Lycopene enhances epigenetic reprogramming and zygotic genome activation in the porcine somatic cell nuclear transfer embryo

Abstract

Pigs are valuable models for human disease research due to their physiological similarities to humans, and somatic cell nuclear transfer (SCNT) is commonly used to generate such models. However, SCNT efficiency is limited by incomplete epigenetic reprogramming and insufficient zygotic genome activation (ZGA). Lycopene, a potent antioxidant carotenoid, was investigated for its potential to improve porcine embryo development during in vitro culture (IVC). Parthenogenetically activated (PA) and SCNT embryos were cultured with various lycopene concentrations, with 0.2 µM showing the most significant benefits. Lycopene treatment significantly improved 4–5-cell cleavage, blastocyst formation, trophectoderm, and total cell numbers, while reducing apoptosis. It also decreased reactive oxygen species (ROS), upregulated the expression of antioxidant enzyme-related genes (CAT, SOD1, SOD2, and HO-1), and increased mitochondrial membrane potential and autophagy in 4-cell embryos. Epigenetically, lycopene reduced H3K4me3, H3K9me3, and 5mC levels and downregulated methyltransferase-related genes (ASH2L, SUV39H2, DNMT1, DNMT3A, and DNMT3B), while upregulating ZGA-related genes (ZSCAN4, UBTFL1, SUPT4H1, MYC, and ELOA). These findings suggest that lycopene treatment during IVC enhances embryonic development by reducing ROS-related mitochondrial dysfunction, inducing autophagy, and improving nuclear reprogramming, thereby improving ZGA in porcine SCNT and PA embryos.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-15672-8.

Introduction

Pigs are similar to humans in their anatomical and physiological features and genetic makeup¹, making them valuable animal disease models and potentially replacing the need for human subjects^2,3. Genetically engineered animals produced through somatic cell nuclear transfer (SCNT) are increasingly utilized in the livestock industry and biomedical research^4,5. The SCNT technique reprograms a somatic cell to a totipotent state by transferring its nucleus into an enucleated oocyte⁶. However, despite the advantages of cloning, its efficiency remains low due to incomplete epigenetic reprogramming^7–9 and the insufficient understanding of ZGA regulation in SCNT embryos¹⁰. ZGA initiates gene expression after fertilization, facilitating the transition from maternal to embryonic genes, and is crucial for preimplantation embryo development¹¹. Through this transition, the embryo becomes totipotent, acquiring the developmental potential to differentiate into any cell lineage¹². Several important factors influence ZGA, including autophagy, which promotes ZGA by degrading maternal transcripts¹³; epigenetic reprogramming, which facilitates the transition to embryonic gene expression¹⁴; and oxidative stress, which disrupts ZGA through cellular damage¹⁵.

Reactive oxygen species (ROS) such as superoxide anions (O₂^–), hydroxyl radicals (OH), and hydrogen peroxide (H₂O₂) are metabolic by-products involved in the regulation of key cellular processes such as proliferation and differentiation¹⁶. However, high ROS levels during in vitro culture (IVC) and SCNT may worsen developmental arrest by interfering with essential reprogramming processes^10,17,18. Porcine embryos undergoing ZGA are particularly sensitive to oxidative stress at the 4-cell stage, often resulting in developmental arrest¹⁹.

Autophagy contributes to the degradation of maternal proteins and cytoplasmic content remodeling, which facilitates proper reprogramming during the oocyte-to-zygote transition^13,20. Inducing autophagy in SCNT embryos enhances their viability, highlighting the critical role of SCNT in preimplantation development²¹. Autophagy appears to play a crucial role in cellular reprogramming by regulating DNA and histone methylation, which are essential for the proper development of cloned embryos²². Moreover, mitochondrial activity during ZGA reprogramming is critical for providing the metabolites necessary for protein and DNA demethylation²³.

Histone modifications critically regulate gene expression during ZGA, while erasing parental DNA methylation patterns in the embryo facilitates the initiation of a new developmental program²⁴. However, mammalian SCNT embryos exhibit abnormal transcriptomic reprogramming compared to IVF embryos during ZGA²⁵. Excessive DNA methylation in SCNT embryos has been specifically linked to reduced developmental potential during the preimplantation stages^26,27. Additionally, the acetylation and methylation of histone 3 lysine 9 (H3K9) are key processes in the epigenetic reprogramming of embryos, such that incomplete H3K9 demethylation restricts the developmental potential of cloned embryos¹⁰. In contrast, H3K9 acetylation may be critical for somatic cell reprogramming²⁸, as it promotes the development of SCNT embryos. Although histone H3 lysine 4 (H3K4) methylation is a transcriptional activator²⁹, excessive accumulation by donor cells interferes with ZGA³⁰. Some studies have shown that antioxidants such as melatonin, vitamin C, and astaxanthin improve epigenetic regulation and ZGA in SCNT embryos^31–33. These findings highlight the need to identify potent antioxidants that enhance reprogramming efficiency and ZGA.

Carotenoids are powerful antioxidants with a remarkable ability to neutralize ROS, particularly singlet oxygen³⁴. Lycopene is a red carotenoid pigment with strong antioxidant-free radical scavenging activity and is the most efficient in vitro singlet oxygen scavenger among carotenoids³⁵. Many studies have suggested that treating various cells with lycopene prevents oxidative stress-induced DNA damage^36,37 and improves mammalian preimplantation development and embryo quality^38,39. However, the effect of lycopene on porcine SCNT embryos remains unclear.

