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. 1996 Aug 1;134(3):637–645. doi: 10.1083/jcb.134.3.637

ZP3-dependent activation of sperm cation channels regulates acrosomal secretion during mammalian fertilization

PMCID: PMC2120933  PMID: 8707844

Abstract

The sperm acrosome reaction is a Ca(2+)-dependent secretory event required for fertilization. Adhesion to the egg's zona pellucida promotes Ca2+ influx through voltage-sensitive channels, thereby initiating secretion. We used potentiometric fluorescent probes to determine the role of sperm membrane potential in regulating Ca2+ entry. ZP3, the glycoprotein agonist of the zona pellucida, depolarizes sperm membranes by activating a pertussis toxin-insensitive mechanism with the characteristics of a poorly selective cation channel. ZP3 also activates a pertussis toxin-sensitive pathway that produces a transient rise in internal pH. The concerted effects of depolarization and alkalinization open voltage-sensitive Ca2+ channels. These observations suggest that mammalian sperm utilize membrane potential-dependent signal transduction mechanisms and that a depolarization pathway is an upstream transducing element coupling adhesion to secretion during fertilization.

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Selected References

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  1. Arnoult C., Villaz M. Differential developmental fates of the two calcium currents in early embryos of the ascidian Ciona intestinalis. J Membr Biol. 1994 Jan;137(2):127–135. doi: 10.1007/BF00233482. [DOI] [PubMed] [Google Scholar]
  2. Babcock D. F., Pfeiffer D. R. Independent elevation of cytosolic [Ca2+] and pH of mammalian sperm by voltage-dependent and pH-sensitive mechanisms. J Biol Chem. 1987 Nov 5;262(31):15041–15047. [PubMed] [Google Scholar]
  3. Babcock D. F., Rufo G. A., Jr, Lardy H. A. Potassium-dependent increases in cytosolic pH stimulate metabolism and motility of mammalian sperm. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1327–1331. doi: 10.1073/pnas.80.5.1327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bailey J. L., Storey B. T. Calcium influx into mouse spermatozoa activated by solubilized mouse zona pellucida, monitored with the calcium fluorescent indicator, fluo-3. Inhibition of the influx by three inhibitors of the zona pellucida induced acrosome reaction: tyrphostin A48, pertussis toxin, and 3-quinuclidinyl benzilate. Mol Reprod Dev. 1994 Nov;39(3):297–308. doi: 10.1002/mrd.1080390307. [DOI] [PubMed] [Google Scholar]
  5. Beebe S. J., Leyton L., Burks D., Ishikawa M., Fuerst T., Dean J., Saling P. Recombinant mouse ZP3 inhibits sperm binding and induces the acrosome reaction. Dev Biol. 1992 May;151(1):48–54. doi: 10.1016/0012-1606(92)90212-y. [DOI] [PubMed] [Google Scholar]
  6. Benoff S., Cooper G. W., Hurley I., Mandel F. S., Rosenfeld D. L., Scholl G. M., Gilbert B. R., Hershlag A. The effect of calcium ion channel blockers on sperm fertilization potential. Fertil Steril. 1994 Sep;62(3):606–617. [PubMed] [Google Scholar]
  7. Blatz A. L. Asymmetric proton block of inward rectifier K channels in skeletal muscle. Pflugers Arch. 1984 Aug;401(4):402–407. doi: 10.1007/BF00584343. [DOI] [PubMed] [Google Scholar]
