Cholesterol Side-chain Cleavage Cytochrome P450scc Research Articles

HC diet inhibited testosterone synthesis by activating endoplasmic reticulum stress in testicular Leydig cells.

Emerging epidemiological studies indicate that hypercholesterolaemia is a risk factor for testosterone deficiency. However, the underlying mechanism is unclear. Testicular Leydig cells are the primary source of testosterone in males. To identify the effect and mechanism of cholesterol overload on Leydig cell function, rats were fed with a HC (HC) diet to induce hypercholesterolaemia. During the 16‐week feeding period, serum testosterone levels were reduced in a time‐dependent manner in rats fed the HC diet. Accordingly, these steroidogenic enzymes within the Leydig cells, including steroidogenic acute regulatory protein (StAR), cholesterol side‐chain cleavage cytochrome P450 (P450scc) and 3β‐hydroxysteroid dehydrogenase (3β‐HSD), were down‐regulated. Notably, the HC‐fed rats showed evident endoplasmic reticulum (ER) stress in the testis, including a dilated ER as an evident pathological change in the Leydig cell ultrastructure, up‐regulated ER stress biomarker (binding immunoglobulin protein) levels and activation of the activating transcription factor 6 (ATF6)‐related unfolded protein response pathway. Further analysis showed that when 4‐phenyl butyric acid (4‐PBA) was used to block ER stress in HC‐fed rats for 8 weeks, the testosterone deficiency was significantly alleviated. Our findings suggested that high dietary cholesterol intake affected serum testosterone levels by down‐regulating steroidogenic enzymes and that activated ER stress might serve as the underlying mechanism.

Morphological rearrangement of the cortical region, in aging ovaries.

The ovary is a structurally dynamic organ that alters with age. Modifications in the paracrine status influence the capacity of aging oocytes to develop normal embryos. Despite the importance of understanding the cellular and molecular mechanism involved in the process of ovarian aging, histological changes remain poorly understood. Correlating the process of folliculogenesis and somatic cell function during ovarian aging is essential to explain the reproductive decline of aged mammalian species, including humans. Here, we performed a morphological and immunohistological study on the ovaries of chinchilla rabbits that varied in age from one to 34-months. The spatiotemporal expression of the cholesterol side-chain cleavage cytochrome P450scc (CYP11A) and the smooth muscle actin (SMA) were analyzed. A significant histological rearrangement of immunodetected cells in theca interna, theca externa and the interstitial tissue around the follicles occurred. The expression of CYP11A1 decreased considerably in antral follicles of aging ovaries. Moreover, we found that the secondary interstitial gland developed extensively, and a remarkable rearrangement of the surface epithelium occurred in aging ovaries. In contrast to ovaries during the reproductive period, the immunohistological changes demonstrate that the interstitial gland became the most abundant tissue during the aging of ovaries. Thus, the current study provides new data for understanding the alteration of somatic cell function in elderly ovaries and how this affects their declined fertility.

Phosphoenolpyruvate Carboxykinase and Glucose-6-phosphatase Are Required for Steroidogenesis in Testicular Leydig Cells

Cyclic AMP (cAMP) induces steroidogenic enzyme gene expression and stimulates testosterone production in Leydig cells. Phosphoenolpyruvate carboxykinase (PEPCK) is expressed in Leydig cells, but its role has not been defined. In this study, we found that PEPCK and glucose-6-phosphatase (Glc-6-Pase) are increased significantly following cAMP treatment of mouse Leydig cells. Moreover, cAMP treatment increased recruitment of the cAMP-response element-binding transcription factor and decreased recruitment of the corepressor DAX-1 on the pepck promoter. Furthermore, cAMP induced an increase in ATP that correlated with a decrease in phospho-AMP-activated protein kinase (AMPK). In contrast, knockdown or inhibition of PEPCK decreased ATP and increased phospho-AMPK. Treatment with an AMPK activator or overexpression of the constitutively active form of AMPK inhibited cAMP-induced steroidogenic enzyme promoter activities and gene expression. Liver receptor homolog-1 (LRH-1) was involved in cAMP-induced steroidogenic enzyme gene expression but was inhibited by AMPK activation in Leydig cells. Additionally, inhibition or knockdown of PEPCK and Glc-6-Pase decreased cAMP-mediated induction of steroidogenic enzyme gene expression and steroidogenesis. Finally, pubertal mouse (8-week-old) testes and human chorionic gonadotropin-induced prepubertal mouse testes showed increased PEPCK and Glc-6-Pase gene expression. Taken together, these results suggest that induction of PEPCK and Glc-6-Pase by cAMP plays an important role in Leydig cell steroidogenesis.

