In mammalian testes, extensive remodeling of the microtubule (MT) and actin cytoskeletons takes place in Sertoli cells across the seminiferous epithelium to support spermatogenesis. However, the mechanism(s) involving regulatory and signaling proteins remains poorly understood. Herein, A-kinase anchoring protein 9 (AKAP9, a member of the AKAP multivalent scaffold protein family) was shown to be one of these crucial regulatory proteins in the rat testis. Earlier studies have shown that AKAP9 serves as a signaling platform by recruiting multiple signaling and regulatory proteins to create a large protein complex that binds to the Golgi and centrosome to facilitate the assembly of the MT-nucleating γ-tubulin ring complex to initiate MT polymerization. We further expanded our earlier studies based on a Sertoli cell-specific AKAP9 knockout mouse model to probe the function of AKAP9 by using the techniques of immunofluorescence analysis, RNA interference (RNAi), and biochemical assays on an in vitro primary Sertoli cell culture model, and an adjudin-based animal model. AKAP9 robustly expressed across the seminiferous epithelium in adult rat testes, colocalizing with MT-based tracks, and laid perpendicular across the seminiferous epithelium, and prominently expressed at the Sertoli-spermatid cell–cell anchoring junction (called apical ectoplasmic specialization [ES]) and at the Sertoli cell–cell interface (called basal ES, which together with tight junction [TJ] created the blood–testis barrier [BTB]) stage specifically. AKAP9 knockdown in Sertoli cells by RNAi was found to perturb the TJ–permeability barrier through disruptive changes in the distribution of BTB-associated proteins at the Sertoli cell cortical zone, mediated by a considerable loss of ability to induce both MT polymerization and actin filament bundling. A considerable decline in AKAP9 expression and a disruptive distribution of AKAP9 across the seminiferous tubules was also noted during adjudin-induced germ cell (GC) exfoliation in this animal model, illustrating AKAP9 is essential to maintain the homeostasis of cytoskeletons to maintain Sertoli and GC adhesion in the testis.

中文翻译：

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AKAP9 通过对大鼠睾丸中的微管和肌动蛋白细胞骨架的影响来支持精子发生

在哺乳动物睾丸中，微管 (MT) 和肌动蛋白细胞骨架的广泛重塑发生在跨生精上皮的支持细胞中，以支持精子发生。然而，涉及调节和信号蛋白的机制仍然知之甚少。在本文中，A-激酶锚定蛋白 9（AKAP9，AKAP 多价支架蛋白家族的成员）被证明是大鼠睾丸中这些关键的调节蛋白之一。早期的研究表明，AKAP9 作为一个信号平台，通过招募多种信号和调节蛋白来创建一个大的蛋白质复合物，该复合物与高尔基体和中心体结合，以促进 MT 成核 γ-微管蛋白环复合物的组装，以启动 MT 聚合。我们进一步扩展了基于支持细胞特异性 AKAP9 敲除小鼠模型的早期研究，通过在体外原代支持细胞培养模型上使用免疫荧光分析、RNA 干扰 (RNAi) 和生化分析技术来探测 AKAP9 的功能，和基于 adjudin 的动物模型。AKAP9 在成年大鼠睾丸的生精上皮中强烈表达，与基于 MT 的轨迹共定位，并垂直放置在生精上皮上，并在支持 - 精子细胞 - 细胞锚定连接处（称为顶端外质特化 [ES]）和在支持细胞 - 细胞界面（称为基底 ES，与紧密连接 [TJ] 一起创造了血 - 睾丸屏障 [BTB]）阶段。发现通过 RNAi 在支持细胞中的 AKAP9 敲低通过在支持细胞皮质区的 BTB 相关蛋白分布的破坏性变化来扰乱 TJ 通透性屏障，这是由诱导 MT 聚合和肌动蛋白丝束束的能力显着丧失介导的. 在该动物模型中，在 adjudin 诱导的生殖细胞 (GC) 脱落过程中，还注意到 AKAP9 表达的显着下降和 AKAP9 在生精小管中的破坏性分布，说明 AKAP9 对于维持细胞骨架的稳态以维持支持和 GC 粘附至关重要在睾丸中。