Porcine Early Embryonic Development Research Articles

ERK5 is essential for early porcine embryonic development by maintaining Endoplasmic Reticulum homeostasis

ERK5 is essential for early porcine embryonic development by maintaining Endoplasmic Reticulum homeostasis

Isoorientin Promotes Early Porcine Embryonic Development by Alleviating Oxidative Stress and Improving Lipid Metabolism

Isoorientin (ISO) is a natural lignan glycoside flavonoid found in various plants, including Charcot and Stonecrop. ISO exhibits diverse physiological and pharmacological effects, such as antioxidative, anti-inflammatory, hepatoprotective, antiviral, antianxiety, and anti-myocardial ischaemic properties, as well as lipid metabolism regulation. This study investigated the impact of ISO supplementation on oxidative stress and lipid accumulation in porcine early embryos, along with its underlying mechanisms. Porcine embryos were cultured in vitro under different concentrations of ISO (0, 1, 10, and 100 nM). The results revealed that 10 nM ISO significantly enhanced the blastocyst rate and total embryonic cell count in vitro. ISO-treated embryos exhibited reduced reactive oxygen species levels and elevated glutathione levels compared to the untreated group. In addition, ISO treatment significantly increased the expression of the key antioxidant regulator Nrf2, improved mitochondrial function, and reduced lipid droplet accumulation. Concurrently, early embryo autophagy and apoptosis levels decreased. Furthermore, ISO treatment upregulated antioxidant-related genes (SOD1, SOD2, and CAT) and mitochondrial biogenesis related genes (NRF1, NRF2, and SIRT1), while downregulating lipid synthesis-related genes (SREBP1 and FASN). Additionally, lipid hydrolysis-related genes (ACADS) were elevated. These findings collectively suggest that ISO may facilitate early embryonic development in pigs by ameliorating oxidative stress and lipid metabolism.

Transcriptome analysis of the effects of high temperature on zygotic genome activation in porcine embryos

Damage to the development of porcine gametes and embryos caused by high temperatures (HT) is one of the main reasons for the decline in the economic benefits of the livestock industry. Zygotic genome activation (ZGA) marks the beginning of gene expression programs in mammalian pre-implantation embryos. In pigs, ZGA occurs at the 4-cell (4 C) stage, indicating that correct gene expression at this stage plays an important regulatory role in embryonic development. However, the effect of the HT environment on early porcine embryonic development and the RNA expression profile of ZGA remain unclear. In this study, we compared the RNA transcription patterns of porcine 4 C embryos under normal and HT conditions using RNA-seq and identified 326 differentially expressed genes (DEGs). These changes were mainly related to DNA polymerase activity, DNA replication, and nucleotidyltransferase activity. In addition, entries for reverse transcription and endonuclease activity were enriched, indicating that ZGA interfered under HT conditions. Further comparison of the experimental results with the porcine ZGA gene revealed 39 ZGA genes among the DEGs. KEGG and GSEA analysis showed that the oxidative phosphorylation pathway was significantly enriched and signaling pathways related to energy metabolism were significantly downregulated. We also found that NDUFA6 and CDKN1A were located at the center of the protein-protein interaction network diagram of the DEGs. In summary, HT conditions affect mitochondrial function and oxidative phosphorylation levels, and lead to changes in the expression pattern of ZGA in early porcine embryos, with its hub genes NDUFA6 and CDKN1A.

