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.

Porcine Granulosa-Cell-Derived Exosomes Enhance Oocyte Development: An In Vitro Study.

Recent studies have established that exosomes (EXs) derived from follicular fluid (FF) can promote oocyte development. However, the specific sources of these EXs and their regulatory mechanisms remain elusive. It is universally acknowledged that oocyte development requires signal communication between granulosa cells (GCs) and oocytes. However, the role of GC-secreted EXs and their functions are poorly understood. This study aimed to investigate the role of porcine granulosa-cell-derived exosomes (GC-EXs) in oocyte development. In this study, we constructed an in vitro model of porcine GCs and collected and identified GC-EXs. We confirmed that porcine GCs can secrete EXs and investigated the role of GC-EXs in regulating oocyte development by supplementing them to cumulus-oocyte complexes (COCs) cultured in vitro. Specifically, GC-EXs increase the cumulus expansion index (CEI), promote the expansion of the cumulus, alleviate reactive oxygen species (ROS), and increase mitochondrial membrane potential (MMP), resulting in improved oocyte development. Additionally, we conducted small RNA sequencing of GC-EXs and hypothesized that miR-148a-3p, the highest-expressed microRNA (miRNA), may be the key miRNA. Our study determined that transfection of miR-148a-3p mimics exerts effects comparable to the addition of EXs. Meanwhile, bioinformatics prediction, dual luciferase reporter gene assay, and RT-qPCR identified DOCK6 as the target gene of miR-148a-3p. In summary, our results demonstrated that GC-EXs may improve oocyte antioxidant capacity and promote oocyte development through miR-148a-3p by targeting DOCK6.

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.

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.

DIA-based quantitative proteomic analysis of porcine endometrium in the peri-implantation phase.

The 12th day of gestation is a critical period for embryo loss and the beginning of imminent implantation in sows. Data independent acquisition (DIA) technology is one of the high-throughput, high-resolution and reproducible proteomics technologies for large-scale digital qualitative and quantitative research. The aim of this study was to identify and characterize the protein abundance landscape of Yorkshire pig endometrium on the 12th day of pregnancy (P12) and estrous cycle (C12) using DIA proteomics. A total of 1251 differentially abundant proteins (DAPs) were identified, of which 882 were up-regulated and 369 were down-regulated at P12. Functional enrichment analysis showed that the identified proteins were related to metabolism, biosynthesis and signaling pathways. Three proteins were selected for Western blot (WB) validation and the results were consistent with the DIA data. Further combined with transcriptome data, fibrinogen like 2 (FGL2) and S100 calcium binding protein A8 (S100A8) were verified to be highly abundant in the P12 endometrial epithelium. In summary, there were significantly different abundance of proteome profiles in C12 and P12 endometrium, suggesting that DAPs are associated with changes in endometrial receptivity, which laid the foundation for further research on related regulatory mechanisms. SIGNIFICANCE: The 12th day of gestation is an important point in the peri-implantation period of pigs, when the endometrium presents a receptive state under the stimulation of estrogen. DIA proteomics technology is an emerging protein identification technology in recent years, which can obtain protein information through comprehensive and unbiased scanning. In this study, DIA technology was used to characterize endometrial proteins in pigs during the peri-implantation period. The results showed that higher protein abundance was detected using the DIA technique, and some of these DAPs may be involved in regulating embryo implantation. This study will help to better reveal the related proteins involved in embryo implantation, and lay a foundation for further research on the mechanism of endometrial regulation of embryo implantation. SIGNIFICANCE OF THE STUDY: The 12th day of gestation is an important point in the peri-implantation period of pigs, when the endometrium presents a receptive state under the stimulation of estrogen. DIA proteomics technology is an emerging protein identification technology in recent years, which can obtain protein information through comprehensive and unbiased scanning. In this study, DIA technology was used to characterize endometrial proteins in pigs during the peri-implantation period. The results showed that higher protein abundance was detected using the DIA technique, and some of these DAPs may be involved in regulating embryo implantation. This study will help to better reveal the related proteins involved in embryo implantation, and lay a foundation for further research on the mechanism of endometrial regulation of embryo implantation.

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.

