Kotula-Balak Małgorzata, Grzmil Paweł, Styrna Józefa, Bilińska Barbara
Laboratory of Endocrinology and Tissue Culture, Department of Animal Physiology, Institute of Zoology, Jagiellonian University, Ingardena 6, Kraków 30-060, Poland.
Acta Histochem. 2004 Feb;106(1):55-64. doi: 10.1016/j.acthis.2003.09.001.
Aromatization of androgens into estrogens is catalyzed by a microsomal enzyme, P450 aromatase. Males of the mouse strain B10.BR and its congenic mutant strain B10.BR-Ydel (with a partial deletion in the long arm of the Y chromosome) were used to identify the cellular source of estrogens within the testis. Immunocytochemistry was applied to localize aromatase in cultured Leydig cells, cytoplasmic droplets attached to flagella of spermatozoa, and sections of testes. The presence of aromatase in testes was checked by means of Western-blot analysis. Steroid hormones secreted by Leydig cells in vitro were measured in homogenates of testes using radioimmunological methods. Additionally, a Southern analysis was performed using the Y353/B probe to check the length of the deletion in the Y chromosome. In sections of testis of B10.BR mice, weak to moderate immunohistochemical staining of aromatase was found in Leydig cells, Sertoli cells, and germ cells. In testicular cells of B10.BR-Ydel mice, stronger immunostaining of aromatase was observed, especially in germ cells and Leydig cells. Positivity for aromatase was also found in the cytoplasm of cultured Leydig cells from both strains, but it was higher in cells derived from mutant males. Western-blot analysis revealed one major band of approx. 55kDa of aromatase in testes from both strains. Lower testosterone levels were found in mutant males in supernatants of culture media and homogenates of testes in comparison with control males. In contrast, estradiol levels were always higher in mutants. Therefore, it seems likely that the increased expression of aromatase and, as a consequence, the higher levels of endogenous estrogens enhance the morphological alterations in testis and affect spermatogenesis in mutant males.
雄激素向雌激素的芳构化由微粒体酶P450芳香化酶催化。使用小鼠品系B10.BR及其同源突变品系B10.BR - Ydel（Y染色体长臂部分缺失）的雄性小鼠来确定睾丸内雌激素的细胞来源。应用免疫细胞化学方法在培养的睾丸间质细胞、附着于精子鞭毛的胞质小滴以及睾丸切片中定位芳香化酶。通过蛋白质免疫印迹分析检查睾丸中芳香化酶的存在情况。使用放射免疫方法在睾丸匀浆中测量体外培养的睾丸间质细胞分泌的类固醇激素。此外，使用Y353/B探针进行Southern分析，以检查Y染色体缺失的长度。在B10.BR小鼠的睾丸切片中，在睾丸间质细胞、支持细胞和生殖细胞中发现了芳香化酶弱阳性至中度免疫组化染色。在B10.BR - Ydel小鼠的睾丸细胞中，观察到芳香化酶的免疫染色更强，尤其是在生殖细胞和睾丸间质细胞中。在两种品系培养的睾丸间质细胞的细胞质中也发现了芳香化酶阳性，但来自突变雄性的细胞中阳性率更高。蛋白质免疫印迹分析显示，两种品系的睾丸中均有一条约55kDa的芳香化酶主要条带。与对照雄性相比，突变雄性的培养基上清液和睾丸匀浆中的睾酮水平较低。相反，突变体中的雌二醇水平总是较高。因此，芳香化酶表达增加以及内源性雌激素水平升高似乎可能会加剧突变雄性睾丸的形态改变并影响精子发生。
