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

Using 2n/3n mixoploid mouse embryos produced by fusion of individual second polar bodies (PB2s) with individual blastomeres of 2-cell embryos, the dynamics of PB2 nuclei in the host blastomeres during mitosis were examined and the fate of the 3n cell line in the mixoploid embryos was followed. Most of the PB2 nuclei were synchronised with the cell cycle of the host blastomeres and all chromosomes were incorporated into a single mitotic spindle. The majority of the mixoploid embryos developed to blastocysts with 3n cells. In conceptuses at Day 11.5 and Day 18.5 of gestation, 3n cells were recognised in both of the embryonic/fetal and placental tissues. When green fluorescent protein (GFP)-transgenic mice were used as a donor of PB2, GFP-positive 3n cells were found in more than 40% of morulae and blastocysts, indicating that the PB2 genome can be reactivated during the pre-implantation stage. GFP-positive 3n cells were non-randomly allocated in trophectoderm in blastocysts. These findings may explain the production mechanism of 2n/3n mixoploid human embryos, that is, a PB2 is incorporated into one daughter blastomere during the early cleavage period.

DOI： 10.1071/RD15081

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

Sirh7/Ldoc1 [sushi-ichi retrotransposon homolog 7/leucine zipper, downregulated in cancer 1, also called mammalian retrotransposonderived 7 (Mart7)] is one of the newly acquired genes from LTR retrotransposons in eutherian mammals. Interestingly, Sirh7/Ldoc1 knockout (KO) mice exhibited abnormal placental cell differentiation/maturation, leading to an overproduction of placental progesterone (P4) and placental lactogen 1 (PL1) from trophoblast giant cells (TGCs). The placenta is an organ that is essential for mammalian viviparity and plays a major endocrinological role during pregnancy in addition to providing nutrients and oxygen to the fetus. P4 is an essential hormone in the preparation and maintenance of pregnancy and the determination of the timing of parturition in mammals; however, the biological significance of placental P4 in rodents is not properly recognized. Here, we demonstrate that mouse placentas do produce P4 in mid-gestation, coincident with a temporal reduction in ovarian P4, suggesting that it plays a role in the protection of the conceptuses specifically in this period. Pregnant Sirh7/Ldoc1 knockout females also displayed delayed parturition associated with a low pup weaning rate. All these results suggest that Sirh7/ Ldoc1 has undergone positive selection during eutherian evolution as a eutherian-specific acquired gene because it impacts reproductive fitness via the regulation of placental endocrine function.

DOI： 10.1242/dev.114520

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

Ca2+ ionophore A23187 is known to induce the acrosome reaction of mammalian spermatozoa, but it also quickly immobilizes them. Although mouse spermatozoa were immobilized by this ionophore, they initiated vigorous motility (hyperactivation) soon after this reagent was washed away by centrifugation. About half of live spermatozoa were acrosome-reacted at the end of 10 min of ionophore treatment; fertilization of cumulus-intact oocytes began as soon as spermatozoa recovered their motility and before the increase in protein tyrosine phosphorylation, which started 30-45 min after washing out the ionophore. When spermatozoa were treated with A23187, more than 95% of oocytes were fertilized in the constant presence of the protein kinase A inhibitor, H89. Ionophore-treated spermatozoa also fertilized 80% of oocytes, even in the absence of HCO3-, a component essential for cAMP synthesis under normal in vitro conditions. Under these conditions, fertilized oocytes developed into normal offspring. These data indicate that mouse spermatozoa treated with ionophore are able to fertilize without activation of the cAMP/PKA signaling pathway. Furthermore, they suggest that the cAMP/PKA pathway is upstream of an intracellular Ca2+ increase required for the acrosome reaction and hyperactivation of spermatozoa under normal in vitro conditions.

DOI： 10.1073/pnas.1317113110

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

Incorporation of a second polar body (PB2) into one of the blastomeres has been considered as a causal mechanism underlying diploid/triploid mixoploidy in humans. Using a mouse model, we examined whether PB2s can participate in the formation of mixoploidy.
Uptake of BrdU was examined to determine DNA synthesis in PB2s up to 28 h after fertilization. PB2s from embryos at 4-6 (1-cell), 24 (2-cell), 48 (4-cell), and 72 h (morula) were fused with MII oocytes to induce premature chromosome condensation. Caspase and TUNEL assays were used to detect apoptotic PB2s at 24, 48, and 72 h. PB2s were fused with one of the blastomeres of the 2-cell embryos to produce mixoploid embryos.
DNA synthesis in the PB2s continued until 22 h after fertilization. At 4-6 h, nearly all of the PB2s showed G1-type chromosomes and there was no significant increase in chromosome damage. At 24, 48, and 72 h, S-type chromatin predominated. Few PB2s showed apoptotic response until 72 h. Regardless of the fusion with the PB2, more than 90 % of the embryos developed to 4-cell stage, and over 80 % of the resultant 4-cell embryos had daughter blastomeres with a morphologically normal nucleus. Some of the daughter blastomeres displayed triploidy.
The PB2 is viable for at least 72 h after fertilization, with slow progression through the cell cycle. Once the PB2 has been incorporated into a blastomere, the cell cycle of the PB2 might be synchronized with that of the host resulting in diploid/triploid mixoploidy.

