JUNO mRNA補完法による精子卵融合の分子機構解明

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I wanted to reveal the mechanism of how and when JUNO (fusion protein in egg membrane) is cleaved. For that, I synthesized a transmembrane type of Juno mRNA, which is originally GPI-anchored protein in vivo. With this, I tried to prove that JUNO does not disappear because it is a transmembrane protein, and as a result, a maximum number of sperm will fuse. I tested this by mRNA injecting into mouse GV stage oocyte. As a result, only 0.13 sperm in average fused with 11.2% of mRNA injected egg, while 1.05 sperm fused with 67.9% of control egg. We concluded that this mRNA does not work well, and better to generate a trans-gene(TG)mice using a special promoter for oocyte-specific expression. The TG mice generation may take long time, so I decided to analyze testis-specific gene knockout(KO)mice.I analyzed 10 KO male mice which could produce pups normally. With these KO mice, I could report that those 10 genes are not essential and thus cannot be used as a candidate gene for further infertility studies. （Biol Reprod. 2020 Aug 4;103(2):195-204）Besides, I found Tbc1d21 KO male mice could not produce pups. KO sperm had abnormal mitochondrial sheath in midpiece, and thus sperm motility was too low to reach ovulated eggs at last. Meanwhile, Armc12 KO had similar phenotype. By immunoprecipitation, we concluded that TBC1D21 and ARMC12 works together in testis and participate in the formation of mitochondrial sheath of sperm. I could show the data of Tbc1d21 KO mice in the following paper. （Proc Natl Acad Sci U S A. 2021 Feb 9;118(6):e2018355118）