线粒体基因ND3、ND4L核苷酸变异与弱精子症相关分析

Association between asthenospermia and mtDNA mutations in ND3 and ND4L genes

目的:对弱精子症(AST)精子线粒体DNAND3、ND4L基因突变检测和分析,探索弱精子症致病的分子机制。方法:收集弱精子症患者50例,年龄匹配的对照42例,用密度梯度离心将弱精子症患者和对照组的不同活力精子进行分离,扩增线粒体ND3、ND4L基因,测序和比对,比较弱精子症组和对照组线粒体ND3、ND4L基因核苷酸变异和单体型差异。结果:在弱精子症组和对照组中共检测出22个变异位点,其中A10157G和A10313C未见报道,G10320A、A10398G、T10609C为错义突变。A10398G和C10400T核苷酸变异率在弱精子症组显著低于对照组(P<0.05),G10310A核苷酸变异率在弱精子症组显著高于对照组(P<0.05);单体型分析可见弱精子症组单体型N百分率(33/50)显著高于对照组(14/42)(P<0.05),单体型R9在弱精子症组(15/50)的比例显著高于对照组(4/42)(P<0.05);单体型F1、F2和R9的前向运动精子百分率显著低于单体型M和Mrest(P<0.05)。对弱精子症同一病例不同活力精子进行检测,有2例不同活力的精子的线粒体单体型存在差异,活力好的精子为单体型M,而活力差的精子为单体型N;在50例弱精子症中有2例活力中等和活力差的精子标本中检测出G10310A异质性突变,而在活力好的精子标本中无G10310A突变。结论:线粒体单体型与精子活力可能存在一定的相关性;线粒体DNA10398G-10400T多态性可能是精子活力的有益因素,线粒体DNAG10310A突变可能是精子活力的有害因素。

AIM： To explore the molecular mechanism of asthenospermia（AST） by preliminary screening of nucleotide sequences from the ND3 and ND4L genes of mitochondrial DNA（mtDNA）. METHODS： Samples from 50 AST patients and 42 age - matched normal controls were collected according to the WHO criteria. Density gradient centrifugation was applied to separate spermatozoa with different vigor. The ND3 and ND4L genes of mtDNA were amplified and sequenced directly from the extracted genomic DNA from AST patients and normal controls. The sequences were compared with revised Cambridge Reference Sequence（rCRS） to analyze the variants. RESULTS： A total of 22 nucleotide variations were found in ND3 and ND4L genes of mtDNA in asthenospermia group and control group. G10320A, A10398G and T10609C were missense mutations, while A10157G and A10313C were the reported for the first time in this study. Haplotype N in patients with AST（33/50） was higher than that in control group（ 14/42, P 〈 0. 05）, and haplotype R9 in patients with AST（15/50） was also higher than that in control group （4/42, P 〈 0. 05 ） through genetic testing of ND3 gene.Rates of sperm progressive motility of haplotype F1, F2 and R9 were significantly lower than those of haplotype M and M rest. Two haplotype differences, haplotype M and N, were found in the same AST patient＇ s spermatozoas which had different vigor. Haplotype M had stronger vigor, while haplotype N had lower vigor. By sequencing ND3 gene of mtDNA from 50 AST patients, we detected G10310A heteroplasmic mutation in 2 specimens of asthenospermia with poor and moderate motility spermatozoa, respectively. No mutation occurred in good motility spermatozoa. CONCLUSION： Haplotype of mito- chondrial may have some correlation with sperm motility. The ntlO398G - 10400T polymorphisms may have benefit for sperm motility, whereas the mutation in ntl0310A may impair sperm motility.