Sperm capacitation, the ultimate maturation event preparing mammalian spermatozoa for fertilization, was first described in 1951, yet its regulatory mechanisms remain poorly understood. The capacitation process encompasses an influx of bicarbonate and calcium ions, removal of decapacitating factors, changes of pH and sperm proteasomal activities, and the increased protein tyrosine phosphorylation. Here, we document a novel biological phenomenon of a unique zinc (Zn2+) ion redistribution associated with mammalian sperm in vitro capacitation (IVC). Using image-based flow cytometry (IBFC), we identified four distinct types of sperm zinc ion distribution patterns (further zinc signature) and their changes during IVC. The zinc signature was altered after sperm capacitation, reduced by proteasomal inhibitors, removed by zinc chelators, and maintained with addition of external ZnCl2. These findings represent a fundamental shift in the understanding of mammalian fertilization, paving the way for improved semen analysis, in vitro fertilization (IVF), and artificial insemination (AI).

Department of Obstetrics Gynecology and Women's Health University of Missouri Columbia MO 65211 5300 USA

Division of Animal Sciences University of Missouri Columbia MO 65211 5300 USA

Laboratory of Reproductive Biology Institute of Biotechnology Czech Academy of Sciences v v i 25242 Vestec Czech Republic

Zobrazit více v PubMed

Que EL, et al. Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks. Nat. Chem. 2015;7:130–139. doi: 10.1038/nchem.2133. PubMed DOI PMC

Zhang N, Duncan FE, Que EL, O’Halloran TV, Woodruff TK. The fertilization-induced zinc spark is a novel biomarker of mouse embryo quality and early development. Sci. Rep. 2016;6:22772. doi: 10.1038/srep22772. PubMed DOI PMC

Lishko PV, Botchkina IL, Fedorenko A, Kirichok Y. Acid extrusion from human spermatozoa is mediated by flagellar voltage-gated proton channel. Cell. 2010;140:327–337. doi: 10.1016/j.cell.2009.12.053. PubMed DOI

Tardif S, Dube C, Bailey JL. Porcine sperm capacitation and tyrosine kinase activity are dependent on bicarbonate and calcium but protein tyrosine phosphorylation is only associated with calcium. Biol. Reprod. 2003;68:207–213. doi: 10.1095/biolreprod.102.005082. PubMed DOI

Boerke A, Tsai PS, Garcia-Gil N, Brewis IA, Gadella BM. Capacitation-dependent reorganization of microdomains in the apical sperm head plasma membrane: functional relationship with zona binding and the zona-induced acrosome reaction. Theriogenology. 2008;70:1188–1196. doi: 10.1016/j.theriogenology.2008.06.021. PubMed DOI

Sutovsky P. Sperm proteasome and fertilization. Reproduction. 2011;142:1–14. doi: 10.1530/REP-11-0041. PubMed DOI

Kerns K, Morales P, Sutovsky P. Regulation of sperm capacitation by the 26S proteasome: an emerging new paradigm in spermatology. Biol. Reprod. 2016;94:117. doi: 10.1095/biolreprod.115.136622. PubMed DOI

Hillman P, Ickowicz D, Vizel R, Breitbart H. Dissociation between AKAP3 and PKARII promotes AKAP3 degradation in sperm capacitation. PLoS ONE. 2013;8:e68873. doi: 10.1371/journal.pone.0068873. PubMed DOI PMC

Yi YJ, et al. Ubiquitin-activating enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm-egg coat penetration during porcine fertilization. Int. J. Androl. 2012;35:196–210. doi: 10.1111/j.1365-2605.2011.01217.x. PubMed DOI

Jaiswal, B. S. & Eisenbach, M. in Fertilization (ed Daniel M. Hardy) Ch. 3, 57–117 (Academic Press, San Diego, 2002).

