BACKGROUND: Protein acetylation has emerged as essential for sperm function, attracting considerable attention recently. Acetylation, typically mediated by lysine acetyltransferases, involves attaching an acetyl group from acetyl-coenzyme A to lysine residues in proteins. Under alkaline conditions, however, acetylation can occur with minimal enzymatic involvement, primarily due to an elevated pH. As sperm migrate towards the ampulla, they experience increasing intracellular pH (pHi) while undergoing two crucial processes for fertilization: capacitation and the acrosome reaction (AR). Whereas the involvement of acetylating enzymes in these events has been partially investigated, the potential for non-enzymatic acetylation driven by the pHi alkalinization remains unknown. RESULTS: This study examined protein acetylation (acLys) levels in sperm incubated under capacitating conditions at pH 7.2 and pH 9.0, the latter condition potentially promoting non-enzymatic acetylation. To more precisely investigate the occurrence of non-enzymatic acetylation events, acetyltransferase activity was selectively attenuated using a specific cocktail of inhibitors. The functional implications of these conditions were assessed by examining key fertilization-related sperm attributes, including motility during capacitation and the ability to initiate the AR. Results demonstrated that alkaline conditions elevated basal acLys levels even with reduced acetyltransferase activity (P < 0.05), indicative of non-enzymatic acetylation. α-tubulin, particularly in the midpiece of the sperm flagellum, was identified as a specific target of this modification, correlating with diminished motility during capacitation. Following the AR, acLys levels in the head and midpiece decreased (P < 0.05) under conditions promoting non-enzymatic acetylation, accompanied by reductions in intracellular and acrosomal pH. In contrast, acLys levels and pH in the sperm head incubated under standard capacitating conditions (pH 7.2) remained stable. Sperm exposed to conditions conducive to non-enzymatic acetylation exhibited an impaired ability to trigger the AR (P < 0.05) compared to those maintained at pH 7.2. Notably, diminished acetylase activity emerged as a key factor impairing the maintenance of intracellular and acrosomal pH levels attained during capacitation, even under a pH of 9.0. CONCLUSION: This study provides novel evidence for the occurrence of non-enzymatic acetylation in sperm, linked to the modulation of α-tubulin acetylation levels and motility during capacitation. Additionally, it suggests that acetyltransferase activity may play a crucial role in regulating intracellular and acrosomal pH levels in capacitated sperm, facilitating the AR.

Biomedical Center Faculty of Medicine in Pilsen Charles University Alej Svobody 76 323 00 Pilsen Czech Republic

Biotechnology of Animal and Human Reproduction Institute of Food and Agricultural Technology University of Girona 17003 Girona Spain

Catalan Institution for Research and Advanced Studies 08010 Barcelona Spain

Department of Histology and Embryology Faculty of Medicine in Pilsen Charles University Alej Svobody 76 323 00 Pilsen Czech Republic

Laboratory of Cell Signal Transduction Networks Instituto de Biología Molecular y Celular de Rosario CONICET UNR Blvd 27 de Febrero 210 Bis S2000EZP Rosario Argentina

Laboratory of Reproductive Medicine Faculty of Biochemical and Pharmaceutical Sciences National University of Rosario Suipacha 531 S2002LRK Rosario Argentina

SaBio IREC Campus Universitario 02071 Albacete Spain

Unit of Cell Biology Department of Biology Faculty of Sciences University of Girona 17003 Girona Spain

Zobrazit více v PubMed

Le-Tian Z, Cheng-Zhang H, Xuan Z, Zhang Q, Zhen-Gui Y, Qing-Qing W, Sheng-Xuan W, Zhong-Jin X, Ran-Ran L, Ting-Jun L, Zhong-Qu S, Zhong-Hua W, Ke-Rong S. Protein acetylation in mitochondria plays critical functions in the pathogenesis of fatty liver disease. BMC Genom. 2020;21(1):1–17. 10.1186/s12864-020-06837-y. PubMed PMC

McTiernan N, Kjosås I, Arnesen T. Illuminating the impact of N-terminal acetylation: from protein to physiology. Nat Commun. 2025;16(1):703. 10.1038/s41467-025-55960-5. PubMed PMC

Xia C, Tao Y, Li M, Che T, Qu J. Protein acetylation and deacetylation: An important regulatory modification in gene transcription (Review). Exp Ther Med. 2020;20:2923–40. 10.3892/etm.2020.9073. PubMed PMC

