The effect of cytochalasin B during cytoplast preparation on the efficiency of somatic cloning in sheep (Ovis aries)

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Background

Somatic cell nuclear transfer (SCNT) is a powerful biotechnological tool aimed at preserving high-value genotypes, accelerating genetic improvement in livestock, generating genome-edited animals, and maintaining species diversity [1–4]. Equally important is the biomedical application of animal cloning, which holds promise for developing more accurate human disease models and therapies, enhancing and expediting drug testing, and enabling xenotransplantation [5–7]. Sheep represent a convenient model for biomedical research. SCNT, combined with genetic engineering techniques, offers new opportunities for genome editing in sheep and for producing biologically active compounds of human interest, particularly recombinant pharmaceutical proteins secreted in milk and monoclonal antibodies [8–10].

Unfortunately, the practical benefits of generating cloned animals, including sheep, are currently limited by the high cost and low efficiency of SCNT, underscoring the need for procedural optimization and greater reproducibility [11–13]. Potential strategies to improve SCNT outcomes include standardizing oocyte quality assessment prior to maturation, optimizing parameters for fusion of the enucleated oocyte and the transferred somatic cell (SC), and refining nuclear reprogramming conditions within the recipient oocyte cytoplasm and the culture conditions for resulting cytoplasmic hybrids [14–17].

The fundamental principle of cloning technology involves the removal of the nucleus from a mature oocyte (enucleation) and its replacement with a SC nucleus containing the genetic information solely from the donor organism [18]. Typically, enucleation is performed using the classical blind aspiration method, which involves removal of the first polar body (PB1) along with adjacent ooplasm containing metaphase chromosomes [19, 20]. During enucleation, the oocyte undergoes assessment for integrity, viability, and developmental competence [21, 22]. Micromanipulations involved in nuclear material removal and transfer—membrane piercing, ooplasm biopsy, somatic cell injection—constitute mechanical stress and may lead to oocyte degeneration due to irreversible membrane damage and cytolysis [23, 24]. Nuclear transfer (NT), defined in English-language publications as the procedure of introducing a somatic cell into the perivitelline space of the enucleated oocyte, may also result in loss or disruption of cellular compartments, ultrastructures, and oocyte-specific molecular factors essential for development [25–27]. Therefore, enhancing oocyte resilience to mechanical manipulation during SCNT is of critical importance [28].

One of the main components of the oocyte cytoskeleton is actin protein subunits, polymerized into double-stranded helical filaments known as microfilaments [29]. These microfilaments accumulate in the cortex during cytoplasmic maturation and fully infiltrate the cortical layer by metaphase II (MII) of meiosis [30, 31]. Microfilaments, closely associated with the oolemma, are vital for cytoskeletal integrity [32].

Cytochalasin B, a fungal metabolite, disrupts actin microfilaments by inducing their depolymerization [33]. This disruption alters the physical properties of oocyte cytoplasm, decreasing rigidity and increasing plasma membrane elasticity [34]. Cytochalasin B stabilizes the oocyte cytoskeleton, preventing structural damage and facilitating reconstruction [35]. It is widely used in mammalian cloning, including during NT [36–39]. However, the necessity of using this chemical agent remains under debate. NT in cattle, sheep, and pigs has been successfully conducted without it [40–42]. In some cases, cytochalasin B adversely affected the development of cloned domestic animal embryos [32, 42].

The aim of this study is to evaluate the efficiency of somatic cell nuclear transfer in sheep (Ovis aries) using cytochalasin B during the preparation of matured oocytes prior to NT, depending on the duration of this procedure.

Methods

Unless otherwise specified, all reagents were obtained from Sigma-Aldrich (USA). Oocyte, SC and embryo culture was carried out at 38.5 °C in a humidified atmosphere of 5% CO₂. All procedures outside the incubator were performed at 37 °C.

Preparation of Donor Cells (Karyoplasts)

Dulbecco’s modified Eagle medium (DMEM; Gibco, USA) was used in two formulations:

• DMEM-P: supplemented with 15% fetal bovine serum (FBS), 50 µg/mL gentamicin, and 1% non-essential amino acids;
• DMEM-M: supplemented with 5% FBS and 50 µg/mL gentamicin.

