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 cells enriched in precursor Leydig cells (PLCs) and Leydig cells (LCs) were isolated from the testis of EDS-treated rats and age-matched controls using a collagenase digestion-Percoll gradient method. Total RNA extracted from the PLC and LC fractions was subjected to reverse transcriptase polymerase chain reaction (RT-PCR) to detect estrogen receptor (ER) mRNA. The RT-PCR results demonstrated that ER mRNA was present in both LC and PLC fractions. Quantitative RT-PCR analysis, using rabbit beta-globin mRNA as the internal standard, showed that ER mRNA in the PLC fraction was 20-fold higher than in the LC fraction in control testis. After EDS treatment, ER mRNA levels in the PLC fraction decreased and reached a nadir at Day 16 posttreatment. Thereafter, ER mRNA in the PLC fraction gradually increased and returned to control PLC levels. In contrast, ER mRNA levels in the LC fraction in controls and at Days 16-45 posttreatment remained constant. To correlate the changes in ER mRNA levels with LC differentiation, in vitro testosterone (T) production 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 fraction was low (1/10th that in the control LC fraction), and hCG addition resulted in only a 1.5-fold stimulation (relative to a 7.5-fold stimulation in LCs). In the PLC fraction, T production was not detectable at Days 2 and 10 after EDS treatments, began to respond to hCG stimulation with increased T production at Day 16, and reached a maximum between 4 and 6 wk after EDS treatment. By Day 60 posttreatment, T production 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 and hCG-responsive T production increased gradually and returned to control LC levels. It is concluded that functional LCs are regenerated from the PLCs and that both these cell types possess ER mRNA. It is interesting to note that PLCs exhibit higher levels of ER mRNA than do LCs. A decrease in ER mRNA in PLCs appears to coincide with the early differentiation process to yield LCs. Thus, estradiol-17 beta produced locally in the testis by the LCs might act via its receptor as a paracrine substance to impede PLC development into LCs. It is therefore possible that either a decrease in E2 production or a decrease in ER and its mRNA in PLCs would then release the PLCs to begin the regeneration process.