Androgen biosynthesis during minipuberty favors the backdoor pathway over the classic pathway: Insights into enzyme activities and steroid fluxes in healthy infants during the first year of life from the urinary steroid metabolome.

The steroid profile changes dramatically from prenatal to postnatal life. Recently, a novel backdoor pathway for androgen biosynthesis has been discovered. However, its role remains elusive. Therefore, we investigated androgen production from birth to one year of life with a focus on minipuberty and on production of androgens through the backdoor pathway. Additionally, we assessed the development of the specific steroid enzyme activities in early life. To do so, we collected urine specimens from diapers in 43 healthy newborns (22 females) at 13 time points from birth to one year of age in an ambulatory setting, and performed in house GC-MS steroid profiling for 67 steroid metabolites. Data were analyzed for androgen production through the classic and backdoor pathway and calculations of diagnostic ratios for steroid enzyme activities were performed. Analysis revealed that during minipuberty androgen production is much higher in boys than in girls (e.g. androsterone (An)), originates largely from the testis (Anboys-Angirls), and uses predominantly the alternative backdoor pathway (An/Et; Δ5<Δ4 lyase activity). Modelling of steroid enzyme activities showed age-related effects for 21-, 11-, 17-hydroxylase and P450 oxidoreductase activities as well as 3ß-hydroxysteroid dehydrogenase, 11ß-hydroxylase type 1/2 and 5α-reductase activities. Sex-related characteristics were found for 21-hydroxylase and 5α-reductase activities. Overall, our study shows that androgen biosynthesis during minipuberty favors the backdoor pathway over the classic pathway. Calculations of specific diagnostic ratios for enzyme activities seem to allow the diagnosis of specific steroid disorders from the urinary steroid metabolome.

Measuring Faecal Epi-Androsterone as an Indicator of Gonadal Activity in Spotted Hyenas (Crocuta crocuta).

Enzyme immunoassays (EIA) that measure faecal testosterone metabolites (fTM) are useful tools to monitor gonadal activity. The aim of this study was to validate an "in-house" epiandrosterone EIA to monitor fTM in spotted hyenas. FTM were characterised in a male and a female hyena that each received an injection of 3H-testosterone. High-performance liquid chromatography (HPLC) analyses revealed a cluster of highly polar enzyme-hydrolysable hormone metabolite conjugates. We performed hydrolysis using ß-glucuronidase to deconjugate metabolites and improve sensitivity of the assay. Because ß-glucuronidase from Helix pomatia has been reported to bias testosterone measurements in some species, we compared the enzymatic activity of the commonly used ß-glucuronidase extracted from H. pomatia with the same enzyme from Escherichia coli. Our results showed that ß-glucuronidases from both sources produced similar results from spotted hyena faeces. We therefore hydrolysed samples with H. pomatia enzymes. HPLC analyses also demonstrated that following hydrolysis the epiandrosterone EIA measured significant amounts of immunoreactive metabolites corresponding to radiolabelled metabolites in both sexes. Additionally, HPLC and GC-MS analyses confirmed the presence of epiandrosterone in faeces of spotted hyenas. The biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM levels in response to a testosterone injection within 16 h, (2) no biological responsiveness to an adrenocorticotropic hormone (ACTH) injection and (3) significant differences in fTM levels between juvenile males and adult immigrant males in a free-ranging wild population. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor gonadal activity in spotted hyenas.

Resveratrol potentiates effects of simvastatin on inhibition of rat ovarian theca-interstitial cells steroidogenesis.

BACKGROUND: Polycystic ovary syndrome (PCOS) is characterized by ovarian enlargement, hyperplastic theca compartment and increased androgen production due to, at least in part, excessive expression of several key genes involved in steroidogenesis. Previously, our group has demonstrated that simvastatin, competitive inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a rate-limiting step of the mevalonate pathway, reduces rat-theca interstitial cell steroidogenesis by inhibiting Cyp17a1 gene expression, the key enzyme of the androgen biosynthesis pathway. Recently, we demonstrated that resveratrol, a bioflavonoid abundant in red grapes, decreases rat theca-interstitial cell steroidogenesis and this suppressive effect is mediated through mechanisms independent of the mevalonate pathway. The present study evaluated the effect of combining simvastatin and resveratrol treatments on rat theca-interstitial cell steroidogenesis. METHODS: Rat theca-interstitial cells isolated from 30 day-old female rats were cultured for up to 48 h with or without simvastatin (1 µM) and/or resveratrol (3-10 µM). Steroidogenic enzymes gene expression was evaluated by quantitative real time PCR and steroid levels were measured by liquid chromatography-mass spectrometry. Comparisons between groups were performed using ANOVA and Tukey test. RESULTS: Resveratrol potentiated inhibitory effects of simvastatin on androstenedione and androsterone production in theca-interstitial cells. This suppressive effect correlated with profound inhibition in Cyp17a1 mRNA expression in the presence of a combination of resveratrol and simvastatin. CONCLUSIONS: The present findings indicate that resveratrol potentiates the simvastatin-induced inhibitory effect on theca-interstitial cell androgen production, raising the possibility of development of novel treatments of PCOS.

