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Heterochromatin protein 1 binding protein 3 (HP1BP3) is a recently described histone H1-related protein with roles in chromatin structure and transcriptional regulation. To explore the potential physiological role of HP1BP3, we have previously described an Hp1bp3(-/-) mouse model with reduced postnatal viability and growth. We now find that these mice are proportionate dwarfs, with reduction in body weight, body length, and organ weight. In addition to their small size, microcomputed tomography analysis showed that Hp1bp3(-/-) mice present a dramatic impairment of their bone development and structure. By 3 weeks of age, mice of both sexes have severely impaired cortical and trabecular bone, and these defects persist into adulthood and beyond. Primary cultures of both osteoblasts and osteoclasts from Hp1bp3(-/-) bone marrow and splenocytes, respectively, showed normal differentiation and function, strongly suggesting that the impaired bone accrual is due to noncell autonomous systemic cues in vivo. One major endocrine pathway regulating both body growth and bone acquisition is the IGF regulatory system, composed of IGF-1, the IGF receptors, and the IGF-binding proteins (IGFBPs). At 3 weeks of age, Hp1bp3(-/-) mice exhibited a 60% reduction in circulating IGF-1 and a 4-fold increase in the levels of IGFBP-1 and IGFBP-2. These alterations were reflected in similar changes in the hepatic transcripts of the Igf1, Igfbp1, and Igfbp2 genes. Collectively, these results suggest that HP1BP3 plays a key role in normal growth and bone development by regulating transcription of endocrine IGF-1 components.

Steroid hormone synthesis is a vital function of the adrenal cortex, serves a critical role in gonadal function, and maintains pregnancy if normally executed in the placenta. The substrate for the synthesis of all steroid hormones is cholesterol, and its conversion to the first steroid, pregnenolone, by the cholesterol side-chain cleavage cytochrome P450 (CYP11A1) enzyme complex takes place in the inner mitochondrial membranes. Steroidogenic acute regulatory protein (STAR) facilitates the rate-limiting transfer of cholesterol from the outer mitochondrial membrane to CYP11A1 located in the inner organelle membranes. The current study explored the mechanisms controlling transcription of the Star gene in primary cell cultures of mouse placental trophoblast giant cells and rat ovarian granulosa cells examined throughout the course of their functional differentiation. Our findings show that the cis-elements required for Star transcription in the rodent placenta and the ovary are centered in a relatively small proximal region of the promoter. In placental trophoblast giant cells, cAMP is required for activation of the Star promoter, and the cis-elements mediating a maximal response were defined as cAMP response element 2 and GATA. EMSA studies show that placental cAMP-responsive element binding protein (CREB)-1 and activating transcription factor-2 (ATF2) bind to a -81/-78 sequence, whereas GATA-2 binds to a -66/-61 sequence. In comparison, patterns of Star regulation in the ovary suggested tissue-specific and developmental controlled modes of Star transcription. During the follicular phase, FSH/cAMP induced CREB-1 dependent activity, whereas upon luteinization STAR expression becomes cAMP and CREB independent, a functional shift conferred by FOS-related antigen-2 displacement of CREB-1 binding, and the appearance of a new requirement for CCAAT enhancer-binding protein beta and steroidogenic factor 1 that bind to upstream elements (-117/-95). These findings suggest that during evolution, the promoters of the Star gene acquired nonconsensus sequence elements enabling expression of a single gene in different organs, or allowing dynamic temporal changes corresponding to progressing phases of differentiation in a given cell type.

The age-related decline in testosterone biosynthesis in testicular Leydig cells has been well documented, but the mechanisms involved in the decline are not clear. Recent studies have described a cyclooxygenase-2 (COX2)-dependent tonic inhibition of Leydig cell steroidogenesis and expression of the steroidogenic acute regulatory protein (StAR). The present study was conducted to determine whether COX2 protein increases with age in rat Leydig cells and whether COX2 plays a role in the age-related decline in testosterone biosynthesis. Our results indicate that from 3 months of age to 30 months, COX2 protein in aged rat Leydig cells increased by 346% over that of young Leydig cells, StAR protein decreased to 33%, and blood testosterone concentration and testosterone biosynthesis in Leydig cells decreased to 41 and 33%, respectively. Further experiments demonstrated that overexpressing COX2 in MA-10 mouse Leydig cells inhibited StAR gene expression and steroidogenesis and that the inhibitory effects of COX2 could be reversed by blocking COX2 activity. Notably, incubation of aged Leydig cells with the COX2 inhibitor NS398 enhanced their testosterone biosynthesis. Blood testosterone concentrations in aged rats fed the COX2 inhibitor DFU, at doses of 5, 10, 15, and 20 mg/kg body weight per day were increased by 15, 23, 56, and 120%, respectively, over the levels in the rats receiving no DFU. The present study suggests a novel mechanism in male aging involving COX2 and a potential application of the mechanism to delay the age-related decline in testosterone biosynthesis.

