Comparison of pregnenolone synthesis by cytochrome P-450scc in mitochondria from porcine corpora lutea and granulosa cells of follicles

Substrate turnover rates by cytochrome P-450scc were measured in mitochondria isolated from corpora lutea and granulosa cells of follicles. Hydroxycholesterol substrates were added to the mitochondria to test the degree of saturation of the cytochrome with endogenous cholesterol during pregnenolone synthesis. 25-Hydroxycholesterol proved unsuitable for this since it was converted into pregnenolone with a maximum velocity of only 25% of that for cholesterol. 20 alpha-Hydroxycholesterol was found to be suitable providing correction was made for the one less hydroxylation required to convert this substrate into pregnenolone, compared to cholesterol. Mitochondria isolated from large follicles and corpora lutea displayed biphasic time courses for pregnenolone synthesis from endogenous cholesterol with a rapid phase lasting for 2-4 min and a slow phase which was linear for at least 30 min. Only a single rapid phase was observed for these mitochondria in the presence of 20 alpha-hydroxycholesterol. From the degree of stimulation of the substrate turnover rate by this steroid, it was concluded that the endogenous cholesterol concentration was saturating during the fast phase for large follicles but subsaturating in luteal mitochondria. Time courses for pregnenolone synthesis by mitochondria isolated from granulosa cells of small and medium follicles were linear for 30 min and gave a substrate turnover rate of 16-18 mol of steroid/min/mol of cytochrome P-450scc, similar to the turnover rates under saturating substrate conditions determined for large follicles and corpora lutea. The substrate turnover rate for cytochrome P-450scc in medium follicles was not increased by the addition of 20 alpha-hydroxycholesterol, indicating that the cholesterol concentration in the steroidogenic pool of these mitochondria was saturating and remained so over the 30-min duration of the incubation. It is therefore unlikely that gonadotropin stimulation of granulosa cells of small to medium follicles could acutely regulate pregnenolone synthesis by increasing the rate of transfer of cholesterol into a steroidogenic pool. This study shows that as the cytochrome P-450scc concentration in porcine ovarian mitochondria increases during follicular growth and luteinization there is a decrease in the fractional saturation of the cytochrome with cholesterol.     

Changes in content and phosphorylation of cytosol proteins in luteinizing ovarian follicles and corpora lutea

Cytosol prepared from rat preovulatory ovarian follicles contained several specific substrates which were phosphorylated by [gamma 32P] ATP in the presence of 2 microM cyclic AMP (cAMP) or 780 nM of highly purified catalytic subunit. These substrates were identified as RII, the regulatory subunit of type II cAMP-dependent protein kinase, an Mr = 43,000 protein presumed to be actin, and four other proteins with Mr = 36,500-15,000. A marked decrease in phosphorylation of these proteins was observed within 6-48 h of human chorionic gonadotropin (hCG)-induced ovulation and luteinization in hormonally primed immature rats. The phosphorylation of these proteins was also low in cytosol of corpora lutea isolated on Days 2, 4, 9, 13 and 23 of pregnancy. The decrease in phosphorylation of RII was associated primarily with a decrease in substrate content as measured by photoaffinity labeling and silver staining techniques, and not to a marked increase in phosphoprotein phosphatase and adenosinetriphosphatase (ATPase) activities. Whereas the decreased phosphorylation of other proteins is also presumed to be related to a decrease in their cytosol content, the data do not exclude the possibility that luteal tissue contains a specific phosphoprotein phosphatase which is not present in granulosa or theca cells of preovulatory follicles. We conclude that luteinizing hormone (LH) or hCG, and thereby cAMP itself, induces the rapid loss of specific phosphoproteins which may be involved in regulating cAMP action in granulosa cells.     

Localization of 3-hydroxy-3-methylglutaryl CoA reductase and 3-hydroxy-3-methylglutaryl CoA synthase in the rat liver and intestine is affected by cholestyramine and mevinolin