New investigations around CYP11A1 and its possible involvement in an androstenone QTL characterised in Large White pigs.

BACKGROUND: Previously, in boars with extreme androstenone levels, differential expression of the CYP11A1 gene in the testes has been characterised. CYP11A1 is located in a region where a QTL influencing boar fat androstenone levels has been detected in a Large White pig population. Clarifying the role of CYP11A1 in boar taint is important because it catalyses the initial step of androstenone synthesis and also of steroid synthesis. RESULTS: A genome-wide association study located CYP11A1 at approximately 1300 kb upstream from SNP H3GA0021967, defining the centre of the region containing the QTL for androstenone variation. In this study, we partially sequenced the CYP11A1 gene and identified several new single nucleotide polymorphisms (SNP) within it. Characterisation of one animal, heterozygous for CYP11A1 testicular expression but homozygous for a haplotype of a large region containing CYP11A1, revealed that variation of CYP11A1 expression is probably regulated by a mutation located downstream from the SNP H3GA0021967. We analysed CYP11A1 expression in LW families according to haplotypes of the QTL region's centre. Effects of haplotypes on CYP11A1 expression and on androstenone accumulation were not concordant. CONCLUSION: This study shows that testicular expression of CYP11A1 is not solely responsible for the QTL influencing boar fat androstenone levels. As a conclusion, we propose to refute the hypothesis that a single mutation located near the centre of the QTL region could control androstenone accumulation in fat by regulating the CYP11A1 expression.

Genetic and metabolic aspects of androstenone and skatole deposition in pig adipose tissue: a review.

High levels of androstenone and skatole in fat tissues are considered the primary causes of boar taint, an unpleasant odour and flavour of the meat from non-castrated male pigs. The aim of this article is to review our current knowledge of the biology and genetic control of the accumulation of androstenone and skatole in fat tissue. Two QTL mapping studies have shown the complexity of the genetic control of these traits. During the last ten years, several authors have taken a more physiological approach to investigate the involvement of genes controlling the metabolism of androstenone and skatole. Although some authors have claimed the identification of candidate genes, it is more appropriate to talk about target genes. This suggests that genes affecting androstenone and skatole levels will have to be sought for among specific or non-specific transcription factors interacting with these target genes.

Development of a cryopreservation protocol for Leydig cells.

BACKGROUND: In the present study, we describe a procedure to cryopreserve the postnatal members of the Leydig cell lineage, including progenitor (PLC), immature (ILC) and adult (ALC) Leydig cells from, respectively 21-, 35- and 90-day-old rats. METHODS: The cells were resuspended in a culture medium supplemented with 1% bovine serum albumin (Dulbecco's Modified Eagle's Medium [DMEM]/F12) to a final concentration of 2 x 10(6)cells/ml and the effects of varying concentrations of dimethylsulfoxide (DMSO) (5, 10, 15 or 20%) were assessed after freezing at -70 degrees C and then storing in liquid nitrogen. After 12 months of frozen storage, these cells were thawed rapidly at 37 degrees C and Trypan Blue exclusion staining and attachment to culture dishes were assessed as measures of viability. RESULTS: The trypan blue exclusion and attachment rates for Leydig cell stages were around 85% in the presence of 15% DMSO. After frozen storage, Leydig cell steroidogenic capacity in response to a range of LH doses, (0.01-100 ng/ml) was unchanged compared with freshly isolated control cells. Furthermore, the steady-state mRNA levels for Leydig cell specific transcripts were maintained. CONCLUSIONS: This study demonstrates that purified rat Leydig cells at a range of developmental stages can be frozen and that the cryopreserved cells retain normal function.

Identification of a single nucleotide polymorphism in porcine testis cytochrome P450-c17 (CYP17) and its effect on steroidogenesis.

