Abstract：
Mammalian target of rapamycin (mTOR) is a crucial signaling protein regulating a range of cellular events. Numerous studies have reported that the mTOR pathway is related to spermatogenesis in mammals. However, its functions and underlying mechanisms in crustaceans remain largely unknown. mTOR exists as two multimeric functional complexes termed mTOR complex 1 (mTORC1) and mTORC2. Herein, we first cloned ribosomal protein S6 (rpS6, a downstream molecule of mTORC1) and protein kinase C (PKC, a downstream effector of mTORC2) from the testis of Eriocheir sinensis. The dynamic localization of rpS6 and PKC suggested that both proteins may be essential for spermatogenesis. rpS6/PKC knockdown and Torin1 treatment led to defects in spermatogenesis, including germ cell loss, retention of mature sperm and empty lumen formation. In addition, the integrity of the testis barrier (similar to the blood-testis barrier in mammals) was disrupted in the rpS6/PKC knockdown and Torin1 treatment groups, accompanied by changing in expression and distribution of junction proteins. Further study demonstrated that these findings may result from the disorganization of filamentous actin (F-actin) networks, which were mediated by the expression of actin-related protein 3 (Arp3) rather than epidermal growth factor receptor pathway substrate 8 (Eps8). In summary, our study illustrated that mTORC1/rpS6 and mTORC2/PKC regulated spermatogenesis via Arp3-mediated actin microfilament organization in E. sinensis.
摘要：
哺乳动物雷帕霉素靶蛋白（mTOR）是调节一系列细胞事件的关键信号蛋白。大量研究报道mTOR通路与哺乳动物精子发生有关。然而,它在甲壳类动物中的功能和潜在机制仍然未知。mTOR作为称为mTOR复合物1(mTORC1)和mTORC2的两种多聚功能复合物存在。在这里，我们首先克隆了核糖体蛋白S6(rpS6,mTORC1的下游分子)和蛋白激酶C(PKC,mTORC2的下游效应物）来自中华绒螯蟹睾丸。rpS6和PKC的动态定位表明这两种蛋白可能对精子发生至关重要。rpS6/PKC敲低和Torin1治疗导致精子发生缺陷，包括生殖细胞损失,保留成熟的精子和空腔形成。此外,在rpS6/PKC敲低和Torin1治疗组中，睾丸屏障的完整性（类似于哺乳动物的血-睾丸屏障）被破坏，伴随着连接蛋白表达和分布的变化。进一步的研究表明，这些发现可能是由于丝状肌动蛋白（F-肌动蛋白）网络的解体，由肌动蛋白相关蛋白3（Arp3）而不是表皮生长因子受体途径底物8（Eps8）的表达介导。总之,我们的研究表明，mTORC1/rpS6和mTORC2/PKC通过Arp3介导的肌动蛋白微丝组织调节了中华绒螯蟹的精子发生。
