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St. Petersburg Polytechnic University Journal: Physics and Mathematics. 2022 Vol. 15, No. 3.2 Научно-технические ведомости СПбГПУ. Физико-математические науки. 15 (3.2) 2022
Conference materials UDC 57.089.38
DOI: https://doi.org/10.18721/JPM.153.258
Oocyte enucleation by 795 nm femtosecond laser is a precise and effective method of recipient cytoplast preparation
A. A. Osychenko H, A. D. Zalessky, U. A. Tochilo, D. Yu. Martirosyan N.N. Semenov Federal Research Center of Chemical Physics, Russian Academy of Sciences, Moscow, Russia
Abstract. Oocyte enucleation by femtosecond (fs) laser radiation is low-invasive and effective method of recipient cytoplast preparation. We have already demonstrated an ability to completely eliminate the metaphase plate by the fs laser without significant oocyte morphology and viability impairment. Considering further development of femtosecond laser oocyte enucleation for artificial reproductive technologies in humans and for the somatic cell nuclear transfer in mammals, the persistence of any DNA fragments after the fs laser enucleation must be studied thoroughly. In this work we revealed the remains of the metaphase plate by super resolution confocal microscopy after the laser enucleation of mouse oocytes at metaphase II stage. The laser was set up on 795 nm in 80 MHz mode with 0,5 nJ pulse energy. By the super resolution microscopy (Airyscan 2) we have detected minor fragments of the metaphase plate in the laser-exposed oocytes. However, the careful laser treatment is able to provide the recipient cytoplast without any detectable DNA fragments.
Keywords: recipient cytoplast, femtosecond laser, super resolution microscopy
Funding: This work is supported by the Russian Science Foundation under grant № 21-7510155. The work was performed on facilities of ACBS Center of the Collective Equipment (no. 506694, FRCCP RAS) and large-scale research facilities № 1440743.
Citation: Osychenko A. A., Zalessky A. D., Tochilo U. A., Martirosyan D. Yu., Oocyte enucleation by 795 nm femtosecond laser is a precise and effective method of recipient cytoplast preparation, St. Petersburg State Polytechnical University Journal. Physics and Mathematics. 15 (3.2) (2022) 317-320. DOI: https://doi.org/10.18721/JPM.153.258
This is an open access article under the CC BY-NC 4.0 license (https://creativecommons. org/licenses/by-nc/4.0/)
Материалы конференции УДК 57.089.38
DOI: https://doi.org/10.18721/JPM.153.258
Энуклеация ооцитов с помощью фемтосекундного лазера с длиной волны 795 нм - точный и эффективный метод подготовки реципиентного цитопласта
А. А. Осыченко н, А. Д. Залесский, У. А. Точило, Д. Ю. Мартиросян
Федеральный исследовательский центр химической физики им. Н.Н. Семенова Российской академии наук, г. Москва, Россия
Аннотация. Энуклеация ооцитов фемтосекундным (фс) лазерным излучением является малоинвазивным и эффективным методом подготовки реципиентного цитопласта. Мы уже продемонстрировали возможность полного удаления метафазной пластинки с помощью фс лазера без значительного ухудшения морфологии и жизнеспособности ооцитов. Учитывая дальнейшее развитие фемтосекундной лазерной энуклеации
© Osychenko A. A., Zalessky A. D., Tochilo U. A., Martirosyan D. Yu., 2022. Published by Peter the Great St. Petersburg Polytechnic University.
^ St. Petersburg Polytechnic University Journal. Physics and Mathematics. 2022 Vol. 15, No. 3.2 ^
ооцитов для искусственных репродуктивных технологий у человека и переноса ядер соматических клеток у млекопитающих, необходимо тщательно изучить присутствие любых фрагментов ДНК после фс лазерной энуклеации. В данной работе мы выявляли остатки метафазной пластинки с помощью конфокальной микроскопии сверхвысокого разрешения после лазерной энуклеации ооцитов мыши на стадии метафазы II. Лазер был настроен на 795 нм в режиме 80 МГц с энергией в импульсе 0,5 нДж. С помощью микроскопии сверхвысокого разрешения (Airyscan 2) мы обнаружили незначительные фрагменты метафазной пластинки в ооцитах, подвергшихся воздействию лазера. Однако тщательная лазерная обработка способна обеспечить реципиентный цитопласт без каких-либо обнаруживаемых фрагментов ДНК.
