Summary 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.
We propose a novel automatic tracking system capable of tracking fast-moving, small objects under extreme conditions by using high-speed visual feedback for assessing sperm chemotaxis. The system shows remarkable performance and versatility, good enough for practical use in biology. Experimental results showed that we successfully achieved continuous stable tracking of swimming ascidian spermatozoa with quality sufficient for assessing sperm motility, indicating the feasibility of our system to tracking almost any type of cell.
Activation state of sperm motility named “hyperactivation” enables mammalian sperm to progress through the oviductal matrix, although a similar state of sperm motility is unknown in non‐mammalian vertebrates at fertilization. Here, we found a high motility state of the sperm in the newt C ynops pyrrhogaster . It was predominantly caused in egg jelly extract ( JE ) and characterized by a high wave velocity of the undulating membrane ( UM ) that was significantly higher at the posterior midpiece. An insemination assay suggested that the high motility state might be needed for sperm to penetrate the egg jelly, which is the accumulated oviductal matrix. Specific characteristics of the high motility state were completely abrogated by a high concentration of verapamil, which blocks the L ‐type and T ‐type voltage‐dependent C a 2+ channels ( VDCC s). Mibefradil, a dominant blocker of T ‐type VDCC s, suppressed the wave of the UM at the posterior midpiece with separate wave propagation from both the anterior midpiece and the posterior principal piece. In addition, nitrendipine, a dominant L ‐type VDCC blocker, weakened the wave of the UM, especially in the anterior midpiece. Live C a 2+ imaging showed that, compared with the intact sperm in the JE , the relative intracellular C a 2+ level changed especially in the anterior and posterior ends of the midpiece of the blocker‐treated sperm. These suggest that different types of C a 2+ channels mediate the intracellular C a 2+ level predominantly in the anterior and posterior ends of the midpiece to maintain the high motility state of the newt sperm.
The reef-building coral Acropora is a broadcast spawning hermaphrodite including more than 110 species in the Indo-Pacific. In addition, many sympatric species show synchronous spawning. The released gametes need to mate with conspecifics in the mixture of the gametes of many species for their species boundaries. However, the mechanism underlying the species recognition of conspecifics at fertilization remains unknown. We hypothesized that rapid molecular evolution (positive selection) in genes encoding gamete-composing proteins generates polymorphic regions that recognize conspecifics in the mixture of gametes from many species. We identified gamete proteins of Acropora digitifera using mass spectrometry and screened the genes that support branch site models that set the “foreground” branches showing strict fertilization specificity. ADAM10, ADAM17, Integrin α9, and Tetraspanin4 supported branch-site model and had positively selected site(s) that produced polymorphic regions. Therefore, we prepared antibodies against the proteins of A. digitifera that contained positively selected site(s) to analyze their functions in fertilization. The ADAM10 antibody reacted only with egg proteins of A. digitifera , and immunohistochemistry showed ADAM10 localized around the egg surface. Moreover, the ADAM10 antibody inhibited only A. digitifera fertilization but not the relative synchronous spawning species A. papillare . This study indicates that ADAM10 has evolved to gain fertilization specificity during speciation and contributes to species boundaries in this multi-species, synchronous-spawning, and species-rich genus.
ABSTRACT A haptonema is an elongated microtubule-based motile organelle uniquely present in haptophytes. The most notable and rapid movement of a haptonema is “coiling”, which occurs within a few milliseconds following mechanical stimulation in an unknown motor-independent mechanism. Here, we analyzed the coiling process in detail by high-speed filming and showed that haptonema coiling was initiated by left-handed twisting of the haptonema, followed by writhing to form a helix from the distal tip. On recovery from a mechanical stimulus, the helix slowly uncoiled from the proximal region. Electron microscopy showed that the seven microtubules in a haptonema were arranged mostly in parallel but that one of the microtubules often wound around the others in the extended state. The persistence lengths calculated from the curvature of the haptonematal microtubules indicated their unusual flexibility. A microtubule stabilizer, paclitaxel, inhibited coiling and induced right-handed twisting of the haptonema in the absence of Ca 2+ , suggesting changes in the microtubule surface lattice. Addition of Ca 2+ caused bend propagation toward the proximal region. These results indicate that switching microtubule conformation with the aid of Ca 2+ -binding microtubule-associated proteins is responsible for rapid haptonematal coiling. Summary Statement Microscopy observations and pharmacological experiments revealed that the rapid coiling of a non-motor microtubule-based motile organelle, the haptonema, is explained by conformational changes of microtubules, including twisting and writhing.
