Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, China.
Reproduction. 2022 Feb 24;163(4):209-218. doi: 10.1530/REP-21-0468.
Desert hedgehog (DHH) signaling has been reported to be involved in spermatogenesis and the self-renewal of spermatogonial stem cells (SSCs). However, the role of DHH in proliferation of spermatogonia including SSCs remains to be elucidated. Here, we report that Dhh from medaka (Oryizas latipes) (named as OlDhh) could directly mediate the proliferation of spermatogonia via Smoothened (Smo) signaling. Oldhh is 1362 bp in length and encodes 453 amino acid (aa) residues with more than 50% identity with the homologs in other species. It has expression predominantly restricted to testis. The soluble and tag-free 176-aa mature OlDhh (named as mOlDhh) were successfully obtained by fusing with the N-terminal tag of cleavable 6-histidine and small ubiquitin-related modifier and then removing the tag. Notably, mOlDhh significantly promoted the proliferation of SG3 (a spermatogonial stem cell line from medaka testis) in a dose-dependent manner and spermatogonia in testicular organ culture. Furthermore, the proliferation of SG3 in the presence of mOlDhh could be inhibited by Smo antagonist (cyclopamine) resulting in apoptosis. Additionally, mOlDhh significantly upregulated the expression of smo as well as the pluripotent-related genes bcl6b and sall4. These data suggest that Smo is an indispensable downstream component in the Dhh signaling pathway. In conclusion, our findings unambiguously demonstrate that Dhh could directly mediate the proliferation of spermatogonia through Smo signaling. This study provides new knowledge about the proliferation regulation of spermatogonia.
沙漠刺猬 (DHH) 信号已被报道参与精子发生和精原干细胞 (SSC) 的自我更新。然而，DHH 在精原细胞包括 SSC 增殖中的作用仍有待阐明。在这里，我们报告来自日本青鳉（Oryizas latipes）的 Dhh（命名为 OlDhh）可以通过 Smoothened (Smo) 信号直接介导精原细胞的增殖。Oldhh 长 1362bp，编码 453 个氨基酸（aa）残基，与其他物种的同源物具有超过 50%的同一性。它主要在睾丸中表达。通过与可切割的 6 组氨酸和小泛素相关修饰物的 N 端标签融合，然后去除标签，成功获得了可溶性和无标签的 176 个氨基酸成熟 OlDhh（命名为 mOlDhh）。值得注意的是，mOlDhh 以剂量依赖性方式显著促进 SG3（来自日本青鳉睾丸的精原干细胞系）和睾丸器官培养中的精原细胞的增殖。此外，在 mOlDhh 存在下，SG3 的增殖可以被 Smo 拮抗剂（环巴胺）抑制，导致细胞凋亡。此外，mOlDhh 显著上调 smo 以及多能相关基因 bcl6b 和 sall4 的表达。这些数据表明 Smo 是 Dhh 信号通路中不可或缺的下游成分。总之，我们的发现明确表明 Dhh 可以通过 Smo 信号直接介导精原细胞的增殖。这项研究为精原细胞增殖调控提供了新的知识。
