Publications by authors named "Manabu Ozawa"

55 Publications

Generation of a p16 Reporter Mouse and Its Use to Characterize and Target p16 Cells In Vivo.

Cell Metab 2020 11 18;32(5):814-828.e6. Epub 2020 Sep 18.

Division of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute of Biomedical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022, Japan.

Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-Cre-tdTomato mouse model to analyze the in vivo characteristics of p16 cells at a single-cell level. We found tdTomato-positive p16 cells detectable in all organs, which were enriched with age. We also found that these cells failed to proliferate and had half-lives ranging from 2.6 to 4.2 months, depending on the tissue examined. Single-cell transcriptomics in the liver and kidneys revealed that p16 cells were present in various cell types, though most dominant in hepatic endothelium and in renal proximal and distal tubule epithelia, and that these cells exhibited heterogeneous senescence-associated phenotypes. Further, elimination of p16 cells ameliorated nonalcoholic steatohepatitis-related hepatic lipidosis and immune cell infiltration. Our new mouse model and single-cell analysis provide a powerful resource to enable the discovery of previously unidentified senescence functions in vivo.
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http://dx.doi.org/10.1016/j.cmet.2020.09.006DOI Listing
November 2020

RNA-binding protein Ptbp1 regulates alternative splicing and transcriptome in spermatogonia and maintains spermatogenesis in concert with Nanos3.

J Reprod Dev 2020 Oct 6;66(5):459-467. Epub 2020 Jul 6.

Laboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.

PTBP1, a well-conserved RNA-binding protein, regulates cellular development by tuning posttranscriptional mRNA modification such as alternative splicing (AS) or mRNA stabilization. We previously revealed that the loss of Ptbp1 in spermatogonia causes the dysregulation of spermatogenesis, but the molecular mechanisms by which PTBP1 regulates spermatogonium homeostasis are unclear. In this study, changes of AS or transcriptome in Ptbp1-knockout (KO) germline stem cells (GSC), an in vitro model of proliferating spermatogonia, was determined by next generation sequencing. We identified more than 200 differentially expressed genes, as well as 85 genes with altered AS due to the loss of PTBP1. Surprisingly, no differentially expressed genes overlapped with different AS genes in Ptbp1-KO GSC. In addition, we observed that the mRNA expression of Nanos3, an essential gene for normal spermatogenesis, was significantly decreased in Ptbp1-KO spermatogonia. We also revealed that PTBP1 protein binds to Nanos3 mRNA in spermatogonia. Furthermore, Nanos3;Ptbp1 mice exhibited abnormal spermatogenesis, which resembled the effects of germ cell-specific Ptbp1 KO, whereas no significant abnormality was observed in mice heterozygous for either gene alone. These data implied that PTBP1 regulates alternative splicing and transcriptome in spermatogonia under different molecular pathways, and contributes spermatogenesis, at least in part, in concert with NANOS3.
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http://dx.doi.org/10.1262/jrd.2020-060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593632PMC
October 2020

NELL2-mediated lumicrine signaling through OVCH2 is required for male fertility.

Science 2020 06;368(6495):1132-1135

Immunology Frontier Research Center, Osaka University, Suita, Osaka 5650871, Japan.

The lumicrine system is a postulated signaling system in which testis-derived (upstream) secreted factors enter the male reproductive tract to regulate epididymal (downstream) pathways required for sperm maturation. Until now, no lumicrine factors have been identified. We demonstrate that a testicular germ-cell-secreted epidermal growth factor-like protein, neural epidermal growth factor-like-like 2 (NELL2), specifically binds to an orphan receptor tyrosine kinase, c-ros oncogene 1 (ROS1), and mediates the differentiation of the initial segment (IS) of the caput epididymis. Male mice in which had been knocked out were infertile. The IS-specific secreted proteases, ovochymase 2 (OVCH2) and A disintegrin and metallopeptidase 28 (ADAM28), were expressed upon IS maturation, and OVCH2 was required for processing of the sperm surface protein ADAM3, which is required for sperm fertilizing ability. This work identifies a lumicrine system essential for testis-epididymis-spermatozoa (NELL2-ROS1-OVCH2-ADAM3) signaling and male fertility.
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http://dx.doi.org/10.1126/science.aay5134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7396227PMC
June 2020

Cell-type dependent enhancer binding of the EWS/ATF1 fusion gene in clear cell sarcomas.

Nat Commun 2019 09 5;10(1):3999. Epub 2019 Sep 5.

Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507, Japan.

Clear cell sarcoma (CCS) is a rare soft tissue sarcoma caused by the EWS/ATF1 fusion gene. Here, we established induced pluripotent stem cells (iPSCs) from EWS/ATF1-controllable murine CCS cells harboring sarcoma-associated genetic abnormalities. Sarcoma-iPSC mice develop secondary sarcomas immediately after EWS/ATF1 induction, but only in soft tissue. EWS/ATF1 expression induces oncogene-induced senescence in most cell types in sarcoma-iPSC mice but prevents it in sarcoma cells. We identify Tppp3-expressing cells in peripheral nerves as a cell-of-origin for these sarcomas. We show cell type-specific recruitment of EWS/ATF1 to enhancer regions in CCS cells. Finally, epigenetic silencing at these enhancers induces senescence and inhibits CCS cell growth through altered EWS/ATF1 binding. Together, we propose that distinct responses to premature senescence are the basis for the cell type-specificity of cancer development.
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http://dx.doi.org/10.1038/s41467-019-11745-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728361PMC
September 2019

RNA-binding protein Ptbp1 is essential for BCR-mediated antibody production.

Int Immunol 2019 03;31(3):157-166

Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.

