Master's and Doctoral Program in Biology, Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8577, Japan.
Laboratory of Regulatory Biology, Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Kawasaki 214-8571, Japan.
Biomolecules. 2023 Mar 24;13(4):586. doi: 10.3390/biom13040586.
Proteasomes are highly sophisticated protease complexes that degrade non-lysosomal proteins, and their proper regulation ensures various biological functions such as spermatogenesis. The proteasome-associated proteins, PA200 and ECPAS, are predicted to function during spermatogenesis; however, male mice lacking each of these genes sustain fertility, raising the possibility that these proteins complement each other. To address this issue, we explored these possible roles during spermatogenesis by producing mice lacking these genes (double-knockout mice; dKO mice). Expression patterns and quantities were similar throughout spermatogenesis in the testes. In epididymal sperm, PA200 and ECPAS were expressed but were differentially localized to the midpiece and acrosome, respectively. Proteasome activity was considerably reduced in both the testes and epididymides of dKO male mice, resulting in infertility. Mass spectrometric analysis revealed LPIN1 as a target protein for PA200 and ECPAS, which was confirmed via immunoblotting and immunostaining. Furthermore, ultrastructural and microscopic analyses demonstrated that the dKO sperm displayed disorganization of the mitochondrial sheath. Our results indicate that PA200 and ECPAS work cooperatively during spermatogenesis and are essential for male fertility.
蛋白酶体是高度复杂的蛋白酶复合物，可降解非溶酶体蛋白，其适当的调节可确保各种生物学功能，如精子发生。蛋白酶体相关蛋白 PA200 和 ECPAS 预计在精子发生过程中发挥作用；然而，缺乏这些基因的雄性小鼠仍能保持生育能力，这表明这些蛋白质可能相互补充。为了解决这个问题，我们通过产生缺乏这些基因的小鼠（双敲除小鼠；dKO 小鼠）来探索这些在精子发生过程中可能发挥的作用。在睾丸中的精子发生过程中，PA200 和 ECPAS 的表达模式和数量相似。在附睾精子中，PA200 和 ECPAS 表达，但分别定位于中段和顶体。dKO 雄性小鼠的睾丸和附睾中的蛋白酶体活性明显降低，导致不育。质谱分析显示 LPIN1 是 PA200 和 ECPAS 的靶蛋白，这通过免疫印迹和免疫染色得到了证实。此外，超微结构和显微镜分析表明，dKO 精子显示线粒体鞘的紊乱。我们的结果表明，PA200 和 ECPAS 在精子发生过程中协同工作，对男性生育能力至关重要。
