Protein Dynamic Landscape of Pig Embryos during Peri-Implantation Development.

Properly developed embryos are critical for successful embryo implantation. The dynamic landscape of proteins as executors of biological processes in pig peri-implantation embryos has not been reported so far. In this study, we collected pig embryos from days 9, 12, and 15 of pregnancy during the peri-implantation stage for a PASEF-based quantitative proteomic analysis. In total, approximately 8000 proteins were identified. These proteins were classified as stage-exclusive proteins and stage-specific proteins, respectively, based on their presence and dynamic abundance changes at each stage. Functional analysis showed that their roles are consistent with the physiological processes of corresponding stages, such as the biosynthesis of amino acids and peptides at P09, the regulation of actin cytoskeletal organization and complement activation at P12, and the vesicular transport at P15. Correlation analysis between mRNAs and proteins showed a general positive correlation between pig peri-implantation embryonic mRNAs and proteins. Cross-species comparisons with human early embryos identified some conserved proteins that may be important in regulating embryonic development, such as STAT3, AP2A1, and PFAS. Our study provides a comprehensive overview of the pig embryo proteome during implantation, fills gaps in relevant developmental studies, and identifies some important proteins that may serve as potential targets for future research.

RASGRP1 targeted by H3K27me3 regulates myoblast proliferation and differentiation in mice and pigs.

Skeletal muscle is not only the largest organ in the body that is responsible for locomotion and exercise but also crucial for maintaining the body's energy metabolism and endocrine secretion. The trimethylation of histone H3 lysine 27 (H3K27me3) is one of the most important histone modifications that participates in muscle development regulation by repressing the transcription of genes. Previous studies indicate that the RASGRP1 gene is regulated by H3K27me3 in embryonic muscle development in pigs, but its function and regulatory role in myogenesis are still unclear. In this study, we verify the crucial role of H3K27me3 in RASGRP1 regulation. The gain/loss function of RASGRP1 in myogenesis regulation is performed using mouse myoblast C2C12 cells and primarily isolated porcine skeletal muscle satellite cells (PSCs). The results of qPCR, western blot analysis, EdU staining, CCK-8 assay and immunofluorescence staining show that overexpression of RASGRP1 promotes cell proliferation and differentiation in both skeletal muscle cell models, while knockdown of RASGRP1 leads to the opposite results. These findings indicate that RASGRP1 plays an important regulatory role in myogenesis in both mice and pigs.

Gender Control of Mouse Embryos by Activation of TLR7/8 on X Sperm via Ligands dsRNA-40 and dsRNA-DR.

Gender control technologies are promising for enhancing the production efficiency of the farm animal industry, and preventing sex-linked hereditary diseases in humans. It has been shown that the X sperm of mammalian animals specifically expresses X-chromosome-derived toll-like receptor 7/8 (TLR7/8), and the activation of TLR7/8 on the X sperm by their agonist, R848, can separate X and Y sperm via the specific inhibition of X sperm motility. The use of R848-preselected sperm for fertilization resulted in sex-ratio-skewed embryos or offspring. In this study, we aimed to investigate whether two other TLR7/8 ligands, double-stranded RNA-40 (dsRNA-40) and double-stranded RNA-DR (dsRNA-DR), are also effective in the separation of mouse X and Y sperm and the subsequent generation of gender-ratio-skewed in vitro fertilization (IVF) embryos. Our results indicated that cholesterol modification significantly enhances the transfection of dsRNA-40 and dsRNA-DR into sperm cells. dsRNA-40 and dsRNA-DR incubation with mouse sperm could separate X and Y sperm by the specific suppression of X sperm motility by decreasing its ATP level and mitochondrial activity. The use of a dsRNA-40- or dsRNA-DR-preselected upper layer of sperm, which predominantly contains high-motility Y sperm, for IVF caused a male-biased sex ratio shift in resulting embryos (with 65.90-74.93% of embryos being male). This study develops a simple new method for the efficient separation of mammalian X and Y sperm, enabling the selective production of male or female progenies.

