TMEM232 is required for the formation of sperm flagellum and male fertility in mice

Asthenoteratozoospermia is a major cause of male infertility. Thus far, the identified related genes can explain only a small share of asthenoteratozoospermia cases, suggesting the involvement of other genes. The transmembrane protein TMEM232 is highly expressed in mouse testes. In the present study, to determine its function of TMEM232 in testes, we constructed a Tmem232-null mouse model using CRISPRCas9 technology. Tmem232 knockout (KO) male mice was completely infertile, and their sperm were immotile, with morphological defects of the flagellum. Electron microscopy revealed an aberrant midpiece-principal junction and the loss of the fourth of nine doublet microtubules in the sperm of Tmem232-/-mice. Sperm cells presented an 8+2 conformation and an irregular arrangement of the mitochondrial sheath. Proteomic analysis indicated a downregulation in the expression of proteins associated with flagellar motility, sperm capacitation, and the integrity and stability of sperm structure, as well as an upregulation in the expression of multiple ribosome components in TMEM232-deficient spermatids. Additionally, TMEM232 was observed to be involved in ribophagy by interacting with autophagy-related proteins, such as ATG14 and ARMC3, to regulate ribosome homeostasis during spermiogenesis. These results suggest that TMEM232 is essential for normal sperm flagellum formation and plays an important role in the autophagic elimination of cytosolic ribosomes to provide energy for flagellar motility. To investigate the changes of gene expression in the mice testis after Tmem232 knockout, we collected testes from Tmem232-knockout and wild-type mice testis for transcriptome sequencing analysis弱畸精子症（Asthenoteratozoospermia）是男性不育的主要致病原因之一。迄今为止，已鉴定的相关致病基因仅能解释少数弱畸精子症病例，提示仍存在其他未被探明的致病基因。跨膜蛋白TMEM232在小鼠睾丸中呈高表达。本研究为明确TMEM232在睾丸中的功能，利用CRISPR-Cas9技术构建了Tmem232基因敲除（Tmem232-null）小鼠模型。Tmem232基因敲除（KO）的雄性小鼠完全不育，其精子活动能力丧失，且存在鞭毛形态缺陷。电镜观察显示，Tmem232敲除小鼠的精子鞭毛中段-主段连接区结构异常，9组双联微管中的第4组发生缺失；精子细胞呈现异常的8+2微管构型，线粒体鞘排列紊乱。蛋白质组学分析表明，在TMEM232缺陷的精子细胞中，与鞭毛运动、精子获能以及精子结构完整性和稳定性相关的蛋白表达显著下调，而多种核糖体组分的蛋白表达则显著上调。此外，TMEM232可通过与ATG14、ARMC3等自噬相关蛋白相互作用参与核糖体自噬（ribophagy），以调控精子形成过程中的核糖体稳态。上述结果提示，TMEM232对于精子鞭毛的正常形成至关重要，同时在胞质核糖体的自噬清除以鞭毛运动供能的过程中发挥关键作用。为探究Tmem232基因敲除后小鼠睾丸的基因表达变化，本研究收集了Tmem232敲除型与野生型小鼠的睾丸组织，开展转录组测序分析。