State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu, China.
Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, China.
J Med Genet. 2021 Jan;58(1):56-65. doi: 10.1136/jmedgenet-2019-106598. Epub 2020 May 6.
Infertility affects approximately 15% of couples worldwide with male infertility being responsible for approximately 50% of cases. Although accumulating evidence demonstrates the critical role of the X chromosome in spermatogenesis during the last few decades, the expression patterns and potential impact of the X chromosome, together with X linked genes, on male infertility are less well understood.
We performed X chromosome exome sequencing followed by a two-stage independent population validation in 1333 non-obstructive azoospermia cases and 1141 healthy controls to identify variant classes with high likelihood of pathogenicity. To explore the functions of these candidate genes in spermatogenesis, we first knocked down these candidate genes individually in mouse spermatogonial stem cells (SSCs) using short interfering RNA oligonucleotides and then generated candidate genes knockout mice by CRISPR-Cas9 system.
Four low-frequency variants were identified in four genes (, , and ) associated with male infertility. Functional studies of the mouse SSCs revealed that knocking down or could inhibit SSCs self-renewal and knocking down could repress SSCs differentiation in vitro. Using CRISPR-Cas9 system, and knockout mice were generated. Excitingly, knockout mice were infertile with impaired spermatogenesis. Moreover, knockout mice exhibited impaired sperm motility and sperm cells displayed abnormal mitochondrial structure.
Our data indicate that the X-linked genes are associated with male infertility and involved in regulating SSCs, which provides a new insight into the role of X-linked genes in spermatogenesis.
不孕不育影响着全球约 15%的夫妇，其中男性不育约占 50%。尽管过去几十年的研究积累了大量证据表明 X 染色体在精子发生过程中起着关键作用，但 X 染色体及其连锁基因对男性不育的表达模式和潜在影响还了解甚少。
我们对 1333 例非梗阻性无精子症病例和 1141 例健康对照进行了 X 染色体外显子组测序，并进行了两阶段独立的人群验证，以鉴定具有高致病性可能性的变异类别。为了探索这些候选基因在精子发生中的功能，我们首先使用短发夹 RNA 寡核苷酸在小鼠精原干细胞（SSC）中单独敲低这些候选基因，然后通过 CRISPR-Cas9 系统生成候选基因敲除小鼠。
在四个与男性不育相关的基因（、、和）中发现了四个低频变异。对小鼠 SSCs 的功能研究表明，敲低或可抑制 SSCs 的自我更新，敲低可抑制 SSCs 的体外分化。利用 CRISPR-Cas9 系统，成功生成了和基因敲除小鼠。令人兴奋的是，基因敲除小鼠不育，精子发生受损。此外，基因敲除小鼠的精子运动能力受损，精子细胞显示出异常的线粒体结构。
我们的数据表明，X 连锁基因与男性不育有关，并参与调节 SSCs，这为 X 连锁基因在精子发生中的作用提供了新的见解。