Identification and characterization of circRNAs in peri-implantation endometrium between Yorkshire and Erhualian pigs.

BACKGROUND: One of the most critical periods for the loss of pig embryos is the 12th day of gestation when implantation begins. Recent studies have shown that non-coding RNAs (ncRNAs) play important regulatory roles during pregnancy. Circular RNAs (circRNAs) are a kind of ubiquitously expressed ncRNAs that can directly regulate the binding proteins or regulate the expression of target genes by adsorbing micro RNAs (miRNA). RESULTS: We used the Illumina Novaseq6,000 technology to analyze the circRNA expression profile in the endometrium of three Erhualian (EH12) and three Yorkshire (YK12) pigs on day 12 of gestation. Overall, a total of 22,108 circRNAs were identified. Of these, 4051 circRNAs were specific to EH12 and 5889 circRNAs were specific to YK12, indicating a high level of breed specificity. Further analysis showed that there were 641 significant differentially expressed circRNAs (SDEcircRNAs) in EH12 compared with YK12 (FDR < 0.05). Functional enrichment of differential circRNA host genes revealed many pathways and genes associated with reproduction and regulation of embryo development. Network analysis of circRNA-miRNA interactions further supported the idea that circRNAs act as sponges for miRNAs to regulate gene expression. The prediction of differential circRNA binding proteins further explored the potential regulatory pathways of circRNAs. Analysis of SDEcircRNAs suggested a possible reason for the difference in embryo survival between the two breeds at the peri-implantation stage. CONCLUSIONS: Together, these data suggest that circRNAs are abundantly expressed in the endometrium during the peri-implantation period in pigs and are important regulators of related genes. The results of this study will help to further understand the differences in molecular pathways between the two breeds during the critical implantation period of pregnancy, and will help to provide insight into the molecular mechanisms that contribute to the establishment of pregnancy and embryo loss in pigs.

Uterine luminal-derived extracellular vesicles: potential nanomaterials to improve embryo implantation.

Most pregnancy losses worldwide are caused by implantation failure for which there is a lack of effective therapeutics. Extracellular vesicles are considered potential endogenous nanomedicines because of their unique biological functions. However, the limited supply of ULF-EVs prevents their development and application in infertility diseases such as implantation failure. In this study, pigs were used as a human biomedical model, and ULF-EVs were isolated from the uterine luminal. We comprehensively characterized the proteins enriched in ULF-EVs and revealed their biological functions in promoting embryo implantation. By exogenously supplying ULF-EVs, we demonstrated that ULF-EVs improve embryo implantation, suggesting that ULF-EVs are a potential nanomaterial to treat implantation failure. Furthermore, we identified that MEP1B is important in improving embryo implantation by promoting trophoblast cell proliferation and migration. These results indicated that ULF-EVs can be a potential nanomaterial to improve embryo implantation.

Fisetin Delays Postovulatory Oocyte Aging by Regulating Oxidative Stress and Mitochondrial Function through Sirt1 Pathway.

The quality of oocytes determines the development potential of an embryo and is dependent on their timely fertilization after ovulation. Postovulatory oocyte aging is an inevitable factor during some assisted reproduction technology procedures, which results in poor fertilization rates and impairs embryo development. We found that fisetin, a bioactive flavonol contained in fruits and vegetables, delayed postovulatory oocyte aging in mice. Fisetin improved the development of aged oocytes after fertilization and inhibited the Sirt1 reduction in aged oocytes. Fisetin increased the GSH level and Sod2 transcription level to inhibit ROS accumulation in aged oocytes. Meanwhile, fisetin attenuated aging-induced spindle abnormalities, mitochondrial dysfunction, and apoptosis. At the molecular level, fisetin decreased aging-induced aberrant expression of H3K9me3. In addition, fisetin increased the expression levels of the mitochondrial transcription factor Tfam and the mitochondrial genes Co2 and Atp8 by upregulating Sirt1 in aged oocytes. Finally, inhibition of Sirt1 reversed the anti-aging effects of fisetin. Taken together, fisetin delayed postovulatory oocyte aging by upregulating Sirt1.

