Ocampo Juan, Mondragón Ricardo, Roa-Espitia Ana Lilia, Chiquete-Félix Natalia, Salgado Zaira O, Mújica Adela
Departamento de Biología Celular, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional. Apdo. Postal 14740, 07000 México, D.F., México.
Tissue Cell. 2005 Aug;37(4):293-308. doi: 10.1016/j.tice.2005.03.003.
The nuclear matrix (NM) of somatic cells is an internal nuclear framework structure, with a structural function and participation in DNA replication and transcription. The NM has been described in mouse, hamster and human spermatozoa. In this study, an NM structural component of the guinea pig sperm nucleus was obtained by removing nuclear proteins and DNA from DTT-CTAB nuclei. Removal was achieved with high ionic strength salt and microccocal nuclease treatments including a heparin treatment to cause a slight swelling of the nucleus and facilitate material extraction. Actin, myosin, cytokeratins and spectrin were detected associated to NM by indirect immunofluorescence, immunogold staining and Western blotting analysis using specific antibodies. The presence of NM in guinea pig sperm nucleus is shown for the first time and some of its components are identified. This is also the first report on cytokeratins and myosin presence in guinea pig sperm. A retarding effect of nuclear decondensation caused by heparin is induced after phalloidin and/or diacetyl-monoxime (a myosin ATPase activity inhibitor) treatment, suggesting a role for F-actin and myosin in the maintenance of nuclear stability in sperm. The actin role was supported by the decondensing effect that citochalasin D and gelsolin had on sperm nuclei.
体细胞的核基质(NM)是一种细胞核内的框架结构,具有结构功能并参与DNA复制和转录。在小鼠、仓鼠和人类精子中都有关于核基质的描述。在本研究中,通过用DTT-CTAB核去除核蛋白和DNA,获得了豚鼠精子细胞核的一种核基质结构成分。通过高离子强度盐和微球菌核酸酶处理(包括肝素处理以使细胞核轻微肿胀并促进物质提取)实现了去除。使用特异性抗体,通过间接免疫荧光、免疫金染色和蛋白质印迹分析检测到肌动蛋白、肌球蛋白、细胞角蛋白和血影蛋白与核基质相关。首次证明了豚鼠精子细胞核中存在核基质,并鉴定了其一些成分。这也是关于豚鼠精子中存在细胞角蛋白和肌球蛋白的首次报道。在鬼笔环肽和/或双乙酰单肟(一种肌球蛋白ATP酶活性抑制剂)处理后,肝素诱导的核去浓缩有延迟效应,表明F-肌动蛋白和肌球蛋白在维持精子核稳定性中起作用。细胞松弛素D和凝溶胶蛋白对精子核的去浓缩作用支持了肌动蛋白的作用。