The adiponectin paralog C1q/TNF-related protein 3 (CTRP3) stimulates testosterone production through the cAMP/PKA signaling pathway

CTRP3, a paralog of adiponectin, is a member of the C1q and tumor necrosis factor (TNF)-related protein (CTRP) superfamily. It is expressed at high levels in adipose tissue and has recently emerged as a novel adipokine. In the present study, we provide the first evidence for a physiological role of the new adipokine, CTRP3, in the reproductive system. CTRP3 was specifically expressed in interstitial Leydig cells, where testosterone is produced, in the adult mouse testis. CTRP3 increased testosterone production by TM3 mouse Leydig cells in a dose-dependent manner. The increased testosterone production was linked to upregulation of steroidogenic proteins expression, such as steroidogenic acute regulatory (StAR) protein and cholesterol side-chain cleavage cytochrome P450 (P450scc). Moreover, increases in intracellular cyclic AMP (cAMP) concentrations and the phosphorylation of cAMP-response element binding protein (CREB) in CTRP3-stimulated TM3 Leydig cells were observed. Inhibition of this signaling pathway by a specific protein kinase A (PKA) inhibitor, H89, blocked testosterone production in CTRP3-stimulated Leydig cells, suggesting that the stimulatory effect of CTRP3 on testosterone production is associated with activation of the cAMP/PKA signaling pathway. Thus, our results demonstrate a physiological role for CTRP3 in testicular steroidogenesis and provide novel insights in the intracellular mechanisms activated by this protein.

Differentiation of Steroid-Producing Cells and Folliculogenesis in the Developing Ovary of the Nile TilapiaOreochromis niloticus

Differentiation and development of steroid-producing cells (SPCs) and folliculogenesis during ovarian differentiation in the Nile tilapia Oreochromis niloticus were immunohistochemically and ultrastructurally examined. Clusters of immunopositive cells (IPCs) against antibodies (ABs) of cholesterol side-chain cleavage cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase (3βHSD), and cytochrome P450aromatase (P450arom) only appeared in the area near blood vessels in the fish ovaries at 50-60 days after hatching (dah). Ultrastructural results showed that differentiation and development of SPCs from undifferentiated to maturation occurred in the area near blood vessels, indicating that it would be the original site of SPCs. At 70-80 dah, IPC clusters invaded the interstices among oocytes at the perinucleolar stage from the area near the blood vessels. IPCs increased in number in the interstices among the previtellogenic oocytes, and some clusters began to enclose the outer thecal layer of the previtellogenic oocytes at 90 dah. The process of folliculogenesis was ultrastructurally observed. SPCs enclosed by fibroblastic cells invaded the interstitial areas among oocytes and some reached the surfaces of oocytes. The upper portions of these elongations opened and began to enclose the outer surfaces of developed oocytes to become thecal layer. Later, newly migrated SPCs reach the thecal layer to become thecal cells. These results indicate that steroid-producing thecal cells originate from the SPCs in the area near blood vessels. After thecal layer formation, an immunopositive reaction against P450arom AB, but not against P450scc or 3β-HSD ABs, appeared first in the granulosa cells enclosing the vitellogenic oocytes at 100 dah. At this time, estrogen production in serum levels rapidly increased. Thus, folliculogenesis could be essential for active production of estrogen in the ovary.

