ivmМеждународный вестник ветеринарииInternational Journal of Veterinary Medicine2072-2419SpbGUVM Publishing House10.52419/issn2072-2419.2025.3.445ivm-1839Research ArticleАКУШЕРСТВО, ГИНЕКОЛОГИЯOBSTETRICS, GYNECOLOGYМетодические аспекты выделения внеклеточной ДНК из антральных овариальных фолликулов Sus scrofa domesticusMethodological aspects of extracellular DNA isolation from antrum ovarian follicles of Sus scrofa domesticushttps://orcid.org/0000-0002-4218-6080КузьминаТ. И.KuzminaT. I.

д-р биол. наук, проф., глав. науч. сотрудник, зав. лабораторией биологии развития

Doctor of Biological Sciences, Professor, Chief Researcher, Head of the Laboratory of Developmental Biology

https://orcid.org/0000-0002-2865-9582ПритужаловаА. О.PrituzhalovaA. O.

науч. сотр. лаборатории биологии развития

Researcher, Laboratory of Developmental Biology

https://orcid.org/0009-0004-1980-9333БарановаЕ. И.BaranovaE. I.

лаборант-исследователь лаборатории биологии развития 

Research Assistant at the Developmental Biology Laboratory 

elenabaranova666@gmail.comВсероссийский научно-исследовательский институт генетики и разведения сельскохозяйственных животных – филиал Федерального государственного бюджетного научного учреждения «Федеральный исследовательский центр животноводства – ВИЖ имени академика Л. К. Эрнста»РоссияThe All-Russian Scientific Research Institute of Genetics and Breeding of Farm Animals is a branch of the Federal State Budgetary Scientific Institution Federal Research Center of Animal Husbandry named after Academician L. K. ErnstRussian Federation20250701202603445457Copyright © Кузьмина Т.И., Притужалова А.О., Баранова Е.И., 20252025Кузьмина Т.И., Притужалова А.О., Баранова Е.И.Kuzmina T.I., Prituzhalova A.O., Baranova E.I.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vetjournal.spbguvm.ru/jour/article/view/1839

Оценка качества донорских ооцитов животных и их компетентности к оплодотворению является важной задачей в репродуктивной биотехнологии. Одним из перспективных неинвазивных биомаркеров, который может использоваться для вышеобозначенных целей, является внеклеточная ДНК (внДНК). Этот показатель активно исследуется в медицине, ориентированной на человека, однако его значение в оценке женских гамет продуктивных животных остается недостаточно изученным. В настоящем исследовании проведен анализ содержания внДНК в фолликулярной жидкости свиней, аспирированной из антральных овариальных фолликулов (диаметр ˃3мм, 3-5мм, < 5мм), проанализировано влияние различных режимов центрифугирования на её выделение, а также оценена жизнеспособность клеток гранулезы (доля клеток с пикнотическими ядрами). Уровень клеток в состоянии пикноза определяли методом Тарковского, включающим гипотоническую обработку цитратом натрия, фиксацию уксусно-метанольной смесью и микроскопический анализ после окрашивания по Романовскому–Гимзе. Установлено, что концентрация внДНК в жидкости фолликулов изменяется в зависимости от их диаметра и режима центрифугирования. При увеличении ускорения центрифугирования наблюдалось снижение количества внДНК и рост доли клеток с пикнотическими ядрами, что свидетельствует об их повреждении. Идентифицирован режим центрифугирования (603,72 g в течение 5 минут), обеспечивающий максимальный уровень содержания внДНК при минимальном количестве пикнотических клеток. Кроме того, полученные данные согласуются с гипотезой о том, что внДНК может служить индикатором клеточного стресса, что особенно важно при оценке качества биоматериала в условиях in vitro. Представленные результаты подчеркивают важность выбора параметров центрифугирования для эффективного выделения внДНК с целью ее дальнейшего изучения как потенциального биомаркера качества ооцитов и перспективного прогностического индикатора фертильности продуктивных животных.

