Xenoestrogen Regulation of Leydig Cells

摘要 人们越来越多地在饮食中使用大豆制品，这引起了人们对其生殖毒性的担忧。 因为大豆含有表现出激素活性的异黄酮类物质，可能会干扰生殖 发展。大豆中的主要异黄酮染料木素和大豆苷在 胃肠道中有染料木素和大豆苷元，而大豆苷元又进一步分解为木酚。的 有趣的是，男性的表型主要是由睾丸产生的性类固醇睾酮支持的。 间质细胞。在这个应用中，Aim 1的实验将检验暴露时间影响的假设 以大豆为基础的饮食对睾丸的激素影响。雄性大鼠将被喂养不含大豆的对照组或以大豆为基础的饲料 观察出生后不同时间的饮食情况，分析血清激素水平与出生后体重的相关性。 暴露于对间质细胞和生殖细胞发育影响的时间。在《目标2》中，派假设染料木素， 大豆苷元和马酚都有助于异黄酮类的暴露效应。给断奶的雄性大鼠喂食对照组、 大豆基础日粮，或在21-35日龄添加染料木苷、大豆苷或马酚的对照日粮。至 确定异黄酮类是否干扰下丘脑和脑下垂体的类固醇激素调节， 对照组和暴露于异黄酮组的动物将被去势并植入硅胶胶囊。 含有睾酮、双氢睾酮(一种非芳香化雄激素)或17β-雌二醇(天然 雌激素)，从产后36-56天。将获取样本用于类固醇的免疫组织化学分析 激素受体[雌激素受体(ESR1、ESR2)和雄激素受体(AR)蛋白在视前区 和下丘脑腹内侧核。此外，促性腺激素释放激素(GnRH)受体 类固醇激素受体和促性腺激素(黄体生成素和卵泡刺激素)亚单位(黄体生成素、β和 卵泡刺激素β)的表达将在脑下垂体的蛋白质印迹中进行分析。在第三个目标中，实验将是 目的是确定异黄酮类化合物在间质细胞中的组织(永久性)效应的分子基础。 在原始时期，定时怀孕的母鸡将维持在对照或豆基饲料中。 生殖细胞迁移，即妊娠7-21天。以雄性后代为F1代，交配研究将 取F2代和F3代雄性大鼠。间充质干细胞与完全分化 从7日龄和90日龄雄性大鼠获得的成年间质细胞将被评估其发育潜力和 末端分化。此外，还将分析基因组dna和总rna的DNA甲基化模式。 和microRNA的表达。总而言之，结果将描述异黄酮类效应如何影响间质细胞 睾丸细胞的发育和旁分泌调节，干扰类固醇激素调节 下丘脑和脑下垂体的功能，并确定使异黄酮永久化的表观遗传机制 暴露会影响几代人。这些数据将推动美国国家环境研究所的任务 健康科学，以发现环境如何影响人们，以促进更健康的生活。 1

ABSTRACT The increasing use of soy-based products in the diet has raised concerns regarding their reproductive toxicity because soybeans contain isoflavones which exhibit hormonal activity and may interfere with reproductive development. The predominant isoflavones in soybeans, genistin and daidzin, are hydrolyzed in the gastrointestinal tract to genistein and daidzein aglycones, and daidzein is further broken down to equol. Of interest, the male phenotype is supported primarily by the sex steroid testosterone which is produced by testicular Leydig cells. In this application, Aim 1 experiments will test the hypothesis that timing of exposure influences the hormonal effects of soy-based diets in the testis. Male rats will be fed either a soy-free control or soy-based diet at different times in the postnatal period followed by analysis of serum hormone levels and correlation of the timing of exposure to the impact on Leydig and germ cell development. In Aim 2, PI hypothesizes that genistein, daidzein and equol all contribute to isoflavone exposure effects. Weanling male rats will be fed either the control, soy-based diet, or the control diet supplemented with genistin, daidzin or equol from 21-35 days of age. To determine if isoflavones interfered with steroid hormone regulation of the hypothalamus and pituitary gland, cohorts of control and isoflavone-exposed animals will be castrated and implanted with silastic capsules containing testosterone, dihydrotestosterone (a non-aromatizable androgen) or 17β-estradiol (the natural estrogen) from days 36-56 postpartum. Samples will be obtained for immunohistochemical analysis of steroid hormone receptor [estrogen receptors (ESR1, ESR2) and androgen receptor (AR) protein in the preoptic area and ventromedial nucleus of the hypothalamus. In addition, gonadotropin releasing hormone (GnRH) receptor, steroid hormone receptors and gonadotropin (luteinizing and follicle stimulating hormone) subunit (LHβ and FSHβ) expression will be analyzed in western blots of pituitary glands. In the third aim, experiments will be performed to determine the molecular basis for organizational (permanent) effects of isoflavones in Leydig cells. Timed-pregnant dams will be maintained either on the control or soy-based diet during the period of primordial germ cell migration, i.e., gestational days 7-21. With male offspring as the F1 generation, mating studies will be performed to generate F2 and F3 generations of male rats. Mesenchymal stem cells and fully differentiated adult Leydig cells, obtained from 7- and 90-day old male rats, will be evaluated for developmental potential and terminal differentiation. In addition, genomic DNA and total RNA will be analyzed for DNA methylation patterns and microRNA expression. Altogether, results will describe how isoflavone effects impact Leydig cell development and paracrine regulation of testicular cells, interfere with steroid hormone regulation of hypothalamic and pituitary gland function, and identify epigenetic mechanisms which perpetuate isoflavone exposure effects across generations. These data will advance mission of the National Institute of Environmental Health Sciences to discover how the environment affects people in order to promote healthier lives. 1