ESTROGEN-RECEPTOR MESSENGER-RIBONUCLEIC-ACID CHANGES DURING LEYDIG-CELL DEVELOPMENT

Mature (60-65 days old) male Sprague-Dawley rats received a single i.p . injection of ethane dimethane sulfonate (EDS, 100 mg/kg BW) and were killed at different times from Days 2 to 60 posttreatment. Bands of c ells enriched in precursor Leydig cells (PLCs) and Leydig cells (LCs) were isolated from the testis of EDS-treated rats and age-matched cont rols using a collagenase digestion-Percoll gradient method. Total RNA extracted from the PLC and LC fractions was subjected to reverse trans criptase polymerase chain reaction (RT-PCR) to detect estrogen recepto r (ER) mRNA. The RT-PCR results demonstrated that ER mRNA was present in both LC and PLC fractions. Quantitative RT-PCR analysis, using rabb it beta-globin mRNA as the internal standard, showed that ER mRNA in t he PLC fraction was 20-fold higher than in the LC fraction in control testis. After EDS treatment, ER mRNA levels in the PLC fraction decrea sed and reached a nadir at Day 16 posttreatment. Thereafter, FR mRNA i n the PLC fraction gradually increased and returned to control PLC lev els. In contrast, FR mRNA levels in the LC fraction in controls and at Days 16-45 posttreatment remained constant. To correlate the changes in FR mRNA levels with LC differentiation, in vitro testosterone (T) p roduction by PLC- and LC-enriched fractions in the presence or absence of 50 mIU hCG was measured by RIA. T production in the control PLC fr action was low (1/10th that in the control LC fraction), and hCG addit ion resulted in only a 1.5-fold stimulation (relative to a 7.5-fold st imulation in LCs). In the PLC fraction, T production was not detectabl e at Days 2 and 10 after EDS treatment, began to respond to hCG stimul ation with increased T production at Day 16, and reached a maximum bet ween 4 and 6 wk after EDS treatment. By Day 60 posttreatment, T produc tion in the PLC fraction decreased and returned to control PLC levels. Testosterone production in the LC fraction was not detectable at Days 2 and 10 posttreatment. From Days 16 to 60 posttreatment, LC basal an d hCG-responsive T production increased gradually and returned to cont rol LC levels. It is concluded that functional LCs are regenerated fro m the PLCs and that both these cell types possess ER mRNA. It is inter esting to note that PLCs exhibit higher levels of ER mRNA than do LCs. A decrease in FR mRNA in PLCs appears to coincide with the early diff erentiation process to yield LCs. Thus, estradiol-17 beta produced loc ally in the testis by the LCs might act via its receptor as a paracrin e substance to impede PLC development into LCs. It is therefore possib le that either a decrease in E(2) production or a decrease in ER and i ts mRNA in PLCs would then release the PLCs to begin the regeneration process.