Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1262/jrd.2022-065.

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-022-22850-5.

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.pone.0270781.

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.pone.0260645.

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.xpro.2021.100933.

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Freeze-drying techniques allow the preservation of mammalian spermatozoa without using liquid nitrogen. However, the current method requires the use of glass ampoules, which are breakable, expensive, and bulky to store or transport. In this study, we evaluated whether mouse freeze-dried (FD) spermatozoa can be preserved and transported on thin materials. In this study, we demonstrated that FD sperm can be preserved in thin plastic sheets. Its DNA integrity was comparable to that of glass ampoule spermatozoa, and healthy offspring were obtained after preservation at -30°C for more than 3 months. We attached preserved FD sperm to postcards, and transported these to other laboratory inexpensively at room temperatures without any protection. This method will facilitate the preservation of thousands of mouse strains in a single card holder, promote collaboration between laboratories, conservation of genetic resources, and assisted reproductive technology.

DOI： 10.1016/j.isci.2021.102815

PubMed

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1126/sciadv.abg5554.

Language：English   Publishing type：Research paper (scientific journal)  

We examined whether peripheral leukocytes of mice derived from in vitro αMEM-cultured embryos and exhibiting type 2 diabetes had higher expression of inflammatory-related genes associated with the development of atherosclerosis. Also, we examined the impact of a barley diet on inflammatory gene expression. Adult mice were produced by embryo transfer, after culturing two-cell embryos for 48 h in either α minimal essential media (α-MEM) or potassium simplex optimized medium control media. Mice were fed either a barley or rice diet for 10 weeks. Postprandial blood glucose and mRNA levels of several inflammatory genes, including Tnfa and Nox2, in blood leukocytes were significantly higher in MEM mice fed a rice diet compared with control mice. Barley intake reduced expression of S100a8 and Nox2. In summary, MEM mice exhibited postprandial hyperglycemia and peripheral leukocytes with higher expression of genes related to the development of atherosclerosis, and barley intake reduced some gene expression.

DOI： 10.1093/bbb/zbab023

PubMed

Language：Japanese   Publisher：The Society for Reproduction and Development  

DOI： 10.14882/jrds.114.0_p-24

CiNii Research

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1242/dev.190777

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Artificial oocyte activation is important for assisted reproductive technologies, such as fertilization with round spermatids (ROSI) or the production of cloned offspring by somatic cell nuclear transfer (SCNT). Recently, phospholipase Cζ (PLCζ)-cRNA was used to mimic the natural process of fertilization, but this method required the serial injection of PLCζ-cRNA and was found to cause damage to the manipulated oocytes. Here we tried to generate offspring derived from oocytes that were fertilized using round spermatid or somatic cell nuclear transfer with the co-injection of PLCζ-cRNA. After co-injecting round spermatids and 20 ng/µL of PLCζ-cRNA into the oocytes, most of them became activated, but the activation process was delayed by more than 1 h. With the co-injection method, the rate of blastocyst formation in ROSI embryos was higher (64%) compared with that of the serial injection method (55%). On another note, when SCNT was performed using the co-injection method, the cloned offspring were obtained with a higher success rate compared with the serial-injection method. However, in either ROSI or SCNT embryos, the birth rate of offspring via the co-injection method was similar to the Sr activation method. The epigenetic status of ROSI and SCNT zygotes that was examined showed no significant difference among all activation methods. The results indicated that although the PLCζ-cRNA co-injection method did not improve the production rate of offspring, this method simplified oocyte activation with less damage, and with accurate activation time in individual oocytes, it can be useful for the basic study of oocyte activation and development.

DOI： 10.1530/REP-20-0054

PubMed

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Animal Reproduction  

Mouse oocytes are generally collected after euthanasia. However, if oocytes were collected without euthanasia, then mice could be used to collect oocytes again after recovery. This condition is especially useful for mice that are genotypically rare. In this study, we examined the reusability of mice after collecting oocytes via a surgical operation. When oocytes were collected using medetomidine/midazolam/butorphanol combination anesthesia and examined for the quality of oocytes after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), they could develop to full term at the same rate as controls. When oocytes were collected from those mice a second time, the average number of oocytes was reduced by nearly 1/3. However, the blastocyst and offspring rates of those oocytes after IVF or ICSI were the same as those of the control regardless of the recovery day period. Although germinal vesicle (GV) oocytes can be collected from all reused mice, the final number of offspring did not increase. Interestingly, when oocytes were collected from the front position of the ampulla, 76% of the oviducts possessed oocytes after reuse, and the average number of oocytes significantly increased to a level comparable to that of the control. Finally, we examined whether reused mice can be used as recipient females, and then healthy offspring were obtained similarly as the control recipients. In conclusion, we provide a new method to collect a sufficient number of oocytes from reused mice without concern.

DOI： 10.1262/jrd.2020-059

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/rmb2.12312.

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1262/jrd.2019-043.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41467-019-13830-x.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.celrep.2019.04.028.

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1262/jrd.2019-058.

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1038/s41598-019-42062-8.

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1038/s41598-018-33304-2.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1371/journal.pone.0202663.

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1038/s41598-018-28896-8.

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.3791/57068.

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Oocytes without a zona pellucida (ZP) often occur as a result of congenital or operational effects, but they are difficult to handle, and embryonic survival is low. Such zona-free (ZF) oocytes are therefore not used in clinics or laboratories. Furthermore, in the laboratory, removal of the ZP is often necessary for genetic manipulation by viral infection or twin production by blastomere separation, but adverse effects on development have been reported. It would therefore be extremely valuable if the embryo could be covered with a structure similar to that of the ZP. In this study, we sought to determine whether an agarose capsule could serve as a substitute for the ZP. Our results indicate that embryos derived from these agarose capsules were able to develop normally, and could be transplanted to obtain viable offspring, without having to remove the agarose capsule. Furthermore, before compaction, the agarose capsule embryos exhibited good freezing tolerance, and survival rate was extremely high compared to ZF embryos. Thus, agarose capsules represent a valuable tool for utilizing oocytes and embryos that lack a ZP, both in a clinical and livestock setting.