Raising uncastrated male pigs could have significant economic benefits for pig production. Uncastrated male pigs can accumulate high levels of 16-androstene steroids, however, resulting in boar taint, which is highly objectionable to consumers. Cytochrome P450-c17 (CYP17) interacts with cytochrome b5 in the biosynthesis of the 16-androstene steroids and the sex steroids from pregnenolone. Amino acid substitutions in CYP17 could therefore affect the ability of this enzyme to catalyze the reactions leading to the production of androstenone and the sex steroids. In this study, we established a sensitive and flexible single-stranded conformational polymorphism technique capable of detecting a single nucleotide polymorphism. We then used this method to identify a substitution from T to A at nucleotide 1317 of CYP17, which caused a change in the amino acid sequence from Leu(439) to His(439). This mutation, however, did not alter the enzyme activity of CYP17 in the biosynthesis of androstenone or sex steroids. Other polymorphisms previously suggested for CYP17, which are vital for the functional interaction of CYP17 with CYB5 in human, were not observed. This study suggests that the synthesis of androstenone in pig testis is not directly affected by any polymorphisms in the coding region of the porcine CYP17 gene.

17 beta-hydroxysteroid dehydrogenase type XI localizes to human steroidogenic cells.

We searched expressed sequence tag databases with conserved domains of the short-chain alcohol dehydrogenase superfamily and identified another isoform of 17 beta-hydroxysteroid dehydrogenase, 17 beta HSDXI. This enzyme converts 5 alpha-androstane-3 alpha, 17 beta-diol to androsterone. The substrate has been implicated in supporting gestation and modulating gamma-aminobutyric acid receptor activity. 17 beta HSDXI is colinear with human retinal short-chain dehydrogenase/reductase retSDR2, a protein with no known biological activity (accession no. AAF06939). Of the proteins with known function, 17 beta HSDXI is most closely related to the retinol-metabolizing enzyme retSDR1, with which it has 30% identity. There is a polymorphic stretch of 15 adenosines in the 5' untranslated region of the cDNA sequence and a silent polymorphism at C719T. A 17 beta HSDXI construct with a stretch of 20 adenosines was found to produce significantly more enzyme activity than constructs containing 15 or less adenosines (43% vs. 26%, P < 0.005). The C719T polymorphism is present in 15% of genomic DNA samples. Northern blot analysis showed high levels of 17 beta HSDXI expression in the pancreas, kidney, liver, lung, adrenal, ovary, and heart. Immunohistochemical staining for 17 beta HSDXI is strong in steroidogenic cells such as syncytiotrophoblasts, sebaceous gland, Leydig cells, and granulosa cells of the dominant follicle and corpus luteum. In the adrenal 17 beta HSDXI, staining colocalized with the distribution of 17 alpha-hydroxylase but was stronger in the mid to outer cortex. 17 beta HSDXI was also found in the fetus and increased after birth. Liver parenchymal cells and epithelium of the endometrium and small intestine also stained. Regulation studies in mouse Y1 cells showed that cAMP down-regulates 17 beta HSDXI enzymatic activity (40% vs. 32%, P < 0.05) and reduces gene expression to undetectable levels. All-trans-retinoic acid did not affect 17 beta HSDXI expression or activity, but addition of the retinoid together with cAMP significantly decreased activity over cAMP alone (32% vs. 23%, P < 0.05). Cloning and sequencing of the 17 beta HSDXI promoter identified the potential nuclear receptor steroidogenic factor-1 half-site TCCAAGGCCGG, and a cluster of three other potential steroidogenic factor-1 half-sites were found in the distal part of intron 1. Collectively, these results suggest a role for 17 beta HSDXI in androgen metabolism during steroidogenesis and a possible role in nonsteroidogenic tissues including paracrine modulation of 5 alpha-androstane-3 alpha, 17 beta-diol levels. 17 beta HSDXI could act by metabolizing compounds that stimulate steroid synthesis and/or by generating metabolites that inhibit it.

Use of bioconversion for the preparation of [4-14C]-labeled 7 alpha- and 7 beta-hydroxylated derivatives of dehydroepiandrosterone and epiandrosterone.