CircHIRA sponges miR-196b-5p to promote porcine early embryonic development

Circular RNA (circRNA) is abundantly expressed in preimplantation embryos and embryonic stem cells in mice and humans. However, its function and mechanism in early development of mammalian embryos remain unclear. Here, we showed that circHIRA mediated miR-196b-5p to regulate porcine early embryonic development. We verified the circular feature of circHIRA by sanger sequencing, and proved the authenticity of circHIRA by enzyme digestion test. HIRA and circHIRA were expressed in porcine early embryos, and its expression levels significantly increased from 8-cell stage onwards and reached the maximum at the blastocyst stage. Functional studies revealed that circHIRA knockdown not only significantly reduced the developmental efficiency of embryos from 8-cell stage to blastocyst stage, but also impaired the blastocyst quality. Mechanistically, integrated analysis of miRNA prediction and gene expression showed that circHIRA knockdown significantly increased the expression of miR-196b-5p in porcine early embryos. Furthermore, miR-196b-5p inhibitor injection could rescue the early development of circHIRA knockdown embryos. Taken together, the findings reveal that circHIRA regulates porcine early embryonic development via inhibiting the expression of miR-196b-5p.

Lysine-specific demethylase 1 (LSD1) participate in porcine early embryonic development by regulating cell autophagy and apoptosis through the mTOR signaling pathway

Lysine-specific demethylase 1 (LSD1) stands as the pioneering histone demethylase uncovered, proficient in demethylating H3K4me1/2 and H3K9me1/2, thereby governing transcription and participating in cell apoptosis, proliferation, or differentiation. Nevertheless, the complete understanding of LSD1 during porcine early embryonic development and the underlying molecular mechanism remains unclear. Thus, we investigated the mechanism by which LSD1 plays a regulatory role in porcine early embryos. This study revealed that LSD1 inhibition resulted in parthenogenetic activation (PA) and in vitro fertilization (IVF) embryo arrested the development, and decreased blastocyst quality. Meanwhile, H3K4me1/2 and H3K9me1/2 methylase activity was increased at the 4-cell embryo stage. RNA-seq results revealed that autophagy related biological processes were highly enriched through GO and KEGG pathway analyses when LSD1 inhibition. Further studies showed that LSD1 depletion in porcine early embryos resulted in low mTOR and p-mTOR levels and high autophagy and apoptosis levels. The LSD1 deletion-induced increases in autophagy and apoptosis could be reversed by addition of mTOR activators. We further demonstrated that LSD1 inhibition induced mitochondrial dysfunction and mitophagy. In summary, our research results indicate that LSD1 may regulate autophagy and apoptosis through the mTOR pathway and affect early embryonic development of pigs.

Mangiferin improves early porcine embryonic development by reducing oxidative stress

Mangiferin improves early porcine embryonic development by reducing oxidative stress

IDH2 and GLUD1 depletion arrests embryonic development through an H4K20me3 epigenetic barrier in porcine parthenogenetic embryos.

Isocitrate dehydrogenase 2 (IDH2) and glutamate dehydrogenase 1 (GLUD1) are key enzymes involved in the production of α-ketoglutarate (α-KG), a metabolite central to the tricarboxylic acid cycle and glutamine metabolism. In this study, we investigated the impact of IDH2 and GLUD1 on early porcine embryonic development following IDH2 and GLUD1 knockdown (KD) via double-stranded RNA (dsRNA) microinjection. Results showed that KD reduced α-KG levels, leading to delayed embryonic development, decreased blastocyst formation, increased apoptosis, reduced blastomere proliferation, and pluripotency. Additionally, IDH2 and GLUD1 KD induced abnormally high levels of trimethylation of lysine 20 of histone H4 (H4K20me3) at the 4-cell stage, likely resulting in transcriptional repression of embryonic genome activation (EGA)-related genes. Notably, KD of lysine methyltransferase 5C ( KMT5C) and supplementation with exogenous α-KG reduced H4K20me3 expression and partially rescued these defects, suggesting a critical role of IDH2 and GLUD1 in the epigenetic regulation and proper development of porcine embryos. Overall, this study highlights the significance of IDH2 and GLUD1 in maintaining normal embryonic development through their influence on α-KG production and subsequent epigenetic modifications.

High Temperature-Induced m6A Epigenetic Changes Affect Early Porcine Embryonic Developmental Competence in Pigs.

