Single-Cell Analysis of the Endometrial Characteristics of Meishan Pigs Across the Estrous Cycle

Single-Cell Analysis of the Endometrial Characteristics of Meishan Pigs Across the Estrous Cycle | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Single-Cell Analysis of the Endometrial Characteristics of Meishan Pigs Across the Estrous Cycle Nengjing Jiang, Wei xiao, Qingbo Zhao, Chenxi Liu, Jinfen Ma, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4582781/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: The Meishan pig, native to China, is renowned for its superior reproductive capabilities, including a high ovulation rate, substantial uterine capacity, and an impressively high rate of embryo implantation. The endometrium plays a pivotal role in facilitating embryo implantation and sustaining pregnancy. It is regulated by ovarian hormones and uterine prostaglandins and undergoes a complex series of coordinated processes across the estrous cycle, including proliferation, differentiation, shedding, and regeneration. A detailed examination of the intricate sow endometrial gene expression patterns during this cycle can yield valuable insights into creating ideal conditions for successful embryo implantation and early embryonic development. To gain a comprehensive understanding of the Meishan pig endometrial biological functions across the estrous cycle, we specifically used uterine tissues in the proliferative and secretory phases for single-cell transcriptomic sequencing. Results: The comprehensive transcriptional profile of uterine cells was elucidated throughout the estrous cycle in Meishan pigs. We identified 7 distinct cell types within the primary cell categories, with 4 subpopulations specifically discerned among the endometrial epithelial cells. Considerable variability was observed in the types and quantities of epithelial cell subpopulations spanning the proliferative and secretory phases of the estrous cycle. Significantly, SOX9-expressing epithelial cells were characterised as potential endometrial epithelial stem cells in Meishan pigs. NURP1 and HES1 were identified as potential marker genes for these stem cells. Pseudotime analysis indicated that these SOX9-expressing epithelial cells can differentiate into glandular epithelial (GE) or luminal epithelial (LE) cells. We also observed that SOX9-expressing epithelial cells may differentiate into ciliated epithelial (CE) cells. There was a marked increase in the number of GE and CE cells during the secretory phase compared to the proliferative phase. GE cells are vital for processes such as glycolysis, amino acid biosynthesis, and N-glycan biosynthesis, all of which are crucial for supplying essential nutrients required for embryo implantation and early stages of embryonic development. Conclusions: We reveal the integrated transcriptional profile of uterine cells in sexually mature Meishan pigs and delineate the gene expression patterns within the uterine horns throughout the estrous cycle. These findings provide potential new diagnostic indicators for determining the estrous cycle in sows. Meishan pig endometrium endometrial epithelial progenitor cells single-cell transcriptomic sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background The Meishan breed of pigs, which originated in the Taihu Lake region in China, is one of the most globally renowned native pig breeds [ 1 ] . Renowned for its exceptional fertility, it can produce three to five more piglets per litter than traditional U.S. or European pig varieties [ 2 ] . The litter size of a pig is limited by the pig’s uterine capacity, which is influenced by factors such as the size of the uterus, dimensions of the placenta, and degree of vascularization [ 3 ] . Previous studies regarding the fecundity of Meishan sow pigs have indicated that their prolificacy stems from lower embryonic mortality than European sows with similar ovulation rates [ 4 ] . The Meishan pig possesses a greater uterine capacity than does the Large White pig and sustains a greater number of implanted embryos throughout gestation [ 5 ] . Between gestation days 70 and 110, a twofold increase in blood vessel density in both the placenta and endometrium of Landrace pigs was observed. Conversely, the blood vessel densities in the placentae and endometria of Large White pigs remains relatively stable [ 6 ] . Meishan pigs have been exported into many countries, including the U.S. and Japan [ 7 ] . It is regarded as an outstanding model animal for hybridization and the improvement of reproductive traits by genetic manipulation. The preservation of Meishan pigs is not only crucial for maintaining their exceptional traits but also offers valuable insights for improving the reproductive capabilities