Effects of overexpression of histone H3K9me3 demethylase on development of porcine cloned embryos.

The abnormal modification of histone is an important factor restricting development of porcine cloned embryos. Overexpression of histone H3K9me3 demethylase KDM4 family can effectively improve the developmental efficiency of cloned embryos. In order to explore the effects of overexpression of H3K9me3 demethylase on the development of porcine cloned embryos, KDM4A mRNA and KDM4D mRNA were injected respectively into porcine cloned embryos at the 1-cell stage and 2-cell stage to detect the blastocyst rate; 2-cell stage cloned embryos injected with KDM4A mRNA and embryo injection water (the control group) at the 1-cell stage were collected to detect the expression level of H3K9me3, and 4-cell stage cloned embryos were collected for single cell transcriptome sequencing, then the sequencing data was analyzed with KEGG and GO. The results showed that the blastocyst rate of porcine cloned embryos injected with KDM4A mRNA at 1-cell stage was significantly higher than that of the control group (25.32 ± 0.74% vs 14.78 ± 0.87%), while cloned embryos injected with KDM4D mRNA had a similar blastocyst rate with cloned embryos in control group (16.27 ± 0.77% vs 14.78 ± 0.87%). Porcine cloned embryos injected with KDM4A mRNA and KDM4D mRNA at 2-cell stage had a similar blastocyst rate with cloned embryos in control group (32.18 ± 1.67%, 30.04 ± 0.91% vs 31.22 ± 1.40%). The expression level of H3K9me3 in cloned embryos injected with KDM4A mRNA at 1-cell stage was lower than that in control group. There were 133 differentially expressed genes detected by transcriptome sequencing, including 52 up-regulated genes and 81 down-regulated genes. Pathways enriched by GO analyses were mainly related to protein localization. Pathways enriched by KEGG analyses were related to cellular senescence and acute myeloid leukemia. These results suggest that overexpression of histone H3K9me3 demethylase KDM4A can significantly improve the developmental efficiency of porcine cloned embryos.

Comprehensive analysis of pre-mRNA alternative splicing regulated by m6A methylation in pig oxidative and glycolytic skeletal muscles.

BACKGROUND: Different types of skeletal myofibers exhibit distinct physiological and metabolic properties that are associated with meat quality traits in livestock. Alternative splicing (AS) of pre-mRNA can generate multiple transcripts from an individual gene by differential selection of splice sites. N6-methyladenosine (m6A) is the most abundant modification in mRNAs, but its regulation for AS in different muscles remains unknown.  RESULTS: We characterized AS events and m6A methylation pattern in pig oxidative and glycolytic muscles. A tota1 of 1294 differential AS events were identified, and differentially spliced genes were significantly enriched in processes related to different phenotypes between oxidative and glycolytic muscles. We constructed the regulatory network between splicing factors and corresponding differential AS events and identified NOVA1 and KHDRBS2 as key splicing factors. AS event was enriched in m6A-modified genes, and the methylation level was positively correlated with the number of AS events in genes. The dynamic change in m6A enrichment was associated with 115 differentially skipping exon (SE-DAS) events within 92 genes involving in various processes, including muscle contraction and myofibril assembly. We obtained 23.4% SE-DAS events (27/115) regulated by METTL3-meditaed m6A and experimentally validated the aberrant splicing of ZNF280D, PHE4DIP, and NEB. The inhibition of m6A methyltransferase METTL3 could induce the conversion of oxidative fiber to glycolytic fiber in PSCs. CONCLUSION: Our study suggested that m6A modification could contribute to significant difference in phenotypes between oxidative and glycolytic muscles by mediating the regulation of AS. These findings would provide novel insights into mechanisms underlying muscle fiber conversion.