In the normal fed rat, both 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) synthase and HMG-CoA reductase are found in high concentrations in hepatocytes that are localized periportally. The majority of the liver cells show little or no evidence of either enzyme. Addition of cholestyramine and mevinolin to the diet resulted in all liver cells showing strong positive staining for both HMG-CoA reductase and HMG-CoA synthase. These two drugs increased the hepatic HMG-CoA reductase and HMG-CoA synthase activities 92- and 6-fold, respectively, and also increased the HMG-CoA reductase activity in intestine, heart, and kidney 3- to 15-fold. We used immunofluorescence and avidin-biotin labeled antibody to localize HMG-CoA reductase in the rat intestine. In rats fed a normal diet, the most HMG-CoA reductase-positive cells were the villi of the ileum greater than jejunum greater than duodenum. Crypt cells showed no evidence of HMG-CoA reductase. Addition of cholestyramine and mevinolin to the diet led to a dramatic increase in the concentration of HMG-CoA reductase in the apical region of the villi of the ileum and jejunum and in the crypt cells of the duodenum. Hence these two drugs affected both the relative concentration and distribution of intestinal HMG-CoA reductase. Cholestyramine and mevinolin feeding induced in the liver, but not intestine, whorls of smooth endoplasmic reticulum that were proximal to the nucleus and contained high concentrations of HMG-CoA reductase. Administration of mevalonolactone led to the rapid dissolution of the hepatic whorls within 15 min, at a time when there is little or no change in the mass of HMG-CoA reductase. We conclude that the whorls are present in the livers of rats fed cholestyramine and mevinolin because the cells are deprived of a cellular product normally synthesized from mevalonate.     

Adriamycin treatment increases 3-hydroxy-3-methylglutaryl CoA reductase and isoprene biosynthesis in the rat liver

Treatment of rats with Adriamycin caused an increase in the incorporation into hepatic cholesterol of [1-¹⁴C] acetate, but not of [2-¹⁴C] mevalonate. The step affected was found to be 3-hydroxy-3-methylglutaryl CoA reductase whose activity in the liver microsomes increased in Adriamycin-treated animals, but was inhibited when the drug was added in the assay medium. Also, the concentration of ubiquinone in the liver and of cholesterol in the plasma increased.     

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.     

Levels of messenger ribonucleic acid encoding cholesterol side-chain cleavage cytochrome P-450, 17 alpha-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor in bovine follicles and corpora lutea throughout the ovarian cycle

To investigate the molecular basis for the pattern of ovarian steroid production during the bovine estrous cycle, the relative levels of mRNA specific for cholesterol side-chain cleavage cytochrome P-450, 17 alpha-hydroxylase cytochrome P-450, adrenodoxin, and low density lipoprotein receptor were determined in ovarian antral follicles of differing size (less than 3-18 mm) and corpora lutea from the early, early-mid, late-mid, and regressionary stages. Total and poly(A)+ RNA was size-fractionated on agarose-formaldehyde gels, transferred to nylon filters and hybridized to specific 32P-labeled probes. The levels of mRNAs for the rate-limiting enzymes in the conversion of cholesterol into progesterone, namely cholesterol side-chain cleavage cytochrome P-450 and its electron donor, adrenodoxin, were higher in corpora lutea than in follicles. Conversely the levels of mRNA specific for the key regulatory enzyme in the conversion of pregnenolone or progesterone to androgen, namely 17 alpha-hydroxylase cytochrome P-450, were high in all antral follicles examined but were low in young corpora lutea and undetectable in more mature corpora lutea. Low density lipoprotein receptor mRNA was detectable in antral follicles and corpora lutea but the levels were greater in corpora lutea. These results suggest that the pattern of changes in steroid hormone biosynthesis during the bovine estrous cycle and in the ovarian content of steroidogenic enzymes is related to and probably dependent upon the pattern of change in levels of mRNAs for steroidogenic enzymes and related proteins.     

The interaction of cytochrome P450 17alpha with NADPH-cytochrome P450 reductase, investigated using chemical modification and MALDI-TOF mass spectrometry

The lysine residues of guinea pig P450 17alpha were acetylated by acetic anhydride in the absence and presence of NADPH cytochrome P450 reductase (CPR). Eight acetylated peptides were identified in the MALDI-TOF mass spectra of the tryptic fragments from the P450 acetylated without CPR in the limited reaction time of 15 min at ice temperature. The presence of CPR during the acetylation of P450 17alpha prevented double acetylations at K326 and K327 in the J-helix. The activity of P450 17alpha was decreased to 35% by the acetylation, but almost no inactivation was detected in the P450 after acetylation in the presence of CPR. This protection from inactivation shows the importance of K326 and/or K327 in the J-helix of P450 17alpha in the interaction between the two enzymes. Our results provided the first experimental evidence for the importance of the J-helix of P450 in the interaction with CPR. The interaction of P450 17alpha with CPR on the membrane is discussed based on the results of this study, which used molecular modeling.     