Integrated Analysis of DNA Methylation and Gene Expression in Porcine Placental Development.

Proper placental development is crucial for the conceptus to grow and survive, because the placenta is responsible for transporting nutrients and oxygen from the pregnant female to the developing fetus. However, the processes of placental morphogenesis and fold formation remain to be fully elucidated. In this study, we used whole-genome bisulfite sequencing and RNA sequencing to produce a global map of DNA methylation and gene expression changes in placentas from Tibetan pig fetuses 21, 28, and 35 days post-coitus. Substantial changes in morphology and histological structures at the uterine-placental interface were revealed via hematoxylin-eosin staining. Transcriptome analysis identified 3959 differentially expressed genes (DEGs) and revealed the key transcriptional properties in three stages. The DNA methylation level in the gene promoter was negatively correlated with gene expression. We identified a set of differentially methylated regions associated with placental developmental genes and transcription factors. The decrease in DNA methylation level in the promoter was associated with the transcriptional activation of 699 DEGs that were functionally enriched in cell adhesion and migration, extracellular matrix remodeling, and angiogenesis. Our analysis provides a valuable resource for understanding the mechanisms of DNA methylation in placental development. The methylation status of different genomic regions plays a key role in establishing transcriptional patterns from placental morphogenesis to fold formation.

RACK1 may participate in placental development at mid-gestation via regulating trophoblast cell proliferation and migration in pigs.

Intrauterine growth restriction (IUGR) is a severe complication in swine production. Placental insufficiency is responsible for inadequate fetal growth, but the specific etiology of placental dysfunction-induced IUGR in pigs remains poorly understood. In this work, placenta samples supplying the lightest weight (LW) and mean weight (MW) pig fetuses in the litter at Day 65 (D65) of gestation were collected, and the relationship between fetal growth and placental morphologies and functions was investigated using histomorphological analysis, RNA sequencing, quantitative polymerase chain reaction, and in vitro experiment in LW and MW placentas. Results showed that the folded structure of the epithelial bilayer of LW placentas followed a poor and incomplete development compared with that of MW placentas. A total of 654 differentially expressed genes (DEGs) were screened out between the LW and MW placentas, and the gene encodes receptor for activated C kinase 1 (RACK1) was found to be downregulated in LW placentas. The DEGs were mainly enriched in translation, ribosome, protein synthesis, and mammalian target of rapamycin (mTOR) signaling pathway according to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. In vitro experiments indicated that the decreased RACK1 in LW placentas may be involved in abnormal development of placental folds (PFs) by inhibiting the proliferation and migration of porcine trophoblast cells. Taken together, these results revealed that RACK1 may be a vital regulator in the development of PFs via regulating trophoblast cell proliferation and migration in pigs.

Interaction between Microbes and Host in Sow Vaginas in Early Pregnancy.

Extensive research has explored the causes of embryo losses during early pregnancy by analyzing interaction mechanisms in sows' uterus, ignoring the importance of the lower reproductive tract in pregnancy development regulation. Despite recent progress in understanding the diversity of vaginal microbes under different physiological states, the dynamic of sows' vaginal microbiotas during pregnancy and the interaction between vaginal microbes and the host are poorly understood. Here, we performed a comprehensive analysis of sows' vaginal microbial communities in early pregnancy coupled with overall patterns of vaginal mucosal epithelium gene expression. The vaginal microbiota was analyzed by 16s rRNA or metagenome sequencing, and the vaginal mucosal epithelium transcriptome was analyzed by RNA sequencing, followed by integration of the data layers. We found that the sows' vaginal microbiotas in early pregnancy develop dynamically, and there is a homeostasis balance of Firmicutes and Proteobacteria. Subsequently, we identified two pregnancy-specific communities, which play diverse roles. The microbes in the vagina stimulate the epithelial cells, while vaginal epithelium changes its structure and functions in response to stimulation. These changes produce specific inflammation responses to promote pregnancy development. Our findings demonstrate the interaction between microbes and host in the sow vagina in early pregnancy to promote pregnancy development, meanwhile providing a reference data set for the study of targeted therapies of microbial homeostasis dysregulation in the female reproductive tract. IMPORTANCE This work sheds light on the dynamics of the sow vaginal microbiotas in early pregnancy and its roles in pregnancy development. Furthermore, this study provides insight into the functional mechanisms of reproductive tract microbes by outlining vaginal microbe-host interactions, which might identify new research and intervention targets for improving pregnancy development by modulating lower reproductive tract microbiota.

