Methods of karyoplast cell cycle synchronization for increasing the efficiency of somatic cloning of farm animals

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INTRODUCTION

Somatic cell nuclear transfer (SCNT) involves the production of multicellular organisms using oocytes whose genetic material is replaced by the contents of donor cell nuclei (karyoplasts). In 1952, the first cloned animals were produced from blastocyst stage cells in amphibians [1]. This was followed by studies using mammals of different species and the use of early embryonic blastomeres as karyoplasts [2]. Then, in 1996, the first cloned offspring using somatic cells from an adult animal was obtained [3]. Currently, several mammalian species, including monkeys, have been cloned by SCNT [2]. This method has potential applications in various fields of agriculture and medicine with proven therapeutic potential: production of genetically modified animals [4], reproduction of valuable and conservation of endangered species, and derivation of embryonic stem cells [5]. Despite the demand for this procedure, its efficiency remains low [6–8]. This is largely explained by the fact that SCNT includes a set of consecutive stages and suboptimal conditions, at any of which can have a significant negative impact on the development of cloned embryos and offspring. Moreover, in a number of cases, recipient animals show disorders of placenta formation, and cloned offspring often die from lethal malformations [9]. The main stages of cloning include the preparation of oocytes and somatic cells, enucleation of mature oocytes (cytoplasts) followed by obtaining oocyte–somatic cell complexes, fusion of cytoplasts with karyoplasts, activation of the resulting cytohybrids, and culturing and transplantation of the embryos [10, 11]. After numerous studies conducted to obtain cloned animals, considerable experience has been accumulated to improve SCNT technology. The modern ways to improve the efficiency of cloning include optimization of each of its stages [11–23]. Most published studies reflect advancements in oocyte research, including the modernization of maturation conditions to decelerate aging processes, mitigate apoptotic alterations, and generate reactive oxygen species [13, 21]. Additionally, advancements have been made in enucleation techniques, with the introduction of robotic approaches [19, 23]. A study on methods to restore cytoplasts after enucleation, including the injection of cytoplasm from donor oocytes, is underway [14]. Furthermore, research is being conducted to regulate mitochondrial DNA inheritance [17]. Of particular significance are studies aimed at enhancing the implantation of cloned embryos at the embryological stage [15] and stage of work with recipient animals. Notably, the conditions optimal for SCNT in one animal species may not be suitable for another species.

The critical event that determines the efficacy of SCNT is the reprogramming of the nuclear material of the karyoplast by the oocyte cytoplasm. This process results in totipotency of the cells [24]. During somatic cell differentiation from stem cells, methylation of specific genome regions occurs, which are responsible for binding to transcription factors that maintain the pluripotent state [25]. Despite the stability of the epigenetic status of somatic cells, including terminally differentiated cells, methylation is not irreversibly fixed; rather, it can be reprogrammed to the embryonic state [26]. The reprogramming process entails epigenetic and metabolic remodeling; however, the molecular regulation underlying this phenomenon remains unclear [27]. The developmental potential of cloned embryos is determined by the capacity of the somatic cell nucleus to be reprogrammed by factors present in the oocyte cytoplasm. Following the fusion of an enucleated oocyte and a donor cell, changes take place. These include the destruction of the nuclear envelope of the karyoplast and premature condensation of chromosomes. This process is mediated by a high concentration of maturation-promoting factor (MPF), a p34^cdc2-cyclin B complex, which is found in the oocyte cytoplasm [28–31]. Moreover, subsequent DNA replication occurs. The replacement of several chromatin structural components with the corresponding components of oocyte origin, which occurs after fusion of the karyoplast and cytoplast membranes, is crucial in the removal of epigenetic marks from DNA, which in turn allows for the reprogramming of donor nuclei into a totipotent state [32–34]. These rearrangements are critical for chromatin decondensation, which is essential for the enhanced binding of transcription regulators to their recognition sites and more efficient reprogramming [35]. Consequently, the intensity of expression and the level of phosphorylation of pivotal histones and chaperones, the degree of acetylation and methylation of oocyte histones, and other factors influence the efficiency of reprogramming [36]. To enhance this index, researchers employ a range of strategies, including the use of inhibitors of DNA deacetylases [37, 38] and methyltransferases [39], induction of demethylase overexpression [40], and correction of aberrant DNA remethylation [41]. However, the cell cycle of the donor somatic cell should be coordinated with that of the recipient oocyte. If not, it may result in chromosome damage due to premature condensation and premature destruction of the nuclear envelope in the donor cells, which are in an inappropriate phase [42, 43].

