Epigenomic analysis of the myometrium during late implantation revealed regulatory elements in genes related to the cellular zinc homeostasis pathway in pigs.

The myometrium, composed of the inner circular muscle (CM) and outer longitudinal muscle (LM), is crucial in establishing and maintaining early pregnancy. However, the molecular mechanisms involved are not well understood. In this study, we identified the transcriptomic features of the CM and LM collected from the mesometrial (M) and anti-mesometrial (AM) sides of the pig uterus on day 18 of pregnancy during the placentation initiation phase. Some genes in the cellular zinc ion level regulatory pathways (MT-1A, MT-1D, MT-2B, SLC30A2, and SLC39A2) were spatially and highly enriched in uterine CM at the mesometrial side. In addition, the histone modification profiles of H3K27ac and H3K4me3 in uterine CM and LM collected from the mesometrial side were characterized. Genomic regions associated with the expression of genes regulating the cellular zinc ion level were detected. Moreover, six highly linked variants in the H3K27ac-enriched region of the pig SLC30A2 gene were identified and found to be significantly associated with the total number born at the second parity (P < 0.05). In conclusion, the genes in the pathways of cellular zinc homeostasis and their regulatory elements identified have implications for pig reproduction trait improvement and warrant further investigations.

A genome-wide association study reveals candidate genes and regulatory regions associated with birth weight in pigs.

Piglet birth weight is associated with preweaning survival, and its related traits have been included in the breeding program. Thus, understanding its genetic basis is essential. This study identified four birth weight-associated genomic regions on chromosomes 2, 4, 5, and 7 through genome-wide association study analysis in 7286 pigs from three different pure breeds using the FarmCPU model. The genetic and phenotypic variance explained by the four candidate regions is 8.42% and 1.85%, respectively. Twenty-eight candidate genes were detected, of which APPL2, TGFBI, MACROH2A1, and SEC22B have been reported to affect body growth or development. In addition, 21 H3K4me3-enriched peaks overlapped with the birth weight-associated genomic regions were identified by integrating the genome-wide association study results with our previous ChIP-seq and RNA-seq data generated in the pig placenta, a fetal organ relevant to birth weight, and three of the regulatory regions influence TGFBI, MACROH2A1, and SEC22B expression. This study provides new insights into understanding the mechanisms for birth weight. Further investigating the variants in the regulatory regions would help identify the functional variants for birth weight in pigs.

Integrating LCM-Based Spatio-Temporal Transcriptomics Uncovers Conceptus and Endometrial Luminal Epithelium Communication that Coordinates the Conceptus Attachment in Pigs.

Attachment of conceptus to the endometrial luminal epithelium (LE) is a critical event for early placentation in Eutheria. Since the attachment occurs at a particular site within the uterus, a coordinated communication between three spatially distinct compartments (conceptus and endometrial LE from two anatomical regions of the uterus to which conceptus attaches and does not attach) is essential but remains to be fully characterized. Using the laser capture microdissection (LCM) technique, we firstly developed an approach that can allow us to pair the pig conceptus sample with its nearby endometrial epithelium sample without losing the native spatial information. Then, a comprehensive spatio-temporal transcriptomic profile without losing the original conceptus-endometrium coordinates was constructed. The analysis shows that an apparent difference in transcriptional responses to the conceptus exists between the endometrial LE from the two anatomically distinct regions in the uterus. In addition, we identified the communication pathways that link the conceptus and endometrial LE and found that these pathways have important roles in conceptus attachment. Furthermore, a number of genes whose expression is spatially restricted in the two different anatomical regions within the uterus were characterized for the first time and two of them (SULT2A1 and MEP1B) may cooperatively contribute to establish conceptus attachment in pigs. The results from our study have implications in understanding of conceptus/embryo attachment in pigs and other large polytocous species.

A miR-18a binding-site polymorphism in CDC42 3'UTR affects CDC42 mRNA expression in placentas and is associated with litter size in pigs.

Increasing evidence suggests that miRNA binding-site polymorphism in the 3'-untranslated region (3'UTR) of a target gene could affect that gene's expression, and can be associated with a variety of complex traits. In this study, we find that miR-18a and cell division cycle 42 (CDC42) mRNA, whose expression was inversely correlated, are differentially expressed in porcine placentas during critical stages of placental development. rs55618224 (T>C), a SNP in the 3'UTR region of CDC42 that is perfectly complementary to the miR-18a seed could influence miR-18a-related regulation of CDC42 gene by altering their binding affinity. In addition, CDC42 mRNA was found to have higher expression level in the homozygous TT placentas as compared to those homozygous CC placentas in pigs. Furthermore, we identified a significant association between rs55618224 and total number born per litter. These results suggest the miR-18a binding-site polymorphism in CDC42 3'UTR may impact litter size by regulation of CDC42 gene in porcine placentas.

Genetic determinants of pig birth weight variability.

