人非梗阻性无精子症相关基因在果蝇睾丸中的功能和机制研究

发布时间：2018-07-13 13:10

【摘要】：男性不育症影响了全世界六分之一的育龄夫妇,而非梗阻性无精子症是男性不育症中比较严重的情况之一。目前,非梗阻性无精子症的致病机理仍非常不清楚。前期,本实验室在中国汉族男性非梗阻性无精子症患者的全基因组关联研究中,鉴定到3个能够达到全基因组显著性关联(P值小于5.0x10-8)的风险位点。但男性不育症是多基因病,仅鉴定到3个风险位点是不足以解释其致病机理的。这可能是由遗传异质性而导致的结果。我们认为那些没有达到全基因组显著关联的单核苷酸多态性位点也可能提示生育的相关基因。现在,通过扩大样本量(3608个非梗阻性无精子症病例和5909个对照),应用人样本的三阶段验证,我们又鉴定到4个与人非梗阻性无精子症显著关联的风险位点。其中一个可疑的风险位点几乎达到全基因组显著关联(1q42.13, rs3000811, P=7.26x10-8)。为了验证这个位点是否影响了男性生育能力,我们通过模式动物果蝇观察了可疑关联位点(位于人基因组1q42.13区域)附近的相关基因CDC42BPA的果蝇同源基因gek在雄性生育方面的表型。为了能够对候选的单核苷酸多态性位点附近的基因进行大规模功能筛选,我们在果蝇体内用dsRNA或者是shRNA对靶基因进行表达沉默的实验。在果蝇中使用这种方法具有工作周期短和高通量筛选的优势。我们的分析结果显示,有31.8%(7/22)的果蝇相关基因是雄性生育必需的。这些基因对应了9个位点。这些基因中,Lar基因主要是在生殖细胞中维持精子发生,而CG12404基因、dmrt11E基因、CG6769基因、ERR基因和sfl基因主要在体细胞中发挥作用。有趣的是,ERR基因和sfl基因对睾丸形态的发生也是必需的。有报道显示,无精子症是精原细胞瘤的一个高发风险因素。精原细胞瘤也是年轻男性最常见的肿瘤之一,但其发病的分子机制并不清楚。为了在全基因组关联研究中鉴定到更多的人非梗阻性无精子症关联位点,我们发现了一个单核苷酸多态性位点(rs1887102, P=2.60x10-7)位于人FOXN3基因中。FOXN3基因是一个进化保守的基因。我们进一步通过模式动物果蝇来检验雄性生育能力是否需要FOXN3基因的果蝇同源基因CHES-1-like的参与。虽然CHES-1-like基因突变果蝇是可活的和可育的,且在生精过程中没有明显的异常,然而在生殖细胞中异位表达CHES-1-like基因却显著降低了雄性果蝇的生育能力。我们通过进一步的研究发现,在异位表达CHES-1-like基因的睾丸中,精原细胞经历了四次有丝分裂后并没有停止分裂,而是继续发育并形成肿瘤样结构。在这些睾丸中,分化因子Bam蛋白的表达水平明显降低,但Bam蛋白表达的区域却明显变大。在CHES-1-like基因异位表达的睾丸中同时降低bam基因的表达可进一步加剧了肿瘤样结构的形成。dad基因作为BMP信号通路的下游基因,可以拮抗dpp基因的表达水平。而dad基因的表达水平在异位表达CHES-1-like基因的睾丸中明显上调。我们在果蝇翅盘中也发现,异位表达CHES-1-like基因可以上调Dpp蛋白的表达水平。在睾丸中,CHES-1-like蛋白可以直接结合dpp基因启动子区域。因此我们提出了一个模型,在生殖细胞中异位表达CHES-1-like基因可以上调Dpp蛋白的表达水平,并抑制生殖细胞的分化,最终导致精原细胞瘤的形成。我们的研究论证了那些没有达到全基因组显著关联的单核苷酸多态性位点也可以为疾病相关基因提供线索,并为其功能研究提供帮助。这些结果深化了我们对人非梗阻性无精子症患者遗传易感性的认识,也对了解其致病机理提供了视野。
[Abstract]:Male infertility affects 1/6 of the world's couples of childbearing age, and non obstructive azoospermia is one of the most serious cases of male infertility. The pathogenesis of non obstructive azoospermia is still unclear. In the early stage, the whole genome association study in the Chinese Han male non stem azoospermia It was identified that 3 risk loci that could reach a genome-wide association (P value less than 5.0x10-8). But male infertility is a polygenic disease, and only 3 risk sites are identified to be insufficient to explain its pathogenic mechanism. This may be a result of genetic heterogeneity. We think those did not reach the significant association of the whole genome. Single nucleotide polymorphisms are also likely to be associated with fertility related genes. Now, by expanding the sample size (3608 cases of non obstructive azoospermia and 5909 controls), we have identified 4 risk sites associated with human non obstructive azoospermia with a three stage test. One of the suspected risk loci is a few. To verify the 1q42.13, rs3000811, P=7.26x10-8. In order to verify whether this site affects male fertility, we have observed the phenotype of the Drosophila homologous gene gek of the suspicious associated locus (located in the human genome 1q42.13 region) by the model animal Drosophila melanogaster homologous gene CDC42BPA in male fertility. In order to perform large-scale functional screening of candidate genes near single nucleotide polymorphisms, we used dsRNA or shRNA to silence the target gene in the Drosophila. The use