Puglisi Rossella, Maccari Irene, Pipolo Simona, Mangia Franco, Boitani Carla
DAHFMO; Section of Histology & Medical Embryology; University of Rome "La Sapienza;" Rome, Italy.
Dept Psychology; Section of Neuroscience; University of Rome"La Sapienza;" Rome, Italy.
Spermatogenesis. 2014 Apr 25;4:e28460. doi: 10.4161/spmg.28460. eCollection 2014.
The testis-specific nuclear form of Phospholipid Hydroperoxide Glutathione Peroxidase (nGPx4) is associated with the nuclear matrix during spermiogenesis and is implicated in sperm chromatin condensation. In this study, we have addressed the question whether nGPx4 directly interacts with protamines by transiently sharing a nuclear matrix localization. We first expressed tagged protamine 1-myc and protamine 2-V5 in HeLa and COS-1 cells and showed by both confocal microscopy and immunoblotting analyses that protamines were produced in vitro and colocalized correctly to the nucleus. Co-transfection experiments demonstrated that protamine 1 was physically associated with flag-nGPx4 specifically at the level of nuclear matrix. The peculiar presence of protamines together with nGPx4 in this subnuclear compartment was also confirmed in mouse elongated spermatids by immunofluorescence, suggesting that nGPx4 is a physiological component of a novel protein complex relevant to chromatin assembly in condensing haploid cells. Also, in epididymal sperm, nGPx4 and protamine 1 co-immunoprecipitated, indicating that nGPx4, although localized to a subnuclear compartment different from that of protamines, represents a constant link between nuclear matrix and chromatin in mammalian male gamete.
磷脂氢过氧化物谷胱甘肽过氧化物酶的睾丸特异性核形式（nGPx4）在精子发生过程中与核基质相关联，并与精子染色质浓缩有关。在本研究中，我们探讨了nGPx4是否通过短暂共享核基质定位而直接与鱼精蛋白相互作用这一问题。我们首先在HeLa和COS-1细胞中表达了带标签的鱼精蛋白1-myc和鱼精蛋白2-V5，通过共聚焦显微镜和免疫印迹分析表明鱼精蛋白在体外产生并正确地共定位于细胞核。共转染实验表明，鱼精蛋白1在核基质水平上与flag-nGPx4发生物理关联。通过免疫荧光在小鼠延长型精子细胞中也证实了鱼精蛋白与nGPx4在这个亚核区室中的特殊存在，这表明nGPx4是与单倍体细胞染色质组装相关的新型蛋白质复合物的生理成分。此外，在附睾精子中，nGPx4和鱼精蛋白1共同免疫沉淀，表明nGPx4虽然定位于与鱼精蛋白不同的亚核区室，但却是哺乳动物雄配子中核基质与染色质之间恒定的连接物。
