Zhai J, Lanclos K D, Abney T O
Department of Physiology and Endocrinology, Medical College of Georgia, August 30912, USA.
Biol Reprod. 1996 Oct;55(4):782-8. doi: 10.1095/biolreprod55.4.782.
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.
选用60 - 65日龄的成年雄性斯普拉格 - 道利大鼠，腹腔注射一次乙烷二甲磺酸盐（EDS，100mg/kg体重），并在处理后的第2天至60天的不同时间点处死。采用胶原酶消化 - Percoll梯度法，从EDS处理大鼠和年龄匹配的对照大鼠的睾丸中分离出富含前体莱迪希细胞（PLCs）和莱迪希细胞（LCs）的细胞带。从PLC和LC组分中提取的总RNA进行逆转录聚合酶链反应（RT-PCR）以检测雌激素受体（ER）mRNA。RT-PCR结果表明，ER mRNA在LC和PLC组分中均有表达。以兔β - 珠蛋白mRNA作为内参进行定量RT-PCR分析，结果显示在对照睾丸中，PLC组分中的ER mRNA水平比LC组分高20倍。EDS处理后，PLC组分中的ER mRNA水平下降，并在处理后第16天达到最低点。此后，PLC组分中的ER mRNA逐渐增加并恢复到对照PLC水平。相比之下，对照和处理后第16 - 45天的LC组分中的ER mRNA水平保持恒定。为了将ER mRNA水平的变化与LC分化相关联，通过放射免疫分析法（RIA）测量在有或无50mIU人绒毛膜促性腺激素（hCG）存在的情况下，富含PLC和LC的组分的体外睾酮（T）生成量。对照PLC组分中的T生成量较低（为对照LC组分的1/10），添加hCG仅导致1.5倍的刺激（相对于LC中的7.5倍刺激）。在PLC组分中，EDS处理后第2天和第10天未检测到T生成，在第16天开始对hCG刺激产生反应，T生成增加，并在EDS处理后4至6周达到最大值。至处理后第60天，PLC组分中的T生成量下降并恢复到对照PLC水平。处理后第2天和第10天，LC组分中未检测到睾酮生成。从处理后第16天至60天，LC的基础和hCG反应性T生成逐渐增加并恢复到对照LC水平。结论是功能性LCs从PLCs再生，并且这两种细胞类型均具有ER mRNA。有趣的是，PLCs中的ER mRNA水平高于LCs。PLCs中ER mRNA的减少似乎与产生LCs的早期分化过程一致。因此，LCs在睾丸局部产生的雌二醇 - 17β可能通过其受体作为旁分泌物质来阻碍PLCs发育成LCs。因此，有可能E2生成的减少或PLCs中ER及其mRNA的减少会使PLCs开始再生过程。
