StAR mRNA Research Articles

Di-(2-ethylhexyl)-phthalate disrupts pituitary and testicular hormonal functions to reduce sperm quality in mature goldfish

Di-(2-ethylhexyl) phthalate (DEHP) interferes with male reproductive endocrine system in mammals, however its effects on fish reproduction are largely unknown. We evaluated sperm quality and investigated reproductive endocrine system in mature goldfish (Carassius auratus) exposed to nominal 1, 10, and 100μg/L DEHP. To examine DEHP estrogenic activity, one group of goldfish was exposed to 17β-estradiol (5μg/L E2) for comparison. Following 30d of exposure, sperm production was decreased and suppressed in DEHP and E2 treated goldfish, respectively. Sperm motility and velocity were decreased in goldfish exposed to 100 and 10μg/L DEHP at 15s post-sperm activation, respectively. Compared to control, 11-ketotestosterone (11-KT) levels were decreased at 10 and 1μg/L DEHP at day 15 and 30, respectively. In E2 treated goldfish, 11-KT levels were decreased compared to control during the period of exposure. E2 levels were increased in goldfish exposed to E2, but remained unchanged in DEHP treated goldfish during the period of exposure. StAR mRNA levels encoding regulator of cholesterol transfer to steroidogenesis were decreased in DEHP and E2 treated goldfish following 15 and 30d of exposure, respectively. Luteinizing hormone (LH) levels were decreased in DEHP and E2 treated goldfish following 15 and 30d of exposure, respectively. In DEHP treated goldfish, gnrh3, kiss1 and its receptor (gpr54) mRNA levels did not change during the experimental period. In E2 treated goldfish, gnrh3 mRNA levels were decreased at day 7, but kiss1 and gpr54 mRNA levels were increased at day 30 of exposure. The mRNA levels of genes encoding testicular LH and androgen receptors remained unchanged in DEHP and E2 treated goldfish. In contrast to E2 treated goldfish, vitellogenin production was not induced in DEHP treated goldfish and mRNA levels of genes with products mediating estrogenic effects remained unchanged or decreased. In conclusion, DEHP interferes with testis and pituitary hormonal functions to reduce sperm quality in goldfish and does not exhibit estrogenic activity.

Adiponectin influences progesterone production from MA-10 Leydig cells in a dose-dependent manner.

Obesity in men is associated with lower testosterone levels, related to reduced sperm concentration and the development of various diseases with aging. Hormones produced by the adipose tissue may have influences on both metabolism and reproductive function. Among them, the production and secretion of adiponectin is inversely correlated to total body fat. Adiponectin receptors (AdipoR1 and AdipoR2) have been found to be expressed in testicular Leydig cells (producing testosterone). Since StAR and Cyp11a1 are essential for testosterone synthesis and adiponectin has been shown to regulate StAR mRNA in swine granulosa cells, we hypothesized that adiponectin might also regulate these genes in Leydig cells. Our objective was to determine whether adiponectin regulates StAR and Cyp11a1 genes in Leydig cells and to better define its mechanisms of action. Methods used in the current study are qPCR for the mRNA levels, transfections for promoter activities, and enzyme-linked immunosorbent assay for the progesterone concentration. We have found that adiponectin cooperates with cAMP-dependent stimulation to activate StAR and Cyp11a1 mRNA expressions in a dose-dependent manner in MA-10 Leydig cells as demonstrated by transfection of a luciferase reporter plasmid. These results led to a significant increase in progesterone production from MA-10 cells. Thus, our data suggest that high doses of adiponectin typical of normal body weight may promote testosterone production from Leydig cells.

Atrazine enhances progesterone production through activation of multiple signaling pathways in FSH-stimulated rat granulosa cells: evidence for premature luteinization.

