Gonadal Steroidogenesis Research Articles

Effect of the NBD-group position on interaction of fluorescently-labeled cholesterol analogues with human steroidogenic acute regulatory protein STARD1

Steroidogenic acute regulatory protein (StAR, STARD1) is a key factor of intracellular cholesterol transfer to mitochondria, necessary for adrenal and gonadal steroidogenesis, and is an archetypal member of the START protein family. Despite the common overall structural fold, START members differ in their binding selectivity toward various lipid ligands, but the lack of direct structural information hinders complete understanding of the binding process and cholesterol orientation in the STARD1 complex in particular. Cholesterol binding has been widely studied by commercially available fluorescent steroids, but the effect of the fluorescent group position on binding remained underexplored. Here, we dissect STARD1 interaction with cholesterol-like steroids bearing 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group in different positions, namely, with 22-NBD-cholesterol (22NC), 25-NBD-cholesterol (25NC), 20-((NBDamino)-pregn-5-en-3-ol (20NP) and 3-(NBDamino)-cholestane (3NC). While being able to stoichiometrically bind 22NC and 20NP with high fluorescence yield and quantitative exhaustion of fluorescence of some protein tryptophans, STARD1 binds 25NC and 3NC with much lower affinity and poor fluorescence response. In contrast to 3NC, binding of 20NP leads to STARD1 stabilization and substantially increases the NBD fluorescence lifetime. Remarkably, in terms of fluorescence response, 20NP slightly outperforms commonly used 22NC and can thus be used for screening of various potential ligands by a competition mechanism in the future.

Social factors and aromatase gene expression during adult male-to-female sex change in captive leopard grouper Mycteroperca rosacea

Social factors and aromatase gene expression in the leopard grouper Mycteroperca rosacea was studied when captive fish were separated by sex during the reproductive (April–June) and post-reproductive (July–September) seasons. Monosex females, monosex males, and mixed-sex, held in social sextet units were analyzed for sex steroids throughout confinement. At the end of the experiment, the gonad-sex was defined by histology, and gonad and brain aromatase gene expressions were quantified. Only males held in the monosex social units changed sex. Histology showed one male remained unchanged, six were found in a transitional sexual stage, in which two had intersex-predominantly-testes, and four had a more defined intersex ovo-testes pattern, and 11 were immature de novo females (neofemales). Neofemales and most intersex fish did not survive. In spring, 11-ketosterone showed a specific male profile, which suggests that male-to-female sex change was not triggered during the reproductive season. The low steroid levels in summer made it impossible to associate the sex change to a gonad hormonal shift; in September, gonad aromatase gene expression was not significantly different among groups. However, brain aromatase expression in intersex fish was significantly higher than monosex females, mixed-sex females, and neofemale groups. These results suggest that in the absence of female hormonal compounds, and at a time when male gonad steroidogenesis was diminished, the brain mediated male-to-male social-behavioral interactions, including stress, by increasing aromatization, resulting in derived intersex-male, which triggered more aromatization, followed by a sex change.

Urinary Cadmium Excretion Is Associated With Increased Synthesis of Cortico- and Sex Steroids in a Population Study.

Urinary cadmium (Cd) excretion is associated with cancer and cardiovascular morbidity. A potential mechanism could be disturbance of steroidogenesis in gonads and adrenal glands. We tested whether urinary excretion of Cd is correlated with that of cortico- and sex steroid metabolites in the general adult population. The Swiss Kidney Project on Genes in Hypertension is a multicentric, family-based population study. Urinary excretions of steroid hormone metabolites and Cd were measured with separate day and night collections. Associations were analyzed by mixed linear models. Urinary Cd and testosterone excretions in men were significantly correlated (respective day and night β values [standard error (SE)], 1.378 [0.242], P < 0.0005; and 1.440 [0.333], P < 0.0005), but not in women [0.333(0.257), P = 0.2; and 0.674 (0.361), P = 0.06]. Urinary Cd and cortisol excretions were positively associated in both sexes [day: β = 0.475 (SE, 0.157), P = 0.0025, and 0.877 (SE, 0.194), P < 0.0005, respectively; night: β = 0.875 (SE, 0.253), P < 0.0005 and 1.183 (SE, 0.277), P = 0.00002, respectively]. Cd excretion was correlated with mineralocorticoid metabolites excretion, except tetrahydroaldosterone, in both sexes (P < 0.01). There was an independent effect of Cd on sex hormone and corticosteroid synthesis and an interdependent effect on gluco- and mineralcorticoid production. Our findings provide evidence for a global stimulating effect on steroid synthesis already at low-dose Cd exposure. These findings might explain the association of Cd with diseases such as steroid-sensitive cancers or metabolic disorders.