  8. Bleil J. D., Wassarman P. M. Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm. J Cell Biol. 1986 Apr;102(4):1363–1371. doi: 10.1083/jcb.102.4.1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bleil J. D., Wassarman P. M. Identification of a ZP3-binding protein on acrosome-intact mouse sperm by photoaffinity crosslinking. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5563–5567. doi: 10.1073/pnas.87.14.5563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bleil J. D., Wassarman P. M. Mammalian sperm-egg interaction: identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm. Cell. 1980 Jul;20(3):873–882. doi: 10.1016/0092-8674(80)90334-7. [DOI] [PubMed] [Google Scholar]
  11. Bleil J. D., Wassarman P. M. Sperm-egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein. Dev Biol. 1983 Feb;95(2):317–324. doi: 10.1016/0012-1606(83)90032-5. [DOI] [PubMed] [Google Scholar]
  12. Bleil J. D., Wassarman P. M. Structure and function of the zona pellucida: identification and characterization of the proteins of the mouse oocyte's zona pellucida. Dev Biol. 1980 Apr;76(1):185–202. doi: 10.1016/0012-1606(80)90371-1. [DOI] [PubMed] [Google Scholar]
  13. Christensen B. N., Hida E. Protonation of histidine groups inhibits gating of the quisqualate/kainate channel protein in isolated catfish cone horizontal cells. Neuron. 1990 Oct;5(4):471–478. doi: 10.1016/0896-6273(90)90086-u. [DOI] [PubMed] [Google Scholar]
  14. Clark E. N., Corron M. E., Florman H. M. Caltrin, the calcium transport regulatory peptide of spermatozoa, modulates acrosomal exocytosis in response to the egg's zona pellucida. J Biol Chem. 1993 Mar 5;268(7):5309–5316. [PubMed] [Google Scholar]
  15. Cook D. L., Ikeuchi M., Fujimoto W. Y. Lowering of pHi inhibits Ca2+-activated K+ channels in pancreatic B-cells. Nature. 1984 Sep 20;311(5983):269–271. doi: 10.1038/311269a0. [DOI] [PubMed] [Google Scholar]
  16. Cox T., Campbell P., Peterson R. N. Ion channels in boar sperm plasma membranes: characterization of a cation selective channel. Mol Reprod Dev. 1991 Oct;30(2):135–147. doi: 10.1002/mrd.1080300210. [DOI] [PubMed] [Google Scholar]
  17. Davies N. W., Standen N. B., Stanfield P. R. The effect of intracellular pH on ATP-dependent potassium channels of frog skeletal muscle. J Physiol. 1992 Jan;445:549–568. doi: 10.1113/jphysiol.1992.sp018939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dixon D. B., Takahashi K., Copenhagen D. R. L-glutamate suppresses HVA calcium current in catfish horizontal cells by raising intracellular proton concentration. Neuron. 1993 Aug;11(2):267–277. doi: 10.1016/0896-6273(93)90183-r. [DOI] [PubMed] [Google Scholar]
  19. Endo Y., Lee M. A., Kopf G. S. Characterization of an islet-activating protein-sensitive site in mouse sperm that is involved in the zona pellucida-induced acrosome reaction. Dev Biol. 1988 Sep;129(1):12–24. doi: 10.1016/0012-1606(88)90157-1. [DOI] [PubMed] [Google Scholar]
  20. Endo Y., Lee M. A., Kopf G. S. Evidence for the role of a guanine nucleotide-binding regulatory protein in the zona pellucida-induced mouse sperm acrosome reaction. Dev Biol. 1987 Jan;119(1):210–216. doi: 10.1016/0012-1606(87)90222-3. [DOI] [PubMed] [Google Scholar]
  21. Florman H. M., Corron M. E., Kim T. D., Babcock D. F. Activation of voltage-dependent calcium channels of mammalian sperm is required for zona pellucida-induced acrosomal exocytosis. Dev Biol. 1992 Aug;152(2):304–314. doi: 10.1016/0012-1606(92)90137-6. [DOI] [PubMed] [Google Scholar]
  22. Florman H. M., First N. L. The regulation of acrosomal exocytosis. I. Sperm capacitation is required for the induction of acrosome reactions by the bovine zona pellucida in vitro. Dev Biol. 1988 Aug;128(2):453–463. doi: 10.1016/0012-1606(88)90307-7. [DOI] [PubMed] [Google Scholar]
