Abstract：
Azoospermia constitutes a significant factor in male infertility, defined by the absence of spermatozoa in the ejaculate, afflicting 15% of infertile men. However, a subset of azoospermic cases remains unattributed to known genetic variants. Prior investigations have identified the chibby family member 2 (CBY2) as prominently and specifically expressed in the testes of both humans and mice, implicating its potential involvement in spermatogenesis. In this study, we conducted whole exome sequencing (WES) on an infertile family to uncover novel genetic factors contributing to azoospermia. Our analysis revealed a homozygous c .355 C>A variant of CBY2 in a non-obstructive azoospermic patient. This deleterious variant significantly diminished the protein expression of CBY2 both in vivo and in vitro, leading to a pronounced disruption of spermatogenesis at the early round spermatid stage post-meiosis. This disruption was characterized by a nearly complete loss of elongating and elongated spermatids. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and co-immunoprecipitation assays demonstrated the interaction between CBY2 and Piwi-like protein 1 (PIWIL1). Immunofluorescence staining further confirmed the co-localization of CBY2 and PIWIL1 in the testes during the spermatogenic process in both humans and mice. Additionally, diminished PIWIL1 expression was observed in the testicular tissue from the affected patient. Our findings suggest that the homozygous c .355 C>A variant of CBY2 compromises CBY2 function, contributing to defective spermatogenesis at the round spermiogenic stage and implicating its role in the pathogenesis of azoospermia.
摘要：
无精子症构成男性不育的重要因素，定义为射精中没有精子，折磨15%的不育男性。然而,一部分无精子症病例仍未归因于已知的遗传变异.先前的研究已经确定了chibby家族成员2（CBY2）在人类和小鼠的睾丸中突出且特异性表达，暗示它可能参与精子发生。在这项研究中,我们对一个不育家庭进行了全外显子组测序(WES),以发现导致无精子症的新遗传因素.我们的分析显示，在非阻塞性无精子症患者中，CBY2的纯合c.355C>A变体。这种有害的变体在体内和体外显着减少了CBY2的蛋白质表达。导致精子发生在减数分裂后的早期精子细胞阶段。这种破坏的特征是伸长和伸长的精子细胞几乎完全丧失。液相色谱-串联质谱（LC-MS/MS）和免疫共沉淀测定证明了CBY2与Piwi样蛋白1（PIWIL1）之间的相互作用。免疫荧光染色进一步证实了CBY2和PIWIL1在人和小鼠生精过程中在睾丸中的共定位。此外,在受影响患者的睾丸组织中观察到PIWIL1表达减少。我们的发现表明，CBY2的纯合c.355C>A变体损害了CBY2的功能，在圆形生精阶段促进精子发生缺陷，并暗示其在无精子症的发病机理中的作用。
