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Background  JUNO and IZUMO1 are the first receptor-ligand protein pairs discovered to be essential for sperm-oocyte 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  Izumo 1 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 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.    Supplementary Information  The online version contains supplementary material available at 10.1186/s40104-021-00548-4.

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