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

GFP小鼠生精障碍评价方法的开发及生殖障碍机制分析

基本信息

批准号：
13670005
负责人：
KOMIYAMA Masatoshi
金额：
$ 2.3万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

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.

为了明确二乙基己烯雌酚（DBS）毒性作用的靶点是生殖细胞还是支持生殖细胞的体细胞，在新生儿暴露于二乙基己烯雌酚所致的精子发生障碍中，将GFP小鼠的生殖细胞通过Busulfan处理移植到游离自身生殖细胞的C57BL/6小鼠精小管中。首先，证实新生儿暴露于0.5 μg/小鼠/天的DES 5天会导致GFP小鼠和C57BL/6小鼠的精子发生障碍。将DES暴露的生殖细胞移植到未暴露DES的无生殖细胞小鼠中，精子发生正常。另一方面，当将正常生殖细胞移植到新生儿暴露于DES的无生殖细胞小鼠体内时，精子发生不正常，很少观察到精子。由此可见，DES毒性作用的靶点不是生殖细胞，而是体细胞。用ICR小鼠研究了新生儿暴露于DES (5 μg/小鼠/d) 5 d后对附睾的影响。暴露于DES后，(1)雄激素受体蛋白的表达延迟；(2)雌激素受体α蛋白表达期变宽，沿附睾小管分布变宽；(3)雌激素应答基因之一乳铁蛋白mRNA表达区近端增宽；(4)附睾小管间隙和管腔增大，上皮增厚等形态学改变。这些影响的机制将使用小鼠附睾cDNA芯片进行分析。