Deletion of Gb3 Synthase in Mice Resulted in the Attenuation of Bone Formation via Decrease in Osteoblasts.

Glycosphingolipids are known to play a role in developing and maintaining the integrity of various organs and tissues. Among glycosphingolipids, there are several reports on the involvement of gangliosides in bone metabolism. However, there have been no reports on the presence or absence of expression of globo-series glycosphingolipids in osteoblasts and osteoclasts, and the involvement of their glycosphingolipids in bone metabolism. In the present study, we investigated the presence or absence of globo-series glycosphingolipids such as Gb3 (globotriaosylceramide), Gb4 (globoside), and Gb5 (galactosyl globoside) in osteoblasts and osteoclasts, and the effects of genetic deletion of Gb3 synthase, which initiates the synthesis of globo-series glycosphingolipids on bone metabolism. Among Gb3, Gb4, and Gb5, only Gb4 was expressed in osteoblasts. However, these glycosphingolipids were not expressed in pre-osteoclasts and osteoclasts. Three-dimensional micro-computed tomography (3D-µCT) analysis revealed that femoral cancellous bone mass in Gb3 synthase-knockout (Gb3S KO) mice was lower than that in wild type (WT) mice. Calcein double labeling also revealed that bone formation in Gb3S KO mice was significantly lower than that in WT mice. Consistent with these results, the deficiency of Gb3 synthase in mice decreased the number of osteoblasts on the bone surface, and suppressed mRNA levels of osteogenic differentiation markers. On the other hand, osteoclast numbers on the bone surface and mRNA levels of osteoclast differentiation markers in Gb3S KO mice did not differ from WT mice. This study demonstrated that deletion of Gb3 synthase in mice decreases bone mass via attenuation of bone formation.

Deficiency of GD3 Synthase in Mice Resulting in the Attenuation of Bone Loss with Aging.

Gangliosides are widely expressed in almost all tissues and cells and are also considered to be essential in the development and maintenance of various organs and tissues. However, little is known about their roles in bone metabolism. In this study, we investigated the effects of genetic deletion of ganglioside D3 (GD3) synthase, which is responsible for the generation of all b-series gangliosides, on bone metabolism. Although b-series gangliosides were not expressed in osteoblasts, these gangliosides were expressed in pre-osteoclasts. However, the expression of these gangliosides was decreased after induction of osteoclastogenesis by receptor activator of nuclear factor kappa-B ligand (RANKL). Three-dimensional micro-computed tomography (3D-µCT) analysis revealed that femoral cancellous bone mass in GD3 synthase-knockout (GD3S KO) mice was higher than that in wild type (WT) mice at the age of 40 weeks, although there were no differences in that between GD3S KO and WT mice at 15 weeks old. Whereas bone formation parameters (osteoblast numbers/bone surface and osteoblast surface/bone surface) in GD3S KO mice did not differ from WT mice, bone resorption parameters (osteoclast numbers/bone surface and osteoclast surface/bone surface) in GD3S KO mice became significantly lower than those in WT mice at 40 weeks of age. Collectively, this study demonstrates that deletion of GD3 synthase attenuates bone loss that emerges with aging.

Conditioned medium from rat dental pulp reduces the number of osteoclasts via attenuation of adhesiveness in osteoclast precursors.

Dental pulp is known to play crucial roles in homeostasis of teeth and periodontal tissue. Although resorption of bone around the roots of nonvital teeth is occasionally observed in clinical practice, little is known about the role of dental pulp in osteoclastogenesis. Here we evaluated the effects of conditioned medium (CM) from rat dental pulp on osteoclastogenesis. It was found that the CM reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts, but did not alter the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 and TRAP. To further understand the mechanism behind these results, we evaluated the effects of CM on osteoclast precursors and found that the CM removed cell processes, resulting in a significant reduction in the number of attached cells and an increase in the number of freely floating cells. Furthermore, the CM suppressed the mRNA levels of focal adhesion kinase and paxillin, which are involved in cell adhesiveness and spreading. Collectively, the present results show that CM from dental pulp serves as an inhibitor of osteoclastogenesis by reducing the number and adhesiveness of osteoclast precursors, suggesting novel therapeutic applicability for osteoporosis.