The 7 alpha- and 7 beta-hydroxylated derivatives of [4-14C]-dehydroepiandrosterone were prepared with use of the yeast-expressed human cytochrome p4507B1. Epiandrosterone (EPIA), 5 alpha-androstane-3beta,17 beta-diol, and 5 alpha-androstane-3,17-dione were obtained after incubation of [4-14C]-5 alpha-dihydrotestosterone with Escherichia coli-expressed (3beta,17 beta)-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. The 7 alpha- and 7 beta-hydroxylated derivatives of [4-14C]-EPIA produced were prepared after incubation with mycelium of Rhizopus nigricans. Each labeled steroid was purified by chromatography and identified by crystallization to constant specific activity after isotopic dilution with each authentic steroid carrier. Production yields and radio-purity measurements allowed the use of such procedures for the preparation of the described radio-steroids for studies of metabolism and mode of action.

Human 17beta-hydroxysteroid dehydrogenase type 5 is inhibited by dietary flavonoids.

Phytoestrogens contained in a vegetarian diet are supposed to have beneficial effects on the development and progression of a variety of endocrine-related cancers. We have tested the effect of a variety of dietary phytoestrogens, especially flavonoids, on the activity of human 17beta-hydroxysteroid dehydrogenase type 5 (17beta-HSD 5), a key enzyme in the metabolism of estrogens and androgens. Our studies show that reductive and oxidative activity of the enzyme are inhibited by many compounds, especially zearalenone, coumestrol, quercetin and biochanin A. Among flavones, inhibitor potency is enhanced with increased degree of hydroxylation. The most effective inhibitors seem to bind to the hydrophilic cofactor binding pocket of the enzyme.

Decrease of core body temperature in mice by dehydroepiandrosterone.

Dietary dehydroepiandrosterone (DHEA) reduces food intake in mice, and this response is under genetic control. Moreover, both food restriction and DHEA can prevent or ameliorate certain diseases and mediate other biological effects. Mice fed DHEA (0.45% w/w of food) and mice pair-fed to these mice (food restricted) for 8 weeks were tested for changes in body temperature. DHEA was more efficient than food restriction alone in causing hypothermia. DHEA injected intraperitoneally also induced hypothermia that reached a nadir at 1 to 2 hr, and slowly recovered by 20 to 24 hr. This effect was dose dependent (0.5-50 mg). Each mouse strain tested (four) was susceptible to this effect, suggesting that the genetics differ for induction of hypophagia and induction of hypothermia. Because serotonin and dopamine can regulate (decrease) body temperature, we treated mice with haloperidol (dopamine receptor antagonist), 5,7-dihydroxytryptamine (serotonin production inhibitor), or ritanserin (serotonin receptor antagonist) prior to injection of DHEA. All of these agents increased rather than decreased the hypothermic effects of DHEA. DHEA metabolites that are proximate (5-androstene-3beta, 17beta-diol and androstenedione) or further downstream (estradiol-17beta) were much less effective than DHEA in inducing hypothermia. However, the DHEA analog, 16alpha-chloroepiandrosterone, was as active as DHEA. Thus, DHEA administered parentally seems to act directly on temperature-regulating sites in the body. These results suggest that DHEA induces hypothermia independent of its ability to cause food restriction, to affect serotonin or dopamine functions, or to act via its downstream steroid metabolites.

Hepatocyte growth factor regulates ovarian theca-interstitial cell differentiation and androgen production.