Effects of different dietary DHA:EPA ratios on gonadal steroidogenesis in the marine teleost, tongue sole (Cynoglossus semilaevis).

The present study was conducted to investigate the effects of dietary DHA and EPA on gonadal steroidogenesis in mature females and males, with a feeding trial on tongue sole, a typical marine teleost with sexual dimorphism. Three experimental diets differing basically in DHA:EPA ratio, that is, 0·68 (diet D:E-0·68), 1·09 (D:E-1·09) and 2·05 (D:E-2·05), were randomly assigned to nine tanks of 3-year-old tongue sole (ten females and fifteen males in each tank). The feeding trail lasted for 90 d before and during the spawning season. Fish were reared in a flowing seawater system and fed to apparent satiation twice daily. Compared with diet D:E-0·68, diet D:E-1·09 significantly enhanced the oestradiol production in females, whereas diet D:E-2·05 significantly enhanced the testosterone production in males. In ovaries, diet D:E-1·09 induced highest mRNA expression of follicle-stimulating hormone receptor (FSHR), steroidogenic acute regulatory protein, 17α-hydroxylase (P450c17) and 3β-hydroxysteroid dehydrogenase (3β-HSD). In testes, diet 2·05 resulted in highest mRNA expression of FSHR, cholesterol side-chain cleavage enzyme, P450c17 and 3β-HSD. Fatty acid profiles in fish tissues reflected closely those of diets. Female fish had more gonadal EPA content but less DHA content than male fish, whereas there was a reverse observation in liver. In conclusion, the dietary DHA:EPA ratio, possibly combined with the dietary EPA:arachidonic acid ratio, differentially regulated sex steroid hormone synthesis in mature female and male tongue soles. Females seemed to require more EPA but less DHA for the gonadal steroidogenesis than males. The results are beneficial to sex-specific nutritive strategies in domestic teleost.

Lipoid congenital adrenal hyperplasia by steroidogenic acute regulatory protein (STAR) gene mutation in an Italian infant: an uncommon cause of adrenal insufficiency

BackgroundLipoid congenital adrenal hyperplasia (CAH) (OMIM n. 201710) is the most severe form of congenital adrenal hyperplasia. It is characterized by severe adrenal and gonadal steroidogenesis impairment due to a defect in the conversion of cholesterol to pregnenolone. Affected infants experience salt loss, but glucocorticoid and mineralocorticoid replacement therapy enables long-term survival. Classic lipoid congenital adrenal hyperplasia is relatively common in Japan and Korea but extremely rare in Caucasian populations.Case presentationA female infant of Italian origin came to our attention in late infancy with a clinical picture of acute adrenal insufficiency.The study of the STAR gene revealed two genomic variants c.562C > T and c.577C > T in compound heterozygosity. At the protein level, the two mutations determine the p.Arg188Cys variant (rs104894090) and the p.Arg193Ter variant (rs387907235), respectively. Sanger sequencing was used to confirm the identified variants and to perform familial study. The mother carried the p.Arg188Cys variant, while the father carried the p.Arg193Ter variant.ConclusionTo our knowledge this is the second case of classic lipoid congenital adrenal hyperplasia reported in the Italian population. STAR mutations resulting in lipoid congenital adrenal hyperplasia should be considered all over the world in the differential diagnosis of newborn babies and infants with primary adrenal insufficiency.