N6-methyladenosine (m6A), the most prevalent modification in eukaryotic messenger RNA (mRNA), plays a key role in various developmental processes in mammals. Three proteins that affect RNA m6A modification have been identified: methyltransferases, demethylases, and m6A-binding proteins, known as "writer," "eraser," and "reader" proteins, respectively. However, changes in the m6A modification when early porcine embryos are exposed to stress remain unclear. In this study, we exposed porcine oocytes to a high temperature (HT, 41°C) for 10 h, after which the mature oocytes were parthenogenetically activated and cultured for 7 days to the blastocyst stage. HT significantly decreased the rates of the first polar body extrusion and blastocyst formation. Further detection of m6A modification found that HT can lead to increased expression levels of "reader," YTHDF2, and "writer," METTL3, and decreased expression levels of "eraser," FTO, resulting in an increased level of m6A modification in the embryos. Additionally, heat shock protein 70 (HSP70) is upregulated under HT conditions. Our study demonstrated that HT exposure alters m6A modification levels, which further affects early porcine embryonic development.

Inhibition of FSP1 impairs early embryo developmental competence in pigs

Ferroptosis suppressor protein 1 (FSP1) is a glutathione-independent ferroptosis inhibitory factor. FSP1 has been found to play a crucial role in the regulation of mitochondrial function and ferroptosis. However, its function in porcine early embryonic development remains unknown. In the present research, we found that FSP1 was expressed at different stages during porcine early embryo development. Compared with the control condition, inhibition of FSP1 reduced the cleavage rate at 24 h and 48 h and the blastocyst rate at 144 h. In addition, inhibiting FSP1 reduced the blastocyst diameter, total cell number, and proliferation capacity. Further analysis showed that inhibition of FSP1 significantly increased the levels of ferrous ions (Fe2+) and MDA but not GPX4. We also found that inhibition of FSP1 significantly decreased mitochondrial membrane potential and ATP levels, which in turn caused excessive accumulation of ROS and decreased the levels of GSH and the activity of the intracellular antioxidant enzymes SOD and CAT in embryos. In conclusion, FSP1, an important regulator, participates in regulating the development and quality of porcine early embryos. Inhibition of FSP1 impairs blastocyst formation, induces glutathione-independent ferroptosis, and further leads to oxidative stress due to mitochondrial dysfunction, ultimately affecting the developmental competence and impairing the quality of porcine early embryos.

18S ribosomal RNA methyltransferase METTL5-mediated CDX2 translation regulates porcine early embryo development1

N6-methyladenosine (m6A) plays a key role in mammalian early embryonic development and cell lineage differentiation. However, the role and mechanisms of 18S ribosomal RNA (rRNA) m6A methyltransferase METTL5 in early embryonic development remain unclear. Here, we found that 18S rRNA m6A methyltransferase METTL5 plays an important role in porcine early embryonic development. METTL5 knockdown and overexpression significantly reduced the developmental efficiency of porcine early embryos and impaired cell lineage allocation. METTL5 knockdown apparently decreased the global translation efficiency in blastocyst, while METTL5 overexpression increased the global translation efficiency. Furthermore, METTL5 knockdown did not affect the abundance of CDX2 mRNA, but resulted in a significant reduction in CDX2 protein levels. Moreover, the low developmental efficiency and abnormal lineage distribution of METTL5 knockdown embryos could be rescued by CDX2 overexpression. Collectively, our results demonstrated that 18S rRNA methyltransferase METTL5 regulates porcine early embryonic development via modulating the translation of CDX2.

Improving the developmental competences of porcine parthenogenetic embryos by Notoginsenoside R1-induced enhancement of mitochondrial activity and alleviation of proapoptotic events.

