1.

Introduction

Since the birth of “Dolly” in 1996 as the first clone of an adult mammal by somatic cell nuclear transfer (SCNT),¹ this technique has been successfully applied to a variety of mammalian species, including cattle,² mice,³ goats,⁴ pigs,⁵ rabbits,⁶ and horses.⁷ When combined with genetically modified donor cells, mammalian cloning offers the possibility to produce transgenic offspring for applications in agriculture and biomedicine.⁸ To overcome the shortage of human organ donors, transgenic pigs are considered to be best suitable as donors of xenografts.^{9, 10}

The production of cloned animals by SCNT involves multiple steps.^{11, 12} At first, the maternal deoxyribonucleic acid (DNA) is usually removed from the recipient oocyte by aspiration of the metaphase II (MII)-spindle and the adjacent first polar body. This procedure is called enucleation. Because of the high amount of lipids in oocytes of farm animals, the metaphase plate must be visualized by staining with Hoechst dye under UV illumination. In less opaque mouse and rhesus monkey oocytes, the spindle can be visualized using polarization microscopy without previous Hoechst staining.^{13, 14} In a second step, a single donor cell is inserted under the zona pellucida as close as possible to the oocyte membrane. Both cells are then electrically fused and the inserted donor cell nucleus is epigenetically reprogrammed to a pluripotent state. Finally, the reconstructed embryo is artificially activated to initiate the embryonic development.

Despite intense research over the past decade, the number of viable offspring compared to the number of reconstructed embryos transferred into surrogate mothers has remained low. Species-dependent success rates between 0.1 and 15% have been reported.⁸ The influencing factors can be divided into biological and technical aspects. Biological factors include the characteristics of oocytes and somatic cells and their epigenetic reprogramming after electrofusion.¹⁵ The removal of maternal DNA during enucleation belongs to the technical aspects. The highly invasive mechanical enucleation requires sophisticated, expensive equipment and considerable micromanipulation skill.¹⁶ To penetrate the oocyte plasma membrane without lysis, oocytes must be pretreated with the microfilament inhibitor cytochalasin B,¹⁵ which is associated with dramatic changes of the cytoskeleton.^{17, 18} DNA staining is usually done with the DNA-specific fluorochrome Hoechst 33342. For localization of the metaphase plate before its aspiration, the oocytes are exposed to UV light for a short time. Although viable offspring have been produced by this technique, it has been suggested that UV irradiation causes damage to the oocyte cytoplasm, especially the mitochondrial DNA.¹⁹ Therefore, possible long-term damaging effects cannot be ruled out.²⁰ During aspiration of the metaphase plate, small amounts of cytoplasm next to the DNA, containing matrix proteins, are inevitably removed, which has detrimental effects on the embryonic development.²¹ To avoid these problems, novel noninvasive methods for oocyte imaging and manipulation must be developed and evaluated.

Over the past four decades, laser pulses have been used in developmental biology for several applications.^{22, 23, 24, 25, 26} Successful laser inactivation (functional enucleation) of metaphase chromosomes was first demonstrated by McKinnell in leopard frog eggs using microsecond pulses.²⁷ Recently, Karmenyan used picosecond pulses for functional enucleation of mouse oocytes by targeted irradiation of the metaphase plate.²⁸ However, these protocols had several major drawbacks. The interaction of microsecond laser pulses with biological tissue is dominated by thermal effects. Depending on the thermal relaxation time, significant thermal damage may occur to the adjacent cytoplasm.²⁹ Karmenyan used conventional UV illumination in combination with Hoechst staining to visualize the metaphase plate in mouse oocytes, which had significant detrimental effects on their developmental potential.²⁸ The metaphase plate was also manually moved into the laser focus prior to manipulation. This time-consuming step significantly slowed down the whole process. Because of targeted irradiation of the metaphase plate, several chromosomes or large chromosome fragments most likely remained in the cytoplasm. The resulting aneuploid embryos after SCNT would still contain residual maternal DNA, which may impede the development to term.⁴ As a consequence of these drawbacks, current methods do not allow for automation of the enucleation procedure.

