A nonsense mutation in Ccdc62 gene is responsible for spermiogenesis defects and male infertility in repro29/repro29 mice
A nonsense mutation in Ccdc62 gene is responsible for spermiogenesis defects and male infertility in repro29/repro29 mice
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Ccdc62 基因的无义突变导致 repro29/repro29 小鼠精子发生缺陷和雄性不育
DOI：
10.1095/biolreprod.116.141408
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发表时间：
2017-03-01
影响因子：
3.6
通讯作者：
Gui, Yaoting
中科院分区：
文献类型：
--
作者：
Li, Yuchi;Li, Cailing;Gui, Yaoting
表型驱动突变是一种无偏的方法，以确定新的基因参与精子发生和其他生殖过程。由杰克逊实验室生殖基因组学项目产生的雄性repo 29/repo 29小鼠不育，精子畸形，活动力差。使用选择性外显子捕获和大规模平行测序技术，我们确定了卷曲螺旋结构域包含62基因（Ccdc 62）的外显子6中的无义突变，其导致形成提前终止密码子和截短的蛋白质。在检测的组织中，通过逆转录-PCR（RT-PCR）和Western blot分析发现CCDC 62在小鼠睾丸中的表达水平最高。免疫荧光染色结果表明，CCDC 62在小鼠睾丸精子细胞质和发育中的顶体中均有表达，在成熟精子中特异定位于顶体。通过将repro 29/+小鼠与Ccdc 62（-/-）小鼠（通过CRISPR-Cas9策略产生）交配的互补分析进一步提供了遗传证据，即repro 29/repro 29小鼠的不育是由Ccdc 62突变引起的。最后，我们发现CCDC 62和高尔基体相关的PDZ和卷曲螺旋基序蛋白的细胞内共定位和相互作用可能对顶体形成很重要。总之，这项研究确定了Ccdc 62中的无义突变，该突变直接导致rep 29/rep 29小鼠的雄性不育。
Phenotype-driven mutagenesis is an unbiased method to identify novel genes involved in spermatogenesis and other reproductive processes. Male repro29/repro29 mice generated by the Reproductive Genomics Program at the Jackson Laboratory were infertile with deformed sperm and poor motility. Using selected exonic capture and massively parallel sequencing technologies, we identified a nonsense mutation in the exon 6 of coiled-coil domain-containing 62 gene (Ccdc62), which results in a formation of a premature stop codon and a truncated protein. Among the tissues examined, CCDC62 was found to be expressed at the highest level in mouse testis by reverse transcriptase-PCR (RT-PCR) and Western blot analysis. With immunofluorescent staining, we demonstrated that CCDC62 was expressed in the cytoplasm and the developing acrosome in the spematids of mouse testis, and was specifically localized at the acrosome in mature sperm. The complementation analysis by mating repro29/+ mice with Ccdc62(-/-) mice (generated by CRISPR-Cas9 strategy) further provided genetic proof that the infertility of repro29/repro29 mice was caused by Ccdc62 mutation. Finally, it was found that intracellular colocalization and interaction of CCDC62 and Golgi-associated PDZ and coiled-coil motif-containing protein may be important for acrosome formation. Taken together, this study identified a nonsense mutation in Ccdc62, which directly results in male infertility in repro29/repro29 mice.