Changes in content and cAMP-dependent phosphorylation of specific proteins in granulosa cells of preantral and preovulatory ovarian follicles and in corpora lutea

The responsiveness of granulosa cells to the gonadotropins and cAMP increases as ovarian follicles mature. To determine if this change in response might be related to either the content or cAMP-dependent phosphorylation of specific proteins, we labeled proteins in 30,000 X g supernatant fractions (cytosol) with [gamma-32P] ATP in the presence or absence of cAMP. Using two-dimensional gel electrophoresis, we observed that granulosa cells of preantral follicles exhibited low amounts of cAMP-dependent phosphorylation of two proteins with apparent molecular weights of 54,000-56,000 and 43,000. Using [32P]8-N3cAMP and photoaffinity labeling procedures, the Mr = 54,000-56,000 protein was identified as RII, the regulatory subunit of type II protein kinase. Polychromatic silver staining, as well as the photoaffinity labeling, revealed that RII exists in three forms, each of which was also labeled by [gamma-32P] ATP. Based on the relative isoelectric points and specific silver staining of highly purified actin and phosphorylated actin, the Mr = 43,000 protein has been provisionally identified as actin. Five proteins (Mr = 37,500, 27,500, 22,500, 19,000, and 15,000), in addition to RII and actin, were phosphorylated in cytosol of granulosa cells from preovulatory follicles. By adding increasing concentrations of exogenous catalytic subunit to the cytosols, we demonstrated that the content, as well as the phosphorylation of these proteins, was increased selectively in granulosa cells of antral follicles. By using hypophysectomized rats, we demonstrated that these five proteins are induced by follitropin (FSH). Because they were not present in cytosols of thecal cells or corpora lutea, they appear to be specific markers for granulosa cells. The content and phosphorylation of RII was also dramatically increased in cytosols of granulosa cells from antral follicles, whereas that of actin remained unchanged. These observations indicate that granulosa cell differentiation involves regulation by FSH of specific proteins which are substrates for cAMP-dependent protein kinase. Thus, FSH and cAMP appear to regulate the intracellular content and phosphorylation of a cAMP response system in granulosa cells. The extent to which RII and the five specific phosphoproteins themselves regulate granulosa cell responsiveness remains to be determined.     

Hormonal regulation of estradiol biosynthesis, aromatase activity, and aromatase mRNA in rat ovarian follicles and corpora lutea

To determine the molecular basis for changes in aromatase (P450arom) activity in rat ovarian follicles and corpora lutea, seven clones for rat P450arom cDNA have been identified and isolated from a rat granulosa cell λgtll cDNA expression library using a 62 mer deoxyoligonucleotide probe (derived from an amino acid sequence of purified human placental aromatase) and a human placental P450arom cDNA probe. One of the rat P450arom cDNA clones contained an insert 1.2 kb in size. Both the human 1.8 kb cDNA and the rat 1.2 kb cDNA probes hybridized to a single species of P450arom mRNA that was 2.6 kb in size. Northern blot analysis revealed that corpora lutea isolated on day 15 of pregnancy contained high amounts of P450arom mRNA, whereas granulosa cells of antral follicles of hormonally primed, hypophysectomized rats (i.e., those from which mRNA was isolated to construct the cDNA library) contained only low amounts of P450arom mRNA. The lower amounts of P450arom in granulosa cells of preovulatory follicles in the estradiol-follicle-stimulating hormone primed hypophysectomized rats were unexpected because follicles incubated in medium containing testosterone substrate produce more estradiol than do corpora lutea isolated on day 15 of pregnancy and incubated under similar conditions. Additional studies will determine the hormonal events responsible for the elevated amounts and constitutive maintenance of P450arom mRNA and aromatase activity in luteal cells in vivo and in vitro.     

Expression of 3beta-hydroxysteroid dehydrogenase, cytochrome p450 17alpha-hydroxylase/17,20-lyase and cytochrome p450 aromatase enzymes in corpora lutea of diestrous and early pregnant mares