DOI： 10.1007/s10815-012-9899-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: During permanent memory formation, recall of acquired place memories initially depends on the hippocampus and eventually become hippocampus-independent with time. It has been suggested that the quality of original place memories also transforms from a precise form to a less precise form with similar time course. The question arises of whether the quality of original place memories is determined by brain regions on which the memory depends.
Results: To directly test this idea, we introduced a new procedure: a non-associative place recognition memory test in mice. Combined with genetic and pharmacological approaches, our analyses revealed that place memory is precisely maintained for 28 days, although the recall of place memory shifts from hippocampus-dependent to hippocampus-independent with time. Moreover, the inactivation of the hippocampal function does not inhibit the precision of remote place memory.
Conclusion: These results indicate that the quality of place memories is not determined by brain regions on which the memory depends.

DOI： 10.1186/1756-6606-5-5

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

We investigated whether the small litter size in the 129 inbred mouse strain results from a reduction in oocyte fertilizability. Sensitivity of the zona pellucida to alpha-chymotrypsin was examined for oocytes collected at 14 h (shortly after ovulation), 17 h, and 20 h after hCG injection. Passage of spermatozoa through the zona pellucida (using an in vitro fertilization (IVF) technique) and the density of cortical granules were examined for oocytes collected at 14 and 17 h after hCG injection. The capability of the oolemma to fuse with the sperm plasma membrane was also evaluated by IVF using zona-free eggs. The zona pellucida became markedly resistant to the enzyme 17 h after hCG injection. IVF rates significantly decreased at this time. In addition, there was a significant reduction in the density of cortical granules. When zona-free oocytes were inseminated, high fertilization rates were obtained at both 17 and 14 h after hCG injection. These results indicate that accelerated modification of the zona pellucida primarily causes a decreased fertilizability of oocytes in 129 mice, resulting in the low reproductive performance of this strain.

DOI： 10.1017/S0967199410000481

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

In general, cell proliferation and differentiation show an inverse relationship, and are regulated in a coordinated manner during development. Embryonic cardiomyocytes must support embryonic life by functional differentiation such as beating, and proliferate actively to increase the size of the heart. Therefore, progression of both proliferation and differentiation is indispensable. It remains unknown whether proliferation and differentiation are related in these embryonic cardiomyocytes. We focused on abnormal phenotypes, such as hyperproliferation, inhibition of differentiation and enhanced expression of cyclin D1 in cardiomyocytes of mice with mutant jumonji (Jmj, Jarid2), which encodes the repressor of cyclin D1. Analysis of Jmj/cyclin D1 double mutant mice showed that Jmj was required for normal differentiation and normal expression of GATA4 protein through cyclin D1. Analysis of transgenic mice revealed that enhanced expression of cyclin D1 decreased GATA4 protein expression and inhibited the differentiation of cardiomyocytes in a CDK4/6-dependent manner, and that exogenous expression of GATA4 rescued the abnormal differentiation. Finally, CDK4 phosphorylated GATA4 directly, which promoted the degradation of GATA4 in cultured cells. These results suggest that CDK4 activated by cyclin D1 inhibits differentiation of cardiomyocytes by degradation of GATA4, and that initiation of Jmj expression unleashes the inhibition by repression of cyclin D1 expression and allows progression of differentiation, as well as repression of proliferation. Thus, a Jmj-cyclin D1 pathway coordinately regulates proliferation and differentiation of cardiomyocytes.

