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

协调的基因表达网络对硬骨鱼有性生殖的调节至关重要。表观基因组，包括甲基组，是基因表达的关键调节器，并对环境刺激作出反应。甲基组在胚胎发生期间被重新编程。由于甲基组在有丝分裂过程中是稳定的，因此在胚胎发生期间暴露于环境刺激所引起的甲基组改变会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上也是稳定的，因此鱼类世代的繁殖可能受到祖先对雄性或雌性生殖系甲基组的改变的影响。因此，甲基组在早期发育阶段的改变有可能对生殖产生长期影响。然而，这一点在硬骨鱼中却知之甚少。本研究的目的是描述甲基组作为暴露于内分泌干扰化学物质的鱼类繁殖性能的关键调节剂的特征。在胚胎发生过程中，将第一代（F1）黑头鲦鱼（Pimephales promelas）暴露于17-乙炔雌二醇（EE2，模型雌激素）或trenbolone（模型雄激素）中，并对生殖生理学，包括性腺发育，性腺功能（类固醇发生）和生殖（繁殖力，受精成功）的影响进行表征。性成熟雄性和雌性胚胎和性腺甲基组的改变将使用靶向（甲基化特异性PCR）和开放格式（增强减少亚硫酸氢盐转换测序）方法进行表征。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。由于甲基组的改变可能遗传，F2和F3代后代的生殖生理将被确定。拟议的研究还将调查甲基组是否介导了在胚胎发育期间暴露于内分泌干扰化学物质的黑头鱼的适应性反应，这使得成年鱼在繁殖过程中更好地应对再次暴露于这些化学物质。这些适应性反应在F2和F3代的遗传力将被确定。最终，将建立暴露于内分泌干扰化学物质的黑头鲦鱼生命早期甲基组改变与成年鱼生殖性能功能变化之间的联系。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。这项研究也有可能改善目前没有考虑暴露于化学压力源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.