Establishment of methods for production of fish sperm in vitro

考虑到生物资源的增殖，了解配子体发生（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。用器官培养系统和种体细胞共培养系统在体外诱导了日本鳗鲡的完全精子发生。因此，日本鳗鱼已成为精子发生分析的良好模型。在本研究中，我们研究了鳗鱼精子发生的分子调控机制，并试图在体外建立大量产生功能精子的培养体系。我们新发现了日本鳗鲡精子发生的两个重要调控因子，一个是阻止精子发生的物质，另一个是精子干细胞更新因子。阻止精子发生的物质是抑制苗勒氏物质的同系物，可以阻止精子发生（精子增殖）的发生。精原干细胞再生因子与血小板来源的内皮生长因子具有同源性，可诱导精原干细胞的有丝分裂。此外，我们认为存在减数分裂诱导激素。据我们所知，这是第一次确定这些与精子发生有关的因素。我们建立了外源基因电移植入睾丸细胞的方法。此外，我们发现生殖细胞和支持细胞之间的三维接触对于体外诱导精子发生是必不可少的。然而，它在体外未能产生“功能性”精子。需要进一步的研究来建立完整的体外精子生产系统。

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.