Collas P, Pinto-Correia C, Poccia D L
Department of Biology, Amherst College, Massachusetts 01002, USA.
Exp Cell Res. 1995 Aug;219(2):687-98. doi: 10.1006/excr.1995.1280.
The dynamics of lamin disassembly and reassembly during sea urchin male pronuclear development in vitro was investigated. Using five anti-lamin antibodies, we monitored by immunofluorescence and immunoblotting the changes in lamins during sperm chromatin decondensation, nuclear envelope (NE) formation, and male pronuclear swelling in fertilized sea urchin egg cytoplasmic extracts. We report the existence of five proteins in sperm nuclei and swollen male pronuclei (p49, p54, p65, p72, p84) which react with the antibodies. The sperm antigens resist membrane permeabilization with lysolecithin and 0.1% Triton X (TX)-100, but are removed from the lateral aspects of the nuclei by 1% TX-100. All five are completely removed from nuclei within 10 min of incubation in egg extracts. Initial chromatin decondensation and NE formation occur without reassembly of the putative lamins, but all lamins or lamin epitope-containing peptides assemble coordinately during pronuclear swelling promoted by adenosine 5'-triphosphate. Of the five pronuclear antigens, p49 and p54 appear to originate exclusively from the sperm. p65, p72, and p84 are also present in the egg cytoplasm and may be contributed to pronuclei by either source. Assembly of putative lamins and nuclear swelling, but not chromatin decondensation and nuclear envelope formation, are prevented in lamin-depleted cytoplasmic extracts. Our results indicate that p49 and p54 are not necessary for nuclear swelling, cytoplasmic p65 is required for swelling, and p72 and p84 are by themselves not sufficient but may be involved with p65 in nuclear swelling and full pronuclear development in vitro.
研究了海胆雄原核在体外发育过程中层粘连蛋白拆卸和重新组装的动态变化。我们使用五种抗层粘连蛋白抗体，通过免疫荧光和免疫印迹法监测了受精海胆卵细胞质提取物中精子染色质解聚、核膜（NE）形成以及雄原核肿胀过程中层粘连蛋白的变化。我们报告了精子细胞核和肿胀的雄原核中存在五种与抗体发生反应的蛋白质（p49、p54、p65、p72、p84）。精子抗原能抵抗溶血卵磷脂和0.1% Triton X（TX）-100引起的膜通透，但会被1% TX-100从细胞核侧面去除。在卵提取物中孵育10分钟内，所有这五种蛋白质都能从细胞核中完全去除。初始染色质解聚和核膜形成过程中假定的层粘连蛋白不会重新组装，但在由5'-三磷酸腺苷促进的原核肿胀过程中，所有层粘连蛋白或含层粘连蛋白表位的肽会协同组装。在这五种原核抗原中，p49和p54似乎仅来源于精子。p65、p72和p84也存在于卵细胞质中，可能由任何一种来源贡献给原核。在缺乏层粘连蛋白的细胞质提取物中，假定层粘连蛋白的组装和核肿胀受到抑制，但染色质解聚和核膜形成不受影响。我们的结果表明，p49和p54对核肿胀不是必需的，细胞质中的p65是肿胀所必需的，p72和p84本身并不充分，但可能与p65一起参与体外核肿胀和完全的原核发育。
