Direct effects of diethylstilbestrol on the gene expression of the cholesterol side-chain cleavage enzyme (P450scc) in testicular Leydig cells.

AIMS: To investigate the precise mechanisms underlying the action of estrogenic endocrine disruptors, we evaluated the direct effects of synthetic estrogen diethylstilbestrol (DES) on steroidogenesis in Leydig cells, with particular emphasis on the expression of the cholesterol side-chain cleavage enzyme P450scc. Furthermore, the mechanism underlying the action of DES was compared with that of endogenous estrogen 17beta-estradiol (E2), which has a potency equivalent to that of DES. MAIN METHODS: TTE1 Leydig cells were treated with 5 x 10(-)(8) microM to 5 microM DES or E2 for 24h, and P450scc gene expression and the histone modifications underlying their transcriptional activation were examined using reverse transcription-polymerase chain reaction (RT-PCR) and chromatin immunoprecipitation (ChIP), respectively. KEY FINDINGS: P450scc mRNA expression in the DES-treated and E2-treated cells reduced in inverse proportion to the dose of DES and E2, respectively; however, cAMP stimulation induced a recovery in the expression to a level approximately equal to those in the controls. In the DES-treated cells, ChIP assay revealed histone deacetylation in the P450scc promoter region. Interestingly, E2 did not cause histone deacetylation. SIGNIFICANCE: In the early stages of steroidogenesis, DES and E2 directly induced a reduction in P450scc mRNA expression in inverse proportion to their doses, and treatment with cAMP restored the decreased P450scc mRNA expression. Furthermore, DES can induce alterations in the histone modification of the P450scc gene, and natural estrogen and synthetic estrogenic compounds such as DES may induce reproductive disorders through different molecular mechanisms.

Activin A and equine chorionic gonadotropin recover reproductive dysfunction induced by neonatal exposure to an estrogenic endocrine disruptor in adult male mice.

We aimed to elucidate the mechanism of action of estrogenic endocrine disruptors and the rescue of reproductive function, particularly the responsiveness of testes to eCG and/or activin A (ACT) after establishing reproductive disorders. Newborn male mice (n = 29) were randomly divided into an untreated group and three treatment groups that received diethylstilbestrol (DES; 100 mug per animal) subcutaneously on Postnatal Day 3 to establish reproductive disorders and daily treatment with PBS (controls: DES + PBS), eCG (eCG group: DES + eCG), or eCG + ACT (eCG + ACT group: DES + eCG + ACT) at 6-8 wk of age prior to mating. After treatment, the controls showed diminished Leydig cells in the testes and thin germ cell layers containing pyknotic germ cells and multinucleated cells. In the eCG and eCG + ACT groups, spermatids and Leydig cells increased markedly. The immunoexpression of androgen receptors in the eCG group and steroidogenic acute regulatory (STAR) protein in the eCG and eCG + ACT groups recovered to approximately the levels in the untreated group; plasma LH and testosterone levels also increased relative to those in the controls. In addition, the cell proliferation index, which is estimated from 5-bromo-2'-deoxyuridine immunoexpression in spermatogonia, increased significantly under eCG treatment, and even more with eCG + ACT. However, the numbers of germ and Leydig cells decreased at 12 wk of age. Thus, ACT and eCG help the testes to recover from the dysfunction induced by neonatal DES administration. Furthermore, the permanent male reproductive disorder induced by neonatal exposure to estrogenic agents may be more likely to result from dysfunction of the hypothalamic-pituitary axis than from dysfunction of the lower reproductive organs.

Cholesterol sulphate affects production of steroid hormones by reducing steroidogenic acute regulatory protein level in adrenocortical cells.

Steroidogenic acute regulatory (StAR) protein plays a crucial role in the intramitochondrial movement of cholesterol, where P450 side chain cleavage enzyme resides. Cholesterol sulphate (CS), which is present ubiquitously in mammalian tissues, is not only a precursor of sulphated adrenal steroids but also an inhibitor of cholesterol biosynthesis. This study was designed to examine the biological roles of CS in steroidogenesis in adrenocortical cells. Human adrenocortical carcinoma H295R cells were cultured with various amounts of CS. To evaluate steroid hormone synthesis, pregnenolone production in cells was assayed. The amount of pregnenolone produced by H295R cells in culture medium, to which over 50 mug/ml CS was added, was significantly (P<0.05) decreased compared with that produced by control cells. Western blot analysis was performed to determine StAR protein level using whole cell extracts from cells. StAR protein level decreased when the concentration of CS in the medium was 50 mug/ml, whereas the level of glyceraldehyde-3-phosphate dehydrogenase did not change. To examine the mechanism by which StAR gene expression is controlled, we performed RT-PCR and measured promoter activity in cells transfected with pGL(2) StAR reporter constructs. StAR mRNA level and promoter activity were decreased in cells. The decrease in StAR protein level is a result of the low StAR gene expression level. In conclusion, CS affects the production of steroid hormones by reducing StAR protein level in adrenocortical cells.

CREM confers cAMP responsiveness in human steroidogenic acute regulatory protein expression in NCI-H295R cells rather than SF-1/Ad4BP.

