Porcine Embryos Research Articles

Comparison Between Electroporation at Different Voltage Levels and Microinjection to Generate Porcine Embryos with Multiple Xenoantigen Knock-Outs

In the context of xenotransplantation, the production of genetically modified pigs is essential. For several years, knock-out pigs were generated through somatic cell nuclear transfer employing donor cells with the desired genetic modifications, which resulted in a lengthy and cumbersome procedure. The CRISPR/Cas9 system enables direct targeting of specific genes in zygotes directly through microinjection or electroporation. However, these techniques require improvement to minimize mosaicism and low mutation rates without compromising embryo survival. This study aimed to determine the gene editing potential of these two techniques to deliver multiplexed ribonucleotide proteins (RNPs) to generate triple-knock-out porcine embryos with a multi-transgenic background. We designed RNP complexes targeting the major porcine xenoantigens GGTA1, CMAH, and B4GALNT2. We then compared the development of mosaicism and gene editing efficiencies between electroporation and microinjection. Our results indicated a significant effect of voltage increase on molecule intake in electroporated embryos, without it notably affecting the blastocyst formation rate. Our gene editing analysis revealed differences among delivery approaches and gene loci. Notably, employing electroporation at 35 V yielded the highest frequency of biallelic disruptions. However, mosaicism was the predominant genetic variant in all RNP delivery methods, underscoring the need for further research to optimize multiplex genome editing in porcine zygotes.

Embryotrophic effect of exogenous protein contained adipose-derived stem cell extracellular vesicles

BackgroundExtracellular vesicles (EVs) regulate cell metabolism and various biological processes by delivering specific proteins and nucleic acids to surrounding cells. We aimed to investigate the effects of the cargo contained in EVs derived from adipose-derived stem cells (ASCs) on the porcine embryonic development.MethodsASCs were isolated from porcine adipose tissue and characterized using ASC-specific markers via flow cytometry. EVs were subsequently extracted from the conditioned media of the established ASCs. These EVs were added to the in vitro culture environment of porcine embryos to observe qualitative improvements in embryonic development. Furthermore, the proteins within the EVs were analyzed to investigate the underlying mechanisms.ResultsWe observed a higher blastocyst development rate and increased mitochondrial activity in early stage embryos in the ASC-EVs-supplemented group than in the controls (24.8% ± 0.8% vs. 28.6% ± 1.1%, respectively). The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of blastocysts also revealed significantly reduced apoptotic cells in the ASC-EVs-supplemented group. Furthermore, through proteomics, we detected the proteins in ASC-EVs and blastocysts from each treatment group. This analysis revealed a higher fraction of proteins in the ASC-EVs-supplemented group than in the controls (1,547 vs. 1,495, respectively). Gene analysis confirmed that ASC-EVs showed a high expression of tyrosine-protein kinase (SRC), whereas ASC-EVs supplemented blastocysts showed a higher expression of Cyclin-dependent kinase 1 (CDK1). SRC is postulated to activate protein kinase B (AKT), which inhibits the forkhead box O signaling pathway and activates CDK1. Subsequently, CDK1 activation influences the cell cycle, thereby affecting in vitro embryonic development.ConclusionASC-EVs promote mitochondrial activity, which is crucial for the early development of blastocysts and vital in the downregulation of apoptosis. Additionally, ASC-EVs supply SRC to porcine blastocysts, thereby elongating the cell cycle.

The splicing factor SF3B1 is essential for proper alternative splicing and zygotic genome activation in early porcine embryos

Alternative splicing (AS) is a pivotal posttranscriptional regulatory mechanism that is involved in embryonic development. However, the roles of AS in specific developmental events, especially the zygotic genome activation (ZGA) of porcine early embryos, remain unclear. In this study, we demonstrated that alternative splicing events (ASEs) were most prevalent in mammalian embryos during ZGA and that skipped exons were the predominant splicing pattern. When splicing factor 3B subunit 1 (SF3B1) was disrupted by the inhibitor pladienolide B (PlaB), we observed that porcine embryos were markedly arrested at the 4-cell stage. Concurrently, the main ZGA genes, namely, DPPA2, EIF6, and SORD, underwent aberrant splicing indicative of the failure of ZGA. Moreover, embryonic metabolic homeostasis was significantly disrupted at the 4-cell stage by SF3B1 inhibition, resulting in increases in the LDHA/LDHB ratio and lactate levels. Interestingly, the levels of the histone lactylation modifications pan-Kla and H4K5la also increased. Our findings enhance our understanding of early mammalian embryonic development, reveal the crucial role of porcine early embryogenesis, and help to resolve reproductive difficulties related to embryonic development.

