BACKGROUND: Hydrogen sulfide (H2S) donors are crucial tools not only for understanding the role of H2S in cellular function but also as promising therapeutic agents for oxidative stress-related diseases. This study aimed to explore the effect of amino acid-derived N-thiocarboxyanhydrides (NTAs), which release physiological H2S levels in the presence of carbonic anhydrase, on porcine sperm function during short-term incubation with and without induced oxidative stress. For this purpose, we employed two H2S-releasing NTAs with release half-lives (t1/2) in the range of hours that derived from the amino acids glycine (Gly-NTA) or leucine (Leu-NTA). Because carbonic anhydrase is crucial for H2S release from NTAs, we first measured the activity of this enzyme in the porcine ejaculate. Then, we tested the effect of Gly- and Leu-NTAs at 10 and 1 nM on sperm mitochondrial activity, plasma membrane integrity, acrosomal status, motility, motile subpopulations, and redox balance during short-term incubation at 38 °C with and without a reactive oxygen species (ROS)-generating system. RESULTS: Our results show that carbonic anhydrase is found both in spermatozoa and seminal plasma, with activity notably higher in the latter. Both Gly- and Leu-NTAs did not exert any noxious effects, but they enhanced sperm mitochondrial activity in the presence and absence of oxidative stress. Moreover, NTAs (except for Leu-NTA 10 nM) tended to preserve the sperm redox balance against the injuries provoked by oxidative stress, which provide further support to the antioxidant effect of H2S on sperm function. Both compounds also increased progressive motility over short-term incubation, which may translate into prolonged sperm survival. CONCLUSIONS: The presence of carbonic anhydrase activity in mammalian spermatozoa makes NTAs promising molecules to investigate the role of H2S in sperm biology. For the first time, beneficial effects of NTAs on mitochondrial activity have been found in mammalian cells in the presence and absence of oxidative stress. NTAs are interesting compounds to investigate the role of H2S in sperm mitochondria-dependent events and to develop H2S-related therapeutic protocols against oxidative stress in assisted reproductive technologies.

Departamento de Fisiología Facultad de Veterinaria Campus de Excelencia Internacional Mare Nostrum Universidad de Murcia 30100 Murcia Spain

Department of Chemistry Virginia Tech Center for Drug Discovery and Macromolecules Innovation Institute Virginia Tech Blacksburg VA 24061 USA

Department of Food Science Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague 16500 Prague Czech Republic

Department of Veterinary Sciences Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague 16500 Prague Czech Republic

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BMC Vet Res. 2023 May 10;19(1):71. doi: 10.1186/s12917-023-03624-1. PubMed

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BMC Vet Res. 2023 May 23;19(1):73. doi: 10.1186/s12917-023-03626-z. PubMed

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Wang R. Physiological implications of hydrogen sulfide: a whiff exploration that blossomed. Physiol Rev. 2012;92:791–896. doi: 10.1152/physrev.00017.2011. PubMed DOI

Cirino G, Szabo C, Papapetropoulos A. Physiological roles of hydrogen sulfide in mammalian cells, tissues, and organs. Physiol. Rev. 2023;103:31–276. 10.1152/physrev.00028.2021. PubMed

Otasevic V, Stancic A, Korac A, Jankovic A, Korac B. Reactive oxygen, nitrogen, and sulfur species in human male fertility. A crossroad of cellular signaling and pathology. BioFactors. 2020;46:206–19. doi: 10.1002/biof.1535. PubMed DOI

Kadlec M, Ros-Santaella JL, Pintus E. The roles of NO and H2S in sperm biology: Recent advances and new perspectives. Int J Mol Sci. 2020;21:2174. doi: 10.3390/ijms21062174. PubMed DOI PMC

Wang J, Wang W, Li S, Han Y, Zhang P, Meng G, et al. Hydrogen sulfide as a potential target in preventing spermatogenic failure and testicular dysfunction. Antioxid Redox Signal. 2018;2018(28):1447–1462. doi: 10.1089/ars.2016.6968. PubMed DOI

Pintus E, Jovičić M, Kadlec M, Ros-Santaella JL. Divergent effect of fast- and slow-releasing H2S donors on boar spermatozoa under oxidative stress. Sci Rep. 2020;10:6508. doi: 10.1038/s41598-020-63489-4. PubMed DOI PMC

Kadlec M, Pintus E, Ros-Santaella JL. The Interaction of NO and H2S in Boar Spermatozoa under Oxidative Stress. Animals. 2022;12:602. doi: 10.3390/ani12050602. PubMed DOI PMC