Suppression of osteoclastogenesis via α2-adrenergic receptors.

The sympathetic nervous system is known to regulate osteoclast development. However, the involvement of α2-adrenergic receptors (α2-ARs) in osteoclastogenesis is not well understood. In the present study, their potential role in osteoclastogenesis was investigated. Guanabenz, clonidine and xylazine were used as agonists of α2-ARs, while yohimbine and idazoxan were employed as antagonists. Using RAW264.7 pre-osteoclast and primary bone marrow cells, the mRNA expression of the osteoclast-related genes nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), tartrate-resistant acid phosphatase (TRAP) and cathepsin K was evaluated following induction with receptor activator of nuclear factor κB ligand (RANKL). TRAP staining was also conducted to assess effects on osteoclastogenesis in mouse bone marrow cells in vitro. Administration of 5-20 µM guanabenz (P<0.01, for RANKL-only treatment), 20 µM clonidine (P<0.05, for RANKL-only treatment) and 20 µM xylazine (P<0.05, for RANKL-only treatment) attenuated RANKL-induced upregulation of NFATc1, TRAP and cathepsin K mRNA. Furthermore, the reductions in these mRNAs by 10 µM guanabenz and 20 µM clonidine in the presence of RANKL were attenuated by 20 µM yohimbine or idazoxan (P<0.05). The administration of 5-20 µM guanabenz (P<0.01, for RANKL-only treatment) and 10-20 µM clonidine (P<0.05, for RANKL-only treatment) also decreased the number of TRAP-positive multi-nucleated osteoclasts. Collectively, the present study demonstrates that α2-ARs may be involved in the regulation of osteoclastogenesis.

Whole-genome amplification-based GenomiPhi for multiple genomic analysis of individual early porcine embryos.

The multiple displacement amplification (MDA) method, which relies on isothermal DNA amplification using the DNA polymerase of the bacteriophage phi29, was recently developed for high-performance, whole-genome amplification (WGA). The objective of the present study was to determine whether a target sequence could be successfully amplified by conventional PCR when the genomic DNA of a single Day-7 porcine blastocyst (derived from SCNT of a gene-engineered fibroblast) was amplified by the MDA method and used as a template. The yield of double-stranded DNA was 103.5 ± 16.0 ng/embryo (range, 75-125), as assessed by a PocoGreen assay. However, non-specific products (20 ± 5 ng/tube) were also generated, even in the negative control. Thus, ∼81% of the 103.5 ng (84 ng) of amplified DNA was estimated to be porcine sequences (2.2 × 10(3)-fold enrichment). In addition, PCR confirmed the presence of transgenes, as well as endogenous α-1,3-galactosyltransferase and homeobox Nanog genes in all embryos. Sequencing of the amplified products verified the fidelity of this system. In conclusion, the MDA-mediated WGA, which was simple, inexpensive, and did not require a thermal cycler, could be a powerful tool for multiple genomic analyses of individual early porcine embryos.

Latrunculin A dramatically improves the developmental capacity of nuclear transfer embryos derived from gene-modified clawn miniature pig cells.

This study was carried out to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerisation inhibitor, on in vitro and in vivo development of somatic cell nuclear transfer (SCNT) embryos derived from gene-modified Clawn miniature pig cells. After the fusion and activation, SCNT embryos were treated with or without a cytoskeletal inhibitor [LatA or 10.4 microM cytochalasin B (CB) for 2 h]. The cleavage rate was significantly higher (p < 0.05) in embryos exposed to 0.5 microM LatA than those in embryos exposed to CB and without a cytoskeletal inhibitor. Moreover, the blastocyst formation rate was significantly higher (p < 0.05) in embryos exposed to 0.5 or 1 microM LatA than those in embryos exposed to CB and without a cytoskeletal inhibitor. In addition, five fetuses were obtained from recipient uteri after transfer of embryos treated with 0.5 muM LatA. The results of this study show for the first time that postactivation treatment with LatA is effective to improve in vitro developmental capacity of gene-modified cloned miniature pig embryos and embryos treated with LatA have the ability to develop into fetuses.

