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High potassium seawater inhibits ascidian sperm chemotaxis, but does not affect the male gamete chemotaxis of a brown alga

  • Nana Kinoshita-Terauchi (a1) (a2), Kogiku Shiba (a2) (a3), Makoto Terauchi (a4) (a5), Francisco Romero (a6), Héctor Vincente Ramírez-Gómez (a6), Manabu Yoshida (a3), Taizo Motomura (a7), Hiroshi Kawai (a4) and Takuya Nishigaki (a6)...


Male gamete chemotaxis towards the female gamete is a general strategy to facilitate the sexual reproduction in many marine eukaryotes. Biochemical studies of chemoattractants for male gametes of brown algae have advanced in the 1970s and 1980s, but the molecular mechanism of male gamete responses to the attractants remains elusive. In sea urchin, a K+ channel called the tetraKCNG channel plays a fundamental role in sperm chemotaxis and inhibition of K+ efflux through this channel by high K+ seawater blocks almost all cell responses to the chemoattractant. This signalling mechanism could be conserved in marine invertebrates as tetraKCNG channels are conserved in the marine invertebrates that exhibit sperm chemotaxis. We confirmed that high K+ seawater also inhibited sperm chemotaxis in ascidian, Ciona intestinalis (robusta), in this study. Conversely, the male gamete chemotaxis towards the female gamete of a brown alga, Mutimo cylindricus, was preserved even in high K+ seawater. This result indicates that none of the K+ channels is essential for male gamete chemotaxis in the brown alga, suggesting that the signalling mechanism for chemotaxis in this brown alga is quite different from that of marine invertebrates. Correlated to this result, we revealed that the channels previously proposed as homologues of tetraKCNG in brown algae have a distinct domain composition from that of the tetraKCNG. Namely, one of them possesses two repeats of the six transmembrane segments (diKCNG) instead of four. The structural analysis suggests that diKCNG is a cyclic nucleotide-modulated and/or voltage-gated K+ channel.


Corresponding author

*Address for correspondence: Takuya Nishigaki. Institute of Biotechnology, National Autonomous University of Mexico (IBT-UNAM), Av. Universidad 2001, Col. Chamilpa, Cuernavaca, Mor. 62210, Mexico. Tel: +52 777 3291709. E-mail:


