Annexin A5 regulates Leydig cell testosterone production via ERK1/2 pathway.

This study was to investigate the effect of annexin A5 on testosterone secretion from primary rat Leydig cells and the underlying mechanisms. Isolated rat Leydig cells were treated with annexin A5. Testosterone production was detected by chemiluminescence assay. The protein and mRNA of Steroidogenic acute regulatory (StAR), P450scc, 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and 17α-hydroxylase were examined by Western blotting and semi-quantitative RT-PCR, respectively. Annexin A5 significantly stimulated testosterone secretion from rat Leydig cells in dose- and time-dependent manners and increased mRNA and protein expression of StAR, P450scc, 3ß-HSD, and 17ß-HSD but not 17α-hydroxylase. Annexin A5 knockdown by siRNA significantly decreased the level of testosterone and protein expression of P450scc, 3ß-HSD, and 17ß-HSD. The significant activation of ERK1/2 signaling was observed at 5, 10, and 30 min after annexin A5 treatment. After the pretreatment of Leydig cells with ERK inhibitor PD98059 (50 µmol l-1 ) for 20 min, the effects of annexin A5 on promoting testosterone secretion and increasing the expression of P450scc, 3ß-HSD, and 17ß-HSD were completely abrogated (P < 0.05). Thus, ERK1/2 signaling is involved in the roles of annexin A5 in mediating testosterone production and the expression of P450scc, 3ß-HSD, and 17ß-HSD in Leydig cells.

Adrenal and Ovarian Phenotype of a Tissue-Specific Urocortin 2-Overexpressing Mouse Model.

Urocortin 2 (UCN2) is a neuropeptide of the CRH family, involved in homeostatic mechanisms, the stress response, and control of anxiety. To elucidate the effects of UCN2 on steroidogenesis, we developed a mouse model that allows a Cre recombinase-determined conditional overexpression of UCN2 (UCN2-COE). In these mice SF1-Cre-driven overexpression of UCN2 was restricted to the adrenal glands, gonads, and parts of the hypothalamus. UCN2-COE animals of both sexes revealed significantly higher plasma UCN2 levels and significantly higher UCN2 expression levels in the adrenals and ovaries. In contrast, the baseline expression of UCN2 was already high in the testes of control mice with no further increase achievable in UCN2-COE animals. Adrenal steroidogenesis of UCN2-COE animals was investigated under baseline conditions, upon an ACTH stimulation test, and following a restraint stress test. A tendency toward lower expression of steroidogenic enzymes was detectable in UCN2-COE animals of both sexes with slight differences between males and females. A similar reduction in the expression levels of the final steps of ovarian steroidogenesis, accompanied by reduced plasma estradiol levels, was observed in female UCN2-COE animals. Thus, adrenal UCN2 overexpression resulted in down-regulation of adrenal steroidogenesis, suggesting a reduction in the stress response in the mouse (stress coping behavior). Similarly, UCN2 overexpression in the ovaries caused a decrease in steroidogenesis and reduction of follicles that had undergone ovulation. Nevertheless, this finding was not associated with reduced fertility.

Bone Morphogenetic Protein-4 (BMP4): A Paracrine Regulator of Human Adrenal C19 Steroid Synthesis.

Bone morphogenetic proteins (BMPs) comprise one of the largest subgroups in the TGF-ß ligand superfamily. We have identified a functional BMP system equipped with the ligand (BMP4), receptors (BMP type II receptor, BMP type IA receptor, also called ALK3) and the signaling proteins, namely the mothers against decapentaplegic homologs 1, 4, and 5 in the human adrenal gland and the human adrenocortical cell line H295R. Microarray, quantitative RT-PCR, and immunohistochemistry confirmed that BMP4 expression was highest in the adrenal zona glomerulosa followed by the zona fasciculata and zona reticularis. Treatment of H295R cells with BMP4 caused phosphorylation of the mothers against decapentaplegic and a profound decrease in synthesis of the C19 steroids dehydroepiandrosterone (DHEA), DHEA sulfate, and androstenedione. Administration of BMP4 to cultures of H295R cells also caused a profound decrease in the mRNA and protein levels of 17α-hydroxylase/17,20-lyase (CYP17A1 and P450c17, respectively) but no significant effect on the mRNA levels of cholesterol side-chain cleavage cytochrome P450 (CYP11A1) or type 2 3ß-hydroxysteroid dehydrogenase (HSD3B2). Furthermore, Noggin (a BMP inhibitor) was able to reverse the negative effects of BMP4 with respect to both CYP17A1 transcription and DHEA secretion in the H295R cell line. Collectively the present data suggest that BMP4 is an autocrine/paracrine negative regulator of C19 steroid synthesis in the human adrenal and works by suppressing P450c17.

