Preliminary Study On The Functions Of Prmt5 In Gonad And Early Embryogenesis Of Medaka

PRMT5,a type II protein arginine methyltransferase that can catalyze the methylation of many compounds,participates in various processes by interacting with multi-subunit complexes,including RNA processing,signal transduction,transcriptional regulation,and germ cell development.PRMT5 is crucial for cell growth,proliferation and development.Based on the completed mutation experiment of the prmt5 gene of the medaka by TALEN technology,this research continues to carry out the work of offspring screening and functional research.This experiment initially explores the function of prmt5 in gonads and early embryonic development.The main results obtained are as follows.1.The homozygous mutant of medaka prmt5 cannot develop normally to the adult stage.In the process of screening adult mutant fish,we still unable to obtain homozygous mutants,which is consistent with the results of previous studies.Subsequently,the average heterozygosity rate of each generation of adult fish was statistically analyzed.It was found that it complied with the Mendel’s laws,which implies that homozygous would be death.Therefore,the homozygous mutant of medaka prmt5 cannot develop normally to the adult stage.2.The development of the gonads of the prmt5 heterozygous mutant of medaka is affected.A paraffin section and hematoxylin-eosin staining was made of the heterozygous gonads,and we found that both ovaries and testis took place at different levels of developmental abnormalities.The results suggest that prmt5 may affect the development of medaka gonads,thereby affecting its reproductive ability.The transcriptome results show that the prmt5 mutant heterozygous and the wild-type gene are significantly different.prmt5 may regulate the function of the gonads by affecting the expression of genes related to the reproductive development of the gonads(such as genes related to gender development,spermatogenesis,and developmental processes).3.The embryonic development of medaka prmt5 heterozygous mutant is affected.In order to explore the function of prmt5 in early embryo development,we performed a statistical analysis on the offspring of five generations of embryos.Interestingly,each generation of embryos’ mortality rate is not the same,and the difference is more evident than in typical situations.The four day embryo morphology observation results show that most of the embryos with high mortality rates cannot complete the cleavage generally in the early embryonic development stage.Besides,during embryonic development,a small probability of abnormalities in developmental morphology will be observed,such as abnormal body axis,development of some tissues,albino eyes,and short tails.WISH and the qRT-PCR experiments showed that the oct4,nanog,vasa,and p53 genes have decreased after the gastrulation stage.Among related genes of apoptosis,caspase3 and caspase8 were significantly up regulated,suggesting that the embryonic death caused by the prmt5 mutant may go through the Caspase pathway.The above results are basically consistent with others animals.So prmt5 has an impact on the growth and proliferation,differentiation and development of organisms.In zebrafish,the deletion of prmt5 cannot obtain homozygous female mutations and can lead to male sterility.But in our study,the deletion of prmt5 not only failed to obtain female mutant homozygous,but also failed to obtain male mutant homozygous.Our results show that medaka prmt5 has varying degrees of influence on the gonads and early embryonic development.