Premature luteinization is a possible cause of infertility in women. It is currently unknown whether environmental chemicals can induce changes associated with premature luteinization. Using rat granulosa cells (GC) in vitro, we demonstrated that exposure to atrazine (ATR), a widely used herbicide, causes GC phenotype that resembles that of human premature luteinization. At the end of the 48-h stimulation with FSH, ATR-exposed GC showed (1) higher levels of progesterone, (2) overexpression of luteal markers (Star and Cyp11a1), and (3) an increase in progesterone:estradiol ratio above 1. Mechanistic experiments were conducted to understand the signaling events engaged by ATR that lead to this phenotype. Western blot analysis revealed prolonged phosphorylation of protein kinase B (AKT) and cAMP response element-binding protein (CREB) in ATR- and FSH-exposed GC. An increased level of ERK1/2-dependent transcriptional factor CCATT/enhancer-binding protein beta (CEBPB) was observed after 4 h of ATR exposure. Inhibitors of PI3K (wortmannin) and MEK (U0126) prevented ATR-induced rise in progesterone level and expression of luteal markers in FSH-stimulated GC. Atrazine intensified AKT and CEBPB signaling and caused Star overexpression in forskolin-stimulated GC but not in epidermal growth factor (EGF)-stimulated GC. In the presence of rolipram, a specific inhibitor of phosphodiesterase 4 (PDE4), ATR was not able to further elevate AKT phosphorylation, CEBPB protein level, and Star mRNA in FSH-stimulated GC, suggesting that ATR inhibits PDE4. Overall, this study showed that ATR acts as a FSH sensitizer leading to enhanced cAMP, AKT, and CEBPB signaling and progesterone biosynthesis, which promotes premature luteinization phenotype in GC.

Autocrine positive regulatory feedback of glucocorticoid secretion: Glucocorticoid receptor directly impacts H295R human adrenocortical cell function

Glucocorticoid receptor (GR), a ubiquitous transcriptional factor, regulates target gene expression upon activation by glucocorticoids, notably cortisol, a corticosteroid hormone synthesized in the adrenal cortex. We thus hypothesized that both GR and cortisol might be involved in the regulation of adrenal physiology and steroidogenesis in an autocrine manner.In a cortisol-secreting human adrenocortical cell line (H295R), the GR-dependent signaling pathway was pharmacologically modulated either by dexamethasone (DEX), a GR agonist or by RU486, a GR antagonist, or was knocked-down by small interfering RNA strategy (SiRNA). We showed that GR activation, elicited by 48 h exposure to DEX, exerts a global positive regulatory effect on adrenal steroidogenesis as revealed by a 1.5- to 2-fold increase in cortisol, 11-deoxycortisol and 17-hydroxyprogesterone secretion associated with a significant enhanced expression of steroidogenesis factors such as StAR, CYP11A1, CYP21A2 and CYP11B1. In sharp contrast, RU486 treatment exerted opposite effects by decreasing both steroid production and expression of these steroidogenic factors. Likewise, GR repression by SiRNA also significantly reduced StAR, CYP11A1, and CYP11B1 mRNA levels. Interestingly, RU486 resulted in a significant CYP21A2 enzymatic blockade as demonstrated by a massive increase in 17-hydroxyprogesterone concentrations in RU486-treated H295R cell supernatants, while cortisol and 11-deoxycortisol secretions were reduced by more than 60%. Consistently, we also demonstrated that metabolic conversion of 17-hydroxyprogesterone into 11-deoxycortisol onto H295R cells was drastically blunted in the presence of RU 486. Finally, steady state levels of MC2R transcripts encoding for the ACTH receptor were significantly induced by DEX, unlikely through a direct GR-mediated transcriptional activation as opposed to CYP11A1 and FKBP5 target genes. These results could account for a higher glucocorticoid-elicited ACTH sensitivity of adrenocortical cells.Our study identifies a positive ultra-short regulatory loop exerted by GR on steroidogenesis in H295R cells, thus supporting a complex intra-adrenal GR-mediated feedback, likely relevant for human adrenocortical pathologies.

Mitochondrial fusion and ERK activity regulate steroidogenic acute regulatory protein localization in mitochondria.

The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR.

Embryo number and periconceptional undernutrition in the sheep have differential effects on adrenal epigenotype, growth, and development.