Therefore, in this study, we assessed whether lycopene treatment during IVC enhances the development of parthenogenetically activated (PA) and SCNT porcine embryos, focusing on its impact on epigenetic modifications. We examined the effects of lycopene on ROS levels, mitochondrial function, autophagy, and subsequent key epigenetic changes, including H3K4 tri-methylation, H3K9 tri-methylation, and DNA methylation. Finally, we determined whether activating organelles and epigenetic changes can promote embryo reprogramming and successfully induce ZGA. The findings of this study could provide valuable insights into improving SCNT efficiency and embryo quality in porcine models, with potential applications in both livestock breeding and biomedical research.

Results

Effects of lycopene on the development of porcine PA embryos

To investigate the effects of lycopene on in vitro development of preimplantation porcine embryos, PA embryos were cultured in post-activation and IVC medium containing 0, 0.02, 0.2, or 2 µM lycopene for the entire IVC period. Lycopene supplementation improved the proportion of 4–5 cell cleavage stage compared to the control, although there were no significant differences in the overall cleavage rate among all groups on day 2. The blastocyst rate in the 0.2 µM lycopene treatment group improved significantly but no differences were detected in the 0.02 µM or 2 µM lycopene treatment groups, compared to the control group (Fig. 1A–D). The number of apoptotic cells decreased significantly in the 0.2 µM lycopene treatment group but no significant difference was observed in the other groups compared to the control group. Similarly, the apoptosis rate decreased significantly in the 0.2 µM lycopene treatment group, but no changes were detected in the 0.02 µM and 2 µM groups (Fig. 1E–G). Total cell numbers increased significantly in the 0.2 µM lycopene treatment group, whereas no differences were detected in the 0.02 µM and 2 µM lycopene treatment groups compared to the control group. A significant increase in the number of trophectoderm (TE) cells was detected in the 0.2 µM lycopene treatment group compared to the other groups. However, no differences in the numbers of inner cell mass (ICM) cells were observed did not differ among the groups (Fig. 1H–J).

Fig. 1.

Effects of lycopene on the development of porcine PA embryos. (A) Representative morphology of day-6 PA embryos in control and 0.02, 0.2, and 2 µM lycopene treatment groups for the entire duration of IVC. (B) Overall cleavage rate, (C) cleavage stage distribution among cleaved embryos and (D) blastocyst formation rates in control and 0.02, 0.2, and 2 µM lycopene treatment groups. At least 114 embryos from each group were used for analysis. (E) Representative images of terminal deoxynucleotidyl transferase-mediated dUTP-digoxygenin nick end-labeling (TUNEL), PA blastocyst staining, (F) apoptotic cell quantification, and (G) blastocyst apoptosis rates in each group. At least 13 blastocysts in each group were analyzed. (H) Representative morphology of PA blastocysts; (I) total cell numbers; (J) ICM and TE cell numbers of blastocysts in the indicated groups. At least 10 blastocysts from each group were analyzed. Superscript letters (a, b) denote significant differences (P < 0.05). Bar = 100 μm.

Effects of lycopene on the development of porcine SCNT embryos

To determine whether lycopene exerts similar effects in porcine SCNT embryos, we cultured them with or without 0.2 µM lycopene for the same duration as the PA embryos. Although the total cleavage rate did not differ significantly between the control and 0.2 µM lycopene groups, supplementation with 0.2 µM lycopene significantly improved both the proportion of 4–5-cell cleavage stage on day 2 and the blastocyst formation rate on day 6 (Fig. 2A–D). The apoptosis rate and the number of apoptotic cells decreased significantly in the 0.2 µM lycopene treatment group compared to the control group (Fig. 2E–G). Total cell numbers were also significantly higher in this group. Moreover, the number of TE cells increased significantly, while no difference was observed in the number of ICM cells across groups (Fig. 2H–J).

Fig. 2.

Effects of lycopene on the development of porcine SCNT embryos. (A) Representative morphology of day 2 and day 6 SCNT embryos in the control and 0.2 µM lycopene treatment groups for the entire duration of IVC. (B) Overall cleavage rate, (C) cleavage stage distribution among cleaved embryos and (D) blastocyst formation rates in the control and 0.2 µM lycopene treatment groups. At least 242 embryos from each group were analyzed. (E) Representative TUNEL images of SCNT blastocyst staining, (F) quantification of apoptotic cells, and (G) apoptosis of blastocysts in the control and the 0.2 μM lycopene treatment groups. At least 17 blastocysts from each group were analyzed. (H) Representative morphology of SCNT blastocysts; (I) total cell numbers; (J) ICM and TE cell numbers of blastocysts in the control and the 0.2 μM lycopene treatment groups. At least 23 blastocysts from each group were analyzed. Superscript letters (a, b) denote significant differences (P < 0.05). Bar = 100 μm.