DOI： 10.1242/dev.059295

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

Background: During fertilization, pronuclear envelope breakdown (PNEB) is followed by the mingling of male and female genomes. Dynamic chromatin and protein rearrangements require posttranslational modification (PTM) for the postfertilization development.
Methodology/Principal Findings: Inhibition of poly(ADP-ribose) polymerase activity (PARylation) by either PJ-34 or 5-AIQ resulted in developmental arrest of fertilized embryos at the PNEB. PARylation inhibition affects spindle bundle formation and phosphorylation of Erk molecules of metaphase II (MII) unfertilized oocytes. We found a frequent appearance of multiple pronuclei (PN) in the PARylation-inhibited embryos, suggesting defective polymerization of tubulins. Attenuated phosphorylation of lamin A/C by PARylation was detected in the PARylation-inhibited embryos at PNEB. This was associated with sustained localization of heterodomain protein 1 (HP1) at the PN of the one-cell embryos arrested by PARylation inhibition.
Conclusions/Significance: Our findings indicate that PARylation is required for pronuclear fusion during postfertilization processes. These data further suggest that PARylation regulates protein dynamics essential for the beginning of mouse zygotic development. PARylation and its involving signal-pathways may represent potential targets as contraceptives.

DOI： 10.1371/journal.pone.0012526

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

A recent study has revealed that fear memory may be vulnerable following retrieval, and is then reconsolidated in a protein synthesis-dependent manner. However, little is known about the molecular mechanisms of these processes. Activin beta A, a member of the TGF-beta superfamily, is increased in activated neuronal circuits and regulates dendritic spine morphology. To clarify the role of activin in the synaptic plasticity of the adult brain, we examined the effect of inhibiting or enhancing activin function on hippocampal long-term potentiation (LTP). We found that follistatin, a specific inhibitor of activin, blocked the maintenance of late LTP (L-LTP) in the hippocampus. In contrast, administration of activin facilitated the maintenance of early LTP (E-LTP). We generated forebrain-specific activin-or follistatin-transgenic mice in which transgene expression is under the control of the Tet-OFF system. Maintenance of hippocampal L-LTP was blocked in the follistatin-transgenic mice. In the contextual fear-conditioning test, we found that follistatin blocked the formation of long-term memory (LTM) without affecting short-term memory (STM). Furthermore, consolidated memory was selectively weakened by the expression of follistatin during retrieval, but not during the maintenance phase. On the other hand, the maintenance of memory was also influenced by activin overexpression during the retrieval phase. Thus, the level of activin in the brain during the retrieval phase plays a key role in the maintenance of long-term memory.

DOI： 10.1101/lm.16659010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：THE SOCIETY OF CHROMOSOME RESEARCH  

To evaluate cytogenetic validity of a simple vitrification technique for embryo cryopreservation, mouse 1-cell embryos were vitrified 4, 6, and 8 h after <i>in vitro</i> fertilization (IVF). In addition, chromosomal damage of spermatozoa treated with methyl methanesulfonate (MMS) was estimated using vitrified 1-cell embryos. More than 90% of embryos survived vitrification regardless of the time after IVF. In the 4-h and 6-h groups, some of the surviving embryos swelled after recovery. The incidence of structural chromosome aberrations and aneuploidy in embryos with morphologically normal features did not significantly increase in any group. The vitrification technique preserved 1-cell embryos derived from MMS-treated spermatozoa without alteration of chromosome damage. This technique will enable us to manage the optimal time for chromosome preparation of mouse 1-cell embryos.

DOI： 10.11352/scr.13.45

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Other Link： http://amcor.asahikawa-med.ac.jp/modules/xoonips/detail.php?id=CS13-3-45

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

Cryopreservation of mouse sperm is useful for maintaining various strains. However, fertility generally decreases after freezing. In particular, the fertility of cryopreserved C57BL/6J sperm is very low. To improve the fertility of frozen sperm, we examined the efficiencies of various media used for sperm preincubation (SP) and in vitro fertilization (IVF) in frozen C57BL/6J sperm. In this study, SP medium was examined for efficiency of fertility with respect to content, especially calcium (Ca2+), phosphate (PO43-) and lactate. In all media containing no Call, including medium lacking Ca2+, lacking Ca2+ and PO43- lacking Ca2+ and lactate and lacking Ca2+, PO43- and lactate, high IVF rates were obtained (79, 69, 76 and 71%, respectively). On the other hand, the rates for media containing Ca2+ were significantly lower (30-38%, P&lt;0.05). After transfer, 41-50% of newborns were obtained in all media containing no Ca2+. In conclusion, preincubation of thawed sperm in medium containing no Ca2+ markedly improved the fertility of cryopreserved C57BL/6J sperm. These results indicate that the present method of IVF using medium with no Ca2+ is practical for use in cryopreserved C57BL/6J sperm.

