Immunocytochemical localization of 4-ene steroid 5 alpha-reductase type 1 along the rat epididymis during postnatal development.

Abstract

Dihydrotestosterone (DHT), the active androgen in many tissues, is synthesized from testosterone by the enzyme 4-ene steroid 5 alpha-reductase (5 alpha-reductase; EC 1.3.1.22). In the epididymis, the maturation and storage of spermatozoa are dependent on the presence of 5 alpha-reduced androgens. The regulation of epididymal 5 alpha-reductase is complex. To date, the regulation of this enzyme has been studied extensively at the level of enzymatic activity and more recently at the mRNA level. Regulation at the level of the protein, however, remains poorly understood. We have raised rabbit polyclonal antibodies to a 24-mer synthetic peptide whose sequence was determined from the predicted amino acid sequence for rat 5 alpha-reductase type 1 to immunolocalize the 5 alpha-reductase type 1 protein in the rat epididymis during postnatal development. Western blot analysis revealed a specific immunoreactive band of 26 kilodaltons in male rat liver, epididymis, and prostate; this apparent molecular size is identical to that obtained when the 5 alpha-reductase type 1 cDNA is expressed in mammalian cells. Furthermore, the relative protein levels, liver > epididymis > prostate, were consistent with the mRNA levels for type 1 rat 5 alpha-reductase. Perfusion-fixed paraffin-embedded epididymal tissue sections were used to immunolocalize type 1 5 alpha-reductase. In the adult rat epididymis, the most intense immunoperoxidase reaction was observed in a discrete lobule of the initial segment of the epididymis. A progressive decrease in staining intensity occurred distally along the tissue to the cauda epididymis. The staining reaction was specific to cytoplasmic elements of epithelial principal cells; no reaction was evident over nuclei. However, specifically in the initial segment, very intense staining was seen in the infranuclear region of the principal cells. In the proximal caput epididymidis, the staining was primarily confined to an oval region above the nuclei, whereas in the remaining epididymal regions, weak staining was seen throughout the cytoplasm. Thus, the intracellular localization of the 5 alpha-reductase type 1 protein changed as one moved down the epididymis. Finally, the pattern of immunolocalization of 5 alpha-reductase type 1 protein was different in the epididymis of rats of different postnatal ages. On day 7, no reactivity was noted; by day 28, a weak apical staining of principal cells was seen throughout the epididymis; by day 47, the adult pattern of staining had been established. Our results revealed that the expression and intracellular localization of the 5 alpha-reductase type 1 protein are both age dependent and epididymal segment specific.