Effects of lycopene on intracellular ROS and antioxidant enzyme-related gene expression levels in porcine SCNT embryos

To investigate the antioxidative effect of lycopene in porcine embryos, we analyzed intracellular ROS levels and the expression of genes related to antioxidant enzymes. Intracellular ROS in SCNT 4-cell embryos and blastocysts treated with 0.2 µM lycopene was significantly lower than that in the corresponding control groups (Fig. 3A, B, D). The mRNA expression levels of antioxidant enzymes and apoptosis-related genes in 4-cell embryos and blastocysts cultured with or without 0.2 µM lycopene were analyzed by qPCR. Supplementing with 0.2 µM lycopene significantly increased the expression levels of CAT, SOD1, SOD2, and HO-1 (antioxidant enzyme-related mRNAs), whereas the BAX/BCL2L ratio (apoptosis-related mRNAs) decreased compared to the control group (Fig. 3C, E). Similar to SCNT embryos, PA 4-cell embryos and blastocysts treated with 0.2 µM lycopene showed significantly lower intracellular ROS levels, along with comparable changes in antioxidant and apoptosis-related mRNA expression, compared to their respective control groups (Supplementary Fig. 1).

Fig. 3.

Effects of lycopene on intracellular ROS, mitochondrial membrane potential, and autophagy in porcine SCNT embryos. (A) Representative fluorescence images of CM-H2DCFDA staining of SCNT 4-cell embryos and blastocysts in the control and 0.2 μM lycopene treatment groups. (B) Quantification of fluorescence intensity and (C) expression levels of antioxidant enzyme-related genes (CAT, SOD1, SOD2, and HO-1) and apoptosis-related genes (BAX and BCL2L) in SCNT 4-cell embryos treated with or without 0.2 μM lycopene. (D) Quantification of fluorescence intensity and (E) expression levels of antioxidant enzyme and apoptosis-related genes in SCNT blastocysts treated with or without 0.2 μM lycopene. At least 30 4-cell embryos and 16 blastocysts from each group were analyzed. (F) Representative JC-1 staining fluorescence images and (G) quantification of the ratio of fluorescence intensity (red/green) of SCNT 4-cell embryos in the control and 0.2 μM lycopene treatment groups. At least 45 embryos from each group were analyzed. (H) Representative fluorescence images of autophagy staining and (I) quantification of fluorescence intensity in SCNT 4-cell embryos treated with or without 0.2 μM lycopene. At least 30 embryos from each group were analyzed. Superscript letters (a, b) denote significant differences (P < 0.05). Bar = 100 μm.

Effects of lycopene on mitochondrial membrane potential and autophagy in porcine SCNT 4-cell embryos

To evaluate the factors that improve the development of early-stage embryos by lycopene treatment, we investigated mitochondrial membrane potential using JC-1 assay and autophagy activity. The JC-1 fluorescent intensity red/green ratio increased significantly in the 0.2 µM lycopene treatment groups compared to the controls (Fig. 3F, G). To investigate the effects of lycopene on autophagy in SCNT 4-cell embryos, both groups were analyzed for green fluorescence in the autophagy assay; the level of fluorescence intensity increased in response to the 0.2 µM lycopene treatment (Fig. 3H, I). Consistent with these findings, PA 4-cell embryos also exhibited an increase in mitochondrial membrane potential and autophagy (Supplementary Fig. 1).

Effects of lycopene on epigenetic modifications, such as histone and DNA methylation, in porcine SCNT 4-cell embryos and blastocysts

To examine whether lycopene affects epigenetic modifications in porcine 4-cell embryos and blastocysts, immunocytochemical staining was performed to assess fluorescence signals. In porcine SCNT blastocysts, the H3K4me3 level decreased significantly in the 0.2 µM lycopene group compared to the controls, whereas no significant change was detected in 4-cell embryos (Fig. 4A–C). In PA embryos, H3K4me3 levels of 4-cell embryos and blastocysts decreased significantly by 0.2 µM lycopene treatment (Fig. S2A–C). The H3K9me3 levels significantly decreased in both porcine SCNT and PA 4-cell embryos and blastocysts (Fig. 4D–F, Supplementary Fig. 2). In contrast, H3K9ac levels increased in porcine blastocysts from both SCNT and PA embryos, with a significant enhancement also observed in PA 4-cell embryos (Supplementary Fig. 3). Treatment with 0.2 µM lycopene also significantly decreased the 5mC levels in SCNT 4-cell embryos and blastocysts (Fig. 4G–I). Similarly, in PA embryos, a reduction in 5mC levels was observed in blastocysts, while no significant change was detected in 4-cell embryos (Fig. 2G–I). In addition, the transcript levels of histone methyltransferase-related genes (ASH2L and SUV39H2) and DNA methyltransferase-related genes (DNMT1, DNMT3A, and DNMT3B) were significantly decreased in SCNT 4-cell embryos and blastocysts following 0.2 µM lycopene treatment (Fig. 4J, K). Consistent with these findings, PA embryos exhibited similar downregulation of these epigenetic regulator genes at both developmental stages (Supplementary Fig. 2).

Fig. 4.

Effects of lycopene on epigenetic modifications in porcine SCNT 4-cell embryos and blastocysts. (A) Representative fluorescence images of H3K4me3 staining of SCNT 4-cell embryos and blastocysts, analyzed with and without 0.2 μM lycopene. (B) Quantification of fluorescence intensity in 4-cell embryos and (C) blastocysts treated with or without 0.2 μM lycopene. At least 23 4-cell embryos and 20 blastocysts from each group were analyzed. (D) Representative fluorescence images of H3K9me3 staining of SCNT 4-cell embryos and blastocysts, analyzed with or without 0.2 μM lycopene. (E) Quantification of fluorescence intensity in 4-cell embryos and (F) blastocysts treated with or without 0.2 μM lycopene. At least 15 4-cell embryos and 18 blastocysts from each group were analyzed. (G) Representative fluorescence images of 5mC staining of SCNT 4-cell embryos and blastocysts, analyzed with or without 0.2 μM lycopene. (H) Quantification of fluorescence intensity in 4-cell embryos and (I) blastocysts treated with or without 0.2 μM lycopene. At least 20 4-cell embryos and blastocysts from each group were analyzed. (J) Quantification of the expression levels of histone methyltransferase-related genes (ASH2L and SUV39H2) and DNA methyltransferase-related genes (DNMT1, DNMT3A, and DNMT3B) in 4-cell embryos and (K) blastocysts from the control and 0.2 μM lycopene treatment groups. Superscript letters (a, b) denote significant differences (P < 0.05). Bar = 100 μm.