Effect of glucocorticoids pretreatment on steroidogenic capacity of adrenocortical cells isolated from Meishan piglets

The present in vitro experiment was designed to test whether 48h of pretreatment with glucocorticoids, cortisol, or dexamethasone (DEX), would affect basal and corticotrophin (ACTH) stimulated (24h) cortisol secretion from primary cultures of pig adrenocortical cells. Cells were divided into six groups: control pretreatment with or without ACTH challenge, cortisol pretreatment with or without ACTH challenge, and DEX pretreatment with or without ACTH. The culture medium and cells were collected at the end of treatment. Cortisol concentration in medium was measured by radioimmunoassay, and protein content of glucocorticoid receptor (GR) and key regulatory factors for steroidogenesis, including melanocortin type 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage cytochrome P450 (P450scc), were detected by Western blot analysis. The results showed that glucocorticoid pretreatment did not affect cortisol secretion under basal condition without ACTH challenge, but significantly enhanced ACTH-stimulated cortisol secretion. Furthermore, the protein content of GR, MC2R, StAR, and P450scc was all increased in groups pretreated with glucocorticoids. These results indicate that adrenocortical cells pretreated with glucocorticoids display higher steroidogenic capacity under ACTH challenge, through the upregulation of GR and other steroidogenic regulatory factors.

Characterization of adrenal ACTH signaling pathway and steroidogenic enzymes in Erhualian and Pietrain pigs with different plasma cortisol levels

Our previous study demonstrated significant difference in the basal plasma cortisol levels between Erhualian (EHL) and Pietrain (PIE) pigs, implicating fundamental breed difference in adrenocortical function. The objectives of the present study were therefore to characterize the expression pattern of proteins involved in adrenal ACTH signaling and, including melanocortin type 2 receptor (MC2R), cAMP response element binding protein (CREB) and phosphorylated CREB (pCREB), steroidogenic acute regulatory protein (StAR), as well as that of the key enzymes involved in steroidogenesis in EHL and PIE pigs, in association with the plasma corticotrophin (ACTH) and cortisol levels. The plasma concentrations of the substrates for adrenal steroidogenesis, cholesterol and low-density lipoprotein (LDL) cholesterol, did not differ between breeds. Plasma concentration of ACTH and the adrenal contents of MC2R mRNA and protein were similar in two breeds of pigs, whereas the basal plasma concentrations of cortisol in EHL pigs were 1.5 folds higher than that in PIE pigs. The higher basal plasma cortisol levels in EHL pigs were found to be accompanied with the higher expression of ACTH post-receptor signaling components, cAMP, pCREB and StAR, as well as the higher expression of cholesterol side-chain cleavage cytochrome P450 (P450scc), 17α-hydroxylase cytochrome P450 (P450 17α), 21-hydroxylase cytochrome P450 (P450c21) and 11β-hydroxylase cytochrome P450 (P450 11β). These results indicated that the enhanced cAMP/PKA/pCREB-signaling system and augmented expression of StAR and steroidogenic enzymes are major attributes to the higher basal plasma cortisol concentrations in pigs.

Immunolocalization of Steroidogenic Enzymes in Equine Fetal Adrenal Glands During Mid-Late Gestation

To elucidate the relationship between steroidogenic hormones and developing adrenal glands, we investigated the immunolocalization of steroidogenic enzymes in equine fetal adrenal glands during mid-late gestation. Fetal adrenal glands were obtained from three horses at 217, 225 and 235 days of gestation. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17) and human placental aromatase cytochrome P450 (P450arom). Histologically, cortex and medulla cells were clearly observed in the three fetal adrenal gland tissue samples. P450scc and P450c17 were identified in cortex cells close to medulla cells and in some medulla cells in the fetal adrenal glands. P450arom was present in both cortex and medulla cells in the fetal adrenal glands. However, 3betaHSD was not found in any of the equine fetal adrenal gland tissue samples. These results suggest that equine fetal adrenal glands have the ability to synthesize androgen and estrogen, which may play an important physiological role in the development of equine fetal adrenal glands.

Immunolocalization of Inhibin/Activin Subunits and Steroidogenic Enzymes in Equine Neonatal Ovaries

The objective of this study was to investigate the cellular immunolocalization of inhibin α and inhibin/activin (βA and βB) subunits and steroidogenic enzymes in neonatal equine ovaries. The ovaries were obtained from two equine neonates of 3 days and 18 days of age. The sections of ovarian tissues were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin α, inhibin/activin βA and inhibin/activin βB and bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3β-hydroxysteroid dehydrogenase (3βHSD), porcine testicular 17α-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). Inhibin α and inhibin/activin βA subunits, but not inhibin/activin βB subunits were expressed in the ovarian interstitial cells of equine neonates. P450scc and P450c17 were also identified in the ovarian interstitial cells and P450arom with weak immunostaining. However, 3βHSD was not found in the ovarian interstitial cells of the equine neonates. These results suggest that equine neonatal ovaries have the ability to synthesize inhibin A and steroid hormones, which may play some important physiological roles in the developing ovaries of equine neonates.

