Using the GEO database and quantitative PCR, we validated our lab’s previous sequencing data (Figure 1). As shown in Figures 1B, C, the expression trend of Myc aligns with our earlier results, which indicated that Myc is highly expressed in four-cell embryos. In four-cell embryos, N-myc is only 1.3% of Myc (Supplementary Figure 1I). Myc mRNA was undetectable in the MII oocytes and two-cell embryos but significantly increased in four-cell embryos (P< 0.01), reaching 23 times the level of GAPDH and five times that of eight-cell embryos (P< 0.01), before rapidly declining thereafter. We further validated MYC protein levels at this stage. As shown in Supplementary Figure 1A, due to the lack of specific antibodies, MYC protein was detected in 1500 MII oocytes and 400 four-cell embryos, providing direct evidence of MYC protein presence in four-cell embryos.

Next, we employed the small molecule inhibitor 10058-F4 and siRNA to interfere with Myc (hereafter referred to as MYCi), which resulted in a significant decline in embryonic development capacity (blastocyst rate). As illustrated in Figures 1D–F, increasing doses of 10058-F4 led to a gradual decrease in the blastocyst rate. At a dose of 25 μM, a statistically significant reduction was observed (P = 0.033); at a dose of 50 μM, more than half of the embryos failed to develop, with a blastocyst rate of only 7.1% ± 1.35%, which was significantly different from the control group (20.6% ± 1.35%, P = 0.016). At a dose of 100 μM, all embryos arrested before reaching the morula stage. To further confirm the effect of the inhibitor, we utilized microinjection of siRNA to interfere with Myc mRNA. As shown in Figures 1F, G, Myc mRNA levels in four-cell embryos decreased by over 82%, indicating substantial interference efficiency. The blastocyst rate also dropped from 28% in the control group (NC) to 2%, consistent with the results obtained from inhibitor treatment.

These findings suggested that the high expression of Myc in four-cell embryos was crucial for early embryonic development.

During the four-cell embryo, porcine embryos undergo maternal-to-zygotic transition and epigenetic reprogramming. Our results showed that the high expression of Myc at this stage suggesting its essential role in these processes (Figure 2). Using EU staining, we found that newly synthesized RNA decreased by over 50% in the Myc inhibition group (MYCi group, Figure 2B), highlighting Myc’s vital importance in transcription regulation. Our previous research showed that Myc inhibition reduces ZGA-related gene expression (Asami et al., 2023).

Subsequently, we further investigated the changes in epigenetic modifications related to transcriptional activity in the MYCi group. Using immunofluorescence, we observed the levels of H3K27me3 and DNA methylation in four-cell embryos. The results showed a significant increase in H3K27me3 and DNA methylation levels in the MYCi group (Figures 2C, D). LINE-1 is a widely distributed repetitive sequence in the genome. The assessment of LINE-1 methylation levels using bisulfite conversion can serve as a representative measure of the overall genomic methylation status (Xu et al., 2020; Yang et al., 2004). To assess global DNA methylation levels, we examined LINE-1 methylation (Tran et al., 2019) and found that LINE-1 methylation levels were higher in the MYCi group compared to the NC group (40.4% ± 10% VS. 22.8% ± 12.8%) (Figures 2E, F). Additionally, we evaluated the modifications of H3K4me3 and H3K9me3 in four-cell embryos and found no significant differences. Moreover, the difference in H3K27me3 modification disappeared at the eight-cell stage (Supplementary Figure 1B).

These results suggested that Myc inhibition regulated gene expression during early embryonic development by affecting DNA and statuses.

To further investigate Myc’s role in four-cell embryos, we conducted transcriptome sequencing analysis of embryos at this stage (Figure 3). The results showed 788 downregulated genes and 170 upregulated genes in the MYCi group compared to the control group (MYCi vs. control, Figure 3B). This is consistent with the role of Myc as a transcription factor that generally promotes gene expression, resulting in the downregulation of most genes upon Myc inhibition.

KEGG and GO pathway analysis of differentially expressed genes (DEGs) indicated a close association with energy metabolism. As shown in Figure 3D, GO pathway analysis of differentially expressed genes (DEGs) revealed significant enrichment in mitochondrial organization and several processes related to the positive regulation of transcription and translation, such as positive regulation of transcription, DNA-templated, and positive regulation of RNA biosynthetic process. The enrichment in mitochondrial organization suggests a potential link to cellular energy dynamics.