Donor cells were fetal sheep fibroblasts at passages IV–V. A few days before NT, cells were thawed in a 37 °C water bath, transferred to a centrifuge tube with 10 mL of DMEM-M, and centrifuged at 1500 rpm Cells were then cultured in DMEM-P until a monolayer formed, followed by a medium change to the same formulation containing 0.5% FBS, and further incubated for 48 hours to arrest fibroblasts at the G0/G1 phase. At the end of serum starvation, coinciding with the start of NT experiments, cells were prepared as previously described [43]. The medium used for culturing fetal fibroblasts was replaced with a trypsin–EDTA solution (Gibco, USA) and incubated at 37°C until cell detachment from the culture dish. The fetal fibroblasts were then transferred to tubes containing TC-199 medium supplemented with 50 µg/mL gentamicin and 0.3% bovine serum albumin. Cells were pelleted by centrifugation at 300 g for 7 minutes, the supernatant was discarded, and the pellet was resuspended in a medium of the same composition until used for NT.

Preparation of Oocytes (Cytoplasts)

Oocytes were obtained from sheep ovaries transported from an abattoir in physiological saline at 30–35 °C within 2.5–3 hours. Dissection, retrieval of cumulus–oocyte complexes (COCs), selection of high-quality COCs, and in vitro maturation (IVM) were performed as previously described [44].

After 19–23 hours of IVM, oocytes were denuded of cumulus cells, followed by selection of those that reached the MII stage of meiosis (matured in vitro), i.e., exhibited a PB1 in their perivitelline space, and those lacking signs of degeneration or lysis [45]. In the experimental group, oocytes with PB1 were incubated for 20 minutes in a medium containing 7.5 µg/mL cytochalasin B before NT. The control group was maintained in an identical medium without cytochalasin B. The culture medium was TC-199 supplemented with 10% FBS and 50 µg/mL gentamicin.

Nuclear Transfer Procedure: Enucleation of Oocytes and Transfer of Somatic Cells Into the Perivitelline Space of the Resulting Cytoplasts

Microsurgical procedures with cells were performed using an inverted Nikon Eclipse Ti microscope (Nikon, Japan) integrated with a system of left- and right-sided coarse (mechanical) and fine (oil-hydraulic) manipulators from Narishige (Japan), along with oil-hydraulic injectors. For NT, 15–20 oocytes were placed in 15 µL droplets of TC-199 medium supplemented with 5% FBS, applied to the bottom of a 60 mm Petri dish (Biomedical, Russia) and covered with light mineral oil. Into the same droplets, 1–2 µL of fetal fibroblast suspension was added. Removal of the PB1 and transfer of single fibroblasts were performed using a micropipette with an inner diameter of 13–15 µm (Origio, USA). Oocytes were positioned using a holding pipette (Origio, USA) in the microscope’s field of view to clearly visualize the PB1 in the perivitelline space, oriented at the 1 or 5 o’clock position of an imaginary clock face. The biopsy micropipette was brought into close contact with the oocyte’s zona pellucida, which was pierced at the PB1 location, and the oocyte chromosomes were blindly aspirated along with the PB1 and 10–20% of adjacent cytoplasm. SC were injected into the perivitelline space of the fixed oocyte using the same micropipette previously used for PB1 biopsy, through the opening created during enucleation. Upon completion of micromanipulations, the efficiency of NT was assessed by selecting morphologically normal (without signs of lysis) oocyte–somatic cell complexes and calculating their proportion relative to the total number of oocytes with a PB1 used for NT.

Generation of Cybrids, Their Activation, and Post-Activation Culturing

To facilitate the integration of SC content into the oocyte, selected oocyte–SC complexes were subjected to electrofusion using an Eppendorf Multiporator (Germany), following a methodology described previously [45]. The complexes were placed between the electrodes of a chamber filled with buffer, aligned using a fine glass capillary, and positioned such that the oocyte’s zona pellucida was adjacent to one electrode, with the SC located near their contact point. Fusion of the oocyte and SC into a cybrid was achieved using direct current pulses. After electrofusion, the complexes were maintained in TC-199 medium supplemented with 10% FBS in an incubator. After 60 minutes of incubation, formed cybrids were identified by the absence of the donor cell in the oocyte’s perivitelline space. Non-fused complexes were subjected to repeated electrical stimulation. Fusion efficiency was assessed by calculating the number of formed cybrids relative to the total number of oocyte–SC complexes used.

