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Takuya Nishigaki

Publications and source records attributed to Takuya Nishigaki.

10 recordsLinked to original sources
PubMed2006

Rho, Rho-kinase, and the actin cytoskeleton regulate the Na+ -H+ exchanger in sea urchin eggs.

At fertilization, the sea urchin egg undergoes an internal pH (pHi) increase mediated by a Na+ -H+ exchanger. We used antibodies against the mammalian antiporters NHE1 and NHE3 to characterize this exchanger. In unfertilized eggs, only anti-NHE3 cross-reacted specifically with a protein of 81-kDa, which localized to the plasma membrane and cortical granules. Cytochalasin D, C3 exotoxin (blocker of RhoGTPase function), and Y-27632 (inhibitor of Rho-kinase) prevented the pHi change in fertilized eggs. These inhibitors blocked the first cleavage division of the embryo, but not the cortical granule exocytosis. Thus, the sea urchin egg has an epithelial NHE3-like Na+ -H+ exchanger which can be responsible for the pHi change at fertilization. Determinants of this pHi change can be: (i) the increase of exchangers in the plasma membrane (via cortical granule exocytosis) and (ii) Rho, Rho-kinase, and optimal organization of the actin cytoskeleton as regulators, among others, of the intrinsic activity of the exchanger.

Actins↗
PubMed2006

Maitotoxin potently promotes Ca2+ influx in mouse spermatogenic cells and sperm, and induces the acrosome reaction.

Maitotoxin (MTX), a potent marine toxin, activates Ca2+ entry via nonselective cation channels in a wide variety of cells. The identity of the channels involved in MTX action remains unknown. In mammalian sperm, Ca2+ entry through store-operated channels regulates a number of physiological events including the acrosome reaction (AR). Here we report that MTX produced an increase in the intracellular concentration of Ca2+ ([Ca2+]i) in spermatogenic cells that depended on extracellular Ca2+. Ni2+ and SKF96365 diminished the MTX-activated Ca2+ uptake, at concentrations they inhibit store-operated channels, and in a similar manner as they inhibit the Ca2+ influx activated following depletion of intracellular stores by thapsigargin (Tpg). In addition, MTX significantly increased [Ca2+]i in single mature sperm and effectively induced the AR with a half-maximal concentration (ED50) of approximately 1.1 nM. Notably, SKF96365 similarly inhibited the MTX-induced increase in sperm [Ca2+]i and the AR triggered by the toxin, Tpg and zona pellucida. These results suggest that putative MTX-activated channels may be involved in the Ca2+ influx required for mouse sperm AR.

Acrosome↗
PubMed2006

Sperm channel diversity and functional multiplicity.

Ion channels are extraordinarily efficient machines that move ions in diversely controlled manners, allowing cells to rapidly exchange information with the outside world and with other cells. Communication is the currency of fertilization, as it is of most fundamental cell signaling events. Ion channels are deeply involved in the dialogue between sperm, its surroundings, and the egg. How sperm swim, find the egg and fertilize it depend on ion permeability changes modulated by environmental cues and components of the egg outer layer. Different ion channels distinctly localized in these tiny, amazing cells perform specific decoding functions that shape the sophisticated behavior of sperm. It is not surprising that certain sperm ion channels are turning out to be unique. New strategies to characterize sperm ion transport have opened exciting possibilities to dissect sperm-egg signaling and unveil novel contraception targets.

Acrosome Reaction↗
PubMed2006

Stroboscopic illumination using light-emitting diodes reduces phototoxicity in fluorescence cell imaging.

Excited fluorophores produce reactive oxygen species that are toxic toward many live cells (phototoxicity) and accelerate bleaching of the fluorophores during the course of extended or repeated measurements (photobleaching). We recently developed an illumination system for fluorescence microscopy using a high power light-emitting diode (LED), which can emit short pulses of light (0.5-2 ms) to excite fluorophores. This system minimizes illumination time, thus reducing phototoxicity and photobleaching artifacts. To demonstrate the usefulness of the new system, we compared images of human sperm loaded with various fluorescent indicators and excited with either a conventional mercury lamp as a continuous excitation light source or the LED as a source of pulsed illumination. We found that sperm motility decreased rapidly and photobleaching was relatively rapid under continuous illumination, whereas under pulsed LED illumination, motility was maintained and photobleaching was much reduced. Therefore, fluorescence microscopy using LED-based pulsed illumination offers significant advantages for long-term live cell imaging, reducing the degree of phototoxicity, and extending the effective lifetime of fluorophores.

Calcium↗
PubMed2005

Real-time analysis of the role of Ca(2+) in flagellar movement and motility in single sea urchin sperm.

