Development of evaluation method for spermatogenesis disorder using GFP mouse and analysis of mechanisms of reproduction disorder

为了明确己烯雌酚（DES）的毒性靶点是生殖细胞还是支持生殖细胞的体细胞，在新生儿暴露于DES引起的精子发生障碍中，将GFP小鼠的生殖细胞移植到通过白消安处理而去除自身生殖细胞的C57 BL/6小鼠的曲细精管中。首先，证实了新生儿暴露于0.5 μg/小鼠/天的DES 5天会导致GFP小鼠以及C57 BL/6小鼠的精子发生障碍。当DES暴露的生殖细胞移植到没有暴露于DES的无生殖细胞小鼠时，精子发生正常。另一方面，当将正常生殖细胞移植到生殖细胞暴露于DES的无生殖细胞小鼠中时，精子发生不正常，很少观察到精子。用ICR小鼠研究了DES（5 μg/mouse/day）染毒5天对仔鼠附睾的影响。结果表明，DES使雄激素受体蛋白表达延迟，雌激素受体α蛋白表达时程增宽，沿附睾管沿着分布变窄，雌激素反应基因乳铁蛋白mRNA表达区向近端增宽，雌激素受体α蛋白表达区向近端增宽，雌激素受体α蛋白表达区向近端增宽。（4）附睾小管间质和管腔扩大，上皮增厚等形态学改变。这些作用的机制将使用内部小鼠附睾cDNA微阵列进行分析。

In order to clarify which are the target of toxicity of diethylstilbestrol (DBS), germ cells or somatic cells supporting the germ cells, in the disorder of spermatogenesis caused by neonatal exposure to DES, germ cells of GFP mice were transplanted to seminiferous tubules of C57BL/6 mice which were free of own germ cells by Busulfan treatment. First, it was confirmed that neonatal exposure to 0.5 μg/mouse/day of DES for 5 days causes spermatogenesis disorder in GFP mice as well as C57BL/6 mice. When DES-exposed germ cells were transplanted to the germ cell-free mice that were not exposed to DES, spermatogenesis occurred normally. On the other hand, when normal germ cells were transplanted to the germ cell-free mice that were neonatally exposed to DES, spermatogenesis was not normal and spermatozoa were rarely observed. From these results, it was concluded that the target of DES toxicity is not germ cells but somatic cells.The effect of neonatal exposure to DES (5 μg/mouse/day) for 5 days on the epididymis was studied using ICR mice. By exposure to DES, (1) expression of androgen receptor protein was delayed ; (2) expression term of estrogen receptor α protein was widened and its distribution along the epididymal ductule was braodened ; (3) area of mRNA expression of lactoferrin, one of the estrogen-responsive genes, was proximally broadened ; and (4) various morphological changes, such as enlargement of interstitial space and lumen of epididymal tubule and thickening of the epithelium, occurred. Mechanisms of these effects will be analyzed using in-house mouse epididymis cDNA microarray.