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Dive into the research topics where Kazuo Onitake is active.

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Featured researches published by Kazuo Onitake.


Development Growth & Differentiation | 1997

Establishment of in vitro spermatogenesis from spermatocytes in the medaka, Oryzias latipes

Atsusi Saiki; Masaru Tamura; Masami Matsumoto; Jun Katowgi; Akihiko Watanabe; Kazuo Onitake

Spermatocytes of the teleost, Oryzias latipes, at meiotic prophase were cultured without contact with somatic cells. They began to divide, progressing through the meiotic divisions and differentiating into round spermatids within 48 h. The chromosome number in both the primary and secondary spermatocytes at metaphase was n= 24. In spermatids, a single flagellum was formed and the release of residual bodies was observed in vitro. The size and shape of the flagellum were the same as those seen in vivo. The expression of protamine mRNA was detected in round spermatids. This result suggests that gene expression, as well as morphological change, is regulated by the progression of spermatogenesis in cell culture. Furthermore, when the eggs of O. latipes were inseminated with germ cells cultured for 10 days, normal embryos developed and hatched out. These results suggest that the spermatocytes of O. latipes develop into fertile sperm in cell culture.


Development Growth & Differentiation | 1991

Time Sequence of Early Events in Fertilization in the Medaka Egg

Takashi Iwamatsu; Kazuo Onitake; Yasuaki Yoshimoto; Yukio Hiramoto

The time sequence of early events in fertilization was examined in eggs of the medaka Oryzias latipes. The mean time after insemination required for sperm attachment to the egg surface through the micropyle depended on sperm concentrations. It was 3 ± 1 sec with a range from 1 to 6 sec after insemination when concentration of spermatozoa was high (about 2 × 108/ml at 23°–25°C). The mean time from sperm attachment until cessation of its movement on the egg surface was 4 ± 1 sec with a range from 1 to 9 sec. Small cortical alveoli at the animal pole region within 15 μm of the sperm attachment point began to undergo exocytosis 9 ± 0.3 sec (range 5–16 sec) after sperm attachment. The velocity at which the exocytosis wave propagated increased from the earliest initiation point of exocytosis up to the 100 μm area, and became constant at about 12 μm/sec from 100 μm to 500 μm from the sperm attachment point. The present results suggest that at the time of fertilization in the fish egg, exocytosis of small cortical alveoli in the area about 15 μm away from the sperm attachment point occurs simultaneously.


Zoological Science | 1999

Substances for the Initiation of Sperm Motility in Egg-Jelly of the Japanese Newt, Cynops pyrrhogaster

Masahiko Ukita; Tokuko Itoh; Toshihiko Watanabe; Akihiko Watanabe; Kazuo Onitake

Abstract The initiation of sperm motility is regulated by certain factors, including a change of osmolality or ion concentration. The cue for the initiation of sperm motility is unique to species and suits the environment in which the fertilization occurs. In the newt, Cynops pyrrhogaster, eggs are fertilized in the cloaca of the female with sperm stored in the sperm reservoir. In this study, we investigated possible factors initiating sperm motility in this unique environment. Sperm of C. pyrrhogaster could be initiated to move by a decrease of osmolality. However, eggs were fertilized with dry sperm and developed to four-cell stage embryos without immersion in solution. They continued to develop normally to the tail-bud stage when placed in Steinbergs salt solution after fertilization. These results indicate that sperm motility was initiated without the change of osmolality around sperm. In egg-jelly extract, the activity for the initiation of sperm motility was strong and heat stable, but disappeared by proteinase treatment. ICP luminescence analysis revealed that sodium, potassium, calcium and magnesium ions were major cations in egg-jelly. The monovalent cations showed the activity for the initiation of sperm motility in high concentration at high pH, and this activity become stronger by the addition of calcium ion. However, the reconstructed ionic solution that was prepared according to the concentrations of these four ions and pH in egg-jelly did not show the activity as strong as egg-jelly extract. These results suggest that the proteinacious factor and cations in egg-jelly are significant to regulate the initiation of sperm motility in C. pyrrhogaster.


The International Journal of Developmental Biology | 2010

Identification of the sperm motility-initiating substance in the newt, Cynops pyrrhogaster, and its possible relationship with the acrosome reaction during internal fertilization

Toshihiko Watanabe; Hideo Kubo; Shinya Takeshima; Mami Nakagawa; Manami Ohta; Saori Kamimura; Eriko Takayama-Watanabe; Akihiko Watanabe; Kazuo Onitake

