Expression,structure and function analysis of the sperm-oocyte fusion genes Juno and Izumo1 in sheep(Ovis aries)  

作　　者：Wenping Hu Xinlong Dong Zhilong Tian Zhuangbiao Zhang Jishun Tang Benmeng Liang Qiuyue Liu Mingxing Chu 

机构地区：[1]Key Laboratory of Animal Genetics and Breeding and Reproduction of Ministry of Agriculture and Rural Affairs,Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China [2]Institute of Animal Husbandry and Veterinary Medicine,Anhui Academy of Agricultural Sciences,Hefei 230031,China [3]Institute of Genetics and Developmental Biology,the Innovation Academy for Seed Design,Chinese Academy of Sciences,Beijing 100101,China

出　　处：《Journal of Animal Science and Biotechnology》2021年第3期917-934,共18页畜牧与生物技术杂志（英文版）

基　　金：This research was funded by National Natural Science Foundation of China,grant number 31501941;Central Public-interest Scientific Institution Basal Research Fund,grant number 2018-YWF-YB-1,and 2015ywf-zd-2;the Earmarked Fund for China Agriculture Research System,grant number CARS-38;the Agricultural Science and Technology Innovation Program of China,grant number ASTIP-IAS13.

摘　　要：Background:JUNO and IZUMO1 are the first receptor-ligand protein pairs discovered to be essential for spermoocyte fusion;their interaction is indispensable for fertilization.Methods:PCR was used to clone the full-length DNA sequence of the Juno gene in sheep.The single nucleotide polymorphism(SNP)loci of Juno were genotyped by Sequenom MassARRAY®.PCR combined with rapid amplification of cDNA Ends were used to clone the full-length cDNA sequence of Juno and Izumo1.Reverse transcriptase-PCR(RT-PCR)and real time-quantitative-PCR(RT-qPCR)were used to analyze the genes’expression in tissues of sheep,and single cell RNA-seq was used to analyze the genes’expression in oocytes,granulosa cells and follicular theca of polytocous and monotocous Small Tail Han ewes.Bioinformatics was used to analyze advanced structure and phylogeny of JUNO and IZUMO1 proteins.Results:The full-length DNA sequence of the Juno gene in sheep was cloned and nine SNPs were screened.We found a significant association between the g.848253 C>A locus of Juno and litter size of Small Tail Han sheep(P<0.05).The full-length cDNA sequence of Juno and Izumo1 genes from Small Tail Han sheep were obtained.We found a new segment of the Izumo1 CDS consisting of 35 bp,and we confirmed the Izumo1 gene has 9 exons,not 8.RT-qPCR showed that Juno and Izumo1 genes were highly expressed in ovarian and testicular tissues,respectively(P<0.01).Single cell RNA-seq showed Juno was specifically expressed in oocytes,but not in granulosa cells or follicular theca,while Izumo1 displayed little to no expression in all three cell types.There was no difference in expression of the Juno gene in oocyte and ovarian tissue in sheep with different litter sizes,indicating expression of Juno is not related to litter size traits.Bioinformatic analysis revealed the g.848253 C>A locus of Juno results in a nonconservative missense point mutation leading to a change from Phe to Leu at position 219 in the amino acid sequence.Conclusions:For the first time,this study systematically analy

关 键 词：FERTILIZATION Izumo1 Juno SHEEP Single cell RNA-seq 

分 类 号：S826[农业科学—畜牧学]