Solution structure of human steroidogenic acute regulatory protein STARD1 studied by small-angle X-ray scattering

Intracellular cholesterol transfer to mitochondria, a bottleneck of adrenal and gonadal steroidogenesis, relies on the functioning of the steroidogenic acute regulatory protein (StAR, STARD1), for which many disease-associated mutations have been described. Despite significant progress in the field, the exact mechanism of cholesterol binding and transfer by STARD1 still remains debatable and often considers significant structural rearrangements to achieve ligand binding. The crystal structure of STARD1, obtained recently at medium resolution, suggests that this protein has the same fold as other members of the START family. However, hydrodynamic properties and solution conformation of STARD1 are insufficiently characterized, partially due to poor solubility of this protein. Here, we used our recent protocol to obtain stable and soluble STARD1 and analyzed its hydrodynamic properties and solution conformation using a previously inapplicable small-angle X-ray scattering (SAXS). The SAXS data obtained exclusively from a monodisperse fraction of the monomeric protein suggest that, apart from movements of the flexible Ω1-loop, STARD1 unlikely undergoes significant spontaneous rearrangements proposed earlier as a gating mechanism for cholesterol binding. The consistency with the previously reported solution NMR structure of STARD6 suggests similarity of hydrodynamic behavior of other STARD-containing proteins.

Effect of fish vitellogenin on the growth of juvenile catfish, Clarias gariepinus (Burchell, 1822)

The effect of heterologous fish vitellogenin (Vg)- implantation on growth performance in the juveniles of the catfish, Clarias gariepinus, was evaluated under ambient photoperiod and temperature during February–April (experiment 1) and again at May–August (experiment 2). Compared to either unimplanted (control) or bovine serum albumin (BSA)-implanted group, juveniles (average body weight: 3.5–4g) implanted (im) with Vg pellets (each containing 100μg of Clarias batrachus Vg, three per fish, at one month interval), revealed significantly (P<0.05) higher specific growth rate (SGR) at 90days during both time frames. Congruent with accelerated somatic growth due to Vg-implantation, a trend of increase in plasma level of growth hormone (GH), estradiol-17β (E2) and testosterone (T) was recorded during expt. 1. While plasma E2 and T did not vary significantly, Vg-implantation promoted significant (P<0.05) increase in plasma GH level in both the sexes during the course of expt. 2. Moreover, Vg-implantation could trigger significant alteration in gonadal growth; while control testis contained primarily spermatogonia (SG) and spermatocytes (SC), appearance of spermatozoa (SZ) was noticed in either Vg-implanted (expt. 1) or both BSA- and Vg-treated juveniles (expt. 2). Conversely, a robust increase in S III yolky oocytes was observed in Vg-implanted ovary during May–Aug; but not during expt. 1 (Feb–Apr). However, oocytes at earlier stages (S I and/or S II) of follicular growth and development were predominant in control ovary. Though the underlying mechanism is not yet clearly understood, present data indicate that implantation of Vg pellets in juvenile C. gariepinus may influence somatic growth indices; e.g., body weight gain and GH synthesis along with precocious gonadal growth in a manner sensitive to gonadal steroidogenesis.

RFamide-related peptide-3 (RFRP-3) suppresses sexual maturation in a eusocial mammal

Neuroendocrine mechanisms underlying social inhibition of puberty are not well understood. Here, we use a model exhibiting the most profound case of pubertal suppression among mammals to explore a role for RFamide-related peptide-3 [RFRP-3; mammalian ortholog to gonadotropin-inhibitory hormone (GnIH)] in neuroendocrine control of reproductive development. Naked mole rats (NMRs) live in sizable colonies where breeding is monopolized by two to four dominant animals, and no other members exhibit signs of puberty throughout their lives unless they are removed from the colony. Because of its inhibitory action on the reproductive axis in other vertebrates, we investigated the role of RFRP-3 in social reproductive suppression in NMRs. We report that RFRP-3 immunofluorescence expression patterns and RFRP-3/GnRH cross-talk are largely conserved in the NMR brain, with the exception of the unique presence of RFRP-3 cell bodies in the arcuate nucleus (Arc). Immunofluorescence comparisons revealed that central expression of RFRP-3 is altered by reproductive status, with RFRP-3 immunoreactivity enhanced in the paraventricular nucleus, dorsomedial nucleus, and Arc of reproductively quiescent NMRs. We further observed that exogenous RFRP-3 suppresses gonadal steroidogenesis and mating behavior in NMRs given the opportunity to undergo puberty. Together, our findings establish a role for RFRP-3 in preserving reproductive immaturity, and challenge the view that stimulatory peptides are the ultimate gatekeepers of puberty.