Ключевые слова: реципиентный цитопласт, фемтосекундный лазер, микроскопия сверхвысокого разрешения
Финансирование: Работа выполнена при поддержке гранта РНФ № 21-75-10155. Работа выполнена на ЦКП № 506694 ФиЦ ХФ РАН и УНУ № 1440743.
Ссылка при цитировании: Осыченко А. А., Залесский А. Д., Точило У. А., Мартиросян Д. Ю. Энуклеация ооцитов с помощью фемтосекундного лазера с длиной волны 795 нм — точный и эффективный метод подготовки реципиентного цитопласта // Научно-технические ведомости СПбГПУ. Физико-математические науки. Т. 15. № 3.2. С. 317-320. DOI: https://doi.org/10.18721/JPM.153.258
Статья открытого доступа, распространяемая по лицензии CC BY-NC 4.0 (https:// creativecommons.org/licenses/by-nc/4.0/)
Introduction
Removing the DNA from the living cell is of relevance for medical and biomedical applications, including somatic cell nuclear transfer (SCNT) in animals and mitochondrial replacement therapy (MRT) in humans. In particular, DNA removal is commonly performed for these purposes, by nucleus or metaphase plate aspiration with a needle, which can be resulted in the loss of reprogramming factors [1]. More, aspiration by a needle implies the cell puncturing, and requires applying cytoskeleton poisons, e.g., cytochalasin B [2]. As an alternative for aspiration, near-infrared femtosecond lasers are able to perform intracellular incisions with the submicron resolution, acting locally in the focal volume of the laser beam [3, 4]. In our recent research we have shown that near-infrared femtosecond laser effectively destroys the metaphase plate but does not affect oocyte viability and development by itself. Double fluorescent staining as well as the disability to develop parthenogenetically after the laser exposure confirm the lack of DNA in enucleated oocytes [5]. However, the question of the persistence of any DNA fragments after the fs laser enucleation must be studied thoroughly. In this work we have studied the remains of the metaphase plate by super resolution confocal microscopy. We have detected minor fragments of the metaphase plate in the laser-exposed oocytes. However, the careful laser treatment is able to provide the recipient cytoplast without any detectable DNA fragments.
Materials and Methods
Oocyte collection. To obtain oocytes at metaphase II stage, C57Bl/6 female mice were induced to superovulate by the standard scheme [5]. Mice were sacrificed and the oviducts were removed. Cumulus-oocyte complexes were purified from cumulus by 0.1% hyaluronidase (H4272, Sigma-Aldrich) in M2 culture medium (M7167, Sigma-Aldrich). For the metaphase plate visualization, the oocytes were stained with 5 ^g/ml Hoechst 33342 dye (B2261, Sigma-Aldrich) for 15 minutes in M2 medium and then washed twice. During the experiment the oocytes were cultured in M2 medium.
Laser parameters and enucleation process. The detailed scheme of the oocyte enucleation is described here [5]. Briefly, in these experiments we used following parameters for the irradiation of the metaphase plate: 1 = 795 nm, и = 80 MHz, pulse energy 0.5 nJ (40 mW power); 100 fs pulse duration, pulse train duration 60 ms. Laser radiation was focused by 60* objective lens (NA = 0.7). Before the enucleation we performed the metaphase plate visualization by UV illumination. Then the metaphase plate was exposed to laser. The exposure was repeated multiple times until the DNA luminescence stopped.
© Осыченко А. А., Залесский А. Д., Точило У. А., Мартиросян Д. Ю., 2022. Издатель: Санкт-Петербургский политехнический университет Петра Великого.