KEGG analysis revealed that five out of the top ten pathways were related to metabolic processes, including glyoxylate and dicarboxylate metabolism, fructose and mannose metabolism, and biosynthesis of amino acids. Protein interaction network analysis of genes enriched in the KEGG pathway for glyoxylate and dicarboxylate metabolism clustered around key TCA cycle enzymes such as CS, ACO2, and IDH2 (Figure 3E).

We assessed mitochondrial membrane potential (MMP), ATP, and reactive oxygen species (ROS) levels as macro metabolic indicators (Figure 4). As shown in Figure 4A, the MYCi group exhibited significant reductions in MMP in both four-cell and eight-cell embryos compared to the control group. Embryonic ATP content in the MYCi group was slightly lower in four-cell embryos and halved compared to the control group in eight-cell embryos (Figure 4B). Conversely, ROS levels were significantly higher in the MYCi group in both four-cell and eight-cell embryos (Figure 4C). This indicated that Myc inhibition caused abnormalities in mitochondrial energy synthesis and elevated ROS due to the upregulation of genes associated with abnormal oxidative phosphorylation.

We measured the expression levels of three metabolic enzymes, CS, IDH2, and ACO2, identified in Figure 3E. As shown in Figure 5, all three enzymes were downregulated in the MYCi group, with CS and IDH2 showing significant statistical differences. Considering that CS, ACO2, and IDH2 are responsible for generating α-Ketoglutarate (α-KG) in the TCA cycle, we collected 500 four-cell embryos to measure α-KG content in the MYCi group. As shown in Figure 5B, α-KG content in the MYCi group decreased by more than two-thirds compared to the control group.

Subsequently, we added α-KG to the MYCi group culture system to observe embryonic development. As shown in Supplementary Figures 1D–H, 2 mM α-KG showed the best rescue effect at 25 and 50 μM inhibitor concentrations. Using a 50 μM inhibitor concentration for further testing, the blastocyst rate in the MYCi group dropped from 18.1% ± 1.1% to 6.7% ± 0.7% (Figure 5D, P = 0.0135), indicating effective inhibition. The MYCi+α-KG group showed a significant recovery in the blastocyst rate to 12.2% ± 1.8%, compared to 6.7% ± 0.7% in the MYCi group (P = 0.024), showing a good rescue effect. The NC+α-KG group also showed a slight decrease in the blastocyst rate, likely due to the addition of high concentration 2 mM α-KG.

We then evaluated the total cell number of blastocysts in each group, and the trend was consistent with the aforementioned changes in blastocyst rate. The average total cell number in the MYCi+α-KG group (22) showed a significant increase compared to the MYCi group (12) (Figure 5E).

The α-KG acts as an intermediate in the TCA cycle, participating in cellular energy metabolism, and as a key cofactor for DNA demethylases, playing an important role in epigenetic modifications. Therefore, in the rescue experiments, we also assessed whether the addition of α-KG improved the abnormal metabolic indicators and DNA methylation levels in the MYCi group (Figures 6, 7).

As shown in Figures 6A–E, 7A, B, compared to the MYCi group, the addition of α-KG significantly improved MMP and ATP content in four-cell and eight-cell embryos, and significantly reduced ROS levels. Specifically, ATP content in the MYCi group was significantly lower than in the normal group, confirming the inhibitor’s effectiveness. In the NC+α-KG group, ATP content decreased, while adding α-KG to the MYCi group significantly increased ATP levels. For ROS detection, ROS levels in the MYCi group were three times higher than those in the normal group, confirming the inhibitor’s effectiveness. Adding α-KG to the NC group did not cause significant changes, but adding α-KG to the MYCi group significantly reduced ROS levels.

In four-cell embryos, we investigated the effects of adding α-KG to the MYCi group in response to the previously observed abnormal elevation of 5 mC and H3K27me3 modifications. Initially, we utilized immunofluorescence techniques to compare the DNA methylation levels between the MYCi group and the MYCi+α-KG group (Figure 7C). The results revealed a notable decrease in DNA methylation levels in the MYCi+α-KG group compared to the MYCi group. To obtain more precise measurements, we further employed bisulfite sequencing to delve deeper into the methylation levels of LINE-1 (Figures 7D, E). The data indicated that the LINE-1 methylation level in the MYCi+α-KG group was significantly reduced from 40.4% ± 10% in the MYCi group to 22.8% ± 12.8%, a change that was statistically significant.

Concurrently, we also observed a marked reduction in H3K27me3 modification levels in the MYCi+α-KG group compared to the MYCi group (Figure 7F). Collectively, these experimental results suggest that the addition of α-KG substantially alleviated the abnormal elevation of DNA and histone methylation levels caused by Myc inhibition.

