Tokalov S V, Gutzeit H O
Institut für Zoologie, TU Dresden, D-01062 Dresden, Germany.
Dev Dyn. 2005 Aug;233(4):1238-47. doi: 10.1002/dvdy.20379.
Spermatogenesis in vertebrates is controlled by endocrine and paracrine factors and involves the communication between somatic and germ line cells. To elucidate some of the relevant factors in the complicated molecular control processes, we established an in vitro test system using primary cultures of tilapia (Oreochromis niloticus) testis cells. The cultures were enriched for germ line cells and Sertoli cells and largely depleted of spermatozoa. By staining the cells with propidium iodide and carboxyfluorescein succinimidyl ester (CFSE), different cell populations could be identified cytologically and, in addition, quantified by flow cytometry. Cells that had gone through one or more divisions could be identified unequivocally based on their CFSE staining intensity. In parallel cultures maintained for up to 16 days in the presence of 11-ketotestosterone (KT), insulin-like growth factor I (IGF), and/or human chorionic gonadotropin (hCG) the initiation of meiotic and mitotic divisions was monitored. Although KT was important for the initiation of meiosis, spermatogonial mitotic divisions between 10 days and 16 days of culture were promoted by IGF and/or hCG in the presence of KT. These results illustrate the potential of the established in vitro test system for the analysis of the molecular control mechanisms of spermatogenesis.
脊椎动物的精子发生受内分泌和旁分泌因子控制，涉及体细胞与生殖细胞之间的通讯。为了阐明复杂分子控制过程中的一些相关因素，我们利用罗非鱼（尼罗罗非鱼）睾丸细胞的原代培养建立了一个体外测试系统。培养物中富含生殖细胞和支持细胞，精子大量减少。通过用碘化丙啶和羧基荧光素琥珀酰亚胺酯（CFSE）对细胞进行染色，可以从细胞学上鉴定不同的细胞群体，此外，还可以通过流式细胞术进行定量分析。根据CFSE染色强度，可以明确鉴定经历过一次或多次分裂的细胞。在含有11-酮睾酮（KT）、胰岛素样生长因子I（IGF）和/或人绒毛膜促性腺激素（hCG）的情况下，在平行培养物中维持长达16天，监测减数分裂和有丝分裂的起始。虽然KT对减数分裂的起始很重要，但在KT存在的情况下，IGF和/或hCG促进了培养10天至16天期间精原细胞的有丝分裂。这些结果说明了所建立的体外测试系统在分析精子发生分子控制机制方面的潜力。
