Seipin基因突变导致雄性生育功能障碍及其分子机制研究

发布时间：2018-07-07 16:39

本文选题：男性不育 + 脂肪营养不良　；参考：《南京医科大学》2014年博士论文

【摘要】：背景：近年来,随着肥胖人群的快速增加,多方临床数据证明肥胖的男性由于机体脂肪含量过多,其生育力受到负面影响,然而,很少有研究关注机体脂肪含量过少对男性生育力有无影响。2004年有文献提及患有遗传性全身脂肪营养不良(Congenital generalized lipodystrophy,CGL)的男性患者生育力正常,但我们前期对CGL2型致病基因Seipin进行研究时,发现全身敲除Seipin基因的雄性小鼠(简称：S-KO)呈现完全不育,这一发现促使我们重新思考机体脂肪组织过少对雄性生殖力的影响。同时,大量数据显示Seipin基因在脂肪组织以及神经系统高表达,Seipin蛋白缺失后导致脂肪分化障碍,Seipin基因突变后出现多种运动神经元病变。本次研究过程中发现Seipin蛋白及mRNA在睾丸表达量明显高于脂肪组织,而其在睾丸组织中的功能尚未有报道。研究方法与结果：我们在临床募集到一例Seipin基因突变家系,其中针对男性患者进行了体脂比例、激素水平和精液分析。同时,在已有的全身性Seipin基因敲除的小鼠(S-KO)基础上,进一步构建了脂肪细胞特异性Seipin基因敲除小鼠(简称：aS-KO)和睾丸生殖细胞特异性Seipin基因敲除小鼠(gS-KO)。继而,对这三种敲除小鼠开展了多方面的检测：机体代谢、生育力测试、精子质量评估等。本次研究结果如下：1.该男性患者因Seipin基因复合型突变导致全身脂肪营养不良的同时,存在严重畸形精子症,精子畸形主要包括头部畸形及多核精子束,且畸形精子内存在异常的大脂滴。2.S-KO和gS-KO雄性小鼠因睾丸精子细胞内出现异常的大脂滴,精子变形发生异常,导致少弱畸形精子症,呈现雄性完全不育,小鼠畸形精子中也存在典型的多核精子束,小鼠睾丸油红染色及电镜观察结果与男性患者精子内异常脂滴相吻合。3. aS-KO并未因脂肪营养不良导致生育力下降,睾丸内精子发生正常。4.脂质组学研究结果提示：在S-KO、gS-KO睾丸内,PA增加明显,且磷脂的多不饱和脂肪酸的比例失衡。5.差异蛋白质组学结果验证了脂质组学的结果,提示S-KO睾丸内脂代谢相关蛋白表达量增加,脂代谢水平处于亢进状态,并且细胞炎症反应及凋亡相关蛋白大大增加,与小鼠表型一致。综上所述：睾丸内Seipin基因能够维持精子变形过程中磷脂稳态,seipin基因突变后导致雄性生殖功能障碍,并且由Seipin突变导致的脂肪营养不良并不能对雄性生育力产生影响,强调了睾丸生殖细胞的Seipin;对维护雄性生殖能力的重要作用。
[Abstract]:Background: in recent years, with the rapid growth of obese people, various clinical data have shown that obese men suffer negative effects on fertility due to excessive body fat content. Few studies have focused on whether low body fat levels have an impact on male fertility. In 2004, there was a reference to normal fertility in men with inherited generalized lipodystrophytic dystrophy. But when we studied Seipin, the pathogenic gene of CGL2, we found that the male mice with Seipin gene knockout showed complete sterility, which prompted us to rethink the effect of too little fat tissue on male fecundity. At the same time, a large number of data showed that Seipin gene in adipose tissue and nervous system overexpression of Seipin protein deletion led to adipose differentiation disorder Seipin gene mutation after the emergence of a variety of motor neuron lesions. In this study, we found that the expression of Seipin protein and mRNA in testis was significantly higher than that in adipose tissue, but the function of Seipin protein and mRNA in testis was not reported. Methods and results: we recruited a family of Seipin gene mutations in a clinical study, in which body fat ratio, hormone levels and semen analysis were performed on male patients. At the same time, based on the existing Seipin knockout mice (S-KO), adipocyte specific Seipin knockout mice (Seipin gene knockout mice) and testicular germ cell specific Seipin gene knockout mice (gS-KO) were further constructed. Then, the three kinds of knockout mice were tested in many aspects: metabolism, fertility test, sperm quality evaluation, etc. The results of this study are as follows: 1. The male patient suffered from systemic fat dystrophy due to Seipin gene complex mutation and had severe sperm malformation, which mainly included head malformation and polynuclear sperm bundles. There were abnormal lipid droplets in abnormal spermatozoa. 2.S-KO and gS-KO male mice showed abnormal lipid droplets in testicular sperm cells, abnormal sperm deformation, resulting in oligodeformed spermatozoa, which showed male complete sterility. Typical polynuclear sperm bundles were also found in abnormal sperm of mice. The results of testicular oil red staining and electron microscopy were in agreement with abnormal lipid droplets in sperm of male patients. AS-KO did not cause fertility decline due to adipose malnutrition. Testicular spermatogenesis is normal. The results showed that PA increased significantly in the testis of S-KOGS-KO, and the proportion of polyunsaturated fatty acids of phospholipid was out of balance. The results of differential proteomics confirmed the results of lipigraphy, suggesting that the expression of lipid metabolism-related proteins in the testis of S-KO was increased, the level of lipid metabolism was in a hyperactive state, and the cellular inflammatory response and apoptosis-related proteins were significantly increased. It is consistent with the mouse phenotype. In conclusion, the Seipin gene in the testis can maintain the mutation of phospholipin homeostasis during sperm deformation and lead to male reproductive dysfunction, and the fatty malnutrition caused by the Seipin mutation has no effect on male fertility. The important role of Seipinin of testicular germ cells in maintaining male reproductive ability was emphasized.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R698

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