1.

Introduction

Inactivation of the oocyte metaphase plate using a near-infrared picosecond or femtosecond laser is an efficient and accurate method to prepare recipient cytoplasts.^1–3 A recipient cytoplast (an oocyte without chromosomes) is required for animal cloning and assisted reproductive technologies in humans, viz., for mitochondrial replacement therapy.^4,5 Removal of chromosomes lined up on the metaphase plate of the oocyte is commonly called enucleation.⁶

Oocytes in metaphase II of meiosis have homogeneous cytoplasm, and the metaphase plate is invisible in bright field and differential interference contrast (DIC). Metaphase II oocytes have typical structure, as shown in Fig. 1(a). In traditional enucleation, the metaphase plate is localized by a polar body⁷ or as a translucent region,^8,9 and then aspirated with a minimal volume of cytoplasm. The spindle as well as reprogramming factors, e.g., cyclin B1 and p34^cdc2,¹⁰ ORF1,¹¹ and soronin,¹² are also aspirated from the cell.

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 $2.46\mu \mathrm{m}$.

Laser enucleation of oocytes appears less harmful compared to the traditional microneedle method of metaphase plate aspiration, since it does not require the cell puncture and avoids the loss of reprogramming factors. The scheme of the laser action on the metaphase plate is given in Fig. 1(b). 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. During nonlinear adsorption of femtosecond laser action, highly localized formation of low-density plasma occurs.¹⁴ Interaction between low-density plasma and biological material leads to desirable localized DNA destruction.¹⁵

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. Enucleation of oocytes using polarized microscopy has already been performed in mice¹⁶ and humans,¹⁷ although this was aspiration with an injection pipette. In this work, we applied polarized light microscopy to detect the spindle and performed stain-free laser enucleation of mouse and human oocytes.

A key advantage of laser enucleation over aspiration is the ability to retain all intracellular compartments surrounding the metaphase plate, i.e., the spindle and reprogramming factors. However, when the metaphase plate is irradiated, the spindle can also be impaired because microtubules tightly surround the chromosomes at this stage of oocyte development. Moreover, it is known that the centromeres of each chromosome together with a kinetochore act as microtubule-organizing centers (MTOCs).¹⁸ Spindle integrity and proper kinetochore-microtubule attachment are essential for successful metaphase-to-anaphase transition (spindle checkpoint).¹⁹ Therefore, it is important to study spindle transformation after laser destruction of chromosomes. This work also studied the effect of laser destruction of chromosomes on the structure and dynamics of the spindle.

2.

Materials and Methods

3.

Results

3.1.

Mouse Oocyte Enucleation: Efficiency and Development

To study the efficiency of the enucleation, we used “stain-free” oocytes and oocytes pre-stained with Hoechst 33342 (the description of the experimental groups is given in Sec. 2.3). The experiments were carried out in two stages: (1) localization of the spindle or metaphase plate and femtosecond laser treatment; (2) observation of treated oocytes. Stage 1 was performed in the main experimental setup; stage 2 was carried out using a confocal microscope Zeiss LSM 980.

The spindle of the “stain-free” oocytes was detected using polarized light microscopy. Depending on the angle of polarization, we observed a light spot on a dark background or a dark spot on a light background at the spindle localization within oocyte [Fig. 2(a)]. The metaphase plate of “pre-stained” oocytes was detected by UV illumination. After detecting the spindle or metaphase plate, the oocytes were exposed to laser radiation and then examined for the presence of chromosomes and spindle. For this purpose, we used Hoechst dye on “stain-free” oocytes and BioTracker 488 Green Microtubule Cytoskeleton dye on both “stain-free” and “pre-stained” oocytes. Further observations were performed using a confocal fluorescent microscope.

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 $5\mu \mathrm{m}$.

