Cytochrome P450 Cholesterol Side-chain Cleavage Research Articles

Steroidogenic factor 1 (SF-1) and SP1 are required for regulation of bovine CYP11A gene expression in bovine luteal cells and adrenal Y1 cells.

Cholesterol side-chain cleavage cytochrome P450 (CYP11A; P450scc) gene expression is regulated by gonadotropins via cAMP in the ovary and by ACTH via cAMP in adrenal cortical cells. Previously, we have characterized a response element located at -118 to -101 bp in the 5'-flanking region of the bovine P450scc gene required for cAMP-stimulated transcription in both mouse adrenocortical Y1 cells and bovine ovarian cells in primary culture. It was shown that this region contains a binding site for the transcription factor Sp1. Deletion of this sequence abolished cAMP-stimulated transcription in both Y1 cells and bovine ovarian luteal cells. Another sequence element located at -57 to -32 bp upstream from the transcription initiation site, which is highly conserved in CYP11A of other species, contains the motif TAGCCTTG, similar to the consensus binding site of steroidogenic factor-1, SF-1 (or Ad4-BP), but in the inverted orientation. In the present study, gel shift analysis using nuclear extracts of either Y1 cells or bovine luteal cells demonstrated that the sequence between -57 and -32 bp bound SF-1. A mutation of the SF-1-binding site that abolished binding of the nuclear protein to DNA reduced markedly the basal transcription of the reporter gene as well as the responsiveness to cAMP, when the mutated fragments containing the region from -186 to +12 bp were cloned into a luciferase construct and transfected into mouse adrenal Y1 cells and bovine luteal cells. The role of SF-1 in P450scc transcription was further confirmed by transactivation of the -186/+12Luc construct employing an SF-1 expression vector after transfection into nonsteroidogenic COS-1 cells. In addition, results obtained employing a double mutation of the Sp1- and SF-1-binding sites, and from a construct containing both Sp1 and SF-1 elements upstream of the CYP11A TATA box, indicated that Sp1 and SF-1 function cooperatively in the transactivation of the bovine CYP11A promoter in both bovine luteal cells and Y1 cells. Finally, a mammalian two-hybrid system was employed to demonstrate that Sp1 and SF-1 can associate in vivo. These results establish that basal and cAMP-stimulated activity of the bovine P450scc promoter in both Y1 cells and bovine luteal cells requires the combined action of at least two transcription factors, Sp1 and SF-1.

Steroidogenic factor-1 expression is transiently repressed and c-myc expression and deoxyribonucleic acid synthesis are induced in rat granulosa cells during the periovulatory period.

The expression of steroidogenic factor 1 (SF-1), cytochrome P450 aromatase (P450arom), and cytochrome P450 cholesterol side-chain cleavage (P450scc) was examined during the periovulatory period. Immature rats were injected with eCG to induce development of ovarian follicles to the preovulatory stage. At 48 h after the eCG injection, the LH surge was simulated by an injection of an ovulatory dose of hCG, and RNA was isolated at 0, 2, 4, 6, 8, and 24 h after hCG injection. The mRNA levels for SF-1, P450arom, and P450scc were relatively high in total ovarian RNA samples from eCG-treated rats. Levels of SF-1 and P450arom mRNA decreased within 2 h after injection of hCG. The SF-1 mRNA levels gradually increased from 4 to 24 h. Aromatase levels remained undetectable until 24 h after hCG. P450scc mRNA levels increased throughout the 24-h period after hCG. Levels of SF-1 and P450arom, but not P450scc, mRNA were also reduced in RNA samples from isolated granulosa cells at 4 h after hCG treatment relative to those from eCG-treated rats. In situ hybridization analysis also revealed that hCG uniformly suppressed SF-1 mRNA levels an all granulosa cells compared to those of eCG-treated controls. The relationship of SF-1 expression to immediate/early gene expression and cell cycle traverse was also examined. C-myc mRNA levels were induced by up to 10-fold at 4 h after hCG injection. Similarly, DNA synthesis, as measured by the percentage of granulosa cells that incorporated 5'-bromodeoxyuridine, was increased from 16 +/- 4% in eCG-treated rats to 61 +/- 7% at 4 h after hCG treatment (p < 0.05). This study provides the novel finding that SF-1 expression is transiently repressed to very low levels in response to the LH surge. Further, these studies suggest that granulosa cells traverse the cell cycle before becoming terminally differentiated luteal cells.