  23. Florman H. M. Sequential focal and global elevations of sperm intracellular Ca2+ are initiated by the zona pellucida during acrosomal exocytosis. Dev Biol. 1994 Sep;165(1):152–164. doi: 10.1006/dbio.1994.1242. [DOI] [PubMed] [Google Scholar]
  24. Florman H. M., Tombes R. M., First N. L., Babcock D. F. An adhesion-associated agonist from the zona pellucida activates G protein-promoted elevations of internal Ca2+ and pH that mediate mammalian sperm acrosomal exocytosis. Dev Biol. 1989 Sep;135(1):133–146. doi: 10.1016/0012-1606(89)90164-4. [DOI] [PubMed] [Google Scholar]
  25. Foresta C., Rossato M., Di Virgilio F. Ion fluxes through the progesterone-activated channel of the sperm plasma membrane. Biochem J. 1993 Aug 15;294(Pt 1):279–283. doi: 10.1042/bj2940279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gong X., Dubois D. H., Miller D. J., Shur B. D. Activation of a G protein complex by aggregation of beta-1,4-galactosyltransferase on the surface of sperm. Science. 1995 Sep 22;269(5231):1718–1721. doi: 10.1126/science.7569899. [DOI] [PubMed] [Google Scholar]
  27. Grantyn R., Lux H. D. Similarity and mutual exclusion of NMDA- and proton-activated transient Na+-currents in rat tectal neurons. Neurosci Lett. 1988 Jun 29;89(2):198–203. doi: 10.1016/0304-3940(88)90381-3. [DOI] [PubMed] [Google Scholar]
  28. Guerrero A., Darszon A. Evidence for the activation of two different Ca2+ channels during the egg jelly-induced acrosome reaction of sea urchin sperm. J Biol Chem. 1989 Nov 25;264(33):19593–19599. [PubMed] [Google Scholar]
  29. Haynes L. W. Block of the cyclic GMP-gated channel of vertebrate rod and cone photoreceptors by l-cis-diltiazem. J Gen Physiol. 1992 Nov;100(5):783–801. doi: 10.1085/jgp.100.5.783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hershlag A., Cooper G. W., Benoff S. Pregnancy following discontinuation of a calcium channel blocker in the male partner. Hum Reprod. 1995 Mar;10(3):599–606. doi: 10.1093/oxfordjournals.humrep.a135996. [DOI] [PubMed] [Google Scholar]
  31. Hinton B. T., Palladino M. A. Epididymal epithelium: its contribution to the formation of a luminal fluid microenvironment. Microsc Res Tech. 1995 Jan 1;30(1):67–81. doi: 10.1002/jemt.1070300106. [DOI] [PubMed] [Google Scholar]
  32. Kaibara M., Kameyama M. Inhibition of the calcium channel by intracellular protons in single ventricular myocytes of the guinea-pig. J Physiol. 1988 Sep;403:621–640. doi: 10.1113/jphysiol.1988.sp017268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kinloch R. A., Mortillo S., Stewart C. L., Wassarman P. M. Embryonal carcinoma cells transfected with ZP3 genes differentially glycosylate similar polypeptides and secrete active mouse sperm receptor. J Cell Biol. 1991 Nov;115(3):655–664. doi: 10.1083/jcb.115.3.655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Klöckner U., Isenberg G. Intracellular pH modulates the availability of vascular L-type Ca2+ channels. J Gen Physiol. 1994 Apr;103(4):647–663. doi: 10.1085/jgp.103.4.647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Laurido C., Candia S., Wolff D., Latorre R. Proton modulation of a Ca(2+)-activated K+ channel from rat skeletal muscle incorporated into planar bilayers. J Gen Physiol. 1991 Nov;98(5):1025–1042. doi: 10.1085/jgp.98.5.1025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lee M. A., Storey B. T. Endpoint of first stage of zona pellucida-induced acrosome reaction in mouse spermatozoa characterized by acrosomal H+ and Ca2+ permeability: population and single cell kinetics. Gamete Res. 1989 Nov;24(3):303–326. doi: 10.1002/mrd.1120240307. [DOI] [PubMed] [Google Scholar]