A network of transient domains for breaking symmetry during anterior-posterior axis formation in the porcine embryo.

Breaking radial symmetry for anterior-posterior axis formation is one of the key developmental steps of vertebrate gastrulation and is established through a succession of transient domains defined by morphology or gene expression. Three such domains were interpreted recently in the rabbit to be part of a "three-anchor-point model" for axis formation. To answer the question as to whether the model is generally applicable to mammals, the dynamic expression patterns of four marker genes were analyzed in the pig, where gastrulating epiblast forms from half the inner cell mass: EOMES and PKDCC transcripts display decreasing expression intensities in the anterior hypoblast and-together with WNT3-increasing intensity in the anterior streak domain and the node; TBX6 expression changes from an initial central expression to exclusive expression in the posterior extremity of the primitive streak. The anterior streak domain has thus a molecular footprint similar to the one in the rabbit, the end node shares TBX6 between the species, while the anterior hypoblast-mirroring specific porcine epiblast derivation and fate-is marked by PKDCC instead of WNT3. The molecular similarities in transient domains point to conserved mechanisms for establishing the mammalian anterior-posterior axis and, possibly, breaking radial symmetry.

Delayed Blastocyst Formation Reduces the Quality and Hatching Ability of Porcine Parthenogenetic Blastocysts by Increasing DNA Damage, Decreasing Cell Proliferation, and Altering Transcription Factor Expression Patterns.

The purpose of this study was to investigate the influence of blastocyst formation timing on the quality of porcine embryos derived from parthenogenetic activation. Newly formed blastocysts at days 6, 7, and 8 of culture [termed formation 6, 7, and 8 blastocysts (F6, F7, and F8 blastocysts)] were obtained, and a series of parameters related to the quality of blastocysts, including apoptosis incidents, DNA replication, pluripotent factors, and blastocyst hatching capacity, were assessed. Delayed blastocyst formation (F7 and/or F8 blastocysts) led to increased levels of ROS, DNA damage, and apoptosis while decreasing the mitochondrial membrane potential, DNA replication, Oct4 levels, and numbers of Sox2-positive cells. F7 blastocysts showed a significantly reduced hatching rate compared to F6 blastocysts; however, F8 blastocysts were unable to develop to the hatching stage. Collectively, our findings suggest a negative correlation between delayed blastocyst formation and blastocyst quality.

Mitoquinone improves porcine embryo development through modulating oxidative stress and mitochondrial function

Oxidative stress caused by excess reactive oxygen species (ROS) is one of the main causes of low efficiency in in vitro production of embryos. These ROS can cause mitochondrial dysfunction and apoptosis, resulting in poor embryo development. Therefore, to prevent mitochondrial damage and apoptosis caused by ROS, we investigated the effects of mitoquinone (MitoQ), a mitochondrial-targeted antioxidant, on the in vitro culture (IVC) of porcine embryos. Various concentrations of MitoQ (0, 0.01, 0.1, or 1 nM) were supplemented during the entire period of IVC. The results showed that supplementation with 0.1 nM MitoQ significantly increased the blastocyst formation rate, with a higher total cell number including trophectoderm cell number and higher transcript expression of lineage-specific transcription factors in blastocysts. In addition, the 0.1 nM MitoQ-treated group showed a significantly lower percentage and number of apoptotic cells in blastocysts with positively regulated transcript expression of apoptosis-related genes. Therefore, 0.1 nM MitoQ was suggested as optimal concentration for porcine IVC and used for further investigations. MitoQ treatment significantly reduced intracellular ROS levels and increased glutathione levels in Day 2 embryos, with upregulated the transcript expression of antioxidant enzymes-related genes. Furthermore, the MitoQ group exhibited a significantly higher mitochondrial quantity, mitochondrial membrane potential, and ATP content in Day 2 embryos, with increased transcript expression of mitochondrial biogenesis-related genes. Taken together, these findings reveal that MitoQ supplementation can enhance the developmental competence of porcine embryos by decreasing oxidative stress and improving mitochondrial function.

