Abstract Background The transcriptional changes around zygotic genome activation (ZGA) in preimplantation embryos are critical for studying mechanisms of embryonic developmental arrest and searching for key transcription factors. However, studies on the transcription profile of porcine ZGA are limited. Results In this study, we performed RNA sequencing in porcine in vivo developed (IVV) and somatic cell nuclear transfer (SCNT) embryo at different stages and compared the transcriptional activity of porcine embryos with mouse, bovine and human embryos. The results showed that the transcriptome map of the early porcine embryos was significantly changed at the 4-cell stage, and 5821 differentially expressed genes (DEGs) in SCNT embryos failed to be reprogrammed or activated during ZGA, which mainly enrichment to metabolic pathways. c-MYC was identified as the highest expressed transcription factor during ZGA. By treating with 10,058-F4, an inhibitor of c-MYC, the cleavage rate (38.33 ± 3.4%) and blastocyst rate (23.33 ± 4.3%) of porcine embryos were significantly lower than those of the control group (50.82 ± 2.7% and 34.43 ± 1.9%). Cross-species analysis of transcriptome during ZGA showed that pigs and bovines had the highest similarity coefficient in biological processes. KEGG pathway analysis indicated that there were 10 co-shared pathways in the four species. Conclusions Our results reveal that embryos with impaired developmental competence may be arrested at an early stage of development. c-MYC helps promote ZGA and preimplantation embryonic development in pigs. Pigs and bovines have the highest coefficient of similarity in biological processes during ZGA. This study provides an important reference for further studying the reprogramming regulatory mechanism of porcine embryos during ZGA. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-022-09015-4. Keywords: Transcriptomics, Zygotic genome activation, Pigs, Comparison analysis, C-MYC Background The early stage of mammalian embryonic development is regulated by maternal factors. Newly expressed mRNAs are continuously accumulated and updated in the embryos, thereby regulating early embryonic development [[39]1]. In the early stages of embryonic development, the first dramatic change in gene transcription occurs during ZGA. The time point of ZGA is species-specific. It has been reported that the activation of zygotic genes in mice occurs at the 2-cell stage, while that in humans, pigs, goats and bovines occurs lightly later, at the 4-cell to 8-cell stage or even later [[40]2–[41]4]. Thus, each species regulates embryonic development through its own unique gene transcription patterns. Whether the embryonic genome can initiate transcription in the early developmental stage depends on key components regulating transcription or their expression levels reaching the threshold level. The embryo needs a certain amount of time to produce these key factors, and transcriptional activation occurs once the threshold level is reached. This has been verified in the Xenopus laevis [[42]5], with continuous cleavage and translation, the TBP transcript increases, reaching a high enough level during genome activation, and then triggers zygotic genome activation [[43]6]. Almouzni et al. found that the lack of gene-specific TFs prior to genome activation causes transcriptional silencing [[44]7]. Zelda (Zld) was the first identified zygotic genome activator in Drosophila [[45]8]. Nanog, Soxb1 and Pou5f3 are important transcription factors during ZGA in zebrafish [[46]9], which that activate the expression of hundreds of ZGA related genes. The Cairns team determined that DUX was the first transcription factor driving the transcription initiation of early mouse embryos [[47]10]. It was proposed that ZSCAN4 is a stabilizer that initiates the ZGA in placental mammals [[48]11, [49]12]. OCT4 is crucial for human major ZGA and enriched in the open chromatin region of human embryos [[50]13, [51]14]. Yamanaka’s team reprogrammed terminally differentiated cells to induce pluripotent stem cells in mice through exogenous introduction of four