Stage-specific effects of osmolarity of a culture medium on development of pig oocytes and miniature pig somatic cell nuclear transfer embryos activated by ultrasound treatment.

Whether high osmolarity of a culture medium at the early culture stage affects the development of pig oocytes and miniature pig somatic cell nuclear transfer (SCNT) embryos activated by ultrasound was examined. When oocytes were cultured in modified porcine zygote medium-3 (mPZM-3) with increased NaCl to 138 mmol/L (mPZM-3+NaCl; 326 mOsm) or 50 mmol/L sucrose (mPZM-3+sucrose; 318 mOsm) for the first 2 days and then cultured in normal mPZM-3 (273 mOsm) for 5 days, the cleavage and blastocyst formation rates were significantly (P < 0.05) higher than those of oocytes cultured in mPZM-3 for 7 days. The cleavage and blastocyst formation rates of SCNT embryos cultured in mPZM-3+NaCl for the first 2 days and then cultured in mPZM-3 for 5 days were also significantly (P < 0.05) higher than those of embryos cultured in mPZM-3 for 7 days. These results showed that the high osmolarity of a culture medium induced by increasing NaCl concentration during the first 2 days improves the development of pig oocytes and miniature pig SCNT embryos activated by ultrasound.

Unusually large hysteresis of temperature-responsive poly(N-ethyl-2-propionamidoacrylamide) studied by microcalorimetry and FT-IR.

We reported here the full characterization of the hysteresis of the phase transition behavior of an aqueous solution of poly(N-ethyl-2-propionamidoacrylamide) (PNEPA), which has a unique alpha,alpha-disubstituted structure, by using microcalorimetry and FT-IR. Phase transition temperatures near the thermodynamic equilibrium were determined by extrapolating the scanning rate of the microcalorimetry to zero. The calculated hysteresis from the phase transition temperature was unusually very large (approximately 8 degrees C). FT-IR analysis indicated that the large hysteresis of PNEPA resulted from a coupling of intra-/intermonomeric unit hydrogen bonds, which is known to occur in a beta-sheet of proteins but has never been reported in temperature-responsive polymers. The effects of the molecular weight and polymer concentration on the hysteresis were studied by using fractionated PNEPAs and it was found that a low molecular weight and a low concentration enhanced the hysteresis.

Design of temperature-responsive polymers with enhanced hysteresis: alpha,alpha-disubstituted vinyl polymers.

Three temperature-responsive polymers which are alpha,alpha-disubstituted vinyl polymers having two amphiphilic groups (ethylamide or ethylester) per monomeric unit were designed. Two of these polymers showed unusually large hysteresis in their phase transition temperatures between a heating and a cooling process. This hysteresis resulted from the extremely slow kinetics of the dissolution process of the aggregated polymer chains in the cooling process due to intra- and interchain interactions including hydrogen bonding and hydrophobic interaction. The high density of the amphiphilic substituents on the polymer chain due to the alpha,alpha-disubstituted structure enhanced these intra- and interchain interactions. The large hysteresis was also observed in the volume change of a corresponding hydrogel. These new classes of temperature-responsive polymers are interesting materials because their large hystereses can be regarded as erasable memory function.

Valproic acid enhances in vitro development and Oct-3/4 expression of miniature pig somatic cell nuclear transfer embryos.

The present study was carried out to examine the effects of valproic acid (VPA), a histone deacetylase inhibitor, on in vitro development of miniature pig somatic cell nuclear transfer (SCNT) embryos and on expression of a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) gene (EGFP expression only detected in Oct-3/4-expressing cells) introduced into donor cells for SCNT during their development. The addition of 4 mM VPA to embryo culture medium for 48 h after activation significantly (p < 0.01) increased the blastocyst formation rate of SCNT embryos compared with the control, whereas VPA did not affect their cleavage rate. The rate of SCNT embryos expressing EGFP at 5 days of culture was not affected by the presence or absence of VPA treatment. At 7 days of culture, however, the addition of 4 mM VPA to embryo culture medium for 48 h after activation significantly (p < 0.05) increased the rate of SCNT embryos expressing EGFP compared with the control. The results indicate that VPA enhances the ability of miniature pig SCNT embryos to develop into blastocysts and maintains the ability of them to express Oct-3/4 gene.

