Development of mouse spermatogonial purification system and its application for fertility restoration in mice

我们试图通过增加供体细胞定殖水平来恢复不育小鼠的生育能力。为此，我们使用了两种方法。首先，我们用未成熟的老鼠作为受体。我们经常使用成熟的成年小鼠进行精子移植恢复生育能力。本实验以5 ~ 10日龄幼犬为受体。这是基于观察到在这一阶段支持细胞之间没有紧密连接。因此，我们假设缺乏紧密连接，从而增加了移植干细胞的定植过程。我们发现幼犬睾丸的定植效率比成年睾丸高10倍。此外，80%到100%的雄性受赠者在与雌性交配后都能生育。令人惊讶的是，受体在与野生型小鼠相当的年龄产生了后代。作为增加供体细胞定植的第二种方法，我们产生了针对未成熟睾丸的单克隆抗体。睾丸含有丰富的未分化精原细胞，包括干细胞。我们在大鼠中免疫了这些睾丸细胞，并获得了几个克隆。其中一个克隆在接受精原细胞移植后，干细胞的富集量达到原来的5倍。我们现在正试图描述抗原的分子结构。综上所述，我们已经建立了一个有效的小鼠精原移植恢复生育能力的系统。我们还在精原干细胞上发现了一种新的抗原。这些结果对人类生育能力恢复技术的发展具有重要意义。

We attempted to restore fertility to infertile mouse by increasing the donor cell colonization level. For this purpose, we used two approaches. First, we used immature mice for recipients. We have regularly used mature adult mice for fertility restoration by spermatogonial transplantation. In the present experiment, we used 5-10 day old pups as recipients. This was based on the observation that there are no tight junction between Sertoli cells at this stage. We therefore hypothesized that the absence of tight junction, thereby increasing the the colonization process of transplanted stem cells.We found that colonization efficiency in the pup testis is 10-fold higher than in the adult testis. Furthermore, 80 to 100% of the recipiet males became fertile upon mating with females. Surprisingly, the recipients sired offspring at age equivalent to wild type mice. As a second approach to increase donor cell colonization, we generated monoclonal antibodies to immature testis. The testes contain enriched population of undifferentiated spermatogonia, including stem cells. We immunized these testis cells in rats, and obtained several clones. One of the clones were able to enrich stem cells by 5-fold after spermatogonial transplantation. We are now trying to characterize the molecular structure of the antigen.In summary, we have established an efficient system for fertility restoration by spermatogonial transplantation in mice. We also found a novel antigen on spermatogonial stem cells. These results have important implications for developing technique for fertility restoration in humans.