神经肽催产素与男性不育关系的研究

目的:分析特发性男性不育患者催产素(oxytocin,OT)水平及其受体的表达。方法:选取特发性少精子症(oligozoospermia group,OG)(含无精子)、弱精子症(asthenozoospermia group,AG)及少精子合并弱精子症(oligoasthenozoospermia group,OAG)男性不育患者共65例,年龄20～45岁;对照组(control group,CG)选取有自然生育史的健康男性志愿者20例(精液参数正常),年龄20～45岁。检测各组血清黄体生成素(luteinizing hormone,LH)、卵泡刺激素(follicle-stimulating hormone,FSH)、睾酮(testosterone,T)及OT含量,进一步通过检测催产素受体启动子功能序列(oxytocin receptor promotor fuctional sequence,OTRP)、催产素受体羧基端部分mRNA序列(OTRmR-NA)以及OTR蛋白构象、组成来分析OTR表达情况。OTR蛋白印迹分析结果经灰度软件处理后转化为计数资料;统计方法采用单因素方差分析。结果:①男性生殖内分泌激素含量分析:OT:各病例组均高于CG(79.30±3.83)pg/ml],具有统计学意义(F0.05/2(2,82)=8.29,P<0.001);LH:AG(4.26±0.31)IU/L]及OAG(4.55±0.40)IU/L]均低于OG(6.77±0.57)IU/L]和CG(7.19±0.50)IU/L](F0.05/2(2,82)=11.64,P<0.01);FSH:AG(5.02±0.39)IU/L]低于CG(8.91±0.91)IU/L]、OG(11.86±1.76)IU/L]及OAG(8.82±1.03)IU/L](F0.05/2(2,82)=7.22,P<0.01);T:各组间无统计学差异(F0.05/2(2,82)=0.42,P=0.739)。②OTR基因转录分析:OTR启动子功能序列和OTR成熟mRNA序列未发现明显的变异。③OTR构象分析:人OTR以单体和寡聚体同时存在,以寡聚体为主,常见为四聚体和六聚体;AG(0.41±0.03)和OAG(0.13±0.01)的单体明显多于OG(0.05±0.004)和CG(0.05±0.003)(F0.05/2(2,82)=115.50,P<0.01);AG中20%病例存在寡聚体明显减少现象。结论:男性不育患者血清OT含量及OTR表达的差异提示OT与男性不育间存在一定的联系,OTR单体表达升高及寡聚体表达降低为进一步探索OT与男性不育间的关系提供了新的方向。

Neuropeptide oxytocin and male infertility

Objective :To analyze the level of the oxytocin (OT) and the expression of oxytocin receptor (OTR) in males with idiopathic infertility. Methods :Sixty-five infertile males aged 20-45 years were divided according to their semen parameters into an idiopathic oligozoospermia group (OG,n = 20),an idiopathic asthenozoospermia group (AG,n = 25),and an idiopathic oligoasthenozoospermia group (OAG,n = 20). Another twenty 20-45 years old healthy male volunteers with a natural childbearing history were included in the control group (CG). All the subjects were detected for the contents of luteinizing hormone (LH),follicle stimulating hormone (FSH),testosterone (T) and OT,and analyzed for the expression of OTR by sequencing the functional region of the OTR promoter (OTRP),OTR-mRNA,and OTR-COOH terminus. The gene sequences were compared using DNASTAR-MegAlign,Western blot values changed into enumeration data,and all the data analyzed by one-way ANOVA and Dunnette's multiple range t-test.Results :A significantly lower content of OT was observed in CG (79.30 ± 3.83] pg/ml) than in OG (118.53 ± 7.69] pg/ml,AG (108.81 ± 5.66]pg/ml) and OAG (103.71 ± 4.54] pg/ml) (F0.05/22,82] = 8.29,P<0.01). The content of LH was significantly lower in AG (4.26 ± 0.31] IU/L) and OAG (4.55 ± 0.40] IU/L) than in OG (6.77 ± 0.57] IU/L) and CG (7.19 ± 0.50] IU/L) (F0.05/22,82] = 11.64,P<0.01),and so was the content of FSH in AG (5.02 ± 0.39] IU/L) than in CG (8.91 ± 0.91] IU/L),OG (11.86 ± 1.76] IU/L) and OAG (8.82 ± 1.03] IU/L) (F0.05/22,82] = 7.22,P<0.01). There were no significant differences in the T content among the four groups (F0.05/22,82] = 0.42,P = 0.739). No evident gene mutation was found in OTRP and OTR-mRNA gene sequencing. Human OTRs in the lymphocytes were monomers and oligomers,mostly tetramers and hexamers. There were obviously more monomers in AG (0.41 ± 0.03) and OAG (0.13 ± 0.01) than in OG (0.05 ± 0.004) and CG (0.05 ± 0.003) (F0.05/22,82] = 115.50,P<0.01),while the number of oligomers was markedly decreased in 20% of the cases in AG. Conclusion :Significant differences in the content of OT and expression of OTR between fertile and infertile men suggested an association of OT with male infertility. The decreased expression of OTR oligomers and increased expression of monomers may be related to idiopathic asthenozoospermia,which has provided a new insight into the pathogenesis and treatment of male infertility.