transcription factors [[52]15], and identified that c-MYC as one of the key transcription factors in the whole reprogramming process. Liu et al. found that TDG was defined as a pig-specific epigenetic regulator for nuclear reprogramming and that transient TDG overexpression promoted DNA demethylation and enhanced the blastocyst-forming rates of porcine SCNT embryos [[53]16]. The whole genome transcript profile of pigs during maternal to zygotic transition is helpful to understand the stage-specific transcriptome, the removal of stored maternal transcripts and newly synthesized transcripts in ZGA, so as to identify key genes and signal pathways. The efficiency of SCNT-mediated cloning in mammals, and especially including pigs, remains very low [[54]17, [55]18]. For those reasons, thus far, a wide panel of investigations have been conducted to recognize, a more extensively, the biological, genetic and epigenetic factors that influence the molecular mechanisms of embryogenesis, ZGA and quality of SCNT-derived oocytes in pigs and different mammalian species [[56]19, [57]20]. The source and provenance of nuclear donor cells is a fundamental prerequisite for determination of the overall outcome of propagating cloned embryos [[58]21–[59]23]. Incomplete ZGA has been shown to be one of the major causes affecting the development of mouse, human and porcine SCNT embryos [[60]24–[61]28]. Scientists have been working to find ways to improve the efficiency of cloning. Yi Zhang Laboratory and Shaorong Gao Laboratory respectively reported that histone demethylase Kdm4d could increase the cloning efficiency in mice from 1.0 to 8.7% [[62]29–[63]31]. Sun Qiang’s lab used the ideal combination of TSA + KDM4D to increase the cloning efficiency of macaque monkeys from 0 to 2.5% [[64]32]. Miao Yiliang’s team and Chen Zhenxia’s team used a combination of KDM4A+ GSK126 to increase the blastocyst rate of pig cloned embryos by nearly twofold [[65]16], but the cloning efficiency was still very low [[66]33]. Referring to the experience of mice and other species and combining multiple high-throughput analysis methods to find the most suitable method for pigs is the direction of research to improve the efficiency of somatic cell cloning in pigs. In our study, we revealed the transcriptional patterns of porcine IVV and SCNT embryos and comparatively analyzed the transcriptional activity of embryos in pigs, mice, bovines and humans. We further identified key genes and transcription factors during ZGA and verified the essential role of the transcription factor in porcine embryonic development, which provided an important reference for further functional investigation. Results Transcriptional activities of porcine MII oocytes and IVV embryos MII oocytes and in vivo fertilized embryos (2-cells, 4-cells, 8-cells and blastocysts) were collected for subsequent RNA-sequencing analysis and differential analysis of gene expression was performed (Fig. [67]1A). Principal component analysis showed that the biological replication in each experimental group was good and MII oocytes were most similar to 2-cell embryos, which proved the reliability of the data (Fig. [68]1B). The profiles of gene expression at various stages (Fold change, FC > 2; False discovery rate, FDR < 0.05) showed dynamic changes (Fig. [69]1C). Fig. 1. [70]Fig. 1 [71]Open in a new tab Global transcriptome assessment of porcine MII oocytes and IVV embryos. A Schematic showing the preparation of porcine MII oocytes and IVV embryos for RNA-seq. For MII oocytes were collected by in vitro maturation; 2-cell, 4-cell, 8-cell and blastocyst embryos were collected by in vivo fertilization (IVV). RNA sequencing was performed using pools of 10 oocytes/embryos (3 replicates per group). B Principal component analysis was performed for all developmental stages with their replicates (MII, n = 3; 2-cell to blastocyst, n = 3). Each point represents a replicate. Developmental stages were clearly separated from each other and replicates within stages were grouped together. C Heatmaps showing the gene expression levels among oocytes, 2-cell, 4-cell, 8-cell