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Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm-oocyte fusion in mice.

ABSTRACT: Sperm-oocyte membrane fusion is one of the most important events for fertilization. So far, IZUMO1 and Fertilization Influencing Membrane Protein (FIMP) on the sperm membrane and CD9 and JUNO (IZUMO1R/FOLR4) on the oocyte membrane have been identified as fusion-required proteins. However, the molecular mechanisms for sperm-oocyte fusion are still unclear. Here, we show that testis-enriched genes, sperm-oocyte fusion required 1 (Sof1/Llcfc1/1700034O15Rik), transmembrane protein 95 (Tmem95), and sperm acrosome associated 6 (Spaca6), encode sperm proteins required for sperm-oocyte fusion in mice. These knockout (KO) spermatozoa carry IZUMO1 but cannot fuse with the oocyte plasma membrane, leading to male sterility. Transgenic mice which expressed mouse Sof1, Tmem95, and Spaca6 rescued the sterility of Sof1, Tmem95, and Spaca6 KO males, respectively. SOF1 and SPACA6 remain in acrosome-reacted spermatozoa, and SPACA6 translocates to the equatorial segment of these spermatozoa. The coexpression of SOF1, TMEM95, and SPACA6 in IZUMO1-expressing cultured cells did not enhance their ability to adhere to the oocyte membrane or allow them to fuse with oocytes. SOF1, TMEM95, and SPACA6 may function cooperatively with IZUMO1 and/or unknown fusogens in sperm-oocyte fusion.

SUBMITTER: Noda T

PROVIDER: S-EPMC7261011 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm-oocyte fusion in mice.

Noda Taichi T Lu Yonggang Y Fujihara Yoshitaka Y Oura Seiya S Koyano Takayuki T Kobayashi Sumire S Matzuk Martin M MM Ikawa Masahito M

Proceedings of the National Academy of Sciences of the United States of America 20200511 21

Sperm-oocyte membrane fusion is one of the most important events for fertilization. So far, IZUMO1 and Fertilization Influencing Membrane Protein (FIMP) on the sperm membrane and CD9 and JUNO (IZUMO1R/FOLR4) on the oocyte membrane have been identified as fusion-required proteins. However, the molecular mechanisms for sperm-oocyte fusion are still unclear. Here, we show that testis-enriched genes, sperm-oocyte fusion required 1 (Sof1/Llcfc1/1700034O15Rik), transmembrane prote ...[more]

PMID: 32393636

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Project description: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.

Dataset Information

Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm-oocyte fusion in mice.

Publications

Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm-oocyte fusion in mice.

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