Ovarian enzyme activities in women with polycystic ovary syndrome

Studies using primary ovarian tissue and cultured human theca and granulosa cells have shown that steroidogenic enzyme activities are up-regulated in theca cells in polycystic ovary syndrome (PCOS). Although granulosa cells in arrested follicles in PCOS fail to express significant amounts of aromatase, there is an overexpression of 5alpha-reductase activity and premature expression of cholesterol side-chain cleavage cytochrome P450.

Immunolocalization of Steroidogenic Enzymes P450scc, 3.BETA.HSD, P450c17, and P450arom in Goettingen Miniature Pig Testes

The objective of this study was to investigate immunolocalization of steroidogenic enzymes in Göttingen miniature (GM) pig testes. Testes of 6 adult GM pigs were obtained in September 1996 (n=2), February (n=2) and June (n=2), 1997. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). Histologically, all types of spermatogenic cells including mature-phase spermatozoa in seminiferous tubules were observed in all testes throughout the year. Moreover, P450scc, 3betaHSD, P450c17and P450arom were identified in Leydig cells but not in Sertoli cells of all testes. These results suggested that adult GM pig testes have the ability to produce germ cells throughout the year, and the synthesis of progestin, androgen and estrogen occurs in the Leydig cells of GM pig testes.

Immunolocalization of Steroidogenic Enzymes in the Corpus Luteum and Placenta of the Japanese Shiba Goat

In this study, we performed immunohistochemistry of cholesterol side-chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase (3betaHSD), cytochrome 17alpha-hydroxylase P450 (P450c17), and cytochrome P450 aromatase (P450arom) in the corpus luteum and placenta of Shiba goats. The aim was to clarify the steroidogenic capability of the corpus luteum and placenta of Shiba goats. Ovaries containing corpora lutea were obtained from four adult Shiba goats during the luteal phase (day10; n=2) and pregnancy (90 and 120 days of gestation). Placenta was obtained from one Shiba goat on day 120 of gestation. The sections of the ovaries and placentae were immunostained using the avidin-biotin-peroxidase complex method (ABC) with polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. All luteal cells expressed P450scc, 3betaHSD, P450c17 and P450arom. The distribution of P450scc, 3betaHSD, P450c17 and P450arom were not different during the luteal phase and pregnancy. P450arom showed a weak positive staining in late pregnancy (120 days). In addition, immunoreactions for P450c17 and P450arom were observed in syncytiotrophoblast of the placenta of one Shiba goat. These results indicate that, in Shiba goats, corpus luteum is not only an important source of progesterone but also has the ability to synthesize androgen and estrogen during the luteal phase and pregnancy. Also the placenta has the ability to synthesize androgen and estrogen in late pregnancy.

Exposure of neonatal rats to diethylstilbestrol affects the expression of genes involved in ovarian differentiation.

Exposing neonatal rats with the synthetic estrogen, diethylstilbestrol (DES), induces morphological and functional abnormalities in the adult ovary. We examined the events that lead to this condition using female rats that were exposed to DES for the first five days after birth. The expression of steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage cytochrome P450 (P450scc), which are both required for steroidogenesis in the theca/interstitial region was markedly reduced. The expression of Mullerian inhibiting substance (MIS) was transiently increased in small growing follicles in the ovary of DES-treated rats at postnatal day 7 (P7), and the expression profile in the ovary differed between DES- and vehicle oil-treated rats at P14 and P21. The expression of the transcription factor, steroidogenic factor-1 (SF-1), reduced in theca/interstitial cells, but increased in granulosa cells of primary follicles. These results indicate that altered steroidogenesis and MIS production are mechanisms through which DES induces abnormal ovarian development, and support the notion that androgens and MIS are both critical factors in regulating early ovarian differentiation.

Seasonal changes in spermatogenesis and testicular steroidogenesis in wild male raccoon dogs (Nyctereutes procynoides).