Fig. 1. Confocal images of the oocytes before and after the laser enucleation: brightfield + luminescence images (a). The red square shows an area containing the metaphase plate. These areas were further investigated by Airyscan 2. Super resolution image, presented in 2.5 D mode (b). X and Y axes show the distance (5 pm scale), Z axe shows the intensity.
Confocal imaging. Fluorescence imaging was performed using a laser scanning confocal microscope Zeiss LSM 980 (Carl Zeiss Microscopy, Jena, Germany), 63x Plan-Apochromat objective (NA = 1.4; oil immersion). The oocytes were placed in M2 medium drop on a 0.17 mm cover glass (Zeiss). Hoechst 33342 luminescence excitation were performed by 405 nm laser, detection range 410-479 nm. Super resolution was obtained by Airyscan 2 with 5 AU pinhole (251 pm).
Results and Discussion
The oocytes were exposed to the laser radiation as described [5]. In short, the femtosecond laser pulses were applied in the area of the metaphase plate, and fluorescence was observed in the focal spot with each pulse simultaneously. Laser exposure on the metaphase plate was repeated multiple times until the DNA luminescence stopped. Fig. 1 represents the oocytes before and after the femtosecond enucleation. Clearly visible at the left image (Fig. 1,a), DNA luminescence is almost undetectable at the right image, obtained after the laser exposure. The remains of the metaphase plate are slightly visible in super resolution mode (Fig. 1,b). A single peak of the luminescence, shown at right B image, could be easily eliminated by more careful laser exposure and control. The rest of the laser-exposed area has practically zero luminescence signal. Note that the oocytes retain their morphology and viability after the laser exposure.
Conclusion
We have detected minor fragments of the metaphase plate in the oocytes, enucleated by 795 nm femtosecond laser. However, the careful laser treatment is able to provide the recipient cytoplast without any detectable DNA fragments.
Acknowledgments
This work is supported by the Russian Science Foundation under grant 21-75-10155. The work was performed on facilities of ACBS Center of the Collective Equipment (no. 506694, FRCCP RAS) and large-scale research facilities 1440743.
St. Petersburg Polytechnic University Journal. Physics and Mathematics. 2022 Vol. 15, No. 3.2
REFERENCES
1. Ogura A., Inoue K., Wakayama T., Recent advancements in cloning by somatic cell nuclear transfer, Phil Trans R Soc B 368 (2012) 20110329.
2. Ogura A., Cloning Mice, Cold Spring Harb Protoc. 8 (2017) pdb.prot094425.
3. Shakhbazyan A.K., Sviridova-Chailakhyan T.A., Karmenyan A.K., Krivokharchenko A.S., Chiou A., Chailakhyan L.M., The Use of Laser for Obtaining Recipient Cytoplasts for Mammalian Nuclear Transfer, Doklady Biologycal Sciences 428 (2009) 1—4.
4. Kuetemeyer K., Lucas-Hahn A., Petersen B., Lemme E., Hassel P., Niemann H., Heisterkamp A., Combined multiphoton imaging and automated functional enucleation of porcine oocytes using femtosecond laser pulses, Journal of Biomedical Optics 15 (4) (2010) 046006.
5. Osychenko A.A., Zalessky A.D., Tochilo U.A., Martirosyan D.Yu., Silaeva Y.Yu., Nadtochenko V.A., Femtosecond laser oocyte enucleation as a low-invasive and effective method of recipient cytoplast preparation, Biomed. Opt. Express 13 (3) (2022) 1447-1456.
THE AUTHORS
OSYCHENKO Alina
[email protected] ORCID: 0000-0002-7519-2479
[email protected] ORCID: 0000-0002-4511-6109
TOCHILO Uliana
ZALESSKY Alexandr
MARTIROSYAN David
[email protected] ORCID: 0000-0002-2952-8867
[email protected] ORCID: 0000-0002-1992-0574
Received 16.07.2022. Approved after reviewing 19.07.2022. Accepted 20.07.2022.
© Peter the Great St. Petersburg Polytechnic University, 2022