Exposure to poor maternal nutrition around the time of conception results in an early prepartum activation of the fetal pituitary-adrenal axis and in increased adrenal growth and stress response after birth associated with epigenetic changes in a differentially methylated region (DMR) of adrenal IGF2/H19. We have determined the effects of maternal undernutrition during the periconceptional period (PCUN: 70% of control intake from 60 days before until 6 days after conception) and early preimplantation period (PIUN: 70% of control intake for 6 days after conception) on fetal plasma ACTH and cortisol concentrations and fetal adrenal ACTHR, StAR, 3βHSD, CYP11B, CYP17, TGFβ1, IGF1, IGF1R, IGF2, and IGF2R mRNA expression and the methylation level of sites within the DMRs of IGF2/H19 and IGF2R in the adrenal of twin and singleton fetuses at 136-138 days gestation. Being a twin resulted in a delayed prepartum increase in fetal ACTH and in a lower cortisol response to CRH in the control but not PCUN and PIUN groups. PCUN, but not PIUN, resulted in an increase in adrenal weight and CYP17 expression in singletons, a decrease in adrenal IGF2 expression in singletons, and an increase in adrenal IGF2R expression in both twins and singletons. IGF2/H19 and IGF2R DMR methylation levels and ACTHR expression were lower in the twin adrenal. Thus, exposure of the oocyte and embryo to maternal undernutrition or to the environment of a twin pregnancy have differential effects on epigenetic and other factors that regulate fetal adrenal growth and IGF2 and IGF2R expression.

Subdominant hierarchical ovarian follicles are needed for steroidogenesis and ovulation in laying hens (Gallus domesticus)

Ovarian follicle development in avian species is characterized by a strict hierarchical arrangement. The hierarchical follicles secrete progesterone, which induces the LH surge, but the capacity to produce other steroids decreases with development. Our aim was to evaluate the complementary action of subdominant follicles (F4–F6) on ovulation and steroidogenesis of the preovulatory follicles (F1–F3) in domestic laying hens. The first study included four groups: control (C); sham-operated (SO); large hierarchical follicles (LHF) from which F4–F6 follicles were extracted; and subdominant hierarchical follicles (SHF) from which F1–F3 follicles were extracted. Blood samples were collected every 2h from 12h before estimated ovoposition until 2h after ovoposition. Egg laying continued at the same rates in C and SO hens, with normal preovulatory surges of oestradiol, testosterone, progesterone and LH. In contrast, in LHF and SHF groups, ovoposition was blocked; oestradiol concentrations were not affected; but no preovulatory surges of testosterone, progesterone or LH were seen. Further, the testosterone surge was required for the occurrence of progesterone and LH surges. In the second study StAR and steroidogenic enzyme mRNA expression was evaluated within F1–F3 follicles from a LHF group and C-14 and C-8 controls groups, in which follicles were collected 14h and 8h before expected ovoposition, respectively. Extraction of F4–F6 follicles caused a significant reduction in StAR and 3β-HSD expressions within theca, but not in granulosa cells. In conclusion, subdominant hierarchical follicles (F4–F6) are required for the preovulatory release of testosterone, progesterone and LH, which are highly inter-correlated.

In vitro exposure of Leydig cells to an environmentally relevant mixture of organochlorines represses early steps of steroidogenesis.

Leydig cell steroidogenesis is mainly regulated by LH via increased cAMP production leading to STAR protein activation. STAR is essential for cholesterol shuttling inside mitochondria where steroidogenesis is initiated. Accumulating evidence suggest that persistent organochlorine compounds disrupt testicular function, but the mechanism of action remains poorly characterized. Here we report that in vitro exposure of MA-10 and MLTC-1 Leydig cells to an environmentally relevant mixture of 15 organochlorines impairs steroidogenesis. While having no effect on cell viability and basal steroid production, the organochlorine mixture caused a 50% decrease in cAMP-induced progesterone production. The mixture also reduced cAMP-induced 30 kDa STAR protein by 50% while having no effect on basal STAR protein. Basal or cAMP-induced Star mRNA levels and promoter activity were unaffected by the mixture, indicating that the organochlorine mixture acted at the translational/posttranslational level. Further supporting this is the fact that in COS-7 cells overexpressing STAR, the organochlorine mixture caused a decrease in the 30 kDa form of STAR and an accumulation of the 37 kDa forms. In addition to STAR, we found that the organochlorine mixture also decreases the levels of CYP11A1 and ADXR, two proteins essential for the conversion of cholesterol into pregnenolone. In conclusion, our data show that organochlorine exposure disrupts Leydig cell function by targeting different components of the steroidogenic pathway.