Studies conducted to identify the optimal stage of the karyoplast cell cycle indicate that embryo development is observed when somatic cells in G0, G1, G2, and M phases are used [44, 45]. Considering that the majority of experiments on the production of cloned animals use oocytes in the MII phase of meiosis (owing to the elevated MPF activity) as recipient cells [46] without prior activation, it is recommended that cells with a diploid nucleus in the G0/G1 phase, awaiting replication, be employed for transfer [31, 47]. Various studies demonstrated that SCNT using somatic cells in this phase as a karyoplast is more efficient [48]. The transfer of cells in the G2 phase results in impaired embryo development because of the replication of the genome, which is directed by the oocyte cytoplasm. The use of karyoplast in the S phase causes chromosome fragmentation [42]. If the cell cycle of the recipient cytoplasm enters the G2/M phase when the karyoplast is in the G1/S phase, premature chromosome condensation and nuclear envelope disintegration may be initiated while DNA synthesis is not yet complete, resulting in chromosome loss and aneuploidy [49].

The arrest of cell proliferation in the G0/G1 phase can be achieved through several methods, the most prevalent of which are serum starvation (culturing somatic cells in a medium containing an extremely low concentration of fetal bovine serum — FBS) [50–53], contact inhibition (culturing somatic cells to a monolayer with a confluency approaching 100%) [52–55], and a combination of these techniques [43, 56]. Many researchers employ chemical inhibitors of the cell cycle [57, 58].

SERUM STARVATION

Serum starvation is an effective method for the reversible arrest of somatic cells in the G0/G1 phase. Numerous studies have shown that the cultivation of somatic cells in a medium with an extremely low FBS content (0.2–0.5%) results in the arrest of 70–90% of these cells in the aforementioned phase of the cycle [43, 58–64]. This rate increases with the duration of cell cultivation in conditions of reduced FBS content [43, 59, 61, 65]. Furthermore, in a study by Sadeghian-Nodoushan et al., the proportion of sheep granulosa cells in the G0/G1 phase increased significantly when cultured for 48 and 72 h compared to 24 h under serum starvation conditions [61]. Restoration of serum content in the medium to values sufficient for active proliferation (15–20%) resulted in a notable increase in the number of cells in the S and G2/M phases [59, 66].

Despite the widespread use of this methodology to synchronize the cell cycle of karyoplasts during animal cloning, it should be considered that the cultivation of somatic cells in a medium with reduced serum content for 48 h results in damage to the endoplasmic reticulum [67]. Furthermore, extended cultivation periods ≥72 h result in increased production of reactive oxygen species [57], alterations in mitochondrial morphology [68], increased DNA fragmentation [59], and increased proportion of apoptotic cells [60, 62, 69]. A consequence of apoptosis induction is a change in the membrane potential of cells [70], which can lead to decreased efficiency of electrospraying in cytohybrid production [71]. However, the results of [72] indicate that the use of annexin-positive and annexin-negative cells as a karyoplast does not significantly affect the number of cytohybrids obtained.

The negative effect of using karyoplasts at early apoptosis on the efficiency of SCNT is demonstrated by the results obtained by Miranda et al. According to their study, the use of annexin-positive fibroblasts as donor cells in SCNT reduces the probability of resulting embryos to develop, leads to a reduction in the number of cells in blastocysts, and increases the number of apoptotic cells [72]. In this context, a statistically significant increase in apoptosis during cycle synchronization by serum starvation is observed when somatic cells are cultured in medium containing reduced FBS for 48 and 72 h. Conversely, serum starvation for 24 h does not cause a significant increase in the number of apoptotic cells in culture when compared to cells cultured in medium containing 10% FBS [61].

