Vasoactive intestinal peptide regulates cholesterol side-chain cleavage cytochrome P-450 (P-450scc) gene expression in granulosa cells from immature rat ovaries

Vasoactive intestinal peptide (VIP), a neuropeptide present in ovarian nerves, has been previously shown to induce synthesis of the side-chain cleavage cytochrome P-450 enzyme which catalyzes the conversion of cholesterol to pregnenolone (the rate-limiting step in progesterone synthesis). In the present study we demonstrate, by means of a bovine 3'-specific P-450scc cDNA probe, that this VIP effect is exerted at least partially at the level of gene expression in cultured granulosa cells that were isolated from estrogen-primed, immature rats. The size and level of the 2.0 kilobase P-450scc mRNA species was assessed by Northern blot analysis, while the translatability of this mRNA was assayed by immunoisolation of the 35S-labeled P-450scc precursor protein translated from total RNA of control and stimulated granulosa cells. FSH was much more effective than VIP at increasing P-450scc mRNA concentrations in cultured granulosa cells, whereas secretin treatment was ineffective. The results suggest that, like FSH, the stimulatory effect of the neuropeptide VIP on ovarian progesterone secretion involves regulation of P-450scc gene expression during functional maturation of the prepubertal ovary.     

Role of the peripheral-type benzodiazepine receptor and the polypeptide diazepam binding inhibitor in steroidogenesis

Steroidogenesis begins with the metabolism of cholesterol to pregnenolone by the inner mitochondrial membrane cytochrome P450 side-chain cleavage (P450scc) enzyme. The rate of steroid formation, however, depends on the rate of (i) cholesterol transport from intracellular stores to the inner mitochondrial membrane and (ii) loading of P450scc with cholesterol. We demonstrated that a key element in the regulation of cholesterol transport is the mitochondrial peripheral-type benzodiazepine receptor (PBR) and that the presence of the polypeptide diazepam binding inhibitor (DBI) was vital for steroidogenesis. We also showed that DBI, as the endogenous PBR ligand, stimulates cholesterol transport. In addition, DBI directly promotes loading of cholesterol to P450scc. We review herein our studies on the structure, function, topography and hormonal regulation of PBR and DBI in steroidogenic cells. Based on these data we propose a model where the interaction of DBI with PBR, at the outer/inner membrane contact sites, is the signal transducer of hormone-stimulated and constitutive steroidogenesis at the mitochondrial level. Hormone-induced changes in PBR microenvironment/structure regulate the affinity of the receptor. PBR ligand binding to a higher affinity receptor results in increased cholesterol transport. In addition, hormone-induced release (processing?) of a 30,000 M_W DBI-immunoreactive protein from the inner mitochondrial membrane may result to the intramitochondrial production of DBI which directly stimulates loading of P450scc with cholesterol. Thus, in vivo, hormonal activation of these two mechanisms results in efficient cholesterol delivery and utilization and thus high levels of steroid synthesis.     

Cyclic AMP-dependent and -independent regulation of cholesterol side chain cleavage cytochrome P-450 (P-450scc) in rat ovarian granulosa cells and corpora lutea. cDNA and deduced amino acid sequence of rat P-450scc

