Mutations in AK9 impair male fertility by altering sperm motility in mice and cattle.

Despite passing routine laboratory tests for semen quality, bulls used in artificial insemination (AI) can exhibit a significant variation in fertility. During fertility analysis, a subfertile bull with a low pregnancy rate of 10% was identified. To fully characterize the phenotype, a range of in vitro, in vivo and molecular assays were carried out. Additionally, knockout mice were generated to investigate the function of the identified mutated gene. Sperm from the subfertile bull exhibited reduced motility and severely reduced caffeine-induced hyperactivation compared to control bulls of proven fertility. Ability to penetrate the zona pellucida, cleavage rate, cleavage kinetics, and blastocyst yield after IVF or AI were significantly lower than in control bulls. Whole-genome sequencing and RNA sequencing of testis revealed a critical mutation in AK9 that affect splicing, shaping the majority of AK9 transcripts that leads to a premature termination codon and a severely truncated protein. Transgenic mice deficient in AK9 were generated, resulting in the production of immotile sperm that were unable to fertilize the oocyte. These sperm exhibited abnormalities, including a low ATP concentration. RNA-seq analysis of testis revealed differential gene expression of components of the axoneme and sperm flagella, as well as steroid metabolic processes. Sperm ultrastructural analysis showed a higher percentage of sperm with abnormal flagella. The infertility produced by AK9 mutant bull and in AK9 deficient mice indicates the essential metabolic role of AK9 in sperm motility and/or hyperactivation, which in turn affects sperm binding and penetration of the zona pellucida.尽管用于人工授精（AI）的公牛已通过精液质量的常规实验室检测，但其生育力仍存在显著差异。在生育力分析中，研究人员鉴定出一头妊娠率仅为10%的低生育力公牛。为全面表征该表型，研究人员开展了一系列体外、体内及分子检测实验，并构建了基因敲除小鼠以探究所鉴定突变基因的功能。与经证实生育力正常的对照公牛相比，该低生育力公牛的精子运动能力显著下降，咖啡因诱导的精子超激活作用也大幅减弱。无论是体外受精（IVF）还是人工授精后，精子穿透透明带的能力、卵裂率、卵裂动力学以及囊胚形成率均显著低于对照公牛。对睾丸组织进行全基因组测序与RNA测序后发现，AK9基因存在一处影响剪接的关键突变：该突变会改变绝大多数AK9转录本的结构，产生提前终止密码子，进而形成严重截短的蛋白质。研究人员构建了AK9缺陷型基因敲除小鼠，其精子完全丧失运动能力，无法完成卵母细胞受精。此类精子还存在多种异常，包括三磷酸腺苷（ATP）浓度降低。对睾丸组织的RNA-seq分析显示，轴丝与精子鞭毛的组成成分以及类固醇代谢过程相关的基因表达存在显著差异。精子超微结构分析发现，携带异常鞭毛的精子比例显著升高。AK9突变公牛与AK9缺陷型小鼠所表现出的不育表型，证实AK9在精子运动能力和/或超激活过程中发挥着关键的代谢调控作用，进而影响精子与透明带的结合及穿透过程。