and blastocyst embryos. Each column represents a replicate. D The scatter plots show the upregulation and downregulation DEGs between porcine IVV and SCNT embryo at the 2-cell, 4-cell and 8-cell stage embryos (FC > 2, FDR < 0.05). E Cluster analysis showing the gene expression levels form porcine MII oocytes to blastocysts Compared with other stages, the gene expression patterns of MII oocytes and 2-cell embryos were similar and 380 upregulated DEGs and 1639 downregulated DEGs (Fig. [72]1D) were identified, indicating that the IVV embryos were still under maternal regulation at this time. The gene expression pattern was significantly increased at the 4-cell stage and 3093 upregulated DEGs and 1676 downregulated DEGs were identified when compared with the 2-cell stage, and then decreased from the 8-cell to blastocyst stage, which indicated that the early embryo ZGA of pigs occurred at the 4-cell stage (Fig. [73]1D). The embryos began the transition from maternal to zygotic regulation at this time. The parallel coordinated expression analysis showed the changes in gene expression patterns from 2-cell and 8-cell embryos (Fig. [74]1E). Functional enrichment analysis of differentially expressed genes in porcine IVV embryos GO enrichment analysis of DEGs at various stages was performed. In terms of biological processes, the upregulated DEGs from the 2-cell to 4-cell stage were enriched in 10 biological processes, which were mainly related to nitrogen compound metabolic process. The upregulated DEGs from the 4-cell to 8-cell stage were enriched in 10 biological processes, which also were mainly related to metabolic process. The upregulated DEGs from the 8-cell to blastocyst stage were enriched in 10 biological processes, which were mainly related to single organism process (Fig. [75]2A). Meanwhile, the downregulated DEGs from MII oocytes to 2-cell stage were enriched in 10 biological processes, which were mainly related to metabolic process. The downregulated DEGs from the 2-cell to 4-cell stage were mainly enriched in organelle organization. The downregulated DEGs from the 4-cell to 8-cell stage were mainly enriched in cellular compound organization. The downregulated DEGs from the 8-cell to blastocyst stage were enriched in biosynthetic process and mitochondrial fusion (Fig. [76]2B). Fig. 2. [77]Fig. 2 [78]Open in a new tab Enrichment analysis and PPI network of porcine DEGs between MII oocytes and IVV embryos. A, B The enrichment analysis of DEGs and clustering of the upregulated and downregulated DEGs in biological processes. A Red arrows represent canonical pathways of DEGs with upregulated and (B) green arrows represent canonical pathways of DEGs with downregulated among MII oocytes, 2-cell, 4-cell, 8-cell and blastocyst stages. C, D PPI network of upregulated and downregulated DEGs among MII oocytes and embryos, at the 2-cell, 4-cell, 8-cell and blastocyst stage To further clarify the function of DEGs, the Protein-Protein Interaction network (PPI network) analysis of the DEGs was performed in the STRING database in early porcine embryos. The core gene clusters in the PPI network showed that the most closely interacting genes among the upregulated genes during the 2-cell to 4-cell stage were related to ribosome alternative splicing, and the core gene were NCBP1 and NCBP2 (Fig. [79]2C). The core gene among the downregulated genes during the 2-cell to 4-cell stage was TOP2B (Fig. [80]2D). Comparative analysis of transcriptome activity between porcine SCNT and IVV embryos To compare the transcriptomic profiles of IVV embryos and SCNT embryos during ZAG, SCNT embryos (2-cells, 4-cells and 8-cells) were collected for RNA-sequencing analysis (Fig. [81]3A). Principal component analysis showed that each experimental group was well biologically replicated, which proved the reliability of the data (Fig. [82]3B). The transcriptomic data of SCNT embryos showed that there was little difference in gene expression patterns at the 2-cell and 4-cell stage, which was significant different from the transcriptome dynamic changes in IVV embryos, so there was a ZGA barrier at the 4-cell