Testes of 15 wild adult male raccoon dogs (Nyctereutes procynoides) obtained from September 2000 to April 2001 were studied to clarify seasonal changes in spermatogenesis and testicular steroidogenesis. There were marked seasonal variations in the testis weight and size with values relatively low in September and highest in March. Spermatogonia and primary spermatocytes were observed in September, while spermatogonia, spermatocytes and round spermatids were present in January, and all types of spermatogenic cells including mature spermatozoa were found in the mating season (February and March). The number of spermatogenic cells reached their peak values in February and March. In addition, steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3 betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). P450scc and P450c17 were identified in Leydig cells and spermatids in February, whereas these enzymes were present only in Leydig cells in September. 3betaHSD was found in Leydig cells in September and February with more intense staining in February. The localization of P450arom changed seasonally: no immunostaining in September; more extensive immunostaining in Leydig cells, Sertoli cells, and elongating spermatids in February. These results suggest that seasonal changes in the testis weight and size of wild male raccoon dogs are correlated with changes in spermatogenesis. Seasonal changes in testicular steroidogenesis suggest that the synthesis of androgen and estrogen reaches its peak in the mating season.

Spermatogenesis, serum testosterone levels and immunolocalization of steroidogenic enzymes in the wild male Japanese black bear (Ursus thibetanus japonicus).

Twenty-one wild male Japanese black bears (Ursus thibetanus japonicus) were captured in the summer-autumn of 1998-2000 in the vicinity of Neo Village, Gifu Prefecture. Testes were measured, and testicular samples were biopsied and observed histologically. Four steroidogenic enzymes, i.e., cholesterol side-chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase (3betaHSD), 17-alpha hydroxylase cytochrome P450 (P450c17), and aromatase cytochrome P450 (P450arom) were immunolocalized. Serum testosterone concentrations were measured by radioimmunoassay. Testis size changed little from 1-3 years of age, increased rapidly at 4 years, and attained its peak at 5 years. Serum testosterone concentrations ranged from 0.05 to 1.78 ng/m l, and the mean +/- standard deviation was 0.43 +/- 0.48 ng/ml. Age of sexual maturation in wild male Japanese black bears was estimated to be 3-4 years. Seasonal changes in spermatogenesis were obvious; active in June, July and August, degenerated by September. Leydig cells, Sertoli cells and germ cells have the capability of synthesizing androgen, and Leydig cells, Sertoli cells, spermatids and spermatogonia have the capability of synthesizing estrogen in Japanese black bears.

Transcription of cholesterol side-chain cleavage cytochrome P450 in the placenta: activating protein-2 assumes the role of steroidogenic factor-1 by binding to an overlapping promoter element.

Progesterone is essential to the sustenance of pregnancy in humans and other mammals. From the second trimester on, the human placenta is the sole origin of de novo synthesized steroid hormones. In mice, placentation at midgestation is accompanied by a temporal rise of steroid hormone synthesis commencing in the giant cells of the mouse trophoblast. In doing so, the giant trophoblasts, as any other steroidogenic cell, express high levels of the key steroidogenic enzyme, cholesterol side-chain cleavage cytochrome P450 (P450scc). Because steroidogenic factor 1 (SF-1), the transcription factor required for expression of P450scc in the adrenals and the gonads, is not expressed in the placenta, we hypothesized that placenta-specific nuclear factor(s) (PNF) assumes the role of SF-1 by binding to the same promoter region that harbors the SF-1 recognition site in the P450scc gene. To address this possibility, we used SCC1, a well conserved proximal region in the P450scc genes (-60/-32 in the rat gene) to purify PNF from human term placenta. Sequencing of the purified PNF revealed that it is the alpha isoform of the human activating protein-2 (AP-2alpha). Specific antibodies tested in EMSA confirmed that AP-2alpha is the predominant isoform that binds SCC1 in the human placenta, whereas AP-2gamma is the only mouse placental protein that binds this oligonucleotide. Functional studies showed that coexpression of the rat P450scc promoter (-378/+8 CAT) and AP-2 isoforms (alpha or gamma) in human embryonic kidney 293 cells results in a marked activation of chloramphenicol acetyltransferase (CAT) transcription that is dependent on an intact AP-2 motif, GCCTTGAGC. This motif conforms with consensus sequences previously determined for binding of the AP-2 alpha and gamma isoforms. Mutations of the AP-2 element ablated binding of AP-2 to SCC1, as well as severely diminished the promoter activity in primary mouse giant trophoblasts and human choriocarcinoma JAR cells. Collectively, these studies suggest that expression of placental P450scc is governed by AP-2 factors that bind to a cis-element that largely overlaps the sequence required for recognition of SF-1 in other steroidogenic tissues.

