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.

Abstract. The effects of mechanical vibration during in vitro maturation and/or in vitro culture after artificial activation of pig oocytes on maturation and development were examined. In addition, the optimal conditions were applied to in vitro production of blastocysts derived from miniature pig somatic cell nuclear transfer (SCNT) embryos. Mechanical vibration during in vitro maturation did not affect the rates (60.5 ± 1.9-69.5 ± 2.2%) of oocytes reaching the metaphase-II stage. However, the blastocyst formation rates after activation of oocytes matured with mechanical vibration for 5 sec at intervals of 30-60 min or for 10 sec at intervals of 60 min were significantly (P<0.05) higher than those of oocytes matured without mechanical vibration (25.7 ± 2.0-28.1 ± 2.7% vs. 12.3 ± 1.4% and 25.8 ± 1.8% vs. 15.7 ± 1.9%, respectively). In contrast, mechanical vibration during in vitro culture after activation did not affect the blastocyst formation (11.6 ± 5.2-16.5 ± 3.0%) of oocytes. Mechanical vibration for 5 sec at intervals of 60 min during in vitro maturation of oocytes did not affect fusion (66.8 ± 3.5-72.1 ± 3.1%) with miniature pig somatic cells after enucleation. However, the blastocyst formation rate of SCNT embryos was improved (P<0.05) by mechanically vibrating recipient oocytes for 5 sec at intervals of 60 min during in vitro maturation, regardless of the presence or absence of the same treatment during in vitro culture (17.6 ± 2.5% vs. 9.4 ± 0.9% and 13.0 ± 0.3% vs. 7.4 ± 0.9%, respectively). The results indicated that mechanical vibration enhances the cytoplasmic maturation of in vitro-matured pig oocytes, resulting in improvement of their parthenogenetic development. In addition, it was shown that in vitro maturation of oocytes with mechanical vibration can be applied to efficient production of blastocysts derived from miniature pig SCNT embryos. he low cloning efficiency associated with the development of somatic cell nuclear transfer (SCNT) embryos to offspring remains the major obstacle to use of this technology in various fields of animal science and biomedical applications. Establishment of systems to support efficient production of blastocysts from SCNT embryos in vitro is quite important for basic research to clarify the mechanism controlling the development of SCNT embryos, which will bring about improvement of cloning efficiency. In cattle and pigs, in vitro oocyte maturation systems produce an abundant and stable supply of recipient oocytes for SCNT because immature oocytes can be obtained from slaughtered animals. In vitro-matured oocytes have been commonly used for production of cloned calves [1-3] and pigs [4][5][6][7][8]. The oocyte maturation process is a crucial step for the generation of oocytes capable of being fertilized and undergoing normal embryonic development into blastocysts after in vitro fertilization [9]. Therefore, efficient production of blastocysts from SCNT embryos requires optimization of both recipient oocyte maturation systems and SCNT embryo culture systems.Oocyte ...

No abstract

Transfer of blastocysts derived from embryos that completed first and second divisions within 25.90 and 37.88 hours after culture, respectively, brings about high pregnancy rates.

Abstract. 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. Key words: Enhanced green fluorescent protein, In vitro development, Nuclear transfer, Oct-3/4 promoter, Reprogramming (J. Reprod. Dev. 55: [661][662][663][664][665][666][667][668][669] 2009) uccessful production of cloned animals using somatic cell nuclear transfer (SCNT) has been reported in several mammalian species [1][2][3][4][5][6][7][8][9][10][11][12]. However, the low cloning efficiency associated with development of SCNT embryos to offspring remains the major obstacle to widespread use of this technology in a number of animal science and biomedical applications. A method for evaluating the developmental ability of SCNT embryos before being transferred into recipient females should be explored to optimize the procedures of SCNT and overcome the low cloning efficiency. Evaluation of the in vitro developmental ability of an SCNT embryo to the blastocyst stage would be considered one of the methods for predicting its in vivo development, but it has been found that this does not often assure successful development to term of SCNT embryos [13]. After SCNT, the gene expression pattern in somatic cells is reprogrammed to mimic that in preimplantation embryos [14]. However, there is a difference in gene expression pattern between SCNT embryos and in vitro-fertilized embryos, suggesting that the low efficiency of SCNT is associated with incomplete reprogramming in the donor nuclei transferred into recipient oocytes [15]. Therefore, evaluation of the reprogramming level in the donor nuclei appears to be most essential for predicting the in vivo developmental ability of SCNT embryos.Oct-3/4 is a transcription factor of the Pit-Oct-Unc family [16,17], and...