Alteration of the expression and 17, 20 lyase activity of cytochrome P450 17-hydroxylase/17, 20 lyase and of the expression of other Leydig cells genes in the rat fetal testis directly exposed to Mono-ethyl hexyl phthalate in culture.. Rattus norvegicus

This study aimed to investigate the direct mechanism(s) of action ofMono-(Ethyl-Hexyl) phthalate (MEHP) on the rat fetal testis with particular emphasis to Leydig cell steroidogenesis, by means of an in vitro system based on culture for 3 days of fetal testes from 14.5 days post-coïtum old rats. We first confirmed that an exposure to MEHP led to a dose-dependent decrease of testosterone and demonstrated that dihydrotestosterone (DHT) production was also inhibited. We then demonstrated that in 10µM MEHP-exposed testis basal 17alpha-hydroxy-progesterone (17OHP) production was increased (+40%) while androstenedione levels decreased (-55%). Furthermore, the addition of the latter steroid but none of the other precursors rescued testosterone thus establishing further that MEHP specifically blocked steroidogenesis at the level of the 17,20 lyase activity of the P450c17 enzyme (CYP17), which converts 17OHP to androstenedione. Furthermore and accordingly, both levels of testicular CYP17 protein (western blot) and cyp17a1 gene (qPCR, microarrays) were found to decrease under MEHP exposure. The microarray analysis also showed that among the few other testicular genes cyp17a1 whose expression was inhibited by MEHP were the genes encoding for the Leydig cell insulin-like peptid 3 (INSL3), involved in the control of testicular descent, and for inhibin A (INHA), that regulates follicular-stimulating hormone secretion.Under in vitro conditions where MEHP is not metabolized and remains at low intratesticular concentration, our findings show in particular that this phthalate directly inhibits several important Leydig cell factors involved in testis development and function. Overall design: Transcription profiling of rat fetal testes explants, vehicle or monoethylhexylphthalate (MEHP)-treated (1uM, 10uM) 3 condition experiment, 1-3 biological replicates per treatment. 2 technical replicates per sample.

本研究旨在探究邻苯二甲酸单(乙基己基)酯（Mono-(Ethyl-Hexyl) phthalate, MEHP）对大鼠胎儿睾丸的直接作用机制，重点关注莱迪希细胞（Leydig cell）的类固醇生成过程，实验采用体外培养体系，以交配后14.5天的大鼠胎儿睾丸体外培养3天为模型。我们首先证实，MEHP暴露会导致睾酮水平呈剂量依赖性下降，并证明二氢睾酮（dihydrotestosterone, DHT）的生成也受到抑制。随后我们发现，在10μM MEHP暴露的睾丸组织中，基础17α-羟孕酮（17α-hydroxy-progesterone, 17OHP）的生成量升高了40%，而雄烯二酮（androstenedione）的水平下降了55%。此外，补充该类固醇前体（而非其他前体）可恢复睾酮的生成，进一步证实MEHP特异性阻断了P450c17酶（CYP17）的17,20裂解酶活性——该酶可将17OHP转化为雄烯二酮，从而抑制类固醇生成通路。与之相符的是，蛋白质免疫印迹（western blot）检测到的睾丸CYP17蛋白水平，以及实时定量聚合酶链反应（quantitative real-time PCR, qPCR）、微阵列（microarrays）分析得到的cyp17a1基因表达水平，均在MEHP暴露后出现下降。微阵列分析还显示，受MEHP抑制表达的其他少量睾丸基因中，包括编码莱迪希细胞胰岛素样肽3（insulin-like peptid 3, INSL3，参与睾丸下降调控）的基因，以及编码抑制素A（inhibin A, INHA，可调节促卵泡激素分泌）的基因。在MEHP未发生代谢且睾丸内浓度较低的体外培养条件下，我们的研究结果尤其表明，该邻苯二甲酸酯可直接抑制参与睾丸发育与功能的多种关键莱迪希细胞因子。实验整体设计：大鼠胎儿睾丸外植体的转录谱分析，分为溶剂对照、单乙基己基邻苯二甲酸酯（MEHP）1μM、10μM处理三组，每组设置1-3次生物学重复，每个样本设置2次技术重复。