Prohibitin通过调控精母细胞组蛋白H3Y41磷酸化影响减数分裂的机制研究

It has been evidenced that meiosis of spermatocytes is a critical step for sperm production during spermatogenesis. Our previous studies show that (1) mitochondrial Prohibitin (PHB) is expressed higher in spermatocytes during mouse spermatogenesis. After deletion of PHB specifically in the male germ cells, Phb cKO mouse are male infertile, showing that meiosis of spermatocytes was blocked between the stages of pre-leptotene to leptotene at the prophase of meiosis I and accordingly no sperm are produced in seminiferous tubules. (2) The expression of phosphorylated histone H3Y41 (H3Y41ph) was found to be expressed higher in leptotene spermatocytes of mouse. (3) Knockdown of Phb mRNA significantly downregulate the level of histone H3Y41ph in the GC2 spermatocyte cell lines. Based on above findings, we proposed that mitochondrial PHB may play key roles on meiosis by the regulation of histone H3Y41ph in the spermatocytes. The objectives of this study, therefore, is to further explore and identify the downstream genes and signaling pathways directly regulated by PHB or histone H3Y41ph in the spermatocytes firstly. Secondly, to clarify what the downstream genes regulated by histone H3Y41ph are also controlled by PHB indirectly. Finally, to elaborate clearly the mechanism of PHB’s role on meiosis by the regulation of histone H3Y41ph in the spermatocytes. This study will help us to understand better about the epigenetic mechanism of mitochondrial PHB on meiosis during the development of male germ cells. The findings of this study could also support us theoretically on how to develop new therapy approach for male infertility.

精子发生过程中精母细胞减数分裂的顺利进行是确保单倍体精子生成的关键环节.申请人前期研究发现①线粒体膜蛋白Prohibitin(PHB)在小鼠精母细胞高表达;Phb生殖细胞特异性敲除小鼠雄性不育,无精子生成,精母细胞减数分裂在细线前期至细线期被阻断.②组蛋白H3Y41磷酸化(H3Y41ph)在小鼠细线期精母细胞高表达.③敲减小鼠GC2精母细胞Phb mRNA表达后,H3Y41ph水平显著降低.推测精母细胞表达的PHB可能通过调控H3Y41ph在减数分裂过程中发挥重要作用.因此,本项目将进一步寻找和鉴定精母细胞PHB所调控的下游相关基因和信号通路,以及PHB间接调控的H3Y41ph介导的下游相关基因,阐明精母细胞PHB通过调控H3Y41ph而影响精母细胞减数分裂的分子机制.本研究将有助于理解线粒体膜蛋白PHB调控生殖细胞发育和精母细胞减数分裂的表观遗传机制,为开发男性不育治疗方法提供理论支持.

精子发生过程中精母细胞减数分裂的顺利进行是确保单倍体精子生成的关键环节。本课题组前期研究结果表明，线粒体膜蛋白Prohibitin(PHB)在小鼠精母细胞高表达，提示PHB对精母细胞减数分裂有重要作用。进一步研究发现，精母细胞特异性Phb敲除小鼠雄性不育，无单倍体精子细胞生成，精母细胞减数分裂在粗线期停滞。敲减小鼠GC2精母细胞Phb mRNA表达后，精母细胞组蛋白H3Y41磷酸化水平显著降低，我们推测精母细胞表达的PHB可能通过调控H3Y41磷酸化水平在减数分裂过程中发挥重要作用。因此，本项目进一步寻找和鉴定精母细胞PHB所调控的下游相关基因和信号通路，以及PHB间接调控H3Y41磷酸化所介导的下游相关基因，以阐明精母细胞PHB通过调控H3Y41磷酸化而影响精母细胞减数分裂的分子机制。在完成上述研究计划并取得预期成果的基础上，进一步检测分析PHB缺失对精母细胞线粒体结构和功能的影响，以及对精母细胞染色质凝集状态的影响。研究结果表明，精母细胞PHB缺失所致的雄性不育，无单倍体精子细胞生成，主要是与PHB缺失所致的精母细胞减数分裂同源重组相关基因STAG3的表达降低有关，以致同源重组异常，减数分裂在粗线期停滞，进而精母细胞凋亡。PHB缺失也同样影响小鼠第一波生精过程中的精母细胞减数分裂进程，以致在粗线期停滞，精母细胞凋亡。同时研究还发现PHB对减数分裂过程中精母细胞线粒体结构和功能完整性的维持有重要作用。进一步开展的分子调控机制研究结果证实，精母细胞具有一种新型的PHB-JAK表观调控轴，可通过调控组蛋白H3Y41磷酸化水平和改变异染色质结构，而影响减数分裂同源重组相关黏着蛋白Stag3基因的转录与表达，进而调控减数分裂过程中DSB修复和同源重组，从而在精子发生过程中发挥重要调控作用。上述研究成果为深入理解线粒体膜蛋白PHB调控生殖细胞发育和精母细胞减数分裂的表观遗传机制，提供了新的理论基础，同时也为临床开发非梗阻性无精症所致男性不育的诊疗方法提供了新的分子靶标。