Motility initiation is a key event during internal fertilization of female-stored sperm, although the underlying mechanisms remain unclear. In internally fertilizing urodeles, quiescent sperm initiate motility on the surface of the egg-jelly, a thick extracellular matrix that accumulates around the egg in oviduct. By immunizing mice with egg-jelly extracts, we successfully generated an alpha34 monoclonal antibody (mAb) which neutralized sperm motility-initiating activity in the egg-jelly of the newt, Cynops pyrrhogaster, in a dose-dependent manner. The alpha34 mAb recognized an unglycosylated 34 kDa protein in the outermost of the six layers that comprise egg-jelly. Under nonreducing conditions, immunoblotting with alpha34 mAb produced many bands in addition to the 34 kDa protein, suggesting that the 34 kDa protein associates not only with the jelly matrix itself, but also with additional substances present in the matrix. Our current results are compatible with the supposed features of sperm motility-initiating substance (SMIS), indicating that the 34 kDa protein itself, or a complex consisting of the 34 kDa protein and some other molecules, is the SMIS in C. pyrrhogaster. Immunofluorescence staining further indicated that SMIS was distributed in a dot-like pattern in the outermost jelly layer and was fully covered with acrosome reaction-inducing substance (ARIS). Immunocytochemical and scanning electron microscopic examinations of the outermost jelly layer strongly suggests that the 34 kDa protein localized in granules (2 microm) and that ARIS was distributed covering the granules and in the sheet-like structure above the granules. These data suggest that the initiation of sperm motility is mediated by the acrosome reaction.


Development Growth & Differentiation | 1994

Activation of Xenopus Eggs by an Extract of Cynops Sperm

Yasuhiro Iwao; Akiko Miki; Michiko Kobayashi; Kazuo Onitake

An extract obtained from Cynops sperm induced the activation of both Cynops and Xenopus eggs with accompanying changes in the potential of the egg membrane that were quite similar to those caused by the Cynops sperm. The activation‐inducing properties of the extract were abolished by treatment with proteinase K or by heating (60°C, 15 min) and were associated with a protease activity against peptidyl Arg‐MCA substrates. The activation of Xenopus eggs by the extract was inhibited by those substrates, or by protease inhibitors, aprotinin or leupeptin. The protease activity was localized in the acrosomal region of Cynops sperm. The activation of Xenopus eggs by the extract was prevented when the exterior concentration of Ca2+ions, [Ca2+]0, was reduced to 1.5 μM, but it was enhanced when [Ca2+]0 was increased to 340 μM. The activation of Xenopus eggs by the extract was not affected by positive clamping when [Ca2+]0 was 340 μM. These results suggest that the sperm extract contains a protease that causes an increase in the influx of Ca2+ions that results in voltage‐insensitive activation of the egg.


Zygote | 2002

The activity for the induction of the sperm acrosome reaction localises in the outer layers and exists in the high-molecular-weight components of the egg-jelly of the newt, Cynops pyrrhogaster

Takahiro Sasaki; Saori Kamimura; Hiroyuki Takai; Akihiko Watanabe; Kazuo Onitake

Localisation of the acrosome reaction inducing activity in egg-jelly was examined in the newt, Cynops pyrrhogaster. The jelly has six layers: the J0, J1, J2, J3, J4 and st layers. Jelly was mechanically dissected and placed on a Millipore filter. When sperm were added from the outer surface side of the jelly, most of them exhibited the acrosome reaction after passing through the jelly. When egg-jelly was divided into four layers, strong activity for the induction of acrosome reaction was detected in the outer layers, J4+st. These findings suggest that the acrosome reaction is induced by a substance in the outer layers of the egg-jelly. Among jelly components separated by SDS-PAGE, a fraction of more than 500 kDa in molecular weight induced the acrosome reaction. Wheat germ agglutinin (WGA), Griffonia simplicifoliar agglutinin 1 (GS-1), Maclura pomifera agglutinin (MPA) and Arachis hypogaea agglutinin (PNA) inhibited the induction of the acrosome reaction by jelly extract, and WGA did so in a dose-dependent manner. Those lectins precipitated some molecules of over 500 kDa. These results suggest that the acrosome reaction is induced by the high molecular-weight components of egg-jelly in C. pyrrhogaster.


Molecular and Biochemical Parasitology | 1991

Vaccination against Taenia taeniaeformis infection in rats using a recombinant protein and preliminary analysis of the induced antibody response

Akira Ito; Henrik O. Bøgh; Marshall W. Lightowlers; Graham F. Mitchell; Tsuyoshi Takami; Masao Kamiya; Kazuo Onitake; M. D. Rickard

