多不饱和脂肪酸对Δ15 Des转基因小鼠睾丸结构和功能的影响及机制

Effect and mechanism of polyunsaturated fatty acids on testicular structure and function of transgenic mice withΔ15 Des

目的探讨多不饱和脂肪酸(PUFAs)对Δ15脂肪酸去饱和酶(Δ15 Des)转基因小鼠睾丸结构和功能的影响及机制。方法取C57BL/6野生型(WT)雄性小鼠和Δ15 Des转基因雄性小鼠分别作为对照组(n=5,WT组)和实验组(n=5,TG组),均饲喂含6%花生四烯酸(ARA)饲料8周,麻醉处死,分离睾丸组织及附睾组织,采用气相色谱(GC)检测睾丸组织中脂肪酸的组成及含量,采用精子质量分析仪(CASA)检测附睾组织中精子形态及运动活力[精子总活力、直线运动精子活力、精子平均路径速度(VAP)、精子曲线速度(VCL)及精子直线速度(VSL)],采用苏木精-伊红(HE)染色观察睾丸组织的形态学特征,采用实时荧光定量PCR(RT-qPCR)检测2组小鼠睾丸组织中游离脂肪酸受体1(FFAR1)、FFAR2、FFAR3、FFAR4及过氧化物酶体增殖物激活受体α(PPARα)、PPARβ、PPARγ信使RNA(mRNA)的表达,采用蛋白质印迹法检测2组小鼠睾丸组织中FFAR4和PPARγ蛋白的水平。结果与WT组相比,TG组小鼠睾丸组织中ARA与二十二碳四烯酸(DTA)含量降低(P<0.05或P<0.01),亚油酸(LA)、二十碳五烯酸(EPA)、二十二碳六烯酸(DHA)含量升高(P<0.05或P<0.01);2组小鼠的精子形态及数量无明显差异,TG组小鼠精子的直线运动精子活力、VSL较WT组升高(P<0.05),精子总活力、VAP、VCL无差异(P>0.05);与WT组相比,TG组小鼠睾丸组织曲细精管内空泡较少,成熟精子增多;与WT组相比,TG组小鼠睾丸组织中FFAR4、PPARαmRNA表达上调(P<0.01或P<0.05),PPARβmRNA表达下调(P<0.05),FFAR4蛋白水平增加(P<0.05)。结论n-3 PUFAs可改善Δ15 Des转基因小鼠睾丸组织的结构和功能,其机制可能与结合FFAR4、激活下游信号分子有关。

Objective To investigate the effect and mechanism of polyunsaturated fatty acids(PUFAs)on the structure and function of testis in transgenic mice with Δ15 fatty acid desaturase(Δ15 Des).Methods C57BL/6 wild-type(WT)male mice and Δ15 Des transgenic male mice were chosen as control group(n=5,WT group)and experimental group(n=5,TG group).They were all fed with 6% arachidonic acid(ARA)feed for 8 weeks,and were executed under anesthesia.Testicular and epididymal tissues were separated,and the composition and fatty acid content of testicular tissues were detected by gas chromatography(GC).Sperm quality analyzer(CASA)was used to detect sperm morphology and motility[total sperm motility,linear sperm motility,mean sperm path velocity(VAP),curve sperm velocity(VCL),and linear sperm velocity(VSL)]in epididymis;hematoxylin-eosin(HE)staining was used to observe morphological characteristics of testicular tissue.The expression of free fatty acid receptor 1(FFAR1),FFAR2,FFAR3,FFAR4,and peroxisome proliferator-activated receptorα(PPARα),PPARβ,and PPARγmessenger RNA(mRNA)in testis of both groups were detected by real-time fluorescence quantitative PCR(RT-qPCR).The levels of FFAR4 and PPARγprotein in testis of both groups were detected by Western blot.Results Compared with WT group,ARA and docosatetraenoic acid(DTA)levels in testis of TG group decreased(P<0.05 or P<0.01),while linoleic acid(LA),eicosapentaenoic acid(EPA),and docosahexaenoic acid(DHA)levels increased(P<0.05 or P<0.01).There was no significant difference in sperm morphology and quantity between both groups.Linear sperm motility and VSL of TG group were higher than those of WT group(P<0.05),but there was no difference in total sperm motility,VAP,and VCL(P>0.05).Compared with WT group,there were fewer vacuoles and more mature sperm in seminiferous tubules in TG group.Compared with WT group,the expressions of FFAR4 and PPARαmRNA in the testis of TG group up-regulated(P<0.01 or P<0.05),PPARβmRNA down-regulated(P<0.05),and FFAR4 protein level increased(P<0.05).Conclusion n-3 PUFAs can improve the structure and function of testicular tissue inΔ15 Des transgenic mice,and its mechanism might be related to the activation of downstream signal molecules by binding FFAR4.