of other pig breeds and for developing new breeds. Therefore, investigating the physiological functions associated with reproductive traits in Meishan pigs is imperative. The uterus is a key component in the reproductive processes of female animals, and the endometrium is a pivotal site for embryo implantation [ 8 ] . Establishing a particular endometrial state is crucial for successful embryo implantation in female animals [ 9 ] . The endometrium is most receptive to embryo implantation during the ovarian luteal phase following ovulation, this is the most favourable phase for successful implantation [ 10 , 11 ] . During the luteal phase, the endometrial glands secrete cytokines, such as adhesins, which facilitate the settling of the embryo. These glands play a vital role in supporting pregnancy by providing necessary nutrients to the developing embryo [ 12 – 14 ] . The uterus is a complex organ with a heterogeneous anatomical structure. During the estrous cycle, the endometrium undergoes coordinated shedding, regeneration and differentiation, which are regulated by the hypothalamic-pituitary-ovarian axis. During the human menstrual cycle, endometrial epithelial cells undergo significant changes, including rapid cell differentiation and endometrial remodelling [ 15 , 16 ] . The uterus, a complex organ, is regulated by a diverse variety of cell types. Single-cell RNA sequencing (scRNA-seq) technology is widely used in heterogeneous tissues to identify various cell types and to construct cellular developmental trajectories at the single-cell level to determine the gene transcription profiles of cells [ 17 ] . Therefore, scRNA-seq is a valuable tool for examining the mechanisms that drive the dynamic alterations in endometrial tissue throughout the estrous cycle after sexual maturation. In 2021, scRNA-seq was employed to compare and analyse the cell types present in the human uterus during the proliferative and secretory phases, allowing for identification of a total of 14 distinct cell subsets within human uterine tissues [ 16 ] . To date, several studies have conducted scRNA-seq on human [ 15 , 16 ] and mouse [ 18 ] uterine tissues to explore the gene transcription characteristics of endometrial epithelial cells during the estrous cycle. However, research on single-cell sequencing of porcine uterine tissues is scarce. The Meishan pig, a high-yielding local pig breed native to the Taihu Lake Basin, is known for its favourable intrauterine environment and high rate of embryo implantation. However, the characteristics and genetic mechanisms of endometrial development after sexual maturity in this pig breed remain largely unknown. In this study, we performed scRNA-seq on uterine tissues during the proliferative and secretory phases. Our goal was to identify the cell types present in the uteri of sexually mature Meishan pigs and to elucidate their growth and development patterns, as well as the transcriptional regulatory networks of endometrial epithelial cells throughout the estrous cycle. Materials and methods Animals and sample collection Uteri were collected from two healthy 8-month-old Meishan pigs (n = 2). The proliferative phase of the uterus was identified by the presence of ovarian follicles with no corpora lutea, while the secretory phase of the uterus was identified by the presence of four to six corpora lutea in the ovary. Approximately 2 cm of tissue was excised from one side of each uterine horn and divided into two pieces: one portion was immersed in 4% paraformaldehyde for histological examination, while the other segment was preserved in MACS Tissue Storage Solution (Miltenyi Biotec) for single-cell RNA sequencing. All the animals were provided by the Kunshan Meishan Pig Breeding Farm. Single-cell dissociation The collected uterine tissues were surgically removed and kept in MACS Tissue Storage Solution (Miltenyi Biotec) until processing. The tissue samples were processed as described below. Briefly, the samples were washed with phosphate-buffered saline (PBS), minced into small pieces (approximately 1 mm 3 ) on ice and enzymatically digested with 2 mg/mL collagenase IV (Worthington), 1 mg/mL dispase II (Worthington) and 50 U/µL DNase I (Worthington) for 40 min at 37°C with agitation. After digestion, the samples were passed through a 70 µm cell strainer and centrifuged at 300 × g for 5 min. After the supernatant was removed, the pelleted cells were suspended in 1× red blood cell lysis buffer (Miltenyi Biotec) to lyse the red blood cells. After washing with PBS containing 0.04% BSA, the cell pellets were resuspended in PBS containing 0.04% BSA and filtered through a 40 µm cell strainer. Dissociated single cells were then stained with AO/PI for viability assessment using a Countstar Fluorescence Cell Analyser. Single-cell sequencing The scRNA-seq libraries were generated using a 10x Genomics Chromium Controller Instrument and Chromium Single Cell 3’ V3.1 Reagent Kits (10x Genomics, Pleasanton, CA). The process was as follows: 1) Cells were concentrated to approximately 1000 cells/µL and loaded into each channel to generate single-cell gel beads-in-emulsion (GEMs). 