Femtosecond (fs) laser are an excellent minimally invasive tool for imaging and precise manipulation of single cells. Compared to continuous UV illumination, the interaction with biological tissue is based on nonlinear absorption. This enables higher penetration depths and impedes out-of-focus absorption and photodamage.³⁰ Because of these advantages, multiphoton microscopy allows long-term imaging of whole embryos without compromising cell viability.³¹ Above a certain pulse intensity threshold, a so-called low-density plasma is produced in the focal volume of the laser beam. It is assumed that these low-density plasmas mediate intracellular dissection by cumulative free-electron-mediated chemical effects.³² In this regime, no significant heat or mechanical energy transfer to surrounding regions occurs.³² On this basis, the ablation of single-cell organelles, such as microtubules or mitochondria, without damaging surrounding structures and other organelles has been successfully accomplished.^{33, 34, 35, 36} Moreover, using the very same fs laser system for both three-dimensional imaging and manipulation of single cells facilitates the automation of the ablation procedure.

In this paper, we show that fs laser pulses offer great potential for combined multiphoton imaging and automated functional enucleation of porcine oocytes. Efficient functional enucleation was achieved by three-dimensional ablation of the metaphase plate in the low-density-plasma regime. Subsequent artificial activation of enucleated oocytes was done to determine their developmental potential in comparison to several control groups.

2.

Materials and Methods

2.1.

Laser System and Microscope

The laser system used in this study was a tunable Ti:sapphire laser (Chameleon Ultra II, Coherent, Santa Clara, California), which generates ultrashort pulses of $140\mathrm{fs}$ at a repetition rate of $80\mathrm{MHz}$ with a $M^2<1.1$ beam quality. The accessible wavelength ranges from $680\text{to}1080\mathrm{nm}$ . For imaging and manipulation of oocytes, the central wavelength was set to $720\mathrm{nm}$ corresponding to the two-photon absorption maximum of the Hoechst 33342 dye in this range.³⁷ At this wavelength, the maximum pulse energy at the laser output is $30\mathrm{nJ}$ . An acousto-optical pulse picker (Pulse Select, APE GmbH, Berlin, Germany) was applied to regulate the pulse frequency for the manipulation of oocytes by selecting pulses of the laser beam with a division ratio between 1:20 and 1:5000 (corresponding to $4\mathrm{MHz}$ and $16\mathrm{kHz}$ ). These pulses are diffracted into the first order with a diffraction angle of $\sim 3.5\mathrm{deg}$ . The diffracted and initial laser beam were used for oocyte manipulation and multiphoton microscopy, respectively (see manipulation and imaging beam in Fig. 1 ).

Fig. 1

Schematic setup for multiphoton imaging and manipulation of single oocytes.

Both laser beams were guided through a mechanical shutter and an attenuator, consisting of a half-wave plate and a polarizing beamsplitter cube, before being superimposed. After entering the tubus of an inverted microscope (Axiovert 100, Carl Zeiss AG, Oberkochen, Germany) via a dichroic mirror, they were focused into the sample by a $0.8\text{-}\mathrm{NA}$ water-immersion objective (C-Achroplan NIR, Carl Zeiss AG). At a central wavelength of $720\mathrm{nm}$ , this results in a theoretical diffraction-limited spot diameter of $\sim 1080\mathrm{nm}$ . Because of dispersion in the optics, the pulse duration in the sample was $\sim 275\mathrm{fs}$ determined with the autocorrelator “CARPE” (APE GmbH). Laser beam scanning in the $x\text{-}y$ plane was achieved by a pair of high-speed galvanometer mirrors (Cambridge Technology, Lexington, Massachusetts). A piezoelectric objective-lens positioning system (nanoMIPOS 400, Piezosystem Jena GmbH, Jena, Germany) was used to move the laser focus along the $z$ -axis. The fluorescence induced by multiphoton excitation at low pulse energies around $0.1\mathrm{nJ}$ passed the objective and the dichroic mirror in the backward direction and was detected by a photomultiplier tube (R6357, Hamamatsu Photonics, Hamamatsu, Japan). A micropipette with an inner diameter of $\sim 50\mu \mathrm{m}$ was attached to the microscope to hold single oocytes in position by applying a slight low pressure.

3.

Results

To demonstrate the suitability of multiphoton microscopy (MPM) for imaging of porcine MII-oocytes, a three-dimensional reconstruction from a stack of multiphoton images is shown in Fig. 2 . In this representation, the autofluorescence of the cytoplasm (levels of gray) and the Hoechst fluorescence of the polar body (orange) are visible, whereas the adjacent metaphase plate is hidden in the cytoplasm. A sharp decrease of the detected fluorescence signal occurred in the deepest layers of these oocytes, which had a diameter of $\sim 150\mu \mathrm{m}$ (see Fig. 2, at the bottom of the images). Therefore, the metaphase plate and polar body were placed in the equatorial plane before metaphase plate ablation in further experiments.

