Dichlorodiphenyldichloroethylene potentiates the effect of protein kinase A pathway activators on progesterone synthesis in cultured porcine granulosa cells.

The insecticide dichlorodiphenyltrichloroethane (DDT) and its major metabolite p,p'-dichlorodiphenyldichloroethylene (DDE) have been implicated as endocrine-modulating chemicals. The DDT metabolite p, p'-DDE has been found contaminating human tissues and follicular fluid because of dietary exposure. We investigated the effects of DDE on progesterone synthesis in a stable porcine granulosa cell line, JC-410, and in primary cultures of porcine granulosa cells. Progesterone synthesis was not affected by 0.1-100 ng/ml DDE in the JC-410 cells. However, 10 ng/ml DDE increased 8-bromo-cAMP (8-Br-cAMP)-stimulated progesterone synthesis 0.4-fold (P < 0.05) over the levels observed with 1 mM 8-Br-cAMP alone. The effect of cholera toxin (CT) on progesterone synthesis was increased 0.7-fold (P < 0.05) by 10 ng/ml DDE over the value observed with 30 ng/ml CT alone. In primary cultures of porcine granulosa cells, 10 ng/ml DDE potentiated CT-stimulated progesterone synthesis 1.2-fold over the value observed with CT alone. In the JC-410 cells, 1 and 10 ng/ml DDE increased CT-stimulated cytochrome P450-cholesterol side-chain cleavage (P450(scc)) mRNA levels 0.3- and 0.4-fold, respectively, over the values obtained with CT alone. Neither basal nor CT-stimulated cAMP levels were changed by DDE. We conclude that DDE affects granulosa cell response to protein kinase A activators by altering the expression of the P450(scc) gene.     

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.     

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.     

Evidence for the presence of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin in fresh granulosa cells. Effects of follicle-stimulating hormone and cyclic AMP on cholesterol side chain cleavage cytochrome P-450 synthesis and activity

The synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450scc) and adrenodoxin was studied both in freshly harvested bovine granulosa cells and in granulosa cells maintained in primary monolayer culture. In addition, the action of follicle-stimulating hormone (FSH) and cyclic AMP analogs to stimulate the synthesis of cytochrome P-450scc was investigated in cultured cells. Precursor forms of cytochrome P-450scc and adrenodoxin were immunoisolated from a cell-free translation system directed by RNA prepared from freshly obtained granulosa cells that were not luteinized. Furthermore, the presence of cytochrome P-450scc in lysates of granulosa cells freshly obtained from very small follicles (containing less than 0.1 ml of follicular fluid) and in mitochondria of freshly obtained granulosa cells was demonstrated by using an immunoblotting technique. Continuous treatment of cultured granulosa cells with FSH or with cyclic AMP analogs (dibutyryl cyclic AMP or 8-bromo cyclic AMP) for 72 h increased incorporation of [35S]methionine into immunoprecipitable cytochrome P-450scc. Moreover, FSH, dibutyryl cyclic AMP, and 8-bromo cyclic AMP stimulated pregnenolone production by cultured granulosa cells (2.3-, 4.0-, and 7.5-fold increase over control, respectively), indicative of an increase in cholesterol side chain cleavage activity. The results of this study demonstrate for the first time the presence of two components of the cholesterol side chain cleavage system in freshly obtained granulosa cells, and provide direct evidence for the trophic effect of FSH and its presumed mediator, cyclic AMP, on the synthesis of cytochrome P-450scc 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.     

Purification and comparative characterization of cytochrome P-450scc from porcine adrenocortical mitochondria.

Cytochrome P-450scc (cholesterol side-chain cleavage enzyme) was purified from porcine adrenocortical mitochondria. 2. The purified cytochrome P-450scc was found to be homogeneous on SDS-polyacrylamide gel electrophoresis. 3. The heme content of the purified enzyme was 20.6 nmol/mg protein. 4. The enzymatic activity of the reconstituted cytochrome P-450scc-linked monooxygenase system amounted to 7.8 nmol of pregnenolone formed per nmole of P-450 per minute, with cholesterol as a substrate. 5. The amino acid sequence of the amino-terminal region of the cytochrome P-450scc and the amino acid residue at the carboxyl terminal were determined and compared with those of other mammalian cytochromes P-450scc.     