The ontogeny and functional role of steroidogenesis during early gestation in rodents is poorly understood. In previous studies, we have shown that expression of messenger RNAs (mRNAs) encoding two key enzymes indispensable for de novo synthesis of steroid hormones, i.e. cholesterol side chain cleavage cytochrome P450 (P450scc) and a newly identified isoform of murine 3beta-hydroxysteroid dehydrogenase/isomerase type VI (3betaHSD VI), is initiated upon decidualization of the uterine wall induced by implantation. In situ hybridization and immunohistochemical visualization of 3betaHSD VI mRNA and protein shows high expression of this enzyme in the antimesometrial cells of the decidua of days 6.5-7.5 post coitum (p.c.). Thereafter, expression of 3betaHSD VI in the decidual zones disappears and is replaced by a high expression of mRNA and protein in the embryonal giant trophoblast cells. At the peak of their development on day 9.5 p.c., the mouse giant trophoblast cells also express Steroidogenic Acute Regulatory (StAR) protein, which is required for steroidogenesis in the gonads and adrenal cortex. Our findings also suggest that the declining levels of P450scc, 3betaHSD VI, and StAR proteins between days 10.5-14.5 p.c. in the developing placenta is consistent with previous reports that the mouse placenta is not involved in de novo synthesis of steroids during the second half of pregnancy. Collectively, the results of the present study suggest that, during early phases of pregnancy, local progesterone synthesis in the maternal decidua and the trophoblast layers surrounding the embryonal cavity is important for successful implantation and/or maintenance of pregnancy. We propose that the local production of progesterone acts as an immunosuppressant at the fetal maternal interface preventing the rejection of the fetal allograft.

It has been proposed that the steroidogenic acute regulatory (StAR) protein controls hormone-stimulated steroid production by mediating cholesterol transfer to the mitochondrial inner membrane. This study was conducted to determine the effect of wild-type StAR and several modified forms of StAR on intramitochondrial cholesterol transfer. Forty-seven N-terminal or 28 C-terminal amino acids of the StAR protein were removed, and COS-1 cells were transfected with pCMV vector only, wild-type StAR, N-47, or the C-28 constructs. Lysates from the transfected COS-1 cells were then incubated with mitochondria from MA-10 mouse Leydig tumor cells that were preloaded with [3H]cholesterol. After incubation, mitochondria were collected and fractionated on sucrose gradients into outer membranes, inner membranes, and membrane contact sites, and [3H]cholesterol content was determined in each membrane fraction. Incubation of MA-10 mitochondria with wild-type StAR containing cell lysate resulted in a significant 34.9% increase in [3H]cholesterol content in contact sites and a significant 32.8% increase in inner mitochondrial membranes. Incubations with cell lysate containing N-47 StAR protein also resulted in a 16.4% increase in [3H]cholesterol in contact sites and a significant 26.1% increase in the inner membrane fraction. In contrast, incubation with the C-28 StAR protein had no effect on cholesterol transfer. The cholesterol-transferring activity of the N-47 truncation, in contrast to that of the C-28 mutant, was corroborated when COS-1 cells were cotransfected with F2 vector (containing cytochrome P450 side-chain cleavage enzyme, ferridoxin, and ferridoxin reductase) and either pCMV empty vector or the complementary DNAs of wild-type StAR, N-47 StAR, or C-28 StAR. Pregnenolone production was significantly increased in both wild-type and N-47-transfected cells, whereas that in C-28-transfected cells was similar to the control value. Finally, immunolocalization studies with confocal image and electron microscopy were performed to determine the cellular location of StAR and its truncated forms in transfected COS-1 cells. The results showed that wild-type and most of the C-28 StAR protein were imported into the mitochondria, whereas most of N-47 protein remained in the cytosol. These studies demonstrate a direct effect of StAR protein on cholesterol transfer to the inner mitochondrial membrane, that StAR need not enter the mitochondria to produce this transfer, and the importance of the C-terminus of StAR in this process.