  37. Leyton L., LeGuen P., Bunch D., Saling P. M. Regulation of mouse gamete interaction by a sperm tyrosine kinase. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11692–11695. doi: 10.1073/pnas.89.24.11692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Leyton L., Saling P. 95 kd sperm proteins bind ZP3 and serve as tyrosine kinase substrates in response to zona binding. Cell. 1989 Jun 30;57(7):1123–1130. doi: 10.1016/0092-8674(89)90049-4. [DOI] [PubMed] [Google Scholar]
  39. McDonald T. F., Pelzer S., Trautwein W., Pelzer D. J. Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells. Physiol Rev. 1994 Apr;74(2):365–507. doi: 10.1152/physrev.1994.74.2.365. [DOI] [PubMed] [Google Scholar]
  40. Miller D. J., Gong X., Shur B. D. Sperm require beta-N-acetylglucosaminidase to penetrate through the egg zona pellucida. Development. 1993 Aug;118(4):1279–1289. doi: 10.1242/dev.118.4.1279. [DOI] [PubMed] [Google Scholar]
  41. Miller D. J., Macek M. B., Shur B. D. Complementarity between sperm surface beta-1,4-galactosyltransferase and egg-coat ZP3 mediates sperm-egg binding. Nature. 1992 Jun 18;357(6379):589–593. doi: 10.1038/357589a0. [DOI] [PubMed] [Google Scholar]
  42. Mohr F. C., Fewtrell C. IgE receptor-mediated depolarization of rat basophilic leukemia cells measured with the fluorescent probe bis-oxonol. J Immunol. 1987 Mar 1;138(5):1564–1570. [PubMed] [Google Scholar]
  43. Moller C. C., Bleil J. D., Kinloch R. A., Wassarman P. M. Structural and functional relationships between mouse and hamster zona pellucida glycoproteins. Dev Biol. 1990 Feb;137(2):276–286. doi: 10.1016/0012-1606(90)90254-g. [DOI] [PubMed] [Google Scholar]
  44. Moody W., Jr Effects of intracellular H+ on the electrical properties of excitable cells. Annu Rev Neurosci. 1984;7:257–278. doi: 10.1146/annurev.ne.07.030184.001353. [DOI] [PubMed] [Google Scholar]
  45. Mortillo S., Wassarman P. M. Differential binding of gold-labeled zona pellucida glycoproteins mZP2 and mZP3 to mouse sperm membrane compartments. Development. 1991 Sep;113(1):141–149. doi: 10.1242/dev.113.1.141. [DOI] [PubMed] [Google Scholar]
  46. Orlowski J., Kandasamy R. A., Shull G. E. Molecular cloning of putative members of the Na/H exchanger gene family. cDNA cloning, deduced amino acid sequence, and mRNA tissue expression of the rat Na/H exchanger NHE-1 and two structurally related proteins. J Biol Chem. 1992 May 5;267(13):9331–9339. [PubMed] [Google Scholar]
  47. Parkkila S., Rajaniemi H., Kellokumpu S. Polarized expression of a band 3-related protein in mammalian sperm cells. Biol Reprod. 1993 Aug;49(2):326–331. doi: 10.1095/biolreprod49.2.326. [DOI] [PubMed] [Google Scholar]
  48. Parrish J. J., Susko-Parrish J. L., First N. L. Capacitation of bovine sperm by heparin: inhibitory effect of glucose and role of intracellular pH. Biol Reprod. 1989 Oct;41(4):683–699. doi: 10.1095/biolreprod41.4.683. [DOI] [PubMed] [Google Scholar]
  49. Pietrobon D., Prod'hom B., Hess P. Interactions of protons with single open L-type calcium channels. pH dependence of proton-induced current fluctuations with Cs+, K+, and Na+ as permeant ions. J Gen Physiol. 1989 Jul;94(1):1–21. doi: 10.1085/jgp.94.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Roos A., Boron W. F. Intracellular pH. Physiol Rev. 1981 Apr;61(2):296–434. doi: 10.1152/physrev.1981.61.2.296. [DOI] [PubMed] [Google Scholar]