2) After the reverse transcription (RT) step, the GEMs were broken, and the resulting barcoded cDNA was purified and amplified. 3) The amplified barcoded cDNA was fragmented, subjected to A-tailing, and ligated with adaptors, followed by index PCR amplification. 4) The final libraries were quantified using a Qubit high-sensitivity DNA assay (Thermo Fisher Scientific), and the size distribution of each library was determined using a high-sensitivity DNA chip on a Bioanalyzer 2200 (Agilent). 5) All libraries were sequenced on an Illumina NovaSeq 6000 (Illumina, San Diego, CA) platform on a 150 bp paired-end run. Single-cell RNA statistical analysis The scRNA-seq data analysis was performed by NovelBio Bio-Pharm Technology Co., Ltd., using the NovelBrain Cloud Analysis Platform (Shanghai, China). The analysis process included the following steps: 1) Quality control and filtering: The raw sequencing reads were processed with fastp using default parameters to filter the adaptor sequence and remove lower-quality reads to obtain clean data. 2) Alignment and quantification: The cleaned reads were then aligned to the pig genome (Sscrofa11.1 Ensemble: version 100) using CellRanger v6.1.1, which generated feature-barcode matrices. 3) Normalization: Down sampling analysis was performed across samples to normalize the data based on the number of mapped barcoded reads per cell for each sample. 4) Aggregation: The normalized data from individual samples were aggregated to form a comprehensive matrix. 5) Quality filtering: Cells with more than 200 expressed genes and a mitochondrial Unique Molecular Identifier (UMI) percentage of less than 20% were considered to have passed the cell quality filtering criteria. 6) Mitochondrial gene removal: Mitochondrial genes were subsequently removed from the expression table to focus on the nuclear transcriptome. The Seurat package (version: 4.0.3, https://satijalab.org/seurat/ ) was used for cell normalization and regression based on the expression table according to the UMI counts of each sample and percentage of mitochondria to obtain the scaled data. PCA was constructed based on the scaled data with the top 2000 highly variable genes, and the top 10 principal components were used for tSNE construction and UMAP construction. Utilising a graph-based clustering method, we acquired the unsupervised cell cluster results based on the top 10 principal components of the PCA, and we calculated the marker genes using the Find All Markers function with the Wilcoxon rank sum test algorithm under the following criteria: lnFC > 0.25, P value 0.1. To identify the cell types in detail, clusters of the same cell type were selected for re-tSNE analysis, graph-based clustering and marker analysis. Pseudotime analysis We applied single-cell trajectory analysis using Monocle2 ( http://cole-trapnell-lab.github.io/monocle-release ) with DDR-Tree and default parameters. Before Monocle analysis, we selected marker genes from the Seurat clustering results and the raw expression counts of the cells that passed filtering. The "plot pseudotime heatmap" function was utilized to generate a heatmap illustrating the signature genes and highly variable genes across pseudotime. Gene ontology (GO) and enrichment analysis Differentially expressed genes (DEGs) across cluster subsets and designated cell types in the proliferative-phase and secretory-phase groups were determined using the Seurat package. The log2 (fold change [FC]) and false discovery rate (FDR) q values for DEGs were ranked after passing the following control thresholds: log2 (FC) > 0.5 and q value < 0.05. The marker genes of each cluster generally presented higher expression levels than did the other functional genes. We averaged gene expression across the specific clusters of each group after normalization by interrogation using the 10× Genomics Loupe Cell Browser software. Volcano plots were generated with the X-axis displaying log2(FC) > 0.26 and the y-axis showing a p value < 0.05 using GraphPad Software. Kyoto Encyclopedia of Genes and KEGG enrichment analysis of cluster DEGs was performed with the cluster R package clusterProfiler. The results were visualized as dot plots using ggplot2. Histology and immunofluorescence staining Full-thickness uterine tissues from Meishan pigs were fixed in 4% paraformaldehyde (w/v) for a minimum of 24 h, dehydrated