The Molecular Mechanism Of Ubiquitin Conjugating Enzyme UBE2B Regulating Meiosis Of Spermatocyte

Meiosis is the unique cell division of eukaryotes in sexual reproduction,in which cells undergo one round of DNA replication,followed by two successive divisions,and eventually produce gametes with half their genetic material.Homologous recombination,the most essential event in meiosis,begins with programmed DNA double stranded breaks(DSBs).With the pairing and synapsis of homologous chromosomes,a small fraction of DSBs are repaired to form crossovers.Crossovers are necessary for the proper separation of homologous chromosomes and the exchange of genetic material.Ubiquitination plays an important role in the regulation of homologous recombination.UBE2B is a ubiquitin conjugating enzyme,it is reported that that genetic variations of UBE2B are highly related to male infertility;and male mice lacking Ube2b are infertile due to apoptosis of spermatocytes in the seminiferous tubules.However,the molecular mechanism of UBE2B in spermatocyte meiosis is unclear.In order to clarify the molecular mechanism of UBE2B in meiotic recombination,we constructed Ube2b knockout mice using CRISPR-Cas9 technology.Through morphological and histological observations,we confirmed that male mice lacking UBE2B were sterile and a large number of germ cells were lost in the seminiferous tubules.Cytological immunofluorescence analysis found that spermatocytes of Ube2b knockout mice were arrest at the diplotene stage.Chromosome axis length increased significantly in the late zygotene,and the number of crossovers also increased.The distribution of MSH4,RNF212 and HEI10 on the chromosome has changed significantly,which indicates that the processing and maturation of the recombination intermediates are abnormal,which ultimately leads to the abnormal crossover.We have not seen the global changes of ubiquitin and SUMO in spermatocytes,which means that UBE2B is not necessary for large-scale ubiquitination and SUMO in cells.Transcriptomics analysis showed that spermatocyte gene expression of Ube2b knockout mice was mainly down-regulated,but the sex chromosome differential genes were significantly up-regulated,which indicated abnormal meiotic sex chromosome inactivation.Yeast two-hybrid results showed that UBE2B interacts with the ubiquitin ligase UBR2,and the absence of UBE2B may cause LINE1-ORF1p unable to be degraded by ubiquitination modification,LINE1 is highly expressed in spermatocytes,which causes abnormal homologous recombination of spermatocytes.Our results demonstrate that UBE2B deletion cause abnormal chromosome structure and homologous recombination.It provides new insights for in-depth understanding of the molecular mechanism of homologous recombination regulation.