睾丸特异性甘油激酶类蛋白在小鼠精子发生过程中的功能研究

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.

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。

伴随人年龄的增长，生殖力发生下降。研究精子发生的调控对于抗衰老具有重要意义。精子发生是一个复杂的生物学过程，其最后步骤是精子的变形成熟，其中包括线粒体在内的细胞器的的重排在其中发挥关键作用。包括少精和弱精症在内的男性不育疾病和线粒体的功能失常密切相关。然而在精子发生过程中线粒体是如何被动态调控的还不是很清楚。我们在研究小鼠的睾丸特异性基因时发现甘油激酶类蛋白Gykl1和Gk2特异性地定位于精子的线粒体。随后我们利用CRISPR/Cas9快速基因敲除技术构建了Gykl1和Gk2分别敲除的基因编辑小鼠。我们发现敲除小鼠雄性不育，精子形态和功能异常，表现出ATP生成上调，线粒体鞘异常以及线粒体排布紊乱，精子尾部异常。经过分子实验我们证明了Gykl1和Gk2的C端对于它们定位到线粒体的外膜是必需的。我们还鉴定出Gykl1和Gk2能够与线粒体PLD6蛋白发生相互作用，诱导磷脂酸依赖的线粒体聚集。总体来说，我们的研究发现了甘油激酶类蛋白在精子发生中的重要作用，为精子异常疾病和雄性不育提出了新的分子机制。同时，在项目的支持下，我们利用CRISPR/Cas9快速基因敲除技术构建了其它的基因编辑小鼠，重点研究了Ccndbp1在骨骼肌生成中的重要作用和分子机制，以及利用涡虫为模型，鉴定并系统研究了端粒保护蛋白同源蛋白SmedOB1对于涡虫稳态维持和再生的重要作用。共发表4篇SCI论文，申请一项专利。我们将会继续研究衰老和干细胞相关的调控机制。