Fig. 2

Three-dimensional reconstruction of a porcine MII oocyte from a stack of multiphoton microscopy images shown at two different angles. The autofluorescence of the cytoplasm and the fluorescence of the polar body (PB) stained with Hoechst 33342 are marked in levels of gray and orange, respectively. The metaphase plate is hidden in the cytoplasm. (Color online only.)

The first set of experiments was made to identify optimal laser parameters for efficient metaphase plate ablation with minimal collateral damage to the cytoplasm. To this end, the repetition rate, scan speed, and pulse energy of the manipulation beam were varied. At a constant spatial pulse overlap, the ablation efficiency was independent of the laser repetition rate. Therefore, a high repetition rate of $1\mathrm{MHz}$ was selected to minimize the duration for the whole procedure and to exclude possible temperature accumulation effects at the same time.³² Figure 3 shows the metaphase plate and polar body both before and after manipulation at $1\mathrm{MHz}$ repetition rate, $2.5\mathrm{nJ}$ pulse energy, and $100\mu \mathrm{m}∕\mathrm{s}$ scan speed. Following metaphase plate irradiation, the Hoechst fluorescence completely vanished in this area [see Fig. 3]. In contrast, the nearby fluorescence of the polar body remained unaffected. Restaining of the oocyte with Hoechst 33342 and SYBR Green I did not result in any detectable fluorescence recovery of the metaphase plate (data not shown). Therefore, the fluorescence decrease after irradiation could be attributed to ablation and not to photobleaching. Numerous repetitions of this experiment at the same parameters resulted in a high ablation efficiency and intact oocyte morphology over at least $3\mathrm{h}$ . In addition, no changes in the mitochondrial distribution and morphology were observed within this period (see Fig. 4 ). Varying the laser pulse energy at $100\mu \mathrm{m}∕\mathrm{s}$ scan speed and $1\text{-}\mathrm{MHz}$ repetition rate exhibited two other regimes. Whereas residual Hoechst fluorescence was detected below $2.5\mathrm{nJ}$ , gas bubble formation in the irradiated area occurred above this value indicating severe cell damage (data not shown).³² Consequently, a pulse energy of $2.5\mathrm{nJ}$ was used in further experiments.

Fig. 3

Multiphoton microscopy images from various depths (a) before and (c) directly after metaphase plate ablation of a porcine MII oocyte stained with Hoechst 33342 at $1\mathrm{MHz}$ laser repetition rate, $2.5\mathrm{nJ}$ pulse energy, and $100\mu \mathrm{m}∕\mathrm{s}$ scan speed (MP: metaphase plate, PB: polar body). The manipulation beam was solely scanned over the metaphase plate along the white lines in (b) whose shape was automatically determined by our software.

Fig. 4

Mitochondrial distribution and morphology of porcine MII oocytes (a) before, (b) directly after and (c) $3\mathrm{h}$ after metaphase plate ablation. The metaphase plate position in (a) is indicated by the red dashed ellipse. No changes in mitochondrial distribution and morphology were observed within this period. (Color online only.)

The next step involved the evaluation of the early development after parthenogenetic activation of all five experimental groups: laser enucleation, sham enucleation, nonirradiation, mechanical enucleation, and control. All experimental groups, including laser-enucleated oocytes, maintained intact morphology $(>95\mathrm{\%})$ over $19\mathrm{h}$ . The efficiency of functional oocyte enucleation was 96%, pronucleus formation occurred in only 2 of 50 oocytes (see Table 1 ), compared to 100% efficiency after mechanical enucleation. Small chromosome fragments were found in $\sim 10\mathrm{\%}$ of laser-enucleated oocytes [see Fig. 5 ], whereas none of them continued the parthenogenetic development and underwent cleavage. After seven days of in vitro culture, cytoplasmic fragmentation without detectable DNA in these fragments was observed in $\sim 35\mathrm{\%}$ of laser-enucleated oocytes [see Fig. 5]. The same observation was made after conventional mechanical enucleation (data not shown). Sham-enucleated oocytes, whose cytoplasm was irradiated at the same parameters, cleaved and developed to the blastocyst stage with no significant differences in comparison with nonirradiated and control oocytes.