The histone variant H3.3 promotes the active chromatin state to repress flowering in Arabidopsis.

The histone H3 family in animals and plants includes replicative H3 and nonreplicative H3.3 variants. H3.3 preferentially associates with active transcription, yet its function in development and transcription regulation remains elusive. The floral transition in Arabidopsis (Arabidopsis thaliana) involves complex chromatin regulation at a central flowering repressor FLOWERING LOCUS C (FLC). Here, we show that H3.3 upregulates FLC expression and promotes active histone modifications histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 36 trimethylation (H3K36me3) at the FLC locus. The FLC activator FRIGIDA (FRI) directly mediates H3.3 enrichment at FLC, leading to chromatin conformation changes and further induction of active histone modifications at FLC. Moreover, the antagonistic H3.3 and H2A.Z act in concert to activate FLC expression, likely by forming unstable nucleosomes ideal for transcription processing. We also show that H3.3 knockdown leads to H3K4me3 reduction at a subset of particularly short genes, suggesting the general role of H3.3 in promoting H3K4me3. The finding that H3.3 stably accumulates at FLC in the absence of H3K36me3 indicates that the H3.3 deposition may serve as a prerequisite for active histone modifications. Our results reveal the important function of H3.3 in mediating the active chromatin state for flowering repression.

Cap-binding complex assists RNA polymerase II transcription in plant salt stress response.

Adaptive response to stress involves an extensive reprogramming of gene expression. Under stressful conditions, the induction of efficient changes in messenger RNA (mRNA) production is crucial for maximized plant survival. Transcription and pre-mRNA processing are two closely related steps in mRNA biogenesis, yet how they are controlled in plant stress response remains elusive. Here, we show that the Arabidopsis nuclear cap-binding complex (CBC) component CBP20 directly interacts with ELF7, a subunit of the transcription elongation factor RNA Pol II-associated factor 1 complex (PAF1c) to promote RNA Pol II transcription in plant response to salt stress. CBP20 and ELF7 coregulate the expression of a large number of genes including those crucial for salt tolerance. Both CBP20 and ELF7 are required for enhanced RNA Pol II elongation at salt-activated genes. Though CBP20 also regulates intron splicing, this function is largely independent of ELF7. Our study reveals the function of an RNA processing regulator CBC in assisting efficient RNA Pol II transcription and pinpoints the complex roles of CBC on mRNA production in plant salt stress resistance.

ITGB6 inhibits the proliferation of porcine skeletal muscle satellite cells.

The formation of embryonic muscle fibers determines the amount of postnatal muscles and is regulated by a variety of signaling pathways and transcription factors. Previously, by using chromatin immunoprecipitation-sequencing and RNA-Seq techniques, we identified a large number of genes that are regulated by H3K27me3 in porcine embryonic skeletal muscles. Among these genes, we found that ITGB6 is regulated by H3K27me3. However, its function in muscle development is unknown. In this study, we first verified that ITGB6 was differentially regulated by H3K27me3 and that its expression levels were upregulated in porcine skeletal muscles at embryonic Days 33, 65, and 90. Then, we performed gain- or loss-of-function studies on porcine skeletal muscle satellite cells to study the role of ITGB6 in porcine skeletal muscle development. The proliferation of porcine skeletal muscle satellite cells was studied through real-time polymerase chain reaction, Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine staining, Western blot, and flow cytometry analyses. We found that the ITGB6 gene was regulated by H3K27me3 during muscle development and had an inhibitory effect on the proliferation of porcine skeletal muscle satellite cells.

Early Gonadal Development and Sex Determination in Mammal.