stage of SCNT embryos (Fig. [83]3C, D). Fig. 3. [84]Fig. 3 [85]Open in a new tab Comparative analysis of transcriptome activity between porcine SCNT and IVV embryos. A Schematic showing the preparation of porcine SCNT embryos for RNA-seq. For 2-cell, 4-cell and 8-cell embryos were collected by SCNT. RNA sequencing was performed using pools of 10 oocytes/embryos (3 replicates per group). B Principal component analysis was performed for all developmental stages with their replicates. Each point represents a replicate. C, D Heatmaps and cluster analysis showing the global gene expression levels between SCNT and IVV embryos at the 2-cell, 4-cell and 8-cell stages. E Volcano plot of differentially expressed genes (DEGs) between porcine IVV 2-cell and MII oocytes stage, 4-cell and 2-cell stage embryos, 8-cell and 4-cell stage embryos, and blastocyst and 8-cell stage embryos (FC > 2, FDR < 0.05) Transcriptome data at the 2-cell stage showed that 1484 genes had more than two-fold differences in expression between the IVV and SCNT embryos (FC > 2, FDR < 0.05) (Fig. [86]3E). There were 5821 genes with differential expression levels of more than 2 times (FC > 2, FDR < 0.05) at the 4-cell stage and 2234 upregulated DEGs and 3587 downregulated DEGs were identified in SCNT embryos, compared with IVV embryos (Fig. [87]3F). In addition, 5605 genes with more than 2-fold expression levels were screened between the two embryos at the 8-cell stage (Fig. [88]3G). The results indicated that abnormal zygotic gene activation occurred in the 4-cell stage of porcine SCNT embryos, so we focused on the analysis of the DEGs at the 4-cell stage. Biological functions analysis of differentially expressed genes at the 4-cell stage To gain insight into the biological functions of the DEGs, a canonical KEGG pathway enrichment analysis was conducted. The persistent difference between IVV and SCNT embryos at the 4-cell stage were illustrated by an enrichment of four canonical pathways (Fig. [89]4). Except for a higher expression in SCNT versus IVV embryo of DEGs involved in ubiquitin mediated proteolysis, purine metabolism and endocytosis (Fig. [90]4A), the DEGs involved in metabolic pathways were lower expressed in SCNT than in IVV embryos (Fig. [91]4B). Fig. 4. [92]Fig. 4 [93]Open in a new tab KEGG enrichment analysis of porcine DEGs between SCNT and IVV embryos at the 4-cell stage. A Red arrows represent canonical pathways of DEGs that were significantly higher expressed and B green arrows represent canonical pathways of DEGs that were significantly lower expressed between SCNT and IVV embryos at the 4-cell stages Identification of critical transcription factors in porcine embryos during ZGA We analyzed the TFs of the DEGs during the ZGA stage, and found 107 upregulated and 90 downregulated TFs (Fig. [94]5A). The probability distribution of fold change in the expression of all activated transcription factors was conducted (Fig. [95]5B), and the top 20 TFs were listed according to their expression multiple, namely c-MYC, KLF4, EED, RBMX, CRABP2, ATF3, TGIF1, MYCN, SRSF, URL1, GSC, FUS, GATA6, ILF2, TFAP2C, EWSR1, PA2G4, CTCF, SNIP1 and FOS. We found that the expression levels of c-MYC, KLF4, EED, RBMX, CRABP2, ATF3 and TGIF1 were 5-fold higher and that c-MYC showed the highest fold expression change in upregulated TFs (Fig. [96]5C), which might play a critical role in porcine ZGA. The expression levels of c-MYC in IVV and SCNT embryos were verified by qPCR, and c-MYC had the significantly lower expression level in SCNT embryos, especially in the 4-cell stage (Fig. [97]5D). It is speculated that c-MYC may be an important transcription factor affecting zygotic gene activation. Fig. 5. [98]Fig. 5 [99]Open in a new tab Expression patterns of transcription factors in porcine embryos during ZGA. A Volcano plot of upregulated and downregulated DEGs and transcription factors (TFs) of porcine embryos during the ZGA. B Probability distribution of expression fold changes of all activated TFs in porcine embryos. C List of the top 20 activated TFs in porcine embryos. D Relative abundance of c-MYC in the