Calicin在精子变形过程中的功能及机制研究

The successful progress of spermiogenesis is the prerequisite for the production of sperm with normal morphology and function. Once the abnormality occurs, it will cause teratozoospermia, leading to male infertility. The applicant collected a family of consanguineous marriage. The two patients were infertile for many years after marriage. Their sperm motility and concentration were normal. Sperm staining and transmission electron microscopy showed malformed sperm head, abnormal acrosome and nuclear morphology. The mutation of CCIN His42Leu was suggested to be a suspicious pathogenic factor by whole exon sequencing and bioinformatics analysis. Calicin, as a CCIN-encoded sperm perinuclear skeleton protein, may be involved in the maintenance of sperm morphology. To further study the mechanism, point mutation mice were constructed and obtained. Previous results showed that the mutant mice had the same clinical phenotype with male infertility, including malformed sperm head, abnormal acrosome and nuclear morphology. In the latter part of this project, we intend to find the protein interacting with calicin by immunoprecipitation, mass spectrometry and proteomics and analyze the crystal structure of calicin in order to elucidate the function and molecular mechanism of calicin in the process of spermiogenesis. It will not only help us to understand the complex biological process of spermiogenesis, but also provide important theoretical support for clinical diagnosis and treatment of such malformed spermatozoa.

精子变形过程的顺利进行是产生形态与功能正常精子的前提。一旦发生异常，将引起畸形精子症，导致男性不育。申请者收集了一例近亲家系，兄弟二人结婚多年不育，精子活力和密度均正常，染色及透射电镜显示精子头部畸形、顶体及核形态异常。通过全外显子测序和生物信息学分析，提出CCIN His42Leu 突变是可疑致病因素。Calicin作为CCIN编码的精子核周骨架蛋白，可能参与精子形态的维持。为进一步研究机制，本课题构建并获得了点突变小鼠。前期结果发现，突变体小鼠和患者的临床表型一致，均表现为雄性不育，精子头部畸形，顶体及核形态异常。本课题后期拟通过免疫共沉淀结合质谱鉴定及蛋白质组学，寻找与calicin相互作用蛋白，联合解析calicin晶体结构，以阐明calicin在精子变形过程中的功能及分子机制，不仅有助于深入了解精子变形这个复杂的生物学过程，而且将为此类畸形精子症患者的临床诊疗提供重要的理论支持。

精子变形过程的顺利进行是产生形态与功能正常精子的前提。该过程的异 常会导致临床畸形精子症，引起男性不育。畸形精子症是一种具有很大遗传异质性的疾病，尽管之前的研究已经鉴定了一些与畸形精子症相关的基因，但仍然有很多病因未知。.课题组在上海第九人民医院辅助生殖科收集了一例患有畸形精子症的近亲家系，患者是两兄弟，均结婚多年不育，精液常规参数正常，精子染色及透射电镜显示精子头部畸形、细胞核不规则凹陷、顶体形态异常且脱落于核膜、颈部胞质残留等异常表型，提示该精子畸形是一种类似于II型圆头精子症的病理形式。全外显子组测序和生物信息学分析推测CCINH42L是其致病因素。随后在126例散发畸形精子症患者中，我们还筛查到一例新发的CCINR432W/C447*复合杂合突变。.CCIN编码精子细胞骨架蛋白Calicin，参与哺乳动物精子花萼结构的形成和维持，可能在精子变形过程发挥作用。为了进一步研究Calicin在精子变形过程的功能及机制，本课题利用CRISPR-Cas9技术分别构建了CcinH42L/H42L纯合突变和CcinR432W/C447*复合杂合突变小鼠模型，成功地再现了患者的不育表型。对Calicin的结构预测和分析及体外的表达验证，推测点突变破坏了Calicin蛋白质的结构稳定性，导致精子细胞中Calicin蛋白水平急剧降低。对小鼠模型的研究表明Calicin的缺陷影响了顶体期细胞核与顶体结构的正常形成，以及manchette 结构的动态变化，引起精子头部畸形。.值得注意的是，体外受精实验表明来自人类患者和小鼠模型的CCIN突变精子都未能粘附到卵子的透明带，这是CCIN突变导致雄性不育的主要原因。使用卵胞浆内单精子注射可以成功地克服这一缺陷，让患者及突变体小鼠模型获得健康子代。综上所述，本研究确定了Calicin在精子头部变形和男性生育中的功能，为遗传咨询和辅助生殖治疗提供了重要指导。