Benzo[a]pyrene disrupts LH/hCG-dependent mouse Leydig cell steroidogenesis through receptor/Gαs protein targeting.

Steroidogenesis of gonadal cells is tightly regulated by gonadotropins. However, certain polycyclic aromatic hydrocarbons, including Benzo[a]pyrene (BaP), induce reproductive toxicity. Several existing studies have considered higher than environmentally relevant concentrations of BaP on male and female steroidogenesis following long-term exposure. Also, the impact of short-term exposure to BaP on gonadotropin-stimulated cells is understudied. Therefore, we evaluated the effect of 1 nM and 1 µM BaP on luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e. the mouse tumor Leydig cell line mLTC1, and the human primary granulosa lutein cells (hGLC) post 8- and 24-h exposure. Cell signalling studies were performed by homogeneous time-resolved fluorescence (HTRF) assay, bioluminescence energy transfer (BRET) and Western blotting, while immunostainings and immunoassays were used for intracellular protein expression and steroidogenesis analyses, respectively. BaP decreased cAMP production in gonadotropin-stimulated mLTC1 interfering with Gαs activation. Therefore, decrease in gonadotropin-mediated CREB phosphorylation in mLTC1 treated with 1 µM BaP was observed, while StAR protein levels in gonadotropin-stimulated mLTC1 cells were unaffected by BaP. Further, BaP decreased LH- and hCG-mediated progesterone production in mLTC1. Contrastingly, BaP failed to mediate any change in cAMP, genes and proteins of steroidogenic machinery and steroidogenesis of gonadotropin-treated hGLC. Our results indicate that short-term exposure to BaP significantly impairs steroidogenic signalling in mLTC1 interfering with Gαs. These findings could have a significant impact on our understanding of the mechanism of reproductive toxicity by endocrine disruptors.

Short-Term Exposure to Bisphenol A Does Not Impact Gonadal Cell Steroidogenesis In Vitro.

Bisphenol A (BPA) is a ubiquitous, synthetic chemical proven to induce reproductive disorders in both men and women. The available studies investigated the effects of BPA on male and female steroidogenesis following long-term exposure to the compound at relatively high environmental concentrations. However, the impact of short-term exposure to BPA on reproduction is poorly studied. We evaluated if 8 and 24 h exposure to 1 nM and 1 µM BPA perturbs luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e., the mouse tumour Leydig cell line mLTC1, and human primary granulosa lutein cells (hGLC). Cell signalling studies were performed using a homogeneous time-resolved fluorescence (HTRF) assay and Western blotting, while gene expression analysis was carried out using real-time PCR. Immunostainings and an immunoassay were used for intracellular protein expression and steroidogenesis analyses, respectively. The presence of BPA leads to no significant changes in gonadotropin-induced cAMP accumulation, alongside phosphorylation of downstream molecules, such as ERK1/2, CREB and p38 MAPK, in both the cell models. BPA did not impact STARD1, CYP11A1 and CYP19A1 gene expression in hGLC, nor Stard1 and Cyp17a1 expression in mLTC1 treated with LH/hCG. Additionally, the StAR protein expression was unchanged upon exposure to BPA. Progesterone and oestradiol levels in the culture medium, measured by hGLC, as well as the testosterone and progesterone levels in the culture medium, measured by mLTC1, did not change in the presence of BPA combined with LH/hCG. These data suggest that short-term exposure to environmental concentrations of BPA does not compromise the LH/hCG-induced steroidogenic potential of either human granulosa or mouse Leydig cells.

Two human menopausal gonadotrophin (hMG) preparations display different early signaling in vitro.