Beneficial effects of reversine on in vitro development of miniature pig somatic cell nuclear transfer embryos.

Reversine, a 2-(4-morpholinoanilino)-6-cyclohexylaminopurine analog, can induce dedifferentiation of myogenic lineage-committed cells into multipotent mesenchymal progenitor cells, from which osteoblasts and adipocytes redifferentiate under lineage-specific inducing conditions. Although the molecular mechanism of how reversine causes dedifferentiation of a differentiated cell has not been fully elucidated, we speculated that it would be involved in reprogramming. In the present study, we examined whether reversine can enhance the development of somatic cell nuclear transfer (SCNT) embryos by improving the reprogramming state of the somatic cell nuclei. As donor cells, we used miniature pig fetal fibroblasts transfected with a plasmid construct containing a mouse Oct-3/4 promoter and enhanced green fluorescent protein (EGFP) cDNA. When the nuclei of these transfected cells are reprogrammed to an undifferentiated state in the SCNT embryos, EGFP expression is expected to commence under the control of the Oct-3/4 promoter. After SCNT, the resulting embryos were treated with 5 muM reversine for different durations (0, 6, 12, 18 and 24 h) or at different concentrations (0, 1, 5 and 10 muM) of reversine for 12 h and then cultured in vitro. When embryos were treated with 5 muM reversine for 12 h, the blastocyst formation rate was significantly (P<0.01) higher than that of embryos without reversine treatment. However, the strength and pattern of EGFP expression in the embryos were not affected by the same treatment. A normal-looking fetus was obtained 21 days after transfer of embryos treated with 5 muM reversine for 12 h into recipients. The present findings indicate that treatment with reversine is beneficial for enhancement of the in vitro development of miniature pig SCNT embryos, although the underlying mechanism is still unclear.

Development of a noninvasive monitoring system for evaluation of Oct-3/4 promoter status in miniature pig somatic cell nuclear transfer embryos.

The present study was carried out to develop a noninvasive monitoring system for evaluation of Oct-3/4 promoter gene status in miniature pig somatic cell nuclear transfer (SCNT) embryos during in vitro development. Miniature pig fetal fibroblasts (MPFFs) were transfected with a gene construct consisting of two expression units, a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) gene (EGFP expression only detected in Oct-3/4-expressing cells) and a neomycin resistance gene. After neomycin selection, MPFFs that did not express EGFP were fused with enucleated pig oocytes, cultured in vitro and assessed for EGFP expression. EGFP expression was detectable in all morulae (at 4-6 days of culture) and 50.0% of blastocysts (at 5-6 days of culture), whereas none of the 1-cell to 16-cell embryos at 1-5 days of culture expressed EGFP. On the other hand, EGFP expression was not maintained in all blastocysts at 7 days of culture. The reactivity with anti-Oct-3/4 antibodies also peaked from the morula to blastocyst stages at 5 days of culture. The results showed that reactivation of the Oct-3/4 promoter gene of donor nuclei occurs in the morula to blastocyst stages at 4-6 days after SCNT and that this noninvasive monitoring system using Oct-3/4 promoter-driven EGFP gene would be useful for evaluation of the reprogramming status of donor nuclei.

Regioregulated Syntheses of Poly(aminopyridine)s by Pd-catalyzed Amination Reaction.

Regioregulated poly(aminopyridine)s were synthesized by a Pd-catalyzed CN coupling reaction. The polymerization using Pd(0) and a bulky monodentate phosphine ligand distinctively produced the para-linked and meta-linked poly(aminopyridine)s, without the need for a protection process. The regioregularity of the polymer was confirmed by (1) H NMR spectroscopy. Model reactions were studied to evaluate the possibility of crosslinkage in the polymer. A large difference in reactivity was observed between 5-amino-2-bromopyridine and 2-amino-5-bromopyridine, which should have afforded same product. Density functional theory (DFT) calculations indicated that electron densities of the Br-bound carbon atom and the pyridine-nitrogen atom determine the reactivity of the monomers.