Evans EPP, Scholten JTM, Mzyk A, Reyes-San-Martin C, Llumbet AE, Hamoh T, et al. Male subfertility and oxidative stress. Redox Biol. 2021;46:102071. doi: 10.1016/j.redox.2021.102071. PubMed DOI PMC

Pintus E, Ros-Santaella JL. Impact of oxidative stress on male reproduction in domestic and wild animals. Antioxidants. 2021;10:1154. doi: 10.3390/antiox10071154. PubMed DOI PMC

Powell CR, Dillon KM, Matson JB. A review of hydrogen sulfide (H2S) donors: Chemistry and potential therapeutic applications. Biochem Pharmacol. 2018;149:110–123. doi: 10.1016/j.bcp.2017.11.014. PubMed DOI PMC

Levinn CM, Cerda MM, Pluth MD. Activatable Small-Molecule H2S Donors. Antioxid Redox Sign. 2019;32:96–109. 10.1089/ars.2019.7841. PubMed PMC

Magli E, Perissutti E, Santagada V, Caliendo G, Corvino A, Esposito G, et al. H2S donors and their use in medicinal chemistry. Biomolecules. 2021;11:1899. doi: 10.3390/biom11121899. PubMed DOI PMC

Song ZL, Zhao L, Ma T, Osama A, Shen T, He Y, et al. Progress and perspective on hydrogen sulfide donors and their biomedical applications. Med. Res. Rev. 2022;42:1–48. 10.1002/med.21913 PubMed

Rose P, Dymock BW, Moore PK. “GYY4137, a novel water-soluble, H2S-releasing molecule” in. Methods in Enzymology. Cadenas E & Packer L, editors. (Elsevier); 2015. p. 554:143–167. 10.1016/bs.mie.2014.11.014. PubMed

Powell CR, Foster JC, Okyere B, Theus MH, Matson JB. Therapeutic delivery of H2S via COS: small molecule and polymeric donors with benign byproducts. J Am Chem Soc. 2016;138:13477–13480. doi: 10.1021/jacs.6b07204. PubMed DOI PMC

Powell CR, Kaur K, Dillon KM, Zhou M, Alaboalirat M, Matson JB. Functional N-Substituted N-Thiocarboxyanhydrides as Modular Tools for Constructing H2S Donor Conjugates. ACS Chem Biol. 2019;14:1129–34. 10.1021/acschembio.9b00248. PubMed PMC

Kaur K, Enders P, Zhu Y, Bratton AF, Powell CR, Kashfi K, et al. Amino acid-based H2S donors: N-thiocarboxyanhydrides that release H2S with innocuous byproducts. Chem Commun. 2021;57:5522. 10.1039/d1cc01309b. PubMed PMC

Steiger AK, Zhao Y, Pluth MD. Emerging roles of carbonyl sulfide in chemical biology: sulfide transporter or gasotransmitter? Antioxid Redox Signal. 2018;2018(28):1516–1532. doi: 10.1089/ars.2017.7119. PubMed DOI PMC

Zhao Y, et al. Hydrogen sulfide and/or ammonia reduces spermatozoa motility through AMPK/AKT related pathways. Sci Rep. 2016;6:37884. doi: 10.1038/srep37884. PubMed DOI PMC

Řimnáčová H, Moravec J, Štiavnická M, Havránková J, Hošek P, Prokešová Š, et al. Evidence of endogenously produced hydrogen sulfide (H2S) and 1 persulfidation in male reproduction. Sci Rep. 2022;12:11426. doi: 10.1038/s41598-022-15360-x. PubMed DOI PMC

Nishita T, Itoh S, Arai S, Ichihara N, Arishima K. Measurement of carbonic anhydrase isozyme VI (CA-VI) in swine sera, colostrums, saliva, bile, seminal plasma and tissues. Anim Sci J. 2011;82:673–678. doi: 10.1111/j.1740-0929.2011.00888.x. PubMed DOI

Zigo M, Kerns K, Sen S, Essien C, Oko R, Xu D, et al. Zinc is a master-regulator of sperm function associated with binding, motility, and metabolic modulation during porcine sperm capacitation. Commun Biol. 2022;5:1–12. doi: 10.1038/s42003-022-03485-8. PubMed DOI PMC

José O, Torres-Rodríguez P, Forero-Quintero LS, Chávez JC, De La Vega-Beltrán JL, Carta F, et al. Carbonic anhydrases and their functional differences in human and mouse sperm physiology. Biochem Biophys Res Commun. 2015;468:713–718. doi: 10.1016/j.bbrc.2015.11.021. PubMed DOI

Wandernoth PM, Mannowetz N, Szczyrba J, Grannemann L, Wolf A, Becker HM, et al. Normal fertility requires the expression of carbonic anhydrases II and IV in sperm. J Biol Chem. 2015;290:29202–29216. doi: 10.1074/jbc.M115.698597. PubMed DOI PMC