Diclofenac can exhibit estrogenic modes of action in male Xenopus laevis, and affects the hypothalamus-pituitary-gonad axis and mating vocalizations

Diclofenac (DCF) is a non-steroidal analgesic and antiphlogistic. Due to its tremendous use, DCF can be found in the environment, especially in sewage, but also surface waters, ground and drinking water. Previous studies indicated that DCF can modulate the reproductive physiology of fish by altering the expression of important key enzymes of the hypothalamus-pituitary-gonad-axis (HPG-axis) and might act as an estrogenic endocrine disrupting chemical (EDC). Other studies, however, demonstrated that DCF does not exhibit any estrogenicity. Thus, in the present study we investigated whether an exposure to DCF can affect reproductive behavior and physiology of adult male X. laevis by analyzing DCF effects on the mate calling behavior of the frogs and on gene expression patterns of key biomarkers of the HPG-axis. In addition, plasma sex steroid levels were determined to gain detailed insights into the mechanisms of DCF action. We could demonstrate that DCF can act as EDC by exhibiting slight estrogenic modes of action. In addition, pharmacological impacts on gonadal steroidogenesis could be revealed leading to imbalances in sex steroid levels and ratios. DCF furthermore altered the calling behavior of exposed males, potentially reducing the mating and reproductive success of the frogs, possibly leading to severe population effects.

Dietary arachidonic acid differentially regulates the gonadal steroidogenesis in the marine teleost, tongue sole (Cynoglossus semilaevis), depending on fish gender and maturation stage

A 3-month feeding trial with tongue sole Cynoglossus semilaevis broodstock was conducted before and during the spawning season to investigate the effects of dietary arachidonic acid (ARA) on the production of sex steroid hormones and gonadal gene expression of key proteins in steroidogenesis. Three isonitrogenous and isolipidic experimental diets were formulated to contain different ARA levels: the control diet without ARA supplementation (C, 0.58% ARA of total fatty acids (TFA)) and two diets with low (5.14% of TFA, ARA-L) or high ARA (15.44% of TFA, ARA-H) supplementation. The diets were randomly assigned to 9 tanks of 3-year-old tongue sole (10 females and 15 males in each tank). Fish were reared in a flowing seawater system and fed to apparent satiation twice daily. At the end of the feeding trial, tissue samples from mature females (MF, with spontaneous ovulation), immature females (IMF, early vitellogenesis), and mature males (MM, expressing milt) were collected to assay the production of sex steroid hormones, gonadal gene expression of sex steroid-synthesizing proteins, as well as the fatty acid profiles of gonad, liver and muscle lipids. Results showed that ARA supplementation significantly reduced the estradiol production in females, but stimulated the testosterone production in males. ARA supplementation significantly reduced the mRNA expression of aromatase in ovaries but significantly increased the gene expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) in testes. In mature ovaries, diet ARA-L significantly reduced the gene expression of follicle stimulating hormone receptor (FSHR), 3β-HSD, and 17β-HSD; however, in immature ovaries, it significantly increased the gene expression of FSHR, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450ssc), 3β-HSD, and 17β-HSD. In all gonads, 17α-hydroxylase (P450c17) responded to dietary ARA differently from other sex steroid-synthesizing proteins. ARA was preferentially accumulated in tongue sole gonad lipids. ARA concentrations were highest in gonad, liver and muscle lipids of MM fish and lowest in MF fish. Compared to female tongue sole, males had higher DHA concentrations in gonad lipids, but lower concentrations in liver lipids. In conclusion, results suggest dietary ARA regulates sex steroid hormone synthesis in tongue sole broodstock, and accumulates in gonad lipids, depending on both fish gender and maturation stage. Dietary ARA supplementation appears more important for male fish than for female fish, and more important for immature females than for mature females. Statement of relevanceThis study is beneficial to the broodstock diet formulation.

