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

Significance: Preparation of a recipient cytoplast by oocyte enucleation is an essential task for animal cloning and assisted reproductive technologies in humans. The femtosecond laser is a precise and low-invasive tool for oocyte enucleation, and it should be an appropriate alternative to traditional enucleation by a microneedle aspiration. However, until recently, the laser enucleation was performed only with applying a fluorescent dye. Aim: This work is aimed to (1) achieve femtosecond laser oocyte enucleation without applying a fluorescent dye and (2) to study the effect of laser destruction of chromosomes on the structure and dynamics of the spindle. Approach: We applied polarized light microscopy for spindle visualization and performed stain-free mouse and human oocyte enucleation with a 1033 nm femtosecond laser. Also, we studied transformation of a spindle after metaphase plate elimination by a confocal microscopy. Results: We demonstrated a fundamental possibility of inactivating the metaphase plate in mouse and human oocytes by 1033 nm femtosecond laser radiation without applying a fluorescent dye. Irradiation of the spindle area, visualized by polarized light microscopy, resulted in partly or complete metaphase plate destruction but avoided the microtubules impairment. After the metaphase plate elimination, the spindle reorganized, however, it was not a complete depolymerization. Conclusions: This method of recipient cytoplast preparation is expected to be useful for animal cloning and assisted reproductive technologies.

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

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.

Probing Intracellular Dynamics Using Fluorescent Carbon Dots Produced by Femtosecond Laser In Situ.

Fluorescent particle tracking is a powerful technique for studying intracellular transport and microrheological properties within living cells, which in most cases employs exogenous fluorescent tracer particles delivered into cells or fluorescent staining of cell organelles. Herein, we propose an alternative strategy, which is based on the generation of fluorescent species in situ with ultrashort laser pulses. Using mouse germinal vesicle oocytes as a model object, we demonstrate that femtosecond laser irradiation produces compact dense areas in the intracellular material containing fluorescent carbon dots synthesized from biological molecules. These dots have tunable persistent and excitation-dependent emission, which is highly advantageous for fluorescent imaging. We further show that tight focusing and tuning of irradiation parameters allow precise control of the location and size of fluorescently labeled areas and minimization of damage inflicted to cells. Pieces of the intracellular material down to the submicrometer size can be labeled with laser-produced fluorescent dots in real time and then employed as probes for detecting intracellular motion activity via fluorescent tracking. Analyzing their diffusion in the oocyte cytoplasm, we arrive to realistic characteristics of active forces generated within the cell and frequency-dependent shear modulus of the cytoplasm. We also quantitatively characterize the level of metabolic activity and density of the cytoskeleton meshwork. Our findings establish a new technique for probing intracellular mechanical properties and also promise applications in tracking individual cells in population or studies of spatiotemporal cell organization.

Femtosecond laser-induced blastomere fusion results in embryo tetraploidy by common metaphase plate formation.

The cell fusion is a widespread process, which takes place in many systems in vivo and in vitro. Fusion of cells is frequently related to tetraploidy, which can be found within natural physiological conditions, e.g., placentation, and in pathophysiological conditions, such as cancer and early pregnancy failure in humans. Here we investigate the mechanism of tetraploidization with help of femtosecond laser-induced mouse blastomere fusion by the means of Hoechst staining, GFP, BODIPY dyes and fluorescent species generated intracellularly by a femtosecond laser. We establish diffusive mixing of cytosol, whereas the large components of a cytoplasm (organelles, cytoskeleton) are poorly diffusible and are not completely mixed after cell fusion and a subsequent division. We show that mechanisms which are responsible for the formation of a common metaphase plate triggered tetraploidization in fused mouse embryos and could be a significant factor in polyploidy formation in vivo. Thus, our results suggest that microtubules play a critical role in tetraploidization.

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

The effect of the laser pulse energy and total expose of the energy incident on the embryo blastomere fusion probability was investigated. The probability of the four different events after laser pulse was determined: the fusion of two blastomeres with the following formation of tetraploid embryo, the destruction of the first blastomere occurs, the second blastomere conservation remains intact, the destruction and the death of both cells; two blastomeres were not fused, and no morphological changes occurred. We report on viability and quality of the embryo after laser surgery as a function of the laser energy incident. To characterize embryo quality, the probability of the blastocyst stage achievement was estimated and the blastocyst cells number was calculated. Blastocoel formation is the only event of morphogenesis in the preimplantation development of mammals, so we assumed it as an indicator of the time of embryonic "clocks" and observed it among fused and control embryos. The blastocoel formation time is the same for fused and control embryos. It indicates that embryo clocks were not affected due to blastomere fusion. Thus, the analysis of the fluorescence microscopic images of nuclei in the fused embryo revealed that nuclei fusion does not occur after blastomere fusion.