Insulin-like growth factors enhance steroidogenic enzyme and corticotropin receptor messenger ribonucleic acid levels and corticotropin steroidogenic responsiveness in cultured human adrenocortical cells.

In several species, including the human fetus, insulin-like growth factors (IGF-I and IGF-II) have been reported to modulate adrenal steroidogenesis, thus contributing to adrenal cortical differentiation. In the present study, we examined the long term effects of IGF-I and -II on human adult adrenal fasciculata-reticularis cells cultured in a chemically defined medium and compared them to the effects of insulin, human GH, and ACTH. Treatment for 3 days with IGF-I or -II at nanomolar concentrations or with insulin at micromolar concentrations slightly increased the production of androstenedione, cortisol, and dehydroepiandrosterone about 1.5-fold over that by control cells. Moreover, the acute steroidogenic response to ACTH of cells pretreated with IGF-I, IGF-II, or insulin was 3- to 6-fold higher than that of control cells. For each hormone, these effects of IGF-I and -II were dose dependent between 0.1-26 nmol/L (1-200 ng/mL). The secretion of androstenedione was more potently stimulated than that of dehydroepiandrosterone and cortisol, and this effect was more clearly yielded by pretreatment with IGF-II than with IGF-I or insulin. Human GH had no effect on these cells. In cells treated with IGF-I or -II, the messenger ribonucleic acid (mRNA) levels of cytochrome P450 17 alpha-hydroxylase and of 3 beta-hydroxysteroid dehydrogenase were increased, and the abundance of ACTH receptor mRNA was also slightly enhanced, but the mRNA of cytochrome P450 cholesterol side-chain cleavage enzyme was unchanged. In conclusion, IGFs enhance the steroidogenesis and ACTH responsiveness of human adrenocortical cells in culture. We speculate, that by this mechanism, IGFs may contribute to clinical states with hyperandrogenemia.

Functional maturation of the primate fetal adrenal in vivo. II. Ontogeny of corticosteroid synthesis is dependent upon specific zonal expression of 3 beta-hydroxysteroid dehydrogenase/isomerase.

Cortisol, produced by the primate fetal adrenal, regulates the maturation of organ systems necessary for extrauterine life. During most of primate pregnancy, however, the fetal adrenal lacks the enzyme 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD), which is essential for cortisol synthesis. Therefore, we used immunohistochemistry and in situ hybridization techniques to investigate the developmental expression of 3 beta HSD in the fetal rhesus monkey adrenal from 109 days' gestation until term (165 +/- 5 days) and assessed the role of ACTH in the induction of its expression and localization. We also examined whether ACTH regulates the expression of two other steroidogenic enzymes, cytochrome P450 cholesterol side-chain cleavage (P450scc) and P450 17 alpha-hydroxylase, 17/20-lyase (P450c17), in the fetal rhesus monkey adrenal. To stimulate ACTH secretion from the fetal pituitary in vivo, we administered metyrapone to late gestation fetal rhesus monkeys for 3-7 days. Adrenals were collected from untreated fetuses at 109-125 days (n = 5), 130-148 days (n = 7), 155-172 days (n = 4), and after metyrapone treatment at 135-137 days (n = 4). The cortical width and total amount of 3 beta HSD staining were measured using an image analysis system. 3 beta HSD was localized primarily in the definitive zone cells of the adrenal from fetuses between 109-148 days, whereas at term (155-172 days), 3 beta HSD was localized in both definitive and transitional zone cells. The cortical width and total amount of 3 beta HSD staining in the adrenal increased significantly (P < 0.05) between 148 days (137 +/- 14 microns and 3,689 +/- 522 grains) and 155 days (315 +/- 61 microns and 7,321 +/- 2,008 grains). Interestingly, in metyrapone-treated fetuses at 135-137 days, 3 beta HSD messenger RNA (mRNA) and protein were localized extensively in both the definitive and transitional zones, a pattern seen only in term fetal adrenals in untreated animals. In addition, metyrapone treatment significantly (P < 0.05) increased cortical width (386 +/- 95 microns) and total 3 beta HSD immunostaining (29,063 +/- 13,692 grains) compared with age-matched controls. In contrast to 3 beta HSD, P450scc mRNA was detected in the definitive, transitional, and fetal zones, and its expression was not altered after metyrapone treatment. P450c17 mRNA was detected in the transitional and fetal zones, and the relative abundance was greater in the transitional zone. The relative abundance of P450c17 mRNA was increased in the fetal zone after metyrapone treatment. In summary, at term or after metyrapone treatment, expression of 3 beta HSD is induced in the transitional zone of the fetal rhesus monkey adrenal gland, an indication of functional maturation of the primate adrenal cortex. These data suggest that the ontogenetic increase in fetal pituitary ACTH secretion plays an important role in the induction of 3 beta HSD expression in the transitional zone.