All chemicals used during the experiments were purchased from Sigma-Aldrich (St. Louis, MO, United States), unless otherwise specifically mentioned.

Ovaries were collected from pigs at a local slaughterhouse (Changchun Huazheng, Jilin, China) and transported to the laboratory within 2 h. They were maintained in 0.9% NaCl solution supplemented with 200 IU/mL penicillin and streptomycin at 35°C–36.5°C. Follicular fluid containing cumulus-oocyte complexes (COCs) was aspirated from 3 to 6 mm ovarian follicles using an 18-gauge needle. COCs with at least three layers of cumulus cells were selected and washed three times in manipulation fluid (TCM-199 supplemented with 0.1% polyvinyl alcohol). These COCs were then cultured in in-vitro maturation medium (TCM-199 supplemented with 10 μg/mL epidermal growth factor, 0.5 μg/mL porcine LH, 0.5 μg/mL porcine FSH, 26 mM sodium bicarbonate, 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.1% PVA, 10% fetal calf serum, 75 mg/mL penicillin G, and 50 mg/mL streptomycin) for 22–24 h. Subsequently, they were cultured in hormone-free maturation medium (identical to the previous medium but without epidermal growth factor, LH, and FSH) for an additional 20 h at 38.5°C and 5% CO₂ and 95% air. Cumulus cells were removed from oocytes using manipulation fluid supplemented with 0.2% hyaluronidase. Oocytes exhibiting the first polar body (PB1) were considered mature and used for subsequent experiments.

Fresh semen was collected from the Jilin University pig farm and washed by density gradient centrifugation. Briefly, 2 mL of semen was added to Percoll (Solarbio, Beijing, China) in concentrations of 90% and 45%, followed by centrifugation at 300 g for 20 min. After removing the supernatant, the sperm pellets were washed with 4 mL Dulbecco’s Phosphate-Buffered Saline (DPBS: 100 mL water with 800 mg NaCl, 20 mg KCl, 112 mg Na₂HPO₄·12H₂O, 20 mg KH₂PO₄, 10 mg CaCl₂, 10 mg MgCl₂·6H₂O, and 100 mg bovine serum albumin (BSA)) and centrifuged at 300 g for 10 min. The spermatozoa were resuspended in porcine gamete medium (100 mL water with 0.6313 g NaCl, 0.07456 g KCl, 0.00477 g KH₂PO₄, 0.00987 g MgSO₄·7H₂O, 0.2106 g NaHCO₃, 0.07707 g CaC₆H₁₀O₆·5H₂O, 0.0187 g D-Glucose, 0.3 g PVA, 0.00242 g Cysteine, 0.04504 g C₇H₈N₄O₂, 0.0022 g C₃H₃NaO₃, and 100 μL/mL penicillin and streptomycin). Sixty mature oocytes (1 h post-siRNA injection) were incubated with spermatozoa in 400 μL porcine gamete medium, with a final sperm concentration of 1.6 × 10⁵ to 5.0 × 10⁵ sperm/mL, at 38.5°C, 5% CO₂ and 95% air for 5–6 h. After washing off the adherent sperm, fertilized oocytes were transferred to PZM-3 medium (50 mL water with 0.3156 g NaCl, 0.0373 g KCl, 0.0024 g KH₂PO₄, 0.0024 g MgSO₄·7H₂O, 0.1055 g NaHCO₃, 0.0011 g Na-pyruvate, 0.0308 g Ca-[lactate]₂·5H₂O, 0.0073 g L-glutamine, 0.0273 g hypotaurine, 0.15 g BSA, 1 mL BME amino acid, 0.5 mL MEM non-essential amino acid). Embryos at the two-cell, four-cell, and eight-cell stages were collected after culturing for 24, 48, and 72 h in PZM-3, respectively. The culture medium was changed to PZM-3 with 10% fetal bovine serum after 5 days, and blastocysts were collected after an additional 2 days of culture.

The Smart-Seq2 method was used to amplify RNA from each sample (six to eight IVF embryos per group) following the manufacturers instructions. RNA concentration and quality were assessed using a Qubit 2.0 Fluorometer (Life Technologies, CA, United States) and an Agilent Bioanalyzer 2100 system. The amplified cDNA was used for transcriptome library construction. Following the library construction, the insert size was assessed using an Agilent Bioanalyzer 2100 system. The accurate insert size was quantified using a Taq-Man fluorescence probe on an AB Step One Plus Real-Time PCR system (library valid concentration >10 nM). The libraries were then clustered using a cBot cluster generation system and sequenced on an Illumina platform by Zhejiang Annoroad Biotechnology (Beijing, China) to generate 150 bp paired-end reads.