Notoginsenoside R1 (NGR1), derived from the Panax notoginseng root and rhizome, exhibits diverse pharmacological influences on the brain, neurons, and osteoblasts, such as antioxidant effects, mitochondrial function protection, energy metabolism regulation, and inhibition of oxygen radicals, apoptosis, and cellular autophagy. However, its effect on early porcine embryonic development remains unclear. Therefore, we investigated NGR1's effects on blastocyst quality, reactive oxygen species (ROS) levels, glutathione (GSH) levels, mitochondrial function, and embryonic development-related gene expression in porcine embryos by introducing NGR1 during the in vitro culture (IVC) of early porcine embryos. Our results indicate that an addition of 1 μM NGR1 significantly increased glutathione (GSH) levels, blastocyst formation rate, and total cell number and proliferation capacity; decreased ROS levels and apoptosis rates in orphan-activated porcine embryos; and improved intracellular mitochondrial distribution, enhanced membrane potential, and reduced autophagy. In addition, pluripotency-related factor levels were elevated (NANOG and octamer-binding transcription factor 4 [OCT4]), antioxidant-related genes were upregulated (nuclear factor-erythroid 2-related factor 2 [NRF2]), and apoptosis- (caspase 3 [CAS3]) and autophagy-related genes (light chain 3 [LC3B]) were downregulated. These results indicate that NGR1 can enhance early porcine embryonic development by protecting mitochondrial function.

BDE-47 Induces Mitochondrial Dysfunction and Endoplasmic Reticulum Stress to Inhibit Early Porcine Embryonic Development.

Widely used as a flame retardant, 2,2'4,4'-tetrabromodiphenyl ether (BDE-47) is a persistent environmental pollutant with toxicological effects, including hepatotoxicity, neurotoxicity, reproductive toxicity, and endocrine disruption. To investigate the toxicological effects of BDE-47 on early porcine embryogenesis in vitro, cultured porcine embryos were exposed to BDE-47 during early development. Exposure to 100 μM BDE-47 decreased the blastocyst rate and mRNA level of pluripotency genes but increased the level of LC3 and the expression of autophagy-related genes. After BDE-47 exposure, porcine embryos' antioxidant capability decreased; ROS levels increased, while glutathione (GSH) levels and the expression of antioxidant-related genes decreased. In addition, BDE-47 exposure reduced mitochondrial abundance and mitochondrial membrane potential levels, downregulated mitochondrial biogenesis-associated genes, decreased endoplasmic reticulum (ER) abundance, increased the levels of GRP78, a marker of ER stress (ERS), and upregulated the expression of ERS-related genes. However, ER damage and low embryo quality induced by BDE-47 exposure were reversed with the ERS inhibitor, the 4-phenylbutyric acid. In conclusion, BDE-47 inhibits the development of early porcine embryos in vitro by inducing mitochondrial dysfunction and ERS. This study sheds light on the mechanisms of BDE-47-induced embryonic toxicity.

Knock-down of YME1L1 induces mitochondrial dysfunction during early porcine embryonic development.

YME1L1, a mitochondrial metalloproteinase, is an Adenosine triphosphate (ATP)-dependent metalloproteinase and locates in the mitochondrial inner membrane. The protease domain of YME1L1 is oriented towards the mitochondrial intermembrane space, which modulates the mitochondrial GTPase optic atrophy type 1 (OPA1) processing. However, during embryonic development, there is no report yet about the role of YME1L1 on mitochondrial biogenesis and function in pigs. In the current study, the mRNA level of YME1L1 was knocked down by double strand RNA microinjection to the 1-cell stage embryos. The expression patterns of YME1L1 and its related proteins were performed by immunofluorescence and western blotting. To access the biological function of YME1L1, we first counted the preimplantation development rate, diameter, and total cell number of blastocyst on day-7. First, the localization of endogenous YME1L1 was found in the punctate structures of the mitochondria, and the expression level of YME1L1 is highly expressed from the 4-cell stage. Following significant knock-down of YME1L1, blastocyst rate and quality were decreased, and mitochondrial fragmentation was induced. YME1L1 knockdown induced excessive ROS production, lower mitochondrial membrane potential, and lower ATP levels. The OPA1 cleavage induced by YME1L1 knockdown was prevented by double knock-down of YME1L1 and OMA1. Moreover, cytochrome c, a pro-apoptotic signal, was released from the mitochondria after the knock-down of YME1L1. Taken together, these results indicate that YME1L1 is essential for regulating mitochondrial fission, function, and apoptosis during porcine embryo preimplantation development.