DOI： 10.1038/s41598-017-18365-z

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PubMed

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：NATL ACAD SCIENCES  

DOI： 10.1073/pnas.1711468114

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Less invasive methods for donor cell collection will facilitate reproduction of wild animals using somatic cell nuclear transfer. Stages of the estrous cycle in mice have long been studies using somatic cells that can be collected from vaginal walls using cotton tipped swabs in a relatively non-invasive manner. In this study, we examined the feasibility of these cells as sources of nuclei for somatic-cell cloning using nuclear transfer. Estrous cycles generally comprise proestrus, estrus, metestrus, and diestrus stages. In the present experiments, more than 60% of cells were nucleated in vaginal smears from all but the estrus stage. However, after somatic-cell nuclear transfer of cells from proestrus, metestrus, and diestrus stages, 66%, 50%, and 72% of cloned embryos developed to the morula/blastocyst, and cloned female mouse birth rates after embryo transfer were 1.5%, 0.3%, and 1%, respectively. These results show that noninvasively collected vaginal smears contain somatic cells that can be used to clone female mice. (C) 2017 Published by Elsevier Inc.

DOI： 10.1016/j.theriogenology.2017.02.012

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PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Cloned mice derived from somatic or ES cells show placental overgrowth (placentomegaly) at term. We had previously analyzed cloned and normal mouse placentae by using two-dimensional gel electrophoresis and mass spectrometry to identify differential protein expression patterns. Cloned placentae showed upregulation of tissue inhibitor of metalloproteinase-2 (TIMP-2), which is involved in extracellular matrix degradation and tissue remodeling, and downregulation of pre-B cell colony enhancing factor 1 (PBEF), which inhibits apoptosis and induces spontaneous labor. Here, we used Western blotting to further analyze the protein expression levels of TIMP-2 and PBEF in cloned placentae derived from cumulus cells, TSA-treated cumulus cells, intracytoplasmic sperm injection (ICSI), and natural mating (NM control). Cloned and TSA-treated cloned placentae had higher expression levels of TIMP-2 compared with NM control and ICSI-derived placentae, and there was a positive association between TIMP-2 expression and the placental weight of cloned mouse concepti. Conversely, PBEF protein expression was significantly lower in cloned and ICSI placentae compared to NM controls. To examine whether the observed differences were due to abnormal gene expression caused by faulty epigenetic reprogramming in clones, we investigated DNA methylation and histone modification in the promoter regions of the genes encoding TIMP-2 and PBEF. Sodium bisulfite sequencing did not reveal any difference in DNA methylation between cloned and NM control placentae. However, ChIP assays revealed that the level of H3-K9/K14 acetylation at the TIMP-2 locus was higher in cloned placentae than in NM controls, whereas acetylation of the PBEF promoter was lower in cloned and ICSI placenta versus NM controls. These results suggest that cloned placentae appear to suffer from failure of histone modification-based reprogramming in these (and potentially other) developmentally important genes, leading to aberrant expression of their protein products. These changes are likely to be involved in generating the abnormalities seen in cloned mouse placentae, including enlargement and/or a lack of proper placental function.

DOI： 10.1371/journal.pone.0166241

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PubMed

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1089/cell.2016.0014

PubMed

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38-77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal.

DOI： 10.1038/srep23808

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Language：English  

Language：English  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Generally, transportation of preimplantation embryos without freezing requires incubators that can maintain an optimal culture environment with a suitable gas phase, temperature, and humidity. Such incubators are expensive to transport. We reported previously that normal offspring were obtained when the gas phase and temperature could be maintained during transportation. However, that system used plastic dishes for embryo culture and is unsuitable for long-distance transport of live embryos. Here, we developed a simple low-cost embryo transportation system. Instead of plastic dishes, several types of microtubes-usually used for molecular analysis-were tested for embryo culture. When they were washed and attached to a gas-permeable film, the rate of embryo development from the 1-cell to blastocyst stage was more than 90%. The quality of these blastocysts and the rate of full-term development after embryo transfer to recipient female mice were similar to those of a dish-cultured control group. Next, we developed a small warm box powered by a battery instead of mains power, which could maintain an optimal temperature for embryo development during transport. When 1-cell embryos derived from BDF1, C57BL/6, C3H/He and ICR mouse strains were transported by a parcel-delivery service over 3 days using microtubes and the box, they developed to blastocysts with rates similar to controls. After the embryos had been transferred into recipient female mice, healthy offspring were obtained without any losses except for the C3H/He strain. Thus, transport of mouse embryos is possible using this very simple method, which might prove useful in the field of reproductive medicine.

DOI： 10.1371/journal.pone.0138854

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Language：English  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

In mammals, VDJ recombination is responsible for the establishment of a highly diversified preimmune antibody repertoire. Acquisition of a functional Ig heavy (H) chain variable (V) gene rearrangement is thought to prevent further recombination at the IgH locus. Here, we describe V(H)Q52(NT); V kappa gr32(NT) Ig monoclonal mice reprogrammed from the nucleus of an intestinal IgA(+) plasma cell. In V(H)Q52(NT) mice, IgA replaced IgM to drive early B-cell development and peripheral B-cell maturation. In V(H)Q52(NT) animals, over 20% of mature B cells disrupted the single productive, non-autoimmune IgH rearrangement through VH replacement and exchanged it with a highly diversified pool of IgH specificities. VH replacement occurred in early pro-B cells, was independent of pre-B-cell receptor signaling, and involved predominantly one adjacent VH germ-line gene. VH replacement was also identified in 5% of peripheral B cells of mice inheriting a different productive VH rearrangement expressed in the form of an IgM H chain. In summary, editing of a productive IgH rearrangement through VH replacement can account for up to 20% of the IgH repertoire expressed by mature B cells.

