Kazuo Inaba

Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor

Changes in membrane potential affect ion channels and transporters, which then alter intracellular chemical conditions. Other signalling pathways coupled to membrane potential have been suggested but their underlying mechanisms are unknown. Here we describe a novel protein from the ascidian Ciona intestinalis that has a transmembrane voltage-sensing domain homologous to the S1–S4 segments of voltage-gated channels and a cytoplasmic domain similar to phosphatase and tensin homologue. This protein, named C. intestinalis voltage-sensor-containing phosphatase (Ci-VSP), displays channel-like ‘gating’ currents and directly translates changes in membrane potential into the turnover of phosphoinositides. The activity of the phosphoinositide phosphatase in Ci-VSP is tuned within a physiological range of membrane potential. Immunocytochemical studies show that Ci-VSP is expressed in Ciona sperm tail membranes, indicating a possible role in sperm function or morphology. Our data demonstrate that voltage sensing can function beyond channel proteins and thus more ubiquitously than previously realized.

Molecular Architecture of the Sperm Flagella: Molecules for Motility and Signaling

Abstract Sperm motility is generated by a highly organized, microtubule-based structure, called the axoneme, which is constructed from approximately 250 proteins. Recent studies have revealed the molecular structures and functions of a number of axonemal components, including the motor molecules, the dyneins, and regulatory substructures, such as radial spoke, central pair, and other accessory structures. The force for flagellar movement is exerted by the sliding of outer-doublet microtubules driven by the molecular motors, the dyneins. Dynein activity is regulated by the radial spoke/central pair apparatus through protein phosphorylation, resulting in flagellar bend propagation. Prior to fertilization, sperm exhibit dramatic motility changes, such as initiation and activation of motility and chemotaxis toward the egg. These changes are triggered by changes in the extracellular ionic environment and substances released from the female reproductive tract or egg. After reception of these extracellular signals by specific ion channels or receptors in the sperm cells, intracellular signals are switched on through tyrosine protein phosphorylation, Ca2+, and cyclic nucleotide-dependent pathways. All these signaling molecules are closely arranged in each sperm flagellum, leading to efficient activation of motility.

A chemoattractant for ascidian spermatozoa is a sulfated steroid

Sperm chemotaxis toward eggs before fertilization has been demonstrated in many animals and plants, and several peptides and small organic compounds acting as chemoattractants have been identified. We previously showed that sperm of the ascidians Ciona intestinalis and Ciona savignyi are activated and then attracted toward the egg by a common factor released from the egg. In this study, we purified sperm-activating and -attracting factor (SAAF) from the egg-conditioning medium of C. intestinalis by using several steps of column chromatography. Determination of the molecular structure by NMR and MS/MS analysis revealed that SAAF is a previously uncharacterized sulfated steroid: 3,4,7,26-tetrahydroxycholestane-3,26-disulfate. Furthermore, it was shown that the SAAF of C. savignyi was indistinguishable from that of C. intestinalis in terms of the chromatographic behavior and molecular weight, indicating that the same compound might be responsible for sperm activation and chemotaxis in both the species. Furthermore, we established a method for quantitative analysis of sperm chemotaxis and showed that the chemotactic behavior of Ciona sperm is controlled by the “chemotactic turn” associated with decrease in the concentration of SAAF.

A web‐based interactive developmental table for the ascidian Ciona intestinalis, including 3D real‐image embryo reconstructions: I. From fertilized egg to hatching larva

The ascidian chordate Ciona intestinalis is an established model organism frequently exploited to examine cellular development and a rapidly emerging model organism with a strong potential for developmental systems biology studies. However, there is no standardized developmental table for this organism. In this study, we made the standard web‐based image resource called FABA: Four‐dimensional Ascidian Body Atlas including ascidians three‐dimensional (3D) and cross‐sectional images through the developmental time course. These images were reconstructed from more than 3,000 high‐resolution real images collected by confocal laser scanning microscopy (CLSM) at newly defined 26 distinct developmental stages (stages 1–26) from fertilized egg to hatching larva, which were grouped into six periods named the zygote, cleavage, gastrula, neurula, tailbud, and larva periods. Our data set will be helpful in standardizing developmental stages for morphology comparison as well as for providing the guideline for several functional studies of a body plan in chordate. Developmental Dynamics 236:1790–1805, 2007.

Sperm flagella: comparative and phylogenetic perspectives of protein components

Sperm motility is necessary for the transport of male DNA to eggs in species with both external and internal fertilization. Flagella comprise several proteins for generating and regulating motility. Central cytoskeletal structures called axonemes have been well conserved through evolution. In mammalian sperm flagella, two accessory structures (outer dense fiber and the fibrous sheath) surround the axoneme. The axonemal bend movement is based on the active sliding of axonemal doublet microtubules by the molecular motor dynein, which is divided into outer and inner arm dyneins according to positioning on the doublet microtubule. Outer and inner arm dyneins play different roles in the production and regulation of flagellar motility. Several regulatory mechanisms are known for both dyneins, which are important in motility activation and chemotaxis at fertilization. Although dynein itself has certain properties that contribute to the formation and propagation of flagellar bending, other axonemal structures-specifically, the radial spoke/central pair apparatus-have essential roles in the regulation of flagellar bending. Recent genetic and proteomic studies have explored several new components of axonemes and shed light on the generation and regulation of sperm motility during fertilization.