Eggs of many marine and mammalian species attract sperm by releasing chemoattractants that modify the bending properties of flagella to redirect sperm paths toward the egg. This process, called chemotaxis, is dependent on extracellular Ca(2+). We used stroboscopic fluorescence imaging to measure intracellular Ca(2+) concentration ([Ca(2+)]i) in the flagella of swimming sea urchin sperm. Uncaging of cyclic GMP induced Ca(2+) entry via at least two distinct pathways, and we identified a nimodipine-sensitive pathway, compartmentalized in the flagella, as a key regulator of flagellar bending and directed motility changes. We found that, contrary to current models, the degree of flagellar bending does not vary in proportion to the overall [Ca(2+)]i. Instead we propose a new model whereby flagella bending is increased by Ca(2+) flux through the nimodipine-sensitive pathway, and is unaffected by [Ca(2+)]i increases through alternative pathways.

Animals↗
PubMed2005

Calcium channels and Ca2+ fluctuations in sperm physiology.

Generating new life in animals by sexual reproduction depends on adequate communication between mature and competent male and female gametes. Ion channels are instrumental in the dialogue between sperm, its environment, and the egg. The ability of sperm to swim to the egg and fertilize it is modulated by ion permeability changes induced by environmental cues and components of the egg outer layer. Ca(2+) is probably the key messenger in this information exchange. It is therefore not surprising that different Ca(2+)-permeable channels are distinctly localized in these tiny specialized cells. New approaches to measure sperm currents, intracellular Ca(2+), membrane potential, and intracellular pH with fluorescent probes, patch-clamp recordings, sequence information, and heterologous expression are revealing how sperm channels participate in fertilization. Certain sperm ion channels are turning out to be unique, making them attractive targets for contraception and for the discovery of novel signaling complexes.

Animals↗
PubMed2005

Sperm-activating peptide induces asymmetric flagellar bending in sea urchin sperm.

Speract, a sperm-activating peptide (SAP) from sea urchin eggs, induces various sperm responses including a transient increase in the intracellular Ca2+ concentration. However, it has not been clarified how speract modulates sperm motility and whether it functions as a chemoattractant. To confirm the effect of speract on sperm motility, we observed the flagellar bending response to speract in sperm of Hemicentrotus pulcherrimus, in experiments using caged speract and a lighting system for a microscope newly developed with a power LED. We found that speract induces increases in curvature of swimming paths and changes flagellar bending shape to asymmetric. These facts show that speract directly regulates flagellar motility, and suggest that speract-induced increases in intracellular Ca2+ concentration play an actual role in regulation of the flagellar movement.

Animals↗
PubMed2004

A sea urchin egg jelly peptide induces a cGMP-mediated decrease in sperm intracellular Ca(2+) before its increase.

Speract, a sperm-activating peptide (SAP) from sea urchin eggs, increases the intracellular concentration of Ca(2+) ([Ca(2+)]i) and modulates sperm motility. We measured the initial sperm response to speract using its caged analog and observed, for the first time, a small but significant decrease in sperm [Ca(2+)]i before the increase. Both directions of the [Ca(2+)]i change were completely blocked in high K(+) seawater. Using membrane-permeant caged cyclic nucleotides (cNMP), only cGMP induced the decrease in [Ca(2+)]i although both cGMP and cAMP increased the [Ca(2+)]i. The decrease in the [Ca(2+)]i induced by cGMP was more notable following a second photolytic event, once [Ca(2+)]i had been elevated by an initial flash. This pattern of [Ca(2+)]i change was confirmed in individual sperm. These results together with pharmacological evidence suggest that the initial [Ca(2+)]i decrease is due to a Na(+)/Ca(2+) exchanger activity, stimulated by hyperpolarization mediated by K(+) efflux through cGMP-regulated K(+) channels.

1-Methyl-3-isobutylxanthine↗
PubMed2002

A caged sperm-activating peptide that has a photocleavable protecting group on the backbone amide.

A backbone-caged sperm-activating peptide (caged speract) that has a 2-nitrobenzyl group at a backbone amide and a vastly reduced affinity for its receptor (IC50=950 nM) was synthesized. UV irradiation of caged speract photocleaves the 2-nitrobenzyl group (tau1/2=26 micros), restoring its affinity (IC50=0.67 nM) and ability to increase sperm intracellular pH and Ca2+, as intact speract. Backbone caging of the biological activity was more efficient than side chain caging, which adds a nitrobenzyl group on the peptide side chain. The backbone caging strategy described can be used as a general procedure to cage biologically active peptides, which have no side chain for introduction of a caging group.

Amino Acid Sequence↗