Effects of monocrotophos pesticide on steroidogenesis and transcription of steroidogenic enzymes in rainbow trout RTG-2 cells involving the protein kinase A signal pathway.

Monocrotophos (MCP) pesticide, listed as a UNEP Prior Informed Consent chemical, has been proved to exert toxic effects on the reproductive system of teleost fishes by changing the balance of sex steroid hormones. To investigate the effects of MCP on steroidogenesis in vitro, the rainbow trout (Oncorhynchus mykiss) gonadal cell line RTG-2 was exposed to different MCP concentrations for 48 h. The levels of 17 ß-estradiol (E(2)) and testosterone in the medium were measured by radioimmunoassay and the expression of steroidogenic acute regulatory protein and cytochrome P450 enzymes CYP11A1, CYP17, and CYP19A was detected by quantitative real-time PCR. The results showed that 1.0 and 10.0 µg/L MCP pesticide induced E(2) levels and promoted steroidogenic enzyme expression. The possible mechanisms of MCP steroidogenic activity were investigated using inhibitors of protein kinase A (PKA) and protein kinase C. The PKA inhibitor H-89 abrogated the 10.0 µg/L MCP-induced transcriptional up-regulation of steroidogenic enzymes, suggesting an involvement of PKA-dependent mechanism in the disruption of steroidogenesis by the MCP pesticide in rainbow trout RTG-2 cells.

Effects of polybrominated diphenyl ethers on steroidogenesis in rat Leydig cells.

BACKGROUND: Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants that have been defined as major environmental pollutants. While previous studies have found that PBDEs may enhance the levels of sex-steroid hormones, their effects on testosterone secretion from rat Leydig cells are unclear. This study investigated the effects and mechanisms of PBDE-710, a mixture of tetra- and penta-PBDEs, on testosterone biosynthesis in rat Leydig cells. METHODS: Leydig cells from adult male rats were challenged with different concentrations of PBDE-710 (0.5-15 ng/ml) to evaluate the effects on testosterone steroidogenesis. Concentrations of testosterone and of cAMP and pregnenolone in medium were measured by radioimmunoassay (RIA) and by enzyme-linked immunosorbent assay, respectively. Nuclear translocation of protein kinase A α (PKAα) was determined by immunofluorence assay and western blot assay, and the mRNA expression of steroidogenic acute regulatory protein (StAR) was analyzed by quantitative real-time polymerase chain reaction. RESULTS: In this in vitro study, PBDE-710 (5 or 15 ng/ml) increased basal testosterone secretion and cAMP production by 3- and 2-fold, respectively. The stimulatory effect was abolished by adenylyl cyclase inhibitor. Enzyme activity of CYP11A1, as determined by the pregnenolone concentration, was stimulated by PBDE-710 treatment. Furthermore, nuclear translocation of PKAα was increased by 20% and StAR gene expression was elevated by 4-fold after PBDE-710 treatment. CONCLUSIONS: These results suggest that low concentrations of PBDE-710 could stimulate testosterone secretion by acting directly on Leydig cells to activate the cAMP pathway and increase expression of StAR.

Effect of methotrexate and leucovorin on female reproductive tract of albino rats.

