Immunodetection of aromatase in mice with a partial deletion in the long arm of the Y chromosome

Abstract

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-Y del (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-Y del 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. 55 kDa 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.