Studer SM, Orens JB, Rosas I, Krishnan JA, Cope KA, Yang S, et al. Patterns and significance of exhaled-breath biomarkers in lung transplant recipients with acute allograft rejection. J Heart Lung Transpl. 2001;20:1158–1166. doi: 10.1016/S1053-2498(01)00343-6. PubMed DOI

Balazy M, Abu-Yousef IA, Harpp DN, Park J. Identification of carbonyl sulfide and sulfur dioxide in porcine coronary artery by gas chromatography/mass spectrometry, possible relevance to EDHF. Biochem Biophys Res Commun. 2003;311:728–734. doi: 10.1016/j.bbrc.2003.10.055. PubMed DOI

Steiger AK, Marcatti M, Szabo C, Szczesny B, Pluth MD. Inhibition of Mitochondrial Bioenergetics by Esterase-Triggered COS/H2S Donors. ACS Chem Biol. 2017;12:2117–2123. doi: 10.1021/acschembio.7b00279. PubMed DOI PMC

Módis K, Bos EM, Calzia E, van Goor H, Coletta C, Papapetropoulos A, et al. Regulation of mitochondrial bioenergetic function by hydrogen sulfide. Part II. Pathophysiological and therapeutic aspects. Br J Pharmacol. 2014;171:2123–46. 10.1111/bph.12368. PubMed PMC

Szabo C, Murghes B, Andriamihaja M, Bouillaud F, Módis K, Olah G, et al. Regulation of mitochondrial bioenergetic function by hydrogen sulfide. Part I. Biochemical and physiological mechanisms. Br. J. Pharmacol. 2014;171:2099–122. doi: 10.1111/bph.12369. PubMed DOI PMC

Borisov VB, Forte E. Impact of hydrogen sulfide on mitochondrial and bacterial bioenergetics. Int J Mol Sci. 2021;22:12688. 10.3390/ijms222312688. PubMed PMC

Storey BT. Mammalian sperm metabolism: oxygen and sugar, friend and foe. Int J Dev Biol. 2008;52:427–437. doi: 10.1387/ijdb.072522bs. PubMed DOI

du Plessis SS, Agarwal A, Mohanty G, van der Linde M. Oxidative phosphorylation versus glycolysis: what fuel do spermatozoa use? Asian J Androl. 2015;17:230–235. doi: 10.4103/1008-682X.135123. PubMed DOI PMC

Amaral A. Energy metabolism in mammalian sperm motility. WIREs Mech. Dis. 2022;114:e1569. 10.1002/wsbm.1569. PubMed

Rodríguez-Gil JE, Bonet S. Current knowledge on boar sperm metabolism: Comparison with other mammalian species. Theriogenology. 2016;85:4–11. doi: 10.1016/j.theriogenology.2015.05.005. PubMed DOI

Nesci S, Spinaci M, Galeati G, Nerozzi C, Pagliarani A, Algieri C, et al. Sperm function and mitochondrial activity: An insight on boar sperm metabolism. Theriogenology. 2020;144:82–88. doi: 10.1016/j.theriogenology.2020.01.004. PubMed DOI

Moraes CR, Meyers S. The sperm mitochondrion: Organelle of many functions. Anim Reprod Sci. 2018;194:71–80. doi: 10.1016/j.anireprosci.2018.03.024. PubMed DOI

Durairajanayagam D, Singh D, Agarwal A, Henkel R. Causes and consequences of sperm mitochondrial dysfunction. Andrologia. 2021;53:e13666. doi: 10.1111/and.13666. PubMed DOI

Guthrie HD, Welch GR, Long JA. Mitochondrial function and reactive oxygen species action in relation to boar motility. Theriogenology. 2008;70:1209–1215. doi: 10.1016/j.theriogenology.2008.06.017. PubMed DOI

Jang HY, Kim YH, Kim BW, Park IC, Cheong HT, Kim JT, et al. Ameliorative effects of melatonin against hydrogen peroxide-induced oxidative stress on boar sperm characteristics and subsequent in vitro embryo development. Reprod Domest Anim. 2010;45:943–950. doi: 10.1111/j.1439-0531.2009.01466.x. PubMed DOI

Fukuzawa K, Saitoh Y, Akai K, Kogure K, Ueno S, Tokumura A, et al. Antioxidant effect of bovine serum albumin on membrane lipid peroxidation induced by iron chelate and superoxide. Biochim Biophys Acta. 2005;1668:145–155. doi: 10.1016/j.bbamem.2004.12.006. PubMed DOI