Methotrexate (MTX) an antifolate drug and leucovorin its antidote, are used in the treatment of both neoplastic and non-neoplastic diseases in young women. We hypothesize that MTX treatment might comprise a deleterious effect on fast proliferating reproductive cells, an unavoidable and unwanted side effect. MTX given dose dependently to rats for 20 days prevented vaginal cyclicity and caused a reduction in serum progesterone and estradiol. External morphology of reproductive tract displayed thinning of organs and reduction in their weights. To reveal mechanism of MTX action, we examined the histology of ovary, oviduct, uterus, cervix and vagina. Results suggested that in a dose-dependent fashion MTX restrained preantral and antral follicular growth in ovary. Epithelium and stroma of oviduct, uterus, cervix and vagina were disrupted and lost their normal structures. Such alterations in ovarian function raised serum follicle stimulating hormone, luteinizing hormonal profiles. Expression of steroidogenic acute regulatory protein and P450 cholesterol side chain cleavage gene, which are both essential for steroidogenesis, markedly decreased in ovary upon MTX treatment. Total RNA, DNA and protein concentrations, glucose 6 phosphate dehydrogenase, lactate dehydrogenase and alkaline phosphatase enzyme activities in ovary were distinctly altered. Leucovorin supplementation and withdrawal of the treatment, improved MTX caused effects partially. These results for the first time indicate that the malfunction of female reproductive organs by MTX treatment in young women is not only correlated to the disrupted circulating levels of hormones and histoarchitecture of tissues but also discrepancies in steroidogenic genes and hormone regulated enzyme activities in ovary.

Reproductive and developmental effects of transovarian exposure to o,p'-DDT in Japanese quails.

Avian species have the possible risk of embryonic exposure to persistent, lipophilic environmental contaminants, such as dichlorodiphenyltrichloroethane (DDT), by transfer of chemicals accumulated in mother birds to eggs. To model developmental and reproductive disorders of wild birds living in contaminated areas, we exposed Japanese quails in ovo to o,p'-DDT prior to incubation. A positive estrogenic substance diethylstilbestrol (DES; 1 and 10 ng/g of egg) and o,p'-DDT (1-100 microg/g of egg) were injected into the yolk before incubation. Treatment with o,p'-DDT (10 or 100 microg/g) but not with DES significantly reduced the hatchability of eggs. After sexual maturation, o,p'-DDT affected eggshell formation in female quails but had little influence on laying; high doses of o,p'-DDT significantly reduced eggshell strength, shell weight, and shell thickness, and several females treated with 100 microg o,p'-DDT/g laid eggs lacking shells. Diethylstilbestrol decreased egg production itself but had little effect on the eggshell. Both o,p'-DDT and DES caused dose-dependent shortening of the left oviduct and abnormal development of the right oviduct in females, while testis asymmetry was observed in males treated with a high dose of DES. In the uterus of the oviduct, the mRNAs for calcium-regulating factors osteopontin and calbindin D28K were reduced by both treatments, particularly that with o,p'-DDT. The results indicated that transovarian exposure to o,p'-DDT could bring about population declines in avian species through loss of fecundity caused by depression of hatchability and dysfunction of the reproductive tract.

Estrogenic effects of selected hydroxy polychlorinated biphenyl congeners in primary culture of Atlantic Salmon (Salmo salar) hepatocytes.

Many persistent organic pollutants are known to have endocrine-disrupting effects in several aquatic and terrestrial species. In this regard, hydroxylated metabolites of polychlorinated biphenyls (OH-PCBs) represent serious health and environmental concern because they are shown to act agonistic or antagonistic at hormone receptors (HRs) or to cause hormone-receptor-mediated responses. In the present study, salmon primary hepatocytes were used to study alterations in an estrogen signaling pathway resulting from exposure to four hydroxylated (4OH-CB 107, 4OH-CB146, 4OH-CB187, and 3OH-CB138) metabolites of PCB at different concentrations using quantitative real-time polymerase chain reaction. The effects of the PCB metabolites were compared to the mRNA expression in 17alpha-ethynylestradiol (EE2)-treated cells. Concentration-specific increase of vitellogenin (Vtg) mRNA transcription after exposure to OH-PCBs was observed. Decreased mRNA transcription was observed for zona radiata protein (Zr-protein) and cytochrome P450 side-chain cleavage (P450scc) enzyme. For estrogen receptor beta (ERbeta), the mRNA expression pattern was OH-PCB-metabolite congener-specific. A novel aspect of this study is that OH-PCBs produced effects on hepatic steroidogenic pathways by targeting the StAR protein and P450scc genes. Given that endocrine toxicology research mainly has focused on estrogenicity involving direct ER-mediated effects and that steroidogenic enzyme and proteins are highly tissue- and cell-type-specific and controlled by different promoters and second-messenger pathways, the present findings provide potential new targets for interaction with xenobiotics such as hydroxylated congeners of certain chemicals. The quantitative expression patterns of hepatic and extrahepatic steroidogenic genes and proteins after exposure to environmental contaminants are the subject of systematic investigations in our laboratory.