Membrane receptor cross talk in gonadotropin-, IGF-I-, and insulin-mediated steroidogenesis in fish ovary: An overview

Gonadal steroidogenesis is critical for survival and reproduction of all animals. The pathways that regulate gonadal steroidogenesis are therefore conserved among animals from the steroidogenic enzymes to the intracellular signaling molecules and G protein-coupled receptors (GPCRs) that mediate the activity of these enzymes. Regulation of fish ovarian steroidogenesis in vitro by gonadotropin (GtH) and GPCRs revealed interaction between adenylate cyclase and calcium/calmodulin-dependent protein kinases (CaMKs) and also MAP kinase pathway. Recent studies revealed another important pathway in GtH-induced fish ovarian steroidogenesis: cross talk between GPCRs and membrane receptor tyrosine kinases. Gonadotropin binding to Gαs-coupled membrane receptor in fish ovary leads to production of cAMP which in turn trans-activate the membrane-bound epidermal growth factor receptor (EGFR). This is followed by activation of ERK1/2 signaling that promotes steroid production. Interestingly, GtH-induced trans-activation of EGFR in the fish ovary uniquely requires matrix-metalloproteinase-mediated release of EGF. Inhibition of these proteases blocks GtH-induced steroidogenesis. Increased cAMP production in fish ovarian follicle upregulate follicular cyp19a1a mRNA expression and aromatase activity leading to increased biosynthesis of 17β-estradiol (E2). Evidence for involvement of SF-1 protein in inducing cyp19a1a mRNA and aromatase activity has also been demonstrated. In addition to GtH, insulin-like growth factor (IGF-I) and bovine insulin can alone induced steroidogenesis in fish ovary. In intact follicles and isolated theca cells, IGF-I and insulin had no effect on GtH-induced testosterone and 17a,hydroxysprogeaterone production. GtH-stimulated E2 and 17,20bdihydroxy-4-pregnane 3-one production in granulosa cells however, was significantly increased by IGF-I and insulin. Both IGF-I and insulin mediates their signaling via receptor tyrosine kinases leading to activation of PI3 kinase/Akt and MAP kinase. These kinase signals then activates steroidogenic enzymes which promotes steroid production. PI3 kinase, therefore considered to be an initial component of the signal transduction pathways which precedes MAP kinase in IGF-1 and insulininduced steroidogenesis in fish ovary. Thus, investigation on the mechanism of signal transduction regulating fish ovarian steroidogenesis have shown that multiple, apparently independent signal transduction pathways are needed to convey the message of single hormone or growth factor.

The Regulation and Function of Fibroblast Growth Factor 8 and Its Function during Gonadotropin-Releasing Hormone Neuron Development.

Over the last few years, numerous studies solidified the hypothesis that fibroblast growth factor (FGF) signaling regulates neuroendocrine progenitor cell proliferation, fate specification, and cell survival and, therefore, is critical for the regulation and maintenance of homeostasis of the body. One important example that underscores the involvement of FGF signaling during neuroendocrine cell development is gonadotropin-releasing hormone (GnRH) neuron ontogenesis. Indeed, transgenic mice with reduced olfactory placode (OP) Fgf8 expression do not have GnRH neurons. This observation indicates the requirement of FGF8 signaling for the emergence of the GnRH neuronal system in the embryonic OP, the putative birth place of GnRH neurons. Mammalian reproductive success depends on the presence of GnRH neurons to stimulate gonadotropin secretion from the anterior pituitary, which activates gonadal steroidogenesis and gametogenesis. Together, these observations are critical for understanding the function of GnRH neurons and their control of the hypothalamus–pituitary–gonadal (HPG) axis to maintain fertility. Taken together, these studies illustrate that GnRH neuron emergence and hence HPG function is vulnerable to genomic and molecular signals that abnormally modify Fgf8 expression in the developing mouse OP. In this short review, we focus on research that is aimed at unraveling how androgen, all-trans retinoic acid, and how epigenetic factors modify control mouse OP Fgf8 transcription in the context of GnRH neuronal development and mammalian reproductive success.