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Beltrán, C, Zapata, O and Darszon, A (1996) Membrane potential regulates sea urchin sperm adenylylcyclase. Biochemistry 35, 75917598.
Böhmer, M, Van, Q, Weyand, I, Hagen, V, Beyermann, M, Matsumoto, M, Hoshi, M, Hildebrand, E and Kaupp, UB (2005) Ca2+ spikes in the flagellum control chemotactic behavior of sperm. EMBO J 24, 27412752.
Bonigk, W, Loogen, A, Seifert, R, Kashikar, N, Klemm, C, Krause, E, Hagen, V, Kremmer, E, Strunker, T and Kaupp, UB (2009) An atypical CNG channel activated by a single cGMP molecule controls sperm chemotaxis. Sci Signal 2, ra68.
Brams, M, Kusch, J, Spurny, R, Benndorf, K and Ulens, C (2014) Family of prokaryote cyclic nucleotide-modulated ion channels. Proc Natl Acad Sci USA 111, 78557860.
Brelidze, TI, Carlson, AE and Zagotta, WN (2009) Absence of direct cyclic nucleotide modulation of mEAG1 and hERG1 channels revealed with fluorescence and electrophysiological methods. J Biol Chem 284, 2798927997.
Brelidze, TI, Carlson, AE, Sankaran, B and Zagotta, WN (2012) Structure of the carboxy-terminal region of a KCNH channel. Nature 481, 530533.
Cock, JM, Sterck, L, Rouzé, P, Scornet, D, Allen, AE, Amoutzias, G, Anthouard, V, Artiguenave, F, Aury, JM, Badger, JH, et al. (2010) The Ectocarpus genome and the independent evolution of multicellularity in brown algae. Nature 465, 6176121.
Darszon, A, Nishigaki, T, Beltran, C and Trevino, CL (2011) Calcium channels in the development, maturation, and function of spermatozoa. Physiol Rev 91, 13051355.
Espinal-Enríquez, J, Priego-Espinosa, DA, Darszon, A, Espinal-Enríquez, J, Priego-Espinosa, DA, Darszon, A, Beltrán, C and Martínez-Mekler, G (2017) Network model predicts that CatSper is the main Ca2+ channel in the regulation of sea urchin sperm motility. Sci Rep 7, 4236.
Fechner, S, Alvarez, L, Bönigk, W, Müller, A, Berger, TK, Pascal, R, Trötschel, C, Poetsch, A, Stölting, G, Siegfried, KR, Kremmer, E, Seifert, R, and Kaupp, UB (2015) A K+-selective CNG channel orchestrates Ca2+ signalling in zebrafish sperm. eLife 4, 125.
Froese, A, Breher, SS, Waldeyer, C, Schindler, RF, Nikolaev, VO, Rinné, S, Wischmeyer, E, Schlueter, J, Becher, J, Simrick, S, Vauti, F, Kuhtz, J, Meister, P, Kreissl, S, Torlopp, A, Liebig, SK, Laakmann, S, Müller, TD, Neumann, J, Stieber, J, Ludwig, A, Maier, SK, Decher, N, Arnold, HH, Kirchhof, P, Fabritz, L, and Brand, T (2012) Popeye domain containing proteins are essential for stress-mediated modulation of cardiac pacemaking in mice. J Clin Invest 122, 11191130.
Fu, G, Nagasato, C, Oka, S, Fu, G, Nagasato, C, Oka, S, Cock, JM and Motomura, T (2014) Proteomics analysis of heterogeneous flagella in brown algae (Stramenopiles). Protist 165, 662675.
Galindo, BE, de la Vega-Beltrán, JL, Labarca, P, Vacquier, VD and Darszon, A (2007) Sp-tetraKCNG: A novel cyclic nucleotide gated K+ channel. Biochem Biophys Res Commun 354, 668675.
Gauss, R, Seifert, R and Kaupp, UB (1998) Molecular identification of a hyperpolarization-activated channel in sea urchin sperm. Nature 393, 583587.
Geller, A and Müller, DG (1981) Anaysis of the flagellar beat pattern of male Ectocarpus siliculosus gametes (Phaeophyta) in relation to chemotactic stimulation by female cells. J Exp Biol 92, 5366.
González-Cota, AL, Silva, , Carneiro, J and Darszon, A (2015) Single cell imaging reveals that the motility regulator speract induces a flagellar alkalinization that precedes and is independent of Ca2+ influx in sea urchin spermatozoa. FEBS Lett 589, 21462154.
Gough, J, Karplus, K, Hughey, R and Chothia, C (2001) Assignment of homology to genome sequences using a library of hidden Markov models that represent all proteins of known structure. J Mol Biol 313, 903919.