Developmental methoxychlor exposure affects multiple reproductive parameters and ovarian folliculogenesis and gene expression in adult rats.

Methoxychlor (MXC) is an organochlorine pesticide with estrogenic, anti-estrogenic, and anti-androgenic properties. To investigate whether transient developmental exposure to MXC could cause adult ovarian dysfunction, we exposed Fischer rats to 20 microg/kg/day (low dose; environmentally relevant dose) or 100 mg/kg/day (high dose) MXC between 19 days post coitum and postnatal day 7. Multiple reproductive parameters, serum hormone levels, and ovarian morphology and molecular markers were examined from prepubertal through adult stages. High dose MXC accelerated pubertal onset and first estrus, reduced litter size, and increased irregular cyclicity (P<0.05). MXC reduced superovulatory response to exogenous gonadotropins in prepubertal females (P<0.05). Rats exposed to high dose MXC had increasing irregular estrous cyclicity beginning at 4 months of age, with all animals showing abnormal cycles by 6 months. High dose MXC reduced serum progesterone, but increased luteinizing hormone (LH). Follicular composition analysis revealed an increase in the percentage of preantral and early antral follicles and a reduction in the percentage of corpora lutea in high dose MXC-treated ovaries (P<0.05). Immunohistochemical staining and quantification of the staining intensity showed that estrogen receptor beta was reduced by high dose MXC while anti-Mullerian hormone was upregulated by both low- and high dose MXC in preantral and early antral follicles (P<0.05). High dose MXC significantly reduced LH receptor expression in large antral follicles (P<0.01), and down-regulated cytochrome P450 side-chain cleavage. These results demonstrated that developmental MXC exposure results in reduced ovulation and fertility and premature aging, possibly by altering ovarian gene expression and folliculogenesis.

Changes in cytochrome P450 side chain cleavage expression in the rat hippocampus after kainate injury.

Our previous study showed an increase in total cholesterol level of the hippocampus after kainate-induced injury, but whether this is further metabolized to neurosteroids is not known. The first step in neurosteroid biosynthesis is the conversion of cholesterol to pregnenolone by the enzyme cytochrome P450 side chain cleavage (P450scc). This study was carried out to elucidate the expression of this enzyme in the kainate-lesioned rat hippocampus. A net decrease in P450scc protein was detected in hippocampal homogenates by Western blots at 2 weeks post-kainate injection (time of peak cholesterol concentration after kainate injury). Immunohistochemistry showed decreased labeling of the enzyme in neurons, but increased expression in a small number of astrocytes. The level of pregnenolone was also analyzed using a newly developed gas chromatography-mass spectrometry (GC-MS) method, optimized for the rat hippocampus. A non-significant tendency to a decrease in pregnenolone level was detected 2 weeks post-lesion. This is in contrast to a large increase in oxysterols in the lesioned hippocampus at this time (He et al. 2006). Together, they indicate that increased cholesterol in the kainate lesioned hippocampus is mostly metabolized to oxysterols, and not neurosteroids.

Hypophyseal corticosteroids stimulate somatotrope differentiation in the embryonic chicken pituitary gland.

