Optimization of Parameters of the Oocyte Enucleation and Fusion With the Somatic Cell During Production of Mammalian Cloned Embryos

Abstract

S u m m a r yParameters of the oocyte enucleation and fusion with somatic cell for producing of cattle cloned cetohybrids were studied. It was established that the electrofusion efficiency depends on strength, duration and set number of electrical pulse. The optimal parameters for Multiporator (Eppendorf, Germany) were determined: two consistent pulses DC electrical field each of 30 V field strength and 15 msec duration followed by the pulse of AC electrical field of 5 V and 10 sec duration. It was found that the first polar body serving as a marker for the chromosome position at «blind» enucleation must not deviate from the chromosome location by more than 10 degrees.Keywords: in vitro culture, oocyte, cattle nuclear transfer, electrofusion, enucleation.Somatic cloning is widely used in biomedical research and in production of transgenic animals (1). Despite a certain success of using this technique in recent years, it is yet inefficient in cattle and other domestic animals owing to high incidence of embryonic abnormalities and low viability of the born cloned offspring (2-4).To create a clone, genetic material of the host's egg cell is replaced by genetic material of a somatic cell (reprogramming). It is often associated with complete removal of the oocyte's chromosomes at metaphase II, after which the somatic donor cell is introduced in the perivitelline space of the (5).The procedure is visualized by staining of the chromosomes with fluorescent vital dye Hoechst 33342 allowing enucleation of the host oocyte under ultraviolet light (6). However, this approach adversely affects viability of cells and future embryos (7). The alternative solution is blind enucleation when metaphase chromosomes are recognized by location of the first polar body that serves as a reference point. This method is considered as more safe for viability of the egg cells and their capacity to future embryonic development (8), though some problems are not excluded too. After isolation of the first spindle pole body, metaphase plate of chromosomes stays for a while in a close proximity to it. Then, with aging of the egg cell, chromosomes disconnect from the spindle pole body that migrates to the perivitelline space. This circumstance complicates blind enucleation of oocytes or interdicts it (9), which necessitates identifying the factors important for efficiency of the method.After enucleation, the fusion of cytoplast and karyoplast is performed to form a new reconstructed egg cell. Following development of cloned embryos directly depends on successful association of the host and donor cells, and viability of the resulting hybrid (10-14).Electrofusion is commonly used technique for karyoplast-cytoplast fusion. A pulsed electric field applied to the cell membrane causes in it a reversible loss of integrity -transmembrane pores, after which the membrane becomes compressed and discharged. This makes possible the fusion of two contacting membranes, whose contact points open cytoplasmic channels for exchange of cytoplasmic colloid. As a result,...