Involvement of protein synthesis in frog pituitary homogenate (FPH)-induced progesterone production andlor accumulation in ovarian follicles was investigated. In amphibians, cycloheximide (C), an inhibitor of protein synthesis, inhibits progesterone and FPH-induced germinal vesicle breakdown (GVBD). However, the site and mechanisms of action of cycloheximide within ovarian follicles have not been elucidated. Intrafollicular progesterone produced by FPH is considered to mediate oocyte maturation; thus, cycloheximide may interfere with production andlor action of progesterone. Simultaneous treatment of FPH-stimulated follicles with cycloheximide inhibited FPH-induced progesterone accumulation (measured by RIA) and the accompanying-GVBD in a dose-dependent fashion. Inhibitory effects of cycloheximide on either FPH-induced progesterone production or GBVD were not reversed when follicles were washed and returned to fresh medium devoid of FPH and cycloheximide. However, subsequent restimulation of washed follicles with FPH resulted in increased progesterone levels and oocyte maturation. The extent of reversibility, in terms of GVBD and progesterone production, after FPH restimulation varied between animals. Pretreatment of follicles with cycloheximide for 6 hours, without FPH, had little or no effect on progesterone production when follicles were washed and treated with FPH. Delayed addition of cycloheximide to follicles following FPH stimulation blocked further progesterone accumulation as indicated by measurement of intrafollicular progesterone at the time of cycloheximide addition and at the end of the incubation period. The results indicate that cycloheximide rapidly inhibits progesterone production and that continuous protein synthesis is required for progesterone accu-muIation. Furthermore, protein synthesis does not appear to be required for progesterone metabolism since intrafollicular progesterone declined with prolonged culture even in the presence of cycloheximide. The nature of protein(s) involved in follicular progesterone production remains to be elucidated. FPH mediation of oocyte maturation within ovarian follicles appears to depend upon protein synthesis in somatic follicle cells, which is required for progesterone production, and in the oocyte, to mediate the response to the steroid trigger.
In amphibians, resumption of meiosis in ovarian foIlicular oocytes can be initiated by the action of hypophysial gonadotropic hormones (Dettlaff, '66; Schuetz, '67). Considerable evidence suggests that a two-step process, involving steroidogenesis, mediates this action (Masui, '67; Smith, et al., '66; Redshaw, '72; Schuetz, '74). First, gonadotropic hormones stimulate follicle cells to produce steroids, which secondarily interact with the oocyte to initiate biochemical modifications that promote germinal vesicle breakdown (GVBD). In Rana pipiens, pituitary extracts (frog pituitary homogenate, FPH) induced a rapid increase in follicular progesterone concentration prior to GVBD (Lessman and Schuetz, '82; Lin and Schuetz, '85). However, the sequence of events and biochemical mechanisms involved in FPHinduced follicular steroid production is poorly understood.
Protein synthesis has been implicated in oocyte maturation with the demonstration that cycloheximide interferes with progesterone and FPH-induced GVBD (DettlafT, '66; Brachet, '67; Wasserman and Masui, '75). If stimulation of intrafollicular progesterone