黄体生成素受体对睾丸间质干细胞增殖分化的影响

Effect of luteinizing hormone/chorionic gonadotropin receptor on the proliferation and differentiation of stem Leydig cells

目的探讨黄体生成素受体(LHCGR)对睾丸间质干细胞(SLC)增殖及分化能力的影响。方法采用实时荧光定量PCR(qRT-PCR)及免疫荧光染色比较LHCGR-/-小鼠与LHCGR^(+/+)小鼠睾丸内SLC标志物的表达情况。通过流式细胞术分选出SLC,进行5-乙炔基-2'-脱氧尿苷(EdU)及CCK-8增殖实验。同时诱导SLC向睾丸间质细胞(LC)分化,14 d后检测上清液睾酮水平。qRT-PCR及免疫荧光检测诱导分化后LC标志物表达水平。结果LHCGR-/-小鼠睾丸内存在SLC,应用CD51标记并通过流式分选可获得SLC。LHCGR-/-小鼠SLC EdU+细胞比例和CCK-8增殖曲线与LHCGR^(+/+)小鼠比较差异均无统计学意义(P均>0.05)。LHCGR-/-小鼠SLC诱导分化后培养基上清液睾酮平均水平仅为0.01 ng/ml,低于LHCGR^(+/+)对照组的2.71 ng/ml(P<0.001),其诱导分化后细胞LC标记物的表达也低于LHCGR^(+/+)小鼠(P均<0.001)。结论敲除LHCGR基因后,睾丸内存在SLC且能维持正常的增殖活性,但是分化为LC受阻。

Objective To evaluate the effect of luteinizing hormone/chorionic gonadotropin receptor(LHCGR)on the proliferation and differentiation of stem Leydig cells(SLCs).Methods The expression levels of SLC markers in the testes between the LHCGR-/-and LHCGR^(+/+)mice were statistically compared by quantitative RT-PCR(qRT-PCR)and immunofluorescent staining.The CD51+SLCs were sorted by flow cytometry and then subject to 5-ethynyl-2’-deoxyuridine(EdU)and CCK8 assay.The SLCs were induced to differentiate into the testicular Leydig cells(LCs).The testosterone level in the supernatant was detected after 14 d.The expression levels of LC markers after induced differentiation were quantitatively measured by qRTPCR and immunofluorescent staining.Results SLCs were found in the testis of LHCGR-/-mice and these cells could be sorted by CD51 labeling and flow cytometry.The proportion of EdU+cells and CCK-8 proliferation curve did not significantly differ between the LHCGR-/-and LHCGR^(+/+)SLCs(both P>0.05).After the induced differentiation of LHCGR-/-SLCs,the average testosterone concentration in the culture supernatant was 0.01 ng/ml,significantly lower than 2.71 ng/ml in the LHCGR^(+/+)mice(P<0.001).The expression levels of LC markers in the LHCGR-/-mice were significantly lower compared with those in the LHCGR^(+/+)mice(all P<0.001).Conclusion After LHCGR knockout,SLCs exist in the testis of LHCGR-/-mice and maintain normal proliferative activity.Nevertheless,the differentiation from SLCs into LCs is suppressed.