During ovarian follicle growth, precise regulation of the onset of androgen production by ovarian theca-interstitial cells (TIC) is necessary for maintaining follicle viability. Thus, temporary suppression of TIC androgen production in preantral follicles is the key to promoting follicle development. Evidence indicates that this process is coordinated via intraovarian growth factors. Hepatocyte growth factor (HGF) can induce granulosa cell (GC) proliferation and suppress follicular atresia, indicating a role for HGF in promoting follicle growth and viability. To determine whether HGF could reversibly suppress androgen production, this study investigated the effect of HGF on TIC differentiation and steroid production. Twenty-six-day-old rats were used in all studies. HGF messenger RNA (mRNA) expression in TIC and GC was determined by reverse transcription-PCR. Agarose gel electrophoresis of the PCR products yielded a single band corresponding to the 290-bp HGF product for both TIC and GC. HGF expression in cultured TIC and GC was not blocked by gonadotropins or HGF. To investigate the effects of HGF on TIC steroidogenesis, TIC were isolated from the ovaries of hypophysectomized rats. TIC (3.0 x 10(4) cells/well) were cultured with LH (0-3 ng/ml) and/or HGF (0-100 ng/ml) for 48 h, and androsterone levels were measured by RIA. HGF did not alter androsterone levels in the absence of LH; however, HGF reversibly impaired LH-dependent androsterone production by as much as 57% (IC50 = 1.5 +/- 0.01 ng/ml). LH (0.3 ng/ml) stimulated progesterone (P4) synthesis by TIC (1201 +/- 190 pg/ml) compared to that by control cells (210 +/- 30 pg/ml). HGF stimulated basal P4 production, and LH-dependent P4 synthesis was augmented 2.6-fold by HGF (ED50 = 0.3 +/- 0.01 ng/ml). The DNA content and cell viability in TIC cultures were not affected by HGF. The effect of HGF on steroidogenic enzyme gene expression in TIC was also investigated via PCR. HGF did not alter the level of basal or LH-induced P450 side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase mRNAs; however, LH-dependent P45017 alpha hydroxylase/C17,20 lyase mRNA content was reduced 4.5 fold in the presence of HGF. Thus, HGF is expressed in both TIC and GC obtained from the immature rat ovary, suggesting its presence in growing follicles. In TIC, HGF stimulated P4 synthesis, but impaired androgen production, concurrent with a down-regulatory effect on P45017 alpha hydroxylase/C17,20 lyase gene expression. Collectively, these results indicate that HGF reversibly impairs LH-stimulated androgen production in TIC. Such effects may help promote folliculogenesis.

Developmental and hormonal regulation of rat theca-cell differentiation factor secretion in ovarian follicles.

We have recently presented data demonstrating that preantral follicles secrete a peptide (or family of peptides) that stimulates ovarian theca-interstitial cell (TIC) androgen production by an LH-independent mechanism. The purpose of the study reported here was to study the gonadotropin and developmental regulation of this thecal differentiating factor(s) (TDF) and to determine whether follicle-conditioned medium (FCM) containing TDF bioactivity could stimulate LH receptor and steroidogenic enzyme mRNA expression in TIC. Preantral follicles devoid of theca were obtained by limited enzymatic dispersal of 26-day-old rat ovaries. Follicles were cultured (5 follicles/well) in 96-well plates containing serum-free medium to generate FCM containing bioactive TDF. To bioassay for TDF activity, isolated TIC were cultured (2 days) with 50% FCM; then androsterone production was measured by RIA. Recombinant FSH (rFSH, 0.3-100 mlU/ml) increased TDF bioactivity in a dose-dependent fashion, stimulating maximum androsterone production (20 ng/ml) at 30 mlU/ml. To determine the time course of the production of TDF bioactivity, FCM was collected from follicle cultures treated with and without rFSH at 1, 6, 12, 18, 24, 30, 36, 42, and 48 h. FCM from follicles cultured without rFSH caused a progressive increase in androsterone production to a peak (8 ng/ml) at 18 h followed by a decline to baseline by 48 h. A similar time course was observed for the first 18 h with the rFSH-treated FCM, but androsterone production continued to increase to a level twice that of the untreated FCM (18 ng/ml) at 36 h of culture. In the presence of 100 mlU/ml of rFSH, TDF bioactivity was produced by preantral follicles with > or = 2 layers of granulosa cells but not by small antral follicles, preovulatory follicles, or corpora lutea, demonstrating that production of TDF bioactivity is developmentally regulated. To determine whether FCM could stimulate mRNA expression in TIC, LH receptor, cholesterol side-chain cleavage (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and 17 alpha-hydroxylase (P450(17) alpha) mRNAs were measured by reverse transcription polymerase chain reaction assays. FCM stimulated LH-receptor, P450scc, 3 beta-HSD, and P450(17) alpha mRNAs above controls. Our data demonstrate that the production of TDF bioactivity is increased by FSH during a specific stage in follicular development when the theca interna is rapidly differentiating, but its production stops when the follicle develops an antrum. Treatment of TIC with FCM stimulates the expression of the mRNAs coding for LH receptors and the steroidogenic enzymes P450scc, 3 beta-HSD, and P450(17) alpha, mimicking the events that occur during normal thecal differentiation. Thus, it seems likely that TDF is involved in the regulation of initial thecal differentiation in preantral follicles.

Growth hormone stimulates androsterone synthesis by rat theca-interstitial cells.