Haitin, Y, Carlson, AE and Zagotta, WN (2013) The structural mechanism of KCNH-channel regulation by the eag domain. Nature 501, 444448.
Harumi, T, Hoshino, K and Suzuki, N (1992) Effects of sperm-activating peptide I on Hemicentrotus pulcherrimus spermatozoa in high potassium sea water. Dev Growth Differ 34, 163172.
Heffernan, R, Dehzangi, A, Lyons, J, Paliwal, K, Sharma, A, Wang, J, Sattar, A, Zhou, Y and Yang, Y (2015a) Highly accurate sequence-based prediction of half-sphere exposures of amino acid residues in proteins. Bioinformatics 32, 843849.
Heffernan, R, Paliwal, K, Lyons, J, Dehzangi, A, Sharma, A, Wang, J, Sattar, A, Yang, Y and Zhou, Y (2015b) Improving prediction of secondary structure, local backbone angles, and solvent accessible surface area of proteins by iterative deep learning. Sci Rep 5, 111.
Izumi, H, Marian, T, Inaba, K, Oka, Y and Morisawa, M (1999) Membrane hyperpolarization by sperm-activating and -attracting factor increases cAMP level and activates sperm motility in the ascidian Ciona intestinalis. Dev Biol 213, 246256.
Jiang, SZ, Xu, W, Emery, AC, Gerfen, CR, Eiden, MV and Eiden, LE (2017) NCS-Rapgef2, the protein product of the neuronal Rapgef2 gene, is a specific activator of D1 dopamine receptor-dependent ERK phosphorylation in mouse brain. eNeuro 4, 117.
Kawai, H, Müller, DG, Fölster, E and Häder, DP (1990) Phototactic responses in the gametes of the brown alga, Ectocarpus siliculosus . Planta 182, 292297.
Kinoshita, N, Nagasato, C, Tanaka, A and Motomura, T (2016a) Chemotaxis in the anisogamous brown alga Mutimo cylindricus (Cutleriaceae, Tilopteridales). Phycologia 55, 359364.
Kinoshita, N, Shiba, K, Inaba, K, Fu, G, Nagasato, C and Motomura, T (2016b) Flagellar waveforms of gametes in the brown alga Ectocarpus siliculosus . Eur J Phycol 51, 139148.
Kinoshita, N, Nagasato, C and Motomura, T (2017a) Phototaxis and chemotaxis of brown algal swarmers. J Plant Res, 111.
Kinoshita, N, Nagasato, C and Motomura, T (2017b) Chemotactic movement in sperm of the oogamous brown algae, Saccharina japonica and Fucus distichus . Protoplasma 254, 547555.
Kirichok, Y, Navarro, B and Clapham, DE (2006) Whole-cell patch-clamp measurements of spermatozoa reveal an alkaline-activated Ca2+ channel. Nature 439, 737740.
Lipinska, AP, D’Hondt, S, Van Damme, EJM and De Clerck, O (2013) Uncovering the genetic basis for early isogamete differentiation: a case study of Ectocarpus siliculosus . BMC Genomics 14, 909.
Lishko, PV, Botchkina, IL, Fedorenko, A and Kirichok, Y (2010) Acid extrusion from human spermatozoa is mediated by flagellar voltage-gated proton channel. Cell 140, 327337.
Maier, I and Müller, DG (1986) Sexual pheromones in algae. Biol Bull 170, 145175.
Maler, I and Calenberg, M (1994) Movement and chemoorientation of male gametes of Ectocarpus siliculosus (Phaeophyceae). Plant Biol 107, 451460.
Meijering, E, Dzyubachyk, O and Smal, I (2012) Chapter 9 − Methods for cell and particle tracking. Imaging and Spectroscopic Analysis of Living Cells. Methods Enzymol 504, 183200.
Miller, RL (1985) Sperm chemo-orientation in the metazoa. In: Biology of Fertilization V2. pp. 275337.
Müller, DG, Jaenicke, L, Donike, M and Akintobi, T (1971) Sex attractant in a brown alga: chemical structure. Science 171, 1132.
Nishigaki, T, Zamudio, FZ, Possani, LD and Darszon, A (2001) Time-resolved sperm responses to an egg peptide measured by stopped-flow fluorometry. Biochem Biophys Res Commun 284, 531535.
Nishigaki, T, Wood, CD, Tatsu, Y, Nishigaki, T, Wood, CD, Tatsu, Y, Yumoto, N, Furuta, T, Elias, D, Shiba, K, Baba, SA and Darszon, A (2004) A sea urchin egg jelly peptide induces a cGMP-mediated decrease in sperm intracellular Ca2+ before its increase. Dev Biol 272, 376388.
Nomura, M and Vacquier, VD (2006) Proteins associated with soluble adenylyl cyclase in sea urchin sperm flagella. Cell Motil Cytoskeleton 63, 582590.
