Fusion of three blastomeres inside 4-cell embryos.

<p>(A) 4-cell embryos with visible contacts between three blastomeres. (B) Embryo with one normal and one big blastomere from three fused blastomeres. (C) Hoechst 33342 staining confirms the presence of three nuclei in one cytoplast. (D) In vitro development of 4-cell embryos with three fused blastomeres. Middle stage and expanded blastocysts.</p

Oocyte-oocyte fusion and oocyte-blastomere fusion.

<p>(A) Two aggregated oocytes before fusion. (B) Fused oocytes, 60 min after laser treatment. (C) Development of fused oocytes after parthenogenetic activation and in vitro cultivation. (D) Laser irradiation of contact site between aggregated oocyte and blastomere (arrow show point of laser beams area of fusion). (E) Fused cells, 60 min after laser treatment. (F) Asymmetric cleavage of fused cells.</p

Fusion of two blastomeres inside 4-cell embryos.

<p>(A) 3-cell embryos as a result of fusion of two blastomeres inside 4-cell embryos. (B) Hoechst 33342 staining demonstrates two nuclei in one cytoplast. (C) Hatching blastocysts.</p

Fusion of two pairs of blastomeres inside 4-cell embryos.

<p>(A) Fusion of first pair of blastomeres. (B) Fusion of second pair of blastomeres. (C) Resulting 2-cell embryos. Hoechst 33342 staining confirms the presence of two nuclei in each blastomere. (D) In vitro development of 4-cell embryos with two fused pairs of blastomeres. Hatching and hatched blastocyst.</p

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

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