Distinct regulation of two flagella by calcium during chemotaxis of male gametes in the brown alga Mutimo cylindricus (Cutleriaceae, Tilopteridales).

Brown algal male gametes show chemotaxis to the sex pheromone that is released from female gametes. The chemotactic behavior of the male gametes is controlled by the changes in the beating of two flagella known as the anterior and posterior flagellum. Our previous study using Mutimo cylindricus showed that the sex pheromone induced an increment in both the deflection angle of the anterior flagellum and sustained unilateral bend of the posterior flagellum, but the mechanisms regulating these two flagellar waveforms were not fully revealed. In this study, we analyzed the changes in swimming path and flagellar waveforms with a high-speed recording system under different calcium conditions. The extracellular Ca2+ concentration at 10-3 M caused an increment in the deflection angle of the anterior flagellum only when ionomycin was absent. No sustained unilateral bend of the posterior flagellum was induced either in the absence or presence of ionomycin in extracellular Ca2+ concentrations below 10-2 M. Real-time Ca2+ imaging revealed that there is a spot near the basal part of anterior flagellum showing higher Ca2+ than in the other parts of the cell. The intensity of the spot slightly decreased when male gametes were treated with the sex pheromone. These results suggest that Ca2+-dependent changes in the anterior and posterior flagellum are regulated by distinct mechanisms and that the increase in the anterior flagellar deflection angle and sustained unilateral bend of the posterior flagellum may not be primarily induced by the Ca2+ concentration.

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

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.

A brown algal sex pheromone reverses the sign of phototaxis by cAMP/Ca2+-dependent signaling in the male gametes of Mutimo cylindricus (Cutleriaceae).

Male gametes of the brown alga Mutimo cylindricus show positive phototaxis soon after spawning in seawater but gradually change the sign of phototaxis with time. This conversion appears to need the decrease of intracellular Ca2+ concentration. In this study, we revealed that the conversion of male gamete phototactic sign, positive to negative, was accelerated by mixing with female gametes. The supernatant after the centrifugation of female gamete suspension showed the same activity to change the phototactic sign, suggesting that a factor released from female gametes was responsible for the reaction. A known brown algal sex pheromone, ectocarpene, induced chemotaxis of male gametes of M. cylindricus. The addition of this compound induced the change of phototactic sign, clearly indicating that a sex pheromone is essential for the reversal. An inhibitor of phosphodiesterase, theophylline, inhibited the chemotaxis and phototactic sign reversion by a factor released from female gametes and ectocarpene. Measurements of cyclic nucleotides showed that the increase in intracellular concentration of cAMP, not cGMP, was parallel to the change of phototactic sign. The inhibition of phototactic sign by theophylline was not observed in low Ca2+ sea water. These results suggest that a signaling pathway mediated by cAMP and Ca2+ concentrations drives the interconversion between two important behaviors of male gametes, phototaxis and chemotaxis.