Use of picosecond infrared laser for micromanipulation of early mammalian embryos

Abstract

A high repetition rate (80 MHz) picosecond pulse (approximately 2 psec) infrared laser was used for the inactivation (functional enucleation) of oocytes and two-cell mouse embryos and also for the fusion of blastomeres of two-cell mouse embryos. The laser inactivation of both blastomeres of two-cell mouse embryos by irradiation of nucleoli completely blocked further development of the embryo. The inactivation of one blastomere, however, did not affect the ability of the second intact blastomere to develop into a blastocyst after treatment. Laser inactivation of oocytes at Metaphase II (MII) stage and parthenogenetically activated pronuclear oocytes also completely blocked their ability for further development. Suitable doses of irradiation in cytoplasm region did not affect the ability of embryos and activated oocytes to development. The efficiency of laser induced fusion for blastomeres of two-cell embryos was 66.7% and all the tetraploid embryos developed successfully into blastocysts in culture. Our results demonstrate unique opportunities of the applications of a suitable infrared periodic pulse laser as a universal microsurgery tool for individual living cells.

Laser Fusion of Mouse Embryonic Cells and Intra-Embryonic Fusion of Blastomeres without Affecting the Embryo Integrity

“…Corresponding data were already published for Drosophila and zebrafish embryos [1]. First results about functional enucleation of mammalian oocytes by laser irradiation of the metaphase plate were obtained in our experiments in mice [2] and repeated later on in pigs [3]. Furthermore, we showed that is possible to perform the entire mammalian cloning procedure at all stages of nuclear transplantation only with the use of laser [4].…”

Noncontact laser microsurgery of three-dimensional living objects for use in reproductive and regenerative medicine

“…In earlier papers it has been shown that nanosecond lasers may be successfully applied for embryo microsurgery [12,13]. At the present time picosecond [14] and femtosecond (fs) lasers [15][16][17] are mainly used. It should be pointed out that femtosecond lasers have several advantages as compared to continuous wave (CW) lasers or long-pulsed lasers.…”

Femtosecond laser-induced fusion of nonadherent cells and two-cell porcine embryos

“…Therefore, laser irradiation had no apparent detrimental effects on the cytoplasm and nuclear DNA, both involved in cell division, which is in very good agreement with previous fusion experiments. 8 A ploidy analysis of embryos developed to the blastocyst stage revealed that tetraploid cells were present in some fused embryos at this stage (data not shown). This indicates that nuclear fusion may occur within a few hours after laser treatment [see Fig.…”

“…For parthenogenetic twocell stage embryos, we achieved a fusion efficiency of 55% with porcine cells which is comparable to previous experiments with mouse cells (65%). 8 Long-term viability of fused two-cell porcine embryos was demonstrated by successful development up to the blastocyst stage with no significant difference to controls [see Fig. 6(a)].…”

“…5,7 Different cell types with volumes ranging from ∼ 0.5 to 4 pl (somatic cells) to 15 to 30 pl (embryonic C.elegans and mouse cells) were successfully fused to this day. 5,7,8 However, fusion of very large cells, such as two-cell stage embryos of farm animals, has not yet been published.…”