小鼠睾丸组织中核受体4A1的表达变化及体外对睾丸间质细胞睾酮合成的影响

摘要

目的：观察核受体（ NR）4A1在不同鼠龄小鼠睾丸组织中的表达变化及NR4A1对体外小鼠睾丸间质细胞睾酮合成的影响，并探讨其机制。方法1周龄、4周龄、12周龄、12月龄雄性小鼠各6只，分别为幼年组、性发育前组、性成熟组及老年组。饲养3 d，适应环境后，各组小鼠全部脱颈处死，取双侧新鲜睾丸组织，采用real-time PCR法检测NR4A1 mRNA，Western blotting法检测NR4A1蛋白。体外培养小鼠睾丸间质细胞系MLTC-1，分别将NR4A1干涉腺病毒（ Ad-siNR4A1）及空载腺病毒（ Ad-CMV）转染MLTC-1。采用放射免疫法检测MLTC-1细胞培养液上清中的睾酮，real-time PCR法检测MLTC-1中的类固醇快速转运蛋白（ StAR）、3β-羟类固醇脱氢酶（ HSD3B） mRNA，Western blotting法检测StAR、HSD3B蛋白。结果幼年组、性发育前组、性成熟组、老年组小鼠睾丸组织中NR4A1 mRNA相对表达量以性发育前组最高，与余3组相比，P均＜0．05；NR4A1蛋白相对表达量以性成熟期组最高，与余3组相比，P均＜0．05。转染Ad-siNR4A1、Ad-CMV的MLTC-1中睾酮水平分别为（2．55±0．23）、（4．50±0．23）nmol／L，两者相比，P＜0．05。转染Ad-siNR4A1的MLTC-1中StAR、HSD3B mRNA及其蛋白相对表达量与转染Ad-CMV的细胞相比，P均＜0．05。结论不同鼠龄小鼠睾丸组织中NR4A1的表达有所差别；NR4A1可能通过调控StAR、HSD3B表达从而参与睾酮的合成。%Objective To observe the expression changes of nuclear receptor ( NR) 4A1 in mouse testis and its effect on testosterone synthesis of Leydig cells in vitro, and to explore its mechanism.Methods The male mice of 1 week old, 4 weeks old, 12 weeks old and 12 months old were respectively divided into the infancy group, before sexual development group, sexual maturity group and the older group with 6 mice in each.After being fed for three days and adapting to the en-vironment, all the mice in each group were sacrificed.We took the fresh bilateral testicular tissue to detect NR4A1 mRNA by using real-time PCR, and NR4A1 protein by using Western blotting.We cultured the mouse Leydig cells MLTC-1 in vitro, and then used NR4A1 interference adenovirus ( Ad-siNR4A1 ) and no-load adenovirus ( Ad-CMV ) to transfect MLTC-1.The radioimmunoassay was used to measure the testosterone level in medium.real-time PCR and Western blotting were applied to determine the mRNA and protein expression of steroidogenic acute regulatory protein ( StAR ) and 3β-hydroxysteroid dehydrogenase (HSD3B).Results The NR4A1 mRNA expression was the highest in the before sexual de-velopment group among the infancy group, before sexual development group, sexual maturity group and the older group;and significant difference was found as compared with the other three groups (all P<0.05).NR4A1 protein expression was the highest in the sexual maturity group as compared with that of the other three groups ( all P<0.05) .The testoster-one levels in MLTC-1 transfected with Ad-siNR4A1 or Ad-CMV were respectively (2.55 ±0.23), (4.50 ±0.23) nmol/L (all P<0.05).The StAR and HSD3B mRNA and protein expression levels in the MLTC-1 transfected with Ad-siNR4A1 were relatively low as compared with those in the Ad-CMV-transfected MLTC-1 (all P<0.05).Conclusion NR4A1 ex-pression is different in testis tissues of mice with different age.NR4A1 is involved in the synthesis of testosterone possibly by regulating the expression of StAR and HSD3B.