Fig. 5

Phase contrast and light-microscopy images of porcine embryos (a) $19\mathrm{h}$ and (b) $7\text{days}$ after parthenogenetic activation, respectively. Chromosome fragments, indicated by the black dashed ellipse in (a), and cytoplasmic fragmentation (b) were observed in some laser-enucleated oocytes. Control oocytes predominantly (a) formed a pronucleus (PN) and (b) developed to the blastocyst stage. Embryos in (b) are stained with Hoechst 33342 (blue). (Color online only.)

Table 1

Early development in parthenogenetically activated control and nonirradiated as well as sham-, laser- and mechanically enucleated porcine MII oocytes. Pronucleus formation and oocyte morphology were evaluated 19h after activation, whereas cleavage and blastocyst rates were examined after 168h .

4.

Discussion and Conclusion

The presented results demonstrated the successful combination of three-dimensional imaging and automated functional oocyte enucleation using fs laser pulses. Compared to previous laser-based functional enucleation protocols, this method had several major advantages.

The generated three-dimensional reconstruction of a Hoechst-stained porcine MII-oocyte showed the feasibility of MPM for three-dimensional oocyte imaging. However, a sharp decrease of the detected fluorescence intensity occurred in the deepest layers of these oocytes (see Fig. 2). Although the use of near-infrared (NIR) wavelengths generally improves the imaging depth of fluorescence microscopy,³⁰ a slightly different behavior is observed in porcine oocytes probably attributed to its high cytoplasmic lipid content. Transmission drops $\sim 30\mathrm{\%}$ from $300\text{to}500\mathrm{nm}$ followed by a constant increase up to the initial value at $1000\mathrm{nm}$ .⁴⁰ Therefore, longer NIR wavelengths combined with other DNA stains such as Sybr14 are promising to further optimize the imaging depth.¹⁹

Using MPM and Hoechst staining for metaphase plate visualization prior to manipulation did not affect the viability and developmental potential of oocytes after parthenogenetic activation with no significant difference to controls (see Table 1). These findings are consistent with the work done by Squirrell, indicating the great potential of multiphoton microscopy for noninvasive long-term imaging of whole embryos.³¹ The great advantage of using fs laser pulses is the negligible multiphoton absorption outside the focal volume and hence reduced damage to adjacent structures including membranes.³⁰ Moreover, no incubation with the microfilament inhibitor cytochalasin B was required prior to metaphase plate ablation (functional enucleation) in contrast to conventional mechanical enucleation. This may positively influence the embryonic developmental potential after nuclear transfer.⁴¹

To the best of our knowledge, this is the first time that the localization, shape acquisition, and subsequent three-dimensional ablation of a cell organelle were fully automated by means of MPM images. This automation of the whole procedure with our self-developed software significantly increased the throughput. Further optimization of the entire experimental protocol allowed for processing 20–25 oocytes per hour. Compared to conventional enucleation methods, this was about two times slower. However, the presented noncontact and noninvasive method using fs laser pulses offers the potential for automation of the enucleation procedure since every step can be done by a microfluidic technique.⁴²

Our experiments determined for the first time the optimal parameters for efficient metaphase plate ablation of porcine MII-oocytes. At a pulse energy of $2.5\mathrm{nJ}$ , the bright fluorescence of the metaphase plate completely vanished (see Fig. 3). Even after restaining with different nucleic acid stains, no fluorescence recovery was observed above the background noise. Because our parameters are $>20\mathrm{\%}$ above the photobleaching threshold,⁴³ we can be sure of metaphase plate ablation.³⁵ However, it is still possible that very small DNA fragments remain that are likely not able to recruit sufficient dye molecules. To disprove this assumption, we scanned a weakly focused amplified Ti:Sa fs laser beam over a small droplet with $100\mathrm{ng}∕\mu \mathrm{l}$ linearized plasmid DNA (pEGFP-C1, Clontech, Mountain View, California) at the same laser fluences. The plasmid encodes a wild-type GFP and has a length of $4.7\mathrm{kbp}$ , which roughly corresponds to the average gene length.⁴⁴ Gel electrophoresis revealed that the intensity of the $4.7\mathrm{kbp}$ band decreased with increasing pulse energy until it completely disappeared. However, no fragments with detectable fluorescence intensity were observed in the range of $4.7\mathrm{kbp}\text{to}20\mathrm{bp}$ by both agarose and polyacrylamid gels.⁴⁵ Therefore, we assume that possible DNA fragments produced by metaphase plate ablation were smaller than genes or transposons whose lengths are $>100\mathrm{bp}$ .⁴⁴ In addition, they were much smaller than the residual chromosomes or large chromosome fragments after previous laser-based functional enucleation protocols. As the cytotoxicity increases with the molecular weight of macromolecules,⁴⁶ our method should be less cytotoxic and may thus improve the embryonic development.

