Marine Biotechnology

, Volume 11, Issue 1, pp 118–123 | Cite as

Roles of Calmodulin and Calcium/Calmodulin-Dependent Protein Kinase in Flagellar Motility Regulation in the Coral Acropora Digitifera

  • Masaya MoritaEmail author
  • Akira Iguchi
  • Akihiro Takemura
Original Article


In the corals Acropora spp., eggs secrete substances that induce sperm motility regulation. An elevation of intracellular pH ([pH]i) and a regulation of intracellular Ca2+ concentration ([Ca2+]) are involved in the sperm motility regulation cascade. However, the detailed molecular aspects of flagellar motility regulation have not been fully demonstrated in Acropora. In this study, we determined the presence and roles of both calmodulin (CaM) and calcium/calmodulin dependent-protein kinase (CaMK) in the sperm flagellar motility regulation of Acropora. A 45Ca2+-overlay assay and an immunoblot analysis showed that sperm contain an acidic 16-kDa protein that was CaM, and an immunoblot analysis revealed the presence of CaMK in coral sperm. In addition, a specific inhibitor of CaMK, KN-93, and a CaM antagonist, W-7, inhibited sperm motility activation induced by NH4Cl treatment. NH4Cl treatment causes an increase in intracellular [pH]i of sperm, suggesting that CaM and CaMK are involved in sperm motility initiation caused by an increase in [pH]i. The involvement of CaM and CaMK in motility regulation in coral highlights the importance of these molecules throughout the animal kingdom.


Flagellar motility Sperm Coral Chemotaxis Protein phosphorylation Calcium-binding proteins 



The authors want to express gratitude to Ms. M Nakamura (University of the Ryukyus) for helpful advice on the manuscript. This study was partially supported by a grant for the 21st Century COE program “The Comprehensive Analyses on Biodiversity in Coral Reef and Island Ecosystems in Asian and Pacific Regions” from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Monbukagakusho) to MM.


