Akira Onishi - Academia.edu (original) (raw)

Papers by Akira Onishi

Experimental Animals, 1988

Experimental Animals, 1985

Nihon Chikusan Gakkaiho, 1991

The effect of mitochondrial DNA (mtDNA) on heterosis in cytochrome c oxidase activity was investi... more The effect of mitochondrial DNA (mtDNA) on heterosis in cytochrome c oxidase activity was investigated using reciprocal crosses in different combinations of

Journal of Reproduction and Development, 1997

In Exp. 1 oocytes were collected from follicles (4-6 mm) in ovaries from prepubertal gilts by asp... more In Exp. 1 oocytes were collected from follicles (4-6 mm) in ovaries from prepubertal gilts by aspiration of the follicles with a syringe or dissection from healthy follicles. After aspiration some oocytes with compact cumulus and granulosa cells were selected. In Exp.2 oocytes were collected from follicles (4 mm, 5-6 mm and 7-8 mm) of gilts on the 18th-19th day of the cycle. Germinal vesicle (GV) stages of oocytes were examined and categorized into GV1, GV2, GV3 and GV4. In Exp. 1 when collected by aspiration, a significantly lower percentage of oocytes (45%) was arrested at the GV1 stage than oocytes from dissected follicles (78%). However, after morphological selection of oocytes, the percentage (58%) significantly increased. In Exp 2, significantly higher percentages (86-90%) of oocytes from follicles of 5-8 mm in diameter were at the GV1 stage than oocytes from follicles in diameter of 4 mm (24%) and atretic follicles (57%). These results indicate that by aspiration some oocytes are collected from follicles that were inadequate to maintain arrest at GV1, but significantly higher percentages of oocytes at GV1 can be obtained after morphological selection of oocytes or isolation from large follicles of gilts on the 18th-19th day of the cycle.

Journal of Reproduction and Development, 2004

Journal of Reproduction and Development, 2011

The aim of the present study was to examine the feasibility of fluorescent in situ hybridization ... more The aim of the present study was to examine the feasibility of fluorescent in situ hybridization (FISH) for detecting a chromosome 1-specific sequence as a means of assessing the ploidy of porcine parthenotes. In vitro-matured oocytes with the first polar body (PB) were electrically activated; some were treated with cytochalasin B to prevent second PB extrusion (1PB embryos), and the others extruded the second PB (2PB embryos). At the 2-cell stage, one and two FISH signals were detected in each nucleus of 2PB and 1PB embryos, respectively. Almost all cells of blastocysts derived from 1PB embryos retained two signals. In contrast, cells of blastocysts derived from 2PB embryos had two signals. These data demonstrate that FISH analysis allows precise ploidy assessment of porcine parthenogenetic embryos, hence providing a practical means of detecting ploidy transition during parthenogenetic embryogenesis.

Japan Agricultural Research Quarterly: JARQ, 2011

In this study, the effects of dibutyryl cyclic AMP (dbcAMP) on the preservation of porcine oocyte... more In this study, the effects of dibutyryl cyclic AMP (dbcAMP) on the preservation of porcine oocytes were investigated. Oocytes were preserved for 24 h or 48 h in modified NCSU37 supplemented with or without 1 mM dbcAMP. After the preservation, 98.3% and 95.2% of oocytes incubated with dbcAMP for 24 h and 48 h, respectively, were at the germinal vesicle stage, whereas about a half of the oocytes incubated without dbcAMP underwent germinal vesicle break down. These preserved oocytes were cultured for 46 h. The in vitro maturation (IVM) rates of oocytes preserved with dbcAMP for 24 h (84.0%) and 48 h (55.3%) were significantly higher than those of oocytes preserved without dbcAMP (48.0% and 21.6%, respectively). IVM oocytes with a visible first polar body were selected, fertilized in vitro (IVF), and cultured for five days. The rate of oocytes preserved for 24 h with dbcAMP and developed to the blastocyst stage after IVF was 21.4% and did not differ from that of oocytes without preservation (23.8%); however, the blastocyst development rate significantly decreased for oocytes preserved with dbcAMP for 48 h (5.2%). These results indicate that dbcAMP is effective for the preservation of porcine oocytes for 24 h without decreasing their developmental ability after IVF.