Rapamycin encourages the maintenance of mitochondrial dynamic balance and mitophagy activity for improving developmental competence of blastocysts in porcine embryos in vitro.

Rapamycin induces autophagosome formation and activity during oocyte maturation, improved fertilization ability of matured oocytes, and early embryonic developmental competence. However, potential changes in mitochondrial fission and mitophagy via regulation of autophagy in early porcine embryonic development have not been previously studied. Here, we investigated embryonic developmental ability and quality of porcine embryos 2 days after in vitro fertilization and following treatment with 1 and 10 nM rapamycin. As a results, 1 nM rapamycin exposure significantly improved (p < 0.05) blastocyst developmental competence compared to that in nontreated embryos (nontreated: 26.2 ± 5.7% vs. 1 nM rapamycin: 35.3 ± 5.1%). We observed autophagic (LC3B) and mitochondrial fission protein expression (dynamin-related protein-1 [DRP1] and pDRP1-Ser616) at the cleavage stage of 1 and 10 nM rapamycin-treated porcine embryos, using Western blot and immunofluorescence analyses. Interestingly, 1 nM rapamycin treatment significantly improved autophagy formation, mitochondrial activation, and mitochondrial fission protein levels (p < 0.05; p-DRP1 [Ser616]) at the cleavage stage of porcine embryos. Additionally, mitophagy was significantly increased in blastocysts treated with 1 nM rapamycin. In conclusion, our results suggest that rapamycin promotes blastocyst development ability in porcine embryos through mitochondrial fission, activation, and mitophagy in in vitro culture.

Zearalenone induces apoptosis and autophagy by regulating endoplasmic reticulum stress signalling in porcine trophectoderm cells.

Zearalenone (ZEA), a mycotoxin produced mainly by fungi belonging to Fusarium species in foods and feeds, causes a serious hazard to humans and animals. Numerous studies have revealed that ingesting ZEA can disrupt the reproductive function and impair the reproductive process in animals. This experiment was to investigate the toxicological effect and the mechanism of ZEA exposure on reproduction in pigs during early stages of pregnancy. In the present study, we treated with 0 to 80μmol/L ZEA for 12 or 24h in trophoblast ectoderm (pTr) cells. The results showed that ZEA had significantly decreased cell proliferation (P<0.05), which was accompanied by DNA damage-related cell cycle arrest at G2/M phase, activation of the apoptosis and endoplasmic reticulum (ER) stress, as well as impairment of barrier function (P<0.05). Western blot analysis and transmission electron microscopy (TEM) showed that exposure to ZEA can activation of autophagy in pTr cells. Importantly, pretreatment with chloroquine (CQ) or 3-methyladenine (3-MA) led to increased apoptosis in pTr cells. Interestingly, pTr cells pretreated with 4-phenylbutyric acid (4-PBA), an inhibitor of ER stress, resulted in reduced cell death in pTr cells, indicating a critical role for ER stress in the activation of autophagy. In conclusion, these results reveal that ZEA-triggered ER stress is critical for the cell fate decision of pTr cells during early porcine embryonic development. Application of small molecules with ability of blocking ER stress might be therapeutic option to reduce the deleterious effect of ZEA in pregnant animals.

ATF6 aggravates apoptosis in early porcine embryonic development by regulating organelle homeostasis under high-temperature conditions.