DOI： 10.1073/pnas.1417988112

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Language：English  

Language：English  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Human chromosome fragments (hCFs) and human artificial chromosomes (HACs) can be transferred into mouse ES cells to produce trans-chromosomic (Tc) mice. Although hCFs and HACs containing large genomic DNAs can be autonomously maintained in Tc mice, their retention rate is variable in mouse ES cell lines and Tc mouse tissues, possibly because of centromere differences between the species. To improve the retention rate of artificial chromosomes in mouse cells, we constructed novel mouse artificial chromosome (MAC) vectors by truncating a natural mouse chromosome at a site adjacent to the centromeric region. We obtained cell clones containing the MAC vectors that were stably maintained in mouse ES cells and various tissues in Tc mice. The MACs possess acceptor sites into which a desired gene or genes can be inserted. Thus, Tc mice harboring the MAC vectors may be valuable tools for functional analyses of desired genes, producing humanized model mice, and synthetic biology.

DOI： 10.1021/sb3000723

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PubMed

Language：English  

Language：English  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Transchromosomic (Tc) technology using human chromosome fragments (hCFs), or human artificial chromosomes (HACs), has been used for generating mice containing Mb-sized segments of the human genome. The most significant problem with freely segregating chromosomes with human centromeres has been mosaicism, possibly due to the instability of hCFs or HACs in mice. We report a system for the stable maintenance of Mb-sized human chromosomal fragments following translocation to mouse chromosome 10 (mChr.10). The approach utilizes microcell-mediated chromosome transfer and a combination of site-specific loxP insertion, telomere-directed chromosome truncation, and precise reciprocal translocation for the generation of Tc mice. Human chromosome 21 (hChr.21) was modified with a loxP site and truncated in homologous recombination-proficient chicken DT40 cells. Following transfer to mouse embryonic stem cells harboring a loxP site at the distal region of mChr.10, a similar to 4 Mb segment of hChr.21 was translocated to the distal region of mChr.10 by transient expression of Cre recombinase. The residual hChr.21/mChr.10ter fragment was reduced by antibiotic negative selection. Tc mice harboring the translocated similar to 4 Mb fragment were generated by chimera formation and germ line transmission. The hChr.21-derived Mb fragment was maintained stably in tissues in vivo and expression profiles of genes on hChr.21 were consistent with those seen in humans. Thus, Tc technology that enables translocation of human chromosomal regions onto host mouse chromosomes will be useful for studying in vivo functions of the human genome, and generating humanized model mice.

DOI： 10.1007/s11248-014-9781-4

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Language：English  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CLINICAL INVESTIGATION INC  

The most common forms of neurocristopathy in the autonomic nervous system are Hirschsprung disease (HSCR), resulting in congenital loss of enteric ganglia, and neuroblastoma (NB), childhood tumors originating from the sympathetic ganglia and adrenal medulla. The risk for these diseases dramatically increases in patients with congenital central hypoventilation syndrome (CCHS) harboring a nonpolyalanine repeat expansion mutation of the Paired-like homeobox 2b (PHOX2B) gene, but the molecular mechanism of pathogenesis remains unknown. We found that introducing nonpolyalanine repeat expansion mutation of the PHOX2B into the mouse Phox2b locus recapitulates the clinical features of the CCHS associated with HSCR and NB. In mutant embryos, enteric and sympathetic ganglion progenitors showed sustained sex-determining region Y (SRY) box10 (Sox10) expression, with impaired proliferation and biased differentiation toward. the glial lineage. Nonpolyalanine repeat expansion mutation of PHOX2B reduced transactivation of wild-type PHOX2B on its known target, dopamine beta-hydroxylase (DBH), in a dominant-negative fashion. Moreover, the introduced mutation converted the transcriptional effect of PHOX2B on a Sox10 enhancer from repression to transactivation. Collectively, these data reveal that nonpolyalanine repeat expansion mutation of PHOX2B is both a dominant-negative and gain-of-function mutation. Our results also demonstrate that Sox10 regulation by PHOX2B is pivotal for the development and pathogenesis of the autonomic ganglia.

DOI： 10.1172/JCI63401

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

To clarify the causes of the poor success rate of somatic cell nuclear transfer (SCNT), we addressed the impact of abnormalities observed at early cleavage stages of development on further full-term development using 'less-damage' imaging technology. To visualize the cellular and nuclear division processes, SCNT embryos were injected with a mixture of mRNAs encoding enhanced green fluorescent protein coupled with alpha-tubulin (EGFP-alpha-tubulin) and monomeric red fluorescent protein 1 coupled with histone H2B (H2B-mRFP1) and monitored until the morula/blastocyst stage three-dimensionally. First, the rate of development of SCNT embryos and its effect on the full-term developmental ability were analyzed. The speed of development was retarded and varied in SCNT embryos. Despite the rate of development, SCNT morulae having more than eight cells at 70 h after activation could develop to term. Next, chromosomal segregation was investigated in SCNT embryos during early embryogenesis. To our surprise, more than 90% of SCNT embryos showed abnormal chromosomal segregation (ACS) before they developed to morula stage. Importantly, ACS per se did not affect the rate of development, morphology or cellular differentiation in preimplantation development. However, ACS occurring before the 8-cell stage severely inhibited postimplantation development. Thus, the morphology and/or rate of development are not significant predictive markers for the full-term development of SCNT embryos. Moreover, the low efficiency of animal cloning may be caused primarily by genetic abnormalities such as ACS, in addition to the epigenetic errors described previously. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2012.01.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