Effects of lycopene on ZGA-related gene expression levels in porcine embryos

To evaluate the effect of lycopene on the ZGA in porcine PA and SCNT embryos, the expression levels of ZGA-related mRNAs in 4-cell embryos were quantified by qPCR. In SCNT 4-cell embryos, the relative mRNA levels of ZSCAN4, UBTFL1, SUPT4H1, MYC, and ELOA were higher in the 0.2 µM lycopene treatment group than in the control group; MYC had more than a two-fold increase (Fig. 5A). Similarly, in PA 4-cell embryos, lycopene supplementation also led to significant increases in the expression levels of the same ZGA-related genes compared to controls (Fig. 5B).

Fig. 5.

Effects of lycopene on ZGA-related gene expression levels in porcine embryos. (A) Quantification of the expression levels of ZGA-related genes (ZSCAN4, UBTFL1, SUPT4H1, MYC, and ELOA) in SCNT 4-cell embryos from the control and 0.2 μM lycopene treatment groups. (B) Quantification of the expression levels of ZGA-related genes in PA 4-cell embryos from the control and 0.2 μM lycopene treatment groups. Superscript letters (a, b) denote significant differences (P < 0.05). Bar = 100 μm.

Discussion

The successful development of SCNT embryos is essential for producing transgenic models⁴⁰. Although SCNT is a valuable technique for generating genetically engineered animals, incomplete somatic cell reprogramming often leads to epigenetic abnormalities, such as defects in DNA and histone methylation^41,42. Such events impair the activation of ZGA, which is crucial for proper embryonic development, leading to developmental arrest and reduced embryo viability¹⁰. Additionally, oxidative stress generated during IVC and experimental procedures related to SCNT disrupts development¹⁷. Consequently, several antioxidants and small molecules have been used to ameliorate SCNT embryonic development^31,32. However, the effects of the potent carotenoid lycopene on SCNT embryonic development remain largely unexplored. Therefore, this study investigated the effects of lycopene on the developmental competence of porcine PA and SCNT embryos during IVC. First, we supplemented PA embryos with various concentrations of lycopene (0, 0.02, 0.2, and 0.2 μM) during IVC to determine the optimal concentration. Our findings indicated that supplementing with 0.2 μM lycopene throughout the entire IVC period significantly enhanced development, such as the 4–5 cell stage cleavage rate on day 2 and the blastocyst formation rate on day 6. Additionally, the 0.2 μM lycopene treatment resulted in a notable improvement in blastocyst quality, as demonstrated by increases in TE and total cell numbers. These enhancements were also observed in SCNT embryos, underscoring the potential of using lycopene as an additive to improve the viability of SCNT-derived embryos. Furthermore, the increased proportion of 4–5-cell stage embryos in the lycopene-treated group led us to hypothesize a potential association with ZGA, a critical event for somatic cell reprogramming in SCNT embryos. Based on this, we focused our investigation on the 4-cell stage.

Optimal ROS levels must be maintained to support embryonic development, whereas excessive ROS production stresses embryos, negatively affecting their developmental competence and quality^44,45. Embryos are generally exposed to high levels of ROS during SCNT compared to in vivo conditions, leading to higher oxidative stress, which can compromise development¹⁷. In particular, excessive ROS levels in SCNT embryos lead to significant damage to the nuclei and mitochondria, resulting in apoptosis, DNA fragmentation, and abnormal metabolic processes¹⁸. The effects of oxidative stress may be particularly detrimental to SCNT embryos as they alter epigenetic status⁴⁶. Oxidative stress leads to abnormal developmental competence, which can cause SCNT embryonic arrest¹⁰, particularly during the ZGA stage when transcript conversion occurs²⁵. Therefore, we evaluated intracellular ROS to investigate the antioxidative effect of lycopene at the 4-cell stage on day 2 and at the blastocyst stage on day 6 on average with normal developmental competence. The results showed that lycopene significantly reduced the intensity of intracellular ROS in PA and SCNT embryos. Additionally, the expression levels of antioxidant enzyme-related genes (SOD1, SOD2, CAT, and HO-1) increased in 4-cell embryos and blastocysts. These results indicate that lycopene effectively mitigates oxidative stress in porcine embryos by scavenging ROS. Moreover, by reducing intracellular ROS, lycopene may help preserve mitochondrial integrity and function, thereby supporting the energy demands necessary for successful ZGA and continued development.