Assessment of the quality of animal donor oocytes and their competence for fertilization is an important task in reproductive biotechnology. Extracellular DNA is one of the promising noninvasive biomarkers that can be used for the above purposes. This indicator is actively being investigated in human-oriented medicine, but its significance in assessing the female gametes of productive animals remains poorly understood. The present study analyzes the content of cDNA in pig follicular fluid aspirated from antral ovarian follicles (diameter 3mm, 3-5mm, < 5mm), analyzes the effect of various centrifugation modes on its isolation, and evaluates the viability of granulosa cells (proportion of cells with pycnotic nuclei). The level of cells in a state of pyknosis was determined by the Tarkovsky method, which included hypotonic treatment with sodium citrate, fixation with an acetic-methanol mixture, and microscopic analysis after Romanovsky–Giemse staining. It was found that the concentration of cDNA in the follicle fluid varies depending on their diameter and mode of centrifugation. With an increase in the acceleration of centrifugation, a decrease in the amount of cDNA and an increase in the proportion of cells with pycnotic nuclei were observed, which indicates their damage. The centrifugation mode (603.72 g for 5 minutes) was identified, providing the maximum level of cDNA content with a minimum number of pycnotic cells. In addition, the data obtained is consistent with the hypothesis that cDNA can serve as an indicator of cellular stress, which is especially important when assessing the quality of biomaterials in vitro. The presented results emphasize the importance of choosing the parameters of centrifugation for the effective isolation of сDNA to further study it as a potential biomarker of oocyte quality and a promising predictive indicator of fertility in productive animals.