Mechanism of action of endosulfan as disruptor of gonadal steroidogenesis in the cichlid fish Cichlasoma dimerus

The organochlorine pesticide endosulfan (ES) is used in several countries as a wide spectrum insecticide on crops with high commercial value. Due to its high toxicity to non-target animals, its persistence in the environment and its ability to act as an endocrine disrupting compound in fish, ES use is currently banned or restricted in many other countries. Previous studies on the cichlid fish Cichlasoma dimerus have shown that waterborne exposure to ES can lead to both decreased pituitary FSH content and histological alterations of testes. As gonadotropin-stimulated sex steroids release from gonads was inhibited by ES in vitro, the aim of the present study was to elucidate possible mechanisms of disruption of ES on gonadal steroidogenesis in C. dimerus, as well as compare the action of the active ingredient (AI) with that of currently used commercial formulations (CF). Testis and ovary fragments were incubated with ES (AI or CF) and/or steroidogenesis activators or precursors. Testosterone and estradiol levels were measured in the incubation media. By itself, ES did not affect hormone levels. Co-incubation with LH and the adenylate cyclase activator forskolin caused a decrease of the stimulated sex steroids release. When co-incubated with precursors dehydroandrostenedione and 17αhydroxyprogesterone, ES did not affect the increase caused by their addition alone. No differences were observed between the AI and CFs, suggesting that the effect on steroidogenesis disruption is mainly caused by the AI. Results indicate that action of ES takes place downstream of LH-receptor activation and upstream of the studied steroidogenic enzymes.

Gonadal macrophage infiltration in congenital lipoid adrenal hyperplasia.

Congenital lipoid adrenal hyperplasia (lipoid CAH) results in impairment of adrenal and gonadal steroidogenesis caused by STAR mutations. Our previous study revealed upregulation of genes associated with inflammatory or immune response and macrophage infiltration in the adrenal cortex of Star-knockout mice. This study aimed at investigating macrophage infiltration in the gonads from human patients with lipoid CAH. This study includes seven patients with lipoid CAH who underwent gonadectomy: two XX women (age, 22 and 40 years) and five XY boys (1 year). Two women with ovarian cysts (32 and 40 years) and six boys with autopsy or tumor (1 year) were examined as controls. Immunohistochemical analysis of their gonads was performed to determine steroidogenic cells by NR5A1 or CYP17A1 and macrophages by IBA1 or CD68. An increased number of macrophages infiltrated into the ovaries of lipoid CAH and consisted of two subpopulations: one scattered within and around a layer of theca cells of maturing follicles and the other massively aggregated in the stroma. Abundant cytoplasmic lipid droplets were observed not only in the theca cells but also in the stromal macrophages. There was no significant difference in the number of macrophages in the testicular interstitium between lipoid CAH (95% confidence interval (95% CI: 19.3-47.7 per 0.2mm(2)) and controls (95% CI: 13.3-25.8 per 0.2mm(2)) (P=0.10). These results demonstrate that macrophages infiltrate the ovaries of lipoid CAH, where the theca cells and the stromal macrophages have abundant cytoplasmic lipid droplets.

Effects of acidic water, aluminum, and manganese on testicular steroidogenesis in Astyanax altiparanae.

