背景:产前尼古丁暴露(PNE)已被证明会对胎儿发育造成许多有害影响。然而,尼古丁暴露对生殖细胞的表观遗传变化仍未得到很好的理解。
目的:在本研究中,我们专注于阐明产前尼古丁暴露对生殖细胞发育重要的调节表观遗传机制的影响。
方法:Sprague-Dawley大鼠在怀孕期间暴露于尼古丁,并在11周龄时分析雄性后代。使用冷冻睾丸切片分析睾丸形态,并通过RT-qPCR检查生殖细胞标志物的表达;通过蛋白质印迹(WB)评估组蛋白修饰。通过亚硫酸氢盐转化的DNA的甲基化特异性PCR进行DNA甲基化分析。使用甲基化DNA免疫沉淀(MeDIP)-seq分析全基因组DNA甲基化。我们还通过RNA-seq对垂体进行了转录组学分析。
结果:我们表明,妊娠期暴露于尼古丁会减少生殖细胞数量,扰乱减数分裂,影响种系重编程响应基因的表达,并影响睾丸中神经系统基因的DNA甲基化。PNE还引起脑垂体中基因表达的扰动。
结论:我们的数据表明,PNE导致男性精子发生的扰动,观察到的效应与周围神经系统信号通路的变化有关。与细胞迁移等多种生物活性相关的基因表达变化,垂体中的细胞粘附和GABA信号强调了怀孕期间尼古丁暴露影响的复杂性。
BACKGROUND: Prenatal nicotine exposure (PNE) has been documented to cause numerous deleterious effects on fetal development. However, the epigenetic changes promoted by nicotine exposure on germ cells are still not well understood.
OBJECTIVE: In this study, we focused on elucidating the impact of prenatal nicotine exposure on regulatory epigenetic mechanisms important for germ cell development.
METHODS: Sprague-Dawley rats were exposed to nicotine during pregnancy and male progeny was analyzed at 11 weeks of age. Testis morphology was analyzed using frozen testis sections and expression of germ cell markers was examined by RT-qPCR; histone modifications were assessed by Western Blot (WB). DNA methylation analysis was performed by methylation-specific PCR of bisulfite converted DNA. Genome-wide DNA methylation was analyzed using Methylated DNA immunoprecipitation (MeDIP)-seq. We also carried out transcriptomics analysis of pituitary glands by RNA-seq.
RESULTS: We show that gestational exposure to nicotine reduces germ cell numbers, perturbs meiosis, affects the expression of germ line reprogramming responsive genes, and impacts the DNA methylation of nervous system genes in the testis. PNE also causes perturbation of gene expression in the pituitary gland of the brain.
CONCLUSIONS: Our data demonstrate that PNE leads to perturbation of male spermatogenesis, and the observed effects are associated with changes of peripheral nervous system signaling pathways. Alterations in the expression of genes associated with diverse biological activities such as cell migration, cell adhesion and GABA signaling in the pituitary gland underscore the complexity of the effects of nicotine exposure during pregnancy.