Sex determination is crucial for the transmission of genetic information through generations. In mammal, this process is primarily regulated by an antagonistic network of sex-related genes beginning in embryonic development and continuing throughout life. Nonetheless, abnormal expression of these sex-related genes will lead to reproductive organ and germline abnormalities, resulting in disorders of sex development (DSD) and infertility. On the other hand, it is possible to predetermine the sex of animal offspring by artificially regulating sex-related gene expression, a recent research hotspot. In this paper, we reviewed recent research that has improved our understanding of the mechanisms underlying the development of the gonad and primordial germ cells (PGCs), progenitors of the germline, to provide new directions for the treatment of DSD and infertility, both of which involve manipulating the sex ratio of livestock offspring.

Differential MicroRNA Expression in Porcine Endometrium Related to Spontaneous Embryo Loss during Early Pregnancy.

Litter size is an important indicator to measure the production capacity of commercial pigs. Spontaneous embryo loss is an essential factor in determining sow litter size. In early pregnancy, spontaneous embryo loss in porcine is as high as 20-30% during embryo implantation. However, the specific molecular mechanism underlying spontaneous embryo loss at the end of embryo implantation remains unknown. Therefore, we comprehensively used small RNA sequencing technology, bioinformatics analysis, and molecular experiments to determine the microRNA (miRNA) expression profile in the healthy and arresting embryo implantation site of porcine endometrium on day of gestation (DG) 28. A total of 464 miRNAs were identified in arresting endometrium (AE) and healthy endometrium (HE), and 139 differentially expressed miRNAs (DEMs) were screened. We combined the mRNA sequencing dataset from the SRA database to predict the target genes of these miRNAs. A quantitative real-time PCR assay identified the expression levels of miRNAs and mRNAs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed on differentially expressed target genes of DEMs, mainly enriched in epithelial development and amino acids metabolism-related pathways. We performed fluorescence in situ hybridization (FISH) and the dual-luciferase report gene assay to confirm miRNA and predicted target gene binding. miR-205 may inhibit its expression by combining 3'-untranslated regions (3' UTR) of tubulointerstitial nephritis antigen-like 1 (TINAGL1). The resulting inhibition of angiogenesis in the maternal endometrium ultimately leads to the formation of arresting embryos during the implantation period. This study provides a reference for the effect of miRNA on the successful implantation of pig embryos in early gestation.

Knockdown of YY1 Inhibits XIST Expression and Enhances Cloned Pig Embryo Development.

The technique of cloning has wide applications in animal husbandry and human biomedicine. However, the very low developmental efficiency of cloned embryos limits the application of cloning. Ectopic XIST-expression-induced abnormal X chromosome inactivation (XCI) is a primary cause of the low developmental competence of cloned mouse and pig embryos. Knockout or knockdown of XIST improves cloning efficiency in both pigs and mice. The transcription factor Yin yang 1(YY1) plays a critical role in XCI by triggering the transcription of X-inactive specific transcript (XIST) and facilitating the localization of XIST RNA on the X chromosome. This study aimed to investigate whether RNA interference to suppress the expression of YY1 can inhibit erroneous XIST expression, rescue abnormal XCI, and improve the developmental ability of cloned pig embryos. The results showed that YY1 binds to the 5' regulatory region of the porcine XIST gene in pig cells. The microinjection of YY1 siRNA into cloned pig embryos reduced the transcript abundance of XIST and upregulated the mRNA level of X-linked genes at the 4-cell and blastocyst stages. The siRNA-mediated knockdown of YY1 altered the transcriptome and enhanced the in vitro and in vivo developmental efficiency of cloned porcine embryos. These results suggested that YY1 participates in regulating XIST expression and XCI in cloned pig embryos and that the suppression of YY1 expression can increase the developmental rate of cloned pig embryos. The present study established a new method for improving the efficiency of pig cloning.

Identification of Important Factors Causing Developmental Arrest in Cloned Pig Embryos by Embryo Biopsy Combined with Microproteomics.

