blocked it. Our results suggest that both spontaneous and FPH‐induced maturation involve an estrogen sensitive process in the follicle wall. Thus, somatic follicle cells appear to serve as a common mediator for both types of maturation, which are linked by some intrafollicular mechanism involving steroidogenesis. Hence, estrogen may play an important role as an endogenous intrafollicular regulator of oocyte meiotic maturation.

Mummichogs (Fundulus heteroclitus) were exposed to various regimens of temperature and photoperiod in order to develop a routine husbandry procedure so as to have throughout the year a population of reproductively healthy fish with a good quantity and quality of prematuration follicles (1.2-1.5 mm) suitable for carrying out homologous bioassays ofF. heteroclitus gonadotropin. During the fall and winter months, wild fish in the field all had regressed ovaries (gonadosomatic index <1). On the other hand, laboratory-maintained fish, with or without cold temperature (15°C) and short photoperiod (10 h light/day) pretreatment, generally had sexually mature ovaries (GSI>10) when maintained on a warm temperature (25°C) and long photoperiod (14 h light) protocol. Ovarian follicles retrieved from laboratory fish were responsive toF. heteroclitus pituitary extract stimulation, and underwent germinal vesicle breakdown normallyin vitro. Hence these ovarian follicles served well as a bioassay forF. heteroclitus gonadotropin even outside of the normal breeding season. The pituitary glands retrieved from laboratory fish in winter also retained high gonadotropic potencies, in terms of maturational and steroidogenic activities. Our results thus demonstrated that active gametogenesis in laboratory-maintained fish can be extended five months beyond the end of the normal breeding season. Apparently,F. heteroclitus in Florida is potentially a continuous breeder when under favorable conditions, but has a reproductive quiescent period imposed upon it by some environmental stressor(s). Although the design of the present experiments did not determine the relative importance of nutritional factors, temperature, and photoperiod on the annual reproductive cycle ofF. heteroclitus, there are indications that diatary factors may play a much more dominant role in the reproductive cycle than previously recognized.