Establishment of methods for production of fish sperm in vitro

考虑到生物资源的增殖，了解配子发生（精子发生和卵子发生）的控制机制并对其进行人工调控是非常重要的。利用器官培养系统和生殖细胞-体细胞共培养系统在体外诱导日本鳗鲡精子发生。因此，日本鳗鲡是研究精子发生的良好模型。本研究以日本鳗鲡为研究对象，探讨其精子发生的分子调控机制，并尝试建立体外培养体系，以获得大量功能性精子，同时从日本鳗鲡中新鉴定出两种重要的精子发生调控因子，一种是精子发生抑制物质，另一种是精原干细胞更新因子。精子发生阻止物质是苗勒氏管抑制物质的鳗鱼同系物，并阻止精子发生的开始（精原细胞增殖）。精原干细胞更新因子是血小板源性内皮生长因子的一种同源物，可诱导精原干细胞的有丝分裂。此外，我们建议存在减数分裂诱导激素。据我们所知，这些精子发生相关因子的鉴定尚属首次。建立了外源基因导入睾丸细胞的方法。此外，我们发现，生殖细胞和支持细胞之间的三维接触是体外诱导精子发生所必需的。然而，它在体外未能产生“功能性”精子。建立完整的体外精子生产系统还需要进一步的研究。

Considering the proliferation of bioresources, it is very important to understand the control mechanisms of gametogenesis (spermatogenesis and oogenesis) and regulate them artificially. In Japanese eel, complete spermatogenesis has been induced in vitro using an organ culture system and a germ-somatic cells coculture system. Thus, Japanese eel has been a good model for analysis of spermatogenesis. In this study, we investigated the molecular regulatory mechanisms of eel spermatogenesis, and tried to develop the culture systems to produce massive amounts of functional sperm in vitro.We newly identified two important regulatory factors of spermatogenesis from Japanese eel, one is spermatogenesis-preventing substance, and another is spermatogonial stem cell renewal factor. The spermatogenesis-preventing substance is an eel homologue of Mullerian-inhibiting substance, and prevents the initiation of spermatogenesis (spermatogonial proliferation). The spermatogonial stem cell renewal factor is an eel homologue of platelet-derived endothelial growth factor, and induces the mitosis of spermatogonial stem cells. In addition, we suggested the presence of the meiosis-inducing hormone. To our knowledge, it is the first time to identify these spermatogenesis related factors. We established the method for transfer of foreign genes into testicular cells by electropolation. Furthermore, we found that the 3-D contact between germ and Sertoli cells is essential for induction of spermatogenesis in vitro. However, it has been failed to produce "functional" sperm in vitro. Further studies are needed to establish the complete system of in vitro sperm production.