porcine IVV and SCNT embryos, quantities were normalized to GAPDH abundance. Data are presented as the mean ± standard deviation. *, P < 0.05; **, P < 0.01 between groups Treatment with 10,058-F4 impeded the developmental competence of porcine embryos To verify the role of c-MYC in porcine embryonic development, porcine IVF embryos were treated with 10,058-F4 at 0, 0.5 uM, 1 uM, 2.5 uM, 5 uM, 7.5 uM, 10 uM, 25 uM, 50 uM or 100 uM for 48 h after fertilization. The cleavage rate and blastocyst rage of porcine IVF embryos were all significantly decreased (Table [100]1, Fig. [101]6A). Next, we studied the effects of the duration (0, 24 h, 48 h or 72 h) and 10,058-F4 treatment concentration (1 uM) on embryonic development capacity. Treatment with 10,058-F4 for 48 h or 72 h remarkably decreased the cleavage rate and blastocyst rate of IVF embryos (Table [102]2). Therefore, we applied 1 uM 10,058-F4 for 48 h treatment in subsequent experiments. Table 1. Effect of different concentrations of 10,058-F4 on the developmental competence of porcine embryo 10,058-F4 Treatment (uM) No. of embryos No. of embryos cleaved (% ± SEM) No. of blastocysts (% ± SEM) 0 150 76(50.8 ± 1.34)^A 51(34.0 ± 0.21)^A 0.5 uM 145 72(49.7 ± 1.58)^B 47(32.4 ± 1.94)^A 1 uM 150 57(38.0 ± 2.34)^B 35(23.3 ± 1.31)^B 2.5 uM 150 50(33.3 ± 0.17)^C 38(25.3 ± 2.47)^B 5 uM 155 63(40.6 ± 2.09)^B 46(29.7 ± 2.11)^B 7.5 uM 155 48(30.9 ± 1.12)^C 38(24.5 ± 0.45)^B 10 uM 150 48(32.0 ± 0.91)^C 31(20.7 ± 1.71)^B 25 uM 150 47(31.3 ± 2.21)^C 25(16.7 ± 2.19)^C 50 uM 150 32(21.3 ± 1.41)^D 8(5.3 ± 0.98)^D 100 uM 140 0(0.0 ± 0.00)^E 0(0.0 ± 0.00)^E [103]Open in a new tab Values in the same column with different superscripts from A to E differ significantly (P < 0.05) Fig. 6. [104]Fig. 6 [105]Open in a new tab Assessment of the development competence of porcine embryo after treatment with 10,058-F4. A Representative images of IVF embryos after culturing for 7 days by treatment with 10,058-F4 at 0, 0.5 uM,1 uM, 2.5 uM, 5 uM, 7.5 uM, 10 uM, 25 uM, 50 uM or 100 uM for 48 h, Scale bars, 50 mm. B Relative abundance of c-MYC (B) and ZGA-related genes ZSCAN4 (C), EIF1AX (D), USP26 (E) and YTHDC2 (F) in the porcine IVF embryos, quantities were normalized to GAPDH abundance. Data are presented as the mean ± standard deviation. *, P < 0.05; **, P < 0.01 between groups, as indicated. BLA, blastocyst. G Representative examples of fragmented nuclei (green), which are indicative of apoptosis, versus total nuclei (blue), Scale bar, 50 um Table 2. Effect of time duration of 10,058-F4(1μM) treatment on the developmental capacity of porcine embryo Time duration (h) No. of embryos No. of embryos cleaved (% ± SEM) No. of blastocysts (% ± SEM) 0 150 77(51.3 ± 2.23)^A 53(35.3 ± 0.67)^A 24 h 150 68(45.3 ± 1.11)^A 47(31.3 ± 1.47)^A 48 h 150 56(37.3 ± 0.43)^B 34(22.7 ± 2.13)^B 72 h 150 58(38.7 ± 0.67)^B 37(24.7 ± 1.63)^B [106]Open in a new tab Values in the same column with different superscripts (A and B) differ significantly (P < 0.05) Treatment with 10,058-F4 interfered with the transcript of ZGA-related genes and damaged the quality of porcine embryos Compared with SCNT embryos, the development process of IVF embryos is closer to that of in vivo embryos [[107]26, [108]34]. Therefore, we selected IVF embryos as a model to verify the effect of 10,058-F4 on early embryo development. We used qPCR to detect the expression changes of c-MYC. Our results showed that c-MYC had a significantly lower expression in the IVF embryos treated with 10,058-F4 compared with control embryos from the 2-cell to blastocyst stage (Fig. [109]6B). Differences in the expression of ZGA-related genes in 4-cell stage were evaluated using qPCR. Our results showed that the expression of ZSCAN4, EIF1AX, USP26 and YTHDC2 (Fig. [110]6C-F) was remarkably decreased in IVF embryos treated with 10,058-F4 compared with the control IVF embryos at the 4-cell stage, indicating that 10,058-F4 treatment impeded embryonic reprogramming during ZGA. To assess the quality of blastocysts, we used the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining to examine the apoptotic cells of