Shang S, Liu J, Hua F. Protein acylation: mechanisms, biological functions and therapeutic targets. Signal Transduct Target Therapy. 2022;7(1):396. 10.1038/s41392-022-01245-y. PubMed PMC

Paik WK, Pearson D, Lee HW, Kim S. Nonenzymatic acetylation of histones with acetyl-CoA. BBA Sect Nucl Acids Protein Synth. 1970;213(2):513–22. 10.1016/0005-2787(70)90058-4. PubMed

Baldensperger T, Glomb MA. Pathways of non-enzymatic lysine acylation. Front Cell Dev Biol. 2021;9(April):1–9. 10.3389/fcell.2021.664553. PubMed PMC

Schastnaya E, Doubleday PF, Maurer L, Sauer U. Non-enzymatic acetylation inhibits glycolytic enzymes in Escherichia coli. Cell Rep. 2023;42(1): 111950. 10.1016/j.celrep.2022.111950. PubMed

Christensen DG, Xie X, Basisty N, Byrnes J, Mcsweeney S, Schilling B, Wolfe AJ. Post-translational protein acetylation : an elegant mechanism for bacteria to dynamically regulate metabolic functions. Front Microbiol. 2019;10(July):1–22. 10.3389/fmicb.2019.01604. PubMed PMC

Wagner GR, Hirschey MD. Nonenzymatic protein acylation as a carbon stress regulated by sirtuin deacylases. Mol Cell. 2014;54(1):5–16. 10.1016/j.molcel.2014.03.027. PubMed PMC

Narita T, Weinert BT, Choudhary C. Functions and mechanisms of non-histone protein acetylation. Nat Rev Mol Cell Biol. 2019;20(3):156–74. 10.1038/s41580-018-0081-3. PubMed

Chávez JC, Carrasquel-Martínez G, Hernández-Garduño S, Matamoros Volante A, Treviño CL, Nishigaki T, Darszon A. Cytosolic and acrosomal pH regulation in mammalian sperm. Cells. 2024;13(10):1–31. 10.3390/cells13100865. PubMed PMC

Xu C, Yuan Y, Zhang C, Zhou Y, Yang J, Yi H, Gyawali I, Lu J, Guo S, Ji Y, Tan C, Wang S, Zhang Y, Jiang Q, Shu G. Smooth muscle AKG/OXGR1 signaling regulates epididymal fluid acid-base balance and sperm maturation. Life Metab. 2022;1(1):67–80. 10.1093/lifemeta/loac012. PubMed PMC

Parrish J. Capacitation of bovine sperm by heparin: inhibitory effect of glucose and role of intracellular pH. Biol Reprod. 1989;41(4):683–99. 10.1095/biolreprod41.4.683. PubMed

Yanagimachi R. In vitro capacitation of hamster spermatozoa by follicular fluid. J Reprod Fertil. 1969;18(2):275–86. 10.1530/jrf.0.0180275. PubMed

Yanagimachi R. Mysteries and unsolved problems of mammalian fertilization and related topics. Biol Reprod. 2022;106(4):644–75. 10.1093/biolre/ioac037. PubMed PMC

Zhou J, Chen L, Li J, Li H, Hong Z, Xie M, Chen S, Yao B, Drevet JR. The semen pH affects sperm motility and capacitation. PLoS ONE. 2015;10(7): e0132974. 10.1371/journal.pone.0132974. PubMed PMC

Visconti PE. Understanding the molecular basis of sperm capacitation through kinase design. Proc Natl Acad Sci USA. 2009;106(3):667–8. 10.1073/pnas.0811895106. PubMed PMC

Aitken RJ, Nixon B. Sperm capacitation: a distant landscape glimpsed but unexplored. Mol Hum Reprod. 2013;19(12):785–93. 10.1093/molehr/gat067. PubMed

Balbach M, Ghanem L, Violante S, Kyaw A, Romarowski A, Cross JR, Visconti PE, Levin LR, Buck J. Capacitation induces changes in metabolic pathways supporting motility of epididymal and ejaculated sperm. Front Cell Dev Biol. 2023;11(June):1–16. 10.3389/fcell.2023.1160154. PubMed PMC

Ritagliati C, Luque GM, Stival C, Baro Graf C, Buffone MG, Krapf D. Lysine acetylation modulates mouse sperm capacitation. Sci Reports. 2018;8(1):13334. 10.1038/s41598-018-31557-5. PubMed PMC