Different responses to soy and estradiol in the reproductive system of prepubertal male rats and neonatal male pigs (373.5)

Concerns have been raised regarding the safety of soy infant formula based on phytochemical components such as genistein, structurally similar to estradiol (E2). To examine potential estrogenic actions on male development, we fed weanling male rats casein‐based or soy protein isolate (SPI)‐based diets (PND21‐PND33) and neonatal male piglets cow’s milk or soy infant formulas (PND2‐PND21) or treated the animals with E2. E2 reduced testis and prostate weight and serum testosterone (P<0.05) in both species. Soy diet had no effects. In the piglet, E2 increased testis tubule area, Sertoli cell and germ cell number (P<0.05) and decreased expression of CYP19 and StAR mRNA. Soy increased germ cell number (P<0.05). The major effect of E2 is to change gene expression in reproductive tissues. E2 changed expression of 2645 genes in rat testis vs. 312 genes changed by SPI >1.5‐fold. 74% of the SPI genes were common to E2. In contrast, 353 genes were changed by E2 and only 116 by feeding soy formula in piglet testis with only 34 genes (29%) in common. These data suggest that soy diets are not estrogenic in the male reproductive system but may interact with a small subset of estrogen‐regulated genes in a species and developmental stage‐specific manner.Grant Funding Source: USDA‐ARS CRIS 6251‐51999‐007‐04S

Levonorgestrel exposure to fathead minnows (Pimephales promelas) alters survival, growth, steroidogenic gene expression and hormone production

Human pharmaceuticals are commonly detected in the environment. Concern over these compounds in the environment center around the potential for pharmaceuticals to interfere with the endocrine system of aquatic organisms. The main focus of endocrine disruption research has centered on how estrogenic and androgenic compounds interact with the endocrine system to elicit reproductive effects. Other classes of compounds, such as progestins, have been overlooked. Recently, studies have investigated the potential for synthetic progestins to impair reproduction and growth in aquatic organisms. The present study utilizes the OECD 210 Early-life Stage (ELS) study to investigate the impacts levonorgestrel (LNG), a synthetic progestin, on fathead minnow (FHM) survival and growth. After 28 days post-hatch, survival of larval FHM was impacted at 462ng/L, while growth was significantly reduced at 86.9ng/L. Further analysis was conducted by measuring specific endocrine related mRNA transcript profiles in FHM larvae following the 28 day ELS exposure to LNG. Transcripts of 3β-HSD, 20β-HSD, CYP17, AR, ERα, and FSH were significantly down-regulated following 28d exposure to 16.3ng/L LNG, while exposure to 86.9ng/L significantly down-regulated 3β-HSD, 20β-HSD, CYP19A, and FSH. At 2392ng/L of LNG, a significant down-regulation occurred with CYP19A and ERβ transcripts, while mPRα and mPRβ profiles were significantly induced. No significant changes occurred in 11β-HSD, CYP11A, StAR, LHβ, and VTG mRNA expression following LNG exposure. An ex vivo steroidogenesis assay was conducted with sexually mature female FHM following a 7 day exposure 100ng/L LNG with significant reductions observed in pregnenolone, 17α,20β-dihydroxy-4-pregnen-3-one (17,20-DHP), testosterone, and 11-ketotestosterone. Together these data suggest LNG can negatively impact FHM larval survival and growth, with significant alterations in endocrine related responses.