The steroidogenic acute regulatory protein (StAR) is a vital mitochondrial protein that is indispensable for the synthesis of steroid hormones in the steroidogenic cells of the adrenal cortex and the gonads. Recent studies have shown that StAR enhances the conversion of the substrate for all steroid hormones, cholesterol, into pregnenolone, probably by facilitating cholesterol entry into the inner compartment of the mitochondria where the steroidogenic cytochrome P450scc complex resides. To study the potential of StAR to affect ovarian steroidogenesis during follicular development, we examined the time-dependent expression of StAR protein and messenger RNA in PMSG/human CG (hCG)-treated immature rats. Western blot analyses and immunohistochemical and RT-PCR methodologies have revealed a biphasic expression of StAR in the ovaries responding to hormones. The first peak of StAR expression was generated by PMSG administration and lasted for 24 h. Furthermore, it was restricted to the entire network of the ovarian secondary interstitial tissue, as well as to a fewer scattered theca-interna cells. The second burst of StAR expression was observed in response to the LH surge, as simulated by hCG. This time, StAR was expressed in the entire theca-interna and interstitial tissue, as well as in those granulosa cells that were confined to periovulatory follicles. Immunoelectron microscopy studies revealed the over 90% of StAR antigenic sites are localized in the inner compartments of the mitochondrion, suggesting a rapid removal of StAR precursor from the mitochondrial surface, where it is believed to exert its activity. Altogether, our observations portray dynamic acute alterations of StAR expression during the process of follicular maturation in this animal model. Furthermore, if StAR indeed determines steroidogenic capacities in the ovary, our findings imply that, in immature rats undergoing hormonally induced first ovulation: 1) the early phases of follicular development are supported by androgen production originating from nonfollicular cells; 2) estrogen production in the granulosa cells of Graafian follicles is nourished by a submaximal androgenic output in the theca-interstitial compartments of the ovary.

The enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD) is a key enzyme in the biosynthesis of steroid hormones. To date, this laboratory has isolated and characterized five distinct 3beta-HSD complementary DNAs (cDNAs) in the mouse (3beta-HSD I through V). These different forms are expressed in a tissue- and developmentally-specific manner and fall into two functionally distinct enzymes. 3beta-HSD I and III, and most likely II, function as dehydrogenase/isomerases, whereas 3beta-HSD IV and V function as 3-ketosteroid reductases. This study describes the isolation, characterization, and tissue-specific expression of a sixth member of this gene family, 3beta-HSD VI. This new isoform functions as an NAD+-dependent dehydrogenase/isomerase exhibiting very low Michaelis-Menten constant (Km) values for pregnenolone (approximately 0.035 microM) and dehydroepiandrosterone (approximately 0.12 microM). 3beta-HSD VI is the earliest isoform to be expressed during embryogenesis in cells of embryonic origin at 7 and 9.5 days postcoitum (pc), and is the major isoform expressed in uterine tissue at the time of implantation (4.5 days pc) and continues to be expressed in uterine tissue at 6.5, 7.5, and 9.5 days pc. 3beta-HSD VI is expressed in giant trophoblasts at 9.5 days pc and is expressed in the placenta through day 15.5 pc. In the adult mouse, 3beta-HSD VI appears to be the only isoform expressed in the skin and also is expressed in the testis, but to a lesser extent than 3beta-HSD I. Mouse 3beta-HSD VI cDNA is orthologous to human 3beta-HSD I cDNA. Human type I 3beta-HSD has been shown to be the only isoform expressed in the placenta and skin. The demonstration that mouse 3beta-HSD VI functions as a dehydrogenase/isomerase and is the predominant isoform expressed during the first half of pregnancy in uterine tissue and in embryonic cells suggests that this isoform may be involved in local production of progesterone, which is needed for successful implantation of the blastocyst and/or maintenance of early pregnancy.

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.

