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Adenylate kinase 9 is essential for sperm function and male fertility in mammals

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journal contribution
posted on 2023-11-10, 10:39 authored by Elena O’Callaghan, Paula Navarrete-Lopez, Miriama StiavnickaMiriama Stiavnicka, José M. Sáncheza, Maria Maroto, Eva Pericuesta, Raul Fernández-González, Ciara O’Meara, Bernard Eivers, Margaret M. Kelleher, Ross D. Evans, Xena M. Mapel, Audald Lloret-Villas, Hubert Pausch, Miriam Balastegui-Alarcón, Manuel Avilés, Ana Sanchez-Rodriguez, Eduardo R. S. Roldan, Michael McDonald, David A. Kenny, Sean FairSean Fair, Alfonso Gutiérrez-Adán, Patrick Lonergan

Despite passing routine laboratory tests for semen quality, bulls used in artificial insemination exhibit significant variation in fertility. Routine analysis of fertility data identified a dairy bull with extreme subfertility (10% pregnancy rate). To characterize the subfertility phenotype, a range of in vitro, in vivo, and molecular assays were carried out. Sperm from the subfertile bull exhibited reduced motility and severely reduced caffeine-induced hyperactivation compared to controls. 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 from semen and RNA sequencing of testis tissue revealed a critical mutation in adenylate kinase 9 (AK9) that impaired splicing, leading to a premature termination codon and a severely truncated protein. Mice deficient in AK9 were generated to further investigate the function of the gene; knockout males were phenotypically indistinguishable from their wild-type littermates but produced immotile sperm that were incapable of normal fertilization. These sperm exhibited numerous abnormalities, including a low ATP concentration and reduced motility. RNA-seq analysis of their testis revealed differential gene expression of components of the axoneme and sperm flagellum as well as steroid metabolic processes. Sperm ultrastructural analysis showed a high percentage of sperm with abnormal flagella. Combined bovine and murine data indicate the essential metabolic role of AK9 in sperm motility and/or hyperactivation, which in turn affects sperm binding and penetration of the zona pellucida. Thus, AK9 has been found to be directly implicated in impaired male fertility in mammals.


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PNAS, 2023, 120 (42) e2305712120


National Academy of Sciences

Other Funding information

This work was funded by Science Foundation Ireland to P.L., S.F. and D.A.K (Project 16/IA/4474). X.M.M., A.L.-V., and H.P. received financial support from Swissgenetics, Zollikofen, Switzerland (https://swissge?, the Swiss NSF (310030 185229), and through an ETH Research Grant. A.G.-A., P.N.-L., M. Maroto, R.F.-G., and E.P. were supported by Grant PID2021-122507OB-I00, and M.B.-A., M.A by Grant PID2021-123091NB-C21, both funded by MCIN/AEI/10.13039/501100011033/ and European Union NextGenerationEU/PRTR. J.M.S. was funded by Talent Attraction Program of the Comunidad de Madrid (ATI2019-T2-BIO-12966). P.N.-L. was supported by a pre-doctoral fellowship from the Spanish Ministry of Science and Innovation (MICINN) (PRE2019-088813).

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