In female mammals, one of two X chromosomes is epigenetically inactivated for gene dosage compensation, known as X inactivation (Xi). Inactivation occurs randomly in either the paternal or maternal X chromosome in all embryonic cell lineages, designated as random Xi. By contrast, in extra-embryonic cell lineages, which are segregated from somatic cell lineages in pre-implantation development, the paternal X chromosome is selectively inactivated, known as imprinted Xi. Although it is speculated that erasure of the imprinted mark on either the maternal or paternal X chromosome in somatic cell lineages might change the mode of Xi from imprinted to random, it is not known when this event is completed in development. Here, we tested the mode of Xi during the differentiation of female mouse embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocyst-stage embryos toward trophectoderm (TE) and primitive endoderm (PrE) lineages induced by artificial activation of transcription factor genes Cdx2 and Gata6, respectively. We found that random Xi occurs in both TE and PrE cells. Moreover, cloned embryos generated by the transfer of nuclei from the female ES cells showed random Xi in TE, suggesting the complete erasure of all X imprints for imprinted Xi in ICM-derived ES cells.

DOI： 10.1242/dev.056606

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J-GLOBAL

Language：English  

A recent paper entitled 'Resurrection of DNA function in vivo from an extinct genome' (Pask et al. 2008
PLoS ONE 3:e2240) suggests that the return to life of extinct animals could be just around the corner. Indeed, every time a mammoth is unearthed, hot debates on the possibility of 'resuscitating' it take place worldwide. However, most of this on-going discussion is purely speculative, and is not backed by rigorous scientific criteria. In the present review we describe a step-by-step approach to test the functionality of nuclei collected from a non-living mammal through nuclear transplantation into enucleated oocytes. © Inter-Research 2011.

DOI： 10.3354/esr00366

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：W B SAUNDERS CO LTD  

Placentomegaly is a common phenotype in cloned mice. To assess differences in protein expression between placentae of cloned and uncloned mice, we used a proteomic approach involving 2-dimensional electrophoresis (DE) and MALDI-TOF MS. Proteins within isoelectric point range of 4-11 separately were analyzed in 2-DE with 3 replications of each sample. A total of approximately 3500 spots were detected in placental 2-DE stained with Coomassie blue. In the comparison of normal and cloned samples, a total of 41 spots were identified as differentially expressed proteins, of which 25 spots were up-regulated proteins such as TIMP-2, glutamate-ammonia, and esterase 10, while 16 spots were down-regulated proteins such as PBEF and annexin A1. The TIMP-2, which is related to extracellular matrix degradation and tissue remodeling processes, is an inhibitor of MMP-2. The PBEF is related to inhibition of apoptosis and induction of spontaneous labor. Western blot analysis confirmed increased TIMP-2 expression and decreased PBEF expression in cloned placentae compared with normal controls. Our results demonstrated composite profiles of key proteins involved in abnormal hypertrophic placenta derived from cloned mice and suggested that the reason for the placentomegaly may be due to abnormal gene expression in cloned mice. (c) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.placenta.2010.07.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

Transgenic (Tg) animals are widely used in researching the characteristics of exogenous genes. Intracytoplasmic sperm injection (ICSI)-mediated transgenesis (ICSI-Tr) has been a useful method for generating Tg animals, especially in the mouse. However, the original methods using freeze-thawed spermatozoa showed severe chromosomal damage and low offspring rates after embryo transfer. Herein, we describe an improved method to generate Tg mice efficiently using a simple pretreatment of spermatozoa with 10 mM NaOH. These spermatozoa lost their plasma membrane and tail, while still maintaining nuclear integrity. Sperm heads were mixed with 0.5-5 ng/mu l of the transgene for enhanced green fluorescent protein (EGFP) for 3 min to 1 h at room temperature and were then microinjected into oocytes by ICSI. The best results were obtained when treated spermatozoa were incubated with 2 ng/mu l of EGFP for 10 min; 55.6% of injected embryos developed to the blastocyst stage, and more than half (56.9%) of them displayed EGFP fluorescence. Under these conditions, 12 pups of 34 offspring were positive for the transgene after transfer at the 2-cell stage into pseudopregnant recipient mice (a high rate [10.2%] from manipulated embryos). This method was found to be suitable for hybrid and inbred strains of mouse such as C57BL/6 and 129x1/Sv. Thus, a simple sperm pretreatment with NaOH before ICSI-Tr resulted in an efficient insertion of an exogenous gene into the host genome. This method allows for easy production of Tg mice, requiring fewer oocytes for micromanipulation than classical methods.

DOI： 10.1095/biolreprod.109.078501

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The life cycle of mammals begins when a sperm enters an egg. Immediately after fertilization, both the maternal and paternal genomes undergo dramatic reprogramming to prepare for the transition from germ cell to somatic cell transcription programs(1). One of the molecular events that takes place during this transition is the demethylation of the paternal genome(2,3). Despite extensive efforts, the factors responsible for paternal DNA demethylation have not been identified(4). To search for such factors, we developed a live cell imaging system that allows us to monitor the paternal DNA methylation state in zygotes. Through short-interfering-RNA-mediated knockdown in mouse zygotes, we identified Elp3 (also called KAT9), a component of the elongator complex(5), to be important for paternal DNA demethylation. We demonstrate that knockdown of Elp3 impairs paternal DNA demethylation as indicated by reporter binding, immunostaining and bisulphite sequencing. Similar results were also obtained when other elongator components, Elp1 and Elp4, were knocked down. Importantly, injection of messenger RNA encoding the Elp3 radical SAM domain mutant, but not the HAT domain mutant, into MII oocytes before fertilization also impaired paternal DNA demethylation, indicating that the SAM radical domain is involved in the demethylation process. Our study not only establishes a critical role for the elongator complex in zygotic paternal genome demethylation, but also indicates that the demethylation process may be mediated through a reaction that requires an intact radical SAM domain.