The RNA-binding protein polypyrimidine tract-binding protein-1 (Ptbp1) binds to the pyrimidine-rich sequence of target RNA and controls gene expression via post-transcriptional regulation such as alternative splicing. Although Ptbp1 is highly expressed in B lymphocytes, its role to date is largely unknown. To clarify the role of Ptbp1 in B-cell development and function, we generated B-cell-specific Ptbp1-deficient (P1BKO) mice. B-cell development in the bone marrow, spleen and peritoneal cavity of the P1BKO mice was nearly normal. However, the P1BKO mice had significantly lower levels of natural antibodies in serum compared with those of the control mice. To investigate the effect of Ptbp1 deficiency on the immune response in vivo, we immunized the P1BKO mice with T-cell-independent type-2 (TI-2) antigen NP-Ficoll and T-cell-dependent (TD) antigen NP-CGG. We found that B-cell-specific Ptbp1 deficiency causes an immunodeficiency phenotype due to defective production of antibody against both TI-2 and TD antigen. This immunodeficiency was accompanied by impaired B-cell receptor (BCR)-mediated B-cell activation and plasmablast generation. These findings demonstrate that Ptbp1 is essential for the humoral immune response.
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http://dx.doi.org/10.1093/intimm/dxy077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400050PMC
March 2019

PTBP1 contributes to spermatogenesis through regulation of proliferation in spermatogonia.

J Reprod Dev 2019 Feb 12;65(1):37-46. Epub 2018 Nov 12.

Laboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.

Polypyrimidine tract-binding protein 1 (PTBP1) is a highly conserved RNA-binding protein that is a well-known regulator of alternative splicing. Testicular tissue is one of the richest tissues with respect to the number of alternative splicing mRNA isoforms, but the molecular role(s) of PTBP1 in the regulation of these isoforms during spermatogenesis is still unclear. Here, we developed a germ cell-specific Ptbp1 conditional knockout (cKO) mouse model by using the Cre-loxP system to investigate the role of PTBP1 in spermatogenesis. Testis weight in Ptbp1 cKO mice was comparable to that in age-matched controls until 3 weeks of age; at ≥ 2 months old, testis weight was significantly lighter in cKO mice than in age-matched controls. Sperm count in Ptbp1 cKO mice at 2 months old was comparable to that in controls, whereas sperm count significantly decreased at 6 months old. Seminiferous tubules that exhibited degeneration in spermatogenic function were more evident in the 2-month-old Ptbp1 cKO mice than in controls. In addition, the early neonatal proliferation of spermatogonia, during postnatal days 1-5, was significantly retarded in Ptbp1 cKO mice compared with that in controls. An in vitro spermatogonia culture model (germline stem cells) revealed that hydroxytamoxifen-induced deletion of PTBP1 from germline stem cells caused severe proliferation arrest accompanied by an increase of apoptotic cell death. These data suggest that PTBP1 contributes to spermatogenesis through regulation of spermatogonia proliferation.
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http://dx.doi.org/10.1262/jrd.2018-109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379764PMC
February 2019

WNT regulation of embryonic development likely involves pathways independent of nuclear CTNNB1.

Reproduction 2017 04 9;153(4):405-419. Epub 2017 Jan 9.

Department of Animal SciencesD.H. Barron Reproductive and Perinatal Biology Research Program, and Genetics Institute, University of Florida, Gainesville, Florida, USA

The bovine was used to examine the potential for WNT signaling to affect the preimplantation embryo. Expression of seven key genes involved in canonical WNT signaling declined to a nadir at the morula or blastocyst stage. Expression of 80 genes associated with WNT signaling in the morula and inner cell mass (ICM) and trophectoderm (TE) of the blastocyst was also evaluated. Many genes associated with WNT signaling were characterized by low transcript abundance. Seven genes were different between ICM and TE, and all of them were overexpressed in TE as compared to ICM, including WNT6, FZD1, FZD7, LRP6, PORCN, APC and SFRP1 Immunoreactive CTNNB1 was localized primarily to the plasma membrane at all stages examined from the 2-cell to blastocyst stages of development. Strikingly, neither CTNNB1 nor non-phospho (i.e., active) CTNNB1 was observed in the nucleus of blastomeres at any stage of development even after the addition of WNT activators to culture. In contrast, CTNNB1 associated with the plasma membrane was increased by activators of WNT signaling. The planar cell polarity pathway (PCP) could be activated in the embryo as indicated by an experiment demonstrating an increase in phospho-JNK in the nucleus of blastocysts treated with the non-canonical WNT11. Furthermore, WNT11 improved development to the blastocyst stage. In conclusion, canonical WNT signaling is attenuated in the preimplantation bovine embryo but WNT can activate the PCP component JNK. Thus, regulation of embryonic development by WNT is likely to involve activation of pathways independent of nuclear actions of CTNNB1.
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http://dx.doi.org/10.1530/REP-16-0610DOI Listing
April 2017

Cell-Type-Specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex.

Cell 2016 Aug;166(5):1147-1162.e15

Division of Genetics and Genomics, Manton Center for Orphan Disease Research, Howard Hughes Medical Institute, Boston Children's Hospital, Boston, MA 02115, USA; Departments of Neurology and Pediatrics, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address:

Alternative splicing is prevalent in the mammalian brain. To interrogate the functional role of alternative splicing in neural development, we analyzed purified neural progenitor cells (NPCs) and neurons from developing cerebral cortices, revealing hundreds of differentially spliced exons that preferentially alter key protein domains-especially in cytoskeletal proteins-and can harbor disease-causing mutations. We show that Ptbp1 and Rbfox proteins antagonistically govern the NPC-to-neuron transition by regulating neuron-specific exons. Whereas Ptbp1 maintains apical progenitors partly through suppressing a poison exon of Flna in NPCs, Rbfox proteins promote neuronal differentiation by switching Ninein from a centrosomal splice form in NPCs to a non-centrosomal isoform in neurons. We further uncover an intronic human mutation within a PTBP1-binding site that disrupts normal skipping of the FLNA poison exon in NPCs and causes a brain-specific malformation. Our study indicates that dynamic control of alternative splicing governs cell fate in cerebral cortical development.
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http://dx.doi.org/10.1016/j.cell.2016.07.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5248659PMC
August 2016

Regulation of gene expression in the bovine blastocyst by colony stimulating factor 2.

BMC Res Notes 2016 Apr 29;9:250. Epub 2016 Apr 29.

Dept. of Animal Sciences, D.H. Barron Reproductive and Perinatal Biology Research Program and Genetics Institute, University of Florida, PO Box 110910, Gainesville, FL, 32611-0910, USA.

