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Cultured granulosa cells from intact immature rats produced large amounts of progestins in response to phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (MIX). MIX alone stimulated up to 9 times higher amounts of 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OH-P) when compared with FSH (100 ng/ml)-stimulated steroidogenesis. Combined action of MIX and FSH did not yield more activity than MIX action alone. MIX activity was demonstrable exclusively in serum-free culture medium (Dulbecco modified Eagle's medium/F-12) supplemented with insulin, transferrin, hydrocortisone, and fibronectin. Serum severely inhibited MIX induction of 20 alpha-OH-P production. MIX also caused dramatic cell shape changes which occurred within 14 h of exposure to the drug. The cells assumed a spherical shape and remained so as long as MIX was maintained in the culture medium. Upon removal of the drug, the cells respread and acquired the morphology of luteinized cells. In contrast to FSH action, MIX failed to induce the appearance of functional LH receptors. However, similar to FSH effect on immature granulosa cells, MIX successfully induced aromatase enzyme throughout 12 days in culture. MIX alone caused only a minute increase in the accumulation of cAMP. cAMP content in cells induced with FSH (100 ng/ml) was 46 times higher than the cyclic nucleotide levels measured under maximal effective doses of MIX. The discrepancy between the intensive steroidogenic activity of MIX and its inability to substantially raise cAMP content suggests a possible cAMP-independent mechanism for steroid production in the ovarian cells.

Immunocytochemical staining methods were used to examine the appearance of cholesterol side-chain cleavage cytochrome P-450 (P-450scc) in mitochondria of cumulus cells during follicular development. The cumulus-oocyte complexes were isolated from pregnant mare serum gonadotropin (PMSG)/human CG (hCG)-treated 25 day rats and examined in culture. It is shown that P-450scc is not expressed in the cumulus cell earlier than 2-3 h before ovulation. After ovulation, the expression of P-450scc rapidly increased, so that postovulatory cumuli contained ample amounts of the cytochrome. RIA of progestins secreted by the cumulus-oocyte complexes in culture corroborated the immunocytochemical observations. A single administration of LH or PMSG treatment of hypophysectomized rat did not result in P-450scc accumulation. However, this failure of hormonal responses in vivo was not due to lack of available receptors, since both FSH and LH could induce cAMP accumulation and P-450scc when added to isolated cumuli in culture. Therefore, these findings suggest the presence of a putative intraovarian suppressive factor(s) which disappears before ovulation and thus render(s) the cumulus cells permissive for P-450scc responsiveness. An additional intriguing aspect of P-450scc responsiveness to gonadotropins was revealed in experiments showing that 60% of the cultured cumulus complexes failed to accumulate P-450scc in response to hormones, if collected from 21 day animals. Interestingly, those P-450scc negative cumuli were always associated with an oocyte which did not resume meiotic maturation. We may therefore suggest that meiotic incompetence of the oocyte is also accompanied by functional incompetence of its embracing cumulus cells which cannot, for yet unclear reasons, acquire their steroidogenic capacity.

Mitochondria in follicular cells from rat ovaries were visualized in culture by indirect immunofluorescence staining of cholesterol side-chain cleavage cytochrome P-450 (P-450scc). The confinement of the immunofluorescence in the conspicuous mitochondria allowed the design of a very sensitive and quantitative assay to study the modulated expression of the cytochrome in primary cultures of granulosa cells. (1) The induction of P-450scc synthesis was totally dependent upon treatment with FSH. Up to 85% of the cells became immunofluorescently labeled in the presence of FSH, and its induced P-450scc synthesis was inhibited by cycloheximide and alpha-amanitin. The induction of FSH was dose dependent (Kmapp = 35 ng/ml) and time dependent. Prolonged incubation with FSH maintained the high levels of the cytochrome content, despite a desensitized steroidogenic response which developed after 60 h of incubation with FSH. Prolonged FSH treatment also resulted in morphological changes in the induced mitochondria, which became fragmented and globular. (2) Inoculum densities, probably by altering cell shape, substantially affected the extent of P-450scc induction; this was suppressed (80%) at lower culture densities. (3) The immunofluorescent staining also revealed various degrees of cellular competence to express P-450scc. Within a single induced cell, all mitochondria emitted a similar fluorescent signal, but the degree of fluorescence per mitochondrion varied with different cells. The cell-specific information gained by the immunofluorescent technique also allowed the detection of ovarian interstitial cells that slightly contaminate the granulosa cell preparations. Unlike granulosa cells, interstitial cells express and maintain high levels of P-450scc without the need for hormonal induction.