Mechanism of Estrogen-Induced Penile Dysfunction and Loss of Fertility

描述(申请人提供)：男性生殖障碍占全球人类不育病例的40%。虽然不孕不育的原因是多因素的，但在发育过程中暴露于雌激素(E)与生殖障碍发病率的增加有关。E诱导的疾病的潜在机制仍不清楚。本研究的目的是确定新生儿暴露E导致不育和阴茎发育不良的分子和细胞机制，其特征是阴茎体内脂肪细胞异常堆积和平滑肌和血管丢失。中心假说是，E暴露，通过雌激素受体(ER)途径和/或雄激素受体(AR)途径，改变阴茎基质细胞(高E，高ER1；低雄激素，高ER1)的ER1表达，然后重新编程，从而导致脂肪细胞分化的关键基因(PPAR3，C/EBP1)异常上调，而平滑肌细胞(1肌动蛋白)和内皮细胞(CD-31)的关键基因下调。具体目标1将检验以下假设：i)E暴露以剂量依赖的方式上调ER1的表达并下调新生儿睾酮激增；ii)ER1上调是时间依赖的，发生在关键的发育时期；iii)ER1上调可被ER拮抗剂ICI182,780(ER途径)以及AR激动剂DHT(AR途径)缓解。《特殊目的2》将检验假设：1)暴露于E可上调PPAR3和C/EBP1的表达，下调1-肌动蛋白和CD-31的表达(因果关系)；2)ICI和DHT可缓解这些改变。具体目标3将验证这样的假设，即暴露于抗雄激素(GnRH拮抗剂和氟他胺)可能由于较低的雄激素作用而上调ER1的表达，但可能不会因为缺乏外源性E暴露而改变PPAR3、C/EBP1、1肌动蛋白和CD-31的表达。具体目标4将检验这样的假设，即使用ER拮抗剂和AR激动剂干预可以防止E诱导的生育丧失。实时聚合酶链式反应将被用来定量mRNAs，免疫组织化学将被用来定位和定量蛋白质。总而言之，结果将揭开ER途径和/或AR途径信号改变导致阴茎发育不良和阴茎功能障碍的机制。这项工作的结果将对人类和野生动物的健康产生重大影响，因为它们将导致更好的战略，以防止E诱导的生殖障碍。

DESCRIPTION (provided by applicant): Male reproductive disorders account for 40% of infertility cases worldwide in humans. Although causes of infertility are multi-factorial, exposure to estrogens (E) during development has been linked with increasing incidence of reproductive disorders. Mechanisms underlying E-induced disorders remain unclear. The goal of this study is to determine molecular and cellular mechanisms whereby neonatal E exposure results in infertility and mal-developed penis characterized by abnormal accumulation of fat cells and loss of smooth muscle and blood vessels in the penis body. The central hypothesis is that E exposure, via estrogen receptor (ER) pathway or androgen receptor (AR) pathway or both, alters ER1 expression in penile stromal cells (higher E, higher ER1; lower androgen, higher ER1), which are then re-programmed so that key genes for differentiation of fat cells (PPAR3, C/EBP1) are undesirably up-regulated and those for smooth muscle cell (1 actin) and endothelial cell (CD-31) are down-regulated. Specific aim 1 will test the hypotheses that I) E exposure up-regulates ER1 expression and down-regulates neonatal testosterone surge in a dose- dependent manner, II) ER1 up-regulation is time-dependent and occurs during a critical developmental period; and III) ER1 up-regulation is mitigated by ER antagonist ICI 182,780 (ER pathway), as well as by AR agonist DHT (AR pathway). Specific aim 2 will test the hypotheses I) that E exposure up-regulates PPAR3 and C/EBP1 expression and down-regulates 1 actin and CD-31 expression (cause and effect relationships); and II) that ICI and DHT mitigate these alterations. Specific aim 3 will test the hypothesis that exposure to anti-androgens (GnRH-antagonist and flutamide) up-regulates ER1 expression because of lower androgenic action, but may not alter PPAR3, C/EBP1, 1 actin, and CD-31 expression because of lack of exogenous E exposure. Specific aim 4 will test the hypothesis that intervention with ER antagonist and AR agonist prevents E-induced loss of fertility. Real-time PCR will be used to quantify mRNAs and immunohistochemistry will be used to localize and quantify proteins. Collectively, results will unravel mechanisms whereby altered signaling in ER pathway or AR pathway or both results in mal-development of the penis and penile dysfunction. Outcomes of this work will have a significant impact on human and wildlife health because they will lead to better strategies to prevent E-induced reproductive disorders.