BACKGROUND: Piglet birth weight variability, a trait also known as the within-litter homogeneity of birth weight, reflects the sow's prolificacy, because it is positively genetically correlated with preweaning mortality but negatively correlated with the mean growth of piglets during sucking. In addition, the maternal additive genetic variance and heritability has been found exist for this trait, thus, reduction in the variability of piglet birth weight to improve the sow prolificacy is possible by selective breeding. RESULTS: We performed a genome wide association study (GWAS) in 82 sows with extreme standard deviation of birth weights within the first parity to identify significant SNPs, and finally 266 genome-wide significant SNPs (p < 0.01) were identified. These SNPs were mainly enriched on chromosome 7, 1, 13, 14, 15 and 18. We further scanned genes of the top 50 SNPs with the lowest p values and found some genes involved in plasma glucose homeostasis (GLP1R) and lipid metabolism as well as maternal-fetal lipid transport (AACS, APOB, OSBPL10 and LRP1B) which may contribute to the birth weight variability trait. CONCLUSIONS: Birth weight variability trait has a low heritability. It is not easy to get significant signal by GWAS using small sample size. Herein, we identified some candidate chromosome regions especially chromosome 7 and suggested five genes which may provide some information for the further study.

Integrating Transcriptomic and ChIP-Seq Reveals Important Regulatory Regions Modulating Gene Expression in Myometrium during Implantation in Pigs.

The myometrium is the outer layer of the uterus. Its contraction and steroidogenic activities are required for embryo implantation. However, the molecular mechanisms underlying its functions remain unknown in pigs. The myometrium includes the inner circular muscle (CM) and the outer longitudinal muscle (LM) layers. In this study, we collected the CM and LM samples from the mesometrial side (named M) of the uterus on days 12 (pre-implantation stage) and 15 (implantation stage) of pregnancy and day 15 of the estrous cycle. The transcriptomic results revealed distinct differences between the uterine CM and LM layers in early pregnancy: the genes expressed in the LM layer were mainly related to contraction pathways, whereas the transcriptional signatures in the CM layer on day 15 of pregnancy were primarily involved in the immune response processes. Subsequent comparisons in the CM layer between pregnant and cyclic gilts show that the transcriptional signatures of the CM layer are implantation-dependent. Next, we investigated the genome-wide profiling of histone H3 lysine 27 acetylation (H3K27ac) and histone H3 lysine 4 trimethylation (H3K4me3) in pig uterine CM and LM layers. The genomic regions that had transcriptional activity and were associated with the expression of genes in the two layers were characterized. Taken together, the regulatory regions identified in the study may contribute to modulating the gene expression in pig uterine CM and LM layers during implantation.

Transcriptomic and ChIP-seq Integrative Analysis Reveals Important Roles of Epigenetically Regulated lncRNAs in Placental Development in Meishan Pigs.

The development of the placental fold, which increases the maternal-fetal interacting surface area, is of primary importance for the growth of the fetus throughout the whole pregnancy. However, the mechanisms involved remain to be fully elucidated. Increasing evidence has revealed that long non-coding RNAs (lncRNAs) are a new class of RNAs with regulatory functions and could be epigenetically regulated by histone modifications. In this study, 141 lncRNAs (including 73 up-regulated and 68 down-regulated lncRNAs) were identified to be differentially expressed in the placentas of pigs during the establishment and expanding stages of placental fold development. The differentially expressed lncRNAs and genes (DElncRNA-DEgene) co-expression network analysis revealed that these differentially expressed lncRNAs (DElncRNAs) were mainly enriched in pathways of cell adhesion, cytoskeleton organization, epithelial cell differentiation and angiogenesis, indicating that the DElncRNAs are related to the major events that occur during placental fold development. In addition, we integrated the RNA-seq (RNA sequencing) data with the ChIP-seq (chromatin immunoprecipitation sequencing) data of H3K4me3/H3K27ac produced from the placental samples of pigs from the two stages (gestational days 50 and 95). The analysis revealed that the changes in H3K4me3 and/or H3K27ac levels were significantly associated with the changes in the expression levels of 37 DElncRNAs. Furthermore, several H3K4me3/H3K27ac-lncRNAs were characterized to be significantly correlated with genes functionally related to placental development. Thus, this study provides new insights into understanding the mechanisms for the placental development of pigs.

Spatial Transcriptomic and miRNA Analyses Revealed Genes Involved in the Mesometrial-Biased Implantation in Pigs.

Implantation failure is a major cause of early embryonic loss. Normally, the conceptus attachment is initiated at mesometrial side of the uterus and then spread to the anti-mesometrial side in pigs, however, the mechanisms that direct the mesometrial-biased attachment are largely unknown. In this study, the histological features of the entire uterine cross-section from gestational days 12 (pre-attachment stage) and 15 (post-attachment stage) were investigated and the differences in histological features between the mesometrial and anti-mesometrial side of the uterus were observed. Then, transcriptomic and miRNA analyses were performed on mesometrial and anti-mesometrial endometrium obtained from gestational days 12 and 15, respectively. Differentially expressed genes (DEGs) and miRNAs (DE-miRs) that were common to both or unique to either of the two anatomical locations of uterus were identified, respectively, indicating that differences in molecular response to the implanting conceptus exist between the two anatomical locations. In addition, we detected DEGs and DE-miRs between the two anatomical locations on the two gestational days, respectively. Of these DEGs, a number of genes, such as chemokine and T cell surface marker genes, were found to be significantly up-regulated mesometrially. Furthermore, we detected the interaction of CXCR4, CXCL11 and miR-9 using dual luciferase reporter assay. Taken together, this study revealed genes and pathways that might play the role of creating a receptive microenvironment at the mesometrial side, which is required to guide a proper positioning of conceptus in the uterus in pigs.