The technique of pig cloning holds great promise for the livestock industry, life science, and biomedicine. However, the prenatal death rate of cloned pig embryos is extremely high, resulting in a very low cloning efficiency. This limits the development and application of pig cloning. In this study, we utilized embryo biopsy combined with microproteomics to identify potential factors causing the developmental arrest in cloned pig embryos. We verified the roles of two potential regulators, PDCD6 and PLK1, in cloned pig embryo development. We found that siRNA-mediated knockdown of PDCD6 reduced mRNA and protein expression levels of the pro-apoptotic gene, CASP3, in cloned pig embryos. PDCD6 knockdown also increased the cleavage rate and blastocyst rate of cloned porcine embryos. Overexpression of PLK1 via mRNA microinjection also improved the cleavage rate of cloned pig embryos. This study provided a new strategy to identify key factors responsible for the developmental defects in cloned pig embryos. It also helped establish new methods to improve pig cloning efficiency, specifically by correcting the expression pattern of PDCD6 and PLK1 in cloned pig embryos.

Ssc-miR-92b-3p Regulates Porcine Trophoblast Cell Proliferation and Migration via the PFKM Gene.

Embryo implantation, the pivotal stage of gestation, is fundamentally dependent on synchronous embryonic development and uterine receptivity. In the early gestation period, the uterus and conceptus secrete growth factors, cytokines, and hormones to promote implantation. Circulating exosomal miRNAs are potential indicators of normal or complicated gestation. Our previous study revealed that pregnant sows' serum exosomes had upregulated miR-92b-3p expression compared to non-pregnant sows, and that the expression level progressively increased during early gestation. The present study's findings indicate that, compared to the ninth day of the estrous cycle (C9), pregnant sows had upregulated miR-92b-3p expression in the endometrium and embryos during the implantation stage ranging from day 9 to day 15 of gestation. Additionally, our results demonstrate that miR-92b-3p promotes the proliferation and migration of Porcine Trophoblast Cells (PTr2). Dual-Luciferase Reporter (DLR) gene assay, real-time fluorescent quantitative PCR (RT-qPCR), and Western blotting (WB) confirmed the bioinformatics prediction that phosphofructokinase-M (PFKM) serves as a target gene of miR-92b-3p. Notably, interference of PFKM gene expression markedly promoted PTr2 proliferation and migration. Furthermore, mice with downregulated uterine miR-92b-3p expression had smaller rates of successful embryo implantation. In summary, miR-92b-3p putatively modulates embryo implantation by promoting PTr2 proliferation and migration via its target gene PFKM.

Supplementation of SDF1 during Pig Oocyte In Vitro Maturation Improves Subsequent Embryo Development.

The quality of in vitro matured oocytes is inferior to that of in vivo matured oocytes, which translates to low developmental capacity of embryos derived from in vitro matured oocytes. The developmental potential of in vitro matured oocytes is usually impaired due to oxidative stress. Stromal cell-derived factor-l (SDF1) can reduce oxidative stress and inhibit apoptosis. The aim of this study was to investigate the effects of SDF1 supplementation during pig oocyte in vitro maturation (IVM) on subsequent embryo development, and to explore the acting mechanisms of SDF1 in pig oocytes. We found that the IVM medium containing 20 ng/mL SDF1 improved the maturation rate of pig oocytes, as well as the cleavage rate and blastocyst rate of embryos generated by somatic cell nuclear transfer, in vitro fertilization, and parthenogenesis. Supplementation of 20 ng/mL SDF1 during IVM decreased the ROS level, increased the mitochondrial membrane potential, and altered the expression of apoptosis-related genes in the pig oocytes. The porcine oocyte transcriptomic data showed that SDF1 addition during IVM altered the expression of genes enriched in the purine metabolism and TNF signaling pathways. SDF1 supplementation during pig oocyte IVM also upregulated the mRNA and protein levels of YY1 and TET1, two critical factors for oocyte development. In conclusion, supplementation of SDF1 during pig oocyte IVM reduces oxidative stress, changes expression of genes involved in regulating apoptosis and oocyte growth, and enhances the ability of in vitro matured pig oocytes to support subsequent embryo development. Our findings provide a theoretical basis and a new method for improving the developmental potential of pig in vitro matured oocytes.

