Zhang Yunfei, Xing Xiaowei, Huang Lihua, Su Yuyan, Liu Gang, Zhang Xinxing, Yang Youbo
Department of Laboratory Medicine, the Third Xiangya Hospital, Central South University, Changsha, China.
Center for Experimental Medicine, the Third Xiangya Hospital, Central South University, Changsha, China.
Heliyon. 2024 Sep 21;10(19):e38263. doi: 10.1016/j.heliyon.2024.e38263. eCollection 2024 Oct 15.
The WD40 repeat (WDR) domain is present in a wide range of proteins, providing sites for protein‒protein interactions. Recent studies have shown that WDR proteins play indispensable roles in spermatogenesis, such as in spermatocyte division, sperm head formation and flagellar assembly. In this study, we identified a novel testis-specific gene, , which has the typical characteristics of WD40 proteins with two β-propellers, and is highly conserved in Mammalia. RT-PCR and Western blot results revealed that was highly expressed in testis. WDR64 protein was weakly expressed at postnatal Day 7, increased substantially at postnatal Day 28 and maintained at high levels thereafter. Further immunofluorescence demonstrated that WDR64 was localized posterior to the nucleus in steps 8-14 spermatids in line with the dynamic localization of manchette, moved to the flagella in steps 15-16 spermatids, and localized at the midpiece of the flagellum in mature spermatozoa. To explore the function of WDR64, we performed immunoprecipitation‒mass spectrometry (IP‒MS) to screen its interacting proteins and found that WDR64 interacted with ODF1 to form a complex. The WDR64/ODF1 complex is located at the manchette during nucleus shaping and finally at the midpiece of the mature spermatozoa tail, suggesting that it may be involved in the assembly of the manchette and flagella during spermiogenesis. Our findings provide the first understanding of the expression pattern of WDR64 and its potential molecular mechanism in spermiogenesis.
WD40重复（WDR）结构域存在于多种蛋白质中，为蛋白质-蛋白质相互作用提供位点。最近的研究表明，WDR蛋白在精子发生过程中发挥着不可或缺的作用，如在精母细胞分裂、精子头部形成和鞭毛组装中。在本研究中，我们鉴定了一个新的睾丸特异性基因，它具有典型的WD40蛋白特征，有两个β-螺旋桨结构，并且在哺乳动物中高度保守。逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹结果显示，该基因在睾丸中高表达。WDR64蛋白在出生后第7天表达较弱，在出生后第28天大幅增加，此后维持在高水平。进一步的免疫荧光显示，WDR64在第8-14步精子细胞中定位于细胞核后方，与精子成形小带的动态定位一致，在第15-16步精子细胞中移至鞭毛，在成熟精子中定位于鞭毛的中段。为了探索WDR64的功能，我们进行了免疫沉淀-质谱分析（IP-MS）以筛选其相互作用蛋白，发现WDR64与ODF1相互作用形成复合物。WDR64/ODF1复合物在细胞核塑形过程中位于精子成形小带，最终位于成熟精子尾部的中段，这表明它可能在精子形成过程中参与精子成形小带和鞭毛的组装。我们的研究结果首次揭示了WDR64在精子形成中的表达模式及其潜在的分子机制。