blastocysts. TUNEL staining showed that the apoptotic rate of IVF blastocysts treated with 10,058-F4 was significantly higher than that of the control IVF blastocysts (Fig. [111]6G), in which 10,058-F4 treatment damaged the quality of blastocysts. Transcriptional activities of preimplantation embryos in mice, human and bovine We further performed cross-species transcription analysis to analyze the gene expression patterns in mouse, human, and bovine preimplantation embryos. In mice, the numbers of differentially expressed genes began to increase at the 2-cell stage, in which 1802 upregulated DEGs and 1641 downregulated DEGs were identified. The subsequent transformation of gene expression patterns occurred at the 8-cell stage, and 1516 upregulated DEGs and 1287 downregulated DEGs were identified (Fig. [112]7A, B). The parallel coordinates expression analysis showed the changes of gene expression from 1-cell to blastocyst using edge R (Fig. [113]7C). Thus, the activation of zygote genes in mice is completed in two stages, 2-cell and 8-cell stage. We named the two ZGAs as mouse-1 and mouse-2 activations in mice. Fig. 7. [114]Fig. 7 [115]Open in a new tab Global transcriptome assessment of preimplantation embryos in mice, human and bovine. A Heatmaps showing the gene expression levels of mouse embryos among 1-cell, 2-cell, 4-cell, 8-cell, morula and blastocyst stages. Each column represents a replicate. B Volcano plot of differentially expressed genes (DEGs) of mouse embryos between 2C vs. 1C, 4C vs. 2C, 8C vs. 4C, Mor vs. 8C and Bla vs. Mor (FC > 2, FDR < 0.05). C The expression of DEGs in mouse embryos during 1-cell to blastocyst stage. D Heatmaps showing the gene expression levels of human embryos among 1-cell, 2-cell, 4-cell, 8-cell, morula and blastocyst stage. Each column represents a replicate. E Volcano plot of differentially expressed genes (DEGs) of human embryos between 2C vs. 1C, 4C vs. 2C, 8C vs. 4C, Mor vs. 8C and Bla vs. Mor (FC > 2, FDR < 0.05). F The expression of DEGs of human embryos from 1-cell to blastocyst stage. G Heatmaps showing the gene expression levels of bovine embryo among oocytes, 1-cell, 2-cell, 4-cell, 8-cell, morula and blastocyst. Each column represents a replicate. H Volcano plot of differentially expressed genes (DEGs) of bovine embryos between 2C vs. 1C, 4C vs. 2C, 8C vs. 4C, 16C vs. 8C, Mor vs. 16C and Bla vs. Mor (FC > 2, FDR < 0.05). I The expression of DEGs in human embryos from oocytes to blastocyst stage. Mor, Morula; Bla, Blastocyst. J Schematic overview of the timing of zygotic genome activation in pigs, mice, humans and bovines For humans, the heatmap and parallel coordinated expression analysis showed that the gene expression range change appeared in 8-cell embryos (Fig. [116]7D-F), and 2158 upregulated DEGs and 2374 downregulated DEGs were identified, indicating that the zygote gene activation of human early embryos occurred at the 8-cell stage. In bovines, the heatmap and parallel coordinated expression analysis showed that the gene expression range changes appeared in 16-cell embryos (Fig. [117]7G-I), and 1156 upregulated DEGs and 1025 downregulated DEGs were identified, in which zygote gene activation of bovine early embryos occurred at the 16-cell stage. Taken together, comparative transcriptome analysis revealed different patterns and timing of zygotic genome activation in different species, with mice even requiring two rounds of zygotic genome activation for the complete reprogramming process (Fig. [118]7J). Functional enrichment analysis of DEGs during ZGA in four species The cross-species gene ontology (GO) enrichment analysis was performed on the DEGs of the ZGA in four species. In terms of biological process, the DEGs of the ZGA stage in pigs were enriched in 12 biological processes, which were mainly related to metabolic process, biological regulation, cellular component organization, response to stimulus, localization and multicellular organismal process. Among the other three species, bovines had the highest correlation with pigs, with a correlation coefficient of 0.972, followed by humans (0.937) and mice (0.934) (Fig. [119]8A). In terms of cellular component, the DEGs at the ZGA stage of pigs were enriched in 21 cellular components, mainly related to nucleus, membrane, macromolecular complexes, membrane-enclosed lumen, cytosol, vesicle and mitochondrion. Among the other three species, bovines and pigs were the most closely related, with a correlation coefficient of 0.956, followed by mice (0.950) and humans (0.936) (Fig. [120]8B). In terms of molecular function, the DEGs at the ZGA stage of porcine embryos were enriched in 15 molecular functions, which were mainly related to protein binding, nucleic acid binding, ion binding, hydrolase activity and nucleotide binding. Among the other three species, bovines had the highest correlation with pigs, with a correlation coefficient of 0.979, followed by mice (0.092) and humans (0.088) (Fig. [121]8C). Fig. 8. [122]Fig. 8 [123]Open in a new tab GO and KEGG enrichment analysis of DEGs in pigs, mice, humans and bovines during ZGA. A Enrichment of DEGs in biological processes and correlation coefficient of enrichment for four mammals. B Enrichment of DEGs in cellular component and correlation coefficient of enrichment for four mammals. C Enrichment of DEGs in molecular function and correlation coefficient of enrichment for four mammals. D Enrichment of DEGs in the KEGG pathways of four mammals The cross-species KEGG pathway enrichment analysis was performed on the differentially expressed genes of the ZGA in four species. The DEGs were enriched in 14 pathways, and ten of these pathways were shared among the four species. They were metabolic pathways, Huntington’s disease, oxidative phosphorylation, RNA transport, Alzheimer’s disease, Parkinson’s disease, non-alcoholic fatty liver disease, spliceosome, ribosome and proteasome. In addition, each of the four species had its own specific enrichment pathways (Fig. [124]8D). Comparative analysis between total transcripts and differentially expressed genes during zygotic gene activation in four species We analyzed the number of DEGs and total transcripts during ZGA in four species. The total number of DEGs in pigs was 4769, accounting for 10.7% of the total transcripts. The total number of DEGs during the first ZGA in mouse embryos was 3442, accounting for 15.2% of the total transcripts, and a total of 2803 DEGs during the second zygotic activation stage, accounting for 12.4% of the total transcripts. There were 4532 DEGs during the ZGA of human early embryos, accounting for 8.4% of the total scripts. A total of 2181 DEGs in bovines accounted for 9.1% of the total transcripts (Fig. [125]9A). Fig. 9. Fig. 9 [126]Open in a new tab Statistical analysis between total transcripts and differentially expressed genes during ZGA in four species. A The numbers of DEGs and total transcripts during the ZGA in human, bovine, pig and mouse. DEGs: Differentially Expressed Genes; ZGA: Zygotic Gene Activation Discussion In the early stage of mammalian embryo development, the dominant role in regulating development gradually shifts from maternal origin to zygote. The depletion of maternal factors and the activation of zygotic genes combine to reprogram terminally differentiated germ cells into totipotent embryos [[127]35]. Due to the rapid development of high-throughput sequencing technology, it is possible to use this technology to carry out transcriptome studies of early embryos, particularly in large mammals such as pigs and bovines, in which embryos are difficult to collect, to reveal the mechanism of mammalian gene transcription more widely and deeply. In the present study, we revealed the transcriptome profiles of porcine IVV and SCNT embryos and performed cross-species analysis of transcriptional activity during ZGA in pigs, mice, bovines and humans. We identified the top 20 transcription factors that were likely key players in activating the porcine zygotic genome and reported conserved features of the transcriptome of the four species during ZGA, which will broaden the current understanding of transcriptional activation during embryo reprogramming and provide references for future studies.