We report the isolating and sequencing of three cDNA clones encoding rat P-450scc, the nucleotide and protein sequences of which are highly homologous to those of bovine and human P-450scc, especially in the putative heme and steroid binding domains. We document that different molecular mechanisms regulate P-450scc in granulosa cells of preovulatory (PO) follicles prior to and after luteinization. Luteinizing hormone/human chorionic gonadotropin (LH/hCG) and cAMP are obligatory to induce P-450scc mRNA in PO granulosa cells in vivo and in vitro. Once P-450scc mRNA is induced as a consequence of the LH/hCG surge it is constitutively maintained by luteinized cells in vivo (0-4 days) and in vitro (0-9 days) in the absence of gonadotropins, is susceptible to modulation by prolactin and is no longer regulated by cAMP. Exposure to elevated concentrations of hCG in vivo for 5-7 h was required for PO granulosa cells to undergo a functional transition establishing the stable luteal cell phenotype. Transient exposure of PO + hCG (7 h) follicles in vitro to the RNA synthesis inhibitor actinomycin D (1 microgram/ml) or the protein synthesis inhibitor cycloheximide (10 micrograms/ml), for 1-5 h prior to culturing the granulosa cells failed to disrupt the induction of P-450scc mRNA, progesterone biosynthesis, and appearance of the luteal cell morphology. Inhibitors of protein kinase A (Rp-cAMPS; 1-500 microM and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8); 1-200 microM) added directly to the luteinized cell cultures also failed to alter P-450scc mRNA in these cells, although the cells contain in vivo amounts of mRNA for RII beta, RI alpha, and C alpha, the primary subunits of protein kinase A found in the rat ovary. These data suggest that expression of the P-450scc gene in rat ovarian follicular cells is regulated in a sequential manner by cAMP-dependent and cAMP-independent mechanisms associated with granulosa cells and luteal cells, respectively.     

Effects of ovarian theca cells on granulosa cell differentiation during gonadotropin-independent follicular growth in cattle

We investigated the effects of theca cells or FSH on granulosa cell differentiation and steroid production during bovine early follicular growth, using a co-culture system in which granulosa and theca cells were cultured on opposite sides of a collagen membrane. Follicular cells were isolated from early antral follicles (2-4 mm) that were assumed to be in gonadotropin-independent phase and just before recruitment into a follicular wave. Granulosa cells were cultured under serum-free conditions with and without theca cells or recombinant human FSH to test their effects on granulosa cell differentiation. Messenger RNA levels for P450 aromatase (aromatase), P450 cholesterol side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), LH receptor (LHr), and steroidogenic acute regulatory protein (StAR) in granulosa cells were measured by real-time quantitative RT-PCR analysis. FSH enhanced aromatase mRNA expression in granulosa cells, but did not alter estradiol production. FSH also enhanced mRNA expression for P450scc, LHr, and StAR in granulosa cells, resulting in an increase in progesterone production. In contrast, theca cells enhanced aromatase mRNA expression in granulosa cells resulting in an increase in estradiol production. Theca cells did not alter progesterone production and mRNA expression in granulosa cells for P450scc, 3beta-HSD, LHr, and StAR. The results of the present study indicate that theca cells are involved in both rate-limiting steps in estrogen production, i.e., androgen substrate production and aromatase regulation, and that theca cell-derived factors regulate estradiol and progesterone production in a way that reflects steroidogenesis during the follicular phase of the estrous cycle.     

Steroid regulation of progesterone synthesis in a stable porcine granulosa cell line: a role for progestins

The objective of this investigation was to determine the effect of steroid hormones on the synthesis of progesterone in a stable porcine granulosa cell line, JC-410. We also examined the effect of steroid hormones on expression of the genes encoding the steroidogenic enzymes, cytochrome P450-cholesterol side chain cleavage (P450scc) and 3β-hydroxy-5-ene steroid dehydrogenase (3β-HSD). We observed that 48 h exposure of the JC-410 cells to estradiol-17β (estradiol), androstenedione, 5-dihydrotestosterone, levonorgestrel, and 5-cholesten-3β, 25-diol (25-hydroxycholesterol) resulted in stimulation of progesterone synthesis. 25-Hydroxycholesterol augmented progesterone synthesis stimulated by estradiol, 5-dihydrotestosterone, levonorgestrel and 8-bromoadenosine 3′:5′-cyclic monophosphate (8-Br-cAMP). This increase in progesterone synthesis was additive with estradiol, 5-dihydrotestosterone and levonorgestrel, and synergistic with 8-Br-cAMP. Cholera toxin, progesterone, levonorgestrel and androstenedione increased P450scc mRNA levels, whereas estradiol had no effect. Cholera toxin, progesterone and levonorgestrel increased 3β-HSD mRNA levels, but estradiol and androstenedione had no effect. The results were interpreted to mean that estrogens, androgens and progestins regulate progesterone synthesis in the JC-410 cells. The effect of androgens appears to be mediated by stimulation of P450scc gene expression while progestins stimulate both P450scc and 3β-HSD gene expression. Our results support the concept that progesterone is an autocrine regulator of its own synthesis in granulosa cells.     