Metals can influence the gonadal steroidogenesis and endocrine systems of fish, thereby affecting their reproduction. The effects of aluminum and manganese in acidic water were investigated on steroidogenesis in sexually mature male Astyanax altiparanae. Whether mature male fish recover from the effects of metals in metal-free water was also assessed. The fish were exposed to 0.5mg L(-1) of isolated or combined aluminum and manganese in acidic pH (5.5) to keep the metals bioavailable. The fish underwent 96h of acute exposure, and samples were taken 24 and 96h after the beginning of the experiment. The fish were then maintained in metal-free water for 96h. Plasma levels of testosterone, 11-ketotestosterone, 17β-estradiol, and cortisol were measured. Acidic water increased the plasma concentration of testosterone and 11-ketotestosterone. Aluminum increased the testosterone levels after 96h of exposure. Manganese increased the 17β-estradiol levels after 24h of exposure and maintained at high levels until the end of the experiment. With the exception of acidic pH, which increased cortisol levels after 24h of exposure, no changes were observed in this corticosteroid during the acute experiment. Aluminum and manganese together also altered steroid levels but without a standard variation. The fish recovered from the effects of most exposure conditions after 96h in metal-free water. A. altiparanae could use reproductive tactics to trigger changes in testicular steroidogenesis by accelerating spermatogenesis and spermiogenesis, which may interfere with their reproductive dynamics.

Overexpression of Anti-Müllerian Hormone Disrupts Gonadal Sex Differentiation, Blocks Sex Hormone Synthesis, and Supports Cell Autonomous Sex Development in the Chicken.

The primary role of Anti-Müllerian hormone (AMH) during mammalian development is the regression of Müllerian ducts in males. This highly conserved function is retained in birds and is supported by the high levels of AMH expression in developing testes. Mammalian AMH expression is regulated by a combination of transcription factors, the most important being Sry-type high-mobility-group box transcription factor-9 (SOX9). In the chicken embryo, however, AMH mRNA expression precedes that of SOX9, leading to the view that AMH may play a more central role in avian testicular development. To define its role in chicken gonadal development, AMH was overexpressed using the RCASBP viral vector. AMH caused the gonads of both sexes to develop as small and undeveloped structures at both embryonic and adult stages. Molecular analysis revealed that although female gonads developed testis-like cords, gonads lacked Sertoli cells and were incapable of steroidogenesis. A similar gonadal phenotype was also observed in males, with a complete loss of both Sertoli cells, disrupted SOX9 expression and gonadal steroidogenesis. At sexual maturity both sexes showed a female external phenotype but retained sexually dimorphic body weights that matched their genetic sexes. These data suggest that AMH does not operate as an early testis activator in the chicken but can affect downstream events, such as sex steroid hormone production. In addition, this study provides a unique opportunity to assess chicken sexual development in an environment of sex hormone deficiency, demonstrating the importance of both hormonal signaling and direct cell autonomous factors for somatic sex identity in birds.

Abstract 12902: Association of Statin Use and Endogenous Serum Sex Hormone Levels in the Multi-ethnic Study of Atherosclerosis(MESA) Cohort

Background: The new 2013 ACC/AHA cholesterol guidelines recommend statin therapy to reduce atherosclerotic cardiovascular disease. Statins inhibit cholesterol biosynthesis which may impair gonadal steroidogenesis. Higher levels of estradiol (E2) and lower levels of sex hormone binding globulin (SHBG) are associated with greater central obesity, metabolic syndrome and diabetes in both men and postmenopausal women. There is however limited information regarding the impact of statins on endogenous sex hormones in large population studies. Methods: We included 3163 men and 1977 women from the baseline examination of the MESA study who had measurement of serum sex hormones: total and bioavailable testosterone (T), E2, dehydroepiandrosterone (DHEA) and SHBG, and excluded those on hormone replacement therapy and non-statin lipid medications. Results: Of the 5140 individuals examined, statin users (n=812) were 67 + 9 years old, 57% males, 41% Whites, 29% Blacks, 11% Chinese and 19% Hispanics. After adjustment for age, gender and race, total cholesterol, HDL-c and BMI, compared to non-users of statins, statin users had significantly lower SHBG by 4.5 nmol/L (95% CI; -6.1, -2.9) and DHEAS by 0.9 nmol/L (95% CI; -1.4, -0.4). No significant gender interaction with statin was seen. No association was noted between statin use and E2, total and bioavailable T. Conclusion: Statin use is associated inversely with serum SHBG and DHEA levels. This is particularly interesting in light of the association of statin use and diabetes, raising the possibility of mediation by endogenous hormones.