In some “stain-free” oocytes the metaphase plate was completely eliminated [Fig. 2(b)], but in others we found DNA luminescence. Thus, to measure the efficiency of femtosecond laser enucleation, we obtained Z-stacks of the spindle area [Fig. 3(a)] and then calculated the total intensity of the Hoechst luminescence along the all Z-stack in “control,” “stain-free,” and “pre-stained” oocytes [Fig. 3(b)]. The total Hoechst intensity was significantly lower in both “stain-free” and “pre-stained” oocytes compared to the control ones (T-test, $p<0.00001$ and $p=0.003$ subsequently). However, the average sum Hoechst intensity in “pre-stained” oocytes was nearly two times lower than in “stain-free” oocytes.

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.

To assess viability of the oocytes as well as the efficiency of the enucleation, we performed diploid parthenogenetic activation, which induced development to the blastocyst stage. Both “pre-stained” and “stain-free” enucleated oocytes were unable to form blastocysts [Fig. 3(c)], whereas “stain-free cytoplasm” and “pre-stained cytoplasm” oocytes (irradiated at any place of the cytoplasm outside the metaphase plate) developed similarly to the control oocytes (“control”). The average number of blastocyst cells did not differ significantly between “cytoplasm” and “control” oocytes [Fig. 3(d)].

3.2.

Human Oocyte Enucleation

We also performed “pre-stained” and “stain-free” enucleation of human oocytes. “Pre-stained” human oocytes were illuminated with UV and exposed to laser radiation in the same way as mouse oocytes [Figs. 4(a)–4(c)]. After laser exposure, the oocytes ($n=11$) were cultured overnight and examined for their viability the following day. We found only one atretic oocyte; another 10 oocytes had normal morphology.

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 $20\mu \mathrm{m}$.

“Stain-free” human oocyte enucleation was complicated by the fact that the oocyte spindle in metaphase II was not visualized by polarization. Therefore, for the enucleation, we irradiated the opaque area near the polar bodies visible by DIC [Fig. 4(d)]. Oocytes of another experimental group (“cytoplasm”) were irradiated beside the metaphase plate ($n=7$). After exposure, we revealed chromosomes using confocal microscopy [Figs. 4(e) and 4(f)] and found that the total intensity of DNA luminescence in enucleated oocytes was almost 35% lower than in control ones ($40.9·{10}^3\pm 13.5·{10}^3$ versus $63.7·{10}^3\pm 23.2·{10}^3$, arbitrary unit). All oocytes were subjected to ICSI. In the “cytoplasm” group, five oocytes formed two pronuclei, one oocyte was three-pronuclear, and another one had no pronuclei. Enucleated oocytes ($n=4$) failed to form two pronuclei; however, two oocytes had one pronucleus each, and another two oocytes had no pronuclei.

3.3.

Spindle Reorganization

We observed the spindle condition in “stain-free” oocytes after femtosecond laser enucleation. The oocytes were stained for tubulin 1 to 2 h after laser exposure. While control oocytes had primarily a typical bipolar structure of the spindle [Fig. 5(a)], enucleated oocytes lost their normal spindle morphology [Fig. 5(b)]. However, this was not a complete depolymerization specific for tubulin poison, namely colchicine [Fig. 5(c)]. After DNA destruction, the most probable outcome for microtubules was polymerization in several (3 to 7) clusters (Table 1).

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 $10\mu \mathrm{m}$.

Table 1

Spindle morphology in control, enucleated (without stain) and colchicine-treated oocytes.

Group	N oocytes	Two poles	Three and more clusters	Depolimerization	Other
Control	22	15 (68%)	2 (9%)	0	5 (23%) – division
Enucleation	23	1 (4%)	14 (61%)	0	4 (17%) – fragmentation and 4 (17%) – division
Colchicine	21	3 (14%)	0	16 (76%)	2 (10%) – division

4.

Discussion and Conclusion

In this work, we performed an effective and low-invasive preparation of a recipient cytoplast using femtosecond 1033 nm laser radiation with or without applying a fluorescent dye. Laser oocyte enucleation has already been performed in mice² and pigs;³ however, laser inactivation of the metaphase plate without a fluorescent dye has not been shown yet. Our study applied polarization microscopy for spindle visualization, and we irradiated the metaphase plate based on the spindle location.