Role of calmodulin-dependent protein kinase II in the acute stimulation of aldosterone production.

Acute aldosterone production in adrenocortical cells is highly dependent on calcium (Ca2+) and calmodulin (CaM) activation. To determine the role of calmodulin-dependent protein kinase II (CaM kinase II) in human adrenal aldosterone production, the action of KN93 (a specific CaM kinase II inhibitor) on human adrenocortical H295R cells was examined. The stimulation of aldosterone, production by angiotensin II (Ang II) and potassium (K+) were inhibited by KN93 in a concentration-dependent manner with an IC50 of approximately 0.9 and approximately 0.5 microM, respectively. Aldosterone production was also stimulated by treatment with the calcium channel activator Bay K 8644 (Bay K) (1 microM). This production was inhibited in a concentration-dependent manner by KN93 with an IC50 of between 1 and 3 microM. No inhibition by KN93 (0.3-3 microM) or by the calmodulin inhibitor calmidazolium (0.03-0.3 microM) was observed for 22R-hydroxycholesterol (22R-OHChol) stimulation of aldosterone production. Because 22R-OHChol is a substrate for the cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) and does not require active transport to the mitochondria, these results indicate that KN93 does not directly inhibit P450scc or later steps leading to aldosterone synthesis. To investigate the site of KN93 action further we examined its effect on agonists induction of steroidogenic acute regulatory (StAR) protein, which was recently shown to regulate the movement of cholesterol from the outer to the inner mitochondrial membranes. Induction of StAR protein in H295R cells by Ang II, or Bay K was not affected by co-treatment with KN93 at concentration which blocked steroidogenesis by 60-80%. These results indicate a direct role of CaM kinase II in Ang II and K+ simulation of aldosterone production and support the hypothesis that CaM kinase II may be involved in the process of cholesterol mobilization to the mitochondria.

A role for epidermal growth factor in the morphological and functional maturation of the adrenal gland in the fetal rhesus monkey in vivo.

We determined the effects of epidermal growth factor (EGF) and beta-methasone on the growth and development of the adrenal gland of the fetal rhesus monkey in vivo between 121-128 days of gestation. The adrenal to body weight ratio was significantly greater (P < 0.05) in EGF-treated fetuses (0.988 +/- 0.046 x 10(-3) g/g) and significantly reduced (P < 0.05) in beta-methasone-treated fetuses (0.401 +/- 0.056 x 10(-3) g/g) compared with that in control fetuses (0.689 +/- 0.050 x 10(-3) g/g). The increase in adrenal weight with EGF administration was due to hypertrophy of definitive zone cells of the adrenal cortex, whereas the reduction in adrenal weight after beta-methasone treatment was due to a decrease in the size of definitive and fetal zone cells of the adrenal cortex. By Western analysis, EGF treatment induced a significant (P < 0.05) 2.8-fold increase in the amount of protein for 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD) in the fetal adrenal. EGF also stimulated the induction of immunocytochemical staining for 3 beta HSD in transitional zone cells of the adrenal cortex. In contrast, beta-methasone resulted in 2.6-, 4.5-, and 6.6-fold significant decreases (P < 0.05) in the amount of protein for cytochrome P450 cholesterol side-chain cleavage, cytochrome P450 17 alpha-hydroxylase/17,20-lyase, and 3 beta HSD in the fetal adrenal. After beta-methasone treatment. 3 beta HSD staining was detected in some of the definitive zone cells, with no 3 beta HSD staining in the transitional zone. In conclusion, growth and functional differentiation of fetal primate adrenal gland can be accelerated prematurely by EGF and inhibited by glucocorticoid negative feedback.