Panner Selvam MK, Agarwal A, Henkel R, Finelli R, Robert KA, Iovine C, et al. The effect of oxidative and reductive stress on semen parameters and functions of physiologically normal human spermatozoa. Free Radic Biol Med. 2020;152:375–385. doi: 10.1016/j.freeradbiomed.2020.03.008. PubMed DOI

Agarwal A, Henkel R, Sharma R, Tadros NN, Sabanegh E. Determination of seminal oxidation–reduction potential (ORP) as an easy and cost-effective clinical marker of male infertility. Andrologia. 2018;50:e12914. doi: 10.1111/and.12914. PubMed DOI

Arafa M, Henkel R, Agarwal A, Majzoub A, Elbardisi H. Correlation of oxidation–reduction potential with hormones, semen parameters and testicular volume. Andrologia. 2019;51:1–7. doi: 10.1111/and.13258. PubMed DOI

Panner Selvam MK, Finelli R, Agarwal A, Henkel R. Evaluation of seminal oxidation–reduction potential in male infertility. Andrologia. 2021;53:e13610. doi: 10.1111/and.13610. PubMed DOI

Agarwal A, Roychoudhury S, Bjugstad KB, Cho CL. Oxidation-reduction potential of semen: What is its role in the treatment of male infertility? Ther Adv Urol. 2016;8:302–318. doi: 10.1177/1756287216652779. PubMed DOI PMC

Kimura Y, Goto Y-I, Kimura H. Hydrogen sulfide increases glutathione production and suppresses oxidative stress in mitochondria. Antioxid Redox Signal. 2010;12:1–13. doi: 10.1089/ars.2008.2282. PubMed DOI

Szczesny B, Módis K, Yanagi K, Coletta C, Le Trionnaire S, Perry A, et al. AP39, a novel mitochondria-targeted hydrogen sulfide donor, stimulates cellular bioenergetics, exerts cytoprotective effects and protects against the loss of mitochondrial DNA integrity in oxidatively stressed endothelial cells in vitro. Nitric Oxide. 2014;41:120–30. doi: 10.1016/j.niox.2014.04.008. PubMed DOI PMC

Wang Y, Shi S, Dong S, Wu J, Song M, Zhong X, et al. Sodium hydrosulfide attenuates hyperhomocysteinemia rat myocardial injury through cardiac mitochondrial protection. Mol Cell Biochem. 2015;399:189–200. doi: 10.1007/s11010-014-2245-6. PubMed DOI

Chauhan P, Gupta K, Ravikumar G, Saini DK, Chakrapani H. Carbonyl Sulfide (COS) Donor Induced Protein Persulfidation Protects against Oxidative Stress. Chem Asian J. 2019;14:4717–4724. doi: 10.1002/asia.201901148. PubMed DOI

Kimura H. Signalling by hydrogen sulfide and polysulfides via protein S-sulfuration. Br J Pharmacol. 2020;177:720–33. 10.1111/bph.14579. PubMed PMC

Peng B, Chen W, Liu C, Rosser EW, Pacheco A, Zhao Y, et al. Fluorescent probes based on nucleophilic substitution-cyclization for hydrogen sulfide detection and bioimaging. Chemistry. 2014;20:1010–1016. doi: 10.1002/chem.201303757. PubMed DOI PMC

Pursel VG, Johnson LA. Freezing of boar spermatozoa: fertilizing capacity with concentrated semen and a new thawing procedure. J Anim Sci. 1975;40:99–102. doi: 10.2527/jas1975.40199x. PubMed DOI

Waberski D, Luther AM, Grünther B, Jäkel H, Henning H, Vogel C, et al. Sperm function in vitro and fertility after antibiotic-free, hypothermic storage of liquid preserved boar semen. Sci Rep. 2019;9:14748. doi: 10.1038/s41598-019-51319-1. PubMed DOI PMC

Thurman AC, Davis JL, Jan M, McCulloch CE, Buelow BD. Development and validation of an app-based cell counter for use in the clinical laboratory setting. J Pathol Inform. 2015;6:2. doi: 10.4103/2153-3539.150252. PubMed DOI PMC

Agarwal A, Gupta S, Sharma R. “Oxidation-Reduction Potential Measurement in Ejaculated Semen Samples” in Andrological Evaluation of Male Infertility, eds. Agarwal A et al.. Publishing Switzerland: Springer International; 2016.

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Correction: N-thiocarboxyanhydrides, amino acid-derived enzyme-activated H2S donors, enhance sperm mitochondrial activity in presence and absence of oxidative stress

Correction to: N-thiocarboxyanhydrides, amino acid-derived enzyme-activated H2S donors, enhance sperm mitochondrial activity in presence and absence of oxidative stress