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

Abstract

Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo’s integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.

Femtosecond laser surgery of two-cell mouse embryos: effect on viability, development, and tetraploidization

“…Gas-vapor bubble formation is necessary but insufficient for the cell fusion. Fused embryo development studies [21][22][23] do not permit separation of the effect of laser impact by itself from the fusion effect. So, the study of not fused (morphologically equal to control) embryo development allows us to understand how the laser impact by itself affects embryo viability and quality.…”

“…19,20 Moreover, only laser nanosurgery allows fusing of selected blastomeres into multicellular embryo (fusing 2 or 3 blastomeres in four-cell embryo) without affecting the whole embryo. 21 In addition to laser-induced intraembryonic blastomere fusion, the effect of femtosecond laser irradiation on embryo viability can be of great interest for science. It was demonstrated that fused embryo viability after pico-and femtosecondlaser treatment remains relatively high compared with control untreated group.…”

“…It was demonstrated that fused embryo viability after pico-and femtosecondlaser treatment remains relatively high compared with control untreated group. [21][22][23] At this time, there is a number of works [13][14][15][16][17][18]21,22,24 considered to the blastomere fusion technique. As far as you can see, all these investigations pay attention only to fused (tetraploid) embryo development.…”

From Zygote to Blastocyst: Application of Ultrashort Lasers in the Field of Assisted Reproduction and Developmental Biology

“…It should also be mentioned that the formation of a long-lasting vapor bubble (4-8 µm in diameter) in the irradiated area, creating a pore in both adjacent cell membranes, has been shown to be a prerequisite for successful blastomere fusion [34]. Apart from blastomere fusion, MII-stage oocyte fusion or fusion of oocyte and blastomere has also been demonstrated in [35,36]. For this purpose, the ZP was removed, and lectin was used for cell aggregation.…”