Paradoxical effect of 3-isobutyl-1-methylxanthine on cytochrome P450 cholesterol side-chain cleavage mRNA accumulation in porcine granulosa cells

Earlier studies in immature porcine granulosa cells cultured in serum-free medium showed dual actions of the protein kinase C activator 12- O-tetradecanoylphorbol-13-acetate (TPA). In cells incubated for 24 h, TPA inhibited follicle-stimulating hormone (FSH)-stimulated cytochrome P450 cholesterol side-chain cleavage (P450 scc) mRNA accumulation. In contrast, at 4 h, TPA increased P450 scc mRNA concentration in the absence and presence of FSH or 8-bromo-cAMP; in addition, TPA augmented FSH-stimulated cAMP accumulation. The actions of TPA were then examined in the presence of the phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX). With IBMX present, TPA caused a smaller relative augmentation of cAMP accumulation during a 4-h incubation period, suggesting that TPA may both increase cAMP synthesis and inhibit its degradation. The stimulatory effect of FSH or 8-bromo-cAMP on P450 scc mRNA concentration was not modified by IBMX. However, TPA no longer augmented the FSH- or 8-bromo-cAMP-stimulated P450 scc mRNA accumulation when IBMX was present. In cells treated with FSH for 24 h, IBMX augmented progesterone production, but paradoxically accentuated the inhibitory effect of TPA on steroidogenesis. These results indicate that IBMX converts TPA from a stimulatory into an inhibitory agent by an action unrelated to cAMP, and points to the need for caution in interpreting experiments with this drug.

Production of extracellular matrix, fibronectin and steroidogenic enzymes, and growth of bovine granulosa cells in anchorage-independent culture.

A proportion of the granulosa cells from bovine antral follicles will survive, like stem cells, in anchorage-independent culture. To study these cells, bovine granulosa cells were isolated from medium-sized follicles (3-5 mm), plated out (in aliquots of 2.5 x 10(4) viable cells) onto a 1 mL agar base, and overlaid with 1 mL of methycellulose solution in culture medium (control). The cells were cultured (14 days) and then processed for histology (n = 14) or Western immunoblotting (n = 5). Under control conditions or after treatment with basic fibroblast growth factor (bFGF; 50 ng mL-1), a proportion of the granulosa cells divided to produce colonies; individual cells remained small. bFGF increased the number of cells harvested (15.8 +/- 7.3-fold, as measured indirectly by the relative amount of the nuclear La antigen), increased the average diameter of the colonies from 88.9 +/- 13.5 microns to 136.5 +/- 4.9 microns and stimulated the production of fibronectin 5.7 +/- 1.5-fold (P < 0.05). An extracellular matrix, which has previously been shown to be a basal lamina, was observed in 19.1% of the colonies (total of 350 colonies examined; n = 8 experiments). Cells treated with dibutyryl cAMP (1 mM) hypertrophied and had 50 +/- 28.7-fold and 102.6 +/- 55.8-fold higher levels of cholesterol side-chain cleavage cytochrome P450 (P < 0.001) and 3 beta-hydroxysteroid dehydrogenase (P < 0.01) respectively (n = 5). Thus, granulosa cells with characteristics of stem cells can divide and produce extracellular matrix, or be induced to differentiate when in culture without anchorage.

Ca(2+)-regulated expression of steroid hydroxylases in H295R human adrenocortical cells.