Cybrids obtained through electrofusion were treated with 5 mM ionomycin for 5 minutes, then placed in an embryonic development medium supplemented with 2 mM 6-(dimethylamino)purine and 10 µg/mL cycloheximide, and incubated for 4 hours. For embryonic development, activated cells were cultured in a medium of the same composition, without these compounds, for an additional two days [45]. Subsequently, the number of cleaved cybrids, their proportion relative to the total number of cybrids obtained, and their ratio to the initial number of oocyte–SC complexes were determined.

Statistical Analysis

Data analysis was performed using the licensed SigmaStat 2.03.0 software (Systat Software Inc., USA). Each experimental group included at least 9 independent replicates. Results are expressed as mean (M) ± standard error of the mean (SEM). Normality was assessed using the Kolmogorov–Smirnov test. Statistical significance between group means was evaluated using Student’s t test. A value of p < 0.05 was considered statistically significant.

Results

The NT procedure in sheep is illustrated in Fig. 1. Experimental data evaluating the efficiency of NT (the number of morphologically normal oocyte–SC complexes relative to the number of oocytes with the PB1), fusion, and cleavage, depending on the use (experimental group) or non-use (control group) of cytochalasin B during the preparation of matured oocytes for NT, are presented in Table 1. At this stage of the study, a total of 520 oocytes with the PB1 were reconstructed. The efficiency of NT did not differ between the compared groups and remained at a high level. Oocytes treated with cytochalasin B and those reconstructed without it also showed no statistically significant differences in cybrid formation (oocytes after enucleation and fusion with SC). However, the proportion of cleaved cybrids on the second day of embryonic culturing, calculated relative to the number of enucleated oocytes fused with SC, was significantly higher—by 22.0%—in the cytochalasin B group compared to the control group (p = 0.0029). This positive effect persisted when the yield of early-stage embryos was calculated relative to the number of oocyte–SC complexes. In the control group, this parameter was 12.1 ± 3.2%, while in the experimental group, it was 20.8 ± 2.6%, which was statistically significantly higher (p = 0.041).

 

Fig. 1. Micrographs of sheep oocytes during the nuclear transfer procedure: a, individual fixation of the oocyte using a holding pipette within the microscope’s field of view, positioned to clearly visualize the first polar body (PB1); b–c, oocyte enucleation; d, somatic cell capture with a micropipette; e, somatic cell transfer into the perivitelline space of the enucleated oocyte; f, completion of the nuclear transfer procedure: formation of the oocyte–somatic cell complex. Microscope: Eclipse Ti-U (Nikon, Japan). PB1 is indicated by white arrows, somatic cell by black arrows; ×200.

 

Table 1. Effect of cytochalasin B¹ on the production of cloned sheep embryos

Experimental group	Number of oocytes with PB1, n	Obtained
		Oocyte–SC complexes		Cytohybrids2		Cleaved сytohybrids3
		n	%, M±SEM	n	%, M±SEM	n	%, M±SEM
Control	201	193	96.3±1.6	62	33.9±5.1	21	35.7±7.7
Сytochalasin B	319	312	97.8±1.7	114	35.6±3.7	65	59.7±6.9*

Note: ¹ oocytes with the first polar body (PB1) were briefly cultured in TC-199 medium with 10% fetal bovine serum in the presence (7.5 µg/mL for 20 min) and absence (control) of cytochalasin B before the nuclear transfer procedure (enucleation of oocytes and transfer of donor somatic cell–derived cytoplasts into the perivitelline space); ² proportion of cytoplasmic hybrids, calculated as the ratio of the number of cytoplasmic hybrids to the number of oocyte–somatic cell complexes; ³ proportion of cleaved cytoplasmic hybrids, calculated as the ratio of the number of cleaved cytoplasmic hybrids to the number of cytoplasmic hybrids; * significant differences compared to the control, p=0.0029.