Although it is known that glucocorticoids induce differentiation of growth hormone (GH)-producing cells in rodents and birds, the effect of mineralocorticoids on GH mRNA expression and the origin of corticosteroids affecting somatotrope differentiation have not been elucidated. In this study, we therefore carried out experiments to determine the effect of mineralocorticoids on GH mRNA expression in the chicken anterior pituitary gland in vitro and to determine whether corticosteroids are synthesized in the chicken embryonic pituitary gland. In a pituitary culture experiment with E11 embryos, both corticosterone and aldosterone stimulated GH mRNA expression and increased the number of GH cells in both lobes of the pituitary gland in a dose-dependent manner. These effects of the corticosteroids were significantly reversed by pretreatment with mifepristone, a glucocorticoid receptor (GR) antagonist, or spironolactone, a mineralocorticoid receptor (MR) antagonist. Interestingly, an in vitro serum-free culture experiment with an E11 pituitary gland showed that the GH mRNA level spontaneously increased during cultivation for 2 days without any extra stimulation, and this increase in GH mRNA level was completely suppressed by metyrapone, a corticosterone-producing enzyme P450C11 inhibitor. Moreover, progesterone, the corticosterone precursor, also stimulated GH mRNA expression in the cultured chicken pituitary gland, and this effect was blocked by pretreatment with metyrapone. We also detected mRNA expression of enzymes of cytochrome P450 cholesterol side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase1 (3beta-HSD1) in the developmental chicken pituitary gland from E14 and E18, respectively. These results suggest that mineralocorticoids as well as glucocorticoids can stimulate GH mRNA expression and that corticosteroids generated in the embryonic pituitary gland by intrinsic steroidogenic enzymes stimulate somatotrope differentiation.

Comparative activity of telavancin against isolates of community-associated methicillin-resistant Staphylococcus aureus.

OBJECTIVES: This study compared the activity of telavancin, a novel multivalent lipoglycopeptide with rapid bactericidal activity, with those of five standard antibiotics for methicillin-resistant Staphylococcus aureus (MRSA) against isolates of community-associated MRSA (CA-MRSA). METHODS: Microdilution tests performed according to CLSI guidelines using cation-adjusted Mueller-Hinton broth were used to determine the MIC values of telavancin, quinupristin/dalfopristin, vancomycin, trimethoprim/sulfamethoxazole, linezolid and daptomycin versus 60 CA-MRSA isolates. MBC values of telavancin were determined according to CLSI guidelines and American Society for Microbiology standards. PFGE was performed using the restriction enzyme SmaI. Samples from three predominant pulsed-field types were typed by multilocus sequence typing. Staphylococcal cassette chromosome mec typing was determined by multiplex PCR. The Panton-Valentine leucocidin (PVL) genes (lukS-PV and lukF-PV) were identified by PCR. RESULTS: The telavancin MIC90 and MBC90 values for this collection of 60 CA-MRSA isolates were 0.5 and 1 mg/L, respectively, with MIC and MBC values both ranging from 0.25 to 1 mg/L. Telavancin was found to be bactericidal in this study, as its MBC was no more than 2-fold higher than its MIC for all CA-MRSA isolates tested except one. (A single isolate yielded an MBC/MIC ratio of 4.) PVL- and non-PVL-producing strains demonstrated similar susceptibility to telavancin and comparator agents. CONCLUSIONS: Based on in vitro activity, telavancin should be an effective agent against CA-MRSA.

Study on the mechanism of trichlorfon-induced inhibition of progesterone synthesis in mouse leydig tumor cells (MLTC-1).

Trichlorfon, a widely used organophosphorus pesticide, has been reported to disrupt reproductive function in humans. However, the mechanisms have not been clearly elucidated. In the present study, mouse Leydig tumor cells (MLTC-1) was used to evaluate the effect of trichlorfon on progesterone synthesis. After the various concentrations of trichlorfon treatment (0, 0.04, 0.2, 1, 5 or 25 micromol/l) for 24h, the progesterone production presented a dose-dependent decrease in the presence of some stimulators such as human chorionic gonadotrophin (hCG), cholera toxin (CT) or forskolin. However, the levels of intracellular cAMP remained unaltered, which suggested that trichlorfon suppressed steroidogenesis occurred after PKA activation along cAMP/PKA pathway. Moreover, trichlorfon suppressed the expression of the steroidogenic acute regulatory protein (StAR) mRNA and protein, and also profoundly inhibited the activity of P450 side chain cleavage enzyme (P450scc), rather than 3beta-hydorxysteroid dehydrogenase (3beta-HSD). The suppressive expression of P450scc mRNA and protein further accounted for the inhibitory action of trichlorfon on steroidogenesis. These results indicated that trichlorfon suppressed progesterone synthesis in MLTC-1 cells, at least in part, via inhibiting StAR expression and P450scc activity.