using an ethanol gradient, embedded in paraffin and then sectioned at a thickness of 4 µm. Then, 4 µm-thick paraffin sections were stained with haematoxylin and eosin. Immunostaining was carried out as follows: The 4 µm paraffin sections were rehydrated, subjected to antigen retrieval, rinsed three times with PBS and treated with blocking solution (1% BSA) for 1 h prior to incubation with primary antibodies at 4°C overnight. A primary antibody against SOX9 (Abcam, ab185966) was used. Goat anti-rabbit Alexa Fluor 546 (Invitrogen, A11035) and DAPI (Beyotime, China) secondary antibodies were used to label the primary antibodies and cell nuclei, respectively. All procedures were performed according to the manufacturer’s instructions. Results A comprehensive transcriptional atlas of single cells in the full-thickness uteri of Meishan pigs The uteri of Meishan pigs, which comprise the endometria, myometria, and perimetria, were utilised for single-cell suspension preparation. To construct a cellular map of the Meishan pig uterus using scRNA-seq analysis, we followed a standard protocol to dissect the uterine tissues and isolate single cells from each experimental group and then conducted scRNA-seq using the 10× Chromium Genomics pipeline. Subsequently, we applied filters based on the number of RNA molecules (n Count_ RNA 500), and mitochondrial content (percent. mt < 5) in each individual cell (Supplementary Fig S1A). After performing computational quality control and RNA removal, we were able to obtain an average of 4,500-6,500 cells per sample. A total of 8,185 high-quality cells were subjected to uniform manifold approximation and projection (UMAP) analysis, leading to the identification of 7 major cell populations (Fig 1D). A histogram illustrates the distribution of various cell types in full-thickness uterine tissue obtained from Meishan pigs (Fig 1E and Additional file 1). Each of these 7 cell types in our dataset has a different specific marker gene: epithelial cells ( CDH1 , KRT18 , KRT8 and EPCAM ); endothelial cells ( PECAM1 , VWF , CD34 , SOX18 , CDH5 and CLDN5 ); fibroblasts ( COL3A1 , HOXA10 , ACTA2 , MYH11 and FN1 ); T-cells ( CD3D , CD3E , CD8A , GZMB , CD4 and PDCD1 ); macrophages ( C1QA , C1QB and C1QC ); neutrophils ( CSF3R ); and mast cells ( KIT and MS4A2 ). The top marker genes for each cell type are presented in a dot plot map (Fig 1F). These clusters can be grouped into four main cellular categories: (1) epithelial; (2) endothelial; (3) fibroblast; and (4) immune (T cells, macrophages, neutrophils and mast cells). Furthermore, we aimed to identify novel biomarkers specific to each cell type. We identified genes that exhibited significantly greater expression levels in the cell type of interest than in other cell types using the Wilcoxon rank sum test ( P value <0.05) (Supplementary Fig S1C, Additional file 2). Endometrial epithelial cells are composed of four cluster subsets based on gene heterogeneity The classification of epithelial cells resulted in the identification of distinct cell groups, with four subpopulations distinguishable based on marker gene expression (Fig 2). (1) SOX9-expressing epithelium ( SOX9 [16] ), (2) ciliated epithelium ( TPPP3 , FOXJ1 , and TP73 [19] ), (3) glandular epithelium (GE) ( DLX5 and DLX6 [20] ) and (4) luminal epithelium (LE) ( LPAR3 [21, 22] ) (Fig 2C and 2D). During the proliferative phase, cell subpopulations predominantly consisted of SOX9-expressing epithelial cells (105, 87.5%), ciliated epithelial cells (9, 7.5%), and GE cells (6, 5%). In contrast, during the secretory phase of the endometrium, the cell subpopulations comprised SOX9-expressing epithelial cells (20, 3.13%), ciliated epithelial cells (337, 52.74%), GE cells (246, 38.5%) and LE cells (36, 5.63%) (Fig 2B, Additional file 3). The results of this experiment revealed a notably greater proportion of SOX9-expressing epithelial cells in the proliferative endometrium than in other cell types. Furthermore, immunofluorescence experiments revealed the specific expression of the SOX9 protein in the proliferative endometria of Meishan pigs, while it was not detected in the endometrium during the secretory phase (Fig 2E). SOX9-expressing epithelial cells are pluripotent in the Meishan pig endometrium The results of the present study revealed significant changes in the abundance of SOX9-expressing epithelial cells within the endometria of Meishan pigs between the proliferative and secretory phases. A subpopulation of SOX9-expressing epithelial cells was identified as the progenitor cell population for the endometrial epithelia in Meishan pigs. Endometrial epithelial progenitor cells are a vital cell population capable of differentiating into specific cells to perform distinct biological