Zimmerman SW, et al. Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization. PLoS ONE. 2011;6:e17256. doi: 10.1371/journal.pone.0017256. PubMed DOI PMC

Yi YJ, et al. Sperm-surface ATP in boar spermatozoa is required for fertilization: relevance to sperm proteasomal function. Syst. Biol. Reprod. Med. 2009;55:85–96. doi: 10.1080/19396360802699074. PubMed DOI

Miles EL, et al. Transgenic pig carrying green fluorescent proteasomes. Proc. Natl Acad. Sci. USA. 2013;110:6334–6339. doi: 10.1073/pnas.1220910110. PubMed DOI PMC

Stoltenberg M, Sørensen M, Danscher G. Histochemical demonstration of zinc ions in ejaculated human semen. Int. J. Androl. 1997;20:229–236. doi: 10.1046/j.1365-2605.1997.00060.x. PubMed DOI

Zimmerman SW, et al. Identification and characterization of RING-finger ubiquitin ligase UBR7 in mammalian spermatozoa. Cell Tissue Res. 2014;356:261–278. doi: 10.1007/s00441-014-1808-x. PubMed DOI

Bianchi F, Rousseaux-Prevost R, Sautiere P, Rousseaux J. P2 protamines from human sperm are zinc -finger proteins with one CYS2/HIS2 motif. Biochem. Biophys. Res. Commun. 1992;182:540–547. doi: 10.1016/0006-291X(92)91766-J. PubMed DOI

Nanassy L, Liu L, Griffin J, Carrell DT. The clinical utility of the protamine 1/protamine 2 ratio in sperm. Protein Pept. Lett. 2011;18:772–777. doi: 10.2174/092986611795713934. PubMed DOI

Ambroggio XI, Rees DC, Deshaies RJ. JAMM: a metalloprotease-like zinc site in the proteasome and signalosome. PLoS Biol. 2003;2:e2. doi: 10.1371/journal.pbio.0020002. PubMed DOI PMC

Nagase H, Woessner JF., Jr. Matrix metalloproteinases. J. Biol. Chem. 1999;274:21491–21494. doi: 10.1074/jbc.274.31.21491. PubMed DOI

Shimokawa Ki K, et al. Matrix metalloproteinase (MMP)-2 and MMP-9 activities in human seminal plasma. Mol. Hum. Reprod. 2002;8:32–36. doi: 10.1093/molehr/8.1.32. PubMed DOI

Beek J, Nauwynck H, Maes D, Van Soom A. Inhibitors of zinc-dependent metalloproteases hinder sperm passage through the cumulus oophorus during porcine fertilization in vitro. Reproduction. 2012;144:687–697. doi: 10.1530/REP-12-0311. PubMed DOI

Bjorndahl L, Kvist U. Human sperm chromatin stabilization: a proposed model including zinc bridges. Mol. Hum. Reprod. 2010;16:23–29. doi: 10.1093/molehr/gap099. PubMed DOI

Kvist U. Importance of spermatozoal zinc as temporary inhibitor of sperm nuclear chromatin decondensation ability in man. Acta Physiol. Scand. 1980;109:79–84. doi: 10.1111/j.1748-1716.1980.tb06567.x. PubMed DOI

Holt WV, North RD. Thermotropic phase transitions in the plasma membrane of ram spermatozoa. J. Reprod. Fertil. 1986;78:447–457. doi: 10.1530/jrf.0.0780447. PubMed DOI

Qi H, et al. All four CatSper ion channel proteins are required for male fertility and sperm cell hyperactivated motility. Proc. Natl Acad. Sci. USA. 2007;104:1219–1223. doi: 10.1073/pnas.0610286104. PubMed DOI PMC

Buffone MG, Calamera JC, Verstraeten SV, Doncel GF. Capacitation-associated protein tyrosine phosphorylation and membrane fluidity changes are impaired in the spermatozoa of asthenozoospermic patients. Reproduction. 2005;129:697–705. doi: 10.1530/rep.1.00584. PubMed DOI

Suarez SS. Regulation of sperm storage and movement in the mammalian oviduct. Int. J. Dev. Biol. 2008;52:455–462. doi: 10.1387/ijdb.072527ss. PubMed DOI