Nihon Chikusan Gakkaiho, 1992

Scientific reports, Jan 5, 2017

Grafting of testicular tissue into immunodeficient mice makes it possible to obtain functional sp... more Grafting of testicular tissue into immunodeficient mice makes it possible to obtain functional sperm from immature donor animals that cannot be used for reproduction. We have developed a porcine model of human haemophilia A (haemophilia-A pigs) by nuclear transfer cloning from foetal fibroblasts after disruption of the X-linked coagulation factor VIII (F8) gene. Despite having a recessive condition, female F8 cloned pigs died of severe bleeding at an early age, as was the case for male F8 cloned pigs, thus making it impossible to obtain progeny. In this study, therefore, we produced sperm from F8 cloned pigs by grafting their foetal testicular tissue into nude mice. Two F8 female pigs were generated from oocytes injected with xenogeneic sperm. Unlike the F8 cloned pigs, they remained asymptomatic, and delivered five F8 and four F8 pigs after being crossed with wild-type boars. The descendant F8 pigs conserved the haemophilia phenotype. Thus, the present F8 pigs show resolution of th...

Nihon Chikusan Gakkaiho

We investigated the association between heterozygosity at 13 marker loci

Science advances, Sep 1, 2016

Genetically modified pigs for biomedical applications have been mainly generated using the somati... more Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential fo...

Journal of Reproduction and Development, 2016

In pigs, the damaged sperm membrane leads to leakage of phospholipase C-ζ (PLCζ), which has been ... more In pigs, the damaged sperm membrane leads to leakage of phospholipase C-ζ (PLCζ), which has been identified as a sperm factor, and a reduction of oocyte-activating ability. In this study, we investigated whether sperm selected by Percoll gradient centrifugation (Percoll) have sufficient PLCζ, and whether the efficiency of fertilization and blastocyst formation after intracytoplasmic sperm injection (ICSI) using Percoll-selected sperm can be improved. Percoll-selected sperm (Percoll group) or sperm without Percoll selection (Control group) were used. A proportion of the oocytes injected with control sperm were subjected to electrical stimulation at 1 h after ICSI (Cont + ES group). It was found that the Percoll group showed a large amount of PLCζ in comparison with the Control group. Furthermore, application of Percoll-selected sperm for ICSI increased the efficiency of fertilization and embryo development. Thus, these results indicate the Percoll-selected sperm have sufficient PLCζ and high oocyte-activating ability after ICSI in pigs.

PloS one, 2016

Several animal models have facilitated the evaluation and pathological understanding of atheroscl... more Several animal models have facilitated the evaluation and pathological understanding of atherosclerosis, but a definitive animal model of coronary atherosclerosis is not available. We therefore developed low density lipoprotein receptor knockout (LDLR-KO) pigs with hypercholesterolemia, a model which rapidly developed coronary atherosclerosis following balloon injury. We deleted LDLR exon regions from cultured porcine fetal fibroblasts and cloned LDLR knockout (LDLR-KO) embryos microinjecting fetal fibroblast nuclei into enucleated oocytes. Twelve LDLR-KO pigs were fed a 2.0% cholesterol and 20% fat diet. Baseline serum LDL cholesterol level was 510.0±86.1 mg/dL. Balloon injury was created in 46 coronary segments and necropsy were obtained 2, 4, 8 and 12 weeks later. Coronary artery sections were reviewed to evaluate lesion progression. We found lipid accumulation with foam cells and inflammatory cells beginning four weeks after balloon injury. The mean ratio of macrophages to plaqu...

The Journal of animal genetics, 1994

日本畜産學會報 the Japanese Journal of Zootechnical Science, Jan 25, 1999

Nihon juigaku zasshi. The Japanese journal of veterinary science, 1988

Transgenic Animal Technology, 2014

Reproduction, Fertility and Development, 2007

The amelogenin (AMEL) gene exists on both X and Y chromosomes in various mammalian species. The n... more The amelogenin (AMEL) gene exists on both X and Y chromosomes in various mammalian species. The non-coding region of this gene is different between X and Y chromosomes. The use of this gene has made sex determination much less complicated, since only one pair of primers is required to amplify the different size fragments of the AMEL gene. Therefore, AMEL had been successfully used to determine the sex in cattle, sheep, and humans. The difference of AMEL genomic sequences between X and Y chromosomes has also been found in pig. In this study, we designed primers that identified AMEL of both chromosomes. The amplicons were isolated and sequenced, and showed a length polymorphism characteristic for the X and Y chromosome in pigs. Furthermore we examined whether a single oocyte or embryo could be sexed. Genomic DNA samples were collected from various breeds of pigs (European breeds: Landrace, Large White, Duroc, Berkshire; Chinese breeds: Meishan, Jinhua). DNA was extracted from ears, ta...

