牦牛和犏牛睾丸CPT1a和ETHE1基因mRNA水平比较研究

Study on Comparison of CPT1a and ETHE1 Genes mRNA Levels in the Testes of Yaks and Cattle-yaks

试验旨在探索牦牛杂交雄性不育后代(犏牛)睾丸脂肪酸氧化特点及其与雄性不育的关联性,阐明犏牛雄性不育的分子机制。以GAPDH和18SrRNA基因为双内参,建立了牦牛和犏牛肝脏型棕榈酰肉碱转移酶1a(CPT1a)和硫双加氧酶1(ETHE1)基因mRNA水平的实时荧光定量PCR检测方法,结果发现,该实时荧光定量PCR方法扩增特异性好,扩增效率为98.5%~107.8%,标准曲线线性关系好。采集成年牦牛(n=9)和雄性不育犏牛(n=7)睾丸和附睾,通过常规组织切片和HE染色检测,证实犏牛睾丸和附睾中无精子,而牦牛睾丸和附睾中有精子。试验进一步从牦牛和犏牛睾丸中提取总RNA,测定睾丸中CPT1a和ETHE1基因的mRNA水平,结果显示,犏牛睾丸中CPT1a和ETHE1基因mRNA水平分别显著和极显著高于牦牛(P<0.05;P<0.01)。CPT1a基因mRNA水平的提高表明犏牛睾丸中脂肪酸β-氧化能力的加强;而ETHE1基因mRNA水平的增加可促进线粒体呼吸链的电子传递和脂肪酸氧化,或影响其他能量代谢通路,这两种基因mRNA水平的提高均有助于睾丸脂肪酸的β-氧化供能。本研究结果表明,与牦牛睾丸相比,雄性不育犏牛睾丸的脂肪酸氧化供能水平加强,可能对脂肪酸燃料分子有更多的依赖性。

This study was aimed to explore characteristics of fatty acid oxidation in the testes of sterile yak hybrids（cattle-yaks）and its association with male sterility,and elucidate the molecular mechanism of male sterility of cattle-yaks.A Real-time quantative PCR method was established to assay the mRNA levels of liver type-carnitine palmitoyl transferase 1 a（CPT1 a）and sulfide dioxygenase 1（ETHE1）genes in yak and cattle-yak testes using GAPDHand 18 SrRNA as reference genes.This Real-time quantative PCR showed high amplification specificity,with amplification rate ranging from 98.5%to 107.8%and qualified standard curves.The testes and epididymides of adult yaks（n=9）and sterile male cattle-yaks（n=7）were collected and detected with conventional histological sections and HE staining.Sperms were found in the testes and epididymides of yaks but not cattle-yaks.Total RNA were extracted from the testes of yaks and cattle-yaks for the analysis of CPT1a and ETHE1 genes mRNA.The results showed that the CPT1a and ETHE1 genes mRNA level of testicular in cattle-yaks were significantly and extremely significantly higherthan those of yaks（P0.05;P0.01）.The increased CPT1 agene mRNA level indicated the higherβ-oxidation capacity in cattle-yak testes;While the elevated ETHE1 gene mRNA level could facilitate the electron transfer and fatty acid oxidation in mitochondria,or influence other energy metabolism pathways.The results preliminarily demonstrated that the testes of sterile cattle-yaks had increased oxidation of fatty acid for energy supply compared to yak testes,and might had more dependent on fatty acids as fuel.