Study On The Mechanism Of Postovulatory Aging Impairs Oocyte Quality In Vitro

Postovulatory aging of oocyte is mainly divided into in vitro aging and in vivo aging.The aged oocytes affect the fertilization efficiency and the subsequent early embryo development,which leads to the lower success rate of assisted reproductive technology.However,the molecular mechanism of the deterioration of oocyte quality caused by oocyte aging after ovulation is still unclear and deserves further investigation.In this study,oocytes from 6-week-old female ICR mice with h CG for 14 h were cultured and aged in vitro 24 h later as experimental subjects.Firstly,the phenotypes of aged oocytes in vitro were investigated by WB,in vitro fertilization,immunofluorescence staining and other experiments.Secondly,protein profiling and transcriptome techniques were used to detect the key proteins of deterioration of oocyte quality due to postovulatory aging in vitro.Finally,the key proteins were overexpressed by microinjection,and then the mechanism of oocytes aging in vitro was investigated.The main results were as follows :(1)through phenotypic exploration experiments,we found that oocytes aging in vitro resulted in significantly increased reactive oxygen content in oocytes,impaired spindle assembly,abnormal mitochondrial distribution and increased aneuploidy rate,significantly increased oocytes fragmentation rate and other phenotypes,which affected the development of early embryos after fertilization.(2)protein spectrum sequencing results showed that 121 differentially expressed proteins in postovulatory-aged oocyte in vitro compared with fresh oocytes.After the GO functional annotations and KEGG pathway enrichment analysis of these differentially expressed proteins was mainly divided into splicing complex of protein,cell cycle and the nuclear mass transportation,protein processing procedure in the endoplasmic reticulum and oocyte meiosis pathway.The result of transcriptome sequencing was consistent with that of protein spectrum.We screened with maternal mRNA degradation related proteins DCP1 A and YBX2;meiotic spindle assembly related proteins SPDL1 and TTK.To explore the mechanism of postovulatory aging of oocyte in vitro in these two aspects.(3)Compared with the fresh oocytes,the expression level of DCP1 A in postovulatoryaged oocytes was significantly increased,while the expression level of YBX2 was significantly decreased.In the overexpression experiment,it was proved that overexpression of DCP1 A could accelerate degradation of maternal mRNA in oocytes.In addition,the expression levels of SPDL1 was decreased in postovulatory-aged oocytes,and the results of overexpression experiment showed that overexpression of SPDL1 protein could to partially restores aberrant spindle assembly and incorrect chromosome alignment in postovulatory-aged oocytes.In conclusion,DCP1 A and YBX2 proteins play an important role in the maternal mRNA degradation of postovulatory-aged oocytes.Secondly,insufficiency of SPDL1 and TTK led to aberrant spindle assembly,incorrect chromosome alignment and aneuploidy in postovulatory-aged oocytes.These results provide a new basis for the study of postovulatory aging of oocyte in vitro and providing fundamental basis for improving the success rate of assisted reproduction.