After confirming that lycopene alleviates oxidative stress, we explored its effects on mitochondrial and autophagic activity in porcine embryos. Mitochondrial dysfunction hinders the supply of metabolites needed for DNA demethylation²³, which is vital for early embryonic development. Normal functioning of the mitochondria is particularly important during ZGA, as additional energy is needed to break down maternal factors⁴⁷. Therefore, we investigated mitochondrial membrane potential in porcine embryos at the 4-cell stage on day 2. Mitochondrial membrane potential is a key marker of mitochondrial function and is associated with the generation of cellular energy⁴⁸. We found that lycopene treatment improved the mitochondrial membrane potential of porcine PA and SCNT embryos at the 4-cell stage. Autophagy is essential for degrading maternal proteins in embryos during ZGA⁴⁹, as it regulates oxidative stress, redox signaling, and protein degradation^50,51. Several studies have shown that reducing autophagy positively impacts embryonic development by alleviating oxidative stress^52,53. However, the inhibition of autophagy led to a significant reduction in developmental efficiency, which was partially rescued by antioxidant treatment⁵⁴. Interestingly, our results showed an increase in autophagy fluorescence intensity in the lycopene treatment group, indicating that lycopene promotes autophagy in porcine PA and SCNT 4-cell embryos. These results suggest that lycopene enhances cellular functions such as mitochondrial and autophagic activity. These findings also imply that lycopene may contribute to a more favorable intracellular environment for ZGA by supporting mitochondrial and autophagic function and also by potentially influencing epigenetic remodeling processes essential for successful reprogramming.

Studies have suggested that developmental abnormalities that occur in cloned animals may be caused primarily by incomplete genomic reprogramming during the SCNT procedure^27,55. Moreover, ROS affects epigenetic factors such as DNA methylation and histone modifications⁴⁶, while antioxidants reduce chromosome aberrations through their activity⁵⁶. Aberrant levels of H3K9me3, H3K4me3, and DNA methylation act as barriers that severely impede the development of SCNT embryos⁵⁵. The critical barrier to complete ZGA lies in epigenetic modifications, including H3K9 and H3K4 methylation, in the donor cell genome^10,57. Global DNA methylation plays a key role in epigenetic reprogramming and mammalian embryonic development⁵⁸. Therefore, we investigated the cellular levels of H3K4me3, H3K9me3, and 5mC in the nucleus through immunocytochemistry staining. A previous study showed that H3K4me3 levels are abnormally high in porcine 4-cell SCNT embryos compared to in vitro fertilized embryos⁵⁹, and another study suggested that the knockdown of H3K4-specific demethylase in porcine embryos disrupts ZGA and impairs embryonic development⁶⁰, indicating that the persistence of H3K4 methylation reduces viability. In this study, supplementing with lycopene downregulated H3K4me3 in PA 4-cell embryos and blastocysts. Additionally, the expression of H3K4me3-specific methyltransferase-related genes, such as ASH2L, decreased. This effect was also observed in SCNT embryos. H3K9me3 is a crucial epigenetic barrier during SCNT-mediated reprogramming¹⁰. The upregulation of H3K9-specific demethylase in bovine and porcine embryos enhanced the cloning efficiency of SCNT^61,62, highlighting its role as a critical epigenetic regulator of ZGA. Our results showed that lycopene treatment decreased H3K9me3 levels in PA and SCNT embryos, and downregulated SUV39H2, which is an H3K9me3-specific methyltransferase-related gene. The restoration of histone acetylation is another key factor in epigenetic reprogramming⁶³, as shown by the marked increase in H3K9ac levels following lycopene treatment in this study. Due to the use of highly methylated somatic cells, SCNT embryos exhibited abnormal DNA methylation, with high levels of 5mC residues that impede preimplantation development²⁷. Consistent with the findings of previous results, we found that lycopene treatment significantly reduced 5mC levels and the expression of DNA methyltransferase-related genes (DNMT, DNM3A, and DNMT3B) in porcine PA and SCNT embryos. These results provide the first evidence that lycopene regulates epigenetic modifications such as H3K4me3, H3K9me3, and DNA methylation, reduces the expression of methyltransferase-related genes, and promotes epigenetic reprogramming in PA and SCNT embryos. Specifically, lycopene restored the activating mark H3K9ac and reduced repressive marks like H3K4me3, H3K9me3, and 5mC. This comprehensive epigenetic modulation appears to create a chromatin environment that is more favorable for the essential transcriptional activation during ZGA, contributing to the alleviation of developmental blockages caused by incomplete reprogramming in SCNT. Thus, our findings highlight the potential of lycopene supplementation as a valuable strategy to improve cloning efficiency and embryonic viability.”

As a crucial step during early embryonic development, ZGA is regulated by maternal factors that promote gene transcription for later development stages and degrade maternal transcripts²⁵. Typically, the first developmental arrest in porcine SCNT embryos occurs at the 4-cell stage, attributed to incomplete ZGA resulting from aberrant reprogramming of somatic cells^7,10. Our data showed that lycopene increased mitochondrial and autophagic activity, and positively modulated epigenetic modifications, including H3K4me3, H3K9me3, and 5mC. These enhancements induced successful ZGA in PA and SCNT embryos after lycopene supplementation, as demonstrated by the improved expression levels of ZGA-related genes such as ZSCAN4, UBTFL1, SUPT4H1, MYC, and ELOA. These results suggest that lycopene alleviates oxidative stress and enhances mitochondrial activity, autophagy, and epigenetic reprogramming, thereby promoting ZGA activation and improving embryonic developmental competence. By orchestrating improvements in these interconnected processes, lycopene facilitates the proper activation of the embryonic genome, a pivotal step that underlies subsequent developmental competence. These insights not only advance our understanding of the molecular mechanisms governing reprogramming and embryogenesis but also emphasize the need for further investigation into the potential of antioxidant supplementation to enhance cloning efficiency.