Pérez-Patino C, Parrilla I, Li J, Barranco I, Martínez EA, Rodriguez-Martínez H, Roca J. The proteome of pig spermatozoa is remodeled during ejaculation. Mol Cell Proteom. 2019;18(1):41–50. 10.1074/mcp.RA118.000840. PubMed PMC

Chen G, Ren L, Chang Z, Zhao Y, Zhang Y, Xia D, Zhao R, He B. Lysine acetylation participates in boar spermatozoa motility and acrosome status regulation under different glucose conditions. In: Theriogenology, vol. 159. Amsterdam: Elsevier; 2021. PubMed

Awe S, Renkawitz-Pohl R. Histone H4 acetylation is essential to proceed from a histone- to a protamine-based chromatin structure in spermatid nuclei of Drosophila melanogaster. Syst Biol Reprod Med. 2010;56(1):44–61. 10.3109/19396360903490790. PubMed

Coffey K, Blackburn TJ, Cook S, Golding BT, Griffin RJ, Hardcastle IR, Hewitt L, Huberman K, McNeill HV, Newell DR, Roche C, Ryan-Munden CA, Watson A, Robson CN. Characterisation of a Tip60 specific inhibitor, NU9056, in prostate cancer. PLoS ONE. 2012;7(10):1–12. 10.1371/journal.pone.0045539. PubMed PMC

Ji C, Xu W, Ding H, Chen Z, Shi C, Han J, Yu L, Qiao N, Zhang Y, Cao X, Zhou X, Cheng H, Feng H, Luo C, Li Z, Zhou B, Ye Z, Zhao Y. The p300 inhibitor A-485 exerts antitumor activity in growth hormone pituitary adenoma. J Clin Endocrinol Metab. 2022;107(6):e2291–300. 10.1210/clinem/dgac128. PubMed PMC

Chow S, Hedley D. Flow cytometric measurement of intracellular pH. Curr Protocols Cytomet. Chapter 9, Unit 9.3 (2001). 10.1002/0471142956.cy0903s14 PubMed

Yeste M, Recuero S, Maside C, Salas-Huetos A, Bonet S, Pinart E. Blocking nhe channels reduces the ability of in vitro capacitated mammalian sperm to respond to progesterone stimulus. Int J Mol Sci. 2021;22(23):12646. 10.3390/ijms222312646. PubMed PMC

Peris-Frau P, Martín-Maestro A, Iniesta-Cuerda M, Sánchez-Ajofrín I, Cesari A, Garde JJ, Villar M, Soler AJ. Cryopreservation of ram sperm alters the dynamic changes associated with in vitro capacitation. Theriogenology. 2020;145:100–8. 10.1016/j.theriogenology.2020.01.046. PubMed

Iniesta-Cuerda M, Havránková J, Řimnáčová H, García-Álvarez O, Nevoral J. Male SIRT1 insufficiency leads to sperm with decreased ability to hyperactivate and fertilize. Reprod Domest Anim. 2022. 10.1111/rda.14172. PubMed

Bhagwat S, Dalvi V, Chandrasekhar D, Matthew T, Acharya K, Gajbhiye R, Kulkarni V, Sonawane S, Ghosalkar M, Parte P. Acetylated α-tubulin is reduced in individuals with poor sperm motility. Fertil Steril. 2014;101(1):95-104.e3. 10.1016/j.fertnstert.2013.09.016. PubMed

Aparicio IM, Santos AJ. Morphometry of porcine spermatozoa and its functional significance in relation with the motility parameters in fresh semen. Theriogenology. 2009;71:254–63. 10.1016/j.theriogenology.2008.07.007. PubMed

Barboni B, Mattioli M, Seren E. Influence of progesterone on boar sperm capacitation. J Endocrinol. 1995;144(1):13–8. 10.1677/joe.0.1440013. PubMed

Working PK, Meizel S. Correlation of increased intraacrosomal pH with the hamster sperm acrosome reaction. J Exp Zool. 1983;227(1):97–107. 10.1002/jez.1402270114. PubMed

Breitbart H, Cohen G, Rubinstein S. Role of actin cytoskeleton in mammalian sperm capacitation and the acrosome reaction. Reproduction. 2005;129(3):263–8. 10.1530/rep.1.00269. PubMed

Francou MM, Ten J, Bernabeu R, De Juan J. Capacitation and acrosome reaction changes α-tubulin immunodistribution in human spermatozoa. Reprod Biomed Online. 2014;28(2):246–50. 10.1016/j.rbmo.2013.10.001. PubMed