The aim of this study was to assess the possible role of growth hormone (GH) on androsterone synthesis. This effect was analyzed in theca-interstitial cells obtained from immature female rats. The addition of GH to the cultures significantly stimulated androsterone (A) synthesis in a dose- and time-dependent way and this effect was not due to a cellular number increase. When added to the hCG cultures, GH significantly enhanced androgen production even though it did not synergyze with the chorionic gonadotropin. The addition of antibodies anti-IGF-I to the GH cultures did not modify the growth hormone effect suggesting that GH probably does not require IGF-I to achieve its effect on A production. Finally, no effect of GH on cAMP levels were observed in the cultures at the end of the treatment. Our results demonstrate that GH is able to significantly induce A synthesis by rat theca-interstitial cells. Since the presence of GH and its receptors in the ovary is now well established the present data strongly suggest a potential relevance of GH in reproductive biology.

Granulosa cell modulation of luteinizing hormone-dependent androgen production by ovarian theca-interstitial cells: a temporal switch from suppression to augmentation stimulated by follicle-stimulating hormone in vitro.

The production of estradiol-17 beta (E2) by granulosa cells (GC) of the dominant follicle is dependent upon LH-stimulated synthesis of androgens by ovarian theca-interstitial cells (TIC). Recent evidence has pointed toward an intrafollicular paracrine system, regulated by FSH and involving GC, that may modulate the LH-dependent production of androgens by TIC. In the present study, the role of GC and FSH in modulating LH-dependent TIC and androsterone production was examined. In cultures of dispersed whole ovarian cells (containing populations of both GC and TIC) from intact immature rats, LH stimulated a 10-fold increase in androsterone production (maximum androsterone = 21.0 +/- 1.1 ng/ml). By comparison, androsterone production was increased 50-fold in LH-stimulated cultures of dispersed whole ovarian cells from hypophysectomized immature rats (108 +/- 18 ng androsterone/ml). The EC50 for LH (0.02 +/- 0.001 ng/ml) was identical in the two cell preparations. We hypothesized that the lesser androgen production by whole ovarian cell cultures from intact rats was due to suppression by the GC. To investigate the role of GC in modulating TIC androgen production, highly purified TIC from immature hypophysectomized rats were cultured in the presence of GC obtained from intact immature rats. Increasing numbers of GC (2.5-100 x 10(3) GC per well) caused a progressive decrease in LH-dependent androsterone production by TIC. Additionally, LH-dependent androsterone production was suppressed by the conditioned medium from recombinant human FSH (rFSH)-stimulated GC (54% of the value for LH-stimulated TIC controls), indicating the involvement of a GC-secreted paracrine factor or factors.(ABSTRACT TRUNCATED AT 250 WORDS)

An autoregulatory process for androgen production in rat thecal-interstitial cells.

The regulation of androgen production by thecal-interstitial cells (TIC) of the mammalian ovary is a complex process. Although androgen production is primarily controlled by LH, a variety of factors have been demonstrated to alter LH-stimulated androgen production. It is uncertain, however, if an androgen-mediated autoregulatory process for androgen production exists in TIC. To determine the existence of this phenomenon, TIC obtained from ovaries of immature hypophysectomized rats were enriched by Percoll density gradient centrifugation. When TIC (20,000 viable cells/0.2 ml/well) were cultured for 48 h in the presence of a maximal concentration of hCG (0.2 ng/ml), androsterone production was increased 26-fold versus control levels. Treatment with increasing concentrations (5-1000 nM) of the synthetic androgen 17 beta-hydroxy-7 alpha,17 alpha-dimethyl-4-estren-3-one (mibolerone) inhibited hCG-stimulated androsterone production by an average of 32% at every dose tested. Mibolerone (100 nM) alone was without effect on basal levels of androgens. The addition of insulin (100 ng/ml) or insulin-like growth factor I (100 ng/ml) to TIC cultures did not alter the basal accumulation of androsterone but significantly augmented hCG-induced androgen production by 2- and 3-fold, respectively, versus controls. Concomitant treatment with mibolerone (100 nM) decreased the synergistic action of insulin or IGF-I on hCG-stimulated androsterone synthesis by 46% and 40%, respectively. To elucidate the mechanism(s) of action of mibolerone, we investigated the effects on 8-bromo-cAMP-stimulated androgen production. At a dose of 0.1 mM 8-bromo-cAMP, androsterone production was maximally stimulated to levels observed with 0.2 ng/ml hCG. In the presence of mibolerone (100 nM), cAMP-induced androsterone synthesis was inhibited by 41%. This result suggested that mibolerone was acting at a site distal to cAMP formation. Additional evidence revealed that through the use of the combination of cAMP analogs, N6-monobutyryl-cAMP (50 microM) and 8-bromo-cAMP (75 microM)--which are known activators of the cAMP-dependent protein kinase isoenzymes PKA I and II--androsterone synthesis was increased by 130-fold over basal levels. Treatment with mibolerone (100 nM), however, reduced this cAMP-stimulated androgen synthesis by 51%. Therefore, the results demonstrate the existence of an autoregulatory process for androgen production in TIC, which may be important in limiting the overproduction of androgens.

