GENE-EXPRESSION OF LUTEINIZING-HORMONE RECEPTOR AND STEROIDOGENIC ENZYMES DURING LEYDIG-CELL DEVELOPMENT

Testicular Leydig cells (LC) are rapidly and selectively destroyed by an injection of ethane dimethane sulfonate (EDS). LC regeneration occu rs in the testis of the EDS-treated rats from the differentiation of t he precursor Leydig cells (PLC). This study was designed to investigat e the patterns of change in the mRNAs for the luteinizing hormone rece ptor (LHR) and the steroidogenic enzymes, cholesterol side chain cleav age (P-450(scc)) and 17 alpha-hydroxylase (P-450(17 alpha)) during LC regeneration from PLCs. Mature (60 days of age) Sprague-Dawley male ra ts received a single intraperitoneal injection of EDS and were killed at different times between days 2 and 60 post-treatment. PLC- and LC-e nriched fractions were isolated from the testes of the EDS-treated rat s and age-matched control rats using a collagenase digestion-Percoll g radient method. Total RNA was extracted from these cell populations an d subjected to Northern blot analysis. The LC fraction isolated from t estes of control rats expressed four major transcripts of the LHR, siz ed 1.8, 2.5, 4.2 and 7.0 kb. The undifferentiated PLC fraction from co ntrols expressed only a truncated form, the 18 kb transcript. This tru ncated LHR transcript was also the only LHR mRNA species detected in P LCs at day 2 post-EDS treatment. In contrast, all four transcripts of the LHR were detected in the PLC fraction at day 10 post-EDS treatment . The levels of the full length 7.0 kb transcript increased thereafter and reached a peak between days 24 and 36 post-EDS treatment in the P LC fraction. Concomitant with the increase in the 7.0 kb transcript, t he truncated 1.8 kb transcript decreased in amount and reached a nadir between days 16 and 36 post-treatment. The changes observed in this c ell fraction reflect the process of differentiation of PLCs into LCs. At day 45 post-EDS treatment, the level of the 7.0 kb transcript decre ased while the 1.8 kb form increased in the PLC fraction, reflecting t he completion of LC regeneration from this cell fraction. By day 60 po st-EDS treatment, the levels of the 1.8 kb transcript rose to the valu e observed in undifferentiated control PLCs and the other transcripts were no longer detected in the PLC fraction, indicating that cells in the PLC fraction were again in an undifferentiated stage. Messenger RN As for both the steroidogenic enzymes, P-450(scc) and P-450(17 alpha) were expressed in the control LC fraction. Neither of these two mRNAs were detected in the PLC fraction of the control rats. P-450(scc) and P-450(17 alpha) mRNAs were first expressed in the PLC fraction at day 10 post-EDS treatment. Thereafter, the levels of P-450(scc) and P-450( 17 alpha) mRNAs increased in the PLC fraction and reached a peak betwe en days 24 and 36 and days 24 and 45 post-EDS treatment respectively. P-450(scc) and P-450(17 alpha) mRNAs were no longer expressed in the P LC fraction at day 60 post-EDS treatment. These patterns also reflect the process of differentiation of PLCs into functional LCs. These resu lts demonstrate for the first time that PLCs in the control testis are undifferentiated and do not express functional LHR and steroidogenic enzymes or their mRNAs. The PLCs are characterized, however, by the ex pression of a truncated 1.8 kb transcript of the LHR mRNA. Functional LHR and steroidogenic enzymes are expressed in PLCs only during their differentiation into LCs after EDS treatment. Subsequent to LC regener ation, the PLCs return to an undifferentiated stage.