Conclusion

Supplementing PA and SCNT embryos with 0.2 μM lycopene during IVC increased developmental parameters such as 4–5-cell cleavage and blastocyst rates, as well as the total and TE blastocyst cell numbers. Lycopene treatment decreased intracellular ROS levels and increased mitochondrial membrane potential and autophagic activity. Moreover, lycopene downregulated aberrant levels of H3K4me3, H3K9me3, and 5mC, along with histone and DNA methyltransferase-related gene expression, while upregulating the expression of ZGA-related genes. Lycopene significantly improved the developmental potential and epigenetic modifications of PA and SCNT embryos.

Methods

Chemicals and animals

Unless stated otherwise, all chemicals and reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). All procedures involving the use of animals were approved by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Institutional Animal Care and Use Committee (KRIBB-AEC-25016), conducted in strict accordance with institutional guidelines and regulations, and reported in accordance with the ARRIVE guidelines.

Oocyte collection and in vitro maturation

Porcine ovaries were collected from a nearby slaughterhouse and transported to the laboratory in 0.9% saline containing 0.75 μg/mL benzylpenicillin potassium (FUJIFILM Wako, Osaka, Japan) and 0.5 μg/mL streptomycin sulfate, maintained at 38.5 °C. To obtain cumulus–oocyte complexes (COCs), 3–6-mm follicles were aspirated from the ovary using a disposable 10-mL syringe with an 18-gauge needle. The COCs were washed in physiological saline, supplemented with 1 mg/mL bovine serum albumin (BSA), and approximately 50–70 COCs with three or more intact cumulus layers were selected. The COCs were washed in IVM medium consisting of tissue culture medium 199 (TCM-199, Invitrogen, Carlsbad, CA, USA), supplemented with 10% porcine follicular fluid, 0.57 mM cysteine, 25 μM β-mercaptoethanol, 10 ng/mL epidermal growth factor, 10 IU/mL pregnant mare serum gonadotropin (PMSG; Prospec, Rehovot, Israel), and 10 IU/mL human chorionic gonadotropin (hCG; Prospec). The COCs were matured in 500 μL of IVM medium in a 4-well multi-dish (Nunc, Roskilde, Denmark) under 5% CO₂ at 38.5 °C. After 22 h of maturation, the COCs were washed and transferred to IVM medium free of hCG and PMSG for an additional 22 h of maturation.

Parthenogenetic activation and in vitro culture

After 44 h of IVM, expended COCs were transferred to denuding medium, which was Dulbecco’s phosphate-buffered saline (DPBS; Gibco, Grand Island, NY, USA), containing 0.1% hyaluronidase, 60 μg/mL gentamicin sulfate salt, and 50 μg/mL streptomycin sulfate, and then removed after vortexing for 2 min. The denuded oocytes were washed in PB1 medium containing DPBS supplemented with 60 μg/mL gentamicin sulfate salt, 50 μg/mL streptomycin sulfate, and 4 mg/mL BSA; metaphase II (MII) oocytes with visible polar bodies were selected. The MII oocytes were incubated in IVC medium consisting of porcine zygote medium-3 (PZM-3) containing 4 mg/mL BSA. The MII oocytes were subjected to parthenogenetic activation through incubation with 15 μM ionomycin in PB1 medium in the dark on a hot plate for 5 min. The activated oocytes were incubated in IVC medium, supplemented with 2 mM 6-dimethylaminopurine and 5 µg/mL cytochalasin B for 4 h under 5% CO₂ at 38.5 °C. After 4 h, the zygotes were transferred to IVC medium in humidified air containing 5% CO₂ at 38.5 °C for 6 days.

Establishment of primary cells and preparation of donor cells

As donor cells for SCNT, the porcine kidney was obtained from a neonatal pig (mixed breed Landrace, Yorkshire, and Duroc, 2-day-old male) after surgery. Kidney tissues were gathered and stored on ice in DPBS washing buffer containing 10% penicillin/streptomycin (Invitrogen), until isolation. The kidney biopsies (2 cm × 1 cm × 1 cm) were washed three times in washing buffer, diced into 0.3 cm × 0.3 cm × 0.3 cm pieces, and washed with Dulbecco’s modified eagle’s medium (DMEM; Invitrogen). The kidney tissue was transferred to 60-mm culture dishes and cultured at 37 °C under 5% CO₂ in DMEM containing 10 ng/mL basic fibroblast growth factor (R&D Systems, Minneapolis, MN, USA), 10% fetal bovine serum (FBS; Gibco), and 1% penicillin/streptomycin (DMEM-FBS). Donor cells for SCNT were used at passages 4–6. The kidney cells were synchronized at the G0–G1 phase by culturing to confluence, followed by an additional 3-day incubation in culture medium containing 0.5% FBS. Donor cells for SCNT were washed in DPBS and treated with 0.25% trypsin–EDTA for 3 min at 37 °C in 5% CO₂. The trypsin was subsequently neutralized with DMEM-FBS. The cells were centrifuged at low speed (l50 × g) for 2 min, and the supernatant was carefully removed without breaking the pellet. The cell pellet was resuspended in DPBS containing 60 μg/mL gentamicin sulfate salt, 50 μg/mL streptomycin sulfate, and 2 mg/mL BSA.