DOI： 10.1262/jrd.20163

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

Mice with maternal duplication of proximal Chromosome 11 (MatDp(prox11)), where Meg1/Grb10 is located, exhibit pre- and postnatal growth retardation. To elucidate the responsible imprinted gene for the growth abnormality, we examined the precise structure and regulatory mechanism of this imprinted region and generated novel model mice mimicking the pattern of imprinted gene expression observed in the MatDp(prox11) by deleting differentially methylated region of Meg1/Grb10 (Meg1-DMR). It was found that Cobl and Ddc, the neighboring genes of Meg1/Grb10, also comprise the imprinted region. We also found that the mouse-specific repeat sequence consisting of several CTCF-binding motifs in the Meg1-DMR functions as a silencer, suggesting that the Meg1/Grb10 imprinted region adopted a different regulatory mechanism from the H19/Igf2 region. Paternal deletion of the Meg1-DMR (+/Delta DMR) caused both upregulation of the maternally expressed Meg1/Grb10 Type I in the whole body and Cobl in the yolk sac and loss of paternally expressed Meg1/Grb10 Type II and Ddc in the neonatal brain and heart, respectively, demonstrating maternalization of the entire Meg1/Grb10 imprinted region. We confirmed that the +/Delta DMR mice exhibited the same growth abnormalities as the MatDp(prox11) mice. Fetal and neonatal growth was very sensitive to the expression level of Meg1/Grb10 Type I, indicating that the 2-fold increment of the Meg1/Grb10 Type I is one of the major causes of the growth retardation observed in the MatDp(prox11) and +/Delta DMR mice. This suggests that the corresponding human GRB10 Type I plays an important role in the etiology of Silver-Russell syndrome caused by partial trisomy of 7p11-p13.

DOI： 10.1093/hmg/ddp049

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：John Wiley and Sons Ltd  

Purpose: 129 inbred mice show poor reproductive ability, as evidenced by small litters
however, the exact cause of this is unknown. In the present in vivo study we examined fertility and subsequent post-implantation development in an attempt to clarify the cause of small litter size in 129 mice. Methods: 129 or C57BL/6J females that displayed vaginal plugs 1 day after mating with males of the same strain were examined for the presence of fertilized eggs. Reciprocal matings were also performed between 129 and C57BL/6J mice. Subsequent post-implantation development of fertilized eggs was examined by dissecting females 18-19 days after the vaginal plugs were found. Results: Mean numbers of recovered eggs were 7.9 and 8.0 in 129 and C57BL/6J mice, respectively. Half of the recovered eggs were unfertilized in 129 mice, whereas all were fertilized in C57BL/6J mice. Mean numbers of live fetuses 18-19 days after mating were significantly lower in 129 mice (4.7) than in C57BL/6J mice (7.3). In different types of pairings using both strains of mice, the fertility was significantly lower whenever 129 females were used. Conclusions: The small litter size in 129 mice is caused by low fertility resulting from female factors. © 2009 Japan Society for Reproductive Medicine.

DOI： 10.1007/s12522-009-0024-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: The formation of long-term memory (LTM) and the late phase of long-term potentiation (L-LTP) depend on macromolecule synthesis, translation, and transcription in neurons. vesl-1S (VASP/Ena-related gene upregulated during seizure and LTP, also known as homer-1a) is an LTP-induced immediate early gene. The short form of Vesl (Vesl-1S) is an alternatively spliced isoform of the vesl-1 gene, which also encodes the long form of the Vesl protein (Vesl-1L). Vesl-1L is a postsynaptic scaffolding protein that binds to and modulates the metabotropic glutamate receptor 1/5 (mGluR1/5), the IP3 receptor, and the ryanodine receptor. Vesl-1 null mutant mice show abnormal behavior, which includes anxiety-and depression-related behaviors, and an increase in cocaine-induced locomotion; however, the function of the short form of Vesl in behavior is poorly understood because of the lack of short-form-specific knockout mice.
Results: In this study, we generated short-form-specific gene targeting (KO) mice by knocking in part of vesl-1L/homer-1c cDNA. Homozygous KO mice exhibited normal spine number and morphology. Using the contextual fear conditioning test, we demonstrated that memory acquisition and short-term memory were normal in homozygous KO mice. In contrast, these mice showed impairment in fear memory consolidation. Furthermore, the process from recent to remote memory was affected in homozygous KO mice. Interestingly, reactivation of previously consolidated fear memory attenuated the conditioning-induced freezing response in homozygous KO mice, which suggests that the short form plays a role in fear memory reconsolidation. General activity, emotional performance, and sensitivity to electrofootshock were normal in homozygous KO mice.
Conclusion: These results indicate that the short form of the Vesl family of proteins plays a role in multiple steps of long-term, but not short-term, fear memory formation.