фолликулярная жидкостьвнеклеточная ДНКклетки гранулезыSus scrofa domesticusпикнозfollicular fluidextracellular DNAgranulosa cellsSus scrofa domesticuspyknosisМатериалы подготовлены в рамках Государственного задания Министерства науки и высшего образования (№ FGGN-2024–0014).The materials were prepared within the framework of the State Assignment of the Ministry of Science and Higher Education (No. FGGN-2024–0014).ReferencesСафронова Н.А., Калинина Е.А., Донников А.Е., Бурменская О.В., Макарова Н.П., Кулакова Е.В., Алиева К.У. Перспективы исследования маркеров клеток кумулюса для оценки качества ооцитов и эмбрионов в программах вспомогательных репродуктивных технологий // Акушерство и гинекология. – 2015. – № 12.Safronova N.A., Kalinina E.A., Donnikov A.E., Burmenskaya O.V., Makarova N.P., Kulakova E.V., Alieva K.U. Prospects for studying cumulus cell markers to assess the quality of oocytes and embryos in assisted reproductive technology programs // Obstetrics and Gynecology. – 2015. – № 12. (In Russ.)Станиславович Т. И., Кузьмина Т. И., Молчанов А. В. Оценка деструктивных процессов хроматина гранулезных клеток овариальных фолликулов коров и функциональный статус ооцита // Аграрный вестник Урала. 2019. № 12 (191) С. 60‒64. DOI: 10.32417/1997-4868-2019-191-12-60-64.Stanislavovich T. I., Kuzmina T. I., Molchanov A. V. Assessment of the Destructive Processes of Chromatin in Granular Cells of Ovarian Follicles in Cows and the Functional Status of the Oocyte // Agrarian Bulletin of the Urals. 2019. No. 12 (191). Pp. 60‒64. DOI: 10.32417/1997-4868-2019- 191-12-60-64. (In Russ.)Гапоненко А.А., Митюрина Е.В., Франкевич В.Е. Метаболомный профиль фолликулярной жидкости как маркер качества ооцитов // Акушерство и гинекология. – 2021. – № 11. – С. 26–31. DOI:10.18565/aig.2021.11.26-31.Gaponenko A.A., Mityurina E.V., Frankevich V.E. The metabolic profile of follicular fluid as a marker of oocyte quality // Obstetrics and Gynecology. – 2021. – No. 11. – pp. 26-31. DOI:10.18565/aig.2021.11.26-31. (In Russ.)Scalici E, Mulet T, Ferrier Hoa A, et al. Circulating nuclear acids and infertility. Gynecol Obstet Fertil. 2015; DOI: 10.1016/j.gyobfe.2015.07.016.Scalici E., Mulet T., Ferrier Hoa A., Gala A., Lu V., Anahori T. et al. Circulating nuclear acids and infertility. Gynecol Obstetric Fertil. 2015; DOI:10.18565/aig.2021.11.26-31. (in French)Андреева Е. А., Хонина Н. А., Демченко Е. Н., Гаврилова Е. Д., Пасман Н. М., Козлов В. А., Черных Е. Р. Свободная ДНК в фолликулярной жидкости у женщин с различными показателями овариальной функции. Бюллетень сибирской медицины. 2019; 18 (2): 16–23. DOI: 10.20538/1682-0363-2019-2-16-23Andreeva E.A., Honina N.A., Demchenko E.N., Gavrilova E.D., Pasman N.M., Kozlov V.A., Chernykh E.R. Free DNA in follicular fluid in women with different indicators of ovarian function. Bulletin of Siberian medicine. 2019; 18 (2): 16–23. DOI: 10.20538/1682-0363-2019-2-16-23 (In Russ.)De Borre M, Che H, Yu Q, et al. Cell-free DNA methylome analysis for early preeclampsia prediction. Nat Med. 2023;29 (9):2206-15. DOI:10.1038/s41591-023-02510-5.De Borre M, Che H, Yu Q, et al. Cellfree DNA methylome analysis for early preeclampsia prediction. Nat Med. 2023;29 (9):2206-15. DOI:10.1038/s41591-023- 02510-5 (in English)Adil, M., Kolarova, T.R., Doebley, AL. et al. Preeclampsia risk prediction from prenatal cell-free DNA screening. Nat Med (2025). DOI:10.1038/s41591-025-03509-wAdil, M., Kolarova, T.R., Doebley, AL. et al. Preeclampsia risk prediction from prenatal cell-free DNA screening. Nat Med (2025). DOI:10.1038/s41591-025-03509-w (in English)Stroun M, Lyautey J, Olson-Sand A, Anker P. 2001. About the possible origin and mechanism of circulating