functions [23-25] . The marker genes currently used to identify human endometrial epithelial progenitor cells include AXIN2 [26] , CDH2 [27] , SSEA-1 [28] , LGR5 [29] , and SOX9 [24, 30] . In this study, the expression of the LGR5 , AXIN2 , and SOX9 genes in Meishan pig endometrial epithelial stem cells was also analysed. Furthermore, our investigation revealed distinct upregulation of expression of the NUPR1 , HES1 , IFI6 , SLPI , MMP7 , MX1 , CD74 , and S100A2 genes, specifically in SOX9-expressing epithelial cells of the Meishan pig endometria (Fig 3A, Additional file 4). GO enrichment analysis revealed that SOX9-expressing epithelial cells primarily participate in biological processes associated with cilium assembly and movement, as well as those associated with molecular functions involving ATP binding and nucleotide binding (Fig 3B, Additional file 5). This finding suggests that CE cells may differentiate from SOX9-expressing endometrial epithelial stem cells. KEGG functional enrichment analysis revealed significant enrichment in signalling pathways regulating the pluripotency of stem cells, GnRH secretion, the FOXO signalling pathway, the oestrogen signalling pathway, the mTOR signalling pathway, PI3K/Akt signalling pathway, the WNT signalling pathway, TGF-β signalling pathway, and MAPK signalling pathway (Fig 3C, Additional file 6). Endometrial luminal epithelial cells exhibit enriched expression of genes relevant to embryo implantation In the present study, it was observed that LE cells initiate their formation within the endometrium during the secretory phase. The LE of the endometrium plays a crucial role in embryo implantation. LPAR3 , NMU , DEFB1.1 , TSPO , HPGD , PHGDH , and H2AJ were identified as genes with significantly elevated expression in LE cells (Fig 4A, Additional file 7). GO enrichment analysis revealed significant enrichment of genes involved in membrane, integral component of membrane, cytosol, cytoplasm, identical protein binding, oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor, NAD binding, extracellular region, identical protein binding, extracellular space and membrane oxidoreductase activities (Fig 4B, Additional file 8). Interactions between specific genes and signalling pathways were observed in LE cells (Fig 4C, Additional file 9). Based on Fig 3C, it can be inferred that LPAR3 primarily functions in the Rap1 signalling pathway, the PI3K/Akt signalling pathway, the phospholipase D signalling pathway, and the neuroactive ligand‒receptor interaction pathway. The PHGDH gene primarily functions in metabolic pathways, including amino acid biosynthesis, carbon metabolism, cysteine and methionine metabolism, and glycine, serine, and threonine metabolism. Previous studies have reported that the local balance of progesterone and estrogen signalling in the mouse uterus is disrupted in Lpar3-knockout mice during implantation, leading to a delay in embryo implantation [22] . Cytochrome P45026A1 (cyp26a1) is reportedly expressed in the mouse uterus prior to implantation, and inhibition of the activity of this protein reduces the number of implantation sites for mouse embryos [31, 32] . Embryo-derived 15-HPGD expression is necessary for maintaining pregnancy in mice, with 15-HPGD -/- mice experiencing early embryo loss during gestation [33] . H2AJ is a mammal-specific histone variant with cell type-specific expression that is particularly abundant in the LE cells of various glands, including the mammary, prostate, pancreatic, thyroid, gastric, and salivary glands [34] . Biological functions of endometrial glandular epithelial cells in Meishan pigs In the present study, many endometrial glandular epithelial cells were found to be produced during the endometrial secretory phase. The endometrial glands and their secretions are critical for blastocyst survival, implantation, and embryo and placental development during early pregnancy. A total of 2374 genes exhibited significantly and specifically high expression in GE cells (Additional file 10). In this study, we focused on eight genes with significantly elevated expression in GE cells, namely, ST6GALNAC1 , AKR1A1 , MT3 , MT1A , SPDEF , CREB3L4 , AQP3 and MGP (Fig 5A). The GE cells were implicated in the biological processes of protein stabilization, endoplasmic reticulum unfolded protein response, and negative regulation of the apoptotic process. The molecular functions included metal ion binding, transferase activity, protein binding, and DNA binding (Fig 5B, Additional file 11). The SPDEF gene primarily participates in the biological process of regulating transcription via RNA polymerase II and exerts a positive regulatory effect on apoptosis. Its molecular functions include DNA-binding transcription factor