Functional and epigenetic effects of preconceptional EDCs on the female HPG axis

摘要 暴露于环境内分泌干扰物(EDCs)，特别是在生命早期， 与不利的生殖后果有关。多氯联苯(PCB)是一类广泛存在的 EDCS，在工业应用中使用了几十年。虽然几十年前就被禁止了，但人类暴露在多氯联苯中 今天主要是通过食物链。在人类和动物中，较高的多氯联苯身体负担都是相关的 有内分泌和生殖功能障碍。然而，在这方面有几个重要的知识空白 我们建议填充的字段。首先，虽然多氯联苯扰乱了下丘脑-垂体-性腺(HPG)轴的每一级 当单独评估时，这些级别之间的串扰尚未被探索。第二，虽然很早 发育(胎儿、婴儿)被认为是易患内分泌细胞疾病的时期，先入为主的时期是 另一个尚未研究多氯联苯对HPG系统的作用的关键窗口，特别是在雌性系统中， 一些我们打算纠正的事情。第三，绝大多数EDC暴露后的表型分析是进行的 在一个单一的生命阶段，通常是在成年。然而，生殖系统经历了巨大的动态变化 在整个生命的结构和功能方面，强调了采用生命周期方法进行表型分析的必要性。这个 第四，也可能是最大的差距是，先入为主的暴露是否以及如何重新编程生殖系 表观基因组，如果这导致了躯体效应。在这个提案中，我们将开发和利用一部小说 先入性接触多氯联苯的代际模型。我们将喂养怀孕的斯普拉格-道利大鼠水坝 (F0祖母)人类相关水平的多氯联苯混合物或载体，来自E8-18，当生殖细胞表观遗传时 F1胎儿的性腺中的标记被擦除；和/或当F1幼崽在P1-21岁和 当生殖细胞表观遗传标记被重新建立时。F1雌鼠与未经处理的雄鼠交配以 生成F2代。F2雌性后代(孙子)将在3个生殖年龄段进行表型分析 阶段：新生儿、青春期和成人。生理结果、关键HPG基因和蛋白的表达 参与控制生殖能力和潜在的分子表观遗传标记(DNA和组蛋白 甲基化)将被评估。此外，我们将使用一种新的方法分离猪原始生殖细胞(PGC)。 新生F1和F2雌性大鼠的多氯联苯和载体素谱系，以量化基因表达和甲基化 马克。这使我们能够将HPG表型与先前的生殖细胞编程联系起来。因此，这项建议是 预计将为先入为主的多氯联苯暴露对女性HPG的影响提供新的见解 生理、功能和潜在的表观遗传调控机制，其结果是直接相关的 女性的环境健康。

ABSTRACT Exposures to environmental endocrine-disrupting chemicals (EDCs), especially during early life, are strongly linked to adverse reproductive outcomes. Polychlorinated biphenyls (PCBs) are a well-established family of EDCs, used for decades in industrial applications. While banned decades ago, humans are exposed to PCBs today primarily through the food chain. In both humans and animals, higher PCB body burdens are associated with endocrine and reproductive dysfunctions. However, there are several important gaps in knowledge in this field that we propose to fill. First, while PCBs perturb each level of the hypothalamic-pituitary-gonadal (HPG) axis when assessed individually, the cross-talk among these levels has not been explored. Second, while early development (fetus, infant) is recognized as a period of vulnerability to EDCs, the preconceptional period is another critical window that has not been studied for PCB actions on HPG systems, especially in females, something we propose to redress. Third, the vast majority of phenotyping following EDC exposures is conducted at a single life stage, typically in the adult. Yet, the reproductive system undergoes enormous dynamic change in structure and function throughout life, underscoring the need for a lifecycle approach to phenotyping. The fourth and perhaps largest gap is whether and how preconceptional exposures reprogram the germline epigenome, and if that leads to somatic effects. In this proposal, we will develop and utilize a novel intergenerational model of preconceptional exposure to PCBs. We will feed pregnant Sprague-Dawley rat dams (F0 grandmothers) human-relevant levels of a PCB mixture, or vehicle, from E8-18, when germ cell epigenetic marks are being erased in the gonads of the F1 fetuses; and/or during lactation when F1 pups are P1-21 and when germ cell epigenetic marks are re-established. The F1 females are bred with untreated male rats to generate the F2 generation. The F2 female offspring (grandchildren) will be phenotyped at 3 reproductive life stages: neonatal, pubertal, and adult. Physiological outcomes, expression of key HPG genes and proteins involved in the control of reproductive competence, and underlying molecular epigenetic marks (DNA and histone methylation) will be assessed. In addition, we will use a novel method to isolate primordial germ cells (PGCs) of newborn F1 and F2 females rats of the PCB and vehicle lineage, to quantify gene expression and methylation marks. This allows us to relate HPG phenotypes to prior germ cell programming. Thus, this proposal is anticipated to provide novel insights into the effects of preconceptional PCB exposures on female HPG physiology, function, and underlying epigenetic mechanisms of regulation, results of which are directly relevant to women’s environmental health.