Background: Colony stimulating factor 2 can have multiple effects on the function of the preimplantation embryo that include increased potential to develop to the blastocyst stage, reduced apoptosis, and enhanced ability of inner cell mass (ICM) to remain pluripotent after culture. The objective of the current experiment was to identify genes regulated by CSF2 in the ICM and trophectoderm (TE) of the bovine blastocyst with the goal of identifying possible molecular pathways by which CSF2 increases developmental competence for survival. Embryos were produced in vitro and cultured from Day 6 to 8 in serum-free medium containing 10 ng/ml recombinant bovine CSF2 or vehicle. Blastocysts were harvested at Day 8 and ICM separated from TE by magnetic-activated cell sorting. RNA was purified and used to prepare amplified cDNA, which was then subjected to high-throughput sequencing using the SOLiD 4.0 system. Three pools of amplified cDNA were analyzed per treatment.

Results: The number of genes whose expression was regulated by CSF2, using P < 0.05 and >1.5-fold difference as cut-offs, was 945 in the ICM (242 upregulated by CSF2 and 703 downregulated) and 886 in the TE (401 upregulated by CSF2 and 485 downregulated). Only 49 genes were regulated in a similar manner by CSF2 in both cell types. The three significant annotation clusters in which genes regulated by ICM were overrepresented were related to membrane signaling. Genes downregulated by CSF2 in ICM were overrepresented in several pathways including those for ERK and AKT signaling. The only significant annotation cluster containing an overrepresentation of genes regulated by CSF2 in TE was for secreted or extracellular proteins. In addition, genes downregulated in TE were overrepresented in TGFβ and Nanog pathways.

Conclusions: Differentiation of the blastocyst is such that, by Day 8 after fertilization, the ICM and TE respond differently to CSF2. Analysis of the genes regulated by CSF2 in ICM and TE are suggestive that CSF2 reinforces developmental fate and function of both cell lineages.
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http://dx.doi.org/10.1186/s13104-016-2038-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850677PMC
April 2016

The Histone Demethylase FBXL10 Regulates the Proliferation of Spermatogonia and Ensures Long-Term Sustainable Spermatogenesis in Mice.

Biol Reprod 2016 Apr 16;94(4):92. Epub 2016 Mar 16.

Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

The F-box and leucine-rich repeat protein 10 (Fbxl10) gene encodes a protein that catalyzes demethylation of H3K4 and H3K36. In this study, we show the important roles of FBXL10 as a histone demethylase in sustainable sperm production using mice in which the JmjC domain of Fbxl10 was deleted (Fbxl10(DeltaJ/DeltaJ)). In histological analysis, testis sections from 10-wk-old Fbxl10(DeltaJ/DeltaJ) mice appeared normal. On the other hand, testes from 7-mo-old Fbxl10(DeltaJ/DeltaJ) mice contained a greater ratio of seminiferous tubules exhibiting degeneration of spermatogenesis. Further analysis using an in vitro spermatogonia culture system, that is, germline stem cells (GSCs), revealed that Fbxl10(DeltaJ/DeltaJ) GSCs expressed a significantly higher level of P21 and P19 mRNA, cyclin-dependent kinase inhibitors and also known as cellular senescence markers, than wild-type (WT) GSCs. Furthermore, the ratio of Fbxl10(DeltaJ/DeltaJ) GSCs in G0/G1 phase was higher and the ratios in S and G2/M phases were lower than the corresponding ratios of WT GSCs, and the doubling speed of Fbxl10(DeltaJ/DeltaJ) GSCs was significantly slower than that of WT GSCs. In addition to these in vitro results, an in vivo study indicated that recovery of spermatogenesis after a transient reduction in the number of testicular germ cells by busulfan treatment was significantly slower in Fbxl10(DeltaJ/DeltaJ) mice than in WT mice. These data suggest that Fbxl10 plays important roles in long-term sustainable spermatogenesis via regulating cell cycle.
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http://dx.doi.org/10.1095/biolreprod.115.135988DOI Listing
April 2016

The histone demethylase Fbxl11/Kdm2a plays an essential role in embryonic development by repressing cell-cycle regulators.

Mech Dev 2015 Feb 30;135:31-42. Epub 2014 Oct 30.

Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Methylation and de-methylation of histone lysine residues play pivotal roles in mammalian early development; these modifications influence chromatin architecture and regulate gene transcription. Fbxl11 (F-box and leucine-rich repeat 11)/Kdm2a is a histone demethylase that selectively removes mono- and di-methylation from histone H3K36. Previously, two other histone H3K36 demethylases (Jmjd5 or Fbxl10) were analyzed based on the phenotypes of the corresponding knockout (KO) mice; the results of those studies implicated H3K36 demethylases in cell proliferation, apoptosis, and senescence (Fukuda et al., 2011; Ishimura et al., 2012). To elucidate the physiological role of Fbxl11, we generated and examined Fbxl11 KO mice. Fbxl11 was expressed throughout the body during embryogenesis, and the Fbxl11 KO mice exhibited embryonic lethality at E10.5-12.5, accompanied with severe growth defects leading to reduced body size. Furthermore, knockout of Fbxl11 decreased cell proliferation and increased apoptosis. The lack of Fbxl11 resulted in downregulation of the Polycomb group protein (PcG) Ezh2, PcG mediated H2A ubiquitination and upregulation of the cyclin-dependent kinase inhibitor p21Cip1. Taken together, our findings suggest that Fbxl11 plays an essential role in embryonic development and homeostasis by regulating cell proliferation and survival.
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http://dx.doi.org/10.1016/j.mod.2014.10.001DOI Listing
February 2015

The requirement for protein kinase C delta (PRKCD) during preimplantation bovine embryo development.

Reprod Fertil Dev 2016 Mar;28(4):482-90

Department of Animal Sciences, University of Florida, Gainesville, FL 32611, USA.