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. Polarized light microscopy allowed us to reveal the spindle location, which we used as a target for metaphase plate irradiation in mouse oocytes.

The efficiency of stain-free metaphase plate destruction in mouse oocytes varies in a wide range and can achieve almost 100% (Fig. 1). The dye allows for more accurate enucleation as it helps to find the exact location of the metaphase plate and absorb the laser more effectively. Unlike pre-stained oocyte enucleation, enucleation without dye requires higher laser intensities because the dye, acting as a photosensitizer, reduces the breakdown threshold.^14,21 For this reason, enucleation of “pre-stained” oocytes was performed with laser pulse energy 32 nJ (irradiance $4.7\times {10}^{12}\mathrm{W}/{\mathrm{cm}}^2$), which is 1.43 times lower than energy used in experiments with “stain-free” oocytes. For each experimental group (“pre-stained” and “stain-free” oocytes), we determined the irradiance threshold for vapor-gas bubble formation. The vapor-gas bubble is able to damage the cell, so it was important to avoid bubble formation. Therefore, we used irradiance 10% lower than corresponding threshold irradiance, in order to omit vapor-gas bubble formation.

Compared to dye-mediated DNA destruction, stain-free enucleation proved to be nearly half as effective. Nevertheless, this was enough to stop the development of mouse oocytes. At the same time, laser action itself appears low-toxic and low-invasive since affecting the cytoplasm (not the metaphase plate) does not significantly disturb the development. The probability of blastocyst formation, as well as the number of blastocyst cells does not differ significantly between cytoplasm-irradiated oocytes and the control oocytes [Figs. 3(c) and 3(d)]. The presence of a stain does not affect the possibility of reaching the blastocyst stage, nor the number of its cells.

The spindle was successfully visualized in almost all freshly-recovered MII mouse oocytes. If the spindle was not visible, we did not use the oocyte in the experiment. In the experiments with human oocytes, we had a small number of samples, and therefore were not able to choose the best ones. Unfortunately, we did not observe spindle birefringence in human oocytes under polarized microscopy, thus we applied DIC microscopy to improve visualization of the spindle or metaphase plate. To some extent, this helped us to find an approximate location of the metaphase plate [Fig. 4(d)]. Our preliminary results show the principal possibility of human oocyte enucleation by 1033 nm femtosecond laser with applying a dye and even without a dye. However, further investigation of human oocyte enucleation by laser is required.

The appearance of the human oocyte spindle depends on the egg maturity.²² In addition, the spindle is sensitive to many factors, including temperature²³ and osmolarity.²⁴ It was difficult to control all these factors in our experiments because the affiliated medical center and our scientific laboratory are located in different places, and the spindle might have been impaired due to oocyte transfer conditions. Moreover, freezing and thawing also affect the spindle incidence, location and morphology.²⁵ However, spindle visualization by polarized light microscopy is applicable in human clinical practice.^22,26,27 Therefore, using laser enucleation in human oocytes is expected to be effective under accurate visualization of the spindle.

Despite the fact that the spindle and the metaphase plate are located very close, the laser destroyed only the metaphase plate (Fig. 1). It should be noted that microneedle aspiration completely eliminates the spindle.¹⁶ For a short period after enucleation (up to 30 min), the spindle remained intact, but then, within 3 to 4 h, it lost its bipolar structure and reorganized into several small clusters. The laser appears to have destroyed the MTOCs, composed of centromeres and kinetochores, and may have spread undetectable remains of MTOCs throughout the oocyte. Those remains became the attractors of microtubule polymerization.

It is currently unclear what remains of DNA after laser irradiation. The absence of fluorescence under confocal microscope observation only implies the absence of a binding structure for the fluorescent dye, such as DNA double strand in the case of Hoechst dye. There is evidence of single and double strand DNA breaks under femtosecond laser irradiation.¹⁵ However, the extent of DNA destruction after femtosecond treatment should be considered in subsequent investigations. The potential of left DNA fragments to affect further development (if using enucleated cytoplasts for cloning or other assisted reproductive techniques) is also the question of great interest and importance.