The xenoestrogen, 4-nonylphenol, impaired steroidogenesis in previtellogenic oocyte culture of Atlantic cod (Gadus morhua) by targeting the StAR protein and P450scc expressions.

The steroidogenic acute regulatory (StAR) protein and cytochrome P450-mediated cholesterol side-chain cleavage (P450scc) have been localized in most steroidogenic organs and are rapidly synthesized in response to acute tropic hormone stimulation. In this study, we present the development of cod previtellogenic oocyte in vitro culture system, histological and molecular methods for evaluating the effects of endocrine disruptors such as nonylphenol (NP) on steroid hormone levels, the StAR protein and P450scc. In addition, expression pattern of cyclin-B was studied, because of cyclin B's role as an indicator of oocyte growth in fish. The in vitro previtellogenic oocyte culture technique was based on an agarose floating method. Tissue was cultured in a humidified incubator at 10 degrees C for 4, 7, 14 and 21 d with different concentrations of nonylphenol (0 (control), 1, 10, 50 and 100 microM) dissolved in ethanol (0.3%). Gene expressions were detected using validated real-time polymerase chain reaction (PCR) with specific primers. Immunohistochemistry of the StAR protein and P450scc were performed using antisera prepared against synthetic peptide for both proteins. Estradiol-17beta (E2) and 11-ketotestosterone (11-KT) tissue levels were estimated using enzyme immunoassay. Our data show that nonylphenol produced a unique and consistent concentration-specific pattern of modulation for the StAR protein, P450scc and cyclin-B gene expression at day 14 after exposure. This pattern is generally described as increasing from 0 (control) to 1 and 10 microM, and decreased at 50 and 100 microM. The observed changes in the StAR protein, P450scc and cyclin-B levels showed a direct relationship with changes in 11-KT levels at day 14 after exposure. Cellular localization of StAR and P450scc were specific to the follicular cells of previtellogenic oocytes, but with no differences in staining intensities. No significant change in oocyte diameter was observed between the exposure groups. Our data reveal some novel aspects of nonylphenol effects on maturation and oocyte growth in teleosts, suggesting impaired steroidogenesis and hormonal imbalance with potential consequences for the vitellogenic process and overt fecundity.

Bone morphogenetic protein 5 expression in the rat ovary: biological effects on granulosa cell proliferation and steroidogenesis.

Recently, the role of several elements of the bone morphogenetic protein (BMP) family has been studied in the ovary, some of them being crucial for ovarian function. In the present work, we have studied bone morphogenetic protein 5 (BMP5) expression and its biological role in the rat ovary. BMP5 is expressed by rat granulosa cells (GCs) and exerts specific biological effects on proliferation and steroidogenesis of these cells in an autocrine manner. These effects were shown to be associated with an increase in cyclin D2 protein level and a decrease in steroidogenic acute regulatory (StAR) protein expression in GCs in vitro. Ultimately, BMP5 actions were inhibited by follistatin. Overall, these data show that BMP5 is a novel element of the BMP family that might play a fully paracrine role in rodent ovarian folliculogenesis.

Differential steroidogenic gene expression in the fetal adrenal gland versus the testis and rapid and dynamic response of the fetal testis to di(n-butyl) phthalate.

