视黄酸信号通路在腐霉利致青春期小鼠睾丸损伤中的表达特征

Expression of retinoic acid signaling pathway in mouse damaged testes induced by procymidone during adolescence

[背景]腐霉利(PCM)暴露可致雄性小鼠生殖器官损伤,但其是否与视黄酸(RA)信号通路有关尚不明确。[目的]探讨RA信号通路在PCM致青春期小鼠睾丸损伤中的表达特征。[方法]取64只健康雄性ICR小鼠(3周龄)适应性饲养1周后,随机均分为对照和低、中、高剂量染毒组,每组16只,连续21 d经口灌胃,给予PCM剂量分别为0、50、100、200 mg·kg^(-1)。停止给药1周后处死小鼠,取其血清和双侧睾丸,检测血清中睾酮含量,观察睾丸切片中的组织学变化,用实时荧光定量PCR法、Western blotting法分别测定小鼠睾丸组织中RA信号通路相关基因及凋亡基因Casp9、Casp12 mRNA的表达丰度及CYP26A1、ALDH2、CASP9蛋白水平。[结果]与对照组相比,各剂量染毒组小鼠的整体外观、睾丸外观均无明显改变。病理切片观察:低剂量染毒组小鼠睾丸生精小管出现轻微萎缩,精子生成减少,中、高剂量染毒组小鼠睾丸出现明显的病理损伤(如生精小管管腔扩张,生精上皮受损,精原细胞数量下降,支持细胞部分缺如);随着PCM剂量增加,小鼠生精上皮损伤程度逐渐加重,生精小管中精子数量降低。4组小鼠肛门和生殖器距离、睾丸质量、睾丸容积、睾丸脏器系数的差异均无统计学意义(均P>0.05)。低、中、高剂量染毒组小鼠体重分别为(34.91±1.89)、(34.88±1.75)、(32.94±1.37)g其中高剂量染毒组低于对照组的(35.93±1.99)g;血清睾酮质量浓度分别为(313.77±5.32)、(305.31±3.47)、(304.80±5.28)pg·mL^(-1),均低于对照组的(319.05±1.92)pg·mL^(-1)(均P<0.05);随着PCM剂量增加,小鼠体重和血清睾酮质量浓度均有下降趋势。高剂量染毒组小鼠Rbp1、Stra6 mRNA表达丰度均高于对照组(均P<0.05);中、高剂量染毒组小鼠Aldh2、Aldh1a1、Aldh1a3、RA核受体相关基因(Rar和Rxr)mRNA表达丰度高于对照组(均P<0.05);中、高剂量染毒组小鼠Cyp26a1、Cyp26b1 mRNA表达丰度均低于对照组(均P<0.05);中、高剂量染毒组小鼠凋亡基因Casp9、Casp12 mRNA表达丰度均高于对照组(均P<0.05)。各剂量染毒组小鼠CYP26A1蛋白表达量均低于对照组(均P<0.05),其表达量随PCM浓度增加有下降趋势;中、高剂量染毒组小鼠ALDH2蛋白表达量和各剂量染毒组小鼠CASP9蛋白表达量均高于对照组(均P<0.05),其表达量随PCM浓度增加有升高趋势。[结论]PCM可损伤青春期小鼠睾丸组织,其RA信号通路、Casp9基因、Casp12基因和CASP9蛋白被激活。

[Background]Procymidone(PCM)exposure can cause damage to reproductive organs of male mice,but whether its mechanism is related to the retinoic acid(RA)signaling pathway is unclear.[Objective]To explore the possible mechanism of PCM-induced testes damage in adolescent mice.[Methods]Three-week-old ICR mice(n=64)were randomly divided into a control group and three dose groups(low,medium,and high),with 16 mice in each group.PCM was administered orally at 0,50,100,and 200 mg·kg^(-1)·d;for 21 consecutive days.Serum and bilateral testes in each mouse were collected to detect content of testosterone in serum and to observe histological changes in testis section after the mice were sacrificed one week after cessation of drug administration.Real-time fluorescence quantitative PCR and Western blotting were used to detect the mRNA expression abundances of genes related to the RA signaling pathway and apoptosis genes Casp9 and Casp12,and the protein expression levels of CYP26A1,ALDH2,and CASP9 respectively.[Results]Compared with the control group,there was no significant change in the overall appearance and testicular appearance of mice in each dose group after the PCM exposure.According to pathological section observation,the testicular seminiferous tubules of mice in the low-dose group showed slight atrophy and reduced sperm production;the testes of mice in the medium-and the high-dose groups showed obvious pathological damage(e.g.dilated lumen of seminiferous tubules,damaged spermatogenic epithelium,decreased number of spermatogonia,and partial absence of sertoli cells);as the concentration of PCM increased,the degree of spermatogenic epithelial damage in mice gradually increased and the number of spermatozoa in the seminiferous tubules decreased.There were no significant differences in the distance between the anus and the genitals,testicular mass,testicular volume,and testicular organ coefficient among the four groups of mice(P>0.05).The body weights of the mice in the low-,medium-,and high-dose groups were(34.91±1.89),(34.88±1.75),and(32.94±1.37)g respectively,and that in the high-dose group was lower than that in the control group,(35.93±1.99)g,(P<0.05);the serum testosterone concentrations were(313.77±5.32),(305.31±3.47),and(304.80±5.28)pg·mL^(-1)respectively,which were lower than that in the control group,(319.05±1.92)pg·mL^(-1)(P<0.05);as the dose of PCM increased,the body weight and serum testosterone concentration showed decreasing trends.The mRNA expression levels of Stra6 and Rbp1 in the high-dose group were higher than those in the control group(P<0.05);the mRNA expression levels of Aldh2,Aldh1a1,Aldh1a3,Rarα,Rarβ,Rxrαand Rxrβin the medium-and the high-dose groups were higher than those in the control group(P<0.05);the mRNA expression levels of Cyp26a1 and Cyp26b1 in the medium-and high-dose groups were lower than those in the control group(P<0.05);the mRNA expression levels of apoptosis genes Casp9 and Casp12 in the medium-and the high-dose groups were higher than those in the control group(P<0.05).The protein expression level of CYP26A1 in each exposure group was lower than that in the control group(P<0.05),and the expression level decreased with increasing concentration of PCM;the expression level of ALDH2 protein in the medium-and the highdose groups and the protein expression level of CASP9 in each exposure group were higher than those in the control group(P<0.05),and the levels increased with increasing concentration of PCM.[Conclusion]PCM can damage the testis tissues of adolescent mice,where RA signaling pathway,Casp9 and Casp12 genes,and CASP9 protein are activated.