Dicamba affects sex steroid hormone level and mRNA expression of related genes in adult rare minnow (Gobiocypris rarus) at environmentally relevant concentrations

Dicamba is a benzoic acid herbicide that has been detected in surface and ground water. The herbicide has been shown to have cytogeneic and DNA damaging effects and to cause organ damage in mammals; however, little is known about the endocrine disrupting effects of dicamba in fish. In this study, histological changes, plasma vitellogenin (VTG) and sex hormone levels, and mRNA expression of sex steroid hormone-related genes were determined in adult rare minnow exposed to environmentally relevant concentrations of dicamba (0, 0.05, 0.5, 5, and 50 μg/L) for 40 days. The results showed inhibition of spermatogenesis in male testes and ovarian degeneration in females. Plasma 17β-estradiol (E2) levels were significantly increased in both genders, and plasma VTG levels were significantly increased in males (p<0.05). These results indicate that sex hormone homeostasis and normal reproduction of fish could be affected by dicamba. Moreover, the mRNA levels of vtg were significantly upregulated in the livers and gonads of both male and female rare minnows (p < 0.05). The downregulation of cytochrome P450c19a (cyp19a) and steroidogenic acute regulatory protein (star) mRNA levels, and the upregulation of cytochrome P450c17 (cyp17) mRNA levels were observed in the livers and ovaries (p<0.05). The results of the mRNA analysis suggest that changes in steroid hormone-related gene expression could serve as a regulatory mechanism to maintain sex hormone homeostasis. Overall, dicamba exposure could result in histological lesions, plasma VTG increases, changes in sex hormone levels, and alterations of hormone-related gene expression. Therefore, dicamba should be considered to be a potential endocrine disruptor.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on secretion of steroids and STAR, HSD3B and CYP19A1 mRNA expression in chicken ovarian follicles

The aim of the study was to investigate the in vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on steroid hormone secretion by chicken ovarian follicles and mRNA expression of genes involved in steroids synthesis. In the first in vitro experiment, white (WF) and yellowish (YF) follicles and fragments of the theca (TL) and granulosa (GL) layers of the 3 largest yellow preovulatory follicles (F3–F1) were incubated in a medium supplemented with TCDD (0.01–100nM). In the second experiment, they were incubated in a medium with TCDD (10nM), ovine LH (10ng/mL; oLH) or a combination of oLH (10ng/mL) and TCDD (10nM). It was found that TCDD decreased estradiol (E2) secretion by WF and the TL of all preovulatory follicles, testosterone (T) secretion by WF, YF, and the TL of F2 and F1 follicles, and progesterone (P4) secretion by the GL of the preovulatory follicles. It also reduced oLH-stimulated E2 and P4 secretion by all examined follicles and T by WF. Real-time qPCR revealed that TCDD affected basal and oLH-stimulated expression of STAR, HSD3B and CYP19A1 mRNAs in all investigated ovarian follicles. In conclusion, the data obtained indicate that TCDD inhibits sex steroids secretion from chicken ovarian follicles. The effects of TCDD depend on its concentration and the stage of follicle maturation, and are associated with modulation of STAR, HSD3B and CYP19A1 mRNAs expression. These results indicate that the exposure of the laying hen to TCDD by influence of ovarian steroidogenesis may impair the selection of white follicles to preovulatory hierarchy and disturb their growth and preovulatory maturation.

Excessive levels of diverse phytoestrogens can modulate steroidogenesis and cell migration of KGN human granulosa-derived tumor cells

Phytoestrogens are plant-derived estrogen-like compounds that are increasingly used for their suggested health promoting properties, even by healthy, young women. However, scientific concerns exist regarding potential adverse effects on female reproduction. In this study, naringenin (NAR), 8-prenylnaringenin (8-PN), genistein (GEN), coumestrol (COU), quercetin (QUE) and resveratrol (RSV) up-regulated steroidogenic acute regulatory protein (StaR) mRNA levels in KGN human granulosa-like tumor cells. Most of the phytoestrogens tested also increased CYP19A1 (aromatase) mRNA levels via activation of ovary-specific I.3 and II promoters. Yet, only NAR (3 and 10 μM), COU (10 and 30 μM) and QUE (10 μM) also statistically significantly induced aromatase activity in KGN cells after 24 h. 8-PN, aromatase inhibitor letrozole and estrogen receptor antagonist ICI 182,780 concentration-dependently inhibited aromatase activity with IC50 values of 8 nM, 10 nM and 72 nM, respectively. Co-exposure with ICI 182,780 (0.1 μM) statistically significantly attenuated the induction of aromatase activity by QUE and COU, but not NAR. Cell cycle status and proliferation of KGN cells were not affected by any of the phytoestrogens tested. Nonetheless, the migration of KGN cells was significantly reduced with approximately 30% by COU, RSV and QUE and 46% by GEN at 10 μM, but not NAR and 8-PN. Our results indicate that phytoestrogens can affect various pathways in granulosa-like cells in vitro at concentrations that can be found in plasma upon supplement intake. This implies that phytoestrogens may interfere with ovarian function and caution is in place regarding the use of supplements with high contents of phytoestrogens.