Site-directed mutagenesis of cytochrome P450scc. II. Effect of replacement of the Arg425 and Arg426 residues on the structural and functional properties of the cytochrome P450scc.

Cytochrome P450-dependent monooxygenases, in spite of their wide distribution, can be simply divided into a few groups differing in the location of the electron transfer chain and their composition. The two main groups of cytochrome P450-dependent monooxygenases are the mitochondrial and microsomal enzymes. While in two-component microsomal cytochrome P450-dependent monooxygenases electrons are supplied to cytochrome P450 by a flavoprotein (NADPH-cytochrome P450 reductase), in three-component mitochondrial monooxygenases the electrons are supplied to cytochrome P450 by a low molecular weight protein (ferredoxin). The interaction of cytochrome P450 with NADPH-cytochrome P450 reductase and ferredoxin is the subject of intensive studies. Using chemical modification, chemical cross-linking, and site-directed mutagenesis, we identified surface exposed positively charged residues of cytochrome P450scc which might be important for interaction with adrenodoxin. Theoretical analysis of the distribution of surface electrostatic potential in cytochrome P450 indicates that in contrast to microsomal monooxygenases, cytochromes P450 of mitochondrial type, and cholesterol side-chain cleavage cytochrome P450 (P450scc) in part, carry on the proximal surface an evidently positively charged site that is formed by residues Arg425 and Arg426. In the present work, to estimate the functional role of Arg425 and Arg426 of cytochrome P450scc, we used site-directed mutagenesis to replace these residues with glutamine. The results indicate that residues Arg425 and Arg426 are involved in the formation of a heme-binding center and electrostatic interaction of cytochrome P450scc with its physiological electron-transfer partner, adrenodoxin.

The acute and chronic effects of adrenocorticotropin on the levels of messenger ribonucleic acid and protein of steroidogenic enzymes in rat adrenal in vivo.

The purpose of this study was to evaluate the effects of acute (a single injection) and chronic stimulation (twice daily injection for 9 days) by ACTH on changes occurring in the temporal expression of steroidogenic enzymes in the rat adrenal in vivo. Under acute ACTH stimulation, the level of steroidogenic acute regulatory protein (StAR) messenger RNA (mRNA) was increased within 0.5 h in both zona glomerulosa (ZG) and zona fasciculata-reticularis (ZFR), with maximal increases of 220-370% and 300-350% in the ZG and ZFR, respectively. Increases in the levels of StAR protein in homogenates were also found in the ZG (700%) and the ZFR (300%), but were delayed compared with those of their mRNA. Furthermore, the increase in mitochondrial StAR protein was concomitant with that in the homogenate, indicating that the entry of StAR into mitochondria might not be necessary to increase steroidogenesis during the early stimulatory phase. The levels of c-jun, c-fos, junB, and fosB mRNA in ZG and ZFR were also rapidly maximally elevated within 0.5-1 h after ACTH administration and fell to near control levels 5 h posttreatment. The levels of c-jun protein were already increased in both zones at 1 h, reached 200% at 3 h, and remained elevated 5 h post-ACTH treatment. The levels of c-Fos protein were maximally increased by 240% in both zones after 1 h and decreased thereafter to control values at 5 h. Few changes were observed in the adrenal protein contents of cholesterol side-chain cleavage cytochrome P450 (P450scc), cytochrome P450 11beta-hydroxylase (P450C11), cytochrome P450 21-hydroxylase (P450C21), and 3beta-hydroxysteroid dehydrogenase (3betaHSD). Under chronic stimulation by ACTH, we observed elevations in the levels of plasma corticosteroids and changes in the mRNA and protein levels of many adrenal steroidogenic enzymes in both zones. In the ZG, administration of ACTH for 9 days provoked an increase in the level of StAR mRNA (210-270%) and a decrease in the levels of 3betaHSD, cytochrome P450 aldosterone synthase (P450aldo), and AT1 receptor mRNA (by 40%, 70%, and 90%, respectively), whereas the levels of P450scc and P450C21 mRNA did not differ significantly from the control values. Western blotting analysis showed that the adrenal ZG protein levels of StAR and P450scc were increased (150%), 3betaHSD was not changed, and P450C21 was decreased by 70%. In the ZFR, the levels of P450scc and StAR mRNAs were increased (260% and 570-870%, respectively). The levels of 3betaHSD, P450C21, and P450C11 mRNA did not differ from control values in that zone. Western blotting analysis showed that the ZFR protein level of 3betaHSD was not changed, P450scc and P450C21 were decreased by 40% and 60%, respectively, and StAR was increased by 160%. Although c-fos and fosB mRNAs were undetectable after 9 days of chronic ACTH treatment, c-jun mRNA and its protein were still detectable, suggesting a basic role for this protooncogene in maintaining the integrity and function of the adrenal cortex. When dexamethasone was administered to rats for 5 days to inhibit their ACTH secretion, the mRNA levels of many steroidogenic enzymes were decreased, with the exception of StAR, 3betaHSD, and P450aldo. These results confirm the importance of physiological concentrations of ACTH in maintaining normal levels of adrenocortical enzymes and also indicate that in addition to ACTH, other factors are involved in controlling the expression of StAR, 3betaHSD, and P450aldo. In conclusion, we showed that ACTH acutely increases StAR mRNA followed, after a delay, by an increase in the level of StAR protein; this suggests that posttranslational modifications of the StAR precursor occurred during the early stimulatory phase and before the apparent translation of the newly formed mRNA. The rapid induction of protooncogenes suggests their participation in the action of ACTH to stimulate steroidogenesis. (ABSTRACT TRUNCATED)