 

The effect of cytochalasin B on SCNT efficiency was also investigated in the context of the duration of the NT procedure. Two experimental groups were compared: in the first, oocytes with the PB1, after short-term culturing in the presence of cytochalasin B, underwent NT within a period not exceeding 40 minutes; in the second, micromanipulations took more than 40 minutes (but not exceeding 50 minutes). The results of this experiment are presented in Table 2. The proportion of enucleated oocyte–SC complexes without signs of degeneration did not differ statistically between the groups and, as in the first series of experiments, remained at a high level. However, fusion efficiency depended on the duration of NT: the proportion of formed cybrids relative to the number of oocyte–SC complexes was statistically significantly lower—by 18.8%—when the procedure lasted more than 40 minutes compared to cases where it lasted less than 40 minutes (p = 0.002). No effect of NT duration on the cleavage competence or embryonic development of the resulting cybrids was observed. However, the lower number of cybrids in the group with NT duration >40 minutes during fusion led to a reduced yield of early-stage embryos, calculated relative to the number of oocyte–SC complexes. In the >40-minute group, this parameter was 13.6 ± 3.5%, while in the ≤40-minute group, it was 23.0 ± 2.5%, which was statistically significantly higher (p = 0.042).

 

Table 2. Effect of cytochalasin B¹ on the production of cloned sheep embryos depending on nuclear transfer duration

NT duration, min	Number of oocytes with PB1, n	Obtained
		Oocyte–SC complexes		Cytohybrids2		Cleaved сytohybrids3
		n	%, M±SEM	n	%, M±SEM	n	%, M±SEM
≤40	205	194	94.3±2.5	84	44.0±3.2а	45	55.2±6.8
>40	164	160	97.9±2.1	41	25.2±4.6б	21	54.9±9.2

Note: ¹ — oocytes with the first polar body (PB1) were briefly cultured in TC-199 medium with 10% FBS in the presence (7.5 µg/mL for 20 min) of cytochalasin B before the nuclear transfer procedure (enucleation of oocytes and transfer of donor somatic cell–derived cytoplasts into the perivitelline space); ² proportion of cytoplasmic hybrids, calculated as the ratio of the number of cytoplasmic hybrids to the number of oocyte–somatic cell complexes; ³ proportion of cleaved cytoplasmic hybrids, calculated as the ratio of the number of cleaved cytoplasmic hybrids to the number of cytoplasmic hybrids; ^{a, b} mean values within a single group marked with different letters are statistically significantly different at p=0.002.

 

Discussion

The primary limitation to the widespread application of SCNT in domestic animals, including sheep, remains its low efficiency, which is partly due to the complexity and multistep nature of the technology [46]. One strategy to improve SCNT efficiency involves the use of agents that reduce oocyte degeneration and preserve their potential for subsequent development.

For the NT procedure, oocytes at the MII stage are primarily used [20]. During enucleation, the oocyte sustains damage due to pipette pressure, resulting in a localized rupture of the plasma membrane and loss of a cytoplasm portion [47]. Cytochalasin B, through its function as a reversible inhibitor of microfilament polymerization, can enhance ooplasm fluidity and oocyte membrane plasticity, reducing overall trauma during micromanipulations [48]. However, the use of cytochalasin B in the NT procedure remains a subject of debate.

The duration of oocyte nucleus removal at the MII stage and SC nucleus transfer, among other factors, depends on the chosen NT strategy: for example, partial dissection of the zona pellucida of all oocytes followed by enucleation and SC transfer; piercing the zona pellucida with enucleation of the entire oocyte group and delayed SC nucleus transplantation; SC transfer into the oocyte immediately after nucleus removal; blind enucleation or enucleation under ultraviolet light control. In our experiments, the NT procedure for 15–20 oocytes took between 20 and 50 minutes, which is comparable to data from international studies indicating that, with the blind enucleation method, enucleation of 30 oocytes takes approximately 30 minutes on average, and SC injection takes 20–30 minutes [47, 49].

