JUNO mRNA補完法による精子卵融合の分子機構解明

我想要揭示Juno(卵膜融合蛋白)如何以及何时被切割的机制。为此，我合成了一种跨膜型的Juno mRNA，它最初是体内的GPI锚定蛋白。有了这一点，我试图证明Juno不会消失，因为它是一种跨膜蛋白，结果是最大数量的精子会融合。我通过将信使核糖核酸注射到小鼠的GV期卵母细胞中来测试这一点。结果表明，平均只有0.13个精子与11.2%的mRNA注入的卵子融合，而1.05个精子与67.9%的对照卵子融合。我们的结论是，这种mRNA不能很好地发挥作用，最好是使用一种特殊的卵母细胞特异性表达启动子来产生转基因(TG)小鼠。Tg小鼠的产生可能需要很长时间，所以我决定分析睾丸特异性基因敲除(KO)小鼠。我分析了10只能够正常生产幼崽的KO雄性小鼠。对于这些KO小鼠，我可以报告，这10个基因不是必需的，因此不能用作进一步不孕症研究的候选基因。(生物报告。2020年8月4日；103(2)：195-204)此外，我发现Tbc1d21 KO雄性小鼠不能生育幼崽。KO精子中段线粒体鞘异常，精子活动率低，最终达不到排卵卵。同时，Armc12 KO具有相似的表型。通过免疫沉淀，我们得出结论：TBC1D21和ARMC12在睾丸中协同作用，参与精子线粒体鞘的形成。我可以在下面的论文中展示Tbc1d21KO小鼠的数据。(学报学报U S A.2021，2月9日；118(6)：e2018355118)

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）