Protein kinase C (PKC) delta (PRKCD) is a member of the novel PKC subfamily that regulates gene expression in bovine trophoblast cells. Additional functions for PRKCD in early embryonic development in cattle have not been fully explored. The objectives of this study were to describe the expression profile of PRKCD mRNA in bovine embryos and to examine its biological roles during bovine embryo development. Both PRKCD mRNA and protein are present throughout early embryo development and increases in mRNA abundance are evident at morula and blastocyst stages. Phosphorylation patterns are consistent with detection of enzymatically active PRKCD in bovine embryos. Exposure to a pharmacological inhibitor (rottlerin) during early embryonic development prevented development beyond the eight- to 16-cell stage. Treatment at or after the 16-cell stage reduced blastocyst development rates, total blastomere numbers and inner cell mass-to-trophoblast cell ratio. Exposure to the inhibitor also decreased basal interferon tau (IFNT) transcript abundance and abolished fibroblast growth factor-2 induction of IFNT expression. Furthermore, trophoblast adhesion and proliferation was compromised in hatched blastocysts. These observations provide novel insights into PRKCD mRNA expression profiles in bovine embryos and provide evidence for PRKCD-dependent regulation of embryonic development, gene expression and post-hatching events.
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http://dx.doi.org/10.1071/RD14160DOI Listing
March 2016

Development of FGF2-dependent pluripotent stem cells showing naive state characteristics from murine preimplantation inner cell mass.

Stem Cell Res 2014 Jul 26;13(1):75-87. Epub 2014 Apr 26.

Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Two distinct types of embryonic pluripotent stem cells can be established from either the inner cell mass (ICM) of preimplantation blastocyst (leukemia inhibitory factor (LIF)-dependent embryonic stem cell, ESC, called naive state) or the epiblast of postimplantation fetuses (fibroblast growth factor 2 (FGF2)-dependent epiblast stem cells, EpiSC, called primed state). Here, we report that naive pluripotent stem cell was established from the ICM, but maintained its self-renewal by treatment with FGF2 and mouse embryonic fibroblasts (MEFs) when they were exposed FGF2 during establishment. This cell line is competent to contribute to chimeric animals, including germ cells, at high efficiency. The ERK1/2, SMAD2/3, and JAK/STAT3 pathways are essential to maintain self-renewal. Inhibition of ERK1/2 or SMAD2/3 initiates transition to a naive state ESC-like state, whereas inhibition of JAK/STAT3 promotes a primed EpiSC-like character. Our present results could provide novel insights into understanding the growth factor environment and ICM plasticity, and mechanisms which orchestrate the pluripotency of embryonic stem cells and the capacity for chimeric contributions.
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http://dx.doi.org/10.1016/j.scr.2014.04.012DOI Listing
July 2014

Delay in cleavage of porcine embryos after intracytoplasmic sperm injection (ICSI) shows poorer embryonic development.

J Reprod Dev 2014 1;60(3):256-9. Epub 2014 Apr 1.

Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Ibaraki 305-0901, Japan.

In pigs, the embryonic developmental ability after intracytoplasmic sperm injection (ICSI) is inferior to that resulting from in vitro fertilization (IVF). We evaluated the timing of cell division up to blastocyst formation on embryonic development after ICSI using either whole sperm (w-ICSI) or the sperm head alone (h-ICSI) and IVF as a control. At 10 h after ICSI or IVF, we selected only zygotes, and each of the zygotes/embryos was evaluated for cleavage every 24 h until 168 h. We then observed a delay in the 1st and 2nd cleavages of h-ICSI embryos and also in blastocoele formation by w-ICSI embryos in comparison with IVF embryos. The rate of blastocyst formation and the quality of blastocysts in both ICSI groups were inferior to those in the IVF group. In conclusion, the delay in cleavage of porcine ICSI embryos shows poorer embryonic development.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4085392PMC
http://dx.doi.org/10.1262/jrd.2013-100DOI Listing
April 2015

Regulation of pluripotency of inner cell mass and growth and differentiation of trophectoderm of the bovine embryo by colony stimulating factor 2.

Biol Reprod 2013 Dec 19;89(6):141. Epub 2013 Dec 19.

Department of Animal Sciences, D.H. Barron Reproductive and Perinatal Biology Research Program, and Genetics Institute, University of Florida, Gainesville, Florida.

Colony-stimulating factor 2 (CSF2) enhances competence of the bovine embryo to establish and maintain pregnancy after the embryo is transferred into a recipient. Mechanisms involved could include regulation of lineage commitment, growth, or differentiation of the inner cell mass (ICM) and trophectoderm (TE). Experiments were conducted to evaluate regulation by CSF2 of pluripotency of the ICM and differentiation and growth of the TE. Embryos were cultured with 10 ng/ml recombinant bovine CSF2 or a vehicle control from Days 5 to 7 or 6 to 8 postinsemination. CSF2 increased the number of putative zygotes that developed to blastocysts when the percent of embryos becoming blastocysts in the control group was low but decreased blastocyst yield when blastocyst development in controls was high. ICM isolated from blastocysts by lysing the trophectoderm using antibody and complement via immunosurgery were more likely to survive passage when cultured on mitomycin C-treated fetal fibroblasts if derived from blastocysts treated with CSF2 than if from control blastocysts. There was little effect of CSF2 on characteristics of TE outgrowths from blastocysts. The exception was a decrease in outgrowth size for embryos treated with CSF2 from Days 5 to 7 and an increase in expression of CDX2 when treatment was from Days 6 to 8. Expression of the receptor subunit gene CSF2RA increased from the zygote stage to the 9-16 cell stage before decreasing to the blastocyst stage. In contrast, CSF2RB was undetectable at all stages. In conclusion, CSF2 improves competence of the ICM to survive in a pluripotent state and alters TE outgrowths. Actions of CSF2 occur through a signaling pathway that is likely to be independent of CSF2RB.
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http://dx.doi.org/10.1095/biolreprod.113.113183DOI Listing
December 2013

Changes in the transcriptome of morula-stage bovine embryos caused by heat shock: relationship to developmental acquisition of thermotolerance.

Reprod Biol Endocrinol 2013 Jan 15;11. Epub 2013 Jan 15.

Kyushu-Okinawa Agricultural Research Center, National Agriculture and Food Research Organization, Kumamoto 861-1192, Japan.

Background: While initially sensitive to heat shock, the bovine embryo gains thermal resistance as it progresses through development so that physiological heat shock has little effect on development to the blastocyst stage by Day 5 after insemination. Here, experiments using 3' tag digital gene expression (3'DGE) and real-time PCR were conducted to determine changes in the transcriptome of morula-stage bovine embryos in response to heat shock (40 degrees C for 8 h) that could be associated with thermotolerance.