The low developmental potential of SCNT-derived embryos may be attributed to somatic cell damage caused by culturing under serum starvation conditions. A study conducted by Park et al. demonstrated that the number of porcine embryos developing to the blastocyst stage was lower when karyoplasts were synchronized by serum starvation for 72 h compared to other cell cycle arrest methods [69]. In another study, the number of bovine blastocysts obtained by the SCNT method using karyoplasts cultured for 24 h in a medium containing 0.5% FBS was higher than the corresponding index when fibroblasts were synchronized by the contact inhibition method [65]. These results confirm that an extended duration of karyoplast cultivation under serum starvation conditions negatively affects their condition, which in turn reduces the efficiency of SCNT.

CONTACT INHIBITION

Currently, contact inhibition is the second most prevalent method of inhibiting proliferative activity [54, 73–76]. The formation of a dense monolayer results in an increase in the surface contact area of neighboring cells, which then causes cell cycle inhibition despite sufficient concentration of nutrients and growth factors in the culture medium [77]. Cell arrest in the G0/G1 phase occurs as a result of increased expression of p27Kip1, an inhibitor of cyclin-dependent kinase (CDK) [78] that are involved in cell division, and through the dissociation of cyclin D from the complex with CDK4 [79], which subsequently becomes inactivated. Importantly, contact inhibition reduces the level of intracellular production of reactive oxygen species compared to cells growing at lower densities [80], and activation of PGC1α, a key regulator of energy metabolism involved in reducing the concentration of reactive oxygen species and protecting cells from oxidative stress, is observed [81]. The use of this method allows 65–84% of cells to be arrested in the G0/G1 phase of the cycle [61–63]. Notably, the level of apoptosis in somatic cells synchronized by the contact inhibition method is comparable to that in actively dividing cells [61, 63]. A limitation in the application of this method is that, in some cases, it is not possible to achieve monolayer confluence sufficient for contact inhibition to occur, because individual cultures of cells used as karyoplasts in the SCNT procedure (e.g., after genome editing) may have low proliferative activity.

COMBINED METHOD

Several researchers have shown that somatic cell culturing to a monolayer, followed by the replacement of the culture medium with one containing a reduced FBS content, cab be used to increase the number of cells present in the G0/G1 phase of the cell cycle. Accordingly, the findings of Ma et al. indicate that the proportion of sheep fibroblasts in this phase was significantly higher in the combined method of synchronization than in contact inhibition and serum starvation: 80.07% vs. 66.82% and 71.24%, respectively [82]. However, a study by Gómez et al. comparing methods of cell cycle synchronization of bovine fibroblasts (serum starvation, contact inhibition, and a combined method) did not reveal significant differences in the number of fibroblasts in the G0/G1 phase [65]. The discrepancies may be attributed to the varying durations of cell cultivation under the specified conditions and potential influence of the species of animals from which the cells were obtained. There is evidence indicating that somatic cells from different animal species may exhibit varying susceptibility to the same methods of cycle synchronization [83, 84].

Moreover, the efficiency of somatic cell synchronization is determined by the type of cells used as karyoplast: the results of the study conducted by Yagcioglu et al. in sheep show that fetal and adult fibroblasts respond differently to cell cycle synchronization [63].

A CHEMICAL WAY TO SYNCHRONIZE THE CELL CYCLE OF KARYOPLASTS

The cell cycle is a unidirectional process in which a cell passes sequentially through its various morphologically and biochemically distinct phases, without skipping or returning to previous phases. The cycle is generally divided into the interphase, including the synthetic (S) period when chromosome doubling occurs, and the intervals separating it from division. These are the G1 and G2 phases. In the G1 phase, proteins are synthesized, organelles are amplified, and cell size increases. In the G2 phase, the correctness of DNA replication is analyzed, and mitosis is prepared. Cells in the G1 phase can exit the cell cycle and enter the G0 phase, which is a reversible quiescent state [85]. Cell progression through the phases of the cell cycle is regulated by the ability of activated CDKs to phosphorylate specific substrates, leading to their activation, inactivation, and localization in the cell. CDK activation occurs when they bind to cyclins, the concentration and activity of which is determined by the phase of the cell cycle [85, 86].

