Cell Steroidogenesis Research Articles

Testicular steroid biosynthesis in a boy with a large cell calcifying Sertoli cell tumor producing prepubertal gynecomastia

A study of a large cell calcifying Sertoli cell tumor of the testis associated with bilateral gynecomastia in an 8-year-old boy is presented. Macroscopically, the two testes showed multiple, large, and hard calcified nodules. Histology revealed clusters or cords of tumor cells with foci of calcifications as well as evidences, in the adjacent testicular parenchyma, of initiation of gonadal development, such as early signs of spermatogenesis and sparse Leydig cell differentation. In vivo, serum hormone studies showed gonadotropin-independent gonadal activity. After orchidectomy two macroscopically distinct fractions of the removed testes, tumoral and extratumoral, were processed separately for cell isolation and culture. The secretion of testosterone, androstenedione, and 17-hydroxyprogesterone to the medium on day 6 of culture showed that steroidogenesis in cells of the extratumoral fraction was more active than in the tumoral fraction. On the other hand, tumoral fraction cells showed much higher aromatase activity than extratumoral cells. Furthermore, conditioned medium of tumoral fraction cells was able to stimulate testosterone secretion when it was added to subcultures of testicular cells isolated from a control subject. It is postulated that tumoral cells might have stimulated neighboring interstitial cells to differentiate into Leydig cells and to secrete androgens, which in turn might have been aromatized to estrogens by tumoral cells.

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.

Characterization of gonadal sex cord-stromal tumor cell lines from inhibin-alpha and p53-deficient mice: the role of activin as an autocrine growth factor.

Inhibin-alpha-deficient mutant mice have been generated by a targeted deletion of the inhibin-alpha gene through homologous recombination in murine embryonic stem cells. Essentially all of the homozygous mutants develop gonadal sex cord-stromal tumors. To investigate their endocrine and proliferative characteristics, gonadal tumor cells were maintained in vitro. Cells from inhibin-alpha-deficient mice multiplied poorly; however, cells from mice deficient in both inhibin-alpha and p53 proliferated rapidly and showed higher saturation density and plating efficiency, thus allowing the establishment of clonal tumor cell lines. Although negligible estrogen and testosterone was produced by the clonal cells, high levels of progesterone were secreted. A clonal testis tumor cell line (inhibin-alpha/p53 deficient) showed no response to exogenous FSH, human CG (hCG), or inhibin A but exhibited a 6- to 8-fold increase in progesterone production in response to forskolin treatment. The stimulatory effect of forskolin was, however, partially blocked by activin treatment. Northern blot analysis revealed inhibin beta A and beta B mRNA expression in these cells. Furthermore, Western blot analyses indicated the secretion of the beta A-subunit protein. We further tested the role of activin on tumor cell growth. Treatment with follistatin, an activin-binding protein, inhibited tumor cell replication in a dose-dependent manner. In contrast, treatment with activin A stimulated tumor cell growth by itself and partially blocked follistatin action. Incorporation of thymidine into DNA of these cells was also stimulated by activin. In addition, treatment with antiactivin A serum inhibited tumor cell replication and blocked the stimulatory action of activin on cell growth. The activin action is likely mediated by specific receptors because cross-linking of [125]activin to the 50-55 kilodalton type I and 75-80 kilodalton type II receptors was found using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Northern blot analysis also revealed follistatin mRNA expression in the tumor cells, suggesting these cells are related to granulosa cells. Our findings indicate that activin can act as an autocrine growth factor in stimulating the proliferation of gonadal tumor cell lines derived from inhibin-alpha and p53-deficient mice and inhibits progesterone production. These tumor cell lines are useful for studies on the regulation of gonadal cell proliferation and steroidogenesis as well as the signaling pathway mediating activin action.

Follistatin antagonizes the effects of activin-A on steroidogenesis in human luteinizing granulosa cells.

