MOLECULAR CYTOCHEMICAL ANALYSIS OF GERM CELL DEATH IN SPERMATOGENESIS.

生殖细胞死亡在哺乳动物的睾丸发育和成虫期都很常见。在小鼠睾丸中，已知有少量但数量可观的生殖细胞在成年期自发退化，事实上，在成熟精子发生过程中，预期精子产量的25-75%被认为丢失。人工隐睾和雌激素处理（每头注射30 μ g -雌二醇-3-苯甲酸酯，每5天，连续60天）小鼠生殖细胞的退化更为明显。然而，生殖细胞的死亡究竟是凋亡性的还是坏死性的，我们还不能确定，而且我们对生殖细胞死亡的潜在机制的了解也很有限。因此，在本研究中，我们试图通过电子显微镜和原位缺口翻译和/或TUNEL来阐明在正常和受损小鼠睾丸中发生的细胞死亡类型。结果发现，在正常和损伤睾丸中，大多数变性生殖细胞具有凋亡性质，在正常睾丸中，精原细胞和精母细胞的凋亡在精原上皮周期第12期最为丰富。为了阐明生殖细胞凋亡的诱导机制，我们通过分子组织化学方法研究了Fas/Fas配体表达与细胞凋亡发生的时空关系。在正常睾丸中，我们没有发现它们之间存在正相关，而在雌激素处理的小鼠睾丸中，Fas和Fas配体的表达分别在生殖细胞和Sertoli细胞中显著增加，并且似乎与生殖细胞凋亡密切相关。因此，这些结果表明，Fas/Fas配体系统可能参与了受损睾丸中生殖细胞凋亡的诱导，但不参与正常精子发生。

Germ cell death is very common in mammalian testes during developmental as well as adult period. In mouse testis, it was known that a small, but significant number of germ cells are spontaneously degenerated during adult life and in fact 25-75% of the expected sperm yield are thought to be lost in the mature spermatogenesis. Also, in artificial cryptorchidism and estrogen-treated (i.p.injection of 30 mu g beta-estradiol-3-benzoate/head, every 5 day for 60 days) mice, the degeneration of germ cells are much more prominent. However, we are not sure wether the germ cell death is of apoptotic nature or necrotic nature, and moreover our knowledge on the underlying mechanism to induce the germ cell death is only limited. Therefore, in this study, we attempted to clarify the type of cell death, which occurs in normal as well as the damaged mouse testes, by both electron microscopy and in situ nick translation and/or TUNEL.As a result, we found that most of the degenerating germ cells were of apoptotic nature in normal and damaged testes, and that in the normal testis apoptosis of spermatogonia and spermatocytes was most abundant in the stage XII of seminiferous epithelium cycle. To elucidate the induction mechanism of germ cell apoptosis, we investigated the spatial and temporal relationship of the expression of Fas/Fas ligand with the appearance of apoptosis molecular-histochemically. Then we did not find any positive correlation between them in normal testis, whereas in estrogen-treated mouse testis both expressions of Fas and Fas ligand were markedly increased in germ cells and Sertoli cells, respectively, and seemed to be closely associated with the apoptotic germ cells. Consequently, these results indicate that the Fas/Fas ligand system may be involved in the induction of germ cell apoptosis in damaged testis, but not in normal spermatogenesis.