Shvedunova M, Akhtar A. Modulation of cellular processes by histone and non-histone protein acetylation. Nat Rev Mol Cell Biol. 2022;23(5):329–49. 10.1038/s41580-021-00441-y. PubMed

McBrian MA, Behbahan IS, Ferrari R, Su T, Huang TW, Li K, Hong CS, Christofk HR, Vogelauer M, Seligson DB, Kurdistani SK. Histone acetylation regulates intracellular pH. Mol Cell. 2013;49(2):310–21. 10.1016/j.molcel.2012.10.025. PubMed PMC

Koshland DE. Effect of catalysts on the hydrolysis of acetyl phosphate. Nucleophilic displacement mechanisms in enzymatic reactions. J Am Chem Soc. 1952;74(9):2286–92. 10.1021/ja01129a035.

Dahl HHM, Brown RM, Hutchison WM, Maragos C, Brown GK. A testis-specific form of the human pyruvate dehydrogenase E1α subunit is coded for by an intronless gene on chromosome 4. Genomics. 1990;8(2):225–32. 10.1016/0888-7543(90)90275-Y. PubMed

Korotchkina LG, Sidhu S, Patel MS. Characterization of testis-specific isoenzyme of human pyruvate dehydrogenase. J Biol Chem. 2006;281(14):9688–96. 10.1074/jbc.M511481200. PubMed

Kishimoto A, Ishiguro-Oonuma T, Takahashi R, Maekawa M, Toshimori K, Watanabe M, Iwanaga T. Immunohistochemical localization of GLUT3, MCT1, and MCT2 in the testes of mice and rats: The use of different energy sources in spermatogenesis. Biomed Res (Japan). 2015;36(4):225–34. 10.2220/biomedres.36.225. PubMed

Mannowetz N, Wandernoth P, Wennemuth G. Basigin interacts with both MCT1 and MCT2 in murine spermatozoa. J Cell Physiol. 2012;227(5):2154–62. 10.1002/jcp.22949. PubMed

Ramponi G, Manao G, Camici G. Nonenzymic acetylation of histones with acetyl phosphate and acetyl adenylate. Biochemistry. 1975;14(12):2681–5. 10.1021/bi00683a018. PubMed

Eshun-Wilson L, Zhang R, Portran D, Nachury MV, Toso DB, Löhr T, Vendruscolo M, Bonomi M, Fraser JS, Nogales E. Effects of α-tubulin acetylation on microtubule structure and stability. Proc Natl Acad Sci USA. 2019;116(21):10366–71. 10.1073/pnas.1900441116. PubMed PMC

Piperno G, LeDizet M, Chang XJ. Microtubules containing acetylated alpha-tubulin in mammalian cells in culture. J Cell Biol. 1987;104(2):289–302. 10.1083/jcb.104.2.289. PubMed PMC

Tsujimoto Y, Shimizu S. The voltage-dependent anion channel: An essential player in apoptosis. Biochimie. 2002;84(2–3):187–93. 10.1016/S0300-9084(02)01370-6. PubMed

Waddell J, Banerjee A, Kristian T. Acetylation in mitochondria dynamics and neurodegeneration. Cells. 2021;10(11):1–17. 10.3390/cells10113031. PubMed PMC

Wagner GR, Payne RM. Widespread and enzyme-independent Nε-acetylation and Nε-succinylation of proteins in the chemical conditions of the mitochondrial matrix. J Biol Chem. 2013;288(40):29036–45. 10.1074/jbc.M113.486753. PubMed PMC

Gnad F, Forner F, Zielinska DF, Birney E, Gunawardena J, Mann M. Evolutionary constraints of phosphorylation in eukaryotes, prokaryotes, and mitochondria. Mol Cell Proteom. 2010;9(12):2642–53. 10.1074/mcp.M110.001594. PubMed PMC

Weinert BT, Wagner SA, Horn H, Henriksen P, Liu WR, Olsen JV, Jensen LJ, Choudhary C. Proteome-wide mapping of the Drosophila acetylome demonstrates a high degree of conservation of lysine acetylation. Sci Signal. 2011;4(183):48. 10.1126/scisignal.2001902. PubMed

Swietach P, Vaughan-Jones RD, Harris AL, Hulikova A. The chemistry, physiology and pathology of pH in cancer. Philos Trans R Soc B Biol Sci. 2014;369(1638):20130099. 10.1098/rstb.2013.0099. PubMed PMC