Genome-Wide mRNA and Long Non-Coding RNA Analysis of Porcine Trophoblast Cells Infected with Porcine Reproductive and Respiratory Syndrome Virus Associated with Reproductive Failure.

Porcine reproductive and respiratory syndrome (PRRS) is a vertically transmitted reproductive disorder that is typically characterized by miscarriage, premature birth, and stillbirth in pregnant sows after infection. Such characteristics indicate that PRRSV can infect and penetrate the porcine placental barrier to infect fetus piglets. The porcine trophoblast is an important component of the placental barrier, and secretes various hormones, including estrogen and progesterone, to maintain normal pregnancy and embryonic development during pregnancy. It is conceivable that the pathogenic effects of PRRSV infection on porcine trophoblast cells may lead to reproductive failure; however, the underlying detailed mechanism of the interaction between porcine trophoblast (PTR2) cells and PRRSV is unknown. Therefore, we conducted genome-wide mRNA and long non-coding RNA (lncRNA) analysis profiling in PRRSV-infected PTR2. The results showed that 672 mRNAs and 476 lncRNAs were significantly different from the control group after viral infection. Target genes of the co-expression and co-location of differential mRNAs and lncRNAs were enriched by GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, revealing that most of the pathways were involved in cell nutrient metabolism, cell proliferation, and differentiation. Specifically, the estrogen signaling pathway, the PI3K (PhosphoInositide-3 Kinase)-Akt (serine/threonine kinase) signaling pathway, and the insulin secretion related to embryonic development were selected for analysis. Further research found that PRRSV inhibits the expression of G-protein-coupled estrogen receptor 1 (GPER1), thereby reducing estrogen-induced phosphorylation of AKT and the mammalian target of rapamycin (mTOR). The reduction in the phosphorylation of AKT and mTOR blocks the activation of the GPER1- PI3K-AKT-mTOR signaling pathway, consequently restraining insulin secretion, impacting PTR2 cell proliferation, differentiation, and nutrient metabolism. We also found that PRRSV triggered trophoblast cell apoptosis, interrupting the integrity of the placental villus barrier. Furthermore, the interaction network diagram of lncRNA, regulating GPER1 and apoptosis-related genes, was constructed, providing a reference for enriching the functions of these lncRNA in the future. In summary, this article elucidated the differential expression of mRNA and lncRNA in trophoblast cells infected with PRRSV. This infection could inhibit the PI3K-AKT-mTOR pathway and trigger apoptosis, providing insight into the mechanism of the vertical transmission of PRRSV and the manifestation of reproductive failure.

Integrating Analysis to Identify Differential circRNAs Involved in Goat Endometrial Receptivity.

Endometrial receptivity is one of the main factors underlying a successful pregnancy, with reports substantiating the fact that suboptimal endometrial receptivity accounts for two-thirds of early implantation event failures. The association between circRNAs and endometrial receptivity in the goat remains unclear. This study aims to identify potential circRNAs and regulatory mechanisms related to goat endometrial receptivity. Therefore, the endometrial samples on day 16 of pregnancy and day 16 of the estrous cycle were analyzed using high-throughput RNA-seq and bioinformatics. The results show that 4666 circRNAs were identified, including 7 downregulated and 11 upregulated differentially expressed circRNAs (DE-circRNAs). Back-splicing and RNase R resistance verified the identified circRNAs. We predicted the competing endogenous RNA (ceRNA) regulatory mechanism and potential target genes of DE-circRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of these predicted target genes suggest that DE-circRNAs were significantly involved in establishing endometrial receptivity. Furthermore, Sanger sequencing, qPCR, correlation analysis and Fluorescence in Situ Hybridization (FISH) show that circ_MYRF derived from the host gene myelin regulatory factor (MYRF) might regulate the expression of interferon stimulating gene 15 (ISG15), thereby promoting the formation of endometrial receptivity. These novel findings may contribute to a better understanding of the molecular mechanisms regulating endometrial receptivity and promoting the maternal recognition of pregnancy (MRP).

