阴囊热激致子代糖代谢异常且跨代遗传的表观遗传机制研究

IVF/ICSI子代表观遗传相关疾病较自然生育增加，但原因和机制不明，并非都由于辅助生殖技术本身引起，基于“配子源性疾病”理论，更可能是致不孕不育的因素本身引起配子遗传/表观遗传等异常而影响子代健康。我们前期发现阴囊热激小鼠生育子代糖代谢有明显异常，且初步表明有跨代遗传。本课题拟进一步明确子代糖代谢异常和跨代遗传的发生规律，转录组学研究其糖代谢靶器官基因表达；全基因组甲基化测序分析阴囊热激致精子DNA甲基化的改变，分析差异甲基化区域（DMRs）直接调控糖代谢靶器官表达差异基因，和／或通过转录因子间接调节的关联，筛选和研究主要的精子DMRs和靶器官糖代谢基因，然后在子代小鼠精子、囊胚、胚胎及靶器官中分析主要DMRs和基因表达和传递，从而研究精子表观遗传变化致子代糖代谢异常及跨代遗传的机制。课题将为阴囊热激相关男性不育患者生育健康子代提供依据，为辅助生殖技术子代健康影响因素研究提供新角度。

Epigenetic disorders remain more common in IVF/ICSI offsprings compared with non-IVF/ICSI offsprings. However the causes and mechanisms remain largely unknown. Based on the theory of gamete and embryo-fetal origins of adult diseases, it is reasonable to postulate that factors lead to infertility more likely to be the potential cause of some diseases in IVF/ICSI offspring, other than IVF/ICSI technology, because some of these factors can directly cause epigenetic abnormalities in the gamete. Scrotal heat stress (SHS) is a common cause of male infertility, and fmSHS exists in men with some lifestyles, occupations and pathological factors. All these factors leading to SHS can impair spermatogenesis or sperm quality, thus, a large numbers of suffered people have to seek assisted reproductive technology to obtain their own offspring. For those “heated” males who are seeking assisted reproductive technology, the heat they exposed to is usually consecutive rather than transient; and heat temperature is not too high, because frequent and strong SHS led to azoospermia. To maximally mimic clinical conditions, we have established a mouse model with frequent and mild scrotal heat stress. Using this model, our preliminary research observed glucose metabolism disorder (GMD) and transgenerational inheritance in the offsprings of “heated” mice. The present proposal aims to reveal the epigenetic mechanism of the GMD and its transgenerational inheritance. We will reveal patterns of the occurrence, trangenerational inheritance, and reversibility of GMD. Gene expression profiles of organs related to glucose metabolism will be determined by RNA-seq to identify differentiated expressed genes. Based on our preliminary results of promoter methylation of some genes related to glucose metabolism in organs of GMD offspring, we chose the DNA methylation for the epigenetic mechanism research. We will analyze sperm DNA methylation by whole genome bisulfite sequencing to screen differentiated methylation regions (DMRs). The mechanism of how sperm epigenetic changes (DNA methylation) cause gene expression abnormalities in GMD offspring will be elucidated by the overlaps of sperm DMRs and differentiated expressed genes in GMD offspring organs, and sperm DMRs related to transcriptional factors. The regulation of Key DMRs and genes will be validated in cells in vitro. Transgenerational inheritance will be explored by determination of methylation of key DMRs in offspring sperm, and expression of genes responsible for GMD in embryos and offspring organs. This study will provide reference for offspring health of “heated” males, and provide a novel aspect for the research of offspring health of assisted reproductive techonology.