Adverse outcome pathway: Framework, application, and challenges in chemical risk assessment

The environmental estrogen 17α-ethinylestradiol (EE2) will depress or completely inhibit egg production in many common model teleosts at low concentrations (≤0.5 ng/L; Runnalls et al., 2015). This inhibition is not seen in the estuarine killifish, or mummichog (Fundulus heteroclitus), even when exposed to 100 ng/L EE2. This relative insensitivity to EE2 exposure indicates species-specific mechanisms for compensating for exogenous estrogenic exposure. This review compares various reproductive responses elicited by EE2 in mummichog to other common model teleosts, such as zebrafish (Danio rerio) and fathead minnow (Pimephales promelas), identifying key endpoints where mummichog differ from other studied fish. For example, EE2 accumulates primarily in the liver/gall bladder of mummichog, which is different than zebrafish and fathead minnow in which accumulation is predominantly in the carcass. Despite causing species-specific differences in fecundity, EE2 has been shown to consistently induce hepatic vitellogenin in males and cause feminization/sex reversal during gonadal differentiation in larval mummichog, similar to other species. In addition, while gonadal steroidogenesis and plasma steroid levels respond to exogenous EE2, it is generally at higher concentrations than observed in other species. In mummichog, production of 17β-estradiol (E2) by full grown ovarian follicles remains high; unlike other teleost models where E2 synthesis decreases as 17α,20β-dihydroxy-4-prenen-3-on levels increase to induce oocyte maturation. New evidence in mummichog indicates some dissimilarity in gonadal steroidogenic gene expression responses compared to gene expression responses in zebrafish and fathead minnow exposed to EE2. The role of ovarian physiology continues to warrant investigation regarding the tolerance of mummichog to exogenous EE2 exposure. Here we present a comprehensive review, highlighting key biological differences in response to EE2 exposure between mummichog and other commonly used model teleosts.

AMPK: a master energy regulator for gonadal function.

From C. elegans to mammals (including humans), nutrition and energy metabolism significantly influence reproduction. At the cellular level, some detectors of energy status indicate whether energy reserves are abundant (obesity), or poor (diet restriction). One of these detectors is AMPK (5′ AMP-activated protein kinase), a protein kinase activated by ATP deficiency but also by several natural substances such as polyphenols or synthetic molecules like metformin, used in the treatment of insulin resistance. AMPK is expressed in muscle and liver, but also in the ovary and testis. This review focuses on the main effects of AMPK identified in gonadal cells. We describe the role of AMPK in gonadal steroidogenesis, in proliferation and survival of somatic gonadal cells and in the maturation of oocytes or spermatozoa. We discuss also the role of AMPK in germ and somatic cell interactions within the cumulus-oocyte complex and in the blood testis barrier. Finally, the interface in the gonad between AMPK and modification of metabolism is reported and discussion about the role of AMPK on fertility, in regards to the treatment of infertility associated with insulin resistance (male obesity, polycystic ovary syndrome).

Genetic and functional study of StAR (steroidogenic acute regulatory protein) deficiency

Congenital lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH, impairing adrenal and gonadal steroidogenesis. Most cases of lipoid CAH are caused by recessive mutations in the gene encoding steroidogenic acute regulatory protein (StAR), a protein that plays an essential role in cholesterol transfer from the outer to inner mitochondrial membrane, thus providing the substrate for steroid hormone biosynthesis. Affected children typically present with life-threatening adrenal insufficiency in early infancy due to a failure of glucocorticoid (cortisol) and mineralocorticoid (aldosterone) biosynthesis, and 46,XY genetic males have complete lack of androgenization and appear phenotypically female due to impaired testicular androgen secretion in utero. Previously, the Q258X mutation of StAR was shown to account for about 70% of affected alleles in most patients of Japanese and Korean ancestry. However, it is more prevalent (92.3%) in the Korean population. These results suggest that the genetic defect in the StAR gene in Korean patients with lipoid CAH is highly homogeneous, probably reflecting a founder effect. Recently, some patients have been reported that they showed late and mild clinical presentation. These cases and studies establish a new entity of ‘non-classic lipoid CAH’ showing that the phenotypic spectrum of StAR mutations is substantially broader than previously appreciated.