Activated bovine cytoplasts prepared by demecolcine-induced enucleation support development of nuclear transfer embryos in vitro

Russell, Deanna; Ibáñez, Elena; Albertini, David F.; Overström, E.W.

doi:10.1002/mrd.20356

Cited by 20 publications

(9 citation statements)

References 34 publications

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“…Denuded oocytes were fixed for nuclear maturation assessment as previously described (Russell et al, 2005) with some modifications. In brief, oocytes were fixed in 4% (v/v) paraformaldehyde (PAF) for 15 min at 288C followed by a 25-min incubation at 288C in a Microtubule Stabilization Buffer-Extraction Fixative (MTSB-XF; Wickramasinghe and Albertini, 1992) containing 5% (v/v) Triton X-100.…”

Section: Oocyte Fixation and Immunostainingmentioning

confidence: 99%

The impact of oocyte maturation media on early bovine embryonic development

Russell

Baqir

Bordignon

et al. 2006

Molecular Reproduction Devel

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Successful production of high quality blastocysts in vitro depends on the use of a culture system that ensures the acquisition of developmental competence by the maturing oocyte. It is now clear that the in vitro maturation environment has a major influence on the oocyte's ability to acquire the potential to develop into blastocysts. In this work we examine the impact of oocyte culture media on the quality of blastocysts by comparing developmental rates, cell number and their allocation to embryonic cell lineages, apoptosis, and expression of developmentally important genes. Higher total cell count and ICM:TCN ratio, which are indicative of embryo viability, were observed in embryos derived from oocyte maturation in TCM-199 supplemented with serum when compared to blastocysts derived from oocyte maturation in SOF BSA. Moreover, oocyte maturation in TCM-199 supplemented with serum-generated embryos of higher morphological quality and producing higher levels of Interferon Tau transcripts when compared to embryos derived from oocyte maturation in SOF BSA. In conclusion, the oocyte maturation regimen affected the morphological feature of blastocysts, including total cell count and allocation of cells to trophectoderm (TE) and inner cell mass (ICM) lineages and the expression profiles of genes involved in various embryo functions such as early embryonic growth, regulation of gene transcription, trophoblast differentiation and function, embryo-maternal communication, and stress response. Our results show that the oocyte culture media have strong impact on the quality of embryos produced in vitro and emphasize the need for more in depth evaluation of oocyte maturation protocols.

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Section: Oocyte Fixation and Immunostainingmentioning

confidence: 99%

The impact of oocyte maturation media on early bovine embryonic development

Russell

Baqir

Bordignon

et al. 2006

Molecular Reproduction Devel

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“…This simple method has garnered much attention and has been successfully used to clone pigs by SCNT. Furthermore, rabbit fetuses [12], cloned mice [13], cloned cows [14], cloned transgenic cats [15], second-generation cloned cats [16], and miniature pigs [17]–[18] have all been generated using oocytes that were enucleated by DEM treatment. This indicates that DEM-treated oocytes can be used for SCNT, although the mechanisms by which DEM elicits its effects are unclear.…”

Section: Introductionmentioning

confidence: 99%

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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“…As an alternative approach to enucleate the MII plate, treating the oocytes with demicoline that induces a membrane protrusion where the MII chromosomes are located, has been successfully used in MII mouse and pig oocytes (Yin et al. 2002; Russell et al. 2005) and anaphase/telophase I ovine oocytes (Lee and Campbell 2006).…”

Section: Introductionmentioning

confidence: 99%

In Vitro Developmental Potential of Nuclear Transfer Embryos Cloned with Enucleation Methods using Pre‐denuded Bovine Oocytes

et al. 2011

Reprod Domestic Animals

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Enucleation of a recipient oocyte is an important essential process in the procedure of somatic cell nuclear transfer (SCNT). The present study investigated a method for the improvement of enucleation efficiency. Oocytes were denuded of cumulus cells before the completion of nuclear maturation (pre-denuded) after 12 h of culture at MI stage and subsequently cultured for additional 6 h until the completion of nuclear maturation and extrusion of the first polar body (PB1). The extrusion rate of PB1 was not significantly different in the pre-denuded oocyte group, compared with control oocyte group matured for 18 h. However, the number of oocytes showing the metaphase II (MII) located just underneath the PB1 was significantly higher (p<0.05) in the pre-denuded oocyte group than those in control oocyte group. To test the effect of pre-denuding on the enucleation rate and developmental potential of embryos to blastocyst stage, subsequent somatic cell nuclear transfer comparisons were made with three different methods of enucleation at MII stage using vital dyes (demicoline and Hoescht) or the PB1 (blind enucleation) to localize the chromosome plate. Enucleation rate of the oocytes with demicoline, Hoechst and pre-denuding enucleation groups were significantly higher (p<0.05) than those of blind enucleation groups. However, cleavage rate to two-cell stage and, developmental rate to blastocyst and hatched blastocyst stage, the mean numbers of total and ICM cells in the SCNT embryos with Hoechst enucleation groups were significantly decreased (p<0.05), compared to those of blind, demicoline and pre-denuding enucleation groups. Moreover, the level of telomerase activity was also significantly (p<0.05) decreased in SCNT blastocysts of Hoechst enucleation group, compared to those of blind, demicoline and pre-denuding enucleation groups. Taken together, pre-denuding enucleation group using pre-denuded oocytes was a useful and simple enucleation method for bovine SCNT embryos.

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Activated bovine cytoplasts prepared by demecolcine-induced enucleation support development of nuclear transfer embryos in vitro

Cited by 20 publications

References 34 publications

The impact of oocyte maturation media on early bovine embryonic development

The impact of oocyte maturation media on early bovine embryonic development

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

In Vitro Developmental Potential of Nuclear Transfer Embryos Cloned with Enucleation Methods using Pre‐denuded Bovine Oocytes

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