In the present study (Table 1), cytochalasin B, used during the preparation of matured oocytes for reconstruction, had no significant effect on NT efficiency or fusion efficiency but increased the yield of early-stage cloned embryos both relative to the number of obtained cybrids (p = 0.0029) and relative to oocyte–SC complexes (p = 0.041). Presumably, the effect of cytochalasin B on oocyte cytoarchitecture manifests over time, i.e., it has a long-term effect. It is known that cytoskeleton damage leads to serious consequences for cell viability, including cessation of cytokinesis and the mitotic cycle [30]. Likely, cytoskeleton stabilization by cytochalasin B, followed by microfilament repolymerization, enables the oocyte to better withstand deformations caused by micromanipulations and promotes its more stable transformation into a cloned embryo after fusion with an SC.

Nevertheless, the effect of cytochalasin B treatment on cloning efficiency depended on the duration of NT: a reduction in fusion efficiency (p = 0.002) and, consequently, the proportion of cloned embryos formed relative to the number of oocyte–SC complexes was observed when the NT procedure exceeded 40 minutes (p = 0.042), with no negative effect of this factor on cybrid cleavage rates. We hypothesize that extending the duration of micromanipulations beyond acceptable time limits (in our conditions, no more than 40 minutes) has a greater impact on the electrical properties of the oocyte and SC than on the functional processes supporting the transition of the resulting cybrid to embryonic development.

Conclusion

Short-term culturing of matured oocytes in the presence of cytochalasin B prior to the NT procedure enhances the yield of early-stage cloned sheep embryos. Prolonging the duration of oocyte enucleation and SC nucleus transfer beyond 40 minutes in oocytes pre-incubated with cytochalasin B reduces the efficiency of sheep SCNT. Overall, given the pronounced long-term effect of cytochalasin B on oocytes, the results and patterns identified in this study can be considered preliminary, as further research remains relevant both for obtaining embryos at later developmental stages and for assessing their quality.

Additional information

Authors' contributions. A.V. Lopukhov — review and analysis of literature sources, participation in experiments, processing and analysis of the obtained results, preparation of the article for publication; E.N. Shedova — participation in experiments; E.V. Tsyndrina — participation in experiments; G.N. Singina — general management, participation in experiments, preparation of the article for publication. All authors approved the manuscript (version for publication) and agreed to take responsibility for all aspects of the work, ensuring the proper consideration and resolution of any issues related to the accuracy and integrity of any part of the study.

Ethics approval. Not applicable.

Funding sources. This work was financially supported by the Ministry of Science and Higher Education of the Russian Federation (State assignment No. FGGN-2024–0014).

Disclosure of interests. The authors declare no relationships, activities, or interests over the past three years involving third parties (commercial or non-commercial) whose interests could be affected by the content of this article.

Statement of originality. The authors did not use any previously published material (text, illustrations, or data) in the preparation of this work.

Data availability statement. All data obtained in the present study are available in the article.

Generative AI. No generative artificial intelligence technologies were used in the preparation of this article.

Provenance and peer-review. This work was submitted to the journal on the authors’ initiative and underwent the standard review process. The manuscript was reviewed by three members of the editorial board and the journal’s scientific editor.

About the authors

Alexandr V. Lopukhov

Federal Research Center for Animal Husbandry named after Academy Member L.K. Ernst

Author for correspondence.
Email: vubi_myaso@mail.ru
ORCID iD: 0000-0002-1284-1486
SPIN-code: 1092-2523
Russian Federation, Village Dubrovitsy, Podolsk, Moscow Region

Ekaterina N. Shedova

Federal Research Center for Animal Husbandry named after Academy Member L.K. Ernst

Email: shedvek@yandex.ru
ORCID iD: 0000-0002-9642-2384
SPIN-code: 1067-8115
Russian Federation, Village Dubrovitsy, Podolsk, Moscow Region

Evgeniya V. Tsyndrina

Federal Research Center for Animal Husbandry named after Academy Member L.K. Ernst

Email: kiril04kina@yandex.ru
ORCID iD: 0000-0002-3263-2358
Russian Federation, Village Dubrovitsy, Podolsk, Moscow Region

Galina N. Singina

Federal Research Center for Animal Husbandry named after Academy Member L.K. Ernst

Email: g_singina@mail.ru
ORCID iD: 0000-0003-0198-9757
SPIN-code: 4118-2990

Cand. Sci. (Biology)

Russian Federation, Village Dubrovitsy, Podolsk, Moscow Region

References

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