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.

Vitamin C enhances the in vitro development of early porcine embryos by improving mitochondrial function

Mammalian embryos often suffer from oxidative stress in vitro, as the oxygen in the atmosphere is higher than that in the oviductal environment. Vitamin C (Vc) has been proven to enhance early embryonic development in vitro, but the underlying mechanism remains unclear. In this study, we investigated the pathways of action by which Vc promotes the in vitro development of porcine embryos. Comparative analysis of in vitro and in vivo gene expression profiles of morula found that most of the differentially expressed genes were enriched in pathways related to mitochondrial function. The addition of 12.5 μg/mL Vc to the culture medium significantly increased blastocyst production in a dose- and duration-dependent manner. Moreover, ROS levels were significantly higher in embryos cultured in the air (21% oxygen) than cultured in a hypoxic condition (5% oxygen) and were reduced by Vc supplementation. Vc also significantly increased the mitochondrial membrane potential levels and the expression levels of mitochondrial function-related genes (MFN1 and OPA1) and TCA cycle-related genes (PDHA1 and OGDH) in embryos cultured in vitro. These results suggest that the addition of Vc to the in vitro culture medium can increase the developmental potential and improve the mitochondrial function of early porcine embryos.

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.

Dynamics and necessity of SIRT1 for maternal–zygotic transition

Dynamic changes in maternal‒zygotic transition (MZT) require complex regulation of zygote formation, maternal transcript decay, embryonic genome activation (EGA), and cell cycle progression. Although these changes are well described, some key regulatory factors are still elusive. Sirtuin-1 (SIRT1), an NAD+-dependent histone deacetylase, is a versatile driver of MZT via its epigenetic and nonepigenetic substrates. This study focused on the dynamics of SIRT1 in early embryos and its contribution to MZT. A conditional SIRT1-deficient knockout mouse model was used, accompanied by porcine and human embryos. Embryos across mammalian species showed the prominent localization of SIRT1 in the nucleus throughout early embryonic development. Accordingly, SIRT1 interacts with histone H4 on lysine K16 (H4K16) in both mouse and human blastocysts. While maternal SIRT1 is dispensable for MZT, at least one allele of embryonic Sirt1 is required for early embryonic development around the time of EGA. This role of SIRT1 is surprisingly mediated via a transcription-independent mode of action.

Optimization of lipofection protocols for CRISPR/Cas9 delivery in porcine zona pellucida intact oocytes: A study of coincubation duration and reagent efficacy

A priority to facilitate the application of lipofection to generate genetically modified porcine embryos and animals will be the use of zona pellucida (ZP)-intact oocytes and zygotes. Recently, our group produced genetically modified embryos by lipofection of ZP-intact oocytes during in vitro fertilization (IVF). This study investigates the effect of two commercial lipofection reagents, Lipofectamine 3000 and Lipofectamine CRISPRMAX, on embryo development and mutation efficiency in ZP-intact porcine oocytes. We compared these reagents with the electroporation method and a control group using two sgRNAs targeting the CAPN3 and CD163 genes. The detrimental effects on cleavage rates were observed in both lipofection treatments compared to the control and electroporated groups. However, blastocyst rates were higher in the Lipofectamine 3000 group than in the electroporated group for both genes. Mutation parameters varied by target gene, with Lipofectamine 3000 achieving higher mutation rates for CD163, while all groups were similar for the CAPN3 gene. Overall efficiency was similar for both lipofectamines, confirming their feasibility for use. In addition, we evaluated the effect of coincubation time (4, 8, and 24 h) on IVF outcomes, embryo development, and mutation parameters. Results indicated that an 8-h coincubation period optimized fertilization and mutation efficiency without significant toxic effects. This study demonstrates that lipofection with either Lipofectamine 3000 or CRISPRMAX during IVF is an effective method for generating genetically modified porcine embryos without the need for specialized equipment or trained personnel, with efficiencies similar to or greater than electroporation. This study also highlights the importance of optimizing reagent selection and coincubation times. There is no difference between Lipofectamine 3000 and CRISPRMAXTM in terms of embryo development and mutation efficiency, and under our experimental conditions, the optimal coincubation time with lipofectamine is 8 h.

