ex vivo spermatogenesis from cultured spermatogonial stem cells

培养精原干细胞的离体精子发生

• 批准号: 18591783
• 负责人: OGAWA Takehiko
• 金额: $ 2.51万
• 依托单位: Yokohama City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18591783/
• 关键词: spermatogenesis; spermatovonial stem cell; culture; micro-insemination

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.

1)小鼠精原干细胞系的建立和维持：使用三种转基因小鼠(Tg)系；普遍存在的表达GFP的Tg、在减数分裂中期表达GFP的Acrosin-GFP Tg和在单倍体阶段表达GFP的Haspin-GFP Tg。精原干细胞系，也称为生殖干细胞（GS 细胞）是从这三只 Tg 小鼠中建立的。2）GS 的离体精子发生细胞：为了在体外诱导GS细胞的精子发生，将Acrosin-GFP Tg和Haspin-GFP Tg的GS细胞在不同条件下（例如不同的饲养细胞）进行培养，但迄今为止，减数分裂标志物GFP的表达尚未见。根据我们的经验检测到。在我们的离体精子发生试验中，我们发现生精小管可以从裸体皮下组织中分离的胎儿或新生儿睾丸细胞重新构建。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 细胞分化至精子细胞（单倍体）阶段，用于授精，最终产生健康的幼仔。睾丸组织：我们在精子发生细胞培养方面的经验相当令人沮丧，因此我们显然需要转换为另一种策略，使用顶体蛋白和 haspin-GFP Tg 小鼠睾丸组织，3-14 天。早期，我们评估了使用7天或以上幼仔时，haspin-GFP在培养条件下呈阳性，基本有利的培养条件包括32℃、10%胎牛血清、维生素等。在不久的将来就有可能。

项目成果

「精子幹細胞(GS細胞)からの精子形成」

“精子干细胞（GS 细胞）的精子发生”

• 发表时间: 2007
• 作者: Ogawa;T;Katagiri;K;Kita;K;Kubota;Y;小川 毅彦;小川 毅彦;小川 毅彦;小川 毅彦
• 通讯作者: 小川 毅彦

Testosterone administration promotes regeneration of chemically impaired spermatogenesis in rats

睾酮管理促进大鼠化学损伤的精子发生的再生

• 发表时间: 2006
• 期刊: Int J Urol 13(8)
• 作者: Udagawa;K;Ogawa;T;Watanabe;T;Tamura;Y;Kita;K;Kubota;Y
• 通讯作者: Y

「精子系細胞の体外分化と成熟」〈特集〉精子をめぐる最近の進歩

《精子细胞的体外分化和成熟》（专题）精子的最新进展

• 发表时间: 2008
• 期刊: 産婦人科治療 90
• 作者: Mizukami T;Kuramitsu M;Takizawa K;Momose H;Masumi A;Naito S;Iwama A;Ogawa T;Noce T;Hamaguchi I;Yamaguchi K.;小川 毅彦
• 通讯作者: 小川 毅彦

「精細胞培養の最前線」特集:男性不妊症

《精子细胞培养最前沿》专题：男性不育症

• 发表时间: 2007
• 期刊: [Modern Physician] 27
• 作者: Kata K;Watanabe T;Ohsaka K;Hayashi H;Kubota Y;Nagashima Y;Aoki I;Taniguchi H;Noce T;Inoue K;Miki H;Ogonuki N;Tanaka H;Ogura A;Ogawa T.;小川 毅彦
• 通讯作者: 小川 毅彦

「精子幹細胞と多能性幹細胞」幹細胞技術の現状と展望 No.8

《精子干细胞与多能干细胞》干细胞技术现状与展望No.8

• 发表时间: 2007
• 期刊: 蛋白質 核酸 酵素 52・4
• 作者: Mizukami T;Kuramitsu M;Takizawa K;Momose H;Masumi A;Naito S;Iwama A;Ogawa T;Noce T;Hamaguchi I;Yamaguchi K.;小川毅彦;小川 毅彦
• 通讯作者: 小川 毅彦