Activin-A decreases progesterone secretion and aromatase activity in cultured human luteinizing granulosa cells. Follistatin is a binding protein for activin and inhibin produced by granulosa cells and present in follicular fluid. The present study examines the hypothesis that follistatin reverses the actions of activin-A on human granulosa cells. Granulosa cells from women undergoing ovarian stimulation for in vitro fertilization were cultured in defined medium with recombinant human (rh) activin-A and purified porcine follistatin. Follistatin completely reversed the inhibition of basal progesterone production by rh-activin-A, but only when added in a greater than 2:1 molar ratio to activin-A. Although variable effects of activin-A on aromatase activity were observed in these studies, follistatin also antagonized these effects. Follistatin had no effect in the absence of added activin-A. rh-Inhibin-A did not alter steroidogenesis by granulosa cells either with or without added activin-A. Even when added in a 45-fold molar excess, inhibin-A did not displace sufficient activin-A from follistatin to inhibit progesterone secretion. This suggests that the affinity of inhibin-A for follistatin is much lower than that of activin-A, and/or that activin-A bound to follistatin dissociates slowly. alpha 2-Macroglobulin, another activin-binding protein, did not alter the inhibition of progesterone production by activin-A. We conclude that in human granulosa cells, follistatin, but not alpha 2-macroglobulin or inhibin-A, acts to modulate the actions of activin-A. Follistatin and activin may be viewed as components of an autocrine/paracrine system within the human follicle that regulate the differentiated functions of granulosa cells.

Insulin preincubation enhances insulin-like growth factor-II (IGF-II) action on steroidogenesis in human granulosa cells.

Although abundant mRNA for IGF-II has been detected in the human ovary, a role for IGF-II in steroidogenesis has not yet been established. In rat adipocytes, incubation with insulin greatly increases cell-surface IGF-II receptor (5-fold) and the receptor is rapidly internalised in the absence of insulin. We have therefore investigated the effects of insulin preincubation on the response of granulosa cells from unstimulated ovaries to a range of doses of IGF-II. In the absence of insulin, IGF-II stimulated steroidogenesis in only one of three experiments. After incubation with 10 ng/ml insulin, there was a dose-dependent response to IGF-II in all experiments. Cells incubated with insulin produced 5-10 fold more estradiol in response to IGF-II than those incubated without. In contrast, insulin produced only a small increase of estradiol in response to IGF-I. These results demonstrate a synergistic interaction of insulin with IGF-II in human granulosa cells and suggest that there is an important role for IGF-II in human ovarian steroidogenesis.

The effects of hydrogen peroxide on steroidogenesis in mouse Leydig tumor cells

The effects of hydrogen peroxide on steroidogenesis in mouse Leydig tumor cells

Protein kinase C is a tonic negative regulator of steroidogenesis and steroid hydroxylase gene expression in Y1 adrenal cells and functions independently of protein kinase A

Protein kinase C is a tonic negative regulator of steroidogenesis and steroid hydroxylase gene expression in Y1 adrenal cells and functions independently of protein kinase A

Endothelin-1 is an autocrine/paracrine regulator of porcine granulosa cells

We studied whether a novel vasoconstrictor, endothelin-1 (ET-1), is synthesized by and released from porcine granulosa cells, and whether ET-1 acts directly on granulosa cells in an autocrine/paracrine fashion. The dilution curve of the conditioned medium from cultured porcine granulosa cells was parallel to a standard curve of ET-1 in RIA. Reverse-phase HPLC of the conditioned media from the granulosa cells revealed a major peak of ET-1-like immunoreactivity (ET-1-LI) coeluting with standard ET-1. ET-1-LI was released from cultured porcine granulosa cells as a function of time. Northern blot analysis demonstrated the expression of mRNA for prepro-ET-1 in the granulosa cells. We demonstrated the presence of ET-1 in the follicular fluid of porcine ovaries, and that the concentration of ET-1 in large follicles was higher than that in small-medium follicles. ET-1 dose-dependently stimulated cell growth and DNA synthesis in porcine granulosa cells. ET-1 inhibited the FSH- and hCG stimulated accumulation of progesterone in porcine granulosa cells in long-term incubation. These findings suggest that ET-1 produced by porcine granulosa cells may function as an autocrine/paracrine growth factor and modulator of steroidogenesis in ovarian granulosa cells.

Sites of inhibition of steroidogenesis by activation of protein kinase-C in swine ovarian (granulosa) cells.

