School of Molecular Biosciences, Center for Reproductive Biology, College of Veterinary Medicine, Washington State University, Pullman, WA, USA.
Priority Research Centre for Reproductive Science, Discipline of Biological Sciences, The University of Newcastle, Callaghan, NSW, Australia.
Biol Reprod. 2022 Jun 13;106(6):1175-1190. doi: 10.1093/biolre/ioac048.
Spermatogenic regeneration is key for male fertility and relies on activities of an undifferentiated spermatogonial population. Here, a high-throughput approach with primary cultures of mouse spermatogonia was devised to rapidly predict alterations in functional capacity. Combining the platform with a large-scale RNAi screen of transcription factors, we generated a repository of new information from which pathway analysis was able to predict candidate molecular networks regulating regenerative functions. Extending from this database, the SRCAP-CREBBP/EP300 (Snf2-related CREBBP activator protein-CREB binding protein/E1A binding protein P300) complex was found to mediate differential levels of histone acetylation between stem cell and progenitor spermatogonia to influence expression of key self-renewal genes including the previously undescribed testis-specific transcription factor ZSCAN2 (zinc finger and SCAN domain containing 2). Single cell RNA sequencing analysis revealed that ZSCAN2 deficiency alters key cellular processes in undifferentiated spermatogonia such as translation, chromatin modification, and ubiquitination. In Zscan2 knockout mice, while spermatogenesis was moderately impacted during steady state, regeneration after cytotoxic insult was significantly impaired. Altogether, these findings have validated the utility of our high-throughput screening approach and have generated a transcription factor database that can be utilized for uncovering novel mechanisms governing spermatogonial functions.
精子发生的再生对于男性生育能力至关重要，依赖于未分化精原细胞群体的活动。在这里，我们设计了一种高通量方法，利用小鼠精原细胞的原代培养物，快速预测功能能力的改变。将该平台与转录因子的大规模 RNAi 筛选相结合，我们生成了一个新信息库，通路分析能够预测调节再生功能的候选分子网络。从这个数据库中扩展出来，SRCAP-CREBBP/EP300（Snf2 相关 CREBBP 激活蛋白-CREB 结合蛋白/E1A 结合蛋白 P300）复合物被发现介导干细胞和祖细胞精原细胞之间不同水平的组蛋白乙酰化，以影响关键自我更新基因的表达，包括先前未描述的睾丸特异性转录因子 ZSCAN2（锌指和 SCAN 结构域包含 2）。单细胞 RNA 测序分析显示，ZSCAN2 缺失会改变未分化精原细胞中的关键细胞过程，如翻译、染色质修饰和泛素化。在 Zscan2 敲除小鼠中，虽然在稳态时精子发生受到中度影响，但细胞毒性损伤后的再生明显受损。总之，这些发现验证了我们高通量筛选方法的实用性，并生成了一个转录因子数据库，可用于揭示调节精原细胞功能的新机制。