Antenatal glucocorticoid exposure enhances the inhibition of adrenal steroidogenesis by leptin in a sex-specific fashion

Antenatal treatment with glucocorticoids (GC) poses long-lasting effects on endocrine and cardiovascular function. Given that leptin attenuates adrenal function and the reported sex differences in plasma leptin concentration, we hypothesized that antenatal GC will affect leptin levels and leptin modulation of adrenal function in a sex-specific manner. Pregnant sheep were randomly given betamethasone or vehicle at 80 days of gestational age, and offspring were allowed to deliver at term. Adrenocortical cells (ADC) were studied from male and female animals at 1.5 yr of age. Plasma leptin was increased 66% in male and 41% in female GC-treated animals (P < 0.05), but adrenal leptin mRNA was increased only in GC-treated males (P < 0.05). Whereas mRNA expression of adrenal leptin receptor isoforms showed sex (Ob-Ra and Ob-Rb) and treatment-dependent (Ob-Rb) differences, protein expression remained unchanged. GC-treated females showed greater plasma cortisol and greater ACTH-stimulated cortisol production (P < 0.05) in ADC. Leptin exerted a greater inhibitory effect on basal and stimulated cortisol by ADC from GC-treated males (P < 0.05), with no differences in females. Similarly, greater inhibitory effects on basal and ACTH-stimulated StAR and ACTH-R mRNA expression by leptin were observed in cells from GC males (P < 0.05), with no changes in females. Persistent effects of antenatal GC on leptin levels and leptin modulation of adrenal function are expressed in a sex-specific manner; males are more sensitive than females to the inhibitory influences of leptin on adrenal function, and this effect appears to be mediated by a greater inhibition of StAR and ACTH-R expression in adrenals of adult GC-treated males.

Triazine Herbicides and Their Chlorometabolites Alter Steroidogenesis in BLTK1 Murine Leydig Cells

The triazine herbicides, atrazine (ATR), simazine (SIM), propazine (PRO), terbuthylazine (TBA), and their chlorinated metabolites have been implicated in the etiology of testicular dysgenesis by altering steroidogenesis. To further investigate their effects on testosterone biosynthesis, BLTK1 cells were used to evaluate steroid hormone levels and genome-wide gene expression. BLTK1 cells are a novel murine Leydig cell line possessing an intact steroidogenic pathway with constitutive low basal testosterone (T) levels that can be induced by recombinant human chorionic gonadotropin (rhCG). Triazines (ATR, SIM, PRO, and TBA) and their chlorometabolites (DEA, DIA, and DACT) induced concentration-dependent (1, 3, 10, 30, 100, 300, and 600 µM) increases in progesterone (P) and T levels relative to solvent control at 24h. Temporal analysis (300 µM at 1, 2, 4, 8, 12, 24, or 48h) elicited comparable P and T profiles by all compounds with varying efficacies (ATR > TBA > PRO > DEA > DIA > DACT >> SIM) that were similar to rhCG. ATR and TBA elicited time- and concentration-dependent induction of Star, Hsd3b6, and Hsd17b3 mRNA levels, whereas Hsd3b1, Cyp17a1, and Srd5a1 mRNA expression was repressed. PRO elicited similar albeit weaker effects, whereas SIM had negligible effects consistent with their induction of P and T levels. Whole-genome microarrays identified 797 differentially regulated genes elicited by 300 µM ATR, occurring primarily at later time points (> 12h) with overrepresented functions associated with steroidogenesis and cholesterol metabolism. These results indicate that changes in P and T levels can be partially attributed to triazine-elicited alterations in steroidogenic gene expression.

