ex vivo spermatogenesis from cultured spermatogonial stem cells

1)小鼠精原干细胞系的建立与维持：使用3个转基因小鼠（Tg）系；GFP普遍表达Tg， Acrosin-GFP Tg在减数分裂中期表达GFP， Haspin-GFP Tg在单倍体阶段表达GFP。从这3只Tg小鼠中建立了精原干细胞系，也称为种系干细胞（GS细胞）。2) GS细胞离体生精：为了在体外诱导GS细胞的生精，在不同的饲养细胞等条件下培养了含有Acrosin-GFP Tg和Haspin-GFP Tg的GS细胞。然而，到目前为止，在我们的经验中还没有检测到减数分裂标志GFP的表达。在我们的离体精子发生试验中，我们发现可以用裸鼠皮下分离的胎儿或新生儿睾丸细胞重新构建精管。我们利用未成熟睾丸细胞的重组能力将GS细胞整合到其中。这些GS细胞经过分化直至精子（单倍体）阶段，用于受精，最终产生健康的幼崽。3)睾丸组织的器官培养：我们在精子发生的细胞培养方面的经验相当令人沮丧，显然我们要转向另一种策略。以3 ~ 14日龄的小鼠睾丸组织为研究对象，对器官培养方法进行了评价。当使用7天或更大的幼犬时，haspin-GFP在培养条件下呈阳性。基本有利培养条件为32℃、10%胎牛血清、维生素等。在不久的将来可能会有进一步的改进。

1) Establishement and maintenance of mouse spermatogonial stem cell lines:Three transgenic mice (Tg) lines were used; ubiquitous GFP expressing Tg, Acrosin-GFP Tg expressing GFP at mid-meiosis onward, and Haspin-GFP Tg expressing GFP at haploid stage. Spermatogonial stem cell lines, also called germline stem cells (GS cells) were established from these three Tg mice.2) Ex vivo spermatogenesis from GS cells:In order to induce spermatogenesis form GS cells in vitro, GS cells of Acrosin-GFP Tg and Haspin-GFP Tg were cultured in various conditions such as different feeder cells. So far, however, GFP expression, marker of meiosis, was not detected in our experience.In our trial for ex vivo spermatogenesis, we have found that seminiferous tubules can be reconstituted de novo from dissociated fetal or neonatal testis cells in the subcutis of nude mice. We took advantage of the reconstitution ability of immature testis cells to get GS cells integrated in them. Those GS cells underwent differentiation up to spermatid (haploid) stage which was used for insemination to end up produce healthy pups.3) Organ culture of testis tissues:Our experience of cell culture for spermatogenesis was rather discouraging and it becomes evident for us to convert to another strategy. Using acrosin- and haspin-GFP Tg mice testis tissue, 3-14 days old, we evaluated organ culture method. When 7 or more days old pups were used, haspin-GFP become positive in a culture condition. Basic favorable culture condition includes 32℃, 10% fetal bovine serum, vitamins, and so on. Further improvement could be possible in near future.