Although changes in the expression of key steroidogenic enzymes such as cytochrome P450 cholesterol side-chain cleavage, 17 alpha-hydroxylase (P450c17), aldosterone synthase, and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in the human adrenal cortex are known to be controlled by factors activating the protein kinase A or protein kinase C signaling pathways, little is known concerning the effects of increased intracellular Ca2+. In this study we describe the effects of K+, an agent known to increase intracellular Ca2+ through the opening of voltage-sensitive Ca2+ channels, on steroidogenesis in H295R human adrenocortical cells and corresponding changes in expression of these vital steroidogenic enzymes. Treatment of cells for 48 h with K+ (14 mM) resulted in an increase in aldosterone (3.5-fold) as well as the 17 alpha-hydroxylated steroids cortisol (2.9-fold) and dehydroepiandrosterone (DHEA; 3.7-fold). This action of K+ was accompanied by a dose-dependent (P < 0.05 at 6 mM K+ or above) and time-dependent (P < 0.05 at 24 h and beyond) increase in expression of P450c17 and, to a lesser extent, cytochrome P450 cholesterol side-chain cleavage messenger RNA (mRNA). Treatment with K+ also caused a time-dependent increase in aldosterone synthase mRNA levels, which were detectable by 12 h. Treatment with K+, however, was without effect on 3 beta HSD expression. These effects contrast with those of (Bu)2cAMP, which stimulated a greater increase in cortisol and DHEA secretion as well as P450c17 expression. The effects of K+ treatment also differ from those of AII, which promoted a greater aldosterone secretory response (5.7-fold), but a lesser effect on DHEA secretion (2.2-fold) and P450c17 expression. Although AII and TPA (known activators of protein kinase C) as well as forskolin and (Bu)2cAMP (known activators of protein kinase A) increased the expression of 3 beta HSD mRNA, K+ treatment was without effect, suggesting that elevation of [Ca2+]i in response to K+ did not activate the protein kinase C or protein kinase A signaling pathways. Furthermore, the effects of K+ on steroid secretion and 17 alpha-hydroxylase activity were reproduced by the voltage-sensitive Ca2+ channel activator BAYK 8644, and increases in P450c17 mRNA in response to K+ were reversed by the Ca2+ channel antagonist, nifedipine. We conclude that K+ can modulate the expression of key steroidogenic enzymes in H295R cells through the Ca2+ signaling pathway without involvement of the protein kinase A or protein kinase C pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca(2+)-regulated expression of steroid hydroxylases in H295R human adrenocortical cells

Although changes in the expression of key steroidogenic enzymes such as cytochrome P450 cholesterol side-chain cleavage, 17 alpha-hydroxylase (P450c17), aldosterone synthase, and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in the human adrenal cortex are known to be controlled by factors activating the protein kinase A or protein kinase C signaling pathways, little is known concerning the effects of increased intracellular Ca2+. In this study we describe the effects of K+, an agent known to increase intracellular Ca2+ through the opening of voltage-sensitive Ca2+ channels, on steroidogenesis in H295R human adrenocortical cells and corresponding changes in expression of these vital steroidogenic enzymes. Treatment of cells for 48 h with K+ (14 mM) resulted in an increase in aldosterone (3.5-fold) as well as the 17 alpha-hydroxylated steroids cortisol (2.9-fold) and dehydroepiandrosterone (DHEA; 3.7-fold). This action of K+ was accompanied by a dose-dependent (P < 0.05 at 6 mM K+ or above) and time-dependent (P < 0.05 at 24 h and beyond) increase in expression of P450c17 and, to a lesser extent, cytochrome P450 cholesterol side-chain cleavage messenger RNA (mRNA). Treatment with K+ also caused a time-dependent increase in aldosterone synthase mRNA levels, which were detectable by 12 h. Treatment with K+, however, was without effect on 3 beta HSD expression. These effects contrast with those of (Bu)2cAMP, which stimulated a greater increase in cortisol and DHEA secretion as well as P450c17 expression. The effects of K+ treatment also differ from those of AII, which promoted a greater aldosterone secretory response (5.7-fold), but a lesser effect on DHEA secretion (2.2-fold) and P450c17 expression. Although AII and TPA (known activators of protein kinase C) as well as forskolin and (Bu)2cAMP (known activators of protein kinase A) increased the expression of 3 beta HSD mRNA, K+ treatment was without effect, suggesting that elevation of [Ca2+]i in response to K+ did not activate the protein kinase C or protein kinase A signaling pathways. Furthermore, the effects of K+ on steroid secretion and 17 alpha-hydroxylase activity were reproduced by the voltage-sensitive Ca2+ channel activator BAYK 8644, and increases in P450c17 mRNA in response to K+ were reversed by the Ca2+ channel antagonist, nifedipine. We conclude that K+ can modulate the expression of key steroidogenic enzymes in H295R cells through the Ca2+ signaling pathway without involvement of the protein kinase A or protein kinase C pathways.

Regulation of cholesterol side-chain cleavage cytochrome P450 in mouse testis Leydig cell line I-10.

