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

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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 17α-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.