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类的有性生殖是必不可少的。表观基因组，包括甲基化组，是基因表达的关键调节因子，并对环境刺激作出反应。甲基化组在胚胎发生期间被重编程。因为它是有丝分裂稳定的，在胚胎发生过程中暴露于环境刺激引起的甲基化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。因为甲基化组也是减数分裂稳定的，所以鱼类的世代生殖可以受到雄性或雌性生殖系的甲基化组的祖先改变的影响。因此，在非常早期的发育阶段甲基化组的改变有可能对生殖造成长期影响。然而，这在硬骨鱼中知之甚少。这里描述的研究的目标是表征甲基化作为一个关键的调节器的生殖性能的鱼类暴露于内分泌干扰化学品。第一代（F1）黑头呆鱼（Pimephales promelas）将在胚胎发生期间暴露于17-乙炔基炔雌醇（EE 2，模型雌激素）或群勃龙（模型雄激素），并表征对生殖生理学的影响，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）。将使用靶向（甲基化特异性PCR）和开放格式（增强型还原亚硫酸氢盐转化测序）方法表征性成熟雄性和雌性胚胎和性腺甲基化组的改变。将使用靶向（qPCR）和开放式（RNAseq）转录组学确定由甲基化组改变引起的基因表达的持续变化。由于甲基化组改变的遗传潜力，将确定F2和F3代后代的生殖生理学。拟议的研究还将调查甲基化是否介导胚胎发育过程中暴露于内分泌干扰化学物质的黑头呆鱼的适应性反应，这使得成鱼能够更好地科普繁殖过程中再次暴露于这些化学物质。将确定F2和F3代这些适应性反应的遗传力。最终，将建立在暴露于内分泌干扰化学品的黑头呆鱼的早期生命阶段的甲基化组的改变与成鱼生殖性能的功能变化之间的联系。因此，拟议的研究将解决有关硬骨鱼生殖调控中甲基化作用的知识空白。这项研究也有可能改善环境风险评估，目前没有考虑到暴露于化学压力的世代影响。

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.