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ex vivo spermatogenesis from cultured spermatogonial stem cells

培养精原干细胞的离体精子发生

基本信息

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

项目摘要

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与维持：选用3个转基因小鼠(TG)系，分别在减数分裂中期及以后普遍表达GFP，在减数分裂中期以上表达GFP，在单倍体阶段表达GFP。2)体外精子发生：为了在体外诱导GS细胞发生，将Acrosin-GFP TG和Haspin-GFP TG的GS细胞分别在不同的饲养层细胞中培养。然而，到目前为止，在我们的经验中还没有检测到减数分裂的标志GFP的表达。在我们的体外精子发生试验中，我们发现在裸鼠的皮下组织中，分离的胎儿或新生睾丸细胞可以重新构建生精小管。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细胞经过分化直到精子细胞(单倍体)阶段，用于人工授精，最终产生健康的瞳孔。3)睾丸组织的器官培养：我们在精子发生细胞培养方面的经验相当令人沮丧，显然我们需要转向另一种策略。采用3-14日龄顶体酶和haspin-GFP转基因小鼠睾丸组织，对器官培养方法进行了评价。当使用7日龄或7天以上的幼崽时，haspin-GFP在培养条件下呈阳性。基本适宜的培养条件为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。

项目成果

期刊论文数量（0）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

「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

DOI：
发表时间：
2008
期刊：
産婦人科治療 90
影响因子：
0
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

DOI：
发表时间：
2007
期刊：
[Modern Physician] 27
影响因子：
0
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

Reproductive stem cell research and its application to urology