Knockdown of Y-box-binding protein 2 induces mitochondrial dysfunction to interrupt zygotic genome activation in porcine embryos

Knockdown of Y-box-binding protein 2 induces mitochondrial dysfunction to interrupt zygotic genome activation in porcine embryos

MAT2A is essential for zygotic genome activation by maintaining of histone methylation in porcine embryos

Methionine adenosyltransferase 2A (MAT2A) is an essential enzyme in the methionine cycle that generates S-adenosylmethionine (SAM) by reacting with methionine and ATP. SAM acts as a methyl donors for histone and DNA methylation, which plays key roles in zygotic genome activation (ZGA). However, the effects of MAT2A on porcine ZGA remain unclear. To investigate the function of MAT2A and its underlying mechanism in porcine ZGA, MAT2A was knocked down by double-stranded RNA injection at the 1-cell stage. MAT2A is highly expressed at every stage of porcine embryo development. The percentages of four-cell-stage embryos and blastocysts were lower in the MAT2A-knockdown (KD) group than in the control group. Notably, depletion of MAT2A decreased the levels of H3K4me2, H3K9me2/3, and H3K27me3 at the four-cell stage, whereas MAT2A KD reduced the transcriptional activity of ZGA genes. MAT2A KD decreased embryonic ectoderm development (EED) and enhancer of zeste homolog 2 (EZH2) expression. Exogenous SAM supplementation rescued histone methylation levels and developmental arrest induced by MAT2A KD. Additionally, MAT2A KD significantly increased DNA damage and apoptosis. In conclusion, MAT2A is involved in regulating transcriptional activity and is essential for regulating histone methylation during porcine ZGA.

Diosmetin Promotes Early Embryonic Development in Pigs by Alleviating Oxidative Stress.

Diosmetin (DIOS), a natural flavonoid monomer derived from lemons and present in various plants such as spearmint and spider moss, exhibits antioxidant, anti-inflammatory, and antiaging properties. Nonetheless, its impact on early embryonic development in pigs remains unexplored. This study aimed to determine the influence of DIOS supplementation in an in vitro culture (IVC) medium on porcine embryo development and to elucidate the underlying mechanisms. Findings revealed that embryos cultured in IVC medium with 0.1 μM DIOS demonstrated an increased blastocyst formation rate, higher total cell number, reduced LC3B and CASPASE3 levels, elevated Nrf2 levels, decreased ROS, and enhanced GSH and mitochondrial membrane potential at the 4-cell embryonic stage. Additionally, the expression of proapoptotic genes (CAS3, CAS8, and BAX) and autophagy-related genes (BECLIN1, ATG5, LC3B, and P62) was downregulated, whereas the expression of embryonic development-related genes (CDK1 and CDK2), antioxidant-related genes (SOD1 and SOD2), and mitochondrial biogenesis-related genes (NRF2) was upregulated. These findings suggest that DIOS promotes early embryonic development in pigs by mitigating oxidative stress and enhancing mitochondrial function, thereby reducing autophagy and apoptosis levels.

Oleanolic acid improves the in vitro developmental competence of early porcine embryos by reducing oxidative stress and ameliorating mitochondrial function.

Oleanolic acid (OA) is a pentacyclic triterpenoid with antioxidant activity that can be an effective scavenger of free radicals in cells. This study was designed to investigate the effects of OA on porcine early embryo developmental competence in vitro and its possible mechanisms of action. In the present study, parthenogenetically activated porcine embryos were used as models to assess the effect of OA on the in vitro developmental capacity of early porcine embryos in vitro. Zygotic genome activation, mitochondrial function, oxidative stress, cell proliferation and apoptosis in early porcine embryos were examined after supplementing the culture medium with 5 μM OA. The results showed that 5 μM OA supplementation not only significantly increased the blastocyst diameter in early porcine embryos on day 6 but also increased the total number of blastocysts. Furthermore, OA supplementation increased the blastocyst proliferation rate and decreased blastocyst apoptosis. Moreover, OA supplementation significantly increased the proportion of embryos that developed to the 4-cell stage after 48 h of in vitro culture and upregulated the expression of genes associated with zygotic genome activation (DPPA2 and ZSCAN4). Notably, OA alleviated oxidative stress by reducing the intracellular levels of reactive oxygen species and increasing the intracellular levels of reduced glutathione at the 4-cell stage and increased the activities of superoxide dismutase and catalase. Concurrently, OA significantly increased the mitochondrial membrane potential and intracellular ATP content. These results suggest that OA promotes the in vitro developmental competence of parthenogenetically activated porcine embryos by reducing oxidative stress and improving mitochondrial function during in vitro culture and that OA may contribute to the efficiency of in vitro embryo production.

