STEADY-STATE STEROID 5-ALPHA-REDUCTASE MESSENGER-RIBONUCLEIC-ACID LEVELS AND IMMUNOCYTOCHEMICAL LOCALIZATION OF THE TYPE-1 PROTEIN IN THE RAT TESTIS DURING POSTNATAL-DEVELOPMENT

Steroid 5 alpha-reductase is the rate-limiting enzyme in the production of 5 alpha-reduced steroids in many tissues. Developmental changes in 5 alpha-reductase activity play an important role in regulating the amount of testosterone that is secreted by the testis. To date, the regulation of testicular 5 alpha-reductase has been studied extensively at the level of enzyme activity. Regulation at the messenger RNA (mRNA) and protein levels, however, has not been investigated. The objectives of the present study were to determine the steady state mRNA levels for the 5 alpha-reductase isozymes, types 1 and 2, and to immunolocalize the 5 alpha-reductase type 1 protein in the developing rat testis (7-91 days postpartum). Consistent with previously reported enzyme activity studies, type 1 5 alpha-reductase mRNA levels were most abundant in the immature animal (days 21-28). Unlike 5 alpha-reductase activity, however, type 1 mRNA levels did not decline thereafter to reach nearly undetectable levels in the adult (day 91). In contrast, 5 alpha-reductase type 1 mRNA levels remained relatively constant between days 42-91. The 5 alpha-reductase type 1 transcript size did not remain constant during post natal testicular development. The characteristic 2.5-kilobase type 1 transcript size was detected in immature rats (days 21-28), whereas in the adult (day 91), a slower migrating 2.7-kilobase type 1 mRNA species was observed. An antipeptide antiserum specific to rat 5 alpha-reductase type 1 was used to immunolocalize the 5 alpha-reductase type 1 protein. At all ages examined, the immunoperoxidase reaction was localized predominantly to the interstitial tissue of the testis. On postnatal day 7, clusters of interstitial cells resembling fetal Leydig cells were clearly immunoreactive. The staining intensity increased steadily from day 7 onwards, so that by days 21 and 28, interstitial cells with the appearance of immature Leydig cells were intensely immunoreactive (peak expression). This was followed by a progressive decrease in staining intensity between days 28-91, so that by day 91 (adult) Leydig cell immunoreactivity was barely detectable. Immunocytochemical staining revealed a predominantly cytoplasmic localization; significant nuclear staining was not evident. We conclude that the expression of the 5 alpha-reductase type 1 protein is primarily found in the cytoplasm of Leydig cells, is dependent on age, and that this expression closely parallels 5 alpha-reductase enzyme activity.