Epigenetic Environmental Memories in Plants: Establishment, Maintenance, and Reprogramming.

Plants are immobile and must respond to or endure fluctuating surroundings and diverse environmental challenges. Environmental inputs often induce chromatin modifications at various responsive genes and consequent changes in their expression. Environment-induced chromatin marks at certain loci are transmittable through cell divisions after relief from the original external signals, leading to acquired 'memorization' of environmental experiences in plants, namely epigenetic environmental memories, which enable plants to adapt to environmental changes or to perform better when events recur. Here, we review recent progress in epigenetic or chromatin-mediated environmental memories in plants, including defense priming, stress memories, and 'epigenetic memory of winter cold' or vernalization. Various advances in epigenetic mechanisms underlying plant-environment interactions highlight that plant environmental epigenetics is emerging as an important area in plant biology.

Knockdown of RLIM inhibits XIST expression and improves developmental competence of cloned male pig embryos.

Ectopic expression of Xist on the putative active X chromosome is a primary cause of the low developmental efficiency of cloned mouse and pig embryos. Suppression of abnormal Xist expression via gene knockout or RNA interference (RNAi) can significantly enhance the developmental competence of cloned mouse and pig embryos. RLIM is a Xist expression activator, whereas REX1 is an Xist transcription inhibitor, as RLIM triggers Xist expression by mediating the proteasomal degradation of REX1 to induce imprinted and random X chromosome inactivation in mice. This study aimed to test whether the knockdown of RLIM and overexpression of REX1 can repress aberrant Xist expression and improve the developmental ability of cloned male pig embryos. Results showed that injection of anti-RLIM small interfering RNA significantly decreased Xist messenger RNA abundance, increased REX1 protein level, and enhanced the preimplantation development of cloned male porcine embryos. These positive effects were not observed in cloned male pig embryos injected with REX1 expression plasmid, which might be due to the low expression efficiency of injected REX1 plasmid and/or the short half-life of expressed REX1 protein. The findings from this study indicated that RLIM participated in the ectopic activation of Xist expression in cloned pig embryos by targeting REX1 degradation. Furthermore, this study provided a new method to improve cloned pig embryo development by the inhibition of Xist expression via RNAi of RLIM.

Associations of cord metabolome and biochemical parameters with the neonatal deaths of cloned pigs.

Neonatal cloned pigs generated via somatic cell nuclear transfer (SCNT) have high incidences of malformation and mortality. The mechanisms underlying the massive loss of cloned pig neonates remain unclear. We compared the cord serum metabolic profiles and biochemical indexes of SCNT-derived piglets that died within 4 days (SCNT-DW4), SCNT-derived piglets that survived over 4 days (SCNT-SO4) and artificial insemination (AI)-generated piglets that survived over 4 days (AI-SO4) to investigate the associations of serum metabolomics and biochemical indexes in umbilical cord (UC) sera at delivery with the neonatal loss of cloned pigs. Results showed that compared with SCNT-SO4 and AI-SO4 piglets, SCNT-DW4 piglets had lower birth weight, placental indexes, placental vascularization scores, UC scores, vitality scores, serum glucose and levels but higher creatinine, urea nitrogen and uric acid levels in cord sera. Metabolomics analysis revealed alterations in lipid, glucose and purine metabolism in the cord sera of SCNT-DW4 piglets. These results indicated that the disturbance of the cord serum metabolome might be associated with the low birth weight and malformations of cloned neonates. These effects were likely the consequences of the impaired placental morphology and function of SCNT-derived piglets. This study provides helpful information regarding the potential mechanisms responsible for the neonatal death of cloned pigs and also offers an important basis for the design of effective strategies to improve the survival rate of these animals.