The Influence of Microinjection of Foreign Gene into the Pronucleus of Fertilized Egg on the Preimplantation Development, Cell Number and Diameter of Rabbit Embryos

Abstract

The aim of this in vitro study was to test the effect of microinjection (Mi) of foreign gene into the rabbit egg pronucleus and epidermal growth factor (EGF) addition on the blastocyst rate, the cell number and the diameter of embryos, and to determine possible relationships between embryo cell number and embryo diameter. Blastocyst rate was significantly decreased in gene-Mi (G-Mi/E0) group (63.1%) comparing to intact ones (83.5%, p 1 <0.05). The addition of EGF at 20ng/ml (G-Mi/E20) or 200 ng/ml (G-Mi/E200) to gene-Mi embryos did not affect blastocyst rate (65.6 and 55.2% resp.). As a control for Mi, the eggs were microinjected with the same volume of phosphate-buffered solution (PBS-Mi) instead of the gene construct solution. Cell numbers and embryo diameters were measured from embryo images obtained on confocal laser scanning microscope. Bonferroni-modified LSD test showed that the embryo cell number in PBS-Mi group was significantly lower (p 1 <0.05) and in gene-Mi group was tended to decrease compared with intact embryos. Embryo diameter was not different among experimental groups. No effect of EGF given at any doses both on the cell number and embryo diameter was found. A positive correlation between cell number and embryo diameter was observed in all groups of embryos. Since embryo diameter was not changed under the influence of Mi or EGF addition in this study, this seems to be more conservative characteristics of the embryo morphology. These results suggest that the pronuclear microinjection compromises developmental potential of embryos, decreasing blastocyst rate and embryo cell number, whilst embryo diameter is not affected. No effects of EGF on studied parameters were confirmed. Declined quality of Mi-derived embryos is caused by the microinjection procedure itself, rather than by the gene construct used.

Quality of rabbit vitrified/thawed transgenic embryos

“…We did not detect differences between the transgenic embryos themselves, although EGFP-positive rabbit embryos were obtained by microinjection (in vitro) and hFVIII-positive embryos were derived from mating of transgenic rabbits (in vivo). Microinjection of the gene construct in the study of Makarevich et al (2006) did not affect the cleavage rate of embryos, but blastocyst rate was significantly lowered compared with intact embryos. Popelková et al (2005) reported that the ability to achieve the hatching in microinjected rabbit blastocysts (94.00%) is comparable with the control group of embryos (90.00%).…”

“…Vitrification procedure used in our study caused a decrease in total cell numbers of vitrified hFVIII-positive embryos compared with the control group (117.00 ± 36.00 vs. 141.00 ± 34.80). Procedure for gene microinjection of rabbit embryos, according to the study of Makarevich et al (2006), did not affect embryo diameter. Our results concerning ICM cells are comparable with those of FouladiNashta et al (2005), who determined the proportion of cells in the ICM of bovine embryos (25.50%).…”

“…Embryo diameter including the zona pellucida was measured from the images on the screen of the monitor using a scale bar micrometer, which was previously calibrated on a ×40 objective and a ×10 eyepiece. The diameter of the embryos was the mean of two measurements made perpendicular to each other (Makarevich et al ., 2006).…”

“…The diameter of the embryos was the mean of two measurements made perpendicular to each other (Makarevich et al, 2006).…”

Effect of vitrification technique and assisted hatching on rabbit embryo developmental rate

“…Embryo diameters, excepting ZP, were measured from the same images on the screen of the monitor using scale bar micrometer, which was previously calibrated on a ×40 objective and ×10 eyepieces (Makarevich et al, 2006).…”