ex vivo spermatogenesis from cultured spermatogonial stem cells

1)小鼠精原干细胞系的培养和维持：使用三种转基因小鼠（Tg）系;泛在GFP表达Tg、在减数分裂中期向前表达GFP的顶体蛋白-GFP Tg和在单倍体阶段表达GFP的Haspin-GFP Tg。从这三种转基因小鼠中建立精原干细胞系，也称为生殖系干细胞（GS细胞）。2）GS细胞的体外生精：为了在体外诱导GS细胞的生精，将顶体蛋白-绿色荧光蛋白转基因小鼠和Haspin-绿色荧光蛋白转基因小鼠的GS细胞在不同条件下培养，如不同的饲养细胞。然而，到目前为止，GFP的表达，减数分裂的标志物，没有检测到在我们的exvivo spermatogenesis的试验中，我们发现，生精小管可以重建从头从游离的胎儿或新生儿睾丸细胞在皮下的裸鼠。我们利用未成熟睾丸细胞的重建能力，将GS细胞整合到其中。这些GS细胞经历分化直至精子细胞（单倍体）阶段，该阶段用于授精以最终产生健康的小狗。3）睾丸组织的器官培养：我们用于精子发生的细胞培养的经验是相当令人沮丧的，并且很明显，我们转向另一种策略。采用顶体蛋白-GFP和haspin-GFP转基因小鼠3-14日龄睾丸组织，对器官培养方法进行了评价。当使用7天或更大的幼仔时，haspin-GFP在培养条件下变为阳性。基本的培养条件包括32℃、10%胎牛血清、维生素等，今后可能进一步改进。

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.