DOI： 10.1038/nature08732

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Intracytoplasmic sperm injection (ICSI) is a technique in which sperm are injected directly into unfertilized oocytes, whereby offspring can be obtained even with dysfunctional sperm. Despite its advantages in human and animal reproductive technology, the low rate of resultant live offspring is perturbing. One major cause is thought to be embryonic chromosomal abnormalities. However, there is no direct evidence of how these occur or how they affect pregnancy outcomes.
Chromosomal dynamics during the first mitotic division of mouse embryos were analyzed using a new live-cell imaging technology. After imaging, the embryos' developmental capacities were determined.
When ICSI-generated embryos were monitored for their chromosome integrity, some embryos with apparent normal morphology seen by conventional light microscopy had abnormal chromosome segregation (ACS) at the first mitotic division. Chromosomal fragments were misaligned during the first metaphase and formed micronuclear-like structures at the interphase of the 2-cell stage. Similar ACS was also found in mouse embryos produced by microinjecting round spermatids, with even higher frequency. Giemsa staining and immunostaining revealed that these fragments were derived from double-strand DNA breaks in the paternal genome. About half of the embryos with ACS developed into normal-looking morulae or blastocysts and implanted, but almost all of them aborted spontaneously before embryonic day 7.5.
ACS during first mitosis appears to be a major cause of early pregnancy losses in ICSI-generated mouse embryos. Moreover, this novel imaging technology could be applicable as a method for the assessment of embryo quality.

DOI： 10.1093/humrep/dep236

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Language：English  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Specification of the germ cell lineage is vital to development and heredity. In mice, the germ cell fate is induced in pluripotent epiblast cells by signaling molecules, yet the underlying mechanism remains unknown. Here we demonstrate that germ cell fate in the epiblast is a direct consequence of Bmp4 signaling from the extraembryonic ectoderm (ExE), which is antagonized by the anterior visceral endoderm (AVE). Strikingly, Bmp8b from the ExE restricts AVE development, thereby contributing to Bmp4 signaling. Furthermore, Wnt3 in the epiblast ensures its responsiveness to Bmp4. Serum-free, defined cultures revealed that, in response to Bmp4, competent epiblast cells uniformly expressed key transcriptional regulators Blimp1 and Prdm14 and acquired germ-cell properties, including genome-wide epigenetic reprogramming, in an orderly fashion. Notably, the induced cells contributed to both spermatogenesis and fertility of offspring. By identifying a signaling principle in germ cell specification, our study establishes a robust strategy for reconstituting the mammalian germ cell lineage in vitro.

DOI： 10.1016/j.cell.2009.03.014

Web of Science

PubMed

Language：Japanese  

CiNii Books

Language：Japanese  

CiNii Books

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Male BALB/c mice produce a high proportion of morphologically abnormal sperm. Although the BALB/c male may be a useful model of human male infertility, it remains unclear whether the sperm abnormality rate (SAR) is affected by age or the BALB/c substrain.
SARs (head shape) were assessed in three BALB/c substrains (A, AnN, ByJ) at 7 and 9 weeks and 6-10 months of age (c.100 sperm/male). The functional ability of abnormal sperm produced from 7-week-old and 6-10-month-old males was determined in BALB/c AnN mice by ICSI.
The SAR (quasi-normal plus abnormal sperm) was lower in BALB/c A than in the other two strains (P < 0.05). Further, the SARs of BALB/c AnN and ByJ strains at 7 weeks old were high and decreased rapidly by 9 weeks, suggesting that early spermatogenesis (i.e. the first wave of spermatogenesis) produced low-quality sperm. ICSI experiments indicated that 2-cell stage embryos which developed from morphologically abnormal sperm from both the first wave of spermatogenesis and the older mice (6-10 months) were similar to those from morphologically normal sperm in terms of producing progeny, although the number of 2-cell embryos was slightly lower (P < 0.05, chi(2) test) than with normal sperm.
Although the SARs of BALB/c mice are affected by both age and substrain, the embryos that developed from morphologically abnormal sperm have normal genetic potential in terms of production of progeny.

DOI： 10.1093/humrep/den456

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

We have been able to develop a stable nuclear transfer (NT) method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although the piezo unit is a complex tool, once mastered it is of great help not only in NT experiments, but also in almost all other forms of micromanipulation. Using this technique, embryonic stem (ntES) cell lines established from somatic cell nuclei can be generated relatively easily from a variety of mouse genotypes and cell types. Such ntES cells can be used not only for experimental models of human therapeutic cloning but also as a means of preserving mouse genomes instead of preserving germ cells. Here, we describe our most recent protocols for mouse cloning. © 2009 Humana Press, a part of Springer Science+Business Media, LLC.

DOI： 10.1007/978-1-59745-471-1_13

Scopus

PubMed

Language：English   Publishing type：(MISC) Summary of the papers read (international conference)   Publisher：SOC STUDY REPRODUCTION  

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The causes of frequent abnormal phenotypes and low success rate in mammalian cloning are poorly understood. Although epigenetic aberration is suspected to be a cause, its connection to the phenotypes has yet to be investigated. To measure the level of reprogramming of an epigenetic mark, acetylation at lysine 9 of histone H3 (H3K9Ac), in cloned mice, we examined its conservation between two cloned mice derived from distinct cell nuclei and their natural donors by utilizing whole-genome tiling arrays and quantitative PCR. Pairwise comparison of the H3K9Ac enrichment profile between the four mice revealed that H3K9Ac is less conserved in intergenic regions than in promoter regions of protein-coding genes. Intriguingly, the variation of H3K9Ac enrichment in intergenic regions is the most prominent in comparison of the two clones, possibly reflecting an additive effect of aberrant reprogramming of this epigenetic information occurring specifically in each of the two clones.

