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Establishment of testis in vitro assay system for risk assessment of endocrine disrupters

内分泌干扰物风险评估睾丸体外检测体系的建立

基本信息

批准号：
13460130
负责人：
HAYASHI Yoshihiro
金额：
$ 7.3万
依托单位：
THE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
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

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13460130/
关键词：
spermatogenic cells estrogen Bisphenol-A primary Sertoli cell culture organ culture of undifferentiated gonad testicular cord rat mouse 未分化生殖腺器官培養系セルトリ細胞培養系テストステロン

项目摘要

In order to collect the fundamental information on spermatogenic cells, we made the stage map of seminiferous epithelium in the greater Japanese shrew mole and examined the lectin-histochemical characteristics of their testicular cells. At the same time, we observed the distribution of Smad in hamster testes. As a result, Smad2 and Smad3 mRNA were detected in spermatogonia and spermatocytes. Next, we examined the effect of estrogen on rat testes. After administration of estrogen to 10 week old rats, testosterone decreased to a great degree and testicuiar steroidogenic enzymes also decreased to a degree. Bisphenol-A (BPA), one of endocrine disrupters, was added to the primary Sertoli cell cultre established from 10 day old rat testes at the concentration of 10, 100, lOOOpg/m. At 2 days after treatment, the increase of degenerative Sertoli cells was 7.2, 14.7 and 20.8%, respectively. At 4 days, the increase was 11.4, 19.5 and 28.3%., and at 6 days, the increase was 12.6, 21.3 and 34.9%, respectively. These increases were significant compared with the control. While, BPA was added to the primary Sertoli cell culture established from 1 〜 2 month old goat testes at the concentration of 1,10, lOOum, and the samples was observed at 1,3,5 days after treatment. As a result, similar to the case in rats, the degenerative Sertoli cells increased dose-dependently and time-dependently. Thus, it is possible to postulate that BPA may affect Sertoli cells directly. Then, BPA and 17b estradiol (E2) was added to the organ culture of mouse undifferentiated gonads at the concentration of 0.1, 1, lOum. At 5 days after treatment, the samples were observed. In this case, no obvious changes were detected. In males, normal testicular cord formatin was clearly recognized. Therefore, it is suggested that BPA and E2 do not affect the initial differentiation of gonads directly.

为了收集生精细胞的基本信息，我们制作了大日本地鼠生精上皮的阶段图，并检测了其睾丸细胞的凝集素组织化学特征。同时观察了Smad在仓鼠睾丸中的分布。结果表明，Smad 2和Smad 3 mRNA在精原细胞和精母细胞中均有表达。接下来，我们研究了雌激素对大鼠睾丸的影响。给10周龄大鼠注射雌激素后，睾酮水平明显下降，睾丸类固醇生成酶也有不同程度的下降。将内分泌干扰物双酚A（BPA）以10、100、1000 μ g/m2的浓度添加到从10日龄大鼠睾丸建立的原代支持细胞培养物中。治疗后第2天，退变支持细胞分别增加7.2%、14.7%和20.8%。在第4天，分别增加11.4%、19.5%和28.3%。第6天分别增加12.6%、21.3%和34.9%。与对照组相比，这些增加是显著的。将BPA以1、10、100 μ m的浓度分别加入1 ~ 2月龄山羊睾丸支持细胞原代培养物中，于处理后1、3、5天取样观察。结果，与大鼠中的情况类似，退化的支持细胞呈剂量依赖性和时间依赖性增加。因此，有可能假设BPA可能直接影响支持细胞。然后，将BPA和17 b雌二醇（E_2）以0.1、1、10 μ m的浓度加入到小鼠未分化性腺的器官培养中。在处理后5天，观察样品。在这种情况下，没有发现明显的变化。在雄性中，正常睾丸索形成被清楚地识别。因此，BPA和E2对性腺的初始分化没有直接影响。