We tested the hypothesis that low density lipoprotein (LDL) metabolism and cellular concentrations of gene transcripts of cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc mRNA) are sites of significant protein kinase-C (PKC) action in the long term (48-h) inhibitory modulation of steroid hormone biosynthesis in ovarian granulosa cells. To this end, we used 12-O-tetradecanoylphorbol-13-acetate (TPA) as an activator of PKC and a monolayer culture system of immature swine granulosa cells responsive to insulin and lipoprotein under serum-free conditions. Insulin-regulated LDL metabolism was identified as a major site of TPA-mediated inhibition of steroidogenesis in granulosa cells. Treatment with TPA (30 ng/ml), but not inactive phorbol base, effectively decreased insulin-stimulated [125I]iodo-LDL binding by 75%, internalization by 90%, and degradation by 75%, as well as delivery and utilization of the [3H]cholesterol moiety of LDL in progesterone biosynthesis by intact granulosa cells. Cellular concentrations of P450scc mRNA, as measured by Northern blot hybridization with a 32P-labeled 1-kilobase porcine cDNA clone, were significantly increased by insulin. This insulin effect was virtually abolished by cotreatment with TPA (30 ng/ml). In contrast, accumulation of mRNA transcripts of a non-steroidogenic gene, 3-phosphoglyceraldehyde dehydrogenase, but not 18S ribosomal RNA, was enhanced by TPA. In summary, major inhibitory actions of PKC activation on granulosa cell steroidogenesis are expressed at specific loci of LDL metabolism, including LDL receptor number, internalization, and degradation, as well as the delivery and utilization of the [3H]cholesterol moiety of LDL to intact granulosa cells. Moreover, a PKC activator suppresses the intracellular accumulation of insulin-stimulated P450scc mRNA, but not that of phosphoglyceraldehyde dehydrogenase or 18S ribosomal RNA. The results obtained in this in vitro study suggest that the inhibition by TPA at these different sites along the steroidogenic pathway may be similar to that which occurs via hormones that work through the PKC system, such as prostaglandin F2 alpha.

Regulation of steroidogenesis in NCI-H295 cells: a cellular model of the human fetal adrenal.

NCI-H295 is a recently described human adrenocortical carcinoma cell line that makes a variety of steroid hormones. We sought to determine if steroidogenesis in these cells employs the same enzymes as those used in normal adrenal steroidogenesis, and if the genes encoding those enzymes exhibit characteristic responsiveness to activators of the protein kinase-A and -C pathways of intracellular second messengers. Northern blots show that NCI-H295 cells contain abundant mRNAs for three key steroidogenic enzymes, cytochrome P450scc, cytochrome P450c17, and cytochrome P450c21. These mRNAs accumulated in a time- and dose-dependent fashion in response to 8-bromo-cAMP (8Br-cAMP), forskolin, cholera toxin, and 3-isobutyl-1-methylxanthine, all activators of the protein kinase-A pathway. Nuclear run-on assays and actinomycin-D transcriptional inhibition experiments show that cAMP regulates the expression of all three genes primarily at the transcriptional level. Inhibition of protein synthesis with cycloheximide did not prevent the cAMP-induced accumulation of P450scc or P450c17 mRNAs, but did inhibit accumulation of P450c21 mRNA, suggesting that cAMP is acting through a mechanism dependent on protein synthesis to promote accumulation of P450c21 mRNA. Stimulation of the protein kinase-C pathway with phorbol ester decreased P450scc and P450c17 mRNAs, but stimulated the accumulation of P450c21 mRNA. RNase protection experiments, Northern blot hybridizations, and reverse transcription-polymerase chain reaction show that NCI-H295 cells express both the 11 beta-hydroxylase (P450c11 beta) encoded by the P450c11B1 gene and the aldosterone synthetase (P450c11AS) encoded by the P450c11B2 gene. 8Br-cAMP increased the abundance of both of these mRNAs with similar kinetics, with maximal accumulation of both after about 24 h. NCI-H295 cells also contain the mRNAs for aromatase and insulin-like growth factor-II. 8Br-cAMP increased the abundance of aromatase mRNA and decreased the abundance of IGF-II mRNA. These studies show that NCI-H295 cells express most of the enzymes needed for human adrenal steroidogenesis, and that the genes encoding these enzymes respond to stimulation of second messenger pathways in a manner similar to that of human adrenals. NCI-H295 cells appear to be a good model for studying the molecular regulation of human adrenal steroidogenesis.

Diazepam binding inhibitor is a paracrine/autocrine regulator of Leydig cell proliferation and steroidogenesis: action via peripheral-type benzodiazepine receptor and independent mechanisms

Diazepam binding inhibitor is a paracrine/autocrine regulator of Leydig cell proliferation and steroidogenesis: action via peripheral-type benzodiazepine receptor and independent mechanisms

Tumor necrosis factor-alpha induces clustering in ovarian theca-interstitial cells in vitro.

