PMFBP1基因突变导致无头精子症的分子机制研究

Acephalic Spermatozoa (AS) is a special type of teratozoospermia, which is one of the causes of male infertility. Previous studies suggested that autosomal recessive mutations were the main cause of AS, however, the genetic causes in more than half of cases remain unclear. Our previous work performed whole-exome sequencing on two isolated AS patients from two pedigrees, and found homozygous and compound heterozygous mutations in the PMFBP1 gene. We also noticed that PMFBP1 protein localized in the connecting piece region between the head and tail by immunostaining. Western blot analysis also indicated that the level of PMFBP1 protein in the patient’s sperm was significantly lower than that in the normal control. We further constructed Pmfbp1 gene knock-out mice and found their spermatozoa were acephalic. Therefore, based on the previous work we hypothesized that PMFBP1 genetic mutations could cause acephalic spermatozoa. To prove the hypothesis we would study the ultrastructural analysis on the sperm, use the transcriptome and proteome methods to dissect the molecular changes, and study the interacting proteins with PMFBP1. Ultimately, we will clarify the molecular mechanism and working model of PMFBP1 in connecting sperm head and tail.

无头精子症是特殊类型的畸形精子症，是导致男性不育的原因之一。以往研究认为无头精子症为常染色体隐性遗传，但仍有一半以上病因尚未明确。PMFBP1是人和小鼠睾丸特异表达基因，目前该基因的功能与疾病发生尚未明确。本课题对前期收集的无头精子患者血样行全外显子测序，发现PMFBP1基因突变为无头精子致病候选基因：我们研究发现PMFBP1蛋白精准定位于正常人精子头尾连接处，并在无头精子症的精子中显著低表达。我们通过敲除小鼠Pmfbp1基因发现小鼠成熟精子表型亦为无头精子。因此，我们推断PMFBP1基因突变是无头精子症致病基因。为验证该假说我们拟研究该基因突变导致无头精子亚显微结构及圆形精子发育动态变化，阐明该基因突变引起精子转录组及蛋白质组学变化，通过分子细胞生物学及生物化学方法揭示与PMFBP1相互作用的蛋白，最终阐明人PMFBP1基因突变或基因敲除小鼠导致无头精子的分子机制及工作模型。

基因突变是引起精子发生异常导致男性不育的主要原因，我们前期研究发现人PMFBP1基因突变导致无头精子症的发生及男性不育，但具体分子机制不清楚。本研究在前期基础上发现Pmbp1基因 KO小鼠表型与人类一致，通过野生型小鼠与KO小鼠睾丸组织行蛋白质谱鉴定出124种上调蛋白和35种下调蛋白，鉴定了与这些蛋白相关的几种富集的生物学过程，其中高尔基囊泡运输是上调过程蛋白的最丰富的过程，过度表达相关的基因可能通过影响近端中心粒附着到核仁而扰乱头颈部连接的形成，证实无头精子中涉及的分子机制与高尔基体囊泡运输有关。此外，我们对收集到的特发性男性不育患者行染色体核型及全外显子组筛查时，我们发现嵌合型环状Y染色体垂直传播导致男性少精子症，母源性染色体46,X, t(X; 1) (q28; q21)核型异常导致两兄弟发生46,Y, t(X; 1) (q28; q21)染色体畸变伴无精子症家系，以及X染色体倒位导致两兄弟严重少精子症家系，导致严重少精子症。我们还发现了多个导致男性不育的新基因及新突变位点，如发现CEP112导致无头精子症的新基因，发现DNAH8、CFAP58、CFAP74、SPEF2、DNAH2、DNAH17等基因突变可导致弱畸形精子症，发现CCDC9、ARMC12新候选基因导致弱精子症，PNLDC1基因突变导致无精子症发生，IQCN基因突变导致受精障碍及早期胚胎发育阻滞。以上研究结果发表15篇SCI论文，另在新杂志发表英文论文1篇和国内核心期刊论文1篇，共计发表17篇论文。中国发明专利3项。以上研究结果将为男性不育患者的遗传咨询和临床诊疗提供重要理论依据。