Steroidogenic acute regulatory (StAR) protein plays a critical role in steroid hormone synthesis. Tropic hormones induce human StAR gene expression by a cAMP-dependent pathway. Steroidogenic factor-1/adrenal-4-binding protein (SF-1/Ad4BP) plays an important role in the expression of human StAR gene. We investigated the mechanism of cAMP responsiveness in human StAR gene expression in NCI-H295R cells. The StAR promoter activity and protein levels in cells subjected to various treatments were examined. Anti-SF-1/Ad4BP IgG transfection treatment resulted in decreases in the basal StAR promoter activity and StAR protein levels, but did not affect cAMP-stimulated promoter activity and protein levels. The basal and cAMP-stimulated StAR promoter activity levels were reduced in SF-1/Ad4BP mutant (G35E)-transfected cells, but the cAMP induction of StAR promoter activity in response to 1 mM 8-Br-cAMP was not inhibited when G35E SF-1/Ad4BP mutant expression vectors were co-transfected with cAMP-response element-binding (CREB) expression vectors. Although the basal StAR mRNA expression and protein levels were decreased by SF-1/Ad4BP-siRNA treatment, the cAMP-stimulated StAR mRNA expression and protein levels did not change. The basal StAR promoter activity level was not decreased by cAMP-response element modulator (CREM)-siRNA treatment, but the cAMP-stimulated StAR promoter activity level, the magnitude of cAMP induction of StAR promoter, and the cAMP-stimulated StAR protein level were decreased. The cAMP induction of StAR promoter activity in cells was inhibited when S117ACREM mutant expressionvectors were transfected. We conclude that inhibition of the function of SF-1/Ad4BP does not reduce the cAMP induction of StAR promoter activity and protein level. CREM is needed to confer cAMP responsiveness in human StAR protein expression.

Progression of the dose-related effects of estrogenic endocrine disruptors, an important factor in declining fertility, differs between the hypothalamo-pituitary axis and reproductive organs of male mice.

For the purpose of investigation of working mechanisms in endocrine disruptors, we evaluated the dose-related effects of fetal and/or neonatal exposure to an estrogenic compound on the male reproductive organs in adult mice, particularly with respect to gene expression of steroidogenic acute regulatory protein (StAR). The pregnant ICR mice were given subcutaneous injections of 10 micro g/day/animal of diethylstilbestrol (DES) to subject the fetal mice to in utero exposure (IUE). Subsequently, the newborn male mice were subjected to neonatal exposure (NE) by treatment with vehicle or 0.1-10 micro g/day/animal of DES. Fertility rates of each group were as follows: control, 100%; IUE only, 60%; IUE+NE 0.1 micro g, 25%; IUE+NE 1 micro g, 0%; IUE+NE 10 micro g, 0%. In general histology, germ cell layers in the seminiferous tubules were thinned in the group of IUE+NE 10 micro g. Hypoplasia of the Leydig cells, in which the staining intensity of eosin was diminished, was also observed in the groups of IUE+NE 0.1-10 micro g. The androgen receptor (AR) and estrogen receptor alpha (ERalpha) immunoexpression in the Leydig cells of IUE+NE 1-10 micro g was slightly lower than that in the controls. Long-term dysfunction of the hypothalamo-pituitary-testicular axis, including sustained hypoproduction of gonadotropin and testosterone, and altered expressions of steroid hormone receptors and StAR genes were observed. The hypothalamo-pituitary control of gonadotropin secretion may be affected by the smaller doses of estrogenic agents than the reproductive organs. Furthermore, the fertility rate in the male mice exposed to this estrogenic agent was closely correlated with the testosterone levels, and even more so with the rate-limiting factor of steroidogenesis, StAR. This finding suggests that endocrine disruptors have an important pronounced effect on StAR gene expression.

Development of a simple screening system for endocrine disruptors.

BACKGROUND: An endocrine disruptor is a synthetic chemical, which causes adverse effects in an organism, or its progeny, after causing perturbations in the endocrine system. It is important to know which synthetic chemicals have endocrine-disrupting action. However, an increasing number of synthetic chemicals are being produced by modern synthetic chemistry, and the examination of endocrine disruptor potential has not yet caught up with the advances in synthetic chemistry. In this study, we have developed such a screening system for detecting synthetic chemicals with estrogen-like effects. MATERIAL/METHODS: The system was based on the yeast one-hybrid system. Both HIS3 and lacZ reporter genes connected to three tandem copies of the estrogen response element were prepared. Gal4-estrogen receptor is a fusion protein made from the activation domain (AD) of the yeast GAL4 transactivator gene and then incorporated into a plasmid, which was transfected into the YM4271 yeast cell strain. The estrogen effect was judged by this developed screening system. RESULTS: A dual reporter assay-system was established by transfection of the both HIS3 and lacZ reporter genes into the yeast cells. This screening system enabled the detection of as little as 10-12 mol of beta-estradiol. CONCLUSIONS: These results show that this newly developed dual assay is useful for the screening of endocrine-disruptors that have estrogen-like action.