Tumor necrosis factor-alpha (TNF) has been implicated in the regulation of steroidogenesis in theca-interstitial cells (TIC). The purpose of this study was to evaluate any change in TIC morphology during the time course of TNF-induced inhibition of LH-stimulated androstenedione production. Ovaries from immature hypophysectomized rats were enzymatically digested and highly purified TIC were obtained by density gradient centrifugation. TIC treated with TNF (0.1-10 ng/ml) demonstrated distinct clustering in the presence and absence of LH (50 ng/ml). The number of clusters and the mean area per cluster were greatest after 4 days as a result of treatment with 1 or 10 ng TNF/ml. In addition, a dose-dependent inhibition of LH-supported androstenedione production was induced by TNF. TNF also inhibited LH-induced androstenedione in TIC after 2, 4, or 6 days of continuous LH treatment, and TIC clustering still occurred. TIC clustering was impeded by the protein kinase inhibitor H7 at 10 microM; however, the protein kinase inhibitor, HA 1004 (5 microM), did not inhibit TNF-induced clustering in TIC. Since H7 blocked TNF induced clustering, but did not block TNF inhibition of LH stimulated androstenedione synthesis, it is suggested that alternate signal transduction pathways for TNF induced inhibition of LH-stimulated androstenedione and stimulation of clustering of TIC may exist. The results also indicate that the TNF-induced TIC clustering may be independent of the TNF-induced inhibition of LH-stimulated androstenedione production and states of LH-induced differentiation of TIC.

Direct effect of arachidonic acid on protein kinase C and LH-stimulated steroidogenesis in rat Leydig cells; evidence for tonic inhibitory control of steroidogenesis by protein kinase C

Direct effect of arachidonic acid on protein kinase C and LH-stimulated steroidogenesis in rat Leydig cells; evidence for tonic inhibitory control of steroidogenesis by protein kinase C

Tight coupling of gonadotropin-releasing hormone receptor to stimulated phosphoinositide turnover and antigonadotropic action in granulosa cells.

Gonadotropin-releasing hormone (Gn-RH) stimulates phosphoinositide turnover by binding to its specific receptor and suppresses gonadotropin-dependent maturation and steroidogenesis in granulosa cells. This study was undertaken to determine whether persistent receptor occupancy was necessary for Gn-RH to exert such actions on rat granulosa cells, or whether Gn-RH actions were continued by a first and transient stimulation by Gn-RH, using a competitive antagonist, antide. Gn-RH stimulated [32P]phosphate incorporation into phosphatidylinositol (PtdIns), which could be terminated by displacement of previously bound Gn-RH from its receptor by antide and restarted by reoccupying the receptors with Gn-RH. Antide could prevent Gn-RH-stimulated PtdIns radiolabelling whenever it was added to incubations. An identical effect of antide was observed also in the anti-follicle-stimulating hormone (FSH) action of Gn-RH. FSH markedly stimulated aromatase activity, and Gn-RH caused a time- and dose-dependent inhibition of FSH action. Estrogen production was quenched by Gn-RH and restarted at a time when Gn-RH was removed from its receptor by antide. These two responses associated with the occupancy of Gn-RH receptor provide the evidence in favor of a tight coupling of stimulated PtdIns turnover to suppression of aromatase activation. These data of required continued activation of receptor might exclude the possibility that hypothalamic Gn-RH participated in the control of steroidogenesis in the ovary.

Prostaglandin E2Is a Positive Regulator of Adrenocorticotropin Receptors, 3β-Hydroxysteroid Dehydrogenase, and 17α-Hydroxylase Expression in Bovine Adrenocortical Cells*

