Effects of Demecolcine and Sucrose on the Incidence of Cytoplasmic Protrusions Containing Chromosomes in Pig Oocytes Matured In Vitro

Miyoshi, Katsuhiko; Mori, Hironori; Yamamoto, Hideki; Kishimoto, Mitsumasa; Yoshida, Mitsutoshi

doi:10.1262/jrd.19142

Cited by 10 publications

(6 citation statements)

References 30 publications

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“…Such protrusions are frequently observed in DEM-treated bovine [23] and rabbit [12] oocytes. Sucrose does not affect the formation of a cytoplasmic protrusion containing chromosomes in pig oocytes [26], but does enlarge the perivitelline space. Therefore, the current study investigated the effects of DEM treatment on oocytes.…”

Section: Discussionmentioning

confidence: 81%

Effect of Demecolcine-Assisted Enucleation on the MPF Level and Cyclin B1 Distribution in Porcine Oocytes

Kang

Jin

et al. 2014

PLoS ONE

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Demecolcine (DEM) treatment of oocytes induces formation of a membrane protrusion containing a mass of condensed maternal chromosomes, which can be removed with minimal damage prior to somatic cell nuclear transfer (SCNT). However, the effect of this method on the distribution of maturation-promoting factor (MPF) in porcine oocytes has not been reported. Here, the level of MPF and the distribution of cyclin B1 were assessed in porcine oocytes following DEM treatment. In addition, the efficiencies of DEM-assisted and mechanical enucleation were compared, as were the development (in vitro and in vivo) of these oocytes following SCNT. MPF was uniformly distributed in oocytes that had been treated with 0.4 μg/ml DEM for 1 h. Immunofluorescence microscopy showed that in untreated oocytes, cyclin B1, the regulatory subunit of MPF, accumulated around the spindle, and was lowly detected in the cytoplasm. DEM treatment disrupted spindle microtubules, induced chromosome condensation, and reduced the level of cyclin B1 in the nuclear region. Cyclin B1 was uniformly distributed in DEM-treated oocytes and the level of MPF was increased. The potential of embryos generated from DEM-treated oocytes to develop in vivo was significantly greater than that of embryos generated from mechanically enucleated oocytes. This is the first study to report the effects of DEM-assisted enucleation of porcine oocytes on the distribution of cyclin B1. MPF in mature oocytes is important for the development of reconstructed embryos and for efficient SCNT.

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Section: Discussionmentioning

confidence: 81%

Effect of Demecolcine-Assisted Enucleation on the MPF Level and Cyclin B1 Distribution in Porcine Oocytes

Kang

Jin

et al. 2014

PLoS ONE

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“…This medium was used as the basic medium (BM) for nuclear transfer. In vitro-matured and denuded oocytes were cultured in 100 μl of BM supplemented with 0.2 μg/ml demecolcine (Sigma) and 20 mM sucrose for 0.5-1 h [14]. Oocytes with a protruding cytoplasm were transferred into BM supplemented with 0.2 μg/ml demecolcine and 5 μg/ml cytochalasin B.…”

Section: Nuclear Transfermentioning

confidence: 99%

Enhancement of Cytoplasmic Maturation of In Vitro-Matured Pig Oocytes by Mechanical Vibration

Mizobe

Yoshida

Miyoshi

2010

Journal of Reproduction and Development

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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 ...

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“…In vitro-matured and denuded oocytes were cultured in 100 μl of BM supplemented with 0.2 μg/ ml demecolcine (Sigma) and 20 mM sucrose for 0.5-1 h [35]. Oocytes with a protruding membrane were transferred into BM supplemented with 0.2 μg/ml demecolcine and 5 μg/ml cytochalasin B.…”

Section: Production Of Scnt Embryosmentioning

confidence: 99%

Development of a Noninvasive Monitoring System for Evaluation of Oct-3/4 Promoter Status in Miniature Pig Somatic Cell Nuclear Transfer Embryos

Miyoshi

Mori

Mizobe

et al. 2009

Journal of Reproduction and Development

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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...

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Effects of Demecolcine and Sucrose on the Incidence of Cytoplasmic Protrusions Containing Chromosomes in Pig Oocytes Matured In Vitro

Cited by 10 publications

References 30 publications

Effect of Demecolcine-Assisted Enucleation on the MPF Level and Cyclin B1 Distribution in Porcine Oocytes

Effect of Demecolcine-Assisted Enucleation on the MPF Level and Cyclin B1 Distribution in Porcine Oocytes

Enhancement of Cytoplasmic Maturation of In Vitro-Matured Pig Oocytes by Mechanical Vibration

Development of a Noninvasive Monitoring System for Evaluation of Oct-3/4 Promoter Status in Miniature Pig Somatic Cell Nuclear Transfer Embryos

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