DOI： 10.1007/s00335-008-9146-5

Web of Science

PubMed

Language：English  

Language：English  

Language：English   Publishing type：(MISC) Other article   Publisher：AMER SOC MICROBIOLOGY  

DOI： 10.1128/MCB.01302-07

Web of Science

Language：English  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Carboxypeptidase E (CPE) has important functions in processing of endocrine pro-peptides, such as pro-insulin, pro-opiomelanocortin, or pro-gonadotropin-releasing hormone, as evidenced by the hyperpro-insulinemia, obesity, and sterility of Cpe mutant mice. Down-regulation of Cpe in enlarged placentas of interspecific hybrid (interspecies hybrid placental dysplasia (IHPD)) and cloned mice suggested that reduced CPE enzyme and receptor activity could underlie abnormal placental phenotypes. In this study, we have explored the role of Cpe in murine placentation by determining its expression at various stages of gestation, and by phenotypic analysis of Cpe mutant placentas. Our results show that Cpe and Carboxypeptidase D (Cpd), another carboxypeptidase with a very similar function, are strictly co-localized in the mouse placenta from late mid-gestation to term. We also show that absence of CPE causes a sporadic but striking placental phenotype characterized by an increase in giant and glycogen cell numbers and giant cell hypertrophy. Microarray-based transcriptional pro. ling of Cpe mutant placentas identified only a very small number of genes with altered expression, including Dtprp, which belongs to the prolactin gene family. Concordant deregulation of Cpe and Cpd in abnormal placentas of interspecies hybrids before the onset of IHPD phenotype and recapitulation of some phenotypes of IHPD hyperplastic placentas in Cpe mutant placentas suggests that these two genes are causally involved in IHPD and may function as speciation genes in the genus Mus.

DOI： 10.1111/j.1432-0436.2006.00093.x

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Somatic cell nuclear transfer technology has been applied to produce live clones successfully in several mammalian species, but the success rates are very low. In mice, about half of the nuclear transfer embryos undergo implantation, but very few survive to term. We undertook detailed histological analyses of placentas from cloned mouse embryos generated from cumulus cells at 10.5 dpc of pregnancy, by which stage most clones have terminated their development. At 10.5 dpc, the extraembryonic tissues displayed several defined histological patterns, each reflecting their stage of developmental arrest. The most notable abnormality was the poor development of the spongiotrophoblast layer of diploid cells. This is in contrast to the placental hyperplasia frequently observed in somatic clones at 12.5 dpc or later stages. A variety of structural abnormalities were also observed in the embryos. Both placental and embryonic defects likely contribute to the low success rate of the mouse clones. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2006.08.057

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIO SCIENTIFICA LTD  

We examined the involvement of histone H3 modifications in the chromosome condensation and decondensation of somatic cell nuclei injected into mature pig oocytes. Nuclei of pig granulosa cells were transferred into in vitro matured intact pig oocytes, and histone H3 phosphorylation, acetylation, and methylation were examined by immunostaining with specific antibodies in relation to changes in chromosome morphology. In the condensed chromosomes of pig oocytes at metaphase 11, histone H3 was phosphorylated at serine 10 (H3-S10) and serine 28 (H3-S28), and methylated at lysine 9 (H3-K9), but was not acetylated at lysine 9, 14 and 18 (H3-H9, H3-K14 and H3-K18). During the first 2 h after nuclear transfer, a series of events were observed in the somatic nuclei: nuclear membrane disassembly; chromosome condensation to form a metaphase-like configuration; an increase in histone H3 phosphorylation levels (H3-S10 and H3-S28). Next, pig oocytes injected with nuclei of somatic cells were electroactivated and the chromosome morphology of oocytes and somatic cells was examined along with histone modifications. Generally, chromosomes of the somatic cells showed a similar progression of cell cycle stage to that of oocytes, through anaphase II- and telophase II-like stages then formed pronucleus-like structures, although the morphology of the spindles differed from that of oocyte spindles. The chromosomes of somatic cells also showed changes in histone H3 dephosphorylation and reacetylation, similar to oocytes. In contrast, histone H3 methylation (H3-K9) of somatic cell nuclei did not show any significant change after injection and electroactivation of the oocytes. These results suggest that nuclear remodeling including histone H3 phosphorylation and acetylation of injected somatic nuclei took place in the oocytes under regulation by the oocyte cytoplasm.

DOI： 10.1530/rep.1.00897

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CAMBRIDGE UNIV PRESS  

The phenomenon of the developmental arrest at the 2-cell stage of 1-cell embryos from some mouse strains during in vitro culture is known as the 2-cell block. We investigated the specific factors involved in the 2-cell block of AKR embryos by means of a modified culture system, the production of reconstructed embryos by pronuclear exchange and a cross experiment. In a culture medium with phosphate, 94.6% of 1-cell embryos from the C57BL mouse strain developed to the blastocyst stage, but 95.7% of embryos from the AKR mouse strain showed 2-cell block. Phosphate-free culture medium rescued the 2-cell block of AKR embryos and accelerated the first cell cycle of the embryos. Co-culture with BRL cells and a BRL-conditioned medium fractionated below 30 kDa also rescued the 2-cell block of AKR embryos. Examinations of in vitro development of reconstructed embryos and of embryos from F1 females between AKR and C57BL strains clearly demonstrated that the AKR cytoplast caused the 2-cell block. In the backcrossed female progeny between (AKR x C57BL) F1 males and AKR females, about three-quarters of the embryos were of the 2-cell blocking phenotype and about one-quarter were of the non-blocking phenotype. These results suggest that two genes are responsible for the 2-cell block of AKR embryos.