Somatic cell nuclear transfer

SCNT was performed based on a previously established method reported by our group⁶², with a modification in the cell fusion step. Enucleation was carried out under an inverted microscope (DMi8, Leica Microsystems, Wetzlar, Germany) equipped with a micromanipulator (NT-88-V3; Nikon Narishige, Tokyo, Japan) using a glass pipette, and a single donor cell was injected into the perivitelline space of each enucleated oocyte. Instead of electrical stimulation, Sendai virus (SV)-mediated fusion was employed as described in our prior study⁶⁴. Donor cells were exposed to the virus solution for 1 min prior to injection, and the reconstructed oocyte–cell couplets were incubated for 2 h. Successful fusion was confirmed using an inverted microscope (DMi8, Leica), followed by chemical activation and in vitro culture as previously described. The fused oocyte–cell couplets were activated in PB1 containing 15 μM of ionomycin in the dark for 5 min. The activated oocyte–cell couplets were transferred to post-activation medium comprised of PZM-3 containing 2 mM 6-dimethylaminopurine and 10 μM JNJ-7706621 (Selleckchem, Houston, TX, USA) for 4 h under 5% CO₂ at 38.5 °C. After 4 h, the zygotes were transferred to IVC medium in humidified air with 5% CO₂ at 38.5 °C for 6 days. Cleavage formation rates were evaluated at 48 h, and blastocyst rates were evaluated at 144 h.

Chemical treatment

Lycopene was dissolved in dimethyl sulfoxide (DMSO) and diluted in PZM-3 medium as the culture medium. To confirm the optimal conditions for lycopene treatment during porcine PA embryonic development, activated embryos were cultured in post-activation medium with various concentrations of lycopene (0, 0.02, 0.2, and 2 μM) for 4 h. Then, the zygotes were transferred to IVC medium with the same concentrations of lycopene in 5% CO₂ at 38.5 °C for 6 days. Cleavage formation rates were evaluated at 48 h, and blastocyst rates were evaluated at 144 h.

Terminal deoxynucleotidyl transferase-mediated dUTP-digoxygenin nick end-labeling (TUNEL) assay

Apoptotic cells were detected in blastocysts using the In Situ Cell Death Detection kit (Roche, Basel, Switzerland). Fixed blastocysts were washed three times in DPBS supplemented with 0.1% polyvinyl acetate (PVA) (PVA-PBS) for 10 min each. The blastocysts were incubated in DPBS containing 1% Triton X-100 to permeabilize the membranes for 1 h at room temperature (RT), and then the blastocysts were washed three times in PVA-PBS. The blastocysts were incubated with fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase for 1 h at 38.5 °C. After washing three times in PVA-PBS, the blastocysts were mounted on glass slides with 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories Inc., Burlingame, CA, USA). The numbers of nuclei and apoptotic cells were observed under a fluorescence microscope (DMi8; Leica).

CDX2 staining

Blastocysts were fixed in 4% paraformaldehyde overnight at 4 °C and washed three times with PVA-PBS for 10 min each. The fixed blastocysts were incubated for 1 h at RT in DPBS containing 1% Triton X-100 to permeabilize the membranes. Next, the blastocysts were washed three times in PVA-PBS and transferred to a blocking solution, PBS-PVA supplemented with 2% BSA (BSA-PVA-PBS), where they were blocked for 6 h at 4 °C. The blastocysts were blocked with 10% normal goat serum in DPBS for 1 h and then incubated overnight at 4 °C with a primary antibody, the mouse monoclonal CDX2 antibody (Biogenex Laboratories Inc., San Ramon, CA, USA). Following three washes in blocking solution, the blastocysts were incubated in the dark for 1 h at RT with a secondary antibody, AlexaFluor-488-labeled goat anti-mouse IgG (1:200). Lastly, the blastocysts were washed three times in BSA-PVA-PBS for 10 min each and then mounted on glass slides with DAPI. The CDX2-positive cells were detected under a fluorescence microscope (DMi8, Leica).

Measurement of intracellular ROS levels

Intracellular ROS levels were measured using CM-H2DCFDA (Invitrogen). 4-cell embryos and blastocysts were incubated in PVA-PBS containing 5 μM CM-H2DCFDA for 30 min in 5% CO₂ at 38.5 °C. After washing three times in PVA-PBS, the embryos were placed in 40-μL droplets of PVA-PBS, and fluorescence signals were detected under a fluorescence microscope (DMI4000B, Leica). ROS levels were measured under identical exposure settings for all embryos to ensure consistent fluorescence intensity comparison. Fluorescent intensity levels were analyzed with ImageJ v1.52 software (National Institutes of Health, Bethesda, MD, USA) and normalized to those of control embryos.

Measurement of mitochondrial membrane potential

4-cell embryos on day 2 were fixed in 4% paraformaldehyde for 1 h at 38.5 °C. The fixed embryos were washed three times in PVA-PBS and incubated with JC-1 (1:500, Cayman Chemical, Ann Arbor, MI, USA) for 30 min. To determine the membrane potential, the ratio of red fluorescence, representing high mitochondrial membrane potential (J-aggregates), to green fluorescence, representing low mitochondrial membrane potential (J-monomers), was calculated. Thereafter, the embryos were washed three times in PVA-PBS, and their images were captured under a fluorescence microscope (DMi8, Leica). The fluorescent intensities were assessed with ImageJ software, and the values were normalized against control embryos.