The Acute and Chronic Effects of Adrenocorticotropin on the Levels of Messenger Ribonucleic Acid and Protein of Steroidogenic Enzymes in Rat Adrenal in Vivo

The purpose of this study was to evaluate the effects of acute (a single injection) and chronic stimulation (twice daily injection for 9 days) by ACTH on changes occurring in the temporal expression of steroidogenic enzymes in the rat adrenal in vivo. Under acute ACTH stimulation, the level of steroidogenic acute regulatory protein (StAR) messenger RNA (mRNA) was increased within 0.5 h in both zona glomerulosa (ZG) and zona fasciculata-reticularis (ZFR), with maximal increases of 220–370% and 300–350% in the ZG and ZFR, respectively. Increases in the levels of StAR protein in homogenates were also found in the ZG (700%) and the ZFR (300%), but were delayed compared with those of their mRNA. Furthermore, the increase in mitochondrial StAR protein was concomitant with that in the homogenate, indicating that the entry of StAR into mitochondria might not be necessary to increase steroidogenesis during the early stimulatory phase. The levels of c-jun, c-fos, junB, and fosB mRNA in ZG and ZFR were also rapidly maximally elevated within 0.5–1 h after ACTH administration and fell to near control levels 5 h posttreatment. The levels of c-jun protein were already increased in both zones at 1 h, reached 200% at 3 h, and remained elevated 5 h post-ACTH treatment. The levels of c-Fos protein were maximally increased by 240% in both zones after 1 h and decreased thereafter to control values at 5 h. Few changes were observed in the adrenal protein contents of cholesterol side-chain cleavage cytochrome P450 (P450scc), cytochrome P450 11β-hydroxylase (P450C11), cytochrome P450 21-hydroxylase (P450C21), and 3β-hydroxysteroid dehydrogenase (3βHSD). Under chronic stimulation by ACTH, we observed elevations in the levels of plasma corticosteroids and changes in the mRNA and protein levels of many adrenal steroidogenic enzymes in both zones. In the ZG, administration of ACTH for 9 days provoked an increase in the level of StAR mRNA (210–270%) and a decrease in the levels of 3βHSD, cytochrome P450 aldosterone synthase (P450aldo), and AT1 receptor mRNA (by 40%, 70%, and 90%, respectively), whereas the levels of P450scc and P450C21 mRNA did not differ significantly from the control values. Western blotting analysis showed that the adrenal ZG protein levels of StAR and P450scc were increased (150%), 3βHSD was not changed, and P450C21 was decreased by 70%. In the ZFR, the levels of P450scc and StAR mRNAs were increased (260% and 570–870%, respectively). The levels of 3βHSD, P450C21, and P450C11 mRNA did not differ from control values in that zone. Western blotting analysis showed that the ZFR protein level of 3βHSD was not changed, P450scc and P450C21 were decreased by 40% and 60%, respectively, and StAR was increased by 160%. Although c-fos and fosB mRNAs were undetectable after 9 days of chronic ACTH treatment, c-jun mRNA and its protein were still detectable, suggesting a basic role for this protooncogene in maintaining the integrity and function of the adrenal cortex. When dexamethasone was administered to rats for 5 days to inhibit their ACTH secretion, the mRNA levels of many steroidogenic enzymes were decreased, with the exception of StAR, 3βHSD, and P450aldo. These results confirm the importance of physiological concentrations of ACTH in maintaining normal levels of adrenocortical enzymes and also indicate that in addition to ACTH, other factors are involved in controlling the expression of StAR, 3βHSD, and P450aldo. In conclusion, we showed that ACTH acutely increases StAR mRNA followed, after a delay, by an increase in the level of StAR protein; this suggests that posttranslational modifications of the StAR precursor occurred during the early stimulatory phase and before the apparent translation of the newly formed mRNA. The rapid induction of protooncogenes suggests their participation in the action of ACTH to stimulate steroidogenesis. Under chronic stimulation by ACTH, adrenals were hypertrophied, and the expression of many steroidogenic enzymes was modified, particularly the level of StAR protein was increased in the ZG and ZFR, confirming the importance of this protein in the control of steroidogenesis in a situation similar to that of Cushing’s syndrome.