DOI： 10.1186/1756-6606-2-7

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

Eutherian placenta, an organ that emerged in the course of mammalian evolution, provides essential architecture, the so-called feto-maternal interface, for fetal development by exchanging nutrition, gas and waste between fetal and maternal blood. Functional defects of the placenta cause several developmental disorders, such as intrauterine growth retardation in humans and mice. A series of new inventions and/ or adaptations must have been necessary to form and maintain eutherian chorioallantoic placenta, which consists of capillary endothelial cells and a surrounding trophoblast cell layer(s)(1). Although many placental genes have been identified(2), it remains unknown how the feto-maternal interface is formed and maintained during development, and how this novel design evolved. Here we demonstrate that retrotransposon-derived Rtl1 (retrotransposon-like 1), also known as Peg11 (paternally expressed 11), is essential for maintenance of the fetal capillaries, and that both its loss and its overproduction cause late-fetal and/or neonatal lethality in mice.

DOI： 10.1038/ng.2007.51

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

The proper regulation of microtubule (NIT) structure is important for dendritic and neural circuit development. However, the relationship between the regulation of the MTs in dendrites and the formation of neural function is still unclear. Stathmin is a NIT destabilizer, and we have previously reported that the expression and the activity of stathmin is downregulated during cerebellar Purkinje cell (PC) development. In this study, we generated transgenic mice that specifically overexpress the constitutively active form of stathmin in the PCs. These mutant mice did not show any obvious morphological or excitatory transmission abnormalities in the cerebellum. In contrast, we observed a decline in the expression of MAP2 and KIF5 signal in the PC dendrites and a discoordination of motor function in the mutant mice, although they displayed normal general behavior. These data indicate that the overexpression of stathmin disrupts dendritic MT organization, motor protein distribution, and neural function in PCs. (C) 2007 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/j.neures.2007.06.1464

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：John Wiley and Sons Ltd  

Aim: Cryopreservation of mouse sperm commonly uses raffinose, which is a trisaccharide, plus 3% skim milk. Because of the present lack of knowledge of the effectiveness of any other saccharides, we examined the cryoprotective effects of various saccharides on the viability of mouse sperm from various strains to determine which saccharides are the best cryoprotectants for mouse sperm. Methods: Sperm from the caudae epididymides of mature C57BL/6J mice were frozen with monosaccharides (fructose, glucose, rhamnose, xylose), disaccharides (lactose, maltose, sucrose, trehalose) or trisaccharides (melezitose, raffinose) in a range of concentrations (4-33%). After thawing, the optimal concentration was determined to be the concentration in which there was the highest proportion of motile sperm. In addition, sperm of inbred and hybrid mice were frozen with the saccharides at the optimal concentrations and used for in vitro fertilization. Results: The optimal concentration was 12% for the disaccharides and 18% for the trisaccharides. The fertility of all strains, except C57BL/6J, showed the best cryoprotective effects with maltose, melezitose and raffinose when compared with fresh sperm. Conclusion: Maltose, melezitose and raffinose have the best effects when used as a protectant for cryopreservation of mouse sperm. © 2007 The AuthorsJournal compilation © 2007 Japan Society for Reproductive Medicine.

DOI： 10.1111/j.1447-0578.2007.00190.x

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

By comparing mammalian genomes, we and others have identified actively transcribed Ty3/gypsy retrotransposon-derived genes with highly conserved DNA sequences and insertion sites(1-6). To elucidate the functions of evolutionarily conserved retrotransposon-derived genes in mammalian development, we produced mice that lack one of these genes, Peg10 ( paternally expressed 10)(1-3,7), which is a paternally expressed imprinted gene on mouse proximal chromosome 6. The Peg10 knockout mice showed early embryonic lethality owing to defects in the placenta. This indicates that Peg10 is critical for mouse parthenogenetic development and provides the first direct evidence of an essential role of an evolutionarily conserved retrotransposon-derived gene in mammalian development.

DOI： 10.1038/ng1699

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Language：Japanese   Publisher：(公社)日本繁殖生物学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCIENCE INC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：W B SAUNDERS CO LTD  

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Language：English   Publisher：日本神経化学会  

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Language：Japanese   Publisher：日本発生生物学会  

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Venue：東京都港区  

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Venue：新潟県新潟市  

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Venue：Charlotte、 NC  

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Venue：兵庫県神戸市  

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Journal of Reproduction and Development誌の論文査読（レビュー）

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Journal of Assisted Reproduction and Genetics誌の論文査読（レビュー）

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大会準備委員

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