Total RNA was extracted, and complementary DNA (cDNA) was synthesized using the SuperScript™ IV CellsDirect™ cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s instructions. Quantitative PCR was performed using FastStart Essential DNA Green Master (Roche, Basel, Switzerland) on a Step One Plus Real-Time PCR system. Primer sequences are provided in Supplementary Material 1. Data were analyzed using the 2^−ΔΔCT method, with GAPDH or β-ACTB as internal reference genes. Each quantitative PCR experiment was repeated three times (See Supplementary Table 1 in the Supplementary Material for primer details).

Embryos were washed with PBS containing 0.1% polyvinylpyrrolidone (PVP) and then treated with acidic Tyrode solution (pH 2.5) to dissolve the zona pellucida. After washing, embryos were fixed with 4% paraformaldehyde for 30 min in the dark. Following another wash, embryos were permeabilized with 0.2% Triton X-100/PBS for 20 min and blocked with 2% BSA in PBS for 1 h. For 5 mC/5 hmC staining, embryos were treated with 4M HCl and Tris-HCl for 30 min each, followed by BSA blocking. Primary antibodies were incubated overnight at 4°C, and secondary antibodies were incubated at 37°C for 2 h in the dark. DNA was stained with 10 μg/mL DAPI for 15 min.

Samples were observed under a Nikon Eclipse Ti-U microscope equipped with appropriate filters. Images were captured using a DS-Ri2 CCD camera and analyzed with NIS-Elements BR software. Fluorescence intensity was evaluated using ImageJ software. Background fluorescence intensity was subtracted for further analysis.

The MMP was measured following the protocol provided by Solarbio (CA1310-100, Beijing, China). Four-cell or eight-cell embryos (at least 20 embryos per replicate) were washed three times in PBS-0.1% PVP and fixed with 4% paraformaldehyde for 1 h. After washing, embryos were incubated in PBS-0.1% PVP supplemented with 500 nM BODIPY FL ATP in the dark for 2 h. Fluorescence images were captured using an epifluorescence microscope with excitation and emission wavelengths of 504 nm and 514 nm, respectively.

ROS levels were measured using a ROS assay kit (Beyotime, Shanghai, China). Embryos were incubated in DPBS-0.1% PVA containing 10 μM 2′,7′-dichlorodihydrofluorescein diacetate at 38.5°C in the dark for 15 min. After washing, embryos were placed in microdrops and covered with mineral oil. Fluorescence images were captured using an epifluorescence microscope.

LINE-1 methylation levels were detected according to the method described by Xu et al. (2020).

The α-KG content in early embryos was measured using a Solarbio kit (BC5425, Beijing, China). Briefly, 6,500 four-cell stage embryos were collected, and the zona pellucida was removed using acidic Tyrode’s solution. Embryos were lysed in the kit’s lysis buffer (up to 80 µL) and disrupted by ultrasonic treatment on ice (300 W power, 3-s bursts with 7-s intervals, for a total of 3 min). The lysate was centrifuged at 4°C, and the supernatant was extracted. Reagents were added according to the protocol, mixed thoroughly, and absorbance at 340 nm was measured at 20 s (A1). After incubation at 37°C for 90 min, absorbance at 340 nm was measured again at 90 min and 20 s (A2). Calculations followed the protocol provided with the kit.

For the detection of Myc, we used the Myc antibody (Cell Signaling Technology, Cat# 9402S) at a dilution of 1:1,500. The 5 mC antibody (Epigentek, Cat# BI-MECY-0100) was used at a dilution of 1:100. For the detection of H3K27me3, we used the H3K27me3 antibody (Cell Signaling Technology, Cat# C36B11) at a dilution of 1:200. The Alexa Fluor 488 goat anti-mouse secondary antibody (Thermo Fisher Scientific, Cat# A32723) and the Alexa Fluor 488 goat anti-rabbit secondary antibody (Thermo Fisher Scientific, Cat# A-11008) were used at a dilution of 1:200.

Data were expressed as mean ± standard deviation. Experiments were repeated at least three times. Statistical analysis was performed using GraphPad Prism 10.1.2 software (GraphPad Software, Boston, MA, United States). Comparisons between two data sets were made using t-tests, while one-way analysis of variance (ANOVA) followed by pairwise comparisons was used for comparisons among multiple data sets. P-value of <0.05 was considered statistically significant.