Measurement of autophagy

4-cell embryos on day 2 were washed three times with PVA-PBS and then incubated with the Autophagy Assay Kit (1:500, Abcam, Cambridge, MA, USA) for 30 min in 5% CO₂ at 38.5 °C. The embryos were stored in 4% paraformaldehyde for 1 h at 38.5 °C for optional fixation. Autophagic vesicles were visualized by detecting green fluorescence. After three washes, the embryos were placed in 40-μL droplets of PVA-PBS and captured under a fluorescence microscope (DMi8, Leica). ImageJ software was used to analyze fluorescence intensity, which was normalized to that of control embryos.

Immunocytochemistry staining

All 4-cell embryos and blastocysts were fixed in 4% paraformaldehyde overnight and washed three times in PVA-PBS for 10 min each. For permeable membrane, the samples were incubated with 1% Triton X-100 in DPBS for 1 h at RT. Staining for 5-methylcytosine (5mC) included an additional step to permeabilize the nuclear membranes; the embryos were incubated with 1 N HCl containing 0.1% PVA for 30 min at 38.5 °C. After washing three times in DPBS containing 0.05% Tween 20 (PBST), the samples were incubated with PBST containing 2% BSA (BSA-PBST) for 1 h at RT. Then, 4-cell embryos and blastocysts were incubated overnight with primary antibodies, including histone 3 lysine 4 tri-methylation (H3K4me3; 1:200, Abcam), histone 3 lysine 9 tri-methylation (H3K9me3; 1:1000, Abcam), histone 3 lysine 9 acetylation (H3K9ac; 1:200, Cell Signaling Technology, Danvers, MA, USA), and 5-methylcytosine (5mC; 1:200, Calbiochem, San Diego, CA, USA) at 4 °C. After three washes in PBST, the 4-cell embryos and blastocysts were blocked in BSA-PBST and incubated with secondary antibody, AlexaFluor-488 goat anti-mouse or rabbit IgG (1:200 for 5mC or 1:200 for H3K4me3, H3K9me3, and H3K9ac) for 1 h at RT. Then, the 4-cell embryos and blastocysts were washed three times in BSA-PBST and subjected to DAPI staining. The fluorescent images were viewed under a fluorescence microscope (DMi8, Leica), and intensity was analyzed with ImageJ software; the values were normalized against those of control embryos.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Poly(A) mRNAs were extracted from 25 4-cell embryos and seven blastocysts using the Dynabeads mRNA Direct kit (Invitrogen). The samples were lysed in 200 μL of lysis buffer at RT for 5 min with vortexing. Then, 20 μL of DynaBeads oligo (dT) 25 was added to each sample and incubated for 10 min at RT to isolate the mRNA. A Dynal magnetic bar (Invitrogen) was used to facilitate the separation of the beads from the binding buffer. The poly(A) mRNAs bound to the beads were washed twice in 200 μL of washing buffers A and B individually, and 15 μL Tris–HCl buffer was added to separate the poly(A) mRNAs. The PrimeScript RT Reagent Kit with the gDNA Eraser (Takara Bio, Kusatsu, Japan) was used for reverse transcription to produce cDNA following the manufacturer’s instructions. The reverse transcription reaction was conducted at 37 °C for 15 min, followed by termination of the reaction at 85 °C for 5 s. The synthesized cDNA samples were used as qPCR templates. qPCR was performed using the Mx3000P qPCR system (Agilent Technologies, Santa Clara, CA, USA) with SYBR Premix EX Taq (Takara Bio). qPCR was performed at 95 ℃ for 10 min, followed by 40 cycles at 95 °C for 20 s and 60 °C for 20 s. The expression levels of the target genes were quantified relative to those of GAPDH. The relative expression (R) was calculated using the following equation: R = 2^{–[ΔCt sample – ΔCt control]}. The qPCR primers are listed in Supplementary Table 1.

Statistical analysis

All experiments were conducted in triplicate. Data are expressed as means ± standard error of the mean (SEM). SigmaStat statistical software (SPSS, Chicago, IL, USA) was used for all analyses. All data were subjected to normality and homoscedasticity testing. Student’s t-test was used to detect significant differences between two groups. One-way analysis of variance (ANOVA) was used to compare among multiple groups, followed by Duncan’s test to assess the significant differences. Significance was evaluated using a threshold of P < 0.05.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

BF

Bright field

BSA

Bovine serum albumin

CDX2

Caudal type homeobox protein 2

COC

Cumulus-oocyte complex

hCG

Human chorionic gonadotropin

H3K4me3

Histone H3 lysine 4 tri-methylation

H3K9ac

Histone H3 lysine 9 acetylation

H3K9me3

Histone H3 lysine 9 tri-methylation

ICM

Inner cell mass

IVC

In vitro culture

IVM

In vitro maturation

MII

Metaphase II

PA

Parthenogenetically activated

PMSG

Pregnant mare serum gonadotropin

ROS

Reactive oxygen species

SCNT

Somatic cell nuclear transfer

TE

Trophectoderm

ZGA

Zygotic genome activation

5mC

5-Methylcytosine

Author contributions

JHY and H-GK designed the study performed experiments, analyzed data, and wrote the manuscript. EYC, S-BJ, MJK, P-SJ, and B-SS performed experiments and analyzed data. S-UK acquired funding and discussed study. K-SM and B-WS designed and supervised the study. All authors read and approved the final manuscript.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files. Additional data are available from the corresponding author [Bo-Woong Sim, embryont@kribb.re.kr] on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.