of this method in Drosophila has the advantage of short work cycle and high throughput screening. Our analysis showed that 31.8% (7/22) The genes associated with Drosophila are essential for male reproduction. These genes correspond to 9 loci. In these genes, the Lar gene maintains spermatogenesis in the germ cells, while the CG12404, dmrt11E, CG6769, ERR and SFL genes are mainly used in the somatic cells. Interestingly, the ERR and SFL genes are in the form of the testis. It is also necessary. It has been reported that azoospermia is a risk factor for the high incidence of seminoma. Spermatogonial tumor is also one of the most common tumors in young men, but the molecular mechanism of its pathogenesis is not clear. In order to identify more people with non obstructive azoospermia associated sites in the whole genome association study, we A single nucleotide polymorphic loci (rs1887102, P=2.60x10-7) in human FOXN3 gene was found to be an evolutionary conserved gene. We further examined whether the male fertility was required by the model animal fruit fly to participate in the participation of the fruit fly homologous gene CHES-1-like of the FOXN3 gene. Although the CHES-1-like gene mutated to the Drosophila melanogaster, the CHES-1-like gene mutated to the fruit fly. It is viable and fertile and has no obvious abnormalities during spermatogenesis, but ectopic expression of the CHES-1-like gene in the germ cells significantly reduces the fertility of the male Drosophila. We found that in the testicles of the ectopic expression of the CHES-1-like gene, the spermatogonial cells have undergone four mitosis and did not. In these testicles, the expression level of the differentiation factor Bam protein decreased obviously, but the region of the expression of Bam protein was significantly larger in these testicles. The decrease of the expression of BAM gene in the testicles expressed in the CHES-1-like gene could further aggravate the formation of the tumor like structure of the.Dad gene. As a downstream gene of the BMP signaling pathway, the expression level of the DPP gene can be antagonized. The expression level of the dad gene is obviously up-regulated in the testicles of the ectopic CHES-1-like gene. We also found that the ectopic expression of the CHES-1-like gene in the fly winged disc can increase the level of the expression of the Dpp protein. In the testis, the CHES-1-like protein can be straight. Then we combine the DPP gene promoter region. Therefore, we have proposed a model in which the ectopic expression of the CHES-1-like gene in the germ cells can up-regulate the expression of Dpp protein and inhibit the differentiation of germ cells and eventually lead to the formation of spermatogonial tumor. Acid polymorphic loci can also provide clues for disease related genes and provide help for their functional studies. These results deepen our understanding of the genetic susceptibility to non obstructive azoospermia patients and provide a vision for understanding its pathogenesis.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R698.2

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