Ferredoxin and cytochrome P-450scc concentrations in granulosa cells of porcine ovaries during follicular cell growth and luteinization

The concentrations of cytochrome P-450scc and ferredoxin, two of the three proteins which comprise the mitochondrial steroidogenic electron transport chain, were measured in granulosa and luteal cells from porcine ovaries by an immunoblot procedure. During the follicular phase of the ovarian cycle the concentration of cytochrome P-450scc increased 5-fold and ferredoxin increased 3-fold. When the large follicles developed into corpora lutea the cytochrome P-450scc concentration increased a further 7-fold while ferredoxin increased only 3-fold. These changes were coincident with an overall 4-fold increase in the concentration of ferredoxin reductase during follicular cell development and luteinization. Analysis of the data revealed that the concentration of ferredoxin, which shuttles electrons from ferredoxin reductase to cytochrome P-450scc, was always adequate to saturate both the reductase and cytochrome P-450scc. This came about from a co-ordinate increase in the concentration of cytochrome P-450scc and the concentration of ferredoxin minus ferredoxin reductase.     

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.     

Adrenocortical cytochrome P-450 responsible for cholesterol side chain cleavage (P-450scc) is phosphorylated by the calcium-activated, phospholipid-sensitive protein kinase (protein kinase C)

Purified bovine adrenocortical cytochrome P-450scc (specific for cholesterol side chain cleavage in the inner mitochondrial membrane) was selectively phosphorylated in vitro by a Ca2+-activated, phospholipid-sensitive protein kinase (protein kinase C) preparation, whereas cyclic AMP dependent and two cyclic nucleotide independent kinases were ineffective. Cytochrome P-450scc incorporated a maximum of 4 mol of phosphate in the presence of protein kinase C within 15 min at 30 degrees C, with apparent Km and Vmax of 0.14 mumol and 0.76 pmol/min, respectively. Serine and threonine were the two target aminoacids phosphorylated in a ratio of about 1:1. In the presence of 1 microM Ca2+, a mixture of phosphatidylserine and diolein (or a potent tumor promoter phorbol ester) was required for optimal cytochrome P-450scc phosphorylation. In addition, purified inner mitochondrial membrane preparations from adrenocortical mitochondria were found to contain protein kinase C activity. These findings, together with the previous demonstration that activators of protein kinase C such as a potent phorbol ester activates steroidogenesis of intact adrenocortical cells, suggest that phosphorylation of P-450scc should be examined for its possible role in the regulation of adrenocortical functions.     

Actions of cyclic adenosine monophosphate on the cytodifferentiation of ovarian cells: studies in cultured swine granulosa cells using a novel exogenous adenylate cyclase from Bordetella pertussis

The regulation by cAMP of cholesterol side-chain cleavage activity and the synthesis of immunoisolated cytochrome P-450scc and adrenodoxin proteins was investigated in primary cultures of swine ovarian (granulosa) cells. Administration of a novel adenylate cyclase toxin isolated from Bordetella pertussis increased granulosa-cell cAMP accumulation up to 200-fold over basal. These effects were additive with those of FSH, forskolin, and cholera toxin. In contrast, bacterial extracts BP 347 and BP 348 from mutant strains of B. pertussis that lack either all virulent factors or the adenylate cyclase toxin and hemolysin were devoid of effect. Granulosa-cell cAMP accumulation supported by active bacterial adenylate cyclase was accompanied by 2- to 11-fold, time-dependent increases in [35S]methionine incorporation into immunospecific cytochrome P-450scc and adrenodoxin. These increases in the synthesis of cholesterol side-chain cleavage proteins were associated with enhanced pregnenolone production in response to exogenous sterol substrate, 25-hydroxycholesterol, and augmented progesterone secretion both in the absence and presence of exogenous lipoprotein. Moreover, the effects of Bordetella adenylate cyclase toxin on granulosa cell steroidogenesis were functionally integrated with other regulatory responses, since the non-cAMP dependent effector, estradiol 17-beta, interacted synergistically with bacterial adenylate cyclase in stimulating progesterone production. We conclude that exogenous adenylate cyclase isolated from B. pertussis can be functionally integrated into the cAMP-dependent effector pathway of granulosa cells with a resulting increase in intracellular cAMP concentrations, augmented biosynthesis of progesterone and pregnenolone, enhanced synthesis of immunospecific cytochrome P-450scc and adrenodoxin, and synergistic interactions with a non-cAMP-dependent ovarian effector hormone (estradiol).(ABSTRACT TRUNCATED AT 250 WORDS)     