The phthalate ester di(n-butyl) phthalate (DBP) causes feminization of male rats upon in utero exposure by repressing expression of genes required for testicular steroidogenesis. Previous work in our laboratory has shown that repression of gene expression and steroidogenesis in the fetal testis is apparent within a few hours of DBP exposure. The purpose of this study was to determine the precise timing of DBP-associated gene expression changes in the fetal testis using transcriptional profiling and to determine whether DBP exerts similar effects on steroidogenesis in the fetal adrenal. A DBP time-course experiment showed that testicular steroidogenesis was decreased within 1 h of DBP exposure and that this decrease preceded the repressed transcription of Star (steroidogenic acute regulatory protein); Scarb1 (scavenger receptor class B, member 1; also know as Sr-b1); Cyp11a1 (cytochrome P450, family 11, subfamily a, polypeptide 1; also known as P450SCC); and Cyp17a1 (cytochrome P450 family 17, subfamily a, polypeptide 1; also known as Cyp17). Gene expression profiling demonstrated rapid (within 1 to 3 h) and transient induction of immediate early genes in the fetal testis after administration of DBP to the pregnant dam. There was a statistically insignificant decrease in corticosterone production by the fetal adrenal after in utero exposure to DBP from Gestation Day 12 to Gestation Day 19. The extent of steroidogenesis diminution was much less in the adrenal than in the testis (approximately 45% decrease in the adrenal versus 87% decrease in the testis) and expression of genes required for steroidogenesis in the adrenal was unaffected by DBP. Together, these studies demonstrate that DBP initiates a rapid and dynamic change in gene expression in the fetal testis that likely plays a role in the reduction in steroidogenesis that is unique to the fetal testis relative to the steroidogenically active fetal adrenal.

Modulation of brain steroidogenesis by affecting transcriptional changes of steroidogenic acute regulatory (StAR) protein and cholesterol side chain cleavage (P450scc) in juvenile Atlantic salmon (Salmo salar) is a novel aspect of nonylphenol toxicity.

Gene expression patterns for key brain steroidogenic (StAR, P450scc, CYP11beta) and xenobiotic- and steroid-metabolizing enzymes (CYP1A1 and CYP3A) have been investigated in waterborne nonylphenol (5, 15, and 50 microg/ L) treated juvenile Atlantic salmon (Salmo salar), in addition to carrier vehicle (ethanol) exposed fish, sampled at different time intervals (0, 3, and 7 days) after exposure. Gene expression patterns were studied using the quantitative polymerase chain reaction (real-time PCR). Treatment of juvenile salmon with nonylphenol caused significant induction of steroidogenic acute regulatory (StAR) protein mRNA at day 7 postexposure in the group receiving 15 microg of nonylphenol/L. P450scc was first induced in the group treated with 5 microg of nonylphenol/L at day 7; thereafter, an apparent nonylphenol-concentration-dependent decrease in P450scc mRNA was observed. CYP11beta mRNA was significantly induced at day 3 after exposure to 5 betag of nonylphenol/L; thereafter, CYP11beta mRNA levels were inhibited below control levels in the 15 and 50 microg of nonylphenol/L groups at day 3. At day 7, significant induction of CYP11beta mRNA was observed only in the group exposed to 15 microg of nonylphenol/L. For CYP1A1 mRNA, apparent nonylphenol-concentration-dependent decreases were observed at day 7 postexposure. CYP3A mRNA was significantly induced by all nonylphenol exposure concentrations at day 7. When exposed groups were compared, CYP3A transcript was significantly induced between 5 and 15 microg of nonylphenol/ L, and decreased between 15 and 50 microg of nonylphenol/ L. The ethanol control showed a significant reduction of CYP3A mRNA at day 3 postexposure. The present study has demonstrated variations in three key steroidogenic proteins and xenobiotic- and steroid-metabolizing CYP isoenzyme gene transcripts in the brain of nonylphenol-exposed juvenile salmon. Therefore, the present study represents a novel aspect of neuroendocrine effects of nonylphenol in fish not previously demonstrated and should be studied in more detail.

Effects of a phytosterol mixture on male fish plasma lipoprotein fractions and testis P450scc activity.

