The seminiferous tubule (ST) is the location of spermatogenesis, where mature spermatozoa are produced with the assistance of Sertoli cells. The role of extracellular vesicles in the direct communication between Sertoli-germ cells in the ST is still not fully understood. In this study, we reported multivesicular bodies (MVBs) and their source of CD63-enriched exosomes by light and ultrastructure microscopy during the reproductive phases of turtles. Strong CD63 immunopositivity was detected at the basal region in the early and luminal regions of the ST during late spermatogenesis by immunohistochemistry (IHC), immunofluorescence (IF), and western blot (WB) analysis. Labeling of CD63 was detected in the Sertoli cell cytoplasmic processes that surround the developing germ cells during early spermatogenesis and in the lumen of the ST with elongated spermatids during late spermatogenesis. Furthermore, ultrastructure analysis confirmed the existence of numerous MVBs in the Sertoli cell prolongations that surround the round and primary spermatogonia during acrosome biogenesis and with the embedded heads of spermatids in the cytoplasm of Sertoli cells. Additionally, in spermatids, Chrysanthemum flower centers (CFCs) generated isolated membranes involved in MVBs and autophagosome formation, and their fusion to form amphiosomes was also observed. Additionally, autophagy inhibition by 3-methyladenine (after 24 h) increased CD63 protein signals during late spermatogenesis, as detected by IF and WB. Collectively, our study found MVBs and CD63 rich exosomes within the Sertoli cells and their response to autophagy inhibition in the ST during the spermatogenesis in the turtle.

中文翻译：

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海龟精子发生过程中生精小管内富含 CD63 的外泌体和多泡体的细胞证据

生精小管 (ST) 是精子发生的位置，在支持细胞的帮助下产生成熟的精子。细胞外囊泡在 ST 中的支持生殖细胞之间的直接通讯中的作用仍未完全了解。在这项研究中，我们通过光学和超微结构显微镜在海龟的生殖阶段报告了多泡体 (MVB) 及其富含 CD63 的外泌体的来源。通过免疫组织化学 (IHC)、免疫荧光 (IF) 和蛋白质印迹 (WB) 分析，在晚期精子发生过程中，在 ST 早期和管腔区域的基底区域检测到强 CD63 免疫阳性。在早期精子发生过程中围绕发育中的生殖细胞的支持细胞细胞质过程中检测到 CD63 的标记，在晚期精子发生过程中，在带有细长精子细胞的 ST 腔中检测到 CD63 的标记。此外，超微结构分析证实了在顶体生物发生过程中围绕圆形和初级精原细胞的支持细胞延长物中存在大量 MVB，并且精子细胞的细胞质中嵌入了精子细胞的头部。此外，在精子细胞中，菊花中心 (CFCs) 产生了参与 MVB 和自噬体形成的分离膜，并且还观察到它们融合形成两性体。此外，如 IF 和 WB 检测到的，3-甲基腺嘌呤（24 小时后）对自噬的抑制增加了晚期精子发生过程中的 CD63 蛋白信号。集体，