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

Abstract

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.

Keywords: enucleation; femtosecond laser; recipient cytoplast; spindle.

Fig. 1

(a) Typical structure of metaphase II oocytes. (b) The scheme of the laser action on the metaphase plate. The laser beam is focused on the chromosomes of a metaphase plate (right), and the laser absorption occurs within the beam waist. The beam waist radius $w_0$ is $0.9\mu \mathrm{m}$, and Rayleigh distance $Z_0$ is $2.46\mu \mathrm{m}$.

Fig. 2

(a) Mouse oocytes imaged by polarized light microscopy. The spindle is visible as a dark spot on a light background light (left) or as a light spot on a dark background (right). Scalebar is $15\mu \mathrm{m}$. (b) Spindle of the intact oocyte (left) and enucleated (without stain) oocyte (right). Microtubules are shown in green, and DNA is shown in blue. Scalebar is $5\mu \mathrm{m}$.

Fig. 3

(a) Images of the spindle taken from different depths of a Z-stack. The Hoechst intensity (blue color) was measured within the circle in each slice, its diameter was the same for all oocytes. Scalebar is $10\mu \mathrm{m}$. (b) Average sum intensity of the Hoechst 33342 luminescence in enucleated (“pre-stained” – dye was applied before and after the laser action; “stain-free” – dye was applied only after the laser action) and control oocytes. The number of oocytes is shown within the bars. (c) Parthenogenetic development of oocytes to the blastocyst stage after laser action. The bars show % of blastocyst stage formation for the following groups: “pre-stained enucleation” (pre-st. enucl.), “stain-free enucleation” (st.-free enucl.), “pre-stained cytoplasm” (pre-st. cyt.), “control,” and “spontaneous activation” (spont.act.). The numbers above the bars indicate the exact number of blastocysts/number of irradiated oocytes. (d) The number of cells in blastocysts obtained from oocytes irradiated to the cytoplasm with or without staining. The number of calculated blastocysts is shown within the bars.

Fig. 4

“Pre-stained” (a)–(c) and “stain-free” (d)–(f) human oocyte enucleation. Oocyte (pre-stained with Hoechst 33342) before laser action (a), during (b), and after (c) exposure. (d) Stain-free oocyte visualization by DIC. The opaque area shown by the arrow was irradiated in enucleation experiments. (e) Oocyte with cytoplasm exposed to laser radiation. (f) Oocyte with the metaphase plate exposed to laser radiation. Scalebar is $20\mu \mathrm{m}$.

Fig. 5

Spindle structure in control, enucleated (without stain), and colchicine-treated oocytes: (a) two poles, (b) three and more clusters of polarization, and (c) depolymerization. Scale bar is $10\mu \mathrm{m}$.