Practical Manual of In Vitro Fertilization, 2012

Nihon Yoton Gakkaishi, 1988

Experimental Animals, 1988

Experimental Animals, 1985

Nihon Chikusan Gakkaiho, 1991

The effect of mitochondrial DNA (mtDNA) on heterosis in cytochrome c oxidase activity was investi... more The effect of mitochondrial DNA (mtDNA) on heterosis in cytochrome c oxidase activity was investigated using reciprocal crosses in different combinations of

Journal of Reproduction and Development, 1997

In Exp. 1 oocytes were collected from follicles (4-6 mm) in ovaries from prepubertal gilts by asp... more In Exp. 1 oocytes were collected from follicles (4-6 mm) in ovaries from prepubertal gilts by aspiration of the follicles with a syringe or dissection from healthy follicles. After aspiration some oocytes with compact cumulus and granulosa cells were selected. In Exp.2 oocytes were collected from follicles (4 mm, 5-6 mm and 7-8 mm) of gilts on the 18th-19th day of the cycle. Germinal vesicle (GV) stages of oocytes were examined and categorized into GV1, GV2, GV3 and GV4. In Exp. 1 when collected by aspiration, a significantly lower percentage of oocytes (45%) was arrested at the GV1 stage than oocytes from dissected follicles (78%). However, after morphological selection of oocytes, the percentage (58%) significantly increased. In Exp 2, significantly higher percentages (86-90%) of oocytes from follicles of 5-8 mm in diameter were at the GV1 stage than oocytes from follicles in diameter of 4 mm (24%) and atretic follicles (57%). These results indicate that by aspiration some oocytes are collected from follicles that were inadequate to maintain arrest at GV1, but significantly higher percentages of oocytes at GV1 can be obtained after morphological selection of oocytes or isolation from large follicles of gilts on the 18th-19th day of the cycle.

Journal of Reproduction and Development, 2004

Journal of Reproduction and Development, 2011

The aim of the present study was to examine the feasibility of fluorescent in situ hybridization ... more The aim of the present study was to examine the feasibility of fluorescent in situ hybridization (FISH) for detecting a chromosome 1-specific sequence as a means of assessing the ploidy of porcine parthenotes. In vitro-matured oocytes with the first polar body (PB) were electrically activated; some were treated with cytochalasin B to prevent second PB extrusion (1PB embryos), and the others extruded the second PB (2PB embryos). At the 2-cell stage, one and two FISH signals were detected in each nucleus of 2PB and 1PB embryos, respectively. Almost all cells of blastocysts derived from 1PB embryos retained two signals. In contrast, cells of blastocysts derived from 2PB embryos had two signals. These data demonstrate that FISH analysis allows precise ploidy assessment of porcine parthenogenetic embryos, hence providing a practical means of detecting ploidy transition during parthenogenetic embryogenesis.

Japan Agricultural Research Quarterly: JARQ, 2011

In this study, the effects of dibutyryl cyclic AMP (dbcAMP) on the preservation of porcine oocyte... more In this study, the effects of dibutyryl cyclic AMP (dbcAMP) on the preservation of porcine oocytes were investigated. Oocytes were preserved for 24 h or 48 h in modified NCSU37 supplemented with or without 1 mM dbcAMP. After the preservation, 98.3% and 95.2% of oocytes incubated with dbcAMP for 24 h and 48 h, respectively, were at the germinal vesicle stage, whereas about a half of the oocytes incubated without dbcAMP underwent germinal vesicle break down. These preserved oocytes were cultured for 46 h. The in vitro maturation (IVM) rates of oocytes preserved with dbcAMP for 24 h (84.0%) and 48 h (55.3%) were significantly higher than those of oocytes preserved without dbcAMP (48.0% and 21.6%, respectively). IVM oocytes with a visible first polar body were selected, fertilized in vitro (IVF), and cultured for five days. The rate of oocytes preserved for 24 h with dbcAMP and developed to the blastocyst stage after IVF was 21.4% and did not differ from that of oocytes without preservation (23.8%); however, the blastocyst development rate significantly decreased for oocytes preserved with dbcAMP for 48 h (5.2%). These results indicate that dbcAMP is effective for the preservation of porcine oocytes for 24 h without decreasing their developmental ability after IVF.

