Yamato Mizobe

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.

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.

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.

Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a) in Plasma.

High lipoprotein(a) [Lp(a)] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a) [apo(a)], the unique component of Lp(a), is found only in primates and humans, the study of human Lp(a) has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT) techniques, we produced transgenic miniature pigs expressing human apo(a) in the plasma. First, we placed the hemagglutinin (HA)-tagged cDNA of human apo(a) under the control of the β-actin promoter and cytomegalovirus enhancer, and then introduced this construct into kidney epithelial cells. Immunostaining of cells with anti-HA antibody allowed identification of cells stably expressing apo(a); one of the positive clones was used to provide donor cells for SCNT, yielding blastocysts that expressed apo(a). Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a) is expressed in various tissues/organs including heart, liver, kidney, and intestine. More importantly, a transgenic line exhibited a high level (>400 mg/dL) of Lp(a) in plasma, and the transgenic apo(a) gene was transmitted to the offspring. Thus, we generated a human apo(a)–transgenic miniature pig that can be used as a model system to study advanced atherosclerosis related to human disease. The anatomical and physiological similarities between the swine and human cardiovascular systems will make this pig model a valuable source of information on the role of apo(a) in the formation of atherosclerosis, as well as the mechanisms underlying vascular health and disease.

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.

Osmolarity- and stage-dependent effects of glycine on parthenogenetic development of pig oocytes.

The osmolarities of media that are most effective for in vitro culture of mammalian oocytes and embryos are lower than that of oviductal fluid. Oocytes and embryos can survive the high physiological osmolarity in vivo perhaps owing to the presence of amino acids such as glycine, which serve as organic osmolytes in the female reproductive tract. In the present study, the effects of glycine on the parthenogenetic development of pig oocytes were examined in hypotonic or isotonic media. The results showed that culturing oocytes in isotonic media improved the cleavage rates (P<0.01) at 2 days in culture but inhibited any further development beyond cleavage when compared with the hypotonic media. However, addition of 4 mM glycine to the isotonic media resulted in improved blastocyst formation rates compared with that observed in the hypotonic media (P<0.01), and there was no inhibition of development beyond the cleavage stages in oocytes. The beneficial effects of glycine were observed only when oocytes were cultured in isotonic media and glycine was added at day 2 or 3 in culture. The results from the present study indicate that an isotonic medium with glycine is useful for in vitro culture of pig oocytes and that glycine may protect pig oocytes against the detrimental effects of increased osmolarity.

Synchrony of the first division as an index of the blastocyst formation rate during embryonic development

To devise an uninvasive selection system for human embryos with high developmental potential after a single oocyte retrieval cycle by comparing the in vitro and in vivo effectiveness of first division synchrony against subsequent embryonic developmental stages.