Sexual Dimorphism of Steroidogenesis Regulated by GnIH in the Goldfish, Carassius auratus1

Gonadotropin-inhibitory hormone (GnIH) has been shown to inhibit reproduction in several species. GnIH suppresses gonadotropin synthesis/release at the hypothalamic and pituitary levels; however, increasing evidence suggests that GnIH has a putative function in the gonad. In this study, we demonstrated that GnIH receptors localize to the ovary and testis in goldfish. In situ hybridization illustrated that goldfish GnIHRs were localized exclusively to the oocytes before the cortical alveolus stage and to the interstitial tissue to the testis. Implantation of goldfish GnIH peptides did not affect the serum estradiol levels in female goldfish, but it did enhance the serum testosterone levels in males. Conversely, injecting goldfish GnIH peptides increased the expression of StAR and 3bHSD mRNA and decreased the expression of CYP19 mRNA significantly in the testis, but these genes remained unchanged in the ovary. In addition, goldfish GnIH peptides not only increased the expression of StAR and 3bHSD and decreased CYP19 mRNA, but they also increased the expression of FSHR and LHR mRNA in testicular cells. However, they did not affect the expression of these genes in ovarian cells in vitro. Thus, we suggest that GnIH may contribute to the sexual dimorphism of steroidogenesis in goldfish.

Effects of age on ACTH, corticosterone, glucose, insulin, and mRNA levels during intermittent hypoxia in the neonatal rat

Apnea, the temporary cessation of respiratory airflow, is a common cause of intermittent hypoxia (IH) in premature infants. We hypothesized that IH elicits a stress response and alters glucose homeostasis in the neonatal rat. Rat pups were studied on postnatal day (PD) 2, 8, 10, 12, and 14. Pups were exposed to normoxia (control) or six cycles consisting of 30-s exposures to hypoxia (FiO2 = 3%) over a 60-min period. Blood samples were obtained at baseline, after the third cycle (~30 min), and after the sixth cycle (~60 min). Tissue samples were collected following the sixth cycle. Plasma ACTH, corticosterone, glucose, and insulin were analyzed at all ages. Hypothalamic, pituitary, and adrenal mRNA expression was evaluated by quantitative PCR in PD2, PD8, and PD12 pups. Exposure to IH elicited significant increases in plasma ACTH and corticosterone at all ages studied. The largest increase in corticosterone occurred in PD2 pups, despite only a very small increase in plasma ACTH. This ACTH-independent increase in corticosterone in PD2 pups was associated with increases in adrenal Ldlr and Star mRNA expression. Additionally, IH caused hyperglycemia and hyperinsulinemia at all ages. We conclude that IH elicits a significant pituitary-adrenal response and significantly alters glucose homeostasis. Furthermore, the quantitative and qualitative characteristics of these responses depend on developmental age.

Effects of compound X, a novel potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase, on the adrenal gland of rats

To investigate the adrenal toxicity of a novel inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase, compound X (CX), histopathological examinations, fat staining, adrenal cholesterol measurement, blood biochemistry, plasma corticosterone and ACTH measurement, ACTH-stimulation assay, and adrenal gene-expression analyses were done in rats in repeated-dose studies (experiment 1: 0, 3, 10, 30 and 150mg/kg for 4, 8, 15 and 28 days; experiment 2: 0, 3, 10,30 and 150mg/kg for 28 days; experiment 3: 0, 10, 30, 100 and 300mg/kg for 28 days). CX induced morphologic changes such as vacuolation and hypertrophy in the zona fasciculata (ZF) at ≥10mg/kg, and eosinophilic changes in the ZF at 150mg/kg. Vacuolation decreased in a dose-dependent manner and was replaced by eosinophilic changes. Inflammatory and fibrous changes were observed at ≥30mg/kg. These changes were expressed at early stages of dosing and were not exacerbated by extension of the administration period. Oil-red-O/Filipin staining showed depletion of cholesterol ester in dose-dependent manner and enabled adrenal cholesterol measurement. Filipin staining also revealed vacuoles to be composed of cholesterol esters. No significant changes were observed during the dosing period of CX for plasma corticosterone and ACTH levels. Gene-expression analyses showed up-regulation of Star and Abca1 mRNA levels at 300mg/kg. In conclusion, CX induced adrenal toxicity, but CX did not influence adrenocortical functions, and exacerbation of adrenal toxicities by extension of the administration period was not observed. Up-regulation of genes related to the transport of FC, such as Star and Abca1, were observed in CX groups, and these genes may be involved in the maintenance of adrenal structure and function in rats given CX.