Nihon Chikusan Gakkaiho, 1992

Scientific reports, Jan 5, 2017

Grafting of testicular tissue into immunodeficient mice makes it possible to obtain functional sp... more Grafting of testicular tissue into immunodeficient mice makes it possible to obtain functional sperm from immature donor animals that cannot be used for reproduction. We have developed a porcine model of human haemophilia A (haemophilia-A pigs) by nuclear transfer cloning from foetal fibroblasts after disruption of the X-linked coagulation factor VIII (F8) gene. Despite having a recessive condition, female F8 cloned pigs died of severe bleeding at an early age, as was the case for male F8 cloned pigs, thus making it impossible to obtain progeny. In this study, therefore, we produced sperm from F8 cloned pigs by grafting their foetal testicular tissue into nude mice. Two F8 female pigs were generated from oocytes injected with xenogeneic sperm. Unlike the F8 cloned pigs, they remained asymptomatic, and delivered five F8 and four F8 pigs after being crossed with wild-type boars. The descendant F8 pigs conserved the haemophilia phenotype. Thus, the present F8 pigs show resolution of th...

Nihon Chikusan Gakkaiho

We investigated the association between heterozygosity at 13 marker loci

Science advances, Sep 1, 2016

Genetically modified pigs for biomedical applications have been mainly generated using the somati... more Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential fo...

Journal of Reproduction and Development, 2016

In pigs, the damaged sperm membrane leads to leakage of phospholipase C-ζ (PLCζ), which has been ... more In pigs, the damaged sperm membrane leads to leakage of phospholipase C-ζ (PLCζ), which has been identified as a sperm factor, and a reduction of oocyte-activating ability. In this study, we investigated whether sperm selected by Percoll gradient centrifugation (Percoll) have sufficient PLCζ, and whether the efficiency of fertilization and blastocyst formation after intracytoplasmic sperm injection (ICSI) using Percoll-selected sperm can be improved. Percoll-selected sperm (Percoll group) or sperm without Percoll selection (Control group) were used. A proportion of the oocytes injected with control sperm were subjected to electrical stimulation at 1 h after ICSI (Cont + ES group). It was found that the Percoll group showed a large amount of PLCζ in comparison with the Control group. Furthermore, application of Percoll-selected sperm for ICSI increased the efficiency of fertilization and embryo development. Thus, these results indicate the Percoll-selected sperm have sufficient PLCζ and high oocyte-activating ability after ICSI in pigs.

PloS one, 2016

Several animal models have facilitated the evaluation and pathological understanding of atheroscl... more Several animal models have facilitated the evaluation and pathological understanding of atherosclerosis, but a definitive animal model of coronary atherosclerosis is not available. We therefore developed low density lipoprotein receptor knockout (LDLR-KO) pigs with hypercholesterolemia, a model which rapidly developed coronary atherosclerosis following balloon injury. We deleted LDLR exon regions from cultured porcine fetal fibroblasts and cloned LDLR knockout (LDLR-KO) embryos microinjecting fetal fibroblast nuclei into enucleated oocytes. Twelve LDLR-KO pigs were fed a 2.0% cholesterol and 20% fat diet. Baseline serum LDL cholesterol level was 510.0±86.1 mg/dL. Balloon injury was created in 46 coronary segments and necropsy were obtained 2, 4, 8 and 12 weeks later. Coronary artery sections were reviewed to evaluate lesion progression. We found lipid accumulation with foam cells and inflammatory cells beginning four weeks after balloon injury. The mean ratio of macrophages to plaqu...

The Journal of animal genetics, 1994

日本畜産學會報 the Japanese Journal of Zootechnical Science, Jan 25, 1999

Nihon juigaku zasshi. The Japanese journal of veterinary science, 1988

Transgenic Animal Technology, 2014

Reproduction, Fertility and Development, 2007

The amelogenin (AMEL) gene exists on both X and Y chromosomes in various mammalian species. The n... more The amelogenin (AMEL) gene exists on both X and Y chromosomes in various mammalian species. The non-coding region of this gene is different between X and Y chromosomes. The use of this gene has made sex determination much less complicated, since only one pair of primers is required to amplify the different size fragments of the AMEL gene. Therefore, AMEL had been successfully used to determine the sex in cattle, sheep, and humans. The difference of AMEL genomic sequences between X and Y chromosomes has also been found in pig. In this study, we designed primers that identified AMEL of both chromosomes. The amplicons were isolated and sequenced, and showed a length polymorphism characteristic for the X and Y chromosome in pigs. Furthermore we examined whether a single oocyte or embryo could be sexed. Genomic DNA samples were collected from various breeds of pigs (European breeds: Landrace, Large White, Duroc, Berkshire; Chinese breeds: Meishan, Jinhua). DNA was extracted from ears, ta...

Practical Manual of In Vitro Fertilization, 2012

Nihon Yoton Gakkaishi, 1988