Nomura, M, Beltrán, C, Darszon, A and Vacquier, VD (2005) A soluble adenylyl cyclase from sea urchin spermatozoa. Gene 353, 231238.
Nutting, WH, Rapoport, H and Machlis, L (1968) The structure of sirenin. J Am Chem Soc 90, 64346438.
Oishi, T, Tsuchikawa, H, Murata, M, Oishi, T, Tsuchikawa, H, Murata, M, Yoshida, M and Morisawa, M (2003) Synthesis of endogenous sperm-activating and attracting factor isolated from ascidian Ciona intestinalis . Tetrahedron Lett 44, 63876389.
Oishi, T, Tsuchikawa, H, Murata, M, Yoshida, M and Morisawa, M (2004) Synthesis and identification of an endogenous sperm activating and attracting factor isolated from eggs of the ascidian Ciona intestinalis. An example of nanomolar-level structure elucidation of novel natural compound. Tetrahedron 60, 69716980.
Orta, G, Ferreira, G, José, O, Treviño, CL, Beltrán, C and Darszon, A (2012) Human spermatozoa possess a calcium-dependent chloride channel that may participate in the acrosomal reaction. J Physiol 590, 26592675.
Provasoli, L (1968) Media and prospects for the cultivation of marine algae. In Culture and Collection of Algae US Japan Conference 1966, Hakone, eds. Watanabe A, Hattori A, pp. 6375.
Romero, F, Santana-Calvo, C and Sánchez-Guevara, Y, Nishigaki, T (2017) FRET-based binding assay between a fluorescent cAMP analogue and a cyclic nucleotide-binding domain tagged with a CFP. FEBS Lett 591, 28692878.
Seifert, R, Flick, M, Bönigk, W, Alvarez, L, Trötschel, C, Poetsch, A, Müller, A, Goodwin, N, Pelzer, P, Kashikar, ND, Kremmer, E, Jikeli, J, Timmermann, B, Kuhl, H, Fridman, D, Windler, F, Kaupp, UB, and Strünker, T (2015) The CatSper channel controls chemosensation in sea urchin sperm. EMBO J 34, 379392.
Shiba, K, Baba, SA, Inoue, T and Yoshida, M (2008) Ca2+ bursts occur around a local minimal concentration of attractant and trigger sperm chemotactic response. Proc Natl Acad Sci USA 105, 19312193127.
Strünker, T, Weyand, I, Bönigk, W, Van, Q, Loogen, A, Brown, JE, Kashikar, N, Hagen, V, Krause, E and Kaupp, UB (2006) A K+-selective cGMP-gated ion channel controls chemosensation of sperm. Nat Cell Biol 8, 11491154.
Suzuki, N, Nomura, K, Ohtake, H and Isaka, S (1981) Purification and the primary structure of sperm-activity peptides from the jelly coat of sea urchin eggs. Biochem Biophys Res Commun 99, 12381244.
Wang, D, King, SM, Quill, TA, Doolittle, LK and Garbers, DL (2003) A new sperm-specific Na+/H+ Exchanger required for sperm motility and fertility. Nat Cell Biol 5, 11171122.
Wood, CD, Nisihigaki, T, Furuta, T, Baba, SA and Darszon, A (2005) Real-time analysis of the role of Ca2+ in flagellar movement and motility in single sea urchin sperm. J Cell Biol 169, 725731.
Yoshida, K, Shiba, K, Sakamoto, A, Ikenaga, J, Matsunaga, S, Inaba, K, and Yoshida, M (2018) Ca2+ efflux via plasma membrane Ca2+-ATPase mediates chemotaxis in ascidian sperm. Sci Rep 8, 16622.
Yoshida, M and Yoshida, K (2011) Sperm chemotaxis and regulation of flagellar movement by Ca2 +. Mol Hum Reprod 17, 457465.
Yoshida, M, Inaba, K, Ishida, K and 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, 589595.
Yoshida, M, Murata, M, Inaba, K and Morisawa, M (2002) A chemoattractant for ascidian spermatozoa is a sulfated steroid. Proc Natl Acad Sci USA 99, 1483114836.


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High potassium seawater inhibits ascidian sperm chemotaxis, but does not affect the male gamete chemotaxis of a brown alga

  • Nana Kinoshita-Terauchi (a1) (a2), Kogiku Shiba (a2) (a3), Makoto Terauchi (a4) (a5), Francisco Romero (a6), Héctor Vincente Ramírez-Gómez (a6), Manabu Yoshida (a3), Taizo Motomura (a7), Hiroshi Kawai (a4) and Takuya Nishigaki (a6)...


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