DNA. Apoptosis and active DNA release. Clin Chim Acta. 313(1–2):139–142. DOI:10.1016/s0009-8981(01)00665-9.15Stroun M, Lyautey J, Olson-Sand A, Anker P. 2001. About the possible origin and mechanism of circulating DNA. Apoptosis and active DNA release. Clin Chim Acta. 313(1–2):139–142. DOI:10.1016/s0009-8981(01)00665-9.15 (in English)Aucamp J, Bronkhorst AJ, Badenhorst CPS, Pretorius PJ. 2018. The diverse origins of circulating cell-free DNA in the human body: a critical re-evaluation of the literature. Biol Rev Camb Philos Soc. 93(3):1649, 168. DOI: 10.1111/BRV.12413.Aucamp J, Bronkhorst AJ, Badenhorst CPS, Pretorius PJ. 2018. The diverse origins of circulating cell-free DNA in the human body: a critical re-evaluation of the literature. Biol Rev Camb Philos Soc. 93(3):1649, 168. DOI: 10.1111/BRV.12413. (in English)Fernández-Domínguez, I. J., ManzoMerino, J., Taja-Chayeb, L., DueñasGonzález, A., Pérez-Cárdenas, E., amp; Trejo-Becerril, C. The role of extracellular DNA (exDNA) in cellular processes. Cancer Biology & Therapy, 22(4), 267–278. Fernández-Domínguez, I. J., Manzo-Merino, J., Taja-Chayeb, L., Dueñas-González, A., Pérez-Cárdenas, E., amp; Trejo- Becerril, C. (2021). The role of extracellular DNA (exDNA) in cellular processes. Cancer Biology amp; Therapy, 22(4), 267–278. DOI:10.1080/15384047.2021.1890319Fernández-Domínguez, I. J., ManzoMerino, J., Taja-Chayeb, L., DueñasGonzález, A., Pérez-Cárdenas, E., amp; Trejo-Becerril, C. The role of extracellular DNA (exDNA) in cellular processes. Cancer Biology; Therapy, 22(4), 267–278. Fernández-Domínguez, I. J., Manzo-Merino, J., Taja-Chayeb, L., Dueñas-González, A., Pérez-Cárdenas, E., amp; Trejo- Becerril, C. (2021). The role of extracellular DNA (exDNA) in cellular processes. Cancer Biology amp; Therapy, 22(4), 267–278. DOI:10.1080/15384047.2021.1890319 (in English)Keeling PJ, Palmer JD. 2008. Horizontal gene transfer in eukaryotic evolution. Nat Rev Genet. 9(8):605–618. DOI: 10.1038/nrg2386.Keeling PJ, Palmer JD. 2008. Horizontal gene transfer in eukaryotic evolution. Nat Rev Genet. 9(8):605–618. DOI: 10.1038/ nrg2386. (in English)Kustanovich A, Schwartz R, Peretz T, Grinshpun A. Life and death of circulating cell-free DNA. Cancer Biol Ther. 2019;20 (8):1057–1067. DOI:10.1080/15384047.2019.1598759.Kustanovich A, Schwartz R, Peretz T, Grinshpun A. Life and death of circulating cell-free DNA. Cancer Biol Ther. 2019;20 (8):1057–1067. DOI:10.1080/15384047.2019.1598759. (in English)Костюк С.В. Роль внеклеточной ДНК в функциональной активности генома человека // Вестник РАН. – 2010. – Т. 80, № 9. – С. 746–752.Kostyuk S. V. The role of extracellular DNA in the functional activity of the human genome // Bulletin of the Russian Academy of Sciences. - 2010. — Vol.80, No. 9. — pp. 746-752. (In Russ.)McCarthy C. G., Wenceslau C. F., Goulopoulou S., Ogbi S., Baban B., Sullivan J. C., et al. (2015). Circulating mitochondrial DNA and Toll-like receptor 9 are associated with vascular dysfunction in spontaneously hypertensive rats. Cardiovasc. Res. 107 119– 130. DOI: 10.1093/cvr/cvv137McCarthy C. G., Wenceslau C. F., Goulopoulou S., Ogbi S., Baban B., Sullivan J. C., et al. (2015). Circulating mitochondrial DNA and Toll-like receptor 9 are associated with vascular dysfunction in spontaneously hypertensive rats. Cardiovasc. Res. 107 119– 130. DOI: 10.1093/cvr/cvv137 (in English)Rykova E, Morozkin E, Ponomaryova A, Loseva E, Zaporozhchenko I, Cherdyntseva N, Vlassov V, Laktionov P. 2012. Cell-free and cell-bound circulating nucleic acid complexes: mechanisms of generation, concentration and content. Expert Opin Biol Ther. 12(Suppl sup1):S141–S153. DOI:10.1517/14712598.2012.673577.Rykova E, Morozkin