State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
College of Life Sciences, Qingdao Agricultural University, Qingdao, People's Republic of China.
Reproduction. 2021 May 27;162(1):47-59. doi: 10.1530/REP-21-0079.
Muscarinic acetylcholine receptor (mAChR) antagonists have been reported to decrease male fertility; however, the roles of mAChRs in spermatogenesis and the underlying mechanisms are not understood yet. During spermatogenesis, extensive remodeling between Sertoli cells and/or germ cells interfaces takes place to accommodate the transport of developing germ cells across the blood-testis barrier (BTB) and adluminal compartment. The cell-cell junctions play a vital role in the spermatogenesis process. This study used ICR male mice and spermatogonial cells (C18-4) and Sertoli cells (TM-4). shRNA of control or M5 gene was injected into 5-week-old ICR mice testes. Ten days post-viral grafting, mice were deeply anesthetized with pentobarbital and the testes were collected. One testicle was fresh frozen for RNA-seq analysis or Western blotting (WB). The second testicle was fixed for immunofluorescence staining (IHF). C18-4 or TM-4 cells were treated with shRNA of control or M5 gene. Then, the cells were collected for RNA-seq analysis, WB, or IHF. Knockdown of mAChR M5 disrupted mouse spermatogenesis and damaged the actin-based cytoskeleton and many types of junction proteins in both Sertoli cells and germ cells. M5 knockdown decreased Phldb2 expression in both germ cells and Sertoli cells which suggested that Phldb2 may be involved in cytoskeleton and cell-cell junction formation to regulate spermatogenesis. Our investigation has elucidated a novel role for mAChR M5 in the regulation of spermatogenesis through the interactions of Phldb2 and cell-cell junctions. M5 may be an attractive future therapeutic target in the treatment of male reproductive disorders.
毒蕈碱型乙酰胆碱受体 (mAChR) 拮抗剂已被报道可降低男性生育能力；然而，mAChR 在精子发生中的作用及其潜在机制尚不清楚。在精子发生过程中，塞尔托利细胞和/或生精细胞界面之间发生广泛的重塑，以容纳发育中的生精细胞穿过血睾屏障 (BTB) 和管腔室的转运。细胞-细胞连接在精子发生过程中起着至关重要的作用。本研究使用 ICR 雄性小鼠和精原细胞 (C18-4) 和塞尔托利细胞 (TM-4)。将对照或 M5 基因的 shRNA 注射到 5 周龄 ICR 小鼠的睾丸中。病毒移植后 10 天，用戊巴比妥钠将小鼠深度麻醉，并收集睾丸。一个睾丸用于新鲜冷冻 RNA-seq 分析或 Western blot (WB)。第二个睾丸用于免疫荧光染色 (IHF)。用对照或 M5 基因的 shRNA 处理 C18-4 或 TM-4 细胞。然后收集细胞进行 RNA-seq 分析、WB 或 IHF。mAChR M5 的敲低破坏了小鼠精子发生，并破坏了塞尔托利细胞和生精细胞中基于肌动蛋白的细胞骨架和多种连接蛋白。M5 敲低降低了生精细胞和塞尔托利细胞中 Phldb2 的表达，这表明 Phldb2 可能参与细胞骨架和细胞-细胞连接的形成，以调节精子发生。我们的研究阐明了 mAChR M5 通过 Phldb2 与细胞-细胞连接的相互作用在调节精子发生中的新作用。M5 可能是治疗男性生殖障碍的有吸引力的未来治疗靶点。
