Developmental Gene-Environment Interactions and Premature Ovarian Failure

项目摘要/摘要 今天在世的美国妇女中有150多万人已经或将被诊断为早产儿 卵巢功能衰竭(POF)在他们的一生中，一个未知的，可能更多的人会有早期 未达到POF诊断标准的更年期。POF的特征是加速耗尽 卵泡和卵母细胞质量下降，但90%的病例原因尚不清楚。多环 芳香烃(PAHs)是由有机物质的不完全燃烧形成的。女人是 通过食物、空气污染和烟草烟雾无处不在地暴露在苯并[a]芘(BaP)和其他多环芳烃中。BaP 是一种强效的卵毒剂，而发育中的卵巢尤其敏感。接触烟草烟雾，这是 含有高浓度的BaP和其他多环芳烃，与生殖力下降和更早 怀孕期间吸烟的妇女的女儿更年期。我们已经证明了孕期接触 小鼠在原始生殖细胞通过减数分裂开始迁移到BaP期间导致F1雌性POF 子代的剂量不影响母亲的卵泡数量。我们进一步证明了胚胎 谷氨酸修饰亚基缺失导致抗氧化剂谷胱甘肽合成不足 半胱氨酸连接酶(Gclm)对BaP的经胎盘卵毒性比野生型更敏感。 我们的初步数据进一步表明，BaP可诱导培养的胎儿卵巢生殖细胞凋亡，并且 Gclm缺失卵巢对BaP诱导生殖细胞凋亡更为敏感。在目前的提案中，我们 将验证BaP通过诱导氧化应激来耗尽产前卵巢中生殖细胞的假设 和凋亡，同时诱导存活的生殖细胞发生可遗传的表观遗传变化，从而引起 后代人卵泡加速耗竭，GSH具有保护作用 以对抗这些影响。我们将在两个目标上检验这一假说：1)建立 BaP经胎盘卵子毒性的研究进展及机制探讨 在临界窗口期，谷胱甘肽调节BaP诱导的卵毒性。我们将在体内使用互补性 经胎盘暴露和培养胚胎卵巢模型。我们将测试潜在的保护作用 补充谷胱甘肽和其他抗氧化剂。2)检测孕期暴露的卵巢表型 BaP是跨代遗传的，由生殖系中的表观遗传变化所介导。我们将利用RNA- 测序以检查全基因组的基因表达，并将使用MBD- 暴露于BaP的F1、F2和F3原始生殖细胞的测序结果与对照谱系的比较。

PROJECT SUMMARY/ABSTRACT More than 1.5 million of the American women alive today have been or will be diagnosed with premature ovarian failure (POF) during their lifetimes and an unknown, probably much larger number will have early menopause without meeting the diagnostic criteria for POF. POF is characterized by accelerated depletion of ovarian follicles and decreased oocyte quality, but the causes remain unknown in 90% of cases. Polycyclic aromatic hydrocarbons (PAHs) are formed by the incomplete combustion of organic materials. Women are ubiquitously exposed to benzo[a]pyrene (BaP) and other PAHs via food, air pollution, and tobacco smoke. BaP is a potent ovotoxicant, and the developing ovary is particularly sensitive. Exposure to tobacco smoke, which contains high concentrations of BaP and other PAHs, is associated with decreased fecundity and earlier menopause in the daughters of women who smoked during pregnancy. We have shown that prenatal exposure of mice to BaP during primordial germ cell migration through the onset of meiosis causes POF in the F1 female offspring at doses that do not affect ovarian follicle numbers in the mothers. We further showed that embryos deficient in synthesis of the antioxidant glutathione (GSH) due to deficiency in the modifier subunit of glutamate cysteine ligase (Gclm) are more sensitive to the transplacental ovotoxicity of BaP than wild type littermates. Our preliminary data further show that BaP induces apoptosis in germ cells of cultured fetal ovaries and that Gclm null ovaries are more sensitive to the induction of germ cell apoptosis by BaP. In the current proposal we will test the hypothesis that BaP depletes germ cells in the prenatal ovary by inducing oxidative stress and apoptosis, while inducing heritable epigenetic changes in surviving germ cells to cause accelerated depletion of ovarian follicles in subsequent generations, and that GSH is protective against these effects. We will test this hypothesis in two aims: 1) To establish the critical window of development for and mechanisms of the transplacental ovotoxicity of BaP and to define the mechanism by which GSH modulates BaP-induced ovotoxicity during the critical window. We will use complementary in vivo transplacental exposure and cultured embryonic ovary models. We will test the potential protective effects of supplementation with GSH and other antioxidants. 2) Test whether the ovarian phenotype of prenatal exposure to BaP is transgenerational and is mediated by epigenetic changes in the germ line. We will utilize RNA- sequencing to examine genomewide gene expression and will assess global DNA methylation using MBD- Sequencing in F1, F2, and F3 primordial germ cells from BaP exposed compared to control lineages.