Transforming growth factor-beta 1 inhibits ovarian androgen production: gene expression, cellular localization, mechanisms(s), and site(s) of action.

It is the aim of this study to establish ovarian transforming growth factor-beta 1 (TGF beta 1) gene expression, to reevaluate its cellular localization, and to explore potential interactions of this regulatory peptide on ovarian androgen biosynthesis. Northern analysis of whole ovarian polyadenylated RNA revealed a single 2.5-kilobase transcript corresponding to the TGF beta 1 precursor. Immunohistochemical staining localized the protein to the thecal-interstitial (interfollicular) compartment. To explore potential autocrine effects of TGF beta 1, use was made of whole ovarian dispersates from immature rats the differentiation of which was monitored by the acquisition of androgen biosynthetic capacity. The accumulation of androsterone, the major androgenic steroid detectable in this culture system, increased 5.4-fold over baseline in response to treatment with hCG (1 ng/ml). This effect was further optimized (2- to 4-fold) by supplementation with insulin (1 microgram/ml) and insulin-like growth factor-I (50 ng/ml). In the absence of these optimizing supplements, TGF beta 1 (10 ng/ml) was without effect on basal androsterone accumulation, producing distinct, albeit relatively limited (25%), inhibition of hCG hormonal action. In contrast, supplement-mediated optimization of ovarian androgen biosynthesis revealed TGF beta 1 to be a highly potent inhibitor (greater than 80%) of hCG hormonal action. This reversible TGF beta 1 action proved time and dose dependent, with a minimal time requirement of 72 h and a median inhibitory dose of 2.6 ng/ml. TGF beta 1 action was not due to diminution in the viable cell mass or altered cAMP generation and, therefore, most likely involved a site(s) of action distal to or independent of cAMP generation. Cellular radiolabeling studies of TGF beta 1-treated ovarian cells disclosed the accumulation of steroid intermediates proximal to the 17 alpha-hydroxylation step, suggesting TGF beta 1-mediated blockade at the level of the steroidogenic enzyme 17 alpha-hydroxylase/17-20-lyase. Taken together, these observations are in keeping with the view that TGF beta 1, possibly of thecal-interstitial origin, may not only play a positive paracrine role at the level of the adjacent granulosa cell (as previously reported), but may also constitute one of several autocrine signals concerned with the regulation of ovarian androgen economy. As such, these findings reaffirm the polyfunctional nature of TGF beta 1 action, as manifested by its diametrically opposed effects in different ovarian compartments.

Insulin-like growth factor-I enhances luteinizing hormone binding to rat ovarian theca-interstitial cells.

We tested the hypothesis that insulin-like growth factor-I (IGF-I) stimulates ovarian androgen production by increasing theca-interstitial cell luteinizing hormone (LH) binding affinity and/or binding capacity. We then investigated the role of transcriptional and translational events in mediating these actions of IGF-I. LH bound to saturable, high affinity binding sites on rat ovarian theca-interstitial cells. Preincubation with LH produced a decrease in LH binding capacity with no effect on LH binding affinity. Treatment with IGF-I, both in the absence and presence of LH, increased LH binding capacity 1.5- to 2-fold with no change in LH binding affinity. Androgen production was increased progressively by LH, suggesting that LH-stimulated steroidogenesis is not tightly coupled to LH receptor downregulation. IGF-I increased androgen synthesis in proportion to its upregulation of LH binding capacity. Transcriptional inhibition with dichlorobenzimidazole riboside inhibited the IGF-I-mediated increase in LH binding capacity but had no effect on androgen production. Translational inhibition with cycloheximide inhibited both the IGF-I-mediated increase in LH binding and stimulation of androgen synthesis. We conclude that IGF-I increases theca-interstitial cell LH binding capacity and reverses the LH-induced downregulation of LH binding sites. The enhancement of LH binding by IGF-I is compatible with transcriptional mediation whereas the effect of IGF-I on androgen synthesis appears to be mediated by a direct effect of the peptide on the translational process(es) involved in steroidogenesis.

