人精子体外氧化损伤对线粒体tRNA~(LeuUUR)基因的影响

目的:探讨活性氧(ROS)对人类精子线粒体tRNALeuUUR基因的氧化损伤。方法:采用Percoll梯度离心法筛选具有正常生理功能的精子,作为正常精子模型,并分为损伤组20例和对照组20例,分别加入次黄嘌呤-黄嘌呤氧化酶体系或不予处理,37℃有氧环境中孵育60min。分别提取精子DNA,以Fpg酶切损伤碱基并采用接头介导PCR(LM-PCR)检测线粒体tRNALeuUUR基因的氧化损伤。采用Rhodamine(Rh123)荧光探针标记精子,通过流式细胞仪检测线粒体膜电位,观察精子的功能。结果:与对照组相比,损伤组精子孵育后线粒体膜电位明显降低[(116.27±11.72)%vs(64.00±4.88)%,P<0.05]。Fpg酶切和LM-PCR显示精子线粒体tRNALeuUUR基因损伤。结论:ROS可能通过对精子线粒体tRNALeuUUR基因氧化损伤而影响精子功能(线粒体膜电位明显降低),从而引起不育。

Effect of Oxidative Damage to Human Sperm in vitro on Mitochondrial tRNALeuUUR Gene

OBJECTIVE: To study the oxidative damage to human sperm mitochondrial tRNA LeuUUR gene by reactive oxygen species (ROS) in vitro. METHODS: Spermatozoa of normal physiological function selected from semen samples by Percoll gradient centrifugation technique were used as normal sperm models, which were divided into two groups of 20 cases each, a damage group and a control group, the former treated with hypoxanthine xanthine oxidase system and the latter left untreated, both incubated at 37 degrees C in aerobic environment for 60 minutes. Sperm DAN was extracted, and digestion by the enzymes fpg and ligation-mediated PCR ( LM-PCR) was performed to map the damage to mitochondrial tRNA LeuUUR gene. The spermatozoa were labeled with specific fluorescent probe of Rhodamine 123 to measure mitochondrial membrane potential ( MMP) by flow cytometry and observe sperm function. RESULTS: Compared with the control group, after the normal spermatozoa were incubated with ROS, MMP of the spermatozoa significantly decreased ( 116. 27+/-11.72 vs 64.00+/-4. 88) , P <0.05. Digestion by the enzymes fpg and LM-PCR showed damage to mitochondrial tRNA LeuUUR, gene. CONCLUSION: Reactive oxygen species may inflict oxidative damage on sperm mitochondrial tRNA LeuUUR gene and thus affect sperm function ( as shown by significant decrease in MMP), resulting in infertility.