Integrative proteome analyses implicate aberrant RNA splicing impairs developmental potential of aged mouse oocytes. Integrative proteome analyses implicate aberrant RNA splicing impairs developmental potential of aged mouse oocytes

Aging has many effects on female reproductive system, among which declined oocyte quality and impaired embryo developmental potential are the most important factors affecting female fertility. However, the mechanisms of oocyte aging have not yet been fully understood due to the complex and multifactorial nature of this physiological process. Here, we selected normal reproductively aging female mice as research object and constructed protein expression profile of metaphase II (MII) oocytes from three age groups. A total of 187 differentially expressed (DE) proteins were identified, and bioinformatics analysis showed that these DE proteins were highly enriched in the RNA splicing. Next, RNA-seq was performed on 2-cell embryos from these three age groups, splicing analysis showed that there were a total of 1375 differentially spliced genes (DSGs) and 2363 differentially spliced events (DSEs). DSGs in the reproductive aging groups versus the younger group were enriched in biological processes related to DNA damage repair/response. Binding motif analysis suggested PUF60 may be the core splicing factor that causes the decline of the developmental potential of oocytes in reproductive aging mice, and changing the splicing pattern of its potential downstream DSG Cdk9 could partially mimic phenotypes in the reproductive aging groups. Taken together, our study suggested the abnormal expression of splicing regulation protein in aging MII oocytes will affect the splicing of nascent RNA after zygotic genome activation in 2-cell embryos, leading to the production of abnormally spliced transcripts of some key genes associated with DNA damage repair/response, thus affected the developmental potential of aged oocytes. Overall design: Total 14 samples from 8-10w, 6-8m and 10-12m mice from MII oocytes and 2-cell embryos.

衰老可对雌性生殖系统产生诸多影响，其中卵母细胞质量下降与胚胎发育潜能受损是影响雌性生育能力的核心因素。然而，由于该生理过程复杂且受多因素调控，卵母细胞衰老的分子机制尚未完全阐明。本研究以正常生殖衰老雌性小鼠为研究对象，构建了三个年龄组的减数分裂II期（MII）卵母细胞蛋白质表达谱。共鉴定得到187个差异表达（DE）蛋白，生物信息学分析显示，这些差异蛋白显著富集于RNA剪接通路。随后，我们对三个年龄组的2-细胞期胚胎开展RNA测序（RNA-seq），剪接分析共鉴定出1375个差异剪接基因（DSGs）及2363个差异剪接事件（DSEs）。相较于年轻组，生殖衰老组的差异剪接基因显著富集于DNA损伤修复/应答相关的生物学过程。结合基序分析结果提示，PUF60可能是导致生殖衰老小鼠卵母细胞发育潜能下降的核心剪接因子；改变其潜在下游差异剪接基因Cdk9的剪接模式，可部分模拟生殖衰老组的表型。综上，本研究表明：衰老MII期卵母细胞中剪接调控蛋白的异常表达，会影响2-细胞胚胎合子基因组激活后新生RNA的剪接过程，导致部分与DNA损伤修复/应答相关的关键基因产生异常剪接转录本，进而降低衰老卵母细胞的发育潜能。整体实验设计：本研究共纳入14份样本，分别来自8~10周龄、6~8月龄及10~12月龄小鼠的MII期卵母细胞与2-细胞期胚胎。