In the pregnant mare, luteal estrogen production increases at the onset of equine chorionic gonadotropin (eCG) secretion by endometrial cups. In previous studies, we have demonstrated that eCG stimulates luteal androgen and estrogen production in pregnant mares. To further elucidate the regulation of steroidogenesis within the equine corpus luteum (CL) of pregnancy, we examined the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/17,20 lyase (P450(17alpha)) and cytochrome P450 aromatase (P450(arom)) in luteal tissue samples collected during diestrus (Days 7 to 10) and pregnancy before (Days 29 to 35) and after (Days 42 to 45) the onset of eCG secretion. Immunoblot analyses revealed a single protein per enzyme with molecular weights of 48 kDa (3beta-HSD), 58 kDa (P450(17alpha)) and 56 kDa (P450(arom)). Steady-state levels of 3beta-HSD were lower in luteal tissue of diestrus than pregnancy, but expression did not change during pregnancy. Steady-state expression of P450(17alpha) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, P450(17alpha) expression was significantly higher after the onset of eCG secretion. Steady-state expression of P450(arom) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, luteal expression of P450(arom) was significantly lower after the onset of eCG secretion. These data support the hypotheses that eCG has a differential effect on the expression of luteal steroidogenic enzymes, that the eCG-induced increase in luteal estrogen production is the result of an increase in available aromatizable androgen due to an increase in P450(17alpha) expression and activity, and that increased luteal estrogen production is not due to an increase in aromatase expression.     

Regulatory effects of sterols and bile acids on hepatic 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol 7alpha-hydroxylase in the rat

Specific activities of the hepatic microsomal enzymes 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase and cholesterol 7alpha-hydroxylase were studied in rats fed sterols and bile acids. The administration of bile acids (taurocholate, taurodeoxycholate, taurochenodeoxycholate) at a level of 1% of the diet for 1 wk reduced the activity of HMG CoA reductase. Taurocholate and taurodeoxycholate, but not taurochenodeoxycholate, inhibited cholesterol 7alpha-hydroxylase. Dietary sitosterol produced increases in the specific activity of HMG CoA reductase (3.6-fold) and cholesterol 7alpha-hydroxylase (1.4-fold), and biliary cholesterol concentrations in this group more than doubled. Compared with controls fed the stock diet, the simultaneous administration of sitosterol and taurochenodeoxycholate resulted in a 60% decrease of HMG CoA reductase activity and no change in cholesterol 7alpha-hydroxylase activity or biliary cholesterol concentration. Rats fed sitosterol plus taurocholate had nearly normal HMG CoA reductase activity, but cholesterol 7alpha-hydroxylase was inhibited and biliary cholesterol remained high. Bile acid secretion rates and biliary bile acid composition were similar in controls and sterol-fed animals. In all groups receiving bile acids, biliary secretion of bile acids was nearly doubled and bile acid composition was shifted in the direction of the administered bile acid. It is concluded that the composition of the bile acid pool influences the hepatic concentrations of the rate-controlling enzymes of bile acid synthesis.     

Orange juice increased the bioavailability of pravastatin, 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, in rats and healthy human subjects

Our objective was to investigate the effects of orange juice on the pharmacokinetics of pravastatin in rats and healthy volunteers. The pharmacokinetics of pravastatin (100 mg/kg p.o.) were assessed with water, orange juice, and carbohydrates (12.5 ml/kg over 30 min) and with acetic acid (0.1 M, pH 3.44). The pharmacokinetics of simvastatin (100 mg/kg p.o.) were assessed with water and orange juice. In addition, the pharmacokinetics (based on plasma levels) of pravastatin 80 mg/kg i.v. were assessed with water and orange juice (5 ml/kg) in rats. The pharmacokinetics of oral pravastatin (10 mg) were assessed when administered with water and orange juice (800 ml over 3 h) in a two-way crossover study in 14 healthy volunteers. Orange juice significantly increased the area under the curve (0-150 min) of pravastatin in rats. Orange juice had no effects on the pharmacokinetic parameters of intravenously administered pravastatin in rats. Carbohydrates and acetic acid with pH and concentration equivalent to those of orange juice also resulted in no statistically significant differences in pravastatin pharmacokinetic parameters in rats. Orange juice did not result in any significant differences in the pharmacokinetic parameters of simvastatin in rats. Orange juice significantly increased oatp1 and oatp2 mRNA and protein in the intestine of rats. Orange juice significantly increased the area under the curve (0-240 min) of pravastatin in healthy volunteers. In conclusion, orange juice increases the bioavailability of pravastatin administered orally. Oatp1 and oatp2 may be related to increases of pharmacokinetics of pravastatin by orange juice.     