Commercial hMG drugs are marketed for the treatment of infertility and consist of highly purified hormones acting on receptors expressed in target gonadal cells. Menopur® and Meriofert® are combined preparation of FSH and hCG and are compared in vitro herein. To this purpose, the molecular composition of the two drugs was analyzed by immunoassay. The formation of FSH receptor and LH/hCG receptor (FSHR; LHCGR) heteromer, intracellular Ca2+ and cAMP activation, ß-arrestin 2 recruitment and the synthesis of progesterone and estradiol were evaluated in transfected HEK293 and human primary granulosa lutein cells treated by drugs administered within the pg-mg/ml concentration range. Molecular characterization revealed that Meriofert® has a higher FSH:hCG ratio than Menopur® which, in turn, displays the presence of LH molecules. While both drugs induced similar FSHR-LHCGR heteromeric formations and intracellular Ca2+ increase, Meriofert® had a higher potency than Menopur® in inducing a cAMP increase. Moreover, Meriofert® revealed a higher potency than Menopur® in recruiting ß-arrestin 2, likely due to different FSH content modulating the tridimensional structure of FSHR-LHCGR-ß-arrestin 2 complexes, as evidenced by a decrease in bioluminescence resonance energy transfer signal. This drug-specific activation of intracellular signaling pathways is consistent with the molecular composition of these preparations and impacts downstream progesterone and estradiol production, with Menopur® more potent than Meriofert® in inducing the synthesis of both the steroids. These findings are suggestive of distinct in-vivo activities of these preparations, but require cautious interpretation and further validation from clinical studies.

Heterogeneous hCG and hMG commercial preparations result in different intracellular signalling but induce a similar long-term progesterone response in vitro.

STUDY QUESTION: Are four urinary hCG/menotropin (hMG) and one recombinant preparation characterized by different molecular features and do they mediate specific intracellular signaling and steroidogenesis? SUMMARY ANSWER: hCG and hMG preparations have heterogeneous compositions and mediate preparation-specific cell signaling and early steroidogenesis, although similar progesterone plateau levels are achieved in 24 h-treated human primary granulosa cells in vitro. WHAT IS KNOWN ALREADY: hCG is the pregnancy hormone marketed as a drug for ARTs to induce final oocyte maturation and ovulation, and to support FSH action. Several hCG formulations are commercially available, differing in source, purification methods and biochemical composition. STUDY DESIGN, SIZE, DURATION: Commercial hCG preparations for ART or research purposes were compared in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS: The different preparations were quantified by immunoassay with calibration against the hCG standard (Fifth IS; NIBSC 07/364). Immunoreactivity patterns, isoelectric points and oligosaccharide contents of hCGs were evaluated using reducing and non-reducing Western blotting, capillary isoelectric-focusing immunoassay and lectin-ELISA, respectively. Functional studies were performed in order to evaluate intracellular and total cAMP, progesterone production and ß-arrestin 2 recruitment by ELISA and BRET, in both human primary granulosa lutein cells (hGLC) and luteinizing hormone (LH)/hCG receptor (LHCGR)-transfected HEK293 cells, stimulated by increasing hormone concentrations. Statistical analysis was performed using two-way ANOVA and Bonferroni post-test or Mann-Whitney's U-test as appropriate. MAIN RESULTS AND THE ROLE OF CHANCE: Heterogeneous profiles were found among preparations, revealing specific molecular weight patterns (20-75 KDa range), isoelectric points (4.0-9.0 pI range) and lectin binding (P < 0.05; n = 7-10). These drug-specific compositions were linked to different potencies on cAMP production (EC50 1.0-400.0 ng/ml range) and ß-arrestin 2 recruitment (EC50 0.03-2.0 µg/ml) in hGLC and transfected HEK293 cells (P < 0.05; n = 3-5). In hGLC, these differences were reflected by preparation-specific 8-h progesterone production although similar plateau levels of progesterone were acheived by 24-h treatment (P ≥ 0.05; n = 3). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The biological activity of commercial hCG/hMG preparations is provided in International Units (IU) by in-vivo bioassay and calibration against an International Standard, although it is an unsuitable unit of measure for in-vitro studies. The re-calibration against recombinant hCG,quantified in grams, is based on the assumption that all of the isoforms and glycosylation variants have similar immunoreactivity. WIDER IMPLICATIONS OF THE FINDINGS: hCG/hMG preparation-specific cell responses in vitro may be proposed to ART patients affected by peculiar ovarian response, such as that caused by polycystic ovary syndrome. Otherwise, all the preparations available for ART may provide a similar clinical outcome in healthy women. STUDY FUNDING AND COMPETING INTEREST(S): This study was supported by a grant of the Italian Ministry of Education, University and Research (PRIN 2015XCR88M). The authors have no conflict of interest.