  51. Schoff P. K., Lardy H. A. Effects of fluoride and caffeine on the metabolism and motility of ejaculated bovine spermatozoa. Biol Reprod. 1987 Nov;37(4):1037–1046. doi: 10.1095/biolreprod37.4.1037. [DOI] [PubMed] [Google Scholar]
  52. Smith M. B., Babcock D. F., Lardy H. A. A 31P NMR study of the epididymis and epididymal sperm of the bull and hamster. Biol Reprod. 1985 Dec;33(5):1029–1040. doi: 10.1095/biolreprod33.5.1029. [DOI] [PubMed] [Google Scholar]
  53. Suzuki M., Takahashi K., Ikeda M., Hayakawa H., Ogawa A., Kawaguchi Y., Sakai O. Cloning of a pH-sensitive K+ channel possessing two transmembrane segments. Nature. 1994 Feb 17;367(6464):642–645. doi: 10.1038/367642a0. [DOI] [PubMed] [Google Scholar]
  54. Tiwari-Woodruff S. K., Cox T. C. Boar sperm plasma membrane Ca(2+)-selective channels in planar lipid bilayers. Am J Physiol. 1995 May;268(5 Pt 1):C1284–C1294. doi: 10.1152/ajpcell.1995.268.5.C1284. [DOI] [PubMed] [Google Scholar]
  55. Tomes C. N., McMaster C. R., Saling P. M. Activation of mouse sperm phosphatidylinositol-4,5 bisphosphate-phospholipase C by zona pellucida is modulated by tyrosine phosphorylation. Mol Reprod Dev. 1996 Feb;43(2):196–204. doi: 10.1002/(SICI)1098-2795(199602)43:2<196::AID-MRD9>3.0.CO;2-M. [DOI] [PubMed] [Google Scholar]
  56. Umbach J. A. Changes in intracellular pH affect calcium currents in Paramecium caudatum. Proc R Soc Lond B Biol Sci. 1982 Sep 22;216(1203):209–224. doi: 10.1098/rspb.1982.0071. [DOI] [PubMed] [Google Scholar]
  57. Vredenburgh-Wilberg W. L., Parrish J. J. Intracellular pH of bovine sperm increases during capacitation. Mol Reprod Dev. 1995 Apr;40(4):490–502. doi: 10.1002/mrd.1080400413. [DOI] [PubMed] [Google Scholar]
  58. Ward C. R., Kopf G. S. Molecular events mediating sperm activation. Dev Biol. 1993 Jul;158(1):9–34. doi: 10.1006/dbio.1993.1165. [DOI] [PubMed] [Google Scholar]
  59. Ward C. R., Storey B. T. Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay. Dev Biol. 1984 Aug;104(2):287–296. doi: 10.1016/0012-1606(84)90084-8. [DOI] [PubMed] [Google Scholar]
  60. Ward C. R., Storey B. T., Kopf G. S. Activation of a Gi protein in mouse sperm membranes by solubilized proteins of the zona pellucida, the egg's extracellular matrix. J Biol Chem. 1992 Jul 15;267(20):14061–14067. [PubMed] [Google Scholar]
  61. Wassarman P. M., Mortillo S. Structure of the mouse egg extracellular coat, the zona pellucida. Int Rev Cytol. 1991;130:85–110. doi: 10.1016/s0074-7696(08)61502-8. [DOI] [PubMed] [Google Scholar]
  62. Wassarman P. M. Zona pellucida glycoproteins. Annu Rev Biochem. 1988;57:415–442. doi: 10.1146/annurev.bi.57.070188.002215. [DOI] [PubMed] [Google Scholar]
  63. Zeng Y., Clark E. N., Florman H. M. Sperm membrane potential: hyperpolarization during capacitation regulates zona pellucida-dependent acrosomal secretion. Dev Biol. 1995 Oct;171(2):554–563. doi: 10.1006/dbio.1995.1304. [DOI] [PubMed] [Google Scholar]
  64. Zeng Y., Oberdorf J. A., Florman H. M. pH regulation in mouse sperm: identification of Na(+)-, Cl(-)-, and HCO3(-)-dependent and arylaminobenzoate-dependent regulatory mechanisms and characterization of their roles in sperm capacitation. Dev Biol. 1996 Feb 1;173(2):510–520. doi: 10.1006/dbio.1996.0044. [DOI] [PubMed] [Google Scholar]

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