Based on underlying cell progression through the phases of the cell cycle, CDK inhibitors are used to stop them at a specific phase (roscovitine*, butyrolactone I*, statins: lovastatin*, mevastatin*), inhibitors of DNA replication (aphidicolin*, mimosine*, statins), microtubule polymerization (nocodazole*, colchicine) [87], and synthesis of essential proteins (cycloheximide*) [88]. The main principles of somatic cell cycle synchronization for SCNT are its reversibility and absence of toxic effects. Therefore, it should be considered that the effect of a number of inhibitors, in addition to stopping cells in the necessary phase of the cycle, often results in apoptosis. Table 1 [57–61, 63, 69, 89–93] presents information on chemical inhibitors of the cell cycle used for synchronization of donor cells in SCNT of farm animals.

Table 1. Inhibitors used for synchronizing the karyoplasts cell cycle during somatic cloning of farm animals

According to the literature, among the drugs for somatic cell synchronization used as karyoplast in SCNT, roscovitine is most often used. This drug promotes reversible cell arrest in the G0/G1 phase by inhibiting CDK1, CDK2, CDK5, CDK7, CDK9, and MPF [65, 94–96]. Various studies have shown that the number of fibroblasts in the G0/G1 phase when roscovitine is added to the culture medium is equivalent to the corresponding indices when cells are synchronized by contact inhibition [60, 63, 69]. The study by Selokar et al. shows a higher concentration of fetal fibroblasts in the G0/G1 stage when cultured for 24 h in medium containing 30 μM roscovitine compared to the corresponding indicators in the groups where cycle synchronization was performed by contact inhibition or serum starvation methods [92]. This index was comparable to that of the combined application of contact inhibition and serum starvation methods.

Data regarding the protective effect of roscovitine on cells are inconsistent: its addition to the culture medium of sheep, fetal pig, and fetal fibroblasts led to a decrease in the number of cells in the apoptotic state [60, 63, 69]. However, incubation of puma fibroblasts in the medium containing 15 μM roscovitine did not have a significant effect on this index [62]. Additionally, the use of fibroblasts cultured in the presence of roscovitine as karyoplasts for SCNT resulted in an increase in the relative proportion of embryos that reached the blastocyst stage [69, 92]. In a study by Hwang et al., the number of recipient gestation pigs was higher in the group where fibroblasts cultured in the presence of roscovitine were used as donor cells for SCNT [91]. Accordingly, most studies indicate that the use of this inhibitor to inhibit the proliferation of somatic cells in the G0/G1 phase of the cell cycle is not superior to the mechanisms of contact inhibition and serum starvation. Nevertheless, the extant data on the protective impact of roscovitine on somatic cells and enhanced production of blastocysts when karyoplasts are cultured in its presence substantiate the employment of this inhibitor as a method of somatic cell synchronization in SCNT.

Rapamycin is a macrolide of bacterial origin that has been found to regulate cell growth, control protein translation, influence energy metabolism, and regulate cytoskeleton formation by inhibiting the mTOR target (mammalian target of rapamycin) [97]. Rapamycin plays a role in maintaining cellular physiology by inducing autophagy, which is crucial for the timely removal of aged proteins, damaged organelles, toxic metabolic products, and peroxisomes from the cell [98, 99]. It has been demonstrated to reduce the expression of cyclin D1 [100–102] and genes responsible for the synthesis of glucose and amino acid transporters [58], inhibiting cell proliferation and the cell cycle in the G0/G1 phase. The findings of a study conducted by Hyun et al. indicate that culturing pig fibroblasts in a medium containing 1 μM rapamycin for a period of 3 days resulted in a notable increase in the proportion of cells in the G0/G1 phase, reaching 93.6% compared to 61.0% in the control group. The authors observed that when these cells were used as a karyoplast, the number of fractured embryos and embryos that developed to the blastocyst stage was significantly higher than that in the control group. Conversely, DNA fragmentation was less pronounced, and the expression of development-related genes (CDX2, CDH1) was higher in the rapamycin group than in the control group [58]. A study conducted on buffaloes yielded comparable outcomes. The content of fibroblasts in the G0/G1 phase was 90% when cultured in a medium containing rapamycin. The number of fibroblasts with a normal karyotype was higher, whereas the number of apoptotic cells and the level of reactive oxygen species production were lower in comparison to the group of cells exposed to serum starvation [57].