ACTH induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase, cholesterol biosynthesis, and steroidogenesis in primary cultures of bovine adrenocortical cells

The current studies demonstrate that corticosteroidogenesis can be maintained by primary cultures of bovine adrenocortical cells under lipoprotein-depleted conditions. The cholesterol necessary as substrate for steroid synthesis was found to arise from de novo synthesis within these cells. Adrenocorticotropin (ACTH) increased 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity 5-fold within 12 h after addition to the medium. The increase in activity apparently represented accumulation of enzyme as determined by protein blotting and immunodetection. The predominant immunodetectable species of HMG-CoA reductase from bovine adrenal cells was 97,000 daltons; no higher molecular mass species was detectable. The ACTH induction of HMG-CoA reductase activity could be prevented after inhibition of cholesterol conversion to pregnenolone with clotrimazole. These results are suggestive that ACTH increases adrenocortical cholesterol biosynthesis and HMG-CoA reductase activity after conversion of a cellular pool of cholesterol and/or oxysterol into steroid. The increased rate of cholesterol biosynthesis is then capable of maintaining ACTH-promoted steroid production. This is the first study, in vitro, to demonstrate an ACTH-promoted accumulation of HMG-CoA reductase of adrenocortical cells.     

Effect of melengestrol acetate on ovarian follicles,interstitial gland and corpora lutea in the rat: a quantitative morphological study

Summary Mature virgin rats were injected daily with 50 g melengestrol acetate (17-acetoxy-6-methyl-16-methyIenepregna-4,6-diene-3,20-dione, or MGA) and 0.2 g estradiol benzoate for periods of 1, 2, 3, and 4 weeks. Ovarian structures were examined histologically, histochemically and in the electron microscope. Graafian follicles during treatment did not attain pre-ovulatory size, ovulation was inhibited, and corpora hemorrhagica were not formed. During treatment, the percentage volume of the ovary occupied by atretic Graafian follicles increased, and that occupied by developing Graafian follicles decreased. In atresia, the membrana granulosa degenerated, and the theca cells hypertrophied peripherally to contribute to the surrounding interstitial gland. The structure and the weak lipid reaction of the hypertrophied theca cells of test follicles, when compared with estrus control follicles, suggested a low level of steroid production. The strong lipid reaction and ultrastructure of the interstitial gland suggested a lipid storage rather than secretory function. Corpora lutea, containing cells with a light lipid reaction and organelles suggestive of steroid synthesis, persisted throughout treatment. On the basis of claims, which associate the theca cells with estrogen synthesis and the luteal cells with progesterone synthesis, the morphological features described are consistent with primarily progestational and low-level estrogenic activities of MGA in the rat.This investigation was supported by the Alexander von Humboldt-Stiftung. The author is on leave from the Department of Veterinary Anatomy, University of Minnesota, St. Paul, U.S.A. The melengestrol acetate was supplied by the Upjohn Company, Kalamazoo, U.S.A.     

Changes in the expression of cytochrome P450c17 associated with ovarian cystic follicles. An immunocytochemical and enzymatic analysis of porcine ovaries.

The steroidogenic activity of normal preovulatory and cystic follicles, and corpora lutea of porcine ovaries was investigated by immunocytochemical and radioenzymatic techniques. Using a specific antibody to porcine cytochrome P450c17, immunocytochemical staining was specifically localized in the theca interna layer of normal follicles and undetectable in the granulosa layer. The theca interna layers of non-luteinized cystic follicles were immunoreactive while those of luteinized follicles were not. Corpora lutea cells were essentially negative. The 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase activity was similar in luteinized cystic follicular and corpora lutea tissues, which had 8 times higher activity than found in normal preovulatory follicles. The formation of either corpora lutea or luteinized cysts led to a profound decline (12- to 15-fold) in 17 alpha-hydroxylase and 17,20 lyase activities compared to normal preovulatory follicles. In agreement with these enzyme findings, radioimmunoassays revealed very high levels of progesterone with nearly undetectable levels of androgens in the luteinized cysts. These studies demonstrate the functional similarities between cells of luteinized cysts and those of normal corpora lutea and suggest a pathology associated suppression of P450c17 expression in porcine cystic follicles.     

The effect of glucagon, norepinephrine, and dibutyryl cyclic AMP on cholesterol efflux and on the activity of 3-hydroxy-3-methylglutaryl CoA reductase in rat hepatocytes.

Incubation of rat hepatocytes for 3 hours in a sterol-free medium containing 1.5% albumin resulted in efflux of cellular sterol into the medium and an increased activity of 3-hydroxy-3-methylglutaryl CoA reductase. The secretion of cholesterol was inhibited when cells were incubated with glucagon, norepinephrine, or dibutyryl cyclic AMP. Glucagon and dibutyryl cyclic AMP also inhibited the induction of HMG-CoA reductase. Norepinephrine treatment resulted in a decrease in the synthesis and secretion of proteins but caused an increase in reductase activity. Insulin treatment had no effect either on reductase activity or on sterol efflux from rat hepatocytes.