Hypoxia causes epigenetic changes and transgenerational reproductive impairments in marine medaka (Oryzias melastigma)

Abstract

Aquatic hypoxia (≤ 2.8 mg/l of dissolved oxygen) is one of the most pressing ecological problems in both marine and freshwater environments worldwide. Earlier laboratory and field studies have demonstrated that hypoxia can alter the expression of specific genes along the brain-pituitary-gonad axis, thereby disrupting the homeostasis of sex hormones and ultimately resulting in reproductive impairments in fish.

Certain endocrine disruptors have been shown to cause epigenetic changes and transgenerational effects in mammals and fish, including reproductive dysfunction, infertility, and abnormal development. Using marine medaka (Oryzias melastigma) as a model, this study set out to explore whether hypoxia can induce epigenetic changes and transgenerational effects on male reproduction.

Experiments were conducted to unravel the effects of hypoxia at the epigenetic, genetic, protein and phenotypic levels in male fish from F0 to F2. Sexually mature F0 marine medaka were maintained under normoxia or hypoxia for one month.

Eggs from the normoxic controls were continuously raised in normoxia for two generations (normoxic group). Eggs from the hypoxic treatment were divided into two batches: one batch was continuously raised under hypoxia for two generations (hypoxic group); and the other batch was returned to normoxia and raised for two generations (transgenerational group).

Genomic DNA methylation was examined by liquid chromatography – tandem mass spectrometry (LCMS-MS) and genome-wide methylated DNA immunoprecipitation sequencing (MeDIP-Seq). The transcriptomic and proteomic profiles were investigated using RNA-Seq and isobaric tags for relative and absolute quantification (iTRAQ), respectively. The identified genes or proteins showing transgenerational effects were further validated by real-time qPCR and western blot analysis, respectively. Gonadosomatic index, sperm motility, the number of spermatids, fertilization success and hatching success were also measured and compared among the normoxic, hypoxic and transgenerational groups to assess phenotypic changes.

F0 fish exposed to hypoxia can cause a decline in sperm motility and spermatid quantity in their F1 and F2 progenies, despite they having never been directly exposed to hypoxia. In both F0 and F2, the observed transgenerational phenotypic changes were associated with differential methylation in forkhead box protein P2 (FOXP2), euchromatic histone-lysine N-methyltransferase 2 (EHMT2) and protein tyrosine kinase 2 beta (PTK2B). The transcriptomic analysis provided further evidence that EHMT2 and PTK2B were upregulated. Elevation of dimethylated Histone H3 lysine 9 (H3K9) mediated by EHMT2 was consistently found in F0, F1 and F2, which may account for the observed reproductive impairment. The aberrant and consistent DNA methylation found in fish sperm demonstrated that hypoxia can make epigenetic marks in the germline, which can be passed on to subsequent generations, leading to transgenerational phenotypic changes. The transgenerational reproductive impairments caused by hypoxia, as revealed in this study, suggest that the detrimental effects on fish in aquatic environments have been grossly under-estimated. Because epigenetic changes and regulation of spermatogenesis are highly conserved in animals, the results of this study may imply that hypoxic stresses, resulting from high latitude and sleep apnea, may potentially lead to similar epigenetic changes and transgenerational reproductive impairments in humans.