DOI： 10.1017/S0967199406003674

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Somatic cell nuclear cloning has repeatedly demonstrated striking reversibility of epigenetic regulation of cell differentiation. Upon injection into eggs, the donor nuclei exhibit global chromatin decondensation, which might contribute to reprogramming the nuclei by derepressing dormant genes. Decondensation of sperm chromatin in eggs is explained by the replacement of sperm-specific histone variants with egg-type histones by the egg protein nucleoplasmin (Npm). However, little is known about the mechanisms of chromatin decondensation in somatic nuclei that do not contain condensation-specific histone variants. Here we found that Npm could widely decondense chromatin in undifferentiated mouse cells without overt histone exchanges but with specific epigenetic modifications that are relevant to open chromatin structure. These modifications included nucleus-wide multiple histone H3 phosphorylation, acetylation of Lys 14 in histone H3, and release of heterochromatin proteins HP1 beta and TIF1 beta from the nuclei. The protein kinase inhibitor staurosporine inhibited chromatin decondensation and these epigenetic modifications with the exception of H3 acetylation, potentially linking these chromatin events. At the functional level, Npm pretreatment of mouse nuclei facilitated activation of four oocyte-specific genes from the nuclei injected into Xenopus laevis oocytes. Future molecular elucidation of chromatin decondensation by Npm will significantly contribute to our understanding of the plasticity of cell differentiation.

DOI： 10.1128/MCB.26.4.1259-1271.2006

Web of Science

PubMed

Language：Japanese   Publisher：一般社団法人 日本泌尿器科学会  

DOI： 10.5980/jpnjurol.97.265_1

Language：English  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

The Mongolian gerbil (Meriones unguiculatus) has been used as a laboratory species in many fields of research, including neurology, oncology, and parasitology. Although the cryopreservation of embryos has become a useful means to protect valuable genetic resources, its application to the Mongolian gerbil has not yet been reported. In this study, we investigated the in vitro and in vivo developmental competence of Mongolian gerbil embryos cryopreserved by vitrification, In vivo-fertilized embryos were vitrified on the day of collection using the ethylene glycol (EG)-based solutions EFS20 and EFS40, which contained 20% and 40% EG, respectively, in PB1 containing 30% (w/v) Ficoll 70 and 0.5M sucrose. First, we compared one-step and two-step vitrification protocols. In the one-step method, the embryos were directly transferred into the vitrification solution (EFS40), whereas in the two-step method, the embryos were exposed serially to EFS20 and EFS40 and then vitrified. After liquefying (thawing), late two-cell embryos (collected on day 3) vitrified by the two-step method showed significantly better rates of in vitro development to the morula stage compared to those vitrified by the one-step method (65% vs. 5%, P < 0.0001). We then examined whether the same two-step method could be applied to early two-cell embryos (collected on day 2), four-cell embryos (day 4), morulae (day 5), and blastocysts (day 6). After liquefying, 87%-100% of the embryos were morphologically normal in all groups, and 23% and 96% developed to the compacted morula stage from early two- and four-cell embryos, respectively. After transfer into recipient females, 3% (4/123), 1% (1/102), 5% (4/73), and 10% (15/155) developed to full-term offspring from vitrified and liquefied early two-cell embryos, late two-cell embryos, morulae, and blastocysts, respectively. This demonstrates that Mongolian gerbil embryos can be safely cryopreserved using EG-based vitrification solutions. (C) 2005 Wiley-Liss, Inc.

DOI： 10.1002/mrd.20226

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CAMBRIDGE UNIV PRESS  

In the current widely used round spermatid injection (ROSI) protocol for the mouse, the spermatid nucleus is separated from most of the cytoplasm before ROSI by drawing a spermatid in and out of a pipette. This results in the highest rate of normal fertilization. However, this separation method is not always consistent and can be time-consuming. An alternative separation method that cuts away the cytoplasm using the tip of an injection pipette was developed. After removing the cytoplasm, ROSI was performed following both post- and pre-activation protocols and development in vitro and in vivo were examined. The new method consistently removed the bulk of the cytoplasm, as shown by quantifying mitochondria. ROSI without the cytoplasm resulted in significantly higher rates of fertilization than ROSI with the cytoplasm into either post- or pre-activated oocytes. Furthermore, the offspring production rates of ROSI without the cytoplasm were also high (50% and 49% for the post- and pre-activation protocols, respectively). This new method for separating the cytoplasm is an alternative way of producing offspring using ROSI.

DOI： 10.1017/S0967199404002965

Web of Science

PubMed

Language：English  

Language：English   Publishing type：(MISC) Summary of the papers read (international conference)   Publisher：SOC STUDY REPRODUCTION  

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：COGNIZANT COMMUNICATION CORP  

Mice can now be cloned from cultured and noncultured adult-, fetus-, male-, or female-derived cells. Using the mouse as a model, research is moving towards a comprehensive description of clones generated by somatic cell nuclear transfer. In addition, embryonic stem (ES) cell lines can be generated from adult somatic cells via nuclear transfer (ntES cells). ntES cells contribute to an extensive variety of cell types including neurons in vitro and germ cells in vivo. Recent advances in mouse cloning are reported to illustrate its strengths and promise in the study of mammalian biology and biomedicine.

Web of Science

PubMed

Language：English  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE AMERICA INC  

Mammalian cloning using somatic cells has been accomplished successfully in several species, and its potential basic, clinical and therapeutic applications are being pursued on many fronts. Determining the long-term effects of cloning on offspring is crucial for consideration of future application of the technique. Although full-term development of animals cloned from adult somatic cells has been reported, problems in the resulting progeny indicate that the cloning procedure may not produce animals that are phenotypically identical to their cell donor. We used a mouse model to take advantage of its short generation time and lifespan. Here we report that the increased body weight of cloned B6C3F1 female mice reflects an increase of body fat in addition to a larger body size, and that these mice share many characteristics consistent with obesity. We also show that the obese phenotype is not transmitted to offspring generated by mating male and female cloned mice.