Growth factor modulation of steroidogenic acute regulatory protein and luteinization in the pig ovary.

In vivo and in vitro luteinization were investigated in the porcine ovary, with emphasis on expression of steroidogenic acute regulatory protein (StAR). StAR mRNA and protein as well as cytochrome P450 side-chain cleavage mRNA (P450scc) increased during the luteal phase in the corpus luteum (CL) and were absent in regressed CL. Cytochrome P450 aromatase mRNA (P450arom) was not detectable at any time in CL. In vitro luteinization of granulosa cells occurred over 96 h in culture, during which P450arom mRNA was present at 1 h after cell isolation but not detectable at 6 h; and P450scc and StAR mRNAs were first detectable at 6 h and 48 h, respectively. Incubation of cultures with insulin-like growth factor I (IGF-I, 10 ng/ml), dibutyryl cAMP (cAMP, 300 microM), or their combination, induced measurable StAR mRNA at 24 h (p < 0.05), increased progesterone accumulation at 48 h, and elevated both StAR and P450scc expression through 96 h. Incubation of luteinized granulosa cells with epidermal growth factor (EGF, 10 nM) changed their phenotype from epithelioid to fibroblastic, eliminated steady-state StAR expression, and interfered with cAMP induction of StAR mRNA and progesterone accumulation. EGF had little apparent effect on P450scc mRNA abundance. It is concluded that StAR expression characterizes luteinization, and early luteinization is induced by cAMP and IGF-I in vitro. Further, EGF induces a morphological and functional phenotype that appears similar to an earlier stage of granulosa cell function.     

The physiology of the ovary: Maturation of ovarian granulosa cells and a novel role for antioxidants in the corpus luteum

During folliculogenesis the granulosa cells divide whilst in contact with each other, and so exhibit some of the characteristics of stem cells. In vitro we have shown that bovine granulosa cells from 3–7 mm follicles, like stem cells, divide without the need for a substratum, and produce colonies of cells. Growth factors, bFGF and IGF's, stimulate their division. These cells secrete and assemble a basal lamina, suggesting that the follicular basal lamina is produced by the granulosa cells. They have the morphological characteristics of follicular granulosa cells. Thus this system is ideal for studying the functions of immature granulosa cells because the cells do not spontaneously differentiate or luteinize into luteal cells, as occurs in culture on a substratum. On differentiation into luteal cells in vivo the cells express the steroidogenic enzymes for progesterone production and accumulate β-carotene. During culture of bovine luteal cells we observed that a proportion of the steroidogenic enzyme cholesterol side-chain cleavage cytochrome P450 enzyme became chemically cross-linked to its electron donor, adrenodoxin. P450 enzymes produce oxygen free radicals and oxygen free radicals can cause cross-linking between proteins in close proximity. Cell protect against this damage by the use of antioxidant vitamins. Repleting the cultured luteal cells with β-carotene reduced the amount of cross-linking. We conclude that the high levels of β-carotene in corpora lutea are to protect against damage due to oxygen free radicals generated in the course of progesterone synthesis.     

Regulated expression of sterol carrier protein 2 in the ovary: a key role for cyclic AMP.