Primary screening of a cDNA expression library of Taenia taeniaeformis oncospheres in lambda gt11 bacteriophage was carried out using rabbit anti-T, taeniaeformis oncosphere serum affinity-purified from oncosphere pellets. From approximately 1.6 x 10(5) plaques, 21 single clones that were positive with the affinity-purified antibodies were isolated. Sibling analysis revealed that 17 clones out of the 21 could be assigned to five different antigen families. Only family 1 was strongly recognized by a serum prepared in a rabbit against a partially purified host-protective oncosphere antigen fraction. The fragments of lambda DNA were inserted into a pGEX plasmid vector that encodes glutathione S-transferase (GST) of Schistosoma japonicum. Clones designated TtO-18, -49.53 (family 1), 46 (family 2), 15 (family 3), 40 (family 4) and 66 (family 5) were established as subclones in pGEX-1 plasmid vectors which produced GST fusion proteins. All GST fusion proteins were soluble and recognized by anti-GST and anti-TtO sera. Three vaccination experiments with these fusion proteins using specific-pathogen-free Wistar rats revealed that all three fusion proteins of family 1 were exclusively effective against T. taeniaeformis oncosphere challenge with approximately 95% and 91% reductions in cystic metacestode and total metacestode recoveries, respectively. Rats vaccinated with fusion proteins of family 1 produced antibodies which reacted with a 21-kDa oncosphere antigen component which appeared to be a major oncosphere stage-specific antigen.


Zoological Science | 2002

The urodele egg-coat as the apparatus adapted for the internal fertilization.

Akihiko Watanabe; Kazuo Onitake

Abstract Fertilization is a significant event for reproducing offspring. It is achieved under a species-specific environment, which influences the conditions to assure the successful fertilization in some cases. Several studies about the basic mechanism of fertilization suggest that the fertilization mechanism is modified among species to be suited for the fertilization environment. In amphibians, many anurans undergo external fertilization while most urodeles do internal fertilization. An amphibian egg is surrounded by egg-coats, which are composed of vitelline envelope and layered egg-jelly. They are significant as fields for the sperm-egg interaction at fertilization. The fertilization processes that take place in the egg-coats are supposed to be easily influenced by the fertilization environment, because they, especially egg-jelly, are exposed to the surroundings at fertilization. In the present article, we describe the fertilization system equipped in newt egg-coats. Newt sperm are stored in spermatheca that exists in cloaca of a female and directly inseminated on the surface of egg-jelly. Sperm motility and acrosome reaction are induced in the outermost portion of the egg-jelly. Motion of the moving sperm becomes vigorous in the egg-jelly and sperm are guided to vitelline envelope by the aid of egg-jelly structure. Most of the sperm passing through the egg-jelly, as the result, has been induced acrosome reaction and those sperm can bind to the vitelline envelope to contribute to the successful fertilization. This fertilization system has a distinct feature from the known system in species undergoing external fertilization. The feature of the system in the newt egg-jelly is discussed with the view to achieving the successful fertilization in the internal environment.


Zoological Science | 2003

Characteristics of Sperm Motility Induced on the Egg-Jelly in the Internal Fertilization of the Newt, Cynops pyrrhogaster

Toshihiko Watanabe; Tokuko Itoh; Akihiko Watanabe; Kazuo Onitake

Abstract Most urodeles undergo internal fertilization and sperm are directly inseminated onto the surface of egg-jelly. Feature of sperm motility induced on the egg-jelly was examined in the newt, Cynops pyrrhogaster. When sperm were directly inseminated onto an egg-jelly, sperm motility was immediately induced on its surface. The egg-jelly of C. pyrrhogaster was composed of six sublayers that were added by turns in oviduct. When the eggs with various sets of the sublayers were obtained and sperm were inseminated onto the egg-jelly, the immediate activity for the initiation of sperm motility was observed only on the outermost sublayer. Similarly, the immediate initiation of sperm motility was induced in the sperm suspended in the extract of the egg-jelly (JE). The initiation of sperm motility was affected by the external pH, and the motility was activated in the moving sperm. A K+-channel antagonist, charybdotoxin (CTX), or a Ca2+-channel antagonist, gallopamil inhibited the initiation of sperm motility in a dose dependent manner. These results demonstrated the feature of the mechanism regulating sperm motility under stable surroundings in the internal fertilization of amphibians.


Development Growth & Differentiation | 1999

Sperm surface heparin/heparan sulfate is responsible for sperm binding to the uterine envelope in the newt, Cynops pyrrhogaster

Satoshi Nakai; Akihiko Watanabe; Kazuo Onitake

The sperm‐binding properties of egg envelopes are investigated in the newt, Cynops pyrrhogaster. Sperm binding was only seen on the uterine envelope when acrosome‐reacted sperm were inseminated. No acrosome‐intact sperm bound to the envelopes. By scanning electron microscopic observation, acrosome‐reacted sperm were revealed to bind to a seat‐like structure present on the surface of the uterine envelope. Sperm binding to the uterine envelope was inhibited by treatment of eggs with heparin or heparan sulfate, or treatment of acrosome‐reacted sperm with heparinase prior to insemination. A molecule with a molecular mass of 75 kDa was purified from the uterine envelope by affinity chromatography with heparin‐Sepharose. These results indicated that sperm binding was mediated by heparin‐like molecules expressed on the surface of acrosome‐reacted sperm and the 75 kDa molecule was present as a constituent of uterine envelopes.

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Takashi Iwamatsu

Aichi University of Education

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Akira Ito

Asahikawa Medical University

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