Kogiku Shiba

Ca2+ bursts occur around a local minimal concentration of attractant and trigger sperm chemotactic response.

Ca2+ is known to have important roles in sperm chemotaxis, although the relationship between intracellular Ca2+ concentration ([Ca2+]i) and modulation of the swimming and chemotactic behavior of spermatozoa has not been elucidated. Using a high-speed Ca2+ imaging system, we examined the chemotactic behavior and [Ca2+]i in individual ascidian sperm cells exhibiting chemotactic responses toward sperm activating and attracting factor (SAAF), a chemoattractant released by eggs. In this study, we found that transient [Ca2+]i increased in the flagellum (Ca2+ bursts) concomitantly with a change in the swimming direction in an SAAF gradient field. During the initial phase of the Ca2+ bursts, the flagellum of the spermatozoon exhibited highly asymmetric waveforms enabling the quick turning of the swimming path. However, the flagellum subsequently changed to symmetric beating, causing the spermatozoon to swim straight. Interestingly, during such responses, [Ca2+]i remained higher than the basal level, indicating that the series of responses was not simply determined by Ca2+ concentrations. Also, we found that Ca2+ bursts were consistently evoked at points at which the spermatozoon attained around a temporally minimal value for a given SAAF concentration. We concluded that Ca2+ bursts induced around a local minimal SAAF concentration trigger a sequence of flagellar responses comprising quick turning followed by straight swimming to direct spermatozoa efficiently toward eggs.

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.

Sperm calcineurin inhibition prevents mouse fertility with implications for male contraceptive

Mouse work may lead to male contraceptive Unintended pregnancies are a major health issue worldwide. Although oral contraceptives were developed decades ago for use in women, there are no male oral contraceptives. Miyata et al. show that genetic deletion or drug inhibition of sperm-specific calcineurin enzymes in mice cause male sterility (see the Perspective by Castaneda and Matzuk). Although calcineurin inhibitors resulted in male infertility within 2 weeks, fertility recovered 1 week after halting drug administration. Because the sperm-specific calcineuin complex is also found in humans, its inhibition may be a strategy for developing reversible male contraceptives. Science, this issue p. 442, see also p. 385 Inhibiting the function of a protein involved in sperm maturation may help in the development of future male contraceptives. [Also see Perspective by Castaneda and Matzuk] Calcineurin inhibitors, such as cyclosporine A and FK506, are used as immunosuppressant drugs, but their adverse effects on male reproductive function remain unclear. The testis expresses somatic calcineurin and a sperm-specific isoform that contains a catalytic subunit (PPP3CC) and a regulatory subunit (PPP3R2). We demonstrate herein that male mice lacking Ppp3cc or Ppp3r2 genes (knockout mice) are infertile, with reduced sperm motility owing to an inflexible midpiece. Treatment of mice with cyclosporine A or FK506 creates phenocopies of the sperm motility and morphological defects. These defects appear within 4 to 5 days of treatment, which indicates that sperm-specific calcineurin confers midpiece flexibility during epididymal transit. Male mouse fertility recovered a week after we discontinued treatment. Because human spermatozoa contain PPP3CC and PPP3R2 as a form of calcineurin, inhibition of this sperm-specific calcineurin may lead to the development of a reversible male contraceptive that would target spermatozoa in the epididymis.

Why small males have big sperm: dimorphic squid sperm linked to alternative mating behaviours

BackgroundSperm 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.ResultsLarge consort males transfer smaller (average total length = 73 μm) sperm to a females 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.ConclusionsHere 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.

Calaxin drives sperm chemotaxis by Ca2+-mediated direct modulation of a dynein motor

Sperm chemotaxis occurs widely in animals and plants and plays an important role in the success of fertilization. Several studies have recently demonstrated that Ca2+ influx through specific Ca2+ channels is a prerequisite for sperm chemotactic movement. However, the regulator that modulates flagellar movement in response to Ca2+ is unknown. Here we show that a neuronal calcium sensor, calaxin, directly acts on outer-arm dynein and regulates specific flagellar movement during sperm chemotaxis. Calaxin inhibition resulted in significant loss of sperm chemotactic movement, despite normal increases in intracellular calcium concentration. Using a demembranated sperm model, we demonstrate that calaxin is essential for generation and propagation of Ca2+-induced asymmetric flagellar bending. An in vitro motility assay revealed that calaxin directly suppressed the velocity of microtubule sliding by outer-arm dynein at high Ca2+ concentrations. This study describes the missing link between chemoattractant-mediated Ca2+ signaling and motor-driven microtubule sliding during sperm chemotaxis.

Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2

Behavioral traits of sperm are adapted to the reproductive strategy that each species employs. In polyandrous species, spermatozoa often form motile clusters, which might be advantageous for competing with sperm from other males. Despite this presumed advantage for reproductive success, little is known about how sperm form such functional assemblies. Previously, we reported that males of the coastal squid Loligo bleekeri produce two morphologically different euspermatozoa that are linked to distinctly different mating behaviors. Consort and sneaker males use two distinct insemination sites, one inside and one outside the females body, respectively. Here, we show that sperm release a self-attracting molecule that causes only sneaker sperm to swarm. We identified CO2 as the sperm chemoattractant and membrane-bound flagellar carbonic anhydrase as its sensor. Downstream signaling results from the generation of extracellular H(+), intracellular acidosis, and recovery from acidosis. These signaling events elicit Ca(2+)-dependent turning behavior, resulting in chemotactic swarming. These results illuminate the bifurcating evolution of sperm underlying the distinct fertilization strategies of this species.

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

Abstract 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.

How to track spermatozoa using high-speed visual feedback

In this paper, we report how to track quickly and vigorously swimming ascidian spermatozoa using high-speed visual feedback at a frame rate of 1 kHz. Ascidian spermatozoa swim as fast as 300 μm/s by rotating their flagella 50 times/s. This vigorous swimming style has prevented stable image observation and made it difficult to track them reliably with our previously developed visual tracking system. Here, we describe how we overcame these problems using image processing techniques to achieve stable tracking of fast, small ascidian spermatozoa for more than 180 s using high-speed visual feedback.

ankAT-1 is a novel gene mediating the apical tuft formation in the sea urchin embryo

In sea urchin embryos, the apical tuft forms within the neurogenic animal plate. When FoxQ2, one of the earliest factors expressed specifically in the animal plate by early blastula stage, is knocked down, the structure of the apical tuft is altered. To determine the basis of this phenotype, we identified FoxQ2-dependent genes using microarray analysis. The most strongly down-regulated gene in FoxQ2 morphants encodes a protein with ankyrin repeats region in its N-terminal domain. We named this gene ankAT-1, Ankyrin-containing gene specific for Apical Tuft. Initially its expression in the animal pole region of very early blastula stage embryos is FoxQ2-independent but becomes FoxQ2-dependent beginning at mesenchyme blastula stage and continuing in the animal plate of 3-day larvae. Furthermore, like FoxQ2, this gene is expressed throughout the expanded apical tuft region that forms in embryos lacking nuclear β-catenin. When AnkAT-1 is knocked-down by injecting a morpholino, the cilia at the animal plate in the resulting embryos are much shorter and their motility is less than that of motile cilia in other ectoderm cells, and remains similar to that of long apical tuft cilia. We conclude that AnkAT-1 is involved in regulating the length of apical tuft cilia.

The birth of quail chicks after intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) has been successfully used to produce offspring in several mammalian species including humans. However, ICSI has not been successful in birds because of the size of the egg and difficulty in mimicking the physiological polyspermy that takes place during normal fertilization. Microsurgical injection of 20 or more spermatozoa into an egg is detrimental to its survival. Here, we report that injection of a single spermatozoon with a small volume of sperm extract (SE) or its components led to the development and birth of healthy quail chicks. SE contains three factors – phospholipase Cζ (PLCZ), aconitate hydratase (AH) and citrate synthase (CS) – all of which are essential for full egg activation and subsequent embryonic development. PLCZ induces an immediate, transient Ca2+ rise required for the resumption of meiosis. AH and CS are required for long-lasting, spiral-like Ca2+ oscillations within the activated egg, which are essential for cell cycle progression in early embryos. We also found that co-injection of cRNAs encoding PLCZ, AH and CS support the full development of ICSI-generated zygotes without the use of SE. These findings will aid our understanding of the mechanism of avian fertilization and embryo development, as well as assisting in the manipulation of the avian genome and the production of transgenic and cloned birds.