利用Gpr177基因敲除/转基因小鼠模型研究Wnt蛋白和Wnt信号通路对精子发生的影响与机制

Male infertility is a common health problem which affects a large number of couples of child-bearing age. The fundamental research of spermatogenesis is conductive to explain the genetic etiology of male infertility. Recent studies suggest that Gpr177 encodes a novel transmembrane protein that, intriguingly, represents an ancient partner for Wnts dedicated to promoting their secretion. Gpr177 gene knockout is expected to prohibit Wnt protein secretion and thereby inhibit Wnt signaling pathway, forming an ideal model to study the in vivo function of Wnt proteins and Wnt signaling. In this fund project, we will adopt Gpr177flox/flox, Vasa-Cre (or Stra8-Cre or Amh-Cre) mouse model to specificly knockout Gpr177 in germ cells or Sertoli cells. Then, we are going to take advantage of Gpr177flox/flox, Ctnnb1lox(e3)/+; Cre mouse model and testis organ culture method to investigate the roles and mechanism of Wnt proteins and Wnt signaling. Clinical study of GPR177 gene mutation in azoospermia and oligospermia patients and molecular biology experiment in human cell line will contribute to establish relevance between fundamental research of mouse model and male infertility. Our study should be conducive to reveal the role and mechanism of Wnt proteins and Wnt signaling in spermatogenesis more scientific and may provide new insights into genetic diagnosis and targeted therapy of male infertility.

男性不育业已成为严重影响我国广大育龄男性生殖健康的一类疾病，精子发生的基础研究将有助于揭示其遗传学病因。Wnt信号通路在小鼠精子发生中发挥关键作用，并与男性不育相关，但其作用机制尚不明确。Gpr177是新被发现的Wnt蛋白特异运送受体。敲除Gpr177基因可从源头上阻滞Wnt蛋白分泌从而切断Wnt信号通路，是研究Wnt蛋白和Wnt信号通路在体功能的理想模型。本项目拟建立睾丸支持细胞和生殖细胞特异性的Gpr177基因敲除/转基因小鼠，利用体内与体外补偿实验，重点研究Wnt蛋白和Wnt信号通路对精子发生的影响及作用机理。我们还拟开展GPR177基因突变与无精症、少精症关系的临床研究和人细胞系的体外分子生物学实验，将模式动物的基础研究与男性不育疾病关联起来。本项目的研究有助于更加科学地揭示Wnt蛋白和Wnt信号通路在精子发生中的作用与机制，并可能为男性不育的遗传学诊断及潜在靶向治疗提供理论依据。

WNT/beta-catenin持续激活（Ctnnb1flox(exon3); Cre）在小鼠胚胎期和出生后睾丸中发挥关键作用，但WNT信号通路的缺失是否影响精子发生存在争议。GPR177（WLS）负责多种Wnt配体（Wnts）的分泌，使得研究WNT信号通路（经典与非经典）和WNT配体的“整体作用”成为可能。我们成功构建并获得了四种Gpr177基因特异性敲除小鼠，分别在小鼠睾丸生殖细胞（Mvh-Cre和Stra8-Cre）、Sertoli细胞（Amh-Cre）、管周肌样细胞（Myh11-Cre）、附睾头主细胞（Lcn5-Cre）特异性敲除Gpr177基因。我们发现睾丸生殖细胞敲除Gpr177基因的雄鼠表现出迟发型（8各月后）睾丸萎缩与生育率降低，生殖细胞的活性氧水平和Caspase 3活性显著高于野生型。另外，睾丸Sertoli细胞、管周肌样细胞和附睾头主细胞特异性敲除Gpr177基因并不影响小鼠精子发生和雄性生殖力。综上所述，小鼠精子发生特异地依赖于睾丸生殖细胞中GPR177。该项资助产出了十余篇SCI论文，对WNT信号通路在精子发生中作用的研究产生了推动作用。

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