Epigenetic transgenerational effects of endocrine disruptors via female germ line

描述（由申请人提供）：众所周知，在子宫内和/或新生儿发育期间暴露于环境应激源或毒物（如内分泌干扰物（EDCs））可导致成人发病疾病。这种有害变化与异常的DNA甲基化模式以及其他表观遗传机制有关。最近的研究表明，这种疾病的易感性可以通过表观遗传机制传播给从未直接暴露于EDCs的后代，这是非常令人担忧的。例如，在子宫内暴露于EDC后，睾丸异常通过雄性生殖系传递给F2-F4代雄性。然而，发现这些效应具有性别特异性，因为F2-F3雌性动物表现出较温和的表型，需要父母双方暴露于EDC。在本提案中，我们的目标是解决女性生殖系是否参与EDC暴露的跨代（TG）效应的关键问题。甲氧滴滴涕（MXC）是一种EDC，具有模仿环境中许多其他EDC的作用，并显示会引起性腺的表观遗传变化;因此，在本提案中将其用作模型EDC。我们的假设是，在发育的关键窗口期（此处定义为通过卵母细胞表观遗传重编程的性腺分化期）暴露于MXC，导致卵巢中的TG效应，其由卵母细胞中的表观遗传变化介导。为了验证我们的假设，该提案有两个相互关联但不同的具体目标：（1）确定暴露于F1后F3代卵巢中MXC的体细胞TG表观遗传效应。F1代近交系雌性大鼠将在E11和PND 7之间接受0、20 5 g/kg/天MXC或50-100 mg/kg/天MXC给药。这些雌性动物将与未给药雄性动物交配，并产生F2-F4雌性动物。最初，仅在F3代雌性动物中评估卵巢形态和基因表达以及生育力，因为在F3代中观察到TG效应是确定TG效应的最低要求。基因表达的变化和全球和基因特异性DNA甲基化的变化将使用传统的方法以及基于阵列的方法进行研究。(2)研究MXC在F1/F2卵母细胞中诱导的表观遗传变化，这些变化可能导致F3中的TG效应。将分析印迹基因和寄生DNA序列（如卵母细胞中的反转录转座子）中的DNA甲基化模式，因为这些序列中的异常甲基化模式可能是致病性的。还将检查卵细胞基因表达模式;将阐明基因表达变化与DNA甲基化之间的任何相关性。该研究将首次研究通过F2代以外的雌性生殖系传播的EDC的表观遗传TG效应。我们认识到与这项研究相关的相当大的风险，但考虑到环境中广泛存在的内分泌干扰物，对今世后代的健康影响是重大的。对MXC的TG表观遗传作用的机械理解将拓宽我们对EDC如何破坏女性正常表观遗传编程的知识，并促进预防和治疗EDC诱导的TG异常的策略的发展。 公共卫生相关性：在发育过程中暴露于环境内分泌干扰化学品可能会伤害暴露的个体，这种影响可以通过生殖细胞内的表观遗传改变传递给他们的子孙。本研究的目的是调查女性内分泌干扰物的跨代表观遗传效应，并了解这些化合物影响生殖细胞并将这些影响传递给后代的机制。更好地了解内分泌干扰物的跨代表观遗传效应对公共卫生有直接影响，因为这种了解可以帮助我们预防和改善这些影响，从而减少未来的健康成本。

DESCRIPTION (provided by applicant): It is well established that exposure to environmental stressors or toxicants such as endocrine-disrupting chemicals (EDCs) during in utero and/or neonatal development can result in adult-onset diseases. Such detrimental changes have been linked to aberrant DNA methylation patterns as well as other epigenetic mechanisms. It is of great concern that recent studies have suggested that such disease susceptibilities can be transmitted via epigenetic mechanisms to subsequent generations that were never directly exposed to the EDCs. For example, testicular abnormalities were transmitted to F2-F4 generation males via the male germ line after in utero exposure to an EDC. However, these effects were found to be sex-specific since F2-F3 females exhibited milder phenotypes that required exposure of both parents to the EDC. In this proposal, our objective is to address the critical issue of whether the female germ line is involved in such transgenerational (TG) effects of EDC exposure. Methoxychlor (MXC) is an EDC that has actions mimicking those of many other EDCs in the environment and was shown to cause epigenetic changes in the gonad; it will therefore be used as a model EDC in this proposal. Our hypothesis is that exposure to MXC during the critical window of development, defined here as the period of gonad differentiation through oocyte epigenetic reprogramming, leads to TG effects in the ovary that are mediated by epigenetic changes in the oocyte. To test our hypothesis, the proposal has two interrelated but distinct specific aims: (1) Determine somatic TG epigenetic effects of MXC in the ovary of F3 generation following exposure of F1. F1-generation inbred female rats will be treated with 0, 20 5g/kg/day MXC, or 50-100 mg/kg/day MXC between E11 and PND7. These females will be bred with untreated males, and F2-F4 females will be generated. Initially, ovarian morphology and gene expression and fertility will be assessed only in the F3 females since observation of TG effects in F3 generation is the minimum requirement for establishing TG effects. Gene expression changes and global and gene-specific DNA methylation changes will be investigated using conventional methods as well as array-based approaches. (2) Investigate the epigenetic changes induced by MXC in F1/F2 oocytes that may lead to TG effects in F3. DNA methylation patterns in imprinted genes and parasitic DNA sequences such as retrotransposons in oocytes will be analyzed, as aberrant methylation patterns in these sequences can be pathogenic. Oocytic gene expression patterns will also be examined; any correlation between the changes in gene expression and DNA methylation will be elucidated. The proposed study will be the first to investigate the epigenetic TG effects of EDCs transmitted via the female germ line beyond the F2 generation. We recognize the considerable risk associated with this study, but implications to the health of present and future generations are significant given the widespread presence of EDCs in the environment. A mechanistic understanding of the TG epigenetic actions of MXC will broaden our knowledge of how EDCs, in general, disrupt normal epigenetic programming in females, and facilitate the development of strategies to prevent and treat EDC-induced TG abnormalities. PUBLIC HEALTH RELEVANCE: Exposure to environmental endocrine-disrupting chemicals during development can harm the exposed individuals, and this effect can be passed on to their children and grandchildren through epigenetic alterations within the germ cells. The objectives of this research are to investigate the transgenerational epigenetic effects of endocrine disruptors in females and to understand the mechanisms by which these compounds affect the germ cells and transmit these effects to subsequent generations. A better understanding of transgenerational epigenetic effects of endocrine disruptors has direct implications for public health because such an understanding can help us prevent and ameliorate these effects, thereby reducing future health costs.