SPAG11基因影响隐睾睾丸发育及功能的分子机制研究

Cryptorchidism is the most frequent congenital birth defect in male children.and represents an important risk factor for male infertility. The etiology of cryptorchidism remains mostly unknown and we need to investigate the high risk factors and validate them through new approach. The second-generation sequencing methods of exome sequencing is a cost-effective, reproducible and robust strategy for the sensitive and specific identification of variants causing protein-coding changes in individual human genomes. We have identified testis-specific-expressed SPAG11A gene variant through exome sequencing in human cryptorchidism sample, which need further validation on a proper subject. The optimal research subject of human cryptorchidism is human early embryos. However, human early embryos are hard to obtain for research purpose due to the ethical debate and technical hurdling. In this study, we would investigate the molecular mechanism of SPAG11A on the development and function of testis in cryptorchidism through the in vitro differentiation of cryptorchidism-specific induced pluripotent stem (iPS) cells established in our previous research. Human iPS cells are reprogrammed from human somatic cells to a pluripotent state through introduction of four transcription factors.These cells could be used to model normal development in humans, which could help to understand disease pathogenesis and to develop suitable treatments in some cases. Their differentiation ability into all types of cells including germ cells enable them a cell model to mimic the germ cell determination and differentiation in vitro. Based on the above-mentioned findings, we would differentiate the human cryptorchidism-specific iPS cells to germ cells and investigate the function of gene SPAG11A through gene overexpression, gene RNAi, gene chip array and data analysis, pathway finding and validation, etc. Through these approaches, we are intending to explore the relationship between SPAG11A and the testis development and function in cryptorchidism. These results will facilitate the future research on predictive diagnosis of male infertility and screening for targets of male contraception.

隐睾是小儿泌尿外科常见病，其发生是个体发育过程中睾丸的发育和下降发生异常停顿所致，常导致不育。其发生机制未明，需进一步研究发现和验证相关高危因素。外显子组测序克服了之前在封闭研究系统内针对动物研究的发现进行有限探索的局限，更加经济、高效。本课题组前期通过外显子组测序，在隐睾患者中发现睾丸特异表达的SPAG11A变异，结合其在动物体内的功能，我们推测其变异可能与人隐睾所致的不育有关。由于伦理制约，早期胚胎发育异常很难在人体胚胎上进行研究，本课题组前期建立的隐睾特异性iPS细胞可作为研究隐睾的体外模型。综上，本课题拟以隐睾特异性iPS细胞为模型，利用其向生精细胞的定向分化模拟人早期胚胎期间性腺的发育和分化，通过基因功能实验，研究SPAG11A在这一过程中的调控作用,探讨SPAG11A与隐睾睾丸发育及功能的关系，为开发用于预测或判断生育功能的检测途径、或者提供与生育相关的药物靶点打下基础。

生殖健康和个体发育一直是生命科学研究的重点关注领域，在国家“十三五”规划期间仍然是生命科学领域的重点研究方向。干细胞的研究仍然也是国家“十三五”规划期间重要的研究方向。睾丸是产生精子和分泌激素以维持性器官和第二性征发育的重要脏器官。隐睾是小儿泌尿外科常见的疾病，是指男婴出生后单侧或双侧睾丸末降至阴囊而停留在其正常下降通路之任何一处，其发生是个体在发育过程睾丸的发育和下降发生异常停顿所致，其成年后不育是常见的并发症，发生机制未明，需进一步研究发现和验证相关高危因素。外显子组测序克服了之前在封闭研究系统内针对动物研究的发现进行有限探索的缺陷，更加经济、高效。本课题前期通过外显子组测序，在隐睾患者中发现睾丸组织中特异表达的SPAG11A变异，结合其在动物体内的功能，我们推测其变异可能与人隐睾所致的不育有关。由于伦理制约，早期胚胎发育异常很难在人体胚胎上进行研究，本课题建立了隐睾特异性iPS细胞，以其作为研究隐睾的体外模型，首先采用单层贴壁法，三步法有序添加诱导因子，模拟生殖细胞的体内分化发育过程所需要的信号分子，建立了稳定的诱导隐睾特异性的iPS细胞和正常来源的iPS细胞向生精细胞的定向诱导分化系统；在此基础上，构建SPAG11A基因的表达质粒，设计SPAG11A基因的过表达质粒和shRNA，分别作用于隐睾特异性的iPS细胞和正常来源的iPS细胞，研究两组细胞诱导后的差异，发现隐睾特异性的iPS细胞在诱导第三阶段过表达SPAG11A后可提高其分化效率、而正常来源的iPS细胞在诱导第三阶段转入SPAG11A的shRNA其生精分化的效率有所下降，而在生精分化的早期阶段并未观察到以上差异，提示SPAG11A的功能与生精分化的后期阶段密切相关。综上，本课题以隐睾特异性iPS细胞为模型，利用其向生精细胞的定向分化模拟人早期胚胎期间性腺的发育和分化，通过基因功能实验，发现隐睾患儿SPAG11A的变异可能是导致其成年后不育的原因之一且其影响发生在生精的后期阶段，为将来的进一步研究打下了重要的前期基础。