BIOCHEMICAL, MOLECULAR BIOLOGICAL AND IMMUNOHISTOCHEMICAL STUDIES ON ANDROGENE RECEPTORS IN HUMAN LEYDIG CELLS

Abstract

The possible existence of androgen receptors (ARs) in human Leydig cells was examined by means of a biochemical AR assay, Western blot analysis, Northern blot analysis and immunohistochemistry.
Percoll gradients were utilized to isolate highly purified Leydig cells from human testicular tissue. After these cells were confirmed to retain their morphological and biochemical integrity, they were used for the AR assay, Western blot analysis and Northern blot analysis. ARs in the purified Leydig cells were measured using ³H-methyltrienolone as the labeled ligand. A high affinity AR was detected in total cell extracts of both purified Leydig cells and whole testicular tissues, but the Bmax was less in the former than the latter.
In Western blot analysis with anti-human AR (hAR) monoclonal antibody, two bands, different in molecular weight, were positively stained. These two 97kDa and 80kDa proteins were thought to be the specific AR proteins which were translated from the first and second initiation site of hAR cDNA, respectively.
Northern blot analysis of RNA from purified Leydig cells as well as RNAs from prostatic and total testicular tissues proved the presence of AR using the hAR cDNA probe, and showed that the molecular size of AR mRNA is about 9.5kb.
Immunoreactive ARs in the human testicular tissue with anti-hAR monoclonal antibody were found predominantly in the nuclei of Leydig cells, suggesting that ARs, like other steroid receptors, exist mainly in nuclei of them.
These findings provided the evidence that ARs are present in human Leydig cells and suggested that some local autoregulations may exist in the human testis.