Using the obtained laser parameters for efficient metaphase plate ablation, functional enucleation was achieved in 96% of oocytes with simultaneous maintenance of intact morphology over a long period (see Table 1). This behavior was comparable to that observed after conventional mechanical enucleation. None of the laser-enucleated oocytes underwent cleavage and continued the parthenogenetic development. In striking contrast, UV-C irradiation of the metaphase plate does not block but only delays the first cleavage by $<48\mathrm{h}$ .⁴⁷ The significantly higher peak intensity of fs laser pulses causes frequent simultaneous multiphoton absorption above the ionization threshold of DNA molecules.⁴⁸ We assume that subsequent free electron and free radical formation induce further severe DNA fragmentation and base modifications.⁴⁹ Minor damage by continuous UV-C irradiation is most likely, at least partially, repaired by DNA repair mechanisms.⁵⁰

Following fs laser-based functional enucleation, no changes in the mitochondrial distribution and morphology were observed within $3\mathrm{h}$ (see Fig. 4). As mitochondrial DNA (mtDNA) damage and apoptosis are associated with rapid changes in mitochondrial morphology,⁵¹ we assume that the laser treatment did not damage the mtDNA. Irradiation of the oocyte cytoplasm at the same parameters (sham enucleation) did not compromise the developmental potential compared to controls (see cleavage and blastocyst rates in Table 1). In previous studies using nanosecond, picosecond, and fs laser pulses, subcellular ablation without damaging surrounding organelles or cytoskeletal structures was demonstrated.^{36, 52, 53} Therefore, our results suggest that the metaphase plate ablation did not affect the oocyte cytoplasm and organelles.

In $\sim 10\mathrm{\%}$ of fs laser-enucleated oocytes, small chromosome fragments were observed after $19\mathrm{h}$ [see Fig. 5]. A detailed analysis of the MPM images after metaphase plate ablation and restaining revealed that about the same percentage was incompletely ablated, leaving small fluorescently labeled DNA fragments in the cytoplasm (data not shown). To avoid these fragments in every oocyte, all laser parameters for metaphase plate ablation have to be further optimized. After seven days of in vitro culture, $\sim 35\mathrm{\%}$ of laser-enucleated oocytes showed cytoplasmic fragmentation without detectable DNA in these fragments [see Fig. 5]. Because this behavior was also observed in mechanically enucleated oocytes⁵⁴ cytoplasmic fragmentation is unlikely caused by fs laser irradiation of the metaphase plate.

Compared to the aspiration of the maternal DNA during mechanical enucleation, DNA fragments and free radical-induced DNA base modifications remained in the oocyte cytoplasm after fs laser-based functional enucleation. It has been shown that fs laser irradiation of cell nuclei causes accumulation of several DNA repair factors and proteins at the irradiation sites.^{55, 56} Furthermore, this accumulation results in cell-cycle checkpoint activation and significant delay of mitotic cleavage.⁵⁷ For this reason, further studies have to investigate potential negative consequences of DNA repair mechanisms on embryonic cleavage after fs laser-based metaphase plate ablation and subsequent nuclear transfer.

In conclusion, we demonstrated the suitability of fs laser as a novel tool for oocyte enucleation. The use of ultrashort pulses with NIR wavelengths instead of UV illumination for imaging and metaphase plate localization improved the oocyte viability and developmental potential. The high functional enucleation efficiency of $>95\mathrm{\%}$ combined with a high throughput (20–25 oocytes per hour) is promising for the application of fs laser systems as a fast, easy-to-use, and reliable enucleation tool. Compared to conventional mechanical enucleation, it may improve the efficiency of somatic cell clone production. Further advancement of the presented method combined with microfluidic technique offers the possibility to automate oocyte enucleation and to significantly increase the speed of this step of the cloning protocol.