Generational Effects of Chemical Stressors - Molecular Mechanisms of Altered Reproductive Performance

基因表达的协调网络对于调节硬骨鱼的有性繁殖是必不可少的。表观基因组，包括甲基组，是基因表达的关键调节因子，对环境刺激有反应。在胚胎发育过程中，甲基组被重新编程。由于它在有丝分裂中是稳定的，在胚胎发育过程中暴露在环境刺激下引起的甲基组的改变可以影响整个发育过程中的基因表达，从而影响生殖等生理过程。由于甲基组在减数分裂中也是稳定的，后代鱼类的繁殖可能会受到祖先对雄性或雌性生殖系甲基组的改变的影响。因此，在非常早期的发育阶段，甲基组的改变有可能对生殖造成长期影响。然而，对硬骨鱼来说，这一点却知之甚少。本文描述的研究目的是将甲基组描述为暴露在内分泌干扰化学物质下的鱼类繁殖性能的关键调节因素。在胚胎发生过程中，第一代肥头小鱼(Pimephales Proelas)将暴露于17-乙炔基雌二醇(EE2，模型雌激素)或特伦勃隆(模型雄激素)，并将表征其对生殖生理的影响，包括性腺发育、性腺功能(类固醇生成)和生殖(繁殖力、受精成功)。性成熟男性和女性胚胎和性腺的甲基组的改变将使用目标(甲基化特异性聚合酶链式反应)和开放格式(增强的简化代表性亚硫酸氢盐转化测序)方法来表征。甲基组改变引起的基因表达的持续变化将使用靶向(QPCR)和开放格式(RNAseq)转录组学来确定。由于甲基组改变的遗传可能性，将确定F2和F3后代的生殖生理。这项拟议的研究还将调查甲基组是否在胚胎发育期间暴露于内分泌干扰物的愚头鱼身上调节适应性反应，这使成年鱼能够更好地应对繁殖期间再次暴露于这些化学品。这些适应性反应的遗传力将由F2和F3世代确定。最终，暴露在内分泌干扰物中的愚头鱼早期生命阶段甲基组的变化与成年鱼繁殖性能的功能变化之间的联系将被建立。因此，拟议的研究将解决关于甲基组在硬骨鱼生殖调节中的作用的知识空白。这项研究还有可能改进目前没有考虑化学应激源暴露的代际影响的环境风险评估。

Co-ordinated networks of gene expression are essential for regulation of sexual reproduction of teleost fishes. The epigenome, including the methylome, is a critical regulator of gene expression and is responsive to environmental stimuli. The methylome is reprogrammed during embryogenesis. Because it is mitotically stable, alterations to the methylome caused by exposure to environmental stimuli during embryogenesis can affect gene expression throughout development, and thus physiological processes such as reproduction. Because the methylome also is meiotically stable, reproductive by generations of fishes can be affected by ancestral alterations to the methylome of the male or female germ line. Thus, alterations of the methylome at very early developmental stages has the potential to cause long-term effects on reproduction. However, this is poorly understood in teleost fishes. The goal of the research described here is to characterize the methylome as a critical regulator of the reproductive performance of fishes exposed to endocrine disrupting chemicals. First (F1) generation fathead minnows (Pimephales promelas) will be exposed either to 17-ethynylestradiol (EE2, model estrogen) or trenbolone (model androgen) during embryogenesis and effects on reproductive physiology, including gonad development, gonad function (steroidogenesis), and reproduction (fecundity, fertilization success) will be characterized. Alterations to the methylome of embryos and gonads from sexually mature males and females will be characterized using targeted (methylation specific PCR) and open format (enhanced reduced representation bisulfite conversion sequencing) methods. Persistent changes in gene expression arising from alterations to the methylome will be determined using targeted (qPCR) and open-format (RNAseq) transcriptomics. Because of the potential for inheritance of alterations to the methylome, the reproductive physiology of F2 and F3 generations of progeny will be determined. The proposed research also will investigate whether methylome mediates adaptive responses in fathead minnows exposed to endocrine disrupting chemicals during embryogenesis, which allow adult fish to better cope with re-exposure to these chemicals during reproduction. Heritability of these adaptive responses by F2 and F3 generations will be determined. Ultimately, linkages between alterations to the methylome in early life-stages of fathead minnows exposed to endocrine disrupting chemicals and functional changes in reproductive performance of adult fish will be established. Thus, the proposed research will address knowledge gaps regarding the role of the methylome in regulation of reproduction in teleost fishes. This research also has the potential to improve environmental risk assessments that currently do not consider the generational effects of exposure to chemical stressors.