Femtosecond laser oocyte enucleation as a low-invasive and effective method of recipient cytoplast preparation

Abstract

Recipient cytoplast preparation, commonly performed by DNA aspiration with a needle, inevitably leads to the loss of reprogramming factors. As an alternative to the traditional enucleation technique, femtosecond laser enucleation can eliminate DNA effectively without loss of reprogramming factors and without oocyte puncturing. In this work we have performed oocyte enucleation by destructing the metaphase plate using a 795 nm femtosecond laser. The disability of the enucleated oocytes to develop after the parthenogenetic activation, as well as the lack of DNA staining luminescence, strongly confirms the efficiency of the femtosecond laser enucleation. The parthenogenetic development of oocytes after the cytoplasm treatment suggests a low-invasive effect of the laser enucleation technique.

Stain-free enucleation of mouse and human oocytes with a 1033 nm femtosecond laser

“…Although pre-stained oocyte enucleation appears low-invasive and effective, 13 the application of dye is not suitable for clinical use. Therefore, we examined enucleation of the oocytes without using dye.…”

“…Animal and oocyte preparation was performed according to a standard scheme described in our previous work. 13 6 to 8 week old C57BL6/CBA female mice (Mus musculus) were induced to superovulate by intraperitoneal (i.p.) injection of 10 IU pregnant mare’s serum gonadotropin (A036A02, Intervet) followed by an i.p.…”

“…Using a tightly focused near-infrared femtosecond laser beam, DNA can be eliminated in a living oocyte without significantly affecting cell viability. 13 This becomes possible due to non-linear nature of femtosecond laser absorption by biological material as absorption occurs only in the region of highest laser intensity (laser focal spot). This provides a possibility of highly localized treatment.…”

“…Typically, oocytes are stained with vital DNA-specific dye Hoechst, which has excitation/emission maxima of 350/461 nm and are subjected to UV illumination for visualization. 2 , 3 , 13 In terms of human oocyte enucleation, laser destruction of DNA without dye (hereinafter, stain-free) is of special interest. Enucleation of oocytes using polarized microscopy has already been performed in mice 16 and humans, 17 although this was aspiration with an injection pipette.…”

“…Laser enucleation requires accurate detection of the metaphase plate. Typically, oocytes are stained with vital DNA-specific dye Hoechst, which has excitation/emission maxima of 350/461 nm and are subjected to UV illumination for visualization 2 , 3 , 13 . In terms of human oocyte enucleation, laser destruction of DNA without dye (hereinafter, stain-free) is of special interest.…”