The maintenance of optimal steroidogenesis in adrenocortical cells primarily depends on the chronic action of cAMP. Herein we examine the effects of prostaglandin E2 (PGE2) on the differentiated functions of bovine adrenocortical (BAC) cells in primary culture. PGE2 (10 microM) treatment for 3 h stimulated steroidogenesis and cAMP production by over 100-fold. In addition, the cAMP antagonist Rp-cAMP (1 mM) inhibited PGE2 stimulation of steroidogenesis by 60%. This observation suggests that the cAMP second messenger system is responsible for much of the PGE2-activated steroid hormone synthesis. Chronic treatment of BAC cells with PGE2 caused induction of 3 beta-hydroxysteroid dehydrogenase and steroid 17 alpha-hydroxylase cytochrome P-450 expression as determined by the examination of enzyme activity, enzyme levels by immunoblotting, and specific messenger RNA (mRNA) levels by Northern analysis. The positive effects of PGE2 on expression of 3 beta-hydroxysteroid dehydrogenase and 17 alpha-hydroxylase cytochrome P-450 were similar to the effects seen after ACTH treatment of BAC cells. In addition, treatment of BAC cells with PGE2 for 3 days caused a 3-fold induction of ACTH receptors as determined by increased cell binding of [125I]ACTH. Finally, we determined that BAC cells produced PGE2 and that the level of synthesis increased 10-fold after treatment with the hormone angiotensin II. Taken together these data indicate that PGE2 is a positive regulator of BAC cell differentiation acting on ACTH receptors, steroid metabolizing enzymes, and steroidogenesis. The ability of BAC cells to produce PGE2 leaves open the possibility for paracrine and autocrine regulation within the adrenal.

Preincubation of human granulosa cells with gonadotrophin prevents the cloprostenol-induced inhibition of progesterone production

Human granulosa cells, from women undergoing ovum collection for in-vitro fertilization (IVF), will luteinize in vitro and provide a model for investigating the antigonadotrophic action of a prostaglandin F2 alpha (PGF2 alpha) analogue, cloprostenol, on granulosa-derived luteal cells. The granulosa cells were cultured in a defined medium and exposed to treatments during a preincubation period of 0 to 3 days and a final incubation with low density lipoprotein (LDL) from days 3 to 4. In the absence of human chorionic gonadotrophin (HCG), progesterone production was low, whereas exposure to HCG in the final incubation resulted in a 10-fold increase in progesterone concentrations. The inclusion of cloprostenol with HCG in the final incubation significantly (P less than 0.05) inhibited HCG-stimulated progesterone production. Exposure to HCG during the preincubation prevented the antigonadotrophic action of cloprostenol in the final incubation. The antigonadotrophic action of cloprostenol was retained when the granulosa cells were exposed to cloprostenol during the preincubation. Omission of LDL from the final incubation lowered the production of progesterone but the pattern of responses to HCG and cloprostenol were similar. Prevention of the antigonadotrophic action of cloprostenol after exposure to HCG may be a mechanism through which chorionic gonadotrophin can prevent regression of the corpus luteum in early pregnancy. Cloprostenol does not appear to inhibit LDL-stimulated steroidogenesis in human granulosa cells.

Follicle-Stimulating Hormone Increases Concentrations of Messenger Ribonucleic Acid Encoding Cytochrome P450 Cholesterol Side-Chain Cleavage Enzyme in Primary Cultures of Porcine Granulosa Cells*

FSH is the primary hormonal inducer of ovarian follicle maturation and a critically important regulator of steroidogenesis in granulosa cells. We examined possible molecular mechanisms subserving FSH action by assessing concentrations of cytochrome P450 cholesterol side-chain cleavage (P450scc) mRNA in porcine granulosa cells maintained in serum-free culture. Cellular concentrations of specific P450scc mRNA were measured by Northern blot hybridization using a 32P-labeled 1-kilobase porcine cDNA clone. Specificity was tested by estimating the granulosa cell mRNA content of the constitutively expressed enzyme, glyceraldehyde-3-phosphate dehydrogenase. Steroidogenesis was evaluated by measuring concomitant progesterone accumulation in the culture medium. Treatment with ovine FSH (100 ng/ml) increased P450scc mRNA concentrations in a time-dependent fashion, with significant effects on both P450scc mRNA concentrations and progesterone accumulation by 4 h and a maximal increase (8- to 10-fold) at 48 h. FSH dose-response studies at 48 h revealed a significant stimulatory effect of 30 ng/ml FSH on P450scc mRNA accumulation and progesterone production, with a maximal effect at 100 ng/ml FSH. To examine the role of cAMP in mediating granulosa cell P450scc mRNA accumulation, granulosa cells were treated with forskolin, cholera toxin, 8-bromo-cAMP, 8-bromo-cGMP, 5'AMP, or cAMP analogs that differentially stimulate the two isoenzymes of protein kinase-A. Increased specific P450scc mRNA accumulation and progesterone production occurred in response to each agent except 5'AMP and 8-bromo-cGMP. No effects of these agents were observed on glyceraldehyde-3-phosphate dehydrogenase mRNA. To assess possible feedback effects of steroid or sterol on FSH-stimulated P450scc mRNA concentrations, granulosa cells were treated with aminoglutethimide to block or with low density lipoprotein to stimulate steroid production. Inhibition of sterol utilization by the cholesterol side-chain cleavage enzyme had no effect on basal or FSH-stimulated concentrations of P450scc mRNA, but markedly suppressed progesterone production. Low density lipoprotein, which increases intracellular sterol, also did not alter basal or FSH-stimulated P450scc mRNA accumulation, suggesting that neither the utilization nor the availability of sterol regulates specific P450scc mRNA levels. Estradiol alone did not increase P450scc mRNA accumulation, but did augment progesterone production. Treatment of granulosa cells with estradiol and FSH produced a synergistic increase in progesterone concentrations, but did not affect FSH-stimulated P450scc mRNA accumulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of the human apolipoprotein E gene suppresses steroidogenesis in mouse Y1 adrenal cells.