Results: Using 3'DGE, expression of 173 genes were modified by heat shock, with 94 genes upregulated by heat shock and 79 genes downregulated by heat shock. A total of 38 differentially-regulated genes were associated with the ubiquitin protein, UBC. Heat shock increased expression of one heat shock protein gene, HSPB11, and one heat shock protein binding protein, HSPBP1, tended to increase expression of HSPA1A and HSPB1, but did not affect expression of 64 other genes encoding heat shock proteins, heat shock transcription factors or proteins interacting with heat shock proteins. Moreover, heat shock increased expression of five genes associated with oxidative stress (AKR7A2, CBR1, GGH, GSTA4, and MAP2K5), decreased expression of HIF3A, but did not affect expression of 42 other genes related to free radical metabolism. Heat shock also had little effect on genes involved in embryonic development. Effects of heat shock for 2, 4 and 8 h on selected heat shock protein and antioxidant genes were also evaluated by real-time PCR. Heat shock increased steady-state amounts of mRNA for HSPA1A (P<0.05) and tended to increase expression of HSP90AA1 (P<0.07) but had no effect on expression of SOD1 or CAT.

Conclusions: Changes in the transcriptome of the heat-shocked bovine morula indicate that the embryo is largely resistant to effects of heat shock. As a result, transcription of genes involved in thermal protection is muted and there is little disruption of gene networks involved in embryonic development. It is likely that the increased resistance of morula-stage embryos to heat shock as compared to embryos at earlier stages of development is due in part to developmental acquisition of mechanisms to prevent accumulation of denatured proteins and free radical damage.
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http://dx.doi.org/10.1186/1477-7827-11-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3583805PMC
January 2013

The expression of fibroblast growth factor receptors during early bovine conceptus development and pharmacological analysis of their actions on trophoblast growth in vitro.

Reproduction 2013 Feb 24;145(2):191-201. Epub 2013 Jan 24.

Department of Animal Sciences, University of Florida, Gainesville, Florida 32611, USA.

The overall aim of this work was to examine the expression profiles for fibroblast growth factor receptors (FGFRs) and describe their biological importance during bovine pre- and peri-implantation conceptus development. FGFR1 and FGFR2 mRNAs were detected at 1-, 2-, 8-cell, morula and blastocyst stages whereas FGFR3 and FGFR4 mRNAs were detected after the 8-cell stage but not earlier. The abundance of FGFR1, FGFR3, and FGFR4 mRNAs increased at the morula and blastocyst stages. Immunofluorescence microscopy detected FGFR2 and FGFR4 exclusively in trophoblast cells whereas FGFR1 and FGFR3 were detected in both trophoblast cells and inner cell mass in blastocysts. Neither transcripts for FGF10 nor its receptor (FGFR2b) were temporally related to interferon τ (IFNT) transcript profile during peri- and postimplantation bovine conceptus development. A series of studies used a chemical inhibitor of FGFR kinase function (PD173074) to examine FGFR activation requirements during bovine embryo development. Exposing embryos to the inhibitor (1 μM) beginning on day 5 post-fertilization did not alter the percentage of embryos that developed into blastocysts or blastocyst cell numbers. The inhibitor did not alter the abundance of CDX2 mRNA but decreased (P<0.05) the relative abundance of IFNT mRNA in blastocysts. Exposing blastocysts to the inhibitor from days 8 to 11 post-fertilization reduced (P<0.05) the percentage of blastocysts that formed outgrowths after transfer to Matrigel-coated plates. In conclusion, each FGFR was detected in bovine embryos, and FGFR activation is needed to maximize IFNT expression and permit outgrowth formation.
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http://dx.doi.org/10.1530/REP-12-0220DOI Listing
February 2013

Global gene expression of the inner cell mass and trophectoderm of the bovine blastocyst.

BMC Dev Biol 2012 Nov 6;12:33. Epub 2012 Nov 6.

Department of Animal Sciences and D,H, Barron Reproductive and Perinatal Biology Research Program, PO Box 110910, Gainesville, FL 32611-0910, USA.

Background: The first distinct differentiation event in mammals occurs at the blastocyst stage when totipotent blastomeres differentiate into either pluripotent inner cell mass (ICM) or multipotent trophectoderm (TE). Here we determined, for the first time, global gene expression patterns in the ICM and TE isolated from bovine blastocysts. The ICM and TE were isolated from blastocysts harvested at day 8 after insemination by magnetic activated cell sorting, and cDNA sequenced using the SOLiD 4.0 system.

Results: A total of 870 genes were differentially expressed between ICM and TE. Several genes characteristic of ICM (for example, NANOG, SOX2, and STAT3) and TE (ELF5, GATA3, and KRT18) in mouse and human showed similar patterns in bovine. Other genes, however, showed differences in expression between ICM and TE that deviates from the expected based on mouse and human.

Conclusion: Analysis of gene expression indicated that differentiation of blastomeres of the morula-stage embryo into the ICM and TE of the blastocyst is accompanied by differences between the two cell lineages in expression of genes controlling metabolic processes, endocytosis, hatching from the zona pellucida, paracrine and endocrine signaling with the mother, and genes supporting the changes in cellular architecture, stemness, and hematopoiesis necessary for development of the trophoblast.
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http://dx.doi.org/10.1186/1471-213X-12-33DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3514149PMC
November 2012

Double expression of CD34 and CD117 on bone marrow progenitors is a hallmark of the development of functional mast cell of Callithrix jacchus (common marmoset).

Int Immunol 2012 Sep 25;24(9):593-603. Epub 2012 Jul 25.

Department of Molecular Cell Immunology and Allergology, Nihon University School of Medicine, Tokyo, Japan.