Transfer of cholesterol between phospholipid vesicles mediated by the steroidogenic acute regulatory protein (StAR)

The steroidogenic acute regulatory protein (StAR) mediates the acute stimulation of steroid synthesis by tropic hormones in steroidogenic cells. StAR interacts with the outer mitochondrial membrane and facilitates the rate-limiting transfer of cholesterol to the inner mitochondrial membrane where cytochrome P-450scc converts this cholesterol into pregnenolone. We tested the ability of N-62 StAR to transfer cholesterol from donor vesicles containing cholesterol but no cytochrome P-450scc to acceptor vesicles containing P-450scc but no cholesterol, using P-450scc activity as a reporter of the cholesterol content of synthetic phospholipid vesicles. N-62 StAR stimulated P-450scc activity in acceptor vesicles 5-10-fold following the addition of donor vesicles. Transfer of cholesterol to acceptor vesicles was rapid and sufficient to maintain a linear rate of pregnenolone synthesis for 10 min. The effect of N-62 StAR in stimulating P-450scc activity was specific for cholesterol transfer and was not due to vesicle fusion or P-450scc exchange between vesicles. Maximum stimulation of P-450scc activity in acceptor vesicles required preincubation of N-62 StAR with phospholipid vesicles prior to adding donor vesicles. The amount of N-62 StAR causing half-maximum stimulation of P-450scc activity in acceptor vesicles was 1.9 microm. Half-maximum stimulation required more than a 10-fold higher concentration of R182L N-62 StAR, a mutant associated with congenital lipoid adrenal hyperplasia. N-62 StAR-mediated transfer of cholesterol between vesicles showed low dependence on the cholesterol concentration in the donor vesicles. Thus StAR can transfer cholesterol between synthetic membranes without other protein components found in mitochondria.     

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.     

Induction of synthesis of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin by follicle-stimulating hormone, 8-bromo-cyclic AMP, and low density lipoprotein in cultured bovine granulosa cells

The actions of follicle-stimulating hormone (FSH), 8-bromo-cyclic AMP (8-Br-cAMP), and low density lipoprotein (LDL) to stimulate the production of progesterone and the synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450ssc) and adrenodoxin were investigated in bovine granulosa cells maintained in primary monolayer culture. Treatment of granulosa cells in culture with FSH resulted in an increased incorporation of [35S]methionine into immunoprecipitable cytochrome P-450scc in a concentration-dependent fashion with a maximal effect being obtained at an FSH concentration of 500 ng/ml. Treatment of granulosa cells with FSH also resulted in the induction of synthesis of adrenodoxin. The cyclic AMP analog, 8-Br-cAMP, induced the synthesis of both cytochrome P-450scc and adrenodoxin to a greater extent than did FSH. LDL also stimulated the synthesis of both cytochrome P-450scc and adrenodoxin, when added to cells maintained in the presence of lipoprotein-poor serum. The presence of FSH or 8-Br-cAMP together with LDL resulted in a higher rate of enzyme synthesis than that observed with each effector alone. FSH, 8-Br-cAMP, and LDL also stimulated progesterone production by cultured granulosa cells. The results of this study offer a possible mechanism whereby granulosa cells undergo cytodifferentiation in vivo into luteal cells. The concentration of LDL in follicular fluid is very low. Following ovulation, vascularization of the follicle occurs and thus the granulosa cells are exposed to high levels of LDL, allowing for provision of substrate cholesterol, as well as stimulation of the synthesis of the enzymes involved in cholesterol side chain cleavage.