Leemans B, et al. An alkaline follicular fluid fraction induces capacitation and limited release of oviduct epithelium-bound stallion sperm. Reproduction. 2015;150:193–208. doi: 10.1530/REP-15-0178. PubMed DOI

Giojalas LC, Rovasio RA, Fabro G, Gakamsky A, Eisenbach M. Timing of sperm capacitation appears to be programmed according to egg availability in the female genital tract. Fertil. Steril. 2004;82:247–249. doi: 10.1016/j.fertnstert.2003.11.046. PubMed DOI

Andreychenko SV, et al. Post-chornobyl remote radiation effects on human sperm and seminal plasma characteristics. Exp. Oncol. 2016;38:245–251. PubMed

Nenkova, G., Petrov, L. & Alexandrova, A. Role of trace elements for oxidative status and quality of human sperm. Balkan Med. J. 34, 343–348 (2017). PubMed PMC

Colagar AH, Marzony ET, Chaichi MJ. Zinc levels in seminal plasma are associated with sperm quality in fertile and infertile men. Nutr. Res. 2009;29:82–88. doi: 10.1016/j.nutres.2008.11.007. PubMed DOI

Zhao, J. et al. Zinc levels in seminal plasma and their correlation with male infertility: a systematic review and meta-analysis. Sci. Rep. 6, 22386 (2016). PubMed PMC

Harrison RA, Dott HM, Foster GC. Effect of ionic strength, serum albumin and other macromolecules on the maintenance of motility and the surface of mammalian spermatozoa in a simple medium. J. Reprod. Fertil. 1978;52:65–73. doi: 10.1530/jrf.0.0520065. PubMed DOI

Beyersmann D, Haase H. Functions of zinc in signaling, proliferation and differentiation of mammalian cells. Biometals. 2001;14:331–341. doi: 10.1023/A:1012905406548. PubMed DOI

Kim I, et al. Pyrrolidine dithiocarbamate and zinc inhibit proteasome-dependent proteolysis. Exp. Cell Res. 2004;298:229–238. doi: 10.1016/j.yexcr.2004.04.017. PubMed DOI

Massanyi P, et al. Concentration of copper, iron, zinc, cadmium, lead, and nickel in boar semen and relation to the spermatozoa quality. J. Environ. Sci. Health A Tox Hazard. Subst. Environ. Eng. 2003;38:2643–2651. doi: 10.1081/ESE-120024453. PubMed DOI

Mogielnicka-Brzozowska M, Wysocki P, Strzezek J, Kordan W. Zinc-binding proteins from boar seminal plasma -- isolation, biochemical characteristics and influence on spermatozoa stored at 4 °C. Acta Biochim. Pol. 2011;58:171–177. PubMed

Henkel R, Bittner J, Weber R, Hüther F, Miska W. Relevance of zinc in human sperm flagella and its relation to motility. Fertil. Steril. 1999;71:1138–1143. doi: 10.1016/S0015-0282(99)00141-7. PubMed DOI

Sørensen MB, Stoltenberg M, Danscher G, Ernst E. Chelation of intracellular zinc ions affects human sperm cell motility. Mol. Hum. Reprod. 1999;5:338–341. doi: 10.1093/molehr/5.4.338. PubMed DOI

Que EL, et al. Zinc sparks induce physiochemical changes in the egg zona pellucida that prevent polyspermy. Integr. Biol. 2017;9:135–144. doi: 10.1039/C6IB00212A. PubMed DOI PMC

Guidobaldi HA, et al. Sperm chemorepulsion, a supplementary mechanism to regulate fertilization. Hum. Reprod. 2017;32:1560–1573. doi: 10.1093/humrep/dex232. PubMed DOI

Ferrer M, et al. MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization. Cell Tissue Res. 2012;349:881–895. doi: 10.1007/s00441-012-1429-1. PubMed DOI PMC