Activating transcription factor 6 (ATF6), one of the three sensor proteins in the endoplasmic reticulum (ER), is an important regulator of ER stress-induced apoptosis. ATF6 resides in the ER and, upon activation, is translocated to the Golgi apparatus, where it is cleaved by site-1 protease (S1P) to generate an amino-terminal cytoplasmic fragment. Although recent studies have made progress in elucidating the regulatory mechanisms of ATF6, its function during early porcine embryonic development under high-temperature (HT) stress remains unclear. In this study, zygotes were divided into four groups: control, HT, HT+ATF6 knockdown, and HT+PF (S1P inhibitor). Results showed that HT exposure induced ER stress, which increased ATF6 protein expression and led to a decrease in the blastocyst rate. Next, ATF6 expression was knocked down in HT embryos under microinjection of ATF6 double-stranded RNA (dsRNA). Results revealed that ATF6 knockdown (ATF6-KD) attenuated the increased expression of CHOP, an ER stress marker, and Ca 2+ release induced by HT. In addition, ATF6-KD alleviated homeostasis dysregulation among organelles caused by HT-induced ER stress, and further reduced Golgi apparatus and mitochondrial dysfunction in HT embryos. AIFM2 is an important downstream effector of ATF6. Results showed that ATF6-KD reduced the occurrence of AIFM2-mediated embryonic apoptosis at HT. Taken together, our findings suggest that ATF6 is a crucial mediator of apoptosis during early porcine embryonic development, resulting from HT-induced ER stress and disruption of organelle homeostasis.

ATF7-dependent epigenetic changes induced by high temperature during early porcine embryonic development.

Activating transcription factor 7 (ATF7) is a member of the ATF/cAMP response element (CRE) B superfamily. ATF2, ATF7, and CRE-BPa are present in vertebrates. Drosophila and fission yeast have only one homologue: dATF2 and Atf1, respectively. Under normal conditions, ATF7 promotes heterochromatin formation by recruiting histone H3K9 di- and tri-methyltransferases. Once the situation changes, all members are phosphorylated by the stress-activated kinase P38 in response to various stressors. However, the role of ATF7 in early porcine embryonic development remains unclear. In this study, we found that ATF7 gradually accumulated in the nucleus and then localized on the pericentric heterochromatin after the late 4-cell stage, while being co-localized with heterochromatin protein 1 (HP1). Knockdown of ATF7 resulted in decreases in the blastocyst rate and blastocyst cell number. ATF7 depletion resulted in downregulation of HP1 and histone 3 lysine 9 dimethylation (H3K9me2) expression. These effects were alleviated when P38 activity was inhibited. High temperatures increased the expression level of pP38, while reducing the quality of porcine embryos, and led to ATF7 phosphorylation. The expression level of H3K9me2 and HP1 was decreased and regulated by P38 activity. Stress-induced ATF7-dependent epigenetic changes play important roles in early porcine embryonic development.

Histone Methyltransferase SETD2 Is Required for Porcine Early Embryonic Development.

Simple SummaryNormal early embryonic development is important for ensuring sow fertility. Low quality of in vitro production embryos severely limits extensive application of porcine embryo engineering technologies in animal agriculture and the biomedicine field. Histone H3K36 methyltransferase SETD2 reportedly regulates oocyte maturation and preimplantation embryonic development in mice. However, the specific substrate and function of SETD2 in porcine early embryonic development remains unclear. Here, we show that SETD2 preferentially catalyzes H3K36me3 in porcine early embryos. SETD2 knockdown severely impeded blastocyst cavitation and perturbed normal allocation of inner cell mass and trophectoderm. SETD2 knockdown caused the apoptosis of cells within blastocysts. Therefore, SETD2 is essential for porcine early embryonic development. These findings provide a better understanding of porcine early embryonic development and lay a potential basis for improving the quality of porcine in vitro production embryos.SET domain-containing 2 (SETD2) is a methyltransferase that can catalyze the di- and tri-methylation of lysine 36 on histone H3 (H3K36me2/me3). SETD2 frequently mediates H3K36me3 modification to regulate both oocyte maturation and preimplantation embryonic development in mice. However, the specific substrate and function of SETD2 in porcine early embryonic development are still unclear. In this study, SETD2 preferentially catalyzed H3K36me3 to regulate porcine early embryonic development. SETD2 mRNA is dynamically expressed during early embryonic development. Functional studies using an RNA interference (RNAi) approach revealed that the expression levels of SETD2 mRNA were effectively knocked down by siRNA microinjection. Immunofluorescence analysis indicated that SETD2 knockdown (KD) did not affect H3K36me2 modification but significantly reduced H3K36me3 levels, suggesting a preferential H3K36me3 recognition of SETD2 in porcine embryos. Furthermore, SETD2 KD significantly reduced blastocyst rate and disrupted allocation between inner cell mass (ICM) and trophectoderm (TE) lineage. The expression levels of key genes important for specification of the first two lineages apparently decreased in SETD2 KD blastocysts. SETD2 KD markedly increased the apoptotic percentage of cells within embryos and altered the expression of pro- and anti-apoptotic genes. Therefore, our data indicate that SETD2 is essential for porcine early embryonic development.