Basic fibroblast growth factor inhibits gonadotropin-supported ovarian androgen biosynthesis: mechanism(s) and site(s) of action.

Current evidence favors the hypothesis that granulosa cell-derived basic fibroblast growth factor (bFGF) may be the centerpiece of an intraovarian autocrine loop. In this report we examine the possibility that bFGF may also be involved in paracrine interactions at the level of the ovarian theca-interstitial cell. To this end, whole ovarian dispersates obtained from immature rats were cultured for 96 h under serum-free conditions. The accumulation of androsterone, the major androgenic steroid detected, increased 3- to 5-fold over baseline in response to treatment with hCG (1 ng/ml), an effect further optimized (2- to 4-fold) by supplementation with insulin-like growth factor-I (10 ng/ml), insulin (1 microgram/ml), terbutaline (10(-6) M), or high density lipoprotein (100 micrograms/ml). In the absence of these optimizing supplements, bFGF was without effect on basal androsterone accumulation, but produced a relatively modest (20%) inhibition of hCG hormonal action. In contrast, bFGF proved a highly potent inhibitor (80%) of hCG-stimulated androgen biosynthesis in supplement-enriched cultures. This reversible bFGF action proved to be time and dose dependent, with a minimal time requirement of 48 h and a median inhibitory dose of 2 ng/ml. Unaccounted for by altered (hCG-stimulated) cAMP generation or a diminution in the viable cell mass, the antigonadotropic effect of bFGF may by inference be assumed to involve a site(s) of action distal to or independent of cAMP generation. In this connection, cellular radiolabeling with [3H] pregnanolone (3 alpha-hydroxy-5 alpha-pregnane-20-one) revealed bFGF to be a potent inhibitor of the steroidogenic enzyme 17 alpha-hydroxylase/17-20-lyase. As such, these findings are in keeping with the possibility that locally derived bFGF may exert a dual inhibitory action on (mature) ovarian estrogen production by reducing androgen substrate provision as well as by exercising its now established ability to attenuate granulosa cell aromatase activity.

An improved method for primary culture of ovarian androgen-producing cells in serum-free medium: effect of lipoproteins, insulin, and insulinlike growth factor-I.

Although luteinizing hormone (LH) alone stimulates ovarian interstitial cells cultured in serum-free medium to synthesize large amounts of androgens, there seem to be additional factors in vivo that modulate the time course and magnitude of the cellular responses to LH. In an attempt to develop a more nearly physiologic cell culture model, lipoproteins, insulin, and insulinlike growth factor-I (IGF-I) were added to the serum-free medium. The effects of these modifications on androgen biosynthesis by dispersed cells from ovaries of hypophysectomized immature rats cultured in 96-well tissue culture plates were examined. A saturating dose of LH stimulated a 25-fold increase in androsterone synthesis at 2 d, which decreased at 4 and 6 d. Addition of human high density (hHDL) or human low density lipoprotein (hLDL) caused a 2.5-fold increase in LH-stimulated androsterone synthesis. Cells were approximately twice as sensitive to hHDL (ED50 = 5.5 +/- 0.5 micrograms cholesterol/ml) compared to hLDL (ED50 = 9.1 +/- 1.1 micrograms cholesterol/ml). Surprisingly, rat HDL caused only a 40% increase in LH-stimulated androsterone synthesis. When insulin alone was added to cells cultured with a saturating dose of LH, there was a 2.8-fold increase in androsterone synthesis. Addition of hHDL and insulin together caused a synergistic increase in LH-stimulated androsterone synthesis. In contrast to hHDL, which did not change the time course of LH-stimulated androsterone production, insulin prolonged maximal LH-stimulated androsterone synthesis at 4 and 6 d. Inasmuch as the ED50 for insulin action (1.3 +/- 0.1 micrograms/ml) was supraphysiologic, the effects of IGF-I on LH-stimulated androgen synthesis were examined. IGF-I mimicked all of the effects of insulin, but at a physiologic concentration (ED50 = 2.5 +/- 0.3 ng/ml). Ovarian cells cultured in serum-free medium supplemented with hHDL and insulin or IGF-I exhibit responses that closely approximate the physiologic responses observed in vivo. These results suggest that lipoproteins and IGF-I are important physiologic stimulators of ovarian theca-interstitial cell androgen biosynthesis which, when added to the serum-free medium, make the cellular responses in this in vitro model more nearly approximate the responses in vivo.