We have characterized regulation of steroidogenesis in a mouse testis Leydig cell line, I-10. Progesterone secretion was increased in a time- and dose-dependent fashion by 8-Br-cAMP treatment. The amount of cholesterol side-chain cleavage enzyme (P450scc), the first and rate-limiting enzyme for the synthesis of steroids, as detected by immunoblotting, was also increased. The calcium ionophore A23187 decreased the amount of P450scc, but it did not interfere with cAMP stimulation of the accumulation of P450scc. This regulation of P450scc expression by cAMP and A23187 is at the post-transcriptional level because the amount of P450scc mRNA was not affected by either treatment. This result was further confirmed by direct measurement of transcription in the presence or absence of forskolin treatment. I-10, however, supported cAMP-dependent transcriptional activation of the exogenous gene, as shown by the increased expression of a reporter gene under the control of the -600 to -2,500 fragment of the P450scc gene. The ability for transcriptional activation of the exogenous but not endogenous P450scc gene makes I-10 a unique steroidogenic cell line.

Regulation of 3 beta-hydroxysteroid dehydrogenase delta 5/delta 4- isomerase and cholesterol side-chain cleavage cytochrome P450 by activin in rat granulosa cells

Regulation of 3 beta-hydroxysteroid dehydrogenase delta 5/delta 4- isomerase and cholesterol side-chain cleavage cytochrome P450 by activin in rat granulosa cells

Cholesterol side-chain cleavage cytochrome P450 gene expression in the primitive gut of the mouse embryo does not require steroidogenic factor 1

Cholesterol side-chain cleavage cytochrome P450 gene expression in the primitive gut of the mouse embryo does not require steroidogenic factor 1

Differential Effects of Pulsatile Versus Continuous Follicle-Stimulating Hormone Delivery on the Production of Cyclic Adenosine 5′-Monophosphates and the Accumulation of Cytochrome P450 Cholesterol Side-Chain Cleavage Enzyme mRNA in Perifused Porcine Granulosa Cells1

Although administration of FSH to porcine granulosa cells in static primary cultures increased accumulation of cAMP in the culture medium and cytochrome P450 cholesterol side chain cleavage enzyme (SCC) mRNA in the cells, the importance of the physiologically pulsatile pattern of FSH secretion in regulating the expression of specific genes required for ovarian steroidogenesis is unknown. To evaluate the impact of the mode of FSH delivery on ovarian target cell responses, we examined cAMP production and SCC mRNA accumulation in granulosa cells perifused with continuous or pulsatile FSH stimuli. Porcine granulosa cells were plated onto Cytodex 3 microcarrier beads, placed into perifusion columns, and perifused at 25 or 37 degrees C with 190 microliters/min Eagle's minimum essential medium (MEM) or MEM containing 100 ng/ml ovine (o) FSH delivered continuously, or 100 or 600 ng/ml oFSH delivered in 15-min pulses every 90 or 180 min. After perifusion, total RNA was isolated from granulosa cells for Northern analysis of specific SCC mRNA normalized to the constitutively expressed cyclophilin mRNA. Cyclic AMP in control columns remained basal throughout the perifusion. In contrast, cAMP increased 10- to 15-fold within 30-50 min of the onset of tonic FSH delivery. This increase in cAMP was maintained at 25 degrees C, but gradually decreased to a 2-fold increase in columns perifused at 37 degrees C. Release of cAMP increased 5- to 7-fold in response to 100 ng/ml oFSH pulses given at 90 or 180 min intervals, and 15- to 20-fold in response to 600 ng/ml oFSH pulses, and decreased to near control levels between FSH pulses.(ABSTRACT TRUNCATED AT 250 WORDS)

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 beta-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 beta-carotene reduced the amount of cross-linking. We conclude that the high levels of beta-carotene in corpora lutea are to protect against damage due to oxygen free radicals generated in the course of progesterone synthesis.

Dual actions of phorbol ester on cytochrome P450 cholesterol side-chain cleavage messenger ribonucleic acid accumulation in porcine granulosa cells.

In earlier studies in cultures of porcine granulosa cells prepared from small antral follicles, steroidogenesis-related loci were inhibited by treatment for 48 h with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a potent activator of protein kinase C (PKC). In the present investigation, cells were incubated in serum-free medium for 48 h, with various agents present during the last 2-24 h. With TPA at 30 ng/ml, the FSH-stimulated cAMP accumulation was markedly enhanced at all time points. FSH increased the concentration of cytochrome P450 cholesterol side-chain cleavage (P450scc) mRNA throughout the 24-h incubation. At 4 and 8 h, TPA increased the accumulation of P450scc mRNA, having an additive effect with FSH. However, at 24 h, TPA markedly suppressed the FSH-induced increased in P450scc mRNA. Pretreatment of cells with FSH did not shorten the time required for TPA to become inhibitory. The stimulatory effect of 8-bromo-cAMP on P450scc mRNA also was augmented by TPA at 4 h, but significant inhibition was not observed at 24 h. The concentration of glyceraldehyde-3-phosphate dehydrogenase mRNA, intended to be used for correction of gel loading, was stably increased by both cAMP and TPA. These effects of TPA suggest multiple actions of PKC(s) on the regulation of P450scc expression and other endpoints in ovarian granulosa cells.