Effects of cycloheximide on parthenogenetic development of pig oocytes activated by ultrasound treatment.

The present study was carried out to examine the parthenogenetic development of pig oocytes treated with different concentrations of cycloheximide for different durations following activation by ultrasound stimulation. When oocytes were treated with 10 microg/ml cycloheximide for different durations, the blastocyst formation rate of oocytes treated for 5 h was significantly (P<0.05) higher than those of oocytes treated for 0-2 h. The blastocyst formation rate of oocytes treated with 10 microg/ml cycloheximide for 5 h was significantly (P<0.05) higher than those of oocytes treated with 0-5 or 15-20 microg/ml cycloheximide for the same duration. When oocytes were treated with different concentrations of cycloheximide for 2 h, however, the blastocyst formation rate of oocytes treated with 40 microg/ml cycloheximide was significantly (P<0.05) higher than those of oocytes treated with 0-10 or 50 microg/ml cycloheximide. The blastocyst formation rate of oocytes treated with 10 microg/ml cycloheximide for 5 h was not significantly different from that of oocytes treated with 40 microg/ml cycloheximide for 2 h. These treatments did not affect the activation status of oocytes compared with controls that were not treated with cycloheximide. The results of the present study showed that cycloheximide improves the parthenogenetic development of pig oocytes activated by ultrasound stimulation.

Effects of demecolcine and sucrose on the incidence of cytoplasmic protrusions containing chromosomes in pig oocytes matured in vitro.

The present study was carried out to examine whether demecolcine and sucrose affect the formation of a cytoplasmic protrusion containing chromosomes in pig oocytes independently or in combination. In the presence of 20 mM sucrose, the rates of oocytes with a cytoplasmic protrusion after culture for 60 min with 0.2-1.0 microg/ml demecolcine were significantly higher than those with 0.01-0.05 microg/ml demecolcine. When oocytes were cultured for 15 min in the presence of 0.2 microg/ml demecolcine and 20 mM sucrose, 35.1% of them extruded a cytoplasmic protrusion; this rate was significantly lower than those of oocytes cultured for 30-90 min. In the presence of 0.2 microg/ml demecolcine, significantly fewer oocytes extruded a cytoplasmic protrusion after culture for 30 min with 160 mM sucrose than with 0-80 mM sucrose. Significantly more oocytes extruded a cytoplasmic protrusion after culture for 30 min with 0.2 microg/ml demecolcine than without it, regardless of the presence or absence of 20 mM sucrose. In 88.9-100% of the oocytes, the cytoplasmic protrusions contained chromosomes with no significant differences among the different concentrations of demecolcine and sucrose and among the different treatment times. The results of the present study show that the cytoplasmic protrusion containing chromosomes in the pig oocyte is attributable to demecolcine, but sucrose does not affect its formation.

Activation and parthenogenetic development of pig oocytes exposed to ultrasound in media containing different concentrations of Ca2+.

The present study was carried out to examine the activation and parthenogenetic development of pig oocytes after exposure to ultrasound in sorbitol media supplemented with different concentrations of Ca2+. The activation rates (68.8-75.6%) of oocytes exposed to ultrasound in media containing 0.1-1.0 mM Ca2+ were significantly (P<0.05) higher than those (54.3-58.3%) of oocytes exposed to ultrasound in media containing 0-0.05 mM Ca2+. When oocytes were exposed to ultrasound in media containing 0.1-0.5 mM Ca2+, the blastocyst formation rates (20.5-21.3%) were significantly (P<0.05) higher than those (3.3-6.0%) of oocytes exposed to ultrasound in media containing 0, 0.05 or 1.0 mM Ca2+. The results of the present study showed that the concentration of Ca2+ in the medium used for exposure to ultrasound affects the activation and parthenogenetic development of pig oocytes and showed that the optimal Ca2+ concentration is 0.1-0.5 mM.