Sterol carrier protein 2 (SCP2) is believed to play an important role in the intracellular movement of cholesterol in steroidogenic cells. We examined the distribution of SCP2 gene expression in the rat ovary and the role of gonadotropins and cyclic AMP in the regulation of SCP2 mRNA levels. In situ hybridization revealed that the most steroidogenically active ovarian compartments (e.g., corpora lutea and theca cells) contain significant amounts of SCP2 mRNA whereas granulosa cells have modest levels. Gonadotropins, which promote follicular growth and luteinization, increased the ovarian content of SCP2 mRNA as assessed by Northern blotting along with increases in cytochrome P450scc mRNA. Using steroidogenic transformed rat granulosa cells (Grs-21), a cyclic AMP analogue (8-Br-cAMP) was found to increase SCP2 mRNA and protein levels within 24 h of treatment. P450scc mRNA was also induced whereas actin mRNA levels were not affected. The 8-Br-cAMP stimulation of SCP2 mRNA accumulation was completely inhibited by actinomycin D and cycloheximide. The cyclic AMP analogue also increased SCP2 mRNA levels in a non-steroid hormone producing transformed rat granulosa cell line Gs-8. We conclude that SCP2 gene expression in the ovary is correlated with the state of differentiation of granulosa cells. Gonadotropic hormones which stimulate luteinization of the cells increase SCP2 gene expression. These actions of gonadotropins appear to be mediated at least in part by cyclic AMP through a mechanism requiring ongoing RNA and protein synthesis. However, SCP2 gene expression is not obligatorily coupled to steroidogenic activity, as cyclic AMP analogues can increase SCP2 mRNA in a line of transformed ovarian granulosa cells incapable of synthesizing hormones.     

Rat cholesterol side-chain cleavage enzyme (P-450scc): use of a cDNA probe to study the hormonal regulation of P-450scc mRNA levels in ovarian granulosa cells

A rat ovarian cDNA library was constructed and screened by differential colony hybridization to detect cDNA clones specific for mRNA induced by follicle-stimulating hormone (FSH). The cDNA clone which demonstrated the greatest degree of induction contained a 766-bp insert which was characterized and sequenced. We conclude that this cDNA is specific for the rat gene coding for cholesterol side-chain cleavage enzyme (P-450scc) by virtue of nucleotide sequence homology to the bovine and human P-450scc cDNA sequences. Southern blotting of rat genomic DNA suggests the presence of a single P-450scc gene. Northern blot analysis indicates that P-450scc mRNA is present in steroidogenic tissues (ovary, adrenal, testis), but not in brain, kidney, liver, lung, or heart. The rat P-450scc mRNA is induced by FSH or pregnant mare's serum gonadotropin in ovaries of estrogen-treated immature rats in vivo. In cultured granulosa cells, estradiol treatment alone did not increase P-450scc mRNA levels, but in combination with FSH or 8-Br-cAMP resulted in three- to four-fold increase in this mRNA.     

Regulation of cytochrome P450scc synthesis and activity in the ovine corpus luteum

The rate-limiting step in luteal biosynthesis of progesterone consists of cleavage of the side chain of cholesterol by mitochondrial cytochrome P450 side-chain cleavage enzyme (P450_scc) to form pregnenolone. Luteal mRNA encoding P450_scc, quantitated on selected days of the 16-day ovine estrous cycle, was similar on days 3 and 6, increased by 2-fold on day 9 (P < 0.05) and remained elevated on day 15. Levels of P450_scc mRNA on day 15 of pregnancy were not different from those found on any day of the cycle (P < 0.05). To determine whether levels of mRNA encoding P450_scc are hormonally regulated, ewes on day 10 of the estrous cycle were injected with hCG or prostaglandin F₂ (PGF₂). P450_scc mRNA was not increased for up to 36 h after injection of hCG, nor decreased within 8 h after injection of PGF₂ (P < 0.05). An assay for P450_scc activity was developed which utilized ovine small and large luteal cells in the presence of 22R-hydroxycholesterol and ovine high density lipoprotein. Enzyme activity was quantitated by measurement of progesterone production. In small luteal cells activation of the protein kinase A (PKA) second-messenger system by treatment with LH resulted in 910% increase in progesterone production without altering activity of P450_scc. Activation of the protein kinase C (PKC) second-messenger system with phorbol 12-myristate 13-acetate caused a 51% reduction in progesterone secretion from large luteal cells but did not alter activity of P450_scc. These findings suggest that in mature luteal tissue steady state levels of mRNA encoding P450_scc, and enzyme activity are independent of acute regulation by activation of PKA or PKC second-messenger systems.