E, Ponomaryova A, Loseva E, Zaporozhchenko I, Cherdyntseva N, Vlassov V, Laktionov P. 2012. Cellfree and cell-bound circulating nucleic acid complexes: mechanisms of generation, concentration and content. Expert Opin Biol Ther. 12(Suppl sup1):S141–S153. DOI:10.1517/14712598.2012.673577. (in English)Thierry AR, El Messaoudi S, Gahan PB, Anker P, Stroun M. 2016. Origins, structures, and functions of circulating DNA in oncology. Cancer Metastasis Rev. 35(3):347 –376. DOI:10.1007/s10555-016-9629-x.Thierry AR, El Messaoudi S, Gahan PB, Anker P, Stroun M. 2016. Origins, structures, and functions of circulating DNA in oncology. Cancer Metastasis Rev. 35(3):347 –376. DOI:10.1007/s10555-016-9629-x. (in English)Wang W, Kong P, Ma G, Li L, Zhu J, Xia T, Xie H, Zhou W, Wang S. 2017. Characterization of the release and biological significance of cell-free DNA from breast cancer cell lines. Oncotarget. 8(26):43180– 43191. DOI: 10.18632/oncotarget.17858.Wang W, Kong P, Ma G, Li L, Zhu J, Xia T, Xie H, Zhou W, Wang S. 2017. Characterization of the release and biological significance of cell-free DNA from breast cancer cell lines. Oncotarget. 8(26):43180– 43191. DOI: 10.18632/oncotarget.17858. (in English)Sato Y, Matoba R, Kato K. 2019. Recent advances in liquid biopsy in precision oncology research. Biol Pharm Bull. 42(3):337– 342. DOI: 10.1248/bpb.b18-00804.13Sato Y, Matoba R, Kato K. 2019. Recent advances in liquid biopsy in precision oncology research. Biol Pharm Bull. 42(3):337– 342. DOI: 10.1248/bpb.b18-00804.13 (in English)McCarthy C. G., Wenceslau C. F., Goulopoulou S., Ogbi S., Baban B., Sullivan J. C., et al. (2015). Circulating mitochondrial DNA and Toll-like receptor 9 are associated with vascular dysfunction in spontaneously hypertensive rats. Cardiovasc. Res. 107 119– 130. DOI:10.1093/cvr/cvv137McCarthy C. G., Wenceslau C. F., Goulopoulou S., Ogbi S., Baban B., Sullivan J. C., et al. (2015). Circulating mitochondrial DNA and Toll-like receptor 9 are associated with vascular dysfunction in spontaneously hypertensive rats. Cardiovasc. Res. 107 119– 130. DOI: 10.1093/cvr/cvv137 (in English)Lowes H., Pyle A., Santibanez-Koref M., Hudson G. (2020). Circulating cell-free mitochondrial DNA levels in Parkinson’s disease are influenced by treatment. Mol. Neurodegener. 15:10. DOI: 10.1186/s13024-020-00362-yLowes H., Pyle A., Santibanez-Koref M., Hudson G. (2020). Circulating cell-free mitochondrial DNA levels in Parkinson’s disease are influenced by treatment. Mol. Neurodegener. 15:10. DOI: 10.1186/s13024-020-00362-y (in English)Vakrakou A. G., Boiu S., Ziakas P. D., Xingi E., Boleti H., Manoussakis M. N. (2018). Systemic activation of NLRP3 inflammasome in patients with severe primary Sjögren’s syndrome fueled by inflammagenic DNA accumulations. J. Autoimmun. 91 23 –33. DOI: 10.1016/j.jaut.2018.02.010Vakrakou A. G., Boiu S., Ziakas P. D., Xingi E., Boleti H., Manoussakis M. N. (2018). Systemic activation of NLRP3 inflammasome in patients with severe primary Sjögren’s syndrome fueled by inflammagenic DNA accumulations. J. Autoimmun. 91 23 –33. DOI:10.1016/j.jaut.2018.02.010 (in English)Scalici E., Mulet T., Ferrier Hoa A., Gala A., Lu V., Anahori T. et al. Circulating nuclear acids and infertility. Gynecol Obstetric Fertil. 2015; 43:593-8. DOI: 10.1016/j.gyobfe.2015.07.016.Scalici E., Mulet T., Ferrier Hoa A., Gala A., Lu V., Anahori T. et al. Circulating nuclear acids and infertility. Gynecol Obstetric Fertil. 