Xenoestrogen regulation of Leydig cells

描述（由申请人提供）：大豆异黄酮具有雌激素特性，生殖道组织（包括间质细胞）表达雌激素受体（ER1和ER2）。间质细胞是雄性类固醇激素睾酮的主要来源。在美国，每年大约有15%的婴儿被喂食大豆配方奶粉，他们会接触到植物雌激素染料木素和大黄酮，这是大豆中主要的异黄酮。文献中的一些报告表明，啮齿动物和非人类灵长类动物接触大豆异黄酮会影响睾丸雄激素分泌。然而，大豆异黄酮在间质细胞中的作用机制尚未被研究。P.I.假设血清雄激素水平和间质细胞类固醇生成能力的差异与间质细胞数量的差异有关。与这一假设一致的是，最近的观察表明染料木素调节间质细胞有丝分裂。此外，大豆异黄酮被认为可以调节睾丸中体细胞的发育，例如支持细胞。因此，细胞间的相互作用，例如支持细胞和间质细胞之间的相互作用，受到大豆异黄酮的调节。为了解决这些问题，将进行实验以实现以下目标：1 .大豆异黄酮对间充质间质细胞前体的调节；二世。染料木素在间质细胞中的作用机制三世。支持细胞介导的间质细胞调节。这项拟议的研究与公共卫生有关，并将首次确定雌激素在间质细胞中的作用机制和分子途径。此外，研究结果将促进在饮食中明智地使用大豆异黄酮，特别是在新生儿中，因为发育中的生殖道对激素环境的变化很敏感。此外，本申请将为本科生、专业学生和研究生提供研究训练。公众对农业恐怖主义的日益关注使兽医站在了帮助保卫食品供应的研究的前沿，正如生物医学研究越来越受益于对食用动物物种的调查，例如，利用猪来研究肥胖和糖尿病，利用狗来研究心肺疾病。因此，目前应用程序提供的研究培训将有助于努力准备未来的兽医来迎接这些挑战。公共卫生相关性：大豆异黄酮具有雌激素特性，生殖道组织（包括间质细胞）表达ER1和ER2。由于间质细胞的增殖活性，决定了间质细胞的最终数量，仅限于青春期前，青春期前间质细胞有丝分裂的调节对成年睾丸的间质细胞数量有影响。本应用程序旨在利用大鼠模型确定大豆异黄酮对睾丸雄激素分泌产生持久影响的机制。实验将解决三个具体目标:大豆异黄酮间充质影响睾丸间质细胞前体的产前和/或新生儿期,这改变了睾丸间质细胞增殖和人口(目标);雌激素对间质细胞有丝分裂的调节是通过信号通路的相互作用介导的，其中包括细胞存活因子蛋白Akt（蛋白激酶B）与雌激素受体ER1、表皮生长因子受体（EGFR）、胰岛素生长因子-1受体（IGF-1R）和丝裂原活化蛋白激酶（MAPK） （Aim II）协同作用；大豆异黄酮在支持细胞中的作用影响间质细胞（Aim III）。定时怀孕的母鸭将在围产期饲喂含有低和高异黄酮水平（5,1000 ppm）的饲粮。分析雄性后代青春期前和青春期大鼠间质细胞增殖情况。此外，在90日龄的成年睾丸中，将用体视学方法枚举间质细胞的数量。分析染料木黄酮调节间质细胞有丝分裂的作用将涉及使用药物抑制和基因沉默技术来研究Akt、ER1、EGFR和IGF1-R的作用。大豆异黄酮直接作用于授精上皮的可能性将通过分析支持细胞的分化和成熟来研究。此外，将通过在支持细胞条件培养基中培养间质细胞来评估支持细胞与间质细胞的相互作用。该实验将首次研究大豆异黄酮在间质细胞中的作用机制。特别是，EDS和busulfan处理的大鼠模型提供了在体内研究细胞增殖和细胞间相互作用的机会。这些模型极大地促进了我们对睾丸生物学的理解，但尚未用于毒理学研究。此外，雌激素化合物和其他内分泌活性物质可能通过支持细胞的作用对间质细胞产生间接影响的假设尚未被探索。总之，这些方法将产生以前未描述的植物雌激素在睾丸细胞中的作用的信息。这些信息将有助于持续研究以大豆为基础的饮食的潜在生殖毒性，特别是对新生儿。公众对农业恐怖主义的日益关注对负有保护食品供应责任的兽医构成了挑战。此外，生物医学研究越来越受益于对食用动物物种进行的调查，例如，利用猪来研究肥胖和糖尿病，利用狗来研究心肺疾病。本应用程序为专业兽医学生提供的研究培训将有助于为未来的兽医做好准备，以应对这些挑战。

DESCRIPTION (provided by applicant): Soy isoflavones possess estrogenic properties, and reproductive tract tissues (including Leydig cells) express estrogen receptors (ER1 and ER2). Leydig cells are the predominant source of the male sex steroid hormone testosterone. Approximately 15% of infants fed soy-based formulas in the United States each year are exposed to the phytoestrogens genistein and daidzen, which are the predominant isoflavones in soybeans. Several reports in the literature have shown that exposure of rodents and non human primates to soy isoflavones affect testicular androgen secretion. However, the mechanisms by which soy isoflavones act in Leydig cells have not been investigated. The P.I. hypothesizes that disparities in serum androgen levels and Leydig cell steroidogenic capacity are linked to differences in the numbers of Leydig cells. Consistent with this hypothesis are recent observations indicating that genistein regulates Leydig cell mitosis. Also, soy isoflavones are known to regulate development of somatic cells in the testis, e.g. Sertoli cells. Therefore, cell-cell interactions, e.g., between Sertoli cells and Leydig cells, are subject to regulation by soy isoflavones. To address these issues, experiments will be performed to achieve the following aims: I. Soy isoflavone regulation of mesenchymal Leydig cell precursors; II. Mechanisms of genistein action in Leydig cells; and, III. Sertoli cell-mediated regulation of Leydig cells. The proposed study is relevant to public health and will be the first to define the mechanisms and molecular pathways of estrogen action in Leydig cells. Also, results will promote informed use of soy isoflavones in the diet, especially in neonates, in which the developing reproductive tract is sensitive to changes in the hormonal milieu. Furthermore, the present application will provide research training to undergraduate, professional and graduate students. The growing public concern for agri-terrorism puts veterinarians in the fore-front of research to aid defense of food supplies