  1. Babcock RC, Bull GD, Harrison PL, Heywar AJ, Oliver CC, Wallace CC, Willis BL (1986) Synchronous spawning of 105 scleractinian coral species on the Great Barrier Reef. Mar Biol 90:379–394CrossRefGoogle Scholar
  2. Ball EE, Hayward DC, Saint R, Miller DJ (2004) A simple plan-Cnidarians and the origins of developmental mechanisms. Nature Rev Genet 5:567–577CrossRefGoogle Scholar
  3. Bookbinder H, Moy GW, Vacquier VD (1990) Identification of sea urchin sperm adenylate cyclase. J Cell Biol 111:1859–1866PubMedCrossRefGoogle Scholar
  4. Brokaw CJ, Nagayama SM (1985) Modulation of the asymmetry of sea urchin sperm flagellar bending by calmodulin. J Cell Biol 100:1875–1883PubMedCrossRefGoogle Scholar
  5. Cook SP, Babcock DF (1993) Activation of Ca2+ permeability by cAMP is coordinated through the pHi increase induced by speract. J Biol Chem 268:22408–22413PubMedGoogle Scholar
  6. Cook SP, Brokaw CJ, Muller CH, Babcock DF (1994) Sperm chemotaxis: egg peptides control cytosolic calcium to regulate flagellar responses. Dev Biol 165:10–19PubMedCrossRefGoogle Scholar
  7. Darszon A, Labarca P, Nishigaki T, Espinosa F (1999) Ion channels in sperm physiology. Physiol Rev 79:481–510PubMedGoogle Scholar
  8. Darszon A, Beltrán C, Felix R, Nishigaki T, Treviño CL (2001) Ion transport in sperm signaling. Dev Biol 240:1–14PubMedCrossRefGoogle Scholar
  9. Harrison PL, Babcock RC, Bull GD, Oliver JK, Wallace CC, Willis BL (1984) Mass spawning in tropical reef corals. Science 223:1186–1189PubMedCrossRefGoogle Scholar
  10. Harrison PL, Wallace CC (1990) Reproduction, dispersal and recruitment of scleractinian corals. In: Dubinsky Z (ed) Ecosystem of the world: coral reefs. Elsevier Science, Amsterdam, pp 133–207Google Scholar
  11. Hayashiba T, Shimoike K, Kimura T, Hosoya S, Heyward A, Harrison P, Kudo K, Omori M (1993) Patterns of coral spawning at Akajima Island, Okinawa, Japan. Mar Ecol Prog Ser 101:253–262CrossRefGoogle Scholar
  12. Heyward AJ, Babcock RC (1986) Self- and cross-fertilization in scleractinian corals. Mar Biol 90:191–195CrossRefGoogle Scholar
  13. Hirabayashi T (1981) Two-dimensional gel electrophoresis of chicken skeletal muscle proteins with agarose gels in first dimension. Anal Biochem 117:443–451PubMedCrossRefGoogle Scholar
  14. Hulen D, Baron A, Salisbury A, Clarke M (1991) Production and specificity of monoclonal antibodies against calmodulin from dictyostelium discoideum. Cell Motil Cytoskel 18:113–122CrossRefGoogle Scholar
  15. James P, Vorherr T, Carafoli E (1995) Calmodulin-binding domains: just two faced or multi-faceted. Trend Biochem Sci 20:38–42PubMedCrossRefGoogle Scholar
  16. Kirichok Y, Navarro B, Clapham DE (2006) Whole-cell patch-clamp measurements of spermatozoa reveal an alkaline-activated Ca2+ channel. Nature 439:7373–740CrossRefGoogle Scholar
  17. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  18. Marín-Briggiler CI, Jha KN, Chertihin O, Buffone MG, Herr JC, Vazquez-Levin MH, Visconti PE (2005) Evidence of the presence of calcium/calmodulin-dependent protein kinase IV in human sperm and its involvement in motility regulation. J Cell Sci 118:2013–2022PubMedCrossRefGoogle Scholar
  19. Maruyama K, Miyakawa T, Ebashi S (1984) Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis. J Biochem 95:511–519PubMedGoogle Scholar
  20. Miller RL (1979a) Sperm chemotaxis in the hydromedusae. I. Species-specificity and sperm behavior. Mar Biol 53:99–114CrossRefGoogle Scholar
  21. Miller RL (1979b) Sperm chemotaxis in the hydromedusae. II. Some chemical properties of the sperm attractants. Mar Biol 53:114–124Google Scholar
  22. Miller RL (1985) Demonstration of sperm chemotaxis in echinodermata: Asteroidea, Holothuroidea, Ophiurodea. J Exp Zool 234:383–414CrossRefGoogle Scholar
  23. Morita M, Nishikawa A, Nakajima A, Iguchi A, Sakai K, Takemura A, Okuno M (2006a) Sperm flagellar motility initiation, chemotaxis, and inhibition by eggs in the coral, Acropora digitifera, A. gemmifera, and A. tenuis. J Exp Biol 209:4574–4579PubMedCrossRefGoogle Scholar
  24. Morita M, Takemura A, Nakajima A, Okuno M (2006b) Microtubule sliding movement in tilapia sperm flagella axoneme is regulated by Ca2+/calmodulin-dependent protein phosphorylation. Cell Motil Cytoskeleton 63:459–470PubMedCrossRefGoogle Scholar
  25. Nakajima A, Morita M, Takemura A, Kamimura S, Okuno M (2005) Increase in intracellular pH induces phosphorylation of axonemal proteins for flagellar motility activation in starfish sperm. J Exp Biol 208:4411–4418PubMedCrossRefGoogle Scholar
  26. Nomura M, Inaba K, Morisawa M (2000) Cyclic AMP- and calmodulin-dependent phosphorylation of 21 kDa and 26 kDa proteins in axoneme is a prerequisite for SAAF-induced motile activation in ascidian spermatozoa. Dev Growth Diff 42:129–138CrossRefGoogle Scholar
  27. Nomura M, Inaba K, Morisawa M (2004) Calmodulin/calmodulin-dependent protein kinase II mediates SAAF-induced motility activation of ascidian sperm. Cell Motil Cytoskeleton 59:28–37PubMedCrossRefGoogle Scholar
  28. Riffell JA, Krug PJ, Zimmer RK (2002) Fertilization in the sea: the chemical identity of an abalone sperm attractant. J Exp Biol 205:1439–1450PubMedGoogle Scholar
  29. Smith EF (2002) Regulation of flagellar dynein by calcium and a role for an axonemal calmodulin and calmodulin-dependent kinase. Mol Biol Cell 13:3303–3313PubMedCrossRefGoogle Scholar
  30. Spehr M, Gisselmann G, Poplawski A, Riffell JA, Wetzel CH, Zimmer RK, Hatt H (2003) Identification of a testicular odorant receptor mediating human sperm chemotaxis. Science 301:2054–2058CrossRefGoogle Scholar
  31. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354PubMedCrossRefGoogle Scholar
  32. White D, Lamirande E, Gagnon C (2007) Protein kinase C is an important signaling mediator associated with motility of intact sea urchin spermatozoa. J Exp Biol 210:4053–4064PubMedCrossRefGoogle Scholar
  33. Wargo MJ, Dymek EE, Smith EF (2005) Calmodulin and PF6 are components of a complex that localizes to the C1 microtubule of the flagellar central apparatus. J Cell Sci 118:4655–4665PubMedCrossRefGoogle Scholar
  34. Wood CD, Darszon A, Whitaker M (2003) Speract induces calcium oscillations in the sperm tail. J Cell Biol 161:89–101PubMedCrossRefGoogle Scholar
  35. Yanagimachi R (1957) Some properties of the sperm-activating factor in the micropyle area of the herring egg. Annot Zool Jpn 41:114–119Google Scholar
  36. Yoshida M, Inaba K, Morisawa M (1993) Sperm chemotaxis during the process of fertilization in the Ascidians Ciona Savignyi and Ciona intestinals. Dev Biol 157:497–506PubMedCrossRefGoogle Scholar
  37. Yoshida M, Inaba K, Ishida K, Morisawa M (1994) Calcium and cyclic AMP mediate sperm activation, but Ca2+ alone contributes sperm chemotaxis in the ascidian, Ciona savignyi. Dev Growth Differ 36:589–595CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Masaya Morita
    • 1
    Email author
  • Akira Iguchi
    • 2
    • 3
  • Akihiro Takemura
    • 1
  1. 1.Sesoko Station, Tropical Biosphere Research CenterUniversity of the RyukyusOkinawaJapan
  2. 2.ARC Centre of Excellence for Coral Reef StudiesJames Cook University, AustraliaTownsvilleAustralia
  3. 3.Comparative Genomics CentreJames Cook University, AustraliaTownsvilleAustralia

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