Establishment and Characterization of a Simian Virus 40-Transformed Temperature-Sensitive Rat Luteal Cell Line

The primary culture of rat luteal cells and their long-term maintenance have been difficult. Low cellular yields have limited the possibility for the study of gene regulation in luteal cells. The goal of this study was to develop a cell line to serve as a model by which to study the expression and regulation of various genes specific to luteal cells. We attempted to develop a luteal cell line by transformation of large luteal cells through infection with a temperature-sensitive simian virus (SV-40 tsA209) mutant that has a temperature-sensitive mutation required for the maintenance of cell transformation. We report here the successful establishment of such a cell line, designated GG-CL cells. Large luteal cells were purified to homogeneity by flow cytometry from corpora lutea of day 14 pregnant rats, cultured for 24 h, and then infected with the SV-40 tsA209 mutant virus. Transformed cells were maintained at the permissive temperature (33 C) until colonies were identified. Several colonies of transformed cells were isolated and passaged. They multiplied at 33 C and formed multilayers. At the nonpermissive temperature (40 C), cells reverted to the normal differentiated phenotype similar to the primary luteal cells in culture. To determine whether GG-CL cells express the genes found in normal luteal cells, messenger RNA (mRNA) expression was examined by either Northern analysis or RT-PCR with primers specific to each mRNA. GG-CL cells were found to express receptors for interleukin-6 and glucocorticoid, as well as the newly discovered estrogen receptor-β (ER-β) and the orphan nuclear receptor nur 77. No receptors for ER-α, progesterone, LH, or PRL could be detected. This cell line also expressed 20α-hydroxysteroid dehydrogenase (20α-HSD), but not cholesterol side-chain cleavage cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase, or aromatase cytochrome P450 (P450arom). Although the cells did not express the PRL receptor, they did express Janus kinase (JAK2) and signal transducers and activators of transcription (Stat5b), and, when transfected with the PRL receptor, they responded to PRL with a marked inhibition in 20α-HSD mRNA expression. In addition, estradiol enhanced ER-β expression in a dose-dependent manner whereas cAMP stimulation caused a marked and rapid increase in the expression of the orphan receptor nur 77. In summary, a temperature-sensitive cell line was successfully established from the large luteal cells of rat corpora lutea. These cells express key genes encoding enzymes and receptors inherent to this defined luteal cell population and respond to stimulation by PRL, estradiol, and cAMP.