JUNO mRNA補完法による精子卵融合の分子機構解明

我想揭示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Juno mRNA，它最初是体内的GPI锚定蛋白。有了这个，我试图证明JUNO不会消失，因为它是一种跨膜蛋白，因此，最大数量的精子会融合。我通过将mRNA注射到小鼠GV期卵母细胞中来测试这一点。结果表明，平均只有0.13个精子与注射mRNA的卵细胞融合，融合率为11.2%，而对照组的融合率为67.9%。我们的结论是，这种mRNA不能很好地工作，更好地产生一个转基因（TG）小鼠使用一个特殊的启动子卵母细胞特异性表达。由于转基因小鼠的繁殖需要较长的时间，所以我决定对睾丸特异性基因敲除（KO）小鼠进行分析。对于这些KO小鼠，我可以报告说，这10个基因不是必需的，因此不能用作进一步不育研究的候选基因。(Biol Reprod. 2020年8月4日;103（2）：195-204）此外，我发现Tbc 1d 21 KO雄性小鼠不能产生幼崽。KO精子中段线粒体鞘异常，精子活力低，最终不能到达排卵卵。同时，Armc 12 KO具有相似的表型。免疫沉淀结果表明，TBC 1D 21和ARMC 12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Tbc 1d 21 KO小鼠的数据。(Proc美国国家科学院2021年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）