2015; 43:593-8. DOI: 10.1016/j.gyobfe.2015.07.016. (in English)Traver S, Scalici E, Mullet T, Molinari N, Vincens C, Anahory T, Hamamah S. Cellfree DNA in Human Follicular Microenvironment: New Prognostic Biomarker to Predict in vitro Fertilization Outcomes PLoS ONE. 2015;10(8):e0136172. DOI: 10.1371/journal.pone.0136172.Traver S, Scalici E, Mullet T, Molinari N, Vincens C, Anahory T, Hamamah S. Cellfree DNA in Human Follicular Microenvironment: New Prognostic Biomarker to Predict in vitro Fertilization Outcomes PLoS ONE. 2015;10(8):e0136172. DOI: 10.1371/journal.pone.0136172. (in English)Guan Y., Zhang W., Wang X., Cai P., Jia Q., Zhao W. Cell-free DNA induced apoptosis of granulosa cells by oxidative stress. Clin. Chim. Acta. 2017; 473: 213– 217. DOI: 10.1016/j.cca.2016.11.023.Guan Y., Zhang W., Wang X., Cai P., Jia Q., Zhao W. Cell-free DNA induced apoptosis of granulosa cells by oxidative stress. Clin. Chim. Acta. 2017; 473: 213–217. DOI: 10.1016/j.cca.2016.11.023. (in English)Ichikawa K., Shibahara H., Shirasuna K., Kuwayama T., Iwata H. Cell-free DNA content in follicular fluid: a marker for the developmental ability of porcine oocytes. Reproductive Medicine and Biology. 2019. Vol. 19. P. 95-103. DOI: 10.1002/rmb2.12309.Ichikawa K., Shibahara H., Shirasuna K., Kuwayama T., Iwata H. Cell-free DNA content in follicular fluid: a marker for the developmental ability of porcine oocytes. Reproductive Medicine and Biology. 2019. Vol. 19. P. 95-103. DOI: 10.1002/rmb2.12309. (in English)Tarkowski AK. An air-drying method for chromosome preparation from mouse eggs. Cytogenetics. 1966;5(3):394-400. DOI:10.1159/000129914Tarkowski A. K. An air-drying method for chromosome preparation from mouse eggs. (1966) Cytogenetics, nr. 5, 3, P. 394- 400. DOI:10.1159/000129914 (in English)Woodruff TK, Shea LD. The role of the extracellular matrix in ovarian follicle development. Reprod Sci. 2007 Dec;14(8 Suppl):6 -10. DOI: 10.1177/1933719107309818.Woodruff TK, Shea LD. The role of the extracellular matrix in ovarian follicle development. Reprod Sci. 2007 Dec;14(8 Suppl):6-10. DOI: 10.1177/1933719107309818. (in English)Liu Z, Li F, Xue J, Wang M, Lai S, Bao H, He S. Esculentoside A rescues granulosa cell apoptosis and folliculogenesis in mice with premature ovarian failure. Aging (Albany NY). 2020 Aug 5;12(17):16951- 16962. DOI: 10.18632/aging.103609. Epub 2020 Aug 5.Liu Z, Li F, Xue J, Wang M, Lai S, Bao H, He S. Esculentoside A rescues granulosa cell apoptosis and folliculogenesis in mice with premature ovarian failure. Aging (Albany NY). 2020 Aug 5;12(17):16951- 16962. DOI: 10.18632/aging.103609. Epub 2020 Aug 5. (in English)Xie J, Xu X, Liu S. Intercellular communication in the cumulus-oocyte complex during folliculogenesis: A review. Front Cell Dev Biol. 2023 Jan 19; 11:1087612. DOI: 10.3389/fcell.2023.1087612.Xie J, Xu X, Liu S. Intercellular communication in the cumulus-oocyte complex during folliculogenesis: A review. Front Cell Dev Biol. 2023 Jan 19; 11:1087612. DOI: 10.3389/fcell.2023.1087612. (in English)Avalos PN, Wong LL, Forsthoefel DJ. Extracellular vesicles promote proliferation in an animal model of regeneration. bioRxiv [Preprint]. 2024 Mar 27:2024.03.22.586206. DOI: 10.1101/2024.03.22.586206.Avalos PN, Wong LL, Forsthoefel DJ. Extracellular vesicles promote proliferation in an animal model of regeneration. bioRxiv [Preprint]. 2024 Mar 27:2024.03.22.586206. DOI: 10.1101/2024.03.22.586206. (in English)Chaves MA, Ferst JG, Fiorenza MF, Vit FF, da Silveira JC. The Influence of Ovarian -Derived Extracellular Vesicles in Reproduction. Adv Anat Embryol Cell Biol. 2025 Jan 1. DOI: 10.1007/102_2024_9.Chaves MA, Ferst JG, Fiorenza MF, Vit FF, da Silveira JC. The Influence of Ovarian-Derived Extracellular Vesicles in Reproduction. Adv Anat Embryol Cell Biol. 2025 Jan 1. DOI: 10.1007/102_2024_9. (in English)

The authors declare that there are no conflicts of interest present.