Molecular basis of sperm flagellar axonemes: structural and evolutionary aspects.

Abstract:  The axonemes serve as motile machineries in sperm flagella. Although atypical axonemal structures are observed in some cases, 9 + 2 microtubule structure of the axoneme is predominant in many organisms. Several structures are bound to these microtubules and comprise a highly organized protein network. Extensive proteomic analysis of the axonemes has led to find several repeats, domains, and motifs in axonemal proteins. Molecular comparison of subunit composition of axonemal substructures between the ascidian Ciona intestinalis and the green algae Chlamydomonas reinhardtti leads to an intriguing molecular aspect concerning the evolution of intracellular functional complex: The architecture of the axonemes has been well conserved through evolution, but the molecular structure of each axonemal component is not always conserved. In light of domain structure in the axonemal proteins, substructures like outer arm dynein and radial spoke contain a set of domain structures, although some domain‐containing subunits are different between these two organisms. Thus, conservation of protein domains within a substructure seems to take precedence over that of each protein (“module‐dominant conservation”), which may ultimately result in morphological and functional conservation of the axonemes through evolution.

Molecular cloning and characterization of a thioredoxin/nucleoside diphosphate kinase related dynein intermediate chain from the ascidian, Ciona intestinalis

Flagellar outer arm dynein from the ascidian, Ciona intestinalis, contains five intermediate chains (IC1-5). Molecular cloning of C. intestinalis IC3 shows significant sequence homology to the dynein intermediate chain (IC1) from sea urchin and human NM23-H8 protein. The N-terminal thioredoxin-related region is well conserved in the C. intestinalis IC3, sea urchin IC1, and human NM23-H8 protein. Three NDP kinase (NDPK)-related sequences are present in middle portions of both C. intestinalis IC3 and sea urchin IC1, but the human NM23-H8 protein had only two. A large part of the C-terminal glutamic acid-rich region present in sea urchin IC1 was greatly reduced in C. intestinalis IC3 and completely lost in human NM23-H8. Thus, thioredoxin/NDPK-related dynein intermediate chains (TNDK-DIC) would be a characteristic of metazoan flagella and they have become smaller in size and less acidic during evolution.

Cyclic AMP- and calmodulin-dependent phosphorylation of 21 and 26 kDa proteins in axoneme is a prerequisite for SAAF-induced motile activation in ascidian spermatozoa

Sperm activating and ‐attracting factor (SAAF), derived from the egg of the ascidian Ciona, activates sperm motility through adenosine 3′:5′‐cyclic monophosphate (cAMP)‐synthesis. A demembranated preparation of intact immotile sperm without SAAF was shown to require cAMP for reactivation. However, a demembranated preparation of intact motile sperm treated with SAAF did not require cAMP for reactivation, suggesting that cAMP is a prerequisite factor for SAAF‐dependent activation of sperm motility. Furthermore, a cAMP‐dependent protein kinase (PKA) inhibitor, H‐89, was found to inhibit sperm motility. During in vivo or in vitro activation of sperm motility by SAAF or cAMP, a 26 kDa axonemal protein and 21 kDa dynein light chain were phosphorylated, respectively, suggesting the involvement of PKA‐dependent phosphorylation of these proteins in sperm activation. The calmodulin antagonist, W‐7, and an inhibitor of calmodulin‐dependent myosin light chain kinase, ML‐7, also inhibited the activation of sperm motility. Inhibition was reversed by the addition of phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine. Demembranated preparations of immotile sperm in the presence of W‐7 or ML‐7 were reactivated by cAMP, suggesting that calmodulin participated in sperm activation and that cAMP synthesis was followed by activation of a calmodulin‐dependent mechanism.

Calcium and Cyclic AMP Mediate Sperm Activation, but Ca2+Alone Contributes Sperm Chemotaxis in the Ascidian, Ciona savignyi

Sperm‐activating and ‐attracting factor (SAAF) released from the ascidian, Ciona savignyi, was partially purified from egg seawater with ethanol extraction and separation with the two‐phase system of chloroform and water. SAAF did not activate sperm motility and cAMP synthesis in calcium‐free seawater (CaFSW), but activated the both in the presence of Ca2+. Sperm activation by SAAF in Ca2+‐containing medium was inhibited by flunarizine, a T‐type Ca2+channel antagonist, but L‐type Ca2+channel specific antagonists had no effect. Theophylline, a phosphodiesterase inhibitor, induced the increase of cAMP level and sperm activation in CaFSW without SAAF. On the other hand, the theophylline‐activated sperm in CaFSW did not exhibit chemotaxis toward the tip of glass capillary containing SAAF, but upon the addition of Ca2+they were attracted toward SAAF in the same manner as chemotaxis in normal artificial seawater. These results suggest that sperm activation is induced by the increased cAMP level caused by Ca2+influx through T‐type Ca2+channel, and that Ca2+alone mediates the sperm chemotaxis in Ciona.

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.