activity and sequence-specific DNA binding (Fig 5C). The interplay between target genes and signalling pathways in this network is depicted in Fig 5D. The genes ST6GALNAC1 and AKR1A1 primarily participate in metabolic pathways, encompassing mucin type O-glycan biosynthesis, glycolysis/gluconeogenesis, ascorbate and aldarate metabolism, and pentose and glucuronate interconversions. The metalloenzyme family genes MT3 and MT1A are primarily involved in the mineral absorption pathway. CREB3L4 is a canonical transcription factor primarily involved in the regulation of diverse signalling pathways, including the PI3K/Akt signalling pathway, oestrogen signalling pathways, growth hormone synthesis and secretion, the AMPK signalling pathway, and the cAMP signalling pathway. In addition, CREB3L4 and AQP3 jointly participate in the regulation of the antidiuretic hormone-mediated water reabsorption signalling pathway (Additional file 12). The secretory ciliated cells evolved from proliferative SOX9-expressing epithelial cells according to pseudotime analysis The developmental trajectory of endometrial epithelial cells in Meishan pigs was constructed to study the transcriptional characteristics of endometrial epithelial cells across the estrous cycle (Fig 6). These results indicate that SOX9-expressing epithelial cells initiate differentiation primarily during the early stage of the estrous cycle, which is associated with endometrial hyperplasia. Subsequently, significant production of LE, GE, and CE cells occurs during late differentiation when the endometrium enters its secretory stage (Fig 6A and 6B). The expression patterns of characteristic genes in endometrial epithelial cells can be classified into four modules, each containing subsets that represent gradual transitions from SOX9-expressing epithelial cells to GEs and LEs. This pathway consists of a branch and two cell fates: SOX9-GE (cell fate 1; generation of ciliated epithelia) and SOX9-LE (cell fate 2; generation of luminal and glandular epithelial cells) (Fig 6C). Pseudotime analysis of endometrial epithelial cells revealed four distinct gene expression modules. Module 1 exhibited a distinct set of marker genes in ciliated epithelial cells, including TP73 , TPPP3 , and FOXJ1 . Genes with high expression levels in module 1 included HMGCS1 , PCM1 , CFAP54 , CFAP44 , CFAP45 , CFAP52 , DNAH5 , CIBAR2 , RSPH4A , MNS1 , SPEF2 , SNTN , DYDC2 , CAPS2 , LRRC71 , LRRC10B , LRRC46 , ODAD2 , CAPS2 , CIBAR2 , FAM183A , FAM216B , CETN2 , C20orf85 , MORN5 , RIBC2 , and SP ACA9 . PCM1 binds to CROCC, and this interaction is critical not only for the accumulation of centriolar satellites near centrosomes and basal bodies but also for cilia formation [35] . RSPH4A was found to be associated with primary ciliary dyskinesia [36] . MNS1 and DNAH5 have been implicated in the assembly of axonemes in murine respiratory epithelial cells [37] . MNS1 is essential for spermiogenesis and motile ciliary functions in mice [38] . SPEF2 is essential for the assembly of sperm flagella in humans [39, 40] . The sentinel-cilium apical structural protein (SNTN) is specifically expressed in multiciliated cells of the nasal epithelium [41] . Module 2 exhibited high expression levels of ST6GALNAC1 , CADPS2 , MT3 , MGP , HPGD , CYP26A1 , NMU , MT1A , TSPO and AQP3 , which were specifically highly expressed in GE and LE cells. Additionally, several other genes, such as NBL1 , AKR1A1 , CCL28 , SAT1 , PLA2G7 and MRPS36 , exhibited significant and specific expression within this module. Spermine N’-adenosyltransferase (SAT1) converts spermine into N1-acetylspermine, and polyamine production is crucial for follicular development, ovulation, and reproductive processes in mammals, as well as for the establishment and maintenance of gestation [42] . Module 3 exhibited high expression levels of UBB , RPL8 , RPL 1 7 , RPS3A , RPS1 2 and RPS19 . Finally, module 4 displayed high expression levels of SOX9 , MMP7 , HES1 , NUPR1 and SLPI , which were highly expressed specifically in SOX9-expressing epithelial cells (Fig 6C). The expression patterns of the representative genes were consistent along the pseudotime axis, as illustrated in Fig 6D. As shown in the figure, the expression levels of MMP7 and NURP1 were high during the early stage (proliferative stage), gradually decreased, and then remained stable after reaching their lowest expression levels in the middle stage (secretory stage). The initial expression levels of AQP3 , MGP , NUM and TPSO were low but gradually increased during proliferation before reaching their lowest expression levels during secretion and remaining stable thereafter. Moreover, the expression levels of FOXJ1 and TPPP3 remained consistently low throughout the proliferative