GRK2 is critical for the cleavage of the porcine embryo by regulating HSP90 and the AKT pathway.

GRK2 deficiency disrupts the early embryonic development in pigs. The regulation of GRK2 on HSP90 and AKT may also play an important role during embryo development and tumor formation. Among the family of GPCR kinases (GRKs) that regulate receptor phosphorylation and signaling termination, G-protein-coupled receptor kinase 2 (GRK2) binds to HSP90 in response to hypoxia or other stresses. In this study, we investigated the effects of GRK2 knockdown and inhibition on porcine embryonic development from the zygote stage. Immunofluorescence and western blotting were used to determine the localization and expression, respectively, of GRK2 and related proteins. First, GRK2 and p-GRK2 were expressed in both the cytoplasm and membrane and co-localized with HSP90 on the membrane. The mRNA level of GRK2 increased until the 8C-morula stage, suggesting that GRK2 may play an essential role during the early development of the porcine embryos. GRK2 knockdown reduced porcine embryo development capacity and led to significantly decreased blastocyst quality. In addition, inhibition of GRK2 also induced poor ability of embryo development at an early stage, indicating that GRK2 is critical for embryonic cleavage in pigs. Knockdown and inhibition of GRK2 reduced HSP90 expression, AKT activation, and cAMP levels. Additionally, GRK2 deficiency increased LC3 expression, suggesting enhanced autophagy during embryo development. In summary, we showed that GRK2 binds to HSP90 on the membrane to regulate embryonic cleavage and AKT activation during embryonic development in pigs.

Mitochondria of Porcine Oocytes Synthesize Melatonin, Which Improves Their In Vitro Maturation and Embryonic Development.

The in vitro maturation efficiency of porcine oocytes is relatively low, and this limits the production of in vitro porcine embryos. Since melatonin is involved in mammalian reproductive physiology, in this study, we have explored whether endogenously produced melatonin can help in porcine oocyte in vitro maturation. We have found, for the first time in the literature, that mitochondria are the major sites for melatonin biosynthesis in porcine oocytes. This mitochondrially originated melatonin reduces ROS production and increases the activity of the mitochondrial respiratory electron transport chain, mitochondrial biogenesis, mitochondrial membrane potential, and ATP production. Therefore, melatonin improves the quality of oocytes and their in vitro maturation. In contrast, the reduced melatonin level caused by siRNA to knockdown AANAT (siAANAT) is associated with the abnormal distribution of mitochondria, decreasing the ATP level of porcine oocytes and inhibiting their in vitro maturation. These abnormalities can be rescued by melatonin supplementation. In addition, we found that siAANAT switches the mitochondrial oxidative phosphorylation to glycolysis, a Warburg effect. This metabolic alteration can also be corrected by melatonin supplementation. All these activities of melatonin appear to be mediated by its membrane receptors since the non-selective melatonin receptor antagonist Luzindole can blunt the effects of melatonin. Taken together, the mitochondria of porcine oocytes can synthesize melatonin and improve the quality of oocyte maturation. These results provide an insight from a novel aspect to study oocyte maturation under in vitro conditions.

