Tres L L, Cahn F, Kierszenbaum A L
Department of Cell Biology and Anatomical Sciences, City University of New York Medical School, New York 10031.
Cell Biol Toxicol. 1992 Jul-Sep;8(3):61-71. doi: 10.1007/BF00130512.
A novel collagen-glycosaminoglycan (C-GAG) substrate was developed to overcome the optical opacity of a HATF nitrocellulose substrate and to provide a more physiological permeable substrate for cocultured Sertoli and spermatogenic cells. Cocultures were prepared on optically transparent C-GAG discs attached to a polyester mesh to facilitate handling. Sertoli cells displayed a cuboidal-to-columnar shape; a large number of spermatogonia and primary spermatocytes connected by intercellular bridges were associated with basolateral and apical surfaces of Sertoli cells up to 12 days after plating. Rat Sertoli-spermatogenic cell cocultures have been used for testing the effect of toxicants on rat spermatogenesis in vitro. In our initial studies, we tested the effects of the toxicant gossypol on spermatogenic cells cocultured with Sertoli cells on non-permeable (plastic) and permeable substrates (HATF nitrocellulose) under both standard culture conditions and during perifusion after achieving a continuous electrical-resistant cell monolayer. A selective mitochondrial structural damage was observed in spermatogenic cells (spermatogonia and spermatocytes) but not in the coexisting Sertoli cells. This damage was time- (15-60 min) and dose-dependent (0.1-10 microM) and developed more rapidly under perifusion conditions. Similar mitochondrial damage was reported in the intact animal but required higher concentrations (mg) and longer administration time (months) for detection. Studies are in progress to evaluate the effect of additional toxic chemical agents on functional properties of Sertoli and spermatogenic cells in cocultures prepared on various classes of C-GAG substrates.
为克服HATF硝酸纤维素基质的光学不透明性，并为共培养的支持细胞和生精细胞提供更具生理通透性的基质，开发了一种新型胶原-糖胺聚糖（C-GAG）基质。在附着于聚酯网的光学透明C-GAG圆盘上制备共培养物，以方便操作。支持细胞呈立方形到柱状；接种后长达12天，大量通过细胞间桥连接的精原细胞和初级精母细胞与支持细胞的基底外侧和顶端表面相关联。大鼠支持细胞-生精细胞共培养物已用于体外测试毒物对大鼠精子发生的影响。在我们的初步研究中，我们测试了毒物棉酚在标准培养条件下以及在形成连续电阻细胞单层后进行灌流时，对与支持细胞共培养于非渗透性（塑料）和渗透性基质（HATF硝酸纤维素）上的生精细胞的影响。在生精细胞（精原细胞和精母细胞）中观察到选择性线粒体结构损伤，但共存的支持细胞中未观察到。这种损伤具有时间依赖性（15-60分钟）和剂量依赖性（0.1-10 microM），并且在灌流条件下发展得更快。在完整动物中也报道了类似的线粒体损伤，但检测需要更高的浓度（毫克）和更长的给药时间（数月）。正在进行研究以评估其他有毒化学物质对在各种类型的C-GAG基质上制备的共培养物中支持细胞和生精细胞功能特性的影响。
