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

Abstract

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.

Handmade cloning: recent advances, potential and pitfalls

“…A brief treatment of bovine [ 30 ]; porcine [ 31 ] and caprine [ 32 ] oocytes with demecolcine (DEM) produces a membrane protrusion that contains a mass of condensed chromosomes, which can be removed easily. Other chemicals such as nocodazole, etoposide, caffeine, and MG132, have been used to induce or assist oocyte enucleation [ 31 ]. Tatham et al [ 8 ] utilized density-gradient centrifugation for enucleation of zona-free oocytes.…”

“…Chemically assisted enucleation has been observed as more efficient and reliable method for goat HMC as compared to oriented bisection of oocyte by hand [ 41 ]. DEM treatment disrupts spindle microtubules, induces chromosome condensation and has significantly greater potential than that of embryos generated from mechanically enucleated oocytes in vivo [ 31 ] . The rates of enucleation, cell fusion and blastula formation of goat oocytes has been found to be similar among caffeine- and DEM-assisted enucleation and significantly higher than mechanical enucleation as reported by Wang et al [ 42 ].…”

“…DEM-assisted enucleation is advantageous over mechanical enucleation as it decreases the cytoplasmic volume of the oocyte minimally without reducing the level of MPF. High MPF activity might promote cytoplasmic maturation and improve the developmental competence of oocytes [ 31 ]. Moreover, enucleation with chemicals like DEM obviates the need of inverted fluorescence microscope and destructive chromatin staining and UV irradiation for cytoplast selection.…”

“…Dutta et al [ 54 ] used RVCL media to produce zona-free cloned goat embryos. North Carolina State University (NCSU) medium has been used for porcine embryos [ 26 , 31 ]. Studies on zona-free cloned buffalo [ 52 ] and goat embryos [ 55 ] revealed that flat surfaces (FS) used as culture system yielded significantly higher blastocyst rates than WOW or microdrops (MD).…”

Cytoplasmic volume of recipient oocytes affects the nucleus reprogramming and the developmental competence of HMC buffalo (<i>Bubalus bubalis</i>) embryos

“…The requirement of expensive micromanipulators and skilled expertise was eliminated in the HMC technique, proving that it was a major revolution in the field of embryology [ 42 ]. With the improvement in the enucleation of zona-free oocytes [ 17 , 32 , 33 ] and the culture system [ 1 , 36 , 40 ], HMC, as a more efficient and economical technique in comparison to the micromanipulator-based approach, was successfully used to produce cloned offspring in several livestock species such as cattle [ 22 ], buffalo [ 11 ], sheep [ 50 ], pigs [ 9 ] and horses [ 14 ]. Although HMC has achieved certain success and offers a new route for SCNT, the problems that restrict the success of traditional SCNT, including incomplete nucleus reprogramming, chromosome remodeling failure, embryonic genomic activation delay, and lower in vivo developmental competence, still need to be resolved.…”