A study on fetal piglet fibroblasts [59] demonstrated that butyrolactone I can stop cells in the G0/G1 phase of the cycle by inhibiting CDK1, CDK2, CDK5, and CDC2 [103, 104]. This was achieved by inhibiting the activity of these enzymes, which regulate the cell cycle. The effect of this drug on cell viability is inconclusive. For example, the study of butyrolactone I on tumor cells demonstrated its ability to induce apoptosis [105, 106], whereas the study on granular neurons did not reveal a decrease in their viability [107].

The plant amino acid mimosine has an inhibitory effect on the initiation of DNA replication and is able to arrest cells in the G1 or S phase of the cell cycle in a dose-dependent manner. Application of mimosine at a concentration of 0.5 mM arrested somatic cells in G1 phase, whereas lower concentrations (0.1–0.2 mM) did not prevent entry into S phase [108, 109]. According to the results of these studies, the addition of mimosine to the culture medium allows the arrest of 85.2% of sheep granulosa cells [61] and 85.7% of porcine granulosa cells [93] in the G0/G1 phase, confirming its efficacy in synchronizing karyoplasts during SCNT.

Cell cycle inhibitors with conflicting data include aphidicolin and nocodazole. Aphidicolin is a reversible inhibitor of DNA polymerases. It arrests cells in the early S phase [110]. However, according to the results of the study by Kues et al., the cultivation of porcine fetal fibroblasts in medium containing aphidicolin resulted in a cell concentration of 82% in the G0/G1 phase, whereas only 3.9% of the fibroblasts stopped in the S phase [59]. Additionally, the inconsistency of the available data on the inhibitory activity of nocodazole is highlighted. The findings of Sadeghian-Nodoushan et al. indicate that 74.6% of sheep granulosa cells were in the G0/G1 stage when cultured in the presence of nocodazole [61]. In contrast, another study demonstrated that the sequential application of aphidicolin and nocodazole could effectively synchronize the cell cycle of porcine cells [111]. The use of cine-induced pluripotent stem cells permitted the cessation of 77.6% of cells on day 2 of culturing and 72.2% on day 3, which was observed to be in the G2/M phase. Conversely, the number of cells in the G0/G1 phase was found to be only 9.4 and 11.3% on days 2 and 3, respectively. In this context, nocodazole induces its effect by disrupting microtubule polymerization, which contributes to cell arrest in the G2/M phase [112]. Therefore, this drug is not recommended for the synchronization of somatic cells for their use as karyoplast in SCNT.

CONCLUSION

The success of cloning, in conjunction with the physiological state and viability of the recipient oocyte, largely determines the potential for somatic cell nuclear material reprogramming. It has been demonstrated that karyoplasts at the G0/G1 stage of the cell cycle are most capable of reprogramming. Therefore, selecting optimal conditions for somatic cell cultivation that ensure a high level of synchronization of their cell cycle enhances the efficiency of somatic cloning. Despite various existing methods for cell arrest in the G0/G1 phase of the cell cycle, there is currently no consensus on the most optimal method. In selecting a method of cell cycle synchronization, it is crucial to consider the species of animal from which the somatic cells are derived and the permitted duration of cell cultivation under conditions that induce cell cycle arrest. The impact of the selected method on cell viability and the induction of apoptotic changes in cells is of significant interest. The investigation of the influence of chemical inhibitors on the cell cycle of karyoplasts is constrained to the evaluation of cell content within a specific phase of the cycle. However, the majority of research is directed toward data pertaining to the number of embryos obtained, their engraftment, and the subsequent birth of viable offspring.

ADDITIONAL INFORMATION

Funding source. This work was supported by the Ministry of Science and Higher Education of the Russian Federation (State assignment FGGN-2024-0014).

Competing interests. The author declares that there are no competing interests.

* hereinafter, the drug is not registered in the Russian State Register of Medicines.

About the authors

Anastasia S. Zhukova

Author for correspondence.
Email: anastasia.s.belyaeva@gmail.com
ORCID iD: 0000-0003-1155-014X
SPIN-code: 4590-3971
Scopus Author ID: 57200071274

Cand. Sci. (Biology)

References

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