The lipid transport protein, apolipoprotein E (apoE), is expressed in many peripheral tissues in vivo including the adrenal gland and testes. To investigate the role of apoE in adrenal cholesterol homeostasis, we have expressed a human apoE genomic clone in the Y1 mouse adrenocortical cell line. Y1 cells do not express endogenous apoE mRNA or protein. Expression of apoE in Y1 cells resulted in a dramatic decrease in basal steroidogenesis; secretion of fluorogenic steroid was reduced 7- to greater than 100-fold relative to Y1 parent cells. Addition of 5-cholesten-3 beta,25-diol failed to overcome the suppression of steroidogenesis in these cells. Cholesterol esterification under basal conditions, as measured by the production of cholesteryl [14C]oleate, was similar in the Y1 parent and the apoE-transfected cell lines. Upon incubation with adrenocorticotropin or dibutyryl cAMP, production of cholesteryl [14C]oleate decreased 5-fold in the Y1 parent cells but was unchanged in the apoE-transfected cell lines. These results suggest that apoE may be an important modulator of cholesterol utilization and steroidogenesis in adrenal cells.

Effect of chronic administration of nicotine or cocaine on steroidogenesis in rat adrenocortical cells

Rats were implanted subcutaneously with osmotic mini-pumps containing either 0.9% NaCl, nicotine (1.5 or 4.5 mg kg-1 day-1), or cocaine (30 mg kg-1 day-1), for 14 days. Neither nicotine nor cocaine treatment significantly altered the maximal rate of steroidogenesis in adrenocortical cell preparations from the animals. However, pretreatment with cocaine increased the sensitivity of the preparation to stimulation by ACTH, the ED50 was 5 pM compared with 10 pM from control animals. Addition of nicotine or cocaine at concentrations up to 100 microM to adrenal cell suspensions from naive rats did not stimulate steroidogenesis or increase the sensitivity of cells to ACTH stimulation. These results suggest that the primary chronic effect of nicotine on steroidogenesis is exerted at the level of the hypothalamus and/or pituitary and not directly on adrenocortical cells. On the contrary, pretreatment with cocaine causes persistent changes in adrenocortical cells.

Regulation of steroidogenesis of infant and adult rhesus monkey granulosa cells in vitro.

The present studies have demonstrated that infant monkey granulosa cells, like the adult ones, have the potential of responding markedly in vitro to human FSH, cyclic-AMP and forskolin, resulting in the increase of progesterone and estrogen production. Exogenous hCG was also capable of increasing FSH-stimulated progesterone biosynthesis in both infant and adult granulosa cells, but did not stimulate the infant granulosa cells to secrete estrogen. Addition of a synthetic estrogen, diethylstilbestrol, to the culture of monkey granulosa cells enhanced the FSH-stimulated progesterone and estrogen production. The steroidogenesis of monkey granulosa cells was also dramatically stimulated by a gonadotropin-releasing hormone agonist. Monkey granulosa cells, unlike the other animal cells, secrete measurable amount of estrogen in the absence of androgen substrate. The findings reported here are significant in regard to understanding of the mechanism of hormonal regulation of primate ovarian function.