The Anti-Epileptic Drug Valproic Acid (VPA) Inhibits Steroidogenesis in Bovine Theca and Granulosa Cells In Vitro

Valproic acid (VPA) is used widely to treat epilepsy and bipolar disorder. Women undergoing VPA treatment reportedly have an increased incidence of polycystic ovarian syndrome (PCOS)-like symptoms including hyperandrogenism and oligo- or amenorrhoea. To investigate potential direct effects of VPA on ovarian steroidogenesis we used primary bovine theca (TC) and granulosa (GC) cells maintained under conditions that preserve their ‘follicular’ phenotype. Effects of VPA (7.8–500 µg/ml) on TC were tested with/without LH. Effects of VPA on GC were tested with/without FSH or IGF analogue. VPA reduced (P<0.0001) both basal (70% suppression; IC50 67±10 µg/ml) and LH-induced (93% suppression; IC50 58±10 µg/ml) androstenedione secretion by TC. VPA reduced CYP17A1 mRNA abundance (>99% decrease; P<0.0001) with lesser effects on LHR, STAR, CYP11A1 and HSD3B1 mRNA (<90% decrease; P<0.05). VPA only reduced TC progesterone secretion induced by the highest (luteinizing) LH dose tested; TC number was unaffected by VPA. At higher concentrations (125–500 µg/ml) VPA inhibited basal, FSH- and IGF-stimulated estradiol secretion (P<0.0001) by GC without affecting progesterone secretion or cell number. VPA reversed FSH-induced upregulation of CYP19A1 and HSD17B1 mRNA abundance (P<0.001). The potent histone deacetylase (HDAC) inhibitors trichostatin A and scriptaid also suppressed TC androstenedione secretion and granulosal cell oestrogen secretion suggesting that the action of VPA reflects its HDAC inhibitory properties. In conclusion, these findings refute the hypothesis that VPA has a direct stimulatory action on TC androgen output. On the contrary, VPA inhibits both LH-dependent androgen production and FSH/IGF-dependent estradiol production in this in vitro bovine model, likely by inhibition of HDAC.

Fluorochemicals used in food packaging inhibit male sex hormone synthesis

Polyfluoroalkyl phosphate surfactants (PAPS) are widely used in food contact materials (FCMs) of paper and board and have recently been detected in 57% of investigated materials. Human exposure occurs as PAPS have been measured in blood; however knowledge is lacking on the toxicology of PAPS. The aim of this study was to elucidate the effects of six fluorochemicals on sex hormone synthesis and androgen receptor (AR) activation in vitro. Four PAPS and two metabolites, perfluorooctanoic acid (PFOA) and 8:2 fluorotelomer alcohol (8:2 FTOH) were tested.Hormone profiles, including eight steroid hormones, generally showed that 8:2 diPAPS, 8:2 monoPAPS and 8:2 FTOH led to decreases in androgens (testosterone, dehydroepiandrosterone, and androstenedione) in the H295R steroidogenesis assay. Decreases were observed for progesterone and 17-OH-progesterone as well. These observations indicated that a step prior to progestagen and androgen synthesis had been affected. Gene expression analysis of StAR, Bzrp, CYP11A, CYP17, CYP21 and CYP19 mRNA showed a decrease in Bzrp mRNA levels for 8:2 monoPAPS and 8:2 FTOH indicating interference with cholesterol transport to the inner mitochondria. Cortisol, estrone and 17β-estradiol levels were in several cases increased with exposure. In accordance with these data CYP19 gene expression increased with 8:2 diPAPS, 8:2 monoPAPS and 8:2 FTOH exposures indicating that this is a contributing factor to the decreased androgen and the increased estrogen levels.Overall, these results demonstrate that fluorochemicals present in food packaging materials and their metabolites can affect steroidogenesis through decreased Bzrp and increased CYP19 gene expression leading to lower androgen and higher estrogen levels.