The antioxidant β-carotene prevents covalent cross-linking between cholesterol side-chain cleavage cytochrome P450 and its electron donor, adrenodoxin, in bovine luteal cells

Steroid hormones are an important class of hormones synthesized from cholesterol by a number of endocrine organs; including ovaries, placenta, testes and adrenal glands. The first and rate-limiting step in steroidogenesis is the cleavage of the side-chain of the cholesterol molecule, catalysed by a cytochrome P450 enzyme, cholesterol side-chain cleavage enzyme. This enzyme, as with other P450 enzymes, produces oxygen radicals. Oxygen free radicals can cause deleterious effects such as cross-linking and aggregation of proteins. Cells can protect against such damage with the use of antioxidants. The corpus luteum, or ‘yellow body’, of the ovary is very steroidogenic and is exceedingly rich in the yellow antioxidant, β-carotene. The corpus luteum produces the steroid hormone progesterone that is needed to support pregnancy. Here we have shown that by depleting, or conversely repleting, luteal cells of their β-carotene content in vitro that P450 side-chain cleavage enzyme became covalently non-disulfide cross-linked to its electron donor, adrenodoxin, and hence inactivated. Bovine luteal cells were cultured in 10% fetal calf serum with of without additional treatments for up to 72 h. Under control conditions the cellular levels of β-carotene and α-tocopherol fell by 50% within 24 h and remained low. P450 side-chain cleavage enzyme become non-disulfide covalently cross-linked to its electron donor, adrenodoxin, as determined by Western immunoblotting ( N = 18). Aminoglutethamide inhibited this cross-linking. The addition of β-carotene at levels found in bovine serum, but not α-tocopherol or ascorbic acid, inhibited the degree of the cross-linking. These observations thus offer a good explanation for the very high levels of β-carotene found in bovine corpora lutea, namely that β-carotene protects cholesterol side-chain cleavage P450 against damage from its own oxygen free radicals. They may also explain many observations showing that antioxidant vitamins support the synthesis of steroid hormones, particularly those involved in the regulation of reproduction.

Concentrations of cytochrome P450 cholesterol side-chain cleavage enzyme and 3 beta-hydroxysteroid dehydrogenase during prostaglandin F2 alpha-induced luteal regression in cattle.

Prostaglandin F2 alpha (PGF2 alpha)-induced regression of the corpus luteum causes both plasma progesterone concentrations and luteal concentrations of mRNA encoding the steroidogenic enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) to fall in parallel. To investigate the hypothesis that a decline in the concentrations of mRNA encoding steroidogenic enzymes causes plasma progesterone to fall, the luteal concentrations of the enzymes 3 beta-HSD and cytochrome P450 cholesterol side-chain cleavage were measured during induced luteolysis. Holstein heifers were treated with PGF2 alpha (25 mg Lutalyse) on Day 6 or Day 7 of the oestrous cycle and corpora lutea were collected 0 h, 2 h, 12 h, and 24 h later (n = 6, 4, 4, and 4 respectively). Analyses of the steroidogenic enzymes were carried out by Western immunoblotting. The luteal concentrations of both steroidogenic enzymes did not decrease over the 24-h period. It is concluded that, although the concentrations of mRNA encoding steroidogenic enzymes may decline in response to PGF2 alpha, this does not lead to a sufficiently rapid reduction in the concentrations of the enzymes to precede, and thus cause, the decline in plasma progesterone concentrations. Thus, the mechanism for the initial decline in plasma progesterone concentrations during luteolysis is still not known.