Mitochondria activation factor (MAF) is a high-molecular-weight polyphenol purified from black tea that activates mitochondrial respiration. It increased the mitochondrial membrane potential and motility of sea urchin sperm, by up to 8%, to the same extent as sperm-activating peptides (SAPs) secreted by the egg. Unlike SAPs, MAF had no effect on sperm swimming behavior, suggesting that the mechanism of sperm activation by MAF is different from that of SAPs.
The swimming of male gametes of brown algae is controlled by two heterokont flagella, regardless of reproductive system, but the patterns of flagellar movement can vary. We investigated sperm chemotaxis and phototaxis in four oogamous Australasian fucalean species. Sperm morphology was similar to that of male gametes of isogamous and anisogamous species but their chemotactic behaviours were much closer to those of other oogamous species than those of isogamous and anisogamous species. Moreover, unlike anisogamous species, sperm chemotaxis of the oogamous species, Hormosira banksii, was not inhibited by a phosphodiesterase inhibitor, theophylline, suggesting variability in regulatory mechanisms of sperm chemotaxis amongst brown algae. In our previous work, lower extracellular Ca2+, supernatant of female gametes and theophylline induced the reversal of phototactic signs in anisogamous male gametes. In contrast, lower extracellular Ca2+ and theophylline did not significantly affect phototaxis in sperm of the oogamous H. banksii. Moreover, high extracellular Ca2+ and supernatant of eggs disrupted the negative phototaxis but did not reverse their phototactic sign. These results suggest that the regulation of phototaxis also varies amongst brown algal gametes. Further investigation of brown algal species is warranted to assess the generality of these patterns of chemotaxis and phototaxis among anisogamous and oogamous gametes.
Brown algae are members of the Stramenopiles and their gametes generally have two heterogeneous flagella: a long anterior flagellum (AF) with mastigonemes and a short posterior flagellum (PF). In this study, swimming paths and flagellar waveforms in free-swimming and thigmotactic-swimming male and female gametes and in male gametes during chemotaxis, were quantitatively analysed in the model brown alga Ectocarpus siliculosus. This analysis was performed using a high-speed video camera. It was revealed that the AF plays a role in changing the locomotion of male and female gametes from free-swimming to thigmotactic-swimming and also in changing the swimming path of male gametes from linear to circular during chemotaxis. In the presence of a sex pheromone, male gametes changed their swimming path from linear (swimming path curvature, 0–0.02 µm–1) to middle and small circular path (swimming path curvature, 0.04–0.20 µm–1). The flagellar asymmetry and the deflection angle of the AF became larger, whereas the oscillation pattern of the AF was stable. However, there was no correlation between the flagellar asymmetry and the deflection angle of the AF and the path curvature when the male gametes showed middle to small circular paths. The PF irregularly changed the deflection angle and the oscillation pattern was unstable depending on the gradient of the sex pheromone concentration. AF waveforms were independent of PF locomotion during chemotaxis. This means that the AF has the ability to change the swimming path of male gametes – for example, from a highly linear path to a circular path – while changes in locomotion from a middle circle path to a small circle path is the result of beating of the PF.
Sperm cells are the target of strong sexual selection that may drive changes in sperm structure and function to maximize fertilisation success. Sperm evolution is regarded to be one of the major consequences of sperm competition in polyandrous species, however it can also be driven by adaptation to the environmental conditions at the site of fertilization. Strong stabilizing selection limits intra-specific variation, and therefore polymorphism, among fertile sperm (eusperm). Here we analyzed reproductive morphology differences among males employing characteristic alternative mating behaviours, and so potentially different conditions of sperm competition and fertilization environment, in the squid Loligo bleekeri. Large consort males transfer smaller (average total length = 73 μm) sperm to a female's internal sperm storage location, inside the oviduct; whereas small sneaker males transfer larger (99 μm) sperm to an external location around the seminal receptacle near the mouth. No significant difference in swimming speed was observed between consort and sneaker sperm. Furthermore, sperm precedence in the seminal receptacle was not biased toward longer sperm, suggesting no evidence for large sperm being favoured in competition for space in the sperm storage organ among sneaker males. Here we report the first case, in the squid Loligo bleekeri, where distinctly dimorphic eusperm are produced by different sized males that employ alternative mating behaviours. Our results found no evidence that the distinct sperm dimorphism was driven by between- and within-tactic sperm competition. We propose that presence of alternative fertilization environments with distinct characteristics (i.e. internal or external), whether or not in combination with the effects of sperm competition, can drive the disruptive evolution of sperm size.
