Effects of cycloheximide treatment and interval between fusion and activation on in vitro development of pig nuclear transfer embryos

Abstract

The effects of cycloheximide (CHX) treatment and the interval between fusion and activation on the development of pig nuclear transfer (NT) embryos constructed with enucleated oocytes and serum-starved granulosa/cumulus cells were examined. One group of couplets was fused and activated simultaneously (FAS) by a single electrical pulse (activation pulse). Another three groups of couplets were fused electricaly 1.5, 2.5 or 4.5 h before being subjected to the activation pulse (FBA). Each group was divided into two subgroups and incubated with or without CHX. The NT embryos treated with CHX showed a high and stable cleavage rate, regardless of the interval between fusion and activation; however, development to blastocysts was improved only when the NT embryos were subjected to FAS with CHX. These results indicate that CHX-sensitive events occurring shortly after FAS may be responsible for the development to blastocysts. Fusion pulse rarely activated M II oocytes, but rapidly dropped the p34cdc2 kinase activity in NT embryos. A pronucleus-like structure was observed 2-2.5 h after the activation pulse with CHX in NT embryos of both the FAS and FBA groups. Therefore, successive inactivation of M-phase promoting factor and cytostatic factor at a certain short interval may also play an important role in the development of NT embryos.

Pseudophysiological transcomplementary activation of reconstructed oocytes as a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetal fibroblast cells

“…An optimal time frame to activate gilt or sow oocytes may depend on both the time required for completion of nuclear-cytoplasmic maturation and the time by which aging process of mature oocytes starts. Cytoplasmic maturation is likely to include changes in the properties, size, and density of cytoplasmic Ca 2+ release channels necessary for the oocyte to elicit an increase in intracellular Ca 2+ concentration in response to the activating stimuli and subsequent development (De Sousa et al 2002, Martinez Diaz et al 2002.…”

“…It is known that InsP 3 -mediated calcium signalling pathway is responsible for downregulation of both maturation/meiosis-promoting factor (MPF) and cytostatic factor (CSF), the latter of which is dependent on the complex activity (enzymatic cascade) of mitogen-activated protein kinases, i.e., extracellular-regulated protein kinases (MAPKs/ERKs). In turn, diminishments in the concentrations and activities of MPF and CSF-related MAPKs contribute to such events during oocyte activation as resumption and termination of meiosis, extrusion of the second polar body, pronuclear formation, transition from meiotic to mitotic control of cell cycle and initiation of embryonic cleavage , Martinez Diaz et al 2002, Ito et al 2004).…”

“…In the swine somatic cell cloning technology, commonly used activating stimuli are physical agents such as electric (DC) pulses (Martinez Diaz et al 2002, Im et al 2004, Hölker et al 2005, or chemical agents such as specific ionophore antibiotics (e.g., calcium ionomycin, Ca 2+ ionophore A23187/calcimycin) (Boquest et al 2002, Yin et al 2002, Hyun et al 2003 or thimerosal in combination with dithiothreitol (Im et al 2006, Whitworth et al 2009). The current intensive studies on improving activation methods of porcine nuclear-cytoplasmic hybrids (i.e., clonal cybrids) are chiefly aimed at optimising technical parameters of electrical field involving strength, duration of DC pulses, number of pulses and time interval between them (Martinez Diaz et al 2002, Im et al 2004). Alternatively, and more often, these investigations are focused on combining an activating stimulus/stimuli, most frequently calcium ionomycin or DC pulses with exogenous agents that non-specifically or specifically block the activity of cyclin-dependent protein kinases (CDKs).…”

“…Another strategy is the treatment of reconstructed pig oocytes with activating factors (physical or chemical), followed by their exposure to the agents that reversibly inhibit protein synthesis. Here an example is cycloheximide (CHXM) which suppresses the re-translation of cyclin B (i.e., regulatory subunit of the heterodimeric MPF enzyme complex) following resumption of oocyte meiosis from metaphase II stage-arrest (Yin et al 2002, Martinez Diaz et al 2002, Lee et al 2003a.…”