Regulation by retinoids of luteinizing hormone/chorionic gonadotropin receptor, cholesterol side-chain cleavage cytochrome P-450, 3β-hydroxysteroid dehydrogenase/Δ 5−4-isomerase and 17α-hydroxylase/C 17–20 lyase cytochrome P-450 messenger ribonucleic acid levels in the K9 mouse Leydig cell line

Vitamin A is a potent regulator of testicular function. We have reported that retinol (R) and retinoic acid (RA) induced a down regulation of luteinizing hormone/human chorionic gonadotropin (LH/CG) binding sites in K9 Leydig cells. In the present study we evaluated the effect of R and RA on LH/CG receptors, cholesterol side-chain cleavage cytochrome P-450 (P-450 scc), 17α-hydroxylase/C 17–20 lyase (P-450 17α) and 3β-hydroxysteroid dehydrogenase (3β HSD) mRNA levels in K9 mouse Leydig cells. To validate K9 cells as a model for studying Leydig cell steroidogenesis at the molecular level, we first investigated the effect of hCG on mRNA levels of the steroidogenic enzymes. P-450 scc, 3β HSD and P-450 17α were expressed constitutively. The addition of 10 ng/ml hCG enhanced mRNA levels for the three genes within 2 h. Maximal accumulation of P-450 scc, P-450 17α and 3β HSD mRNA in treated cells represents a 2.5-, 8.5- and 4-fold increase over control values, respectively. P-450 17α expression reached a maximum by 4 h and then declined rapidly to return to control value by 24 h. The pattern of LH/CG receptor mRNAs in K9 cells was very similar to that of MA10 Leydig cells and showed six transcripts of 1.1, 1.6, 1.9, 2.6, 4.2 and 7.0kb. Treatment of cells with R or RA resulted in a time- and dose-dependent decrease in all six species. Comparing the time course of the reduction in mRNA levels and in binding activity, we postulate that retinoids down regulate LH/CG receptors primarily by reducing their mRNAs. Addition of R or RA greatly enhanced P-450 17α mRNA levels in a time- and dose-dependent manner with maximal induction at 12 h (about a 5-fold increase) using 10 −6 M R or RA. The level of P-450 17α mRNA remained high throughout the treatment. Under the same conditions, 3β HSD mRNA levels were repressed but the effect was long term (70% decrease after 8 days with 10 −6 M R or RA). On the contrary, P-450 scc expression was not affected.

Corticosterone regulates cell proliferation and cytochrome P450 cholesterol side-chain cleavage enzyme messenger ribonucleic acid expression in primary cultures of fetal rat adrenals

Glucocorticoids are known to inhibit growth in many different cell types. Although corticosterone is secreted by the adrenal cortex, its direct effect on the growth of different zones is poorly determined. We studied the effects of corticosterone on cell proliferation and cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc; the rate-limiting step in adrenal steroidogenesis) messenger RNA (mRNA) accumulation in primary cultures of fetal rat adrenals. Adrenocortical cells, grown in the absence of ACTH for 3 weeks, possess typical features of zona glomerulosa cells. These cells differentiate into fasciculata-type cells and undergo biphasic proliferation when stimulated with ACTH. The primary antimitogenic phase of 24 h is followed by rapidly increased bromodeoxyuridine incorporation after 72 h of ACTH treatment. If the treatment is continued, the proliferation decreases again, but remains higher than the proliferation of the untreated cells. Undifferentiated zona glomerulosa-type cells secrete very low amounts of corticosterone. The 10% basal proliferation was not affected if exogenous corticosterone was added. However, if corticosterone was combined with ACTH for 3 days, it blocked the stimulatory growth effect of ACTH dose dependently. Etomidate, an inhibitor of steroidogenic enzymes, completely blocked corticosterone secretion. In our cultures it inhibited 50% of the proliferation of the zona glomerulosa-type cells. However, its effect was totally opposite in long term ACTH-treated cultures; in these fasciculata-type cells, etomidate stimulated the proliferation rate 3-fold. P450scc gene expression was low in undifferentiated zona glomerulosa-like cells. ACTH stimulation increased P450scc mRNA expression 10-fold. Exogenous corticosterone inhibited ACTH-induced P450scc mRNA accumulation by 50%, whereas etomidate doubled it. Our data suggest that a low corticosterone concentration supports the proliferation of undifferentiated zona glomerulosa-type cells, whereas a high corticosterone concentration inhibits the proliferation of differentiated zona fasciculata-type cells. In addition, a high corticosterone concentration may inhibit steroidogenesis by reducing P450scc expression. Thus, corticosterone may be an important modulator of adrenocortical cell proliferation and steroidogenesis in different zones of the adrenal cortex.