Steven FS, Griffin MM, Chantler EN. Inhibition of human and bovine sperm acrosin by divalent metal ions. Possible role of zinc as a regulator of acrosin activity. Int. J. Androl. 1982;5:401–412. doi: 10.1111/j.1365-2605.1982.tb00270.x. PubMed DOI

Ciereszko A, Dabrowski K, Mims SD, Glogowski J. Characteristics of sperm acrosin-like activity of paddlefish (Polyodon spathula Walbaum) Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2000;125:197–203. doi: 10.1016/S0305-0491(99)00167-4. PubMed DOI

Backstrom JR, Miller CA, Tokes ZA. Characterization of neutral proteinases from Alzheimer-affected and control brain specimens: identification of calcium-dependent metalloproteinases from the hippocampus. J. Neurochem. 1992;58:983–992. doi: 10.1111/j.1471-4159.1992.tb09352.x. PubMed DOI

Stephenson JL, Brackett BG. Influences of zinc on fertilisation and development of bovine oocytes in vitro. Zygote. 1999;7:195–201. doi: 10.1017/S096719949900057X. PubMed DOI

Noguchi S, Nakano M. Structure of the acidic N-linked carbohydrate chains of the 55-kDa glycoprotein family (PZP3) from porcine zona pellucida. Eur. J. Biochem. 1992;209:883–894. doi: 10.1111/j.1432-1033.1992.tb17361.x. PubMed DOI

Yurewicz EC, Pack BA, Sacco AG. Isolation, composition, and biological activity of sugar chains of porcine oocyte zona pellucida 55K glycoproteins. Mol. Reprod. Dev. 1991;30:126–134. doi: 10.1002/mrd.1080300209. PubMed DOI

Noguchi S, Hatanaka Y, Tobita T, Nakano M. Structural analysis of the N-linked carbohydrate chains of the 55-kDa glycoprotein family (PZP3) from porcine zona pellucida. Eur. J. Biochem. 1992;204:1089–1100. doi: 10.1111/j.1432-1033.1992.tb16733.x. PubMed DOI

Amari S, et al. Essential role of the nonreducing terminal alpha-mannosyl residues of the N-linked carbohydrate chain of bovine zona pellucida glycoproteins in sperm-egg binding. Mol. Reprod. Dev. 2001;59:221–226. doi: 10.1002/mrd.1026. PubMed DOI

Flesch FM, Colenbrander B, van Golde LMG, Gadella BM. Capacitation induces tyrosine phosphorylation of proteins in the boar sperm plasma membrane. Biochem. Biophys. Res. Commun. 1999;262:787–792. doi: 10.1006/bbrc.1999.1300. PubMed DOI

Tardif S, Dube C, Chevalier S, Bailey JL. Capacitation is associated with tyrosine phosphorylation and tyrosine kinase-like activity of pig sperm proteins. Biol. Reprod. 2001;65:784–792. doi: 10.1095/biolreprod65.3.784. PubMed DOI

Dube C, Tardif S, LeClerc P, Bailey JL. The importance of calcium in the appearance of p32, a boar sperm tyrosine phosphoprotein, during in vitro capacitation. J. Androl. 2003;24:727–733. doi: 10.1002/j.1939-4640.2003.tb02734.x. PubMed DOI

Kennedy CE, et al. Protein expression pattern of PAWP in bull spermatozoa is associated with sperm quality and fertility following artificial insemination. Mol. Reprod. Dev. 2014;81:436–449. doi: 10.1002/mrd.22309. PubMed DOI

Bottom-up approach to deciphering the targets of the ubiquitin-proteasome system in porcine sperm capacitation

The Ubiquitin-Proteasome System Does Not Regulate the Degradation of Porcine β-Microseminoprotein during Sperm Capacitation

Perfect date-the review of current research into molecular bases of mammalian fertilization

Fluorescent analysis of boar sperm capacitation process in vitro

Compartmentalization of the proteasome-interacting proteins during sperm capacitation