Sutovský P, Fléchon J E, Fléchon B, Motlik J, Peynot N, Chesné P, Heyman Y
Institute of Physiology and Genetics of Animals, Libĕchov, Czech Republic.
Biol Reprod. 1993 Dec;49(6):1277-87. doi: 10.1095/biolreprod49.6.1277.
Changes in cell-to-cell contact and distribution of cytoskeletal components were investigated during in vitro culture of cattle oocyte cumulus complexes (OCC). Freeze-fracture analysis (FF), microinjections of the fluorescent dye Lucifer Yellow (LY), immunofluorescence, and ultrastructural immunocytochemistry were used. The cumulus cells (CC) remained in close contact via gap junctions (GJ) constituted of connexin43 (Cx43) during the entire culture time. Whereas the GJ decreased in diameter after 24 h of culture, their number was still substantially great at that time. The Cx43-positive GJ, localized between corona radiata cell projections and oolemma, disappeared after 6 h of culture. Concomitantly, the OCC lost the ability to transfer LY from cumulus to oocyte, and connexin32 (Cx32) became detectable in the oocytes. Both the changes in corona-oocyte coupling and cumulus expansion were preceded by the redistribution of F-actin in cytoplasm of CC. These data indicate that functional GJ linked the CC until the second meiotic arrest. However, the removal of Cx43-positive GJ interconnecting cytoplasmic projections of corona radiata cells with the oocyte was temporally correlated with germinal vesicle breakdown. The present results suggest the pivotal role of the cytoskeleton (F-actin) in cumulus expansion.
在牛卵母细胞-卵丘复合体（OCC）的体外培养过程中，研究了细胞间接触和细胞骨架成分分布的变化。采用了冷冻断裂分析（FF）、荧光染料路西法黄（LY）显微注射、免疫荧光和超微结构免疫细胞化学等方法。在整个培养期间，卵丘细胞（CC）通过由连接蛋白43（Cx43）构成的缝隙连接（GJ）保持紧密接触。虽然培养24小时后GJ直径减小，但此时其数量仍然相当多。位于放射冠细胞突起与卵膜之间的Cx43阳性GJ在培养6小时后消失。与此同时，OCC失去了将LY从卵丘转移到卵母细胞的能力，并且在卵母细胞中可检测到连接蛋白32（Cx32）。在放射冠-卵母细胞偶联和卵丘扩展的变化之前，CC细胞质中的F-肌动蛋白均发生了重新分布。这些数据表明，功能性GJ在第二次减数分裂停滞之前连接着CC。然而，将放射冠细胞的细胞质突起与卵母细胞相互连接的Cx43阳性GJ的去除在时间上与生发泡破裂相关。目前的结果表明细胞骨架（F-肌动蛋白）在卵丘扩展中起关键作用。