Mast cells (MCs) are developed from hematopoietic progenitor cells and play an important role in inflammation. Study of the kinetics of development and accumulation of primate MC in vivo is crucial for the control of human inflammatory diseases, as evolution of the immune system is quite rapid and inflammation including MC response is considered to be different between mouse and human. In the present study, we examined the development of MC from hematopoietic progenitors of Callithrix jacchus (common marmoset), an experimental animal of nonhuman primates. Bone marrow cells were fractionated for the expression of CD34 and CD117 by cell sorting. MCs were developed in vitro or by transplanting the cells to NOD/SCID/IL-2γc knockout (NOG) mice. In vitro culture of CD34(+)CD117(+) (double positive, DP) cells with stem cell factor could generate high-affinity Fc epsilon receptor (FcεR)-expressing CD117(+) cells with typical granules. The developed MC released β-hexosaminidase and produced leukotriene C(4) after the stimulation of FcεRI. Transplantation of DP cells gave rise to a marked expansion of CD34(-)CD45(+)CD117(+)FcεR(+) cells in NOG mice. They expressed transcripts encoding chymase 1 and tryptase β. Differentiation of CD34(-)CD117(+) cells to MCs was relatively limited compared with the DP cells, similarly to human MCs. These results suggest that this marmoset system provides a good model for human MC development.
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http://dx.doi.org/10.1093/intimm/dxs070DOI Listing
September 2012

Fibroblast growth factor 2 promotes primitive endoderm development in bovine blastocyst outgrowths.

Biol Reprod 2011 Nov 20;85(5):946-53. Epub 2011 Jul 20.

Department of Animal Sciences, University of Florida, Gainesville, Florida 32611-0910, USA.

Primitive endoderm (PE) is the second extraembryonic tissue to form during embryogenesis in mammals. The PE develops from pluripotent cells of the blastocyst inner cell mass. Experimental results described herein provide evidence that FGF2 stimulates PE development during bovine blastocyst development in vitro. Bovine blastocysts were cultured individually on a feeder layer-free, Matrigel-coated surface in the presence or absence of FGF2. A majority of blastocysts cultures formed outgrowths (76.8%) and the rate of outgrowth formation was not affected by FGF2 supplementation. However, supplementation with FGF2 increased the incidence of PE outgrowths on Days 13 and 15 after in vitro fertilization. Presumptive PE cultures contained cells with a phenotype distinct from trophectoderm (TE). Cell identity was validated by expression of GATA4 and GATA6 mRNA and transferrin protein, all markers of the PE lineage. Expression of GATA4 occurred coincident with blastocyst expansion and hatching. These cells did not express IFNT and CDX2 (TE lineage markers). Profiles of FGF receptor (FGFR) isoforms were distinct between PE and TE cultures. Specifically, FGFR1b and FGFR1c were the predominant FGFR transcripts in PE whereas FGFR2b transcripts were abundant in TE. Supplementation with FGF2 increased the mitotic index of PE but not TE. Moreover, FGF signaling appears important for initiation of PE formation in blastocysts, presumably by lineage committal from NANOG-positive epiblast cells, because chemical disruption of FGFR kinase activity with PD173074 reduces GATA4 expression and increases NANOG expression. Collectively, these results indicate that FGF2 and potentially other FGFs specify PE formation and mediate PE proliferation during early pregnancy in cattle.
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http://dx.doi.org/10.1095/biolreprod.111.093203DOI Listing
November 2011

Factors affecting fertilization and embryonic development during intracytoplasmic sperm injection in pigs.

J Reprod Dev 2011 Apr;57(2):183-7

National Institute of Agrobiological Sciences, Ibaraki, Japan.

In intracytoplasmic sperm injection (ICSI) technique, a sperm was injected into ooplasm directly using a glass pipette. The fertilization physiology in ICSI is considered quite different from that of the natural fertilization. The different mechanisms for fertilization may be the causes of various results in ICSI. In this paper, we focus on the state of sperm membranes, nuclear or DNA integrity during ICSI procedure and discuss the influence of these factors on fertilization and embryonic development. We also introduce some examples in application of ICSI for new technologies in pigs.
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http://dx.doi.org/10.1262/jrd.10-200eDOI Listing
April 2011

A novel method for purification of inner cell mass and trophectoderm cells from blastocysts using magnetic activated cell sorting.

Fertil Steril 2011 Feb 5;95(2):799-802. Epub 2010 Nov 5.

Department of Animal Sciences and DH Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, Florida 32601, USA.

Objective: To develop a simple method to purify blastomeres of inner cell mass (ICM) and trophectoderm (TE) lineage using magnetic activated cell sorting.

Design: Prospective laboratory study.

Setting: Embryology research laboratory.

Patient(s): None.

Intervention(s): Trophectoderm cells of zona-free blastocysts were labeled with concanavalin A conjugated to FITC, and every nucleus in the blastocyst was labeled with Hoechst 33342. The labeled blastocyst was disaggregated to single cells by trypsin treatment followed by pipetting using a finely drawn, flame-polished micropipet. Disaggregated blastomeres were incubated with anti-FITC antibody conjugated to magnetic microbeads and subjected to magnetic cell sorting to separate cells into FITC-positive and -negative fractions.

Main Outcome Measure(s): Purity and gene expression.

Result(s): In the FITC-positive fraction, an average of 91.2% of cells was dual-labeled with FITC and Hoechst, whereas only 7.8% of FITC negative fractions were labeled with FITC. Expression of CDX2, a trophectoderm marker, was significantly higher in the FITC-positive fraction, whereas expression of NANOG, an inner cell mass marker, was significantly higher in the FITC-negative fraction.

Conclusion(s): Highly purified trophectoderm cells or inner cell mass cells can be collected using magnetic activated cell sorting. This method can be useful for understanding differentiation and function of cell lineages in the blastocyst.
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http://dx.doi.org/10.1016/j.fertnstert.2010.10.006DOI Listing
February 2011

Evaluation of developmental competence of in vitro-produced porcine embryos based on the timing, pattern and evenness of the first cleavage and onset of the second cleavage.

J Reprod Dev 2010 Dec 20;56(6):593-600. Epub 2010 Jul 20.

Department of Animal Science, National Institute of Agrobiological Sciences, Ibaraki, Japan.