Small cell aggregates of functional interstitial tissue were isolated from ovaries of 21- to 24-day-old rats, and their biochemical properties were studied in miniature cultures of 500-4000 cells. The isolated interstitial cells expressed high amounts of the mitochondrial cholesterol side-chain cleavage cytochrome P-450, visualized by immunofluorescent staining. Freshly isolated cells also expressed high activities of 17 alpha-hydroxylase and 17:20-lyase, which were assayed by TLC analysis of [3H]progesterone metabolites. The TLC technique revealed the immediate conversion of progesterone to 5 alpha-reduced progestins. Consequently, no aromatizable androgens were produced but, accumulation of androsterone resulted instead from the direct action of 17 alpha-hydroxylase on 3 alpha-hydroxy-5 alpha-pregnane-20-one (pregnanolone). Both the immunoreactive levels of cholesterol side-chain cleavage cytochrome P-450 and the rate of the 17:20-lyase activity declined rapidly during culture. However, addition of LH to the medium restored both enzymes, indicating the presence of functional LH receptors. The latter were also demonstrable by their ability to evoke cAMP formation in response to LH, but not to FSH. The activity of 5 alpha-reductase in the interstitial cells was much higher (3-fold) than its activity in the granulosa cells. Unlike 17:20 lyase, the activity of 5 alpha-reductase did not decay in culture, nor was it affected by LH. We thus established a novel and sensitive experimental method to isolate and study a minute population of ovarian cells which, unlike the follicular granulosa and theca cells, are enigmatically differentiated at the early stages of ovarian development.

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.

Long term cultures of rat granulosa cells were grown in serum-free medium, consisting of Dulbecco's modified Eagle's medium mixed 1:1 with Ham's nutrient F-12 medium and supplemented with insulin, transferrin, hydrocortisone, and fibronectin (4F medium). In sparse cultures (10(4) cells/cm2), the granulosa cells were steroidogenically responsive to ovine FSH (NIADDK-oFSH-15) during days 1-2 and 10-14 (responsive periods). The major steroids produced were 20 alpha-hydroxyprogesterone (20 alpha-OH-P) and 5 alpha-pregnane, 3 alpha,20 alpha-diol (pregnanediol). However, as of day 3, the cells gradually lost their steroidogenic responsiveness which was inhibited by 88% at day 7 (refractory period). Nevertheless, from day 8 onward, the cells regained their responsiveness which was fully restored at day 12. The transient loss of responsiveness was uniquely associated with progestin biosynthesis, since FSH-induced aromatase activity declined to background levels within 12 days and was never restored again. The loss of progestin responsiveness was not due to lack of cAMP because FSH induced increasing levels of cAMP accumulation, reaching maximal values on day 7 in culture. On the other hand, the onset of the refractory period occurred concomitantly with the entry of the cultured cells into a synchronous proliferation phase, during which the cell population doubled. Thereafter, as DNA synthesis ceased, the cells regained their steroidogenic responsiveness. A deliberate arrest of cell replication, in the presence of excess thymidine or in high density cultures, prevented the temporal loss of activity. The data presented favor the notion that cell proliferation and expression of differentiated functions are inversely related. It is suggested that growth-related processes suppress steroidogenesis by an as yet unknown mechanism.

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.

Using an affinity-purified antibody against cholesterol side-chain cleavage P450 (P450scc) and a human P450scc cDNA probe, 11 rat P450scc cDNA clones were identified and isolated from our rat granulosa cell lambda gt11 cDNA expression library. Two clones were plaque purified and subcloned into pBR322. One of these P450scc cDNA clones, approximately 1.2 kilobases (kb) in size, was used as a probe for Northern and filter hybridization assays to analyze the tissue distribution and hormonal regulation of P450scc mRNA in rat ovarian follicles and corpora lutea. Northern transfers revealed a single P450scc mRNA species about 2.0 kb in size. Filter hybridization assays showed that P450scc mRNA was low in granulosa cells and thecal cells of small antral follicles, was increased in both tissues of preovulatory follicles, and was rapidly (within 7 h) and maximally increased (30-fold) during hCG-induced luteinization. P450scc enzyme and mRNA were also elevated in corpora lutea isolated from pregnant rats (days 4-22 of gestation) and rats 1 day after parturition (day 23). The elevation of P450scc enzyme and mRNA was maintained despite the marked decline in serum progesterone concentrations between days 19-22, suggesting that once P450scc mRNA is induced in luteal tissue it may be constitutively expressed. Administering hormones to granulosa cells in culture and to hypophysectomized immature rats in vivo demonstrated that the induction of P450scc mRNA by FSH in granulosa cells was time, dose, and estradiol dependent. High doses of FSH acting on estradiol-primed cells gave the greatest response. The increase in P450scc mRNA in cultured granulosa cells was also stimulated by forskolin and was directly associated with increased synthesis of cAMP and progesterone accumulation. Thus, whereas the induction of P450scc mRNA in granulosa cells was dependent on hormones and cAMP, the maintenance of P450scc mRNA and P450scc protein in corpora lutea appears to involve constitutive expression of P450scc mRNA.

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.