Analysis of spermatogonial proliferation with technique of spermatogonial transplantation-A trial for improving spermatogenic activity

当应用于精子发生的研究时，精原移植有几个独特的优点。例如，可以对生殖细胞和由许多体细胞组成的睾丸环境进行不同的处理，以观察治疗的效果。在移植后菌落发育的早期，也可以清楚地观察到精原细胞的增殖状态。我们首先在该系统中引入了GFP小鼠作为供体小鼠，从而使该系统更加定量。以GFP小鼠为供体，精子干细胞的序贯移植成为可能。到目前为止，我们已经连续4次成功地传代了干细胞。积累的数据表明，精原干细胞随着菌落大小的增加和时间的推移而扩大。在移植后的100天内，估计其扩张率约为30倍。这一扩张速度似乎在一年多的时间里保持不变，没有显示出枯竭的迹象。同时，我们也试图发现睾丸内微环境对精原细胞增殖的影响。LH-RH类似物（leuprorelin）用于改变睾丸激素环境。在leuprorelin处理的小鼠睾丸中，早在移植后4周，移植精原细胞在精小管基底膜上的密度就增加了。这一发现表明，在leuprorelin处理的小鼠睾丸中，精原细胞被刺激增殖或其凋亡减少。本研究建立了精原细胞移植技术作为评价精原细胞增殖、分化和死亡的定量方法。

Spermatogonial transplantation has several unique advantages when applied to the study of spermatogenesis. For instance, germ cells and testis environment composed of many somatic cells can be differently treated to see effects of the treatments. The proliferation status of spermatogonia can be also clearly observed in the early stage of colony development after transplantation. We have first introduced GFP mice in this system as donor mice by which the system became more quantitative. With GFP mice as donor, sequential transplantation of spermatogonial stem cell became possible. We have so far succeeded to passage stem cells for 4 times successively. Accumulated data indicated that spermatogonial stem cells expand as colony size increases, and as time passes by. The expansion rate was estimated to be about 30-fold during 100 days following transplantation. This expansion rate appeared to be constant for more than a year showing no sign of exhaustion. Meanwhile, we also tried to find the effect of intra-testicular microenvironment on spermatogonial proliferation. The LH-RH analogue (leuprorelin) was used to modify hormonal environment of the testis. In the leuprorelin-treated mouse testes, transplanted speramtogonia presented more densely on the basement membrane of the seminiferous tubules as early as 4 weeks after the transplantation. This finding suggests that spermatogonia are stimulated to proliferate or their apoptosis are reduced in the leuprorelin-treated mouse testes. Our present research established the spermatogonial transplantation technique as a quantitative method for evaluating various treatments on spermatogonial proliferation, differentiation, and death.