无精症缺失基因（DAZ）家族蛋白BOULE对于精子发生的分子调控机制

Infertility affects approximately 10% of couples, with half of these cases attributable to the male partner. The DAZ(Deleted in AZoospermia) gene family consists of highly conserved RNA binding proteins essential for fertility among diverse animals, including humans. Boule is the ancestral member of this family, and is a key regulatory factor in the process of male spermatogenesis．The expression change of DAZ genes or the absence of BOULE protein results in spermatogenic failure，leading to azoospermia and male infertility．But the molecular mechanism underlying infertility is unclear, specifically as a RNA-binding protein, its target RNA and downstream pathways remain to be revealed..To figure out the mechanism of post-transcriptional regulation of Boule of spermatogenic genes, the knockout and overexpression animal models were constructed to study the target site and regulatory process of Boule with molecular biology, genetics and bioinformatics methods. First, HITS-CLIP (crosslinking-immunoprecipitation and high-throughput sequencing) technique has been utilized to find the target mRNA binding with Boule, validation of those potential targets will be validated in the cell culture and animal models. On the other hand, density gradient centrifugation technique will be used to isolate the subcellular organelles of testes cells, to identify the location of Boule and its target RNAs, and hence reveal the post-transcriptional regulatory mechanisms. The preliminary experiments showed that we have established these important technologies, supporting the feasibility of our approach. Hence this project should be accomplished in one or two years resulting in one or two high profile papers.

目前约有10%-15%的已婚夫妇不育，其中50%与男性有关。BOULE是无精症缺失基因（DAZ）家族中的最保守的成员。DAZ缺失是导致精子生成障碍最常见的分子缺陷，但其导致男性不育的分子机制尚不明朗，尤其作为RNA结合蛋白，所调控的靶标RNA和下游通路完全不清楚。本项目旨在阐明Boule对于精子形成相关基因的转录后调控机制，希望运用已建立的Boule敲除和过表达动物模型，结合分子生物学、生物信息学等方法深入研究Boule在整个调控过程中的具体靶标位点及后续干预步骤。首先运用代表RNA领域最新技术的紫外交联免疫沉淀结合高通量测序技术得到Boule蛋白特异性结合的mRNA，同时用密度梯度离心技术分离得到睾丸组织细胞亚单位，对Boule的作用途径精确定位。目前已完成预实验，确定了实验步骤可行性和特异性抗体的可靠性，计划在一到两年内完成该项目并在国际高水平期刊上发表一至两篇研究性论文。