Abstract
BackgroundJUNO and IZUMO1 are the first receptor-ligand protein pairs discovered to be essential for sperm-oocyte fusion; their interaction is indispensable for fertilization.MethodsPCR 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.ResultsThe 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.ConclusionsFor the first time, this study systematically analyzed the expression, structure and function of Juno and Izumo1 genes and their encoded proteins in Small Tail Han sheep, providing the basis for future studies of the regulatory mechanisms of Juno and Izumo1 genes.
Highlights
Sexual reproduction results in completely new individuals and ensures continuous reproduction for most eukaryotes
The full-length DNA sequences of the Juno gene in Small Tail Han sheep and Sunite sheep were 2167 bp and 2168 bp, respectively, and those two sequences were more than 95% identity
single nucleotide polymorphism (SNP) identification based on whole genome re-sequencing results Based on whole genome re-sequencing data from our laboratory consisting of 100 individual sheep from 10 different breeds [15], all SNPs near this region of Juno gene were checked, nine SNP loci of Juno were identified: g.844598A > G, g.846206C > T, g.846268A > G, g.847219G > C, g.847810C > G, g.847814C > T, g.847830T > C, g.847927A > C and g.848253C > A
Summary
Sexual reproduction results in completely new individuals and ensures continuous reproduction for most eukaryotes. Fertilization – the encounter and combination of two morphologically distinct male (sperm) and female (oocyte) germ cells – is the most important part of mammalian sexual reproduction. These two haploid germ cells meet in the reproductive tube of the mother (e.g., the oviduct in mammals), recognize each other, fuse and become a diploid that develops into a genetically unique organism. In 2005, Inoue et al identified a sperm surface protein, IZUMO1, that is essential for binding to oocytes [1]. JUNO and IZUMO1 are the first receptor-ligand protein pairs discovered to be essential for spermoocyte fusion; their interaction is indispensable for fertilization
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