Species-Specific Enhancer Activity of OCT4 in Porcine Pluripotency: The Porcine OCT4 Reporter System Could Monitor Pluripotency in Porcine Embryo Development and Embryonic Stem Cells.

The present study examined the activity and function of the pig OCT4 enhancer in the porcine early embryonic development stage and porcine authentic embryonic stem cells. OCT4 is known as a pluripotent regulator, and its upstream regulatory region-based dual-fluorescence protein reporter system controlled by distal and proximal enhancers is broadly used in studies examining the states and mechanism of pluripotency. We analyzed how this reporter system functions during early embryo development and in stem cells using a previously established porcine-specific reporter system. We demonstrated that the porcine OCT4 distal enhancer and proximal enhancer were activated with different expression patterns simultaneously as the expression of pluripotent marker genes changed during the development of in vitro pathenotes and the establishment of porcine embryonic stem cells (ESCs). This work demonstrates the applicability of the porcine OCT4 upstream region-derived dual-fluorescence reporter system, which may be applied to investigations of species-specific pluripotency in porcine-origin cells. These reporter systems may be useful tools for studies of porcine-specific pluripotency, early embryo development, and embryonic stem cells.

Vitrification of Pronuclear Zygotes Perturbs Porcine Zygotic Genome Activation.

Simple SummaryVitrification is a commonly used cryopreservation technique that has played an important role in the preservation of animal genetic resources and assisted reproduction in humans. However, the effect of vitrification on embryonic genome activation remains to be elucidated. This study found that vitrification reduced the developmental efficiency of pig blastocysts. Further studies found that vitrification reduced transcriptional activity during embryonic genome activation and disrupted gene expression. Our findings suggest that vitrification impairs genome activation in pig embryos, and our results can provide new insight into improving the developmental efficiency of vitrified embryos.Zygotic genome activation (ZGA) plays an essential role in early embryonic development. Vitrification is a common assisted reproductive technology that frequently reduces the developmental competence of embryos. However, the effect of vitrification on porcine ZGA and gene expression during ZGA remains largely unclear. Here, we found that vitrification of pronuclear zygotes derived from parthenogenetic activation (PA) and in vitro fertilization (IVF) resulted in a significant reduction in the rates of 2-cell, 4-cell, and blastocysts, but did not affect the quality of blastocysts. Functional research revealed that RNA polymerase II Inhibitor (α-amanitin) treatment significantly reduced global transcriptional activity and developmental efficiency of both 4-cell and 8-cell embryos, implying an essential role of ZGA in porcine early embryonic development. Furthermore, vitrification did not affect the synthesis of nascent mRNA of 2-cell embryos, but significantly inhibited global transcriptional activity of both 4-cell and 8-cell embryos, suggesting an impaired effect of vitrification on porcine ZGA. Correspondingly, the single-cell analysis showed that vitrification caused the downregulation or upregulation expression of maternal genes in 4-cell embryos, also significantly decreased the expression of zygotic genes. Taken together, these results indicated that vitrification of pronuclear zygotes impairs porcine zygotic genome activation.