Vogl A W, Soucy L J
J Cell Biol. 1985 Mar;100(3):814-25. doi: 10.1083/jcb.100.3.814.
We have investigated the arrangement and function of actin filament bundles in Sertoli cell ectoplasmic specializations found adjacent to junctional networks and in areas of adhesion to spermatogenic cells. Tissue was collected, from ground squirrel (Spermophilus spp.) testes, in three ways: seminiferous tubules were fragmented mechanically; segments of intact epithelium and denuded tubule walls were isolated by using EDTA in a phosphate-buffered salt solution; and isolated epithelia and denuded tubule walls were extracted in glycerol. To determine the arrangement of actin bundles, the tissue was fixed, mounted on slides, treated with cold acetone (-20 degrees C), and then exposed to nitrobenzoxadiazole-phallacidin. Myosin was localized using immunofluorescence. To investigate the hypothesis that ectoplasmic specializations are contractile, glycerinated models were exposed to exogenous ATP and Ca++; then contraction was assessed qualitatively by using nitrobenzoxadiazole-phallacidin as a marker. Actin bundles in ectoplasmic specializations adjacent to junctional networks circumscribe the bases of Sertoli cells. When intact epithelia are viewed from an angle perpendicular to the epithelial base, honeycomb staining patterns are observed. Filament bundles in Sertoli cell regions adjacent to spermatogenic cells dramatically change organization during spermatogenesis. Initially, the bundles circle the region of contact between the developing acrosome and nucleus. They then expand to cover the entire head. As the spermatid flattens, filaments on one side of the now saucer-shaped head orient themselves parallel to the germ cell axis while those on the other align perpendicularly to it. Before sperm release, all filaments course parallel to the rim of the head. Contrary to the results we obtained with myoid cells, we could not convincingly demonstrate myosin in ectoplasmic specializations or induce contraction of glycerinated models. Our data are consistent with the hypothesis that actin in ectoplasmic specializations of Sertoli cells may be more skeletal than contractile.
我们研究了在连接网络附近的支持细胞外质特化结构以及与生精细胞黏附区域中肌动蛋白丝束的排列和功能。从地松鼠（黄鼠属）睾丸收集组织，采用了三种方法：机械破碎曲细精管；在磷酸盐缓冲盐溶液中使用乙二胺四乙酸（EDTA）分离完整上皮片段和裸露的管壁；在甘油中提取分离的上皮和裸露的管壁。为了确定肌动蛋白束的排列，将组织固定、装片、用冷丙酮（-20℃）处理，然后用硝基苯并恶二唑 - 鬼笔环肽处理。使用免疫荧光定位肌球蛋白。为了研究外质特化结构具有收缩性这一假说，将甘油化模型暴露于外源三磷酸腺苷（ATP）和钙离子（Ca++）；然后用硝基苯并恶二唑 - 鬼笔环肽作为标记物定性评估收缩情况。连接网络附近的外质特化结构中的肌动蛋白束围绕支持细胞的基部。当从垂直于上皮基部的角度观察完整上皮时，可观察到蜂窝状染色模式。支持细胞与生精细胞相邻区域的丝束在精子发生过程中组织发生显著变化。最初，这些丝束环绕发育中的顶体和细胞核之间的接触区域。然后它们扩展以覆盖整个头部。随着精子细胞变扁平，现在呈碟形头部一侧的细丝平行于生殖细胞轴排列，而另一侧的细丝则垂直于该轴排列。在精子释放之前，所有细丝都平行于头部边缘排列。与我们在肌样细胞中获得的结果相反，我们无法令人信服地在外质特化结构中证明肌球蛋白的存在，也无法诱导甘油化模型收缩。我们的数据与支持细胞外质特化结构中的肌动蛋白可能更具骨架性而非收缩性这一假说一致。