phase but gradually increased during the secretory phase (Fig 6D). Discussion Previous studies have predominantly focused on gene transcription in endometrial tissue using bulk RNA-seq. However, it is crucial to consider the heterogeneity and cellular interactions among distinct cell subpopulations within complex tissues and organs. The use of scRNA-seq technology has become a primary method for exploring the diversity and complexity of RNA transcripts within individual cells. This technique enables the elucidation of cellular compositions and functions in heterogeneous organs or structurally intricate tissues [ 17 ] . In this study, we conducted detailed cellular mapping of the Meishan pig uterus, encompassing the most comprehensive tissue characterization to date. Some studies have identified the transcription factor SOX9 as being crucial for various biological processes, including uterine gland development [ 43 , 44 ] , cartilage differentiation [ 45 , 46 ] , hair growth [ 47 ] , and testis determination [ 48 ] . Interestingly, our study revealed obvious SOX9 protein localization in the basal layer of the Meishan pig endometrium. Hence, the SOX9 gene was identified as a potential marker of endometrial epithelial progenitor cells in Meishan pigs. Moreover, the results of this study revealed that NUPR1 and HES1 may serve as marker genes for identifying endometrial SOX9-expressing epithelial cells. NUPR1 serves as a transcriptional regulator that plays a decisive role in regulating the differentiation of intestinal epithelial cells [ 49 ] . Hes1 , a major target gene in the Notch signalling pathway, regulates the fate and differentiation of various cell types in many developmental systems. Hes1 regulates the development of the mouse cornea and the homeostatic functions of corneal epithelial stem/progenitor cells [ 50 ] . In this study, in addition to identifying marker genes for porcine endometrial epithelial stem cells, several candidate marker genes potentially associated with adenoepithelial and ciliated epithelial cells were also identified. SPDEF was specifically highly expressed in GE cells, which renders it a potential candidate marker gene for GE cells. Researchers have discovered that expression of the Spdef gene regulates the differentiation of both goblet cells and antral mucous gland cells [ 51 ] . In Spdef -knockout mice, there are reduced numbers of goblet cells in the intestinal [ 52 ] , conjunctival [ 19 , 53 ] , and tracheobronchial mucosa, as well as impaired terminal differentiation. Similarly, expression of multiple key genes affecting cilia differentiation were identified in ciliated cells. It was indicated that the protein encoded by C20orf85 is associated with cilia functionality [ 54 ] . CFAP45 regulates adenine nucleotide homeostasis to facilitate mammalian ciliary and flagellar motility, thereby contributing to the maintenance of normal sperm function in asthenospermia patients [ 55 ] . Researchers have discovered that CDHR3 is a transmembrane protein whose expression is increased specifically in the ciliated cells of lung bronchioles [ 56 ] . The colocalization of expression of the FAM183A gene, specifically with the FOXJ1 gene, was observed in human endometrial ciliated epithelial cells [ 15 ] . A study by Kwon revealed that the RIBC2 gene plays a significant role in regulating ciliary motility in airway epithelial cells [ 57 ] . A study conducted by Gui et al. revealed that the microtubule-binding protein SPACA9 can form both spirals and striations within the microtubules of human cilia [ 58 ] . In both humans and mice, endometrial epithelial stem cells have been shown to possess the capacity to differentiate into LE and GE cells. Hapangama et al. [ 59 ] reported that SSEA1 and nuclear SOX9 (nSOX9), which serve as indicators of basal epithelial cells, exhibited certain glandular characteristics in vitro. Jin et al. [ 60 ] found that endometrial epithelial progenitor cells exhibit bidirectional differentiation capabilities, leading to the formation of both GE and LE cells in mice. Remarkably, this study provides a groundbreaking finding that SOX9-expressing epithelial cells in Meishan pigs may also exhibit the potential to differentiate into ciliated cells. Specifically, the MMP7 gene was highly expressed in SOX9-expressing epithelial cells according to the heatmap of gene expression analysis in the proposed time series. Endometrial expression of MMP7 occurs during menstrual breakdown and subsequent estrogen-mediated growth but not during the secretory phase [ 61 ] . MMP7 expression regulates the remodelling of the human endometrium to facilitate blastocyst implantation [ 62 ] . Axin2 is a well-known target gene of the WNT pathway that is expressed in the stem cells