Extracellular vesicles-encapsulated microRNA in mammalian reproduction: A review.

Extracellular vesicles (EVs) are nanoscale cell-derived lipid vesicles that participate in cell-cell communication by delivering cargo, including mRNAs, proteins and non-coding RNAs, to recipient cells. MicroRNA (miRNA), a non-coding RNA typically 22 nucleotides long, is crucial for nearly all developmental and pathophysiological processes in mammals by regulating recipient cells gene expression. Infertility is a worldwide health issue that affects 10-15% of couples during their reproductive years. Although assisted reproductive technology (ART) gives infertility couples hope, the failure of ART is mainly unknown. It is well accepted that EVs-encapsulated miRNAs have a role in different reproductive processes, implying that these EVs-encapsulated miRNAs could optimize ART, improve reproductive rate, and treat infertility. As a result, in this review, we describe the present understanding of EVs-encapsulated miRNAs in reproduction regulation.

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.

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.

Effect of miR-143-3p from Extracellular Vesicles of Porcine Uterine Luminal Fluid on Porcine Trophoblast Cells.

Extracellular vesicles (EVs) in uterine luminal fluid (ULF) can reportedly affect the proliferation and migration function of porcine trophoblast cells (PTr2 cells) by mediating the maternal-fetal exchange of information. miR-143-3p is considered a crucial miRNA in early pregnancy in mammals; however, little is currently known about how it regulates the function of PTr2 cells. This study aimed to investigate the effects of ssc-miR-143-3p in ULF-EVs on the function of PTr2 cells during porcine embryo implantation. The uptake of ULF-EVs by PTr2 cells was confirmed, which significantly increased the expression of ssc-miR-143-3p. Ssc-miR-143-3p was found to facilitate the proliferation and migration of PTr2 cells in the CCK-8, EdU and wound-closure assays, while the opposite findings were observed after the knockdown of ssc-miR-143-3p. Bioinformatics analysis and the luciferase reporter assay showed that glycerol-3 phosphate dehydrogenase 2 (GDP2) was directly targeted by miR-143-3p. Inhibition of miR-143-3p was validated in mice to inhibit embryo implantation. In summary, ssc-miR-143-3p in ULF-EVs affects the proliferation and migration of PTr2 cells by mediating GPD2, thereby affecting embryo implantation.

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.

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.

Extracellular vesicle-encapsulated miR-21-5p in seminal plasma prevents sperm capacitation via Vinculin inhibition.

To penetrate the zona pellucida before sperm-egg binding, sperm must undergo highly time-controlled capacitation and acrosome reaction in the female reproductive tract. Our previous study demonstrated that miR-21-5p is the most abundant miRNA in boar seminal plasma (SP)-derived extracellular vesicles (EVs) and can target Vinculin (VCL) gene, which may participate in boar sperm capacitation. Thus, this study aims to explore the potential role of miR-21-5p from SP-derived EVs in preventing sperm capacitation and its underlying mechanism. We observed that sperm could incorporate miR-21-5p from SP-derived EVs. The roles of SP-derived EVs miR-21-5p in sperm capacitation were then determined using gain- and loss-of-function analyses. In addition, the expression levels of miR-21-5p, VCL, and VCL protein in liquid-preserved boar sperm following transfection were determined using RT-qPCR and Western blotting. Our results revealed that miR-21-5p overexpression inhibited sperm capacitation and acrosome reaction. Similarly, miR-21-5p expression was significantly lower (P < 0.05) in capacitated sperm than un-capacitated sperm. However, the protein level of VCL was also significantly lower (P < 0.05) in capacitated sperm than un-capacitated sperm. Furthermore, immunofluorescence analysis showed that VCL protein mainly located in sperm head and sperm capacitation was inhibited after treating with VCL protein inhibitor (Chrysin). In conclusion, our study provides reasonable evidence that miR-21-5p expression in SP-derived EVs could prevent sperm capacitation via VCL inhibition.