Genes & CellsGenes & CellsГены и КлеткиGenes and Cells2313-18292500-2562Human Stem Cells Institute63812710.17816/gc638127Original Study ArticlesОригинальные исследованияResearch ArticleThe effect of cytochalasin B during cytoplast preparation on the efficiency of somatic cloning in sheep (Ovis aries)Влияние цитохалазина Б в период подготовки цитопластов на эффективность соматического клонирования овец (Ovis aries)https://orcid.org/0000-0002-1284-14861092-2523LopukhovAlexandr V.ЛопуховАлександр Викторович
Russian Federation
vubi_myaso@mail.ruhttps://orcid.org/0000-0002-9642-23841067-8115ShedovaEkaterina N.ШедоваЕкатерина Николаевна
Russian Federation
shedvek@yandex.ruhttps://orcid.org/0000-0002-3263-2358TsyndrinaEvgeniya V.ЦындринаЕвгения Валериевна
Russian Federation
kiril04kina@yandex.ruhttps://orcid.org/0000-0003-0198-97574118-2990SinginaGalina N.СингинаГалина Николаевна
Russian Federation

Cand. Sci. (Biology)

канд. биол. наук

g_singina@mail.ruFederal Research Center for Animal Husbandry named after Academy Member L.K. ErnstФедеральный исследовательский центр животноводства — ВИЖ имени академика Л.К. Эрнста020320250704202520131402510202402122024Copyright ©; 2025, Eco-VectorCopyright ©; 2025, Эко-Вектор2025Eco-VectorЭко-Векторhttps://creativecommons.org/licenses/by-nc-nd/4.0/https://genescells.ru/2313-1829/article/view/638127

BACKGROUND: Somatic cloning of sheep is of great interest for genetic resource conservation, biomedicine, and biopharmaceuticals. However, its efficiency remains extremely low. One way to enhance the efficiency of this technology is to optimize its individual steps, particularly the procedure of oocyte enucleation and the transfer of somatic cell–derived cytoplasts into the perivitelline space — nuclear transfer. The chemical agent cytochalasin B enhances the oocyte’s resistance to external deformation and facilitates micromanipulation procedures. However, its application in cloning technology remains a subject of debate.

AIM: To evaluate the efficiency of somatic cloning in sheep (Ovis aries) using cytochalasin B during the preparation of matured oocytes prior to nuclear transfer, depending on the duration of this procedure.

MATERIALS AND METHODS: Nuclear transfer was performed using in vitro–matured oocytes containing a first polar body (PB1) in their perivitelline space. In the experimental group, oocytes with PB1 were incubated for 20 minutes in a medium containing 7.5 µg/mL cytochalasin B before nuclear transfer. The control group was maintained in an identical medium without cytochalasin B. Nuclear transfer was conducted using an inverted microscope equipped with a Narishige micromanipulation system (Narishige Scientific Inst. Lab., Japan). Oocyte chromosomes were removed blindly by aspirating the PB1 and the adjacent cytoplasm. Somatic cells were injected directly into the perivitelline space of the enucleated oocyte. Electrofusion was used to combine the oocyte–somatic cell complexes. The cytoplasmic hybrids formed as a result of electrical stimulation were activated using ionomycin, subjected to simultaneous treatment with 6-(dimethylamino)purine and cycloheximide, and then cultured for two days for embryonic development.

RESULTS: The nuclear transfer efficiency (the number of oocyte–somatic cell complexes relative to the number of oocytes with PB1) and fusion rate (the number of cytoplasmic hybrids formed relative to the number of oocyte–somatic cell complexes) did not differ between the control and experimental groups, remaining at 96%–97% and 33%–35%, respectively. In the control group, the proportion of cytoplasmic hybrids undergoing cleavage after two days of culture was 35.7±7.7%. Pre-incubation of oocytes in cytochalasin B increased this parameter to 59.7±6.9% (p <0.0029). However, the effect of cytochalasin B treatment on cloning efficiency depended on the duration of nuclear transfer: when the procedure exceeded 40 minutes, a significant decrease in the fusion rate (p=0.002) and a reduction in the proportion of cloned embryos (relative to the number of oocyte–somatic cell complexes) (p=0.041) were observed.

CONCLUSION: Short-term culture of matured oocytes in the presence of cytochalasin B before nuclear transfer increases the yield of cloned embryos, while prolonged nuclear transfer procedures negatively affect the efficiency of somatic cloning in sheep.

Обоснование. Соматическое клонирование овец представляет большой интерес с точки зрения сохранения генетических ресурсов, биомедицины и биофармацевтики, но его эффективность остаётся крайне низкой. Способом повышения результативности этой технологии является оптимизация её отдельных этапов, в частности процедуры энуклеации ооцитов и переноса в перивителлиновое пространство полученных цитопластов соматической клетки (СК) — nuclear transfer (NT). Химический агент цитохалазин Б повышает устойчивость ооцита к внешним деформациям и облегчает проведение микроманипуляций. Однако вопрос его использования в технологии клонирования остаётся дискуссионным.

Цель исследования — оценить эффективность соматического клонирования овец (Ovis aries) при использовании цитохалазина Б в период подготовки созревших ооцитов перед NT в зависимости от продолжительности данной процедуры.

Материалы и методы. Для NT использовали ооциты, созревшие in vitro и имеющие в своем перивителлиновом пространстве первое полярное тельце (ППТ). Первую группу ооцитов с ППТ перед процедурой NT инкубировали в течение 20 мин в среде, содержащей 7,5 мкг/мл цитохалазина Б (опыт); другая часть их хранилась в среде аналогичного состава в отсутствие цитохалазина Б (контроль). NT выполняли с использованием инвертированного микроскопа, совмещённого с микроманипуляционной системой Narishige (Narishige Scientific Inst. Lab., Япония). Хромосомы яйцеклетки удаляли вслепую аспирацией ППТ и прилежащей к нему части цитоплазмы. СК инъецировали непосредственно в перивителлиновое пространство энуклеированного ооцита. Электрослияние являлось способом объединения комплексов ооцит–СК. Образовавшиеся в результате электрического воздействия цитогибриды активировали иономицином, подвергали одновременной обработке 6-(диметиламино)пурином и циклогексимидом, после чего культивировали в течение 2 дней для эмбрионального развития.

Результаты. Результативность NT (количество комплексов ооцит–СК относительно числа ооцитов с ППТ) и уровень слияния (количество образовавшихся цитогибридов относительно числа комплексов ооцит–СК) не различались между контрольной и опытной группами и находились на уровне 96–97 и 33–35% соответственно. В контрольной группе доля раздробившихся цитогибридов после 2 дней культивирования составила 35,7±7,7%. Предварительная инкубация ооцитов в присутствии цитохалазина Б повышала данный показатель до 59,7±6,9% (p <0,0029). Влияние обработки ооцитов цитохалазином Б на показатели эффективности клонирования, тем не менее, зависело от длительности NT: наблюдалось снижение уровня слияния (p=0,002) и уменьшение доли клонированных эмбрионов (от числа комплексов ооцит–СК) в случае превышения продолжительности процедуры более 40 мин (p=0,041).

Заключение. Кратковременное культивирование созревших ооцитов в присутствии цитохалазина Б перед процедурой NT повышает выход клонированных эмбрионов, при этом увеличение продолжительности данной процедуры оказывает негативный эффект на показатели результативности соматического клонирования овец.

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