DOI： 10.1038/nm0302-262

Web of Science

PubMed

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1038/s41598-018-28896-8.

Language：Japanese  

CiNii Books

Language：English  

Language：English  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：MACMILLAN PUBLISHERS LTD  

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Mice have been successfully cloned from cumulus cells, fibroblast cells, embryonic stem cells, and immature Sertoli cells only after direct injection of their nuclei into enucleated oocytes. This technical feature of mouse nuclear transfer differentiates it from that used in domestic species, where electrofusion is routinely used for nuclear transfer. To examine whether nuclear transfer by electrofusion can be applied to somatic cell cloning in the mouse, we electrofused tail tip fibroblast cells with enucleated oocytes, and then assessed the subsequent in vitro and in vivo development of the reconstructed embryos. The rate of successful nuclear transfer (fusion and nuclear formation) was 68.8% (753/1094) and the rate of development into morulae/blastocysts was 40.8% (260/637). After embryo transfer, seven (six males and one female; 2.5% per transfer) normal fetuses were obtained at 17.5-21.5 dpc. These rates of development in vitro and in vivo are not significantly different from those after cloning by injection (44.7% to morulae/blastocysts and 4.8% to term). These results indicate that nuclear transfer by electrofusion is practical for mouse somatic cell cloning and provide an alternative method when injection of donor nuclei into recipient oocytes is technically difficult. (C) 2000 Wiley-Liss, Inc.

DOI： 10.1002/1098-2795(200009)57:1<55::AID-MRD8>3.0.CO;2-W

Web of Science

Language：Japanese  

Language：English  

Language：English  

Language：Japanese  

Language：English  

Language：English  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

To initiate normal embryonic development, an egg must receive a signal to become activated at fertilization. We here report that the ability of demembranated sperm heads to activate is abolished after incubation over the range 20-44 degrees C and is sensitive to reducing agents. On the basis of this observation, we have developed a microinjection-based, trans-complementation assay in order to dissect the heat-inactivated sperm-borne oocyte-activating factor(s) (SOAF). We demonstrate that the failure of heat-inactivated sperm heads to activate an egg is rescued by coinjection with dithiothreitol-solubilized SOAF from demembranated sperm heads. The solubilized SOAF (SOAF(s)) is trypsin sensitive and is liberated from demembranated heads in a temperature-dependent manner that inversely correlates with the ability of sperm heads to activate. This argues that SOAF(s) is a proteinaceous molecular species required to initiate activation. Injection of oocytes with mouse or hamster sperm cytosolic factors, but not SOAF(s) alone, induced resumption of meiosis, further suggesting that these cytosolic factors and SOAF are dis tinct. Collectively, these data strongly suggest that full mammalian oocyte activation is initiated by the coordinated action of one or more heat-sensitive protein constituents of the perinuclear matrix and at least one heat-stable submembrane component.

Web of Science

Language：English  

Language：English  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CAMBRIDGE UNIV PRESS  

Alcohol is known to preserve genomic DNA and the primary structure of sperm protamines. To determine whether alcohol can retain the genetic and reproductive potential of mammalian sperm nuclei, mature mouse spermatozoa were stored in 70% ethanol or propanol for up to 2 months before injection into oocytes. Live offspring were obtained after injection of spermatozoa stored in 70% ethanol for 1 day at -20 degrees C. About 20% of the spermatozoa stored under this condition had normal chromosomes. The remaining 80% of spermatozoa and all the spermatozoa stored in 70% ethanol for 2 months had structurally aberrant chromosomes, and none could support the development of normal embryos. High concentrations of alcohol do not alter the primary structure of either DNA or small-molecular-weight protamines. However, alcohol may modify protamine-protamine or protamine-DNA interactions in a manner that results in the induction of DNA strand breaks during sperm chromatin decondensation within the oocyte. The limited success in obtaining normal offspring with ethanol-stored spermatozoa is encouraging. It may be possible to overcome these problems and develop a simple method for preserving mammalian spermatozoa without freezing.

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CAMBRIDGE UNIV PRESS  

The primary spermatocytes used were male germ cells at prophase I. The present study was undertaken to see whether bivalent chromosomes of mouse primary spermatocytes can undergo. meiotic divisions within maturing oocytes and participate in subsequent embryonic development. Primary spermatocytes (pachytene to diplotene) freshly collected from the testes of mature males were electrofused with immature oocytes shortly before or after germinal vesicle breakdown. After culture in MEM-alpha medium for 15 h, most (&gt; 90%) of the oocytes containing spermatocyte chromosomes underwent maturation and arrested at metaphase II (MII). Among 23 MII oocytes examined, 17 (74%) had one group of chromosomes and one polar body, indicating that male chromosomes had intermingled with those of the females and completed the first meiotic division. Chromosome analyses of these MII oocytes demonstrated their diploidy. The metaphase chromosomes were transferred to enucleated MII oocytes freshly recovered from superovulated mice. After artificial activation, the reconstructed MII oocytes resumed meiosis and developed to the morula/blastocyst stage. However, no pups were born following embryo transfer into recipient females. These findings indicate that the chromosomes of primary spermatocytes undergo meiotic divisions in maturing oocytes and participate in the formation of diploid embryos.

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC LABORATORY ANIMAL SCIENCE  

Web of Science

Event date： 2019.12

Language：Japanese   Presentation type：Oral presentation(invited, special)  

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Event date： 2017.9

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Venue：Okinawa Japan  

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Event date： 2017.9

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Venue：Okinawa Japan  

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Event date： 2015.11

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凍結死体の体細胞からのクローン個体作出に成功

体細胞クローンマウスに関する研究

発生学分野における体細胞クローン技術に関する研究

Audience： High school students

Type：Visiting lecture

Audience： Junior students

Type：Visiting lecture

Audience： High school students

Type：Visiting lecture

Audience： High school students

Type：Visiting lecture