just as biomedical research is increasingly benefiting from investigation conducted in food animal species, e.g., use of pigs to study obesity and diabetes and dogs for cardiopulmonary diseases. Therefore, research training afforded by the present application will contribute to efforts geared to preparing future veterinarians to meet these challenges. PUBLIC HEALTH RELEVANCE: Soy isoflavones possess estrogenic properties, and reproductive tract tissues (including Leydig cells) express ER1 and ER2. Because proliferative activity in Leydig cells, which determines the final number of Leydig cells, is restricted to the prepubertal period, modulation of Leydig cell mitosis in the prepubertal period has consequences for Leydig cell numbers in the adult testis. The present application is designed to identify the mechanisms by which soy isoflavones cause long-lasting effects on testicular androgen secretion using the rat model. Experiments will be performed to address three Specific Aims: Soy isoflavones affect mesenchymal Leydig cell precursors in the prenatal and/or neonatal period, which alters Leydig cell proliferation and population (Aim I); Estrogen regulation of Leydig cell mitosis is mediated by interaction of signaling pathways, which involves the cell survival factor protein Akt (protein kinase B) acting in concert with estrogen receptor ER1, epidermal growth factor receptors (EGFR), insulin growth factor-1 receptors (IGF-1R) and mitogen activated protein kinases (MAPK) (Aim II); and, Action of soy isoflavones in Sertoli cells affects Leydig cells (Aim III). Timed pregnant dams will be fed diets containing low and high isoflavone levels (5, 1000 ppm) in the perinatal period. Male offspring will be analyzed for Leydig cell proliferation in prepubertal and pubertal rats. In addition, the number of Leydig cells will be enumerated by stereology in the adult testis at 90 days of age. Analysis of genistein action regulating Leydig cell mitosis will involve use of pharmacological inhibition and gene silencing techniques to investigate the role of Akt, ER1, EGFR and IGF1-R. The possibility that soy isoflavones act directly in the seminiferous epithelium will be investigated by analysis of Sertoli cell differentiation and maturation. In addition, Sertoli cell-Leydig cell interactions will be evaluated by culture of Leydig cells in Sertoli cell-conditioned media. The proposed experiments will be the first to investigate the mechanisms of soy isoflavone action in Leydig cells. In particular, the EDS- and busulfan-treated rat models provide opportunities to study cell proliferation and cell-cell interactions in vivo. These models have contributed immensely to our understanding of testicular biology but have not been used in toxicological studies. Also, the hypothesis that estrogenic compounds, and perhaps other endocrine-active agents, may exert indirect effects in Leydig cells by action exerted in Sertoli cells has not been explored. Together, these approaches will yield information not previously described for phytoestrogen action in testicular cells. The information will contribute to ongoing about potential reproductive toxicity of soy-based diets especially in neonates. The growing public concern for agri-terrorism represents a challenge to veterinarians who have responsibilities for defense of food supplies. Also, biomedical research is increasingly benefiting from investigation conducted in food animal species, e.g., use of pigs to study obesity and diabetes and dogs for cardiopulmonary diseases. Research training to be afforded by the present application to professional veterinary students will contribute to efforts geared to preparing future veterinarians to meet these challenges.