Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, C.P. 486, 30161-970 Belo Horizonte, Minas Gerais, Brasil.
Cell Tissue Res. 2012 Feb;347(2):467-78. doi: 10.1007/s00441-012-1327-6.
Follicular atresia in fish ovary provides an interesting model for studying autophagy and apoptosis. In order to improve knowledge of the mechanisms regulating ovarian regression, we investigated the immunolocalisation of various proteins involved in the complex network of autophagy and apoptosis. Females of three species of freshwater fish maintained in captivity were sampled after the reproductive period and the main events of follicular atresia were assessed by histology: splits in the zona radiata, yolk degradation and reabsorption, hypertrophy of the follicular cells, accumulation of autophagic vacuoles, closing of the follicular lumen and thickening of the theca. The interplay of apoptosis and autophagy was analysed by TUNEL in situ and by immunocytochemistry for caspase-3, bax, bcl-2, beclin-1 and cathepsin-D. During early and advanced stages of follicular regression, the actin cytoskeleton was well developed and labelling for bcl-2 and cathepsin-D were pronounced in the follicular cells at a stage when they were intensively involved in yolk phagocytosis. Immunofluorescence for beclin-1 was prevalent in the follicular cells, punctate labelling often surrounding autophagic vacuoles during the advanced stage of follicular regression, a critical step towards cell death. TUNEL-positive reaction and immunostaining for bax and caspase-3 demonstrated the participation of apoptosis in late follicular regression. Overall, this study provides evidence that autophagic and apoptotic proteins are activated in a coordinated fashion depending on the stage of follicular regression, with interplay between autophagy and apoptosis being essential in determining the fate of the cell during follicular atresia in fish ovary.
鱼类卵巢中的卵泡闭锁为研究自噬和细胞凋亡提供了一个有趣的模型。为了深入了解调控卵巢退化的机制，我们研究了参与自噬和细胞凋亡复杂网络的各种蛋白的免疫定位。在繁殖期后，从三种淡水鱼类的养殖雌性个体中取样，并通过组织学评估卵泡闭锁的主要事件：放射区分裂、卵黄降解和吸收、卵泡细胞肥大、自噬空泡积累、卵泡腔闭合和囊层变厚。通过原位末端标记法（TUNEL）和免疫细胞化学分析半胱氨酸天冬氨酸蛋白酶-3（caspase-3）、B 淋巴细胞瘤-2 基因（bcl-2）、Bcl-2 相关 X 蛋白（bax）、Bcl-2 样 1（beclin-1）和组织蛋白酶-D（cathepsin-D）来分析细胞凋亡和自噬之间的相互作用。在卵泡退化的早期和晚期，肌动蛋白细胞骨架发育良好，bcl-2 和 cathepsin-D 的免疫标记在卵泡细胞中表达强烈，此时它们正积极参与卵黄的吞噬作用。在卵泡退化的晚期，beclin-1 的免疫荧光很普遍，点状标记常常围绕着自噬空泡，这是细胞死亡的关键步骤。TUNEL 阳性反应和 bax 和 caspase-3 的免疫染色表明细胞凋亡参与了晚期卵泡退化。总的来说，这项研究提供的证据表明，自噬和凋亡蛋白在协调方式下被激活，这取决于卵泡退化的阶段，自噬和凋亡之间的相互作用对于确定鱼类卵巢卵泡闭锁过程中细胞的命运至关重要。
