Pit-1, a tissue-specific POU domain transcription factor, is required for the activation of the prolactin, growth hormone, and Pit-1 promoters that confer regulation by epidermal growth factor, adenosine 3',5'-monophosphate (cAMP), and phorbol esters. Pit-1 is phosphorylated in pituitary cells at two distinct sites in response to phorbol esters and cAMP. Phosphorylation of Pit-1 modifies its conformation on DNA recognition elements and results in increased binding at certain sites and decreased binding at other sites, dependent on DNA sequences adjacent to the core Pit-1 binding motif. One residue (Thr220), located in the POU homeodomain within a sequence conserved throughout the POU-domain family, confers these responses.

Rabbit antiserum to rat cytochrome P-450 cholesterol side chain cleavage (P-450scc) was produced without a previous biochemical purification of the enzyme. Instead, for immunization we used a single protein band of mol wt 53,000, which was isolated from sodium dodecyl sulfate polyacrylamide gel electrophoresis of rat steroidogenic mitochondrial membranes. The resulting antiserum cross-reacted in a protein-blotting test with affinity purified and biologically active bovine adrenocortical P-450scc enzyme. The antiserum to the rat P-450scc also substantially blocked the conversion of cholesterol to pregnenolone in sonicated steroidogenic mitochondria, suggesting a successful cross-reactivity with the native form of the enzyme, despite the fact that the immunizing antigen was sodium dodecyl sulfate-denatured protein. The antiserum was applied for ultrastructural immunocytochemical visualization of the P-450scc in thin sections of adrenal cortex and immature ovary. Immunoreactive enzyme was identified by the protein-A-gold technique which showed that the gold particles concentrated exclusively in the steroidogenic mitochondria of adrenal zona glomerulosa and fasciculata cells. In the immature ovary, the only zone which was heavily stained with colloidal gold was the population of the interstitial cells. Part of the theca cell population contained P-450scc before PMSG treatment. The granulosa cells were devoided of the enzyme in any follicles before the preovulatory stage. The high resolution of the pAg technique allowed to visualize the localization of the P-450scc antigen in the matrix side of the inner mitochondrial membranes. Moreover, a clear coupling could be demonstrated between the morphological and functional maturation of the steroidogenic mitochondrion in the ovary: from a few lamella cristae devoid of P-450scc in the unstimulated granulosa mitochondria, to numerous tubulovesicular inner membranes, heavily loaded with the enzyme, in the mitochondria of the interstitial cells.

Using a specific antiserum against rat cholesterol side-chain cleavage cytochrome P-450 (P-450scc), we examined the expression of this key steroidogenic enzyme during follicular development in PMSG-treated immature rats. The accumulation of the enzyme was monitored in ovary homogenates by quantitative immunodot blot assay, while expression of P-450scc in various cell types was visualized concomitantly by immunofluorescent staining of ovarian cryosections. Before PMSG treatment, no labeling of P-450scc could be observed in follicular granulosa cells. In contrast, steroidogenic cytochrome was markedly expressed in interstitial cells, part of theca interna cells, and hypertrophied theca of atretic follicles. As a result of PMSG treatment, the interstitial thecal cells promptly enriched their P-450scc content within 24 h, whereas the granulosa cells acquired the enzyme at a later time, between 30 and 48 h after hormone administration. After ovulation, many corpora lutea filled most of the ovarian volume, and the ovarian content of P-450scc was 47 times higher than that in control ovaries of untreated rats. In granulosa cell population of a single preovulatory follicle, a downward gradient of P-450scc expression was observed, starting high in the cells abutting the basal lamina and decreasing toward the cells lining the antrum. Cumulus cells failed to express P-450scc. Referring to the basal lamina, theca interna cells exhibited a reverse gradient of P-450scc expression, starting high in peripheral cells close to the theca externa layer and decreasing in cells located near the follicular basement membrane. Immunofluorescent labeling revealed a major difference between P-450scc expression in thecal cells compared to that in granulosa cells. While expression of P-450scc in granulosa cells was restricted exclusively to cells within preovulatory follicles, P-450scc labeling was observed throughout the ovary in thecal and interstitial cells associated with follicles at any phase of follicular maturation. Therefore, it may be proposed that the thecal and interstitial cells represent an all ovarian network which expresses its steroidogenic capacity at early stages of follicular maturation and thereby is able to supply androgens necessary for the follicular development.

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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.