Plant sterols (phytosterols) have been identified as one potential source of reproductive effects in fish living downstream of pulp mills. beta-Sitosterol, the predominant plant sterol in pulp mill effluent, has previously been shown to decrease plasma sex steroid and cholesterol levels and in vitro gonadal steroid production in fish. In this study, male brook trout (Salvelinus fontinalis) and goldfish (Carassius auratus) were exposed to a phytosterol mixture (72% beta-sitosterol) via Silastic intraperitoneal implants to help elucidate the mechanisms of action of phytosterols on steroid depression. As cholesterol is exogenously supplied for gonadal steroidogenesis, changes in plasma cholesterol fractions were examined. In male brook trout, low-density lipoprotein cholesterol and triglyceride levels decreased significantly, 43 and 50%, respectively, in phytosterol-treated fish. It is improbable, however, that these decreases are linked to depressed gonadal steroidogenesis in fish. The activity of P450scc, which converts cholesterol to pregnenolone (the first step in the steroidogenic pathway), was not affected in testis mitochondria isolated from brook trout or goldfish. Further investigation of the mechanisms of action of phytosterols is required.

The effect of glycerol on cytochrome P450scc (CYP11A1) spin state, activity, and hydration.

We examined the effects of glycerol, a stabilizing agent commonly used in cytochrome P450scc purification and analysis, on the spin state, catalytic activity, and molecular volume of the cytochrome. Glycerol induced a sigmoidal low-spin response. The binding of hydroxycholesterol reaction intermediates, but not cholesterol, increased the concentration of glycerol required for the spin transition to be 50% complete (K(1/2)). Glycerol weakened adrenodoxin binding to P450scc but had no effect on CO or 20alpha,22R-dihydroxycholesterol binding. Cytochrome P450scc activity was inhibited by glycerol with the K(1/2) for inhibition being substrate-dependent. The osmotic stress exerted by glycerol on P450scc resulted in decreases in P450scc molecular volume for both the transition to low spin state and the inhibition of activity. From this we determined that two dissociative water molecules are involved in the inhibition of activity with cholesterol as substrate and five or six dissociative waters are involved in the low-spin transition. The dehydration of P450scc by osmotic stress provides an explanation for the effects of glycerol on P450scc spin transition and activity.

Adrenocorticotrophic hormone (ACTH) stimulation of sheep fetal adrenal cortex can occur without increased expression of ACTH receptor (ACTH-R) mRNA.

In the present study, it was hypothesized that the adrenocorticotrophin hormone receptor (ACTH-R) would be up-regulated in the adrenal gland of the sheep fetus following infusion of physiological amounts of ACTH, as shown for adrenal cortical cells in culture. In chronically catheterized sheep, an intravenous infusion of ACTH(1-24) was given to 6 fetuses for 24 h at a rate of 0.5 microg h(-1), starting on Day 126 or 127 of gestation (term approximately 147 days). Four control fetuses received an infusion of vehicle (saline). Total RNA was extracted from the fetal adrenal glands by the guanidinium thiocyanate method. Expression of specific mRNAs was determined by ribonuclease protection assay using cRNA probes directed against: ACTH-R; the steroid enzymes side-chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17apha-hydroxylase (P450c17) and 21beta-hydroxylase (P450c21); and beta-actin. Ratios of mRNA expression to beta-actin mRNA expression (arbitrary units) were calculated to correct for differences in RNA quality between samples. The concentration (mean +/- SEM) of immunoreactive cortisol in fetal plasma was greater after ACTH infusion than after vehicle infusion (47 +/- 3 v. 13 +/- 2 ng mL(-1) respectively; P<0.001). Adrenal expression of P450scc and P450c21 mRNA increased after ACTH infusion (P<0.05), whereas expression of P450c17 and 3beta-HSD mRNA was unchanged. There was no difference in ACTH-R mRNA expression between ACTH- and vehicle-infused fetuses (254 +/- 48 v. 305 +/- 76 arbitrary units respectively). It was concluded that ACTH is able to increase plasma cortisol concentrations in the sheep fetus by up-regulating cortisol synthesis in the adrenal gland, but that in vivo this does not require up-regulation of ACTH-R mRNA.