The coculture of in vitro produced porcine embryos and oviductal epithelial cells improves blastocyst formation and modify embryo quality

The efficiency of in vitro embryo production in mammals is influenced by variables associated with culture conditions during maturation, fertilization, and embryonic development. The embryos obtained often exhibit low quality due to suboptimal in vitro culture conditions compared to the in vivo environment. Co-culturing gametes and embryos with somatic cells has been developed to enhance in vitro culture conditions. This study aimed to assess the impact of coculturing in vitro-produced porcine embryos with porcine oviductal epithelial cells (POEC) on embryo development and quality. Firstly, a pure culture of POEC suitable for coculture systems was established. The epithelial origin of the cells was confirmed by the expression of E-cadherin and cytokeratin. The expression pattern of hormone receptors aligned with the diestrous oviduct, and POEC also secreted oviductal glycoprotein type 1 (OVGP-1). Secondly, POEC from passage 1 (POEC-1) were used to coculture with in vitro-produced porcine embryos. A successful coculture system was established without the addition of fetal bovine serum as a supplement. Coculturing POEC-1 in monolayers with in vitro-produced porcine embryos during the initial two days of culture enhanced the percentage of blastocysts and their hatching. Although the coculture did not alter the number of cells in the blastocysts or apoptosis assessed by TUNEL, it significantly reduced reactive oxygen species (ROS) levels in cleaved porcine embryos. This study represents the first report evaluating the quality of porcine embryos produced by IVF in coculture systems and assessing ROS levels in cleaved porcine embryos obtained by IVF.

Dynamic intrauterine crosstalk promotes porcine embryo implantation during early pregnancy.

Dynamic crosstalk between the embryo and mother is crucial during implantation. Here, we comprehensively profile the single-cell transcriptome of pig peri-implantation embryos and corresponding maternal endometrium, identifying 4 different lineages in embryos and 13 cell types in the endometrium. Cell-specific gene expression characterizes 4 distinct trophectoderm subpopulations, showing development from undifferentiated trophectoderm to polar and mural trophectoderm. Dynamic expression of genes in different types of endometrial cells illustrates their molecular response to embryos during implantation. Then, we developed a novel tool, ExtraCellTalk, generating an overall dynamic map of maternal-foetal crosstalk using uterine luminal proteins as bridges. Through cross-species comparisons, we identified a conserved RBP4/STRA6 pathway in which embryonic-derived RBP4 could target the STRA6 receptor on stromal cells to regulate the interaction with other endometrial cells. These results provide insight into the maternal-foetal crosstalk during embryo implantation and represent a valuable resource for further studies to improve embryo implantation.

Transcriptome analysis of porcine oocytes during postovulatory aging

Decreased oocyte quality is a significant contributor to the decline in female fertility that accompanies aging in mammals. Oocytes rely on mRNA stores to support their survival and integrity during the protracted period of transcriptional dormancy as they await ovulation. However, the changes in mRNA levels and interactions that occur during porcine oocyte maturation and aging remain unclear. In this study, the mRNA expression profiles of porcine oocytes during the GV, MII, and aging (24 h after the MII stage) stages were explored by transcriptome sequencing to identify the key genes and pathways that affect oocyte maturation and postovulatory aging. The results showed that 10,929 genes were coexpressed in porcine oocytes during the GV stage, MII stage, and aging stage. In addition, 3037 genes were expressed only in the GV stage, 535 genes were expressed only in the MII stage, and 120 genes were expressed only in the aging stage. The correlation index between the GV and MII stages (0.535) was markedly lower than that between the MII and aging stages (0.942). A total of 3237 genes, which included 1408 upregulated and 1829 downregulated genes, were differentially expressed during porcine oocyte postovulatory aging (aging stage vs. MII stage). Key functional genes, including ATP2A1, ATP2A3, ATP2B2, NDUFS1, NDUFA2, NDUFAF3, SREBF1, CYP11A1, CYP3A29, GPx4, CCP110, STMN1, SPC25, Sirt2, SYCP3, Fascin1/2, PFN1, Cofilin, Tmod3, FLNA, LRKK2, CHEK1/2, DDB1/2, DDIT4L, and TONSL, and key molecular pathways, such as the calcium signaling pathway, MAPK signaling pathway, TGF-β signaling pathway, PI3K/Akt signaling pathway, FoxO signaling pathway, gap junctions, and thermogenesis, were found in abundance during porcine postovulatory aging. These genes are mainly involved in the regulation of many biological processes, such as oxidative stress, calcium homeostasis, mitochondrial function, and lipid peroxidation, during porcine oocyte postovulatory aging. These results contribute to a more in-depth understanding of the biological changes, key regulatory genes and related biological pathways that are involved in oocyte aging and provide a theoretical basis for improving the efficiency of porcine embryo production in vitro and in vivo.