男性不育影响全球约7%的育龄夫妇，对精子发生障碍的研究将加深我们对不育因素的了解，同时推动相应临床疾病治疗发展。通过分析组织测序数据库和我们对睾丸生殖细胞的高通量测序数据，我们发现小鼠睾丸中的甘油激酶类基因GK2和Gykl1特异性地高表达，而男性不育患者高通量测序疾病数据库显示GK2的表达水平显著低于正常组，提示这类基因的低表达可能与人类男性不育正相关。申请人前期利用小鼠模型验证GK2和Gykl1特异性地在睾丸内的生殖细胞中表达。通过特异抗体制备和组织染色，发现它们特异性定位于圆形精子和长形精子的线粒体。线粒体是参与调控精子发生和成熟的重要细胞器。本项目将通过最新的CRISPR/Cas9技术快速制备Gykl1和GK2基因敲除小鼠模型，系统揭示Gykl1/GK2在精子发生以及精子功能维持中的作用，研究结果将揭示睾丸特异性甘油激酶类基因与雄性不育症的关系，并为临床诊断和治疗提供重要的参考。

Male infertility affects about 7% couples during their reproductive lifespan in the world. The studies on the molecular mechanisms of abnormity in spermatogenesis will deepen our understanding of factors in infertility and largely promote the development of appropriate clinical treatment of diseases. By analysis of the organization sequencing databases and our high-throughput sequencing data with testicular germ cells, we found that mouse testes glycerol kinase-like genes GK2 (glycerol kinase 2) and Gykl1 (glycerol kinase-like 1) were highly expressed in testis only. The database of high-throughput sequencing with tissues from human male infertility disease shows the expression level of GK2 was significantly lower than the control group, suggesting that low expression of GK2 may be positively correlated with male infertility. In our previous experiments, we confirmed GK2 and Gykl1 expressed in testicular germ cells specifically with RT-PCR by using mice model. Using home-made specific antibodies to do testicular tissues immunofluorescence staining, we found that they were specifically located on mitochondria in the round and elongated sperm. Mitochondria are important organelles involved in the regulation of spermatogenesis and sperm mature. Our project will adopt the latest technology CRISPR/Cas9 technique rapid preparation Gykl1 and GK2 knockout mouse models to reveal the role of Gykl1/GK2 in spermatogenesis and in maintaining of sperm functions. The research results will uncover the functions of testis-specific glycerol kinase-like proteins in male fertility and may provide important references for clinical diagnosis and treatment.