The following selection markers for in vitro-produced porcine embryos were investigated: the timing, pattern and evenness of the first cleavage and the timing of the second cleavage. The embryos that cleaved by 30 h post-insemination (hpi) developed to blastocysts at a significantly higher rate (60.9%) and with a significantly higher cell number (33.6 cells) than those of embryos cleaved by 36 hpi (26.4% and 23.6 cells, respectively, P<0.05). Blastocyst proportions derived from 2- and 3-cell embryos cleaved by 30 hpi (68.2 and 65.3%, respectively) were significantly higher than those of 4- and >4-cell embryos (46.3 and 42.6%, respectively, P<0.05). The cell number per blastocyst generated from 2-cell embryos was significantly greater (37.3 cells) than those from 3-, 4- and >4-cell embryos (23.6-27.8 cells, P<0.05). Among embryos cleaved by 30 hpi, the blastocysts derived from evenly cleaved embryos (40.6 cells) were of significantly better quality than those derived from unevenly cleaved embryos (33.2 cells, P<0.05), although their blastocyst rates did not differ. The evenly cleaved embryos that underwent subsequent cleavage within 18 h had significantly higher blastocyst rates (72.7-81.0%) and quality (36.2-40.9 cells) than those without subsequent cleavage (48.3% and 22.5 cells, respectively, P<0.05) during the same period. In conclusion, the timing, pattern and evenness of the first cleavage and the timing of the second cleavage affected the developmental competence and quality of in vitro-produced porcine embryos.
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http://dx.doi.org/10.1262/jrd.10-038mDOI Listing
December 2010

Introduction of various vietnamese indigenous pig breeds and their conservation by using assisted reproductive techniques.

J Reprod Dev 2010 Feb;56(1):31-5

Department of Animal Breeding and Reproduction, National Institute of Livestock and Grassland Science, Japan.

Pigs are one of the most important domesticated animals in Vietnam. They are the main source of meat for the Vietnamese. According to FAO statistics, Vietnam is among the top 5 countries raising pigs in the world, with nearly 27 million hogs. This review article introduces the distribution, morphology, growth potential, meat-producing ability and reproductive efficiency of six Vietnamese indigenous pig breeds: I, Mong Cai, Muong Khuong, Soc, Meo and Co. The collected data showed that these Vietnamese pigs are less effective in comparison with Western pigs in terms of reproductive and meat-producing ability as well as weight growth. However, these Vietnamese indigenous breeds have some special characteristics, such as very early sexual maturity, and good adaptability to harsh raising conditions or poor feeding. Moreover, recent genetic research has shown that Vietnamese pigs are genetically diverse. Thus, conservation of these pig breeds using assisted reproductive techniques is urgent and important.
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http://dx.doi.org/10.1262/jrd.09-165kDOI Listing
February 2010

Production of viable piglets for the first time using sperm derived from ectopic testicular xenografts.

Reproduction 2010 Feb 16;139(2):331-5. Epub 2009 Dec 16.

Division of Animal Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.

Xenografting of testicular tissue into immunodeficient mice is known to be a valuable tool for facilitating the development of immature germ cells present in mammalian gonads. Spermatogenesis in xenografts and/or in vitro embryonic development to the blastocyst stage after ICSI of xenogeneic sperm has already been reported in large animals, including pigs; however, development of the embryos to term has not yet been confirmed. Therefore, in pigs, we evaluated the in vivo developmental ability of oocytes injected after ICSI of xenogeneic sperm. Testicular tissues prepared from neonatal piglets, which contain seminiferous cords consisting of only gonocytes/spermatogonia, were transplanted under the back skin of castrated nude mice. Between 133 and 280 days after xenografting, morphologically normal sperm were recovered, and a single spermatozoon was then injected into an in vitro matured porcine oocyte. After ICSI, the oocytes were electrostimulated and transferred into estrus-synchronized recipients. Two out of 23 recipient gilts gave birth to six piglets. Here, we describe for the first time that oocytes fertilized with a sperm from ectopic xenografts have the ability to develop to viable offspring in large mammals.
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http://dx.doi.org/10.1530/REP-09-0509DOI Listing
February 2010

Cumulus cell-enclosed oocytes acquire a capacity to synthesize GSH by FSH stimulation during in vitro maturation in pigs.

J Cell Physiol 2010 Feb;222(2):294-301

National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.

We investigated (i) follicle stimulating hormone (FSH)-modulated changes in the expression of glutathione (GSH) and its rate-limiting enzyme, glutamate cysteine ligase (GCL), in porcine oocytes and cumulus cells, and (ii) the contribution of gap-junctional communications (GJCs) in cumulus-oocyte complexes (COCs) to intraoocyte GSH accumulation. In experiment (i), COCs were cultured for 48 h with (+FSH group) or without FSH (-FSH group). The GSH content of oocytes increased with cultivation time in the +FSH group, but decreased in the -FSH group. The GSH content of cumulus cells at 48 h was also higher in the +FSH group than that in the -FSH group. Expression of GCL subunit mRNAs in oocytes and cumulus cells was increased by FSH stimulation until 12 h, and then fell to the baseline level. On the other hand, the amount of GCL subunit proteins in oocytes and cumulus cells increased gradually throughout the period of culture with FSH. In experiment (ii), blocking of GJCs in COCs during 0-24 h of culture led to a decrease in the GSH content of oocytes at 24 h of culture, whereas the GSH content at 48 h of culture did not differ even after blocking of the GJCs during 24-48 h of culture. These findings indicate that FSH initiates GSH synthesis in cumulus cells and oocytes by modulating the expression of GCL, and that porcine oocytes are able to synthesize GSH without GJC-mediated support from cumulus cells, at least in the later half of maturation culture.
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http://dx.doi.org/10.1002/jcp.21949DOI Listing
February 2010

Development of monoclonal antibodies for analyzing immune and hematopoietic systems of common marmoset.

Exp Hematol 2009 Nov 26;37(11):1318-29. Epub 2009 Aug 26.

Department of Immunology, Tokai University School of Medicine, Isehara, Japan.

Objective: Common marmosets are considered experimental animals of primates useful for medical research. We developed several monoclonal antibodies (mAbs) directed to CD molecules to gain initial insight into the immune and hematopoietic systems of this organism, and analyzed the basic cellularity and characters of marmoset lymphocytes.

Materials And Methods: Anti-marmoset CD antigen mAbs were prepared using marmoset antigen-expressing transfectants and used for flow cytometric analyses and cell fractionation. Expression of T-cell-related cytokine gene transcripts was examined in response to T-cell receptor stimulation by reverse transcription polymerase chain reaction analyses. Hematopoietic progenitor activities of marmoset bone marrow cells were examined in fractionated cells by mAbs against CD117 (c-kit) and CD34.

Results: CD4 and CD8 expression profiles in T-cell subsets of marmoset were essentially similar to those in mouse and human. CD4(+) and CD8(+) subsets were isolated from marmoset spleens. Detected transcripts after stimulation of T cells included Th1-, Th2-, and Th17-related cytokines in CD4(+) cells and cytotoxic proteases in CD8(+) cells, respectively. Colony-forming abilities were detected mainly in CD117 (c-kit)(+) cells, irrespective of CD34 expression.

Conclusions: Marmoset immune system was basically similar to human and mouse systems.
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http://dx.doi.org/10.1016/j.exphem.2009.08.003DOI Listing
November 2009

Live piglets derived from in vitro-produced zygotes vitrified at the pronuclear stage.

Biol Reprod 2009 Jan 3;80(1):42-9. Epub 2008 Sep 3.

Division of Animal Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.

We report the successful cryopreservation of in vitro-produced porcine zygotes. Follicular oocytes from prepubertal gilts were matured (IVM), fertilized (IVF), and cultured (IVC) in vitro. At 10 or 23 h after IVF, the oocytes were centrifuged to visualize pronuclei. Zygotes with two or three pronuclei were used for solid surface vitrification (SSV). Survival of vitrified-warmed zygotes was determined by their morphology. To assess their developmental competence, vitrified (SSV), cryoprotectant-treated (CPA), and untreated (control) zygotes were subjected to IVC for 6 days. Survival and developmental competence did not differ between control and CPA zygotes. The proportion of live zygotes after SSV and warming (93.4%) was similar to that in the controls (100%). Cleavage and blastocyst formation rates of SSV zygotes after vitrification (71.7% and 15.8%, respectively) were significantly lower than those of controls (86.3% and 24.5%, respectively; ANOVA P<0.05). Blastocyst cell numbers of SSV and control embryos were similar (41.2+/-3.4 and 41.6+/-3.3, respectively). There was no difference in developmental ability between zygotes cryopreserved at an early (10 h after IVF) or late (23 h after IVF) pronuclear stage. Storage in liquid nitrogen had no effect on the in vitro developmental competence of vitrified zygotes beyond the reduction induced by the vitrification itself. When the embryo culture medium was supplemented with 1 muM glutathione, the rate of development of cryopreserved zygotes to the blastocyst stage did not differ significantly from that of control glutathione-treated zygotes (18.6% and 22.1%, respectively). To test their ability to develop to term, vitrified zygotes were transferred to five recipients, resulting in three pregnancies and the production of a total of 17 piglets. These data demonstrate that IVM-IVF porcine zygotes can be cryopreserved at the pronuclear stage effectively without micromanipulation-derived delipation, preserving their full developmental competence to term.
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http://dx.doi.org/10.1095/biolreprod.108.070235DOI Listing
January 2009

Affected homologous chromosome pairing and phosphorylation of testis specific histone, H2AX, in male meiosis under FKBP6 deficiency.

J Reprod Dev 2008 Jun 14;54(3):203-7. Epub 2008 Apr 14.

Reproductive Biology Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Japan.

A gene for FK506 binding protein 6 (Fkbp6) expresses during a specific stage of male and female meiosis. Disruption of the gene influences male reproduction, i.e. arrests spermatogenesis, but not female reproduction. Using the mouse model (targeted disruption), the role of the gene in homologous chromosome pairing has been demonstrated in a previous study. For further understanding the function of Fkbp6 in chromosome synapsis, we evaluated chromosome pairings during male meiosis in the as/as rat, a spontaneous null mutation, and compared them with those of the mouse model. Electron microscopy of the pachytene nuclei unveiled several types of abnormal chromosome pairing in the rat model, as shown in the mouse previously. The frequencies of aberrant pairings in the knockout mice and mutant rats were 42 of 67 nuclei (62.7%) and 20 out of 74 nuclei (27.0%), respectively. In order to clarify the mechanism of male specific infertility in Fkbp6 deficiency, the localization of gammaH2AX, a marker protein of XY chromosome inactivation during male meiosis, was examined. Immunostaining of gammaH2AX unveiled normal localization of the molecule to XY chromosomes (XY body) in both models, showing the independency of FKBP6 in sex chromosome inactivation. Besides the XY body, focal localization of gammaH2AX was observed in accordance with the unsynapsed chromosomes in both types of null animal. These results indicate the fundamental role of Fkbp6 in homologous chromosome synapsis during male meiosis. In conclusion, male specific infertility under Fkbp6 deficiency remains unsolved.
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http://dx.doi.org/10.1262/jrd.19158DOI Listing
June 2008

DL-alpha-tocopherol acetate mitigates maternal hyperthermia-induced pre-implantation embryonic death accompanied by a reduction of physiological oxidative stress in mice.

Reproduction 2008 Apr;135(4):489-96

Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.

Maternal hyperthermia induces pre-implantation embryo death, which is accompanied by enhanced physiological oxidative stress. We evaluated whether the administration of DL-alpha-tocopherol acetate (TA) to hyperthermic mothers mitigated pre-implantation embryo death. Mice were exposed to heat stress (35 degrees C, 60% relative humidity) for 12 h or not heated (25 degrees C) on the day of mating. Twelve hours before the beginning of temperature treatment, TA was injected intraperitoneally at a dose of 1 g/kg body weight. After the treatment, zygotes were recovered and the developmental abilities and intracellular glutathione (GSH) levels were evaluated. Another set of mice, with or without TA treatment, was exposed to heat stress for 12, 24 and 36 h, and the urinary levels of the oxidative stress marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) were measured. Heat stress significantly decreased the blastocyst development rate and the GSH content in zygotes, as compared with the non-heat-stressed embryos, while TA administration significantly mitigated the deleterious effects of heat stress with regard to both parameters. Moreover, although the urinary levels of 8-OHdG gradually increased according to the duration of heat exposure, with or without TA administration, the levels were lower in the TA-administered group than in the placebo-injected mice. These results suggest that heat stress enhances physiological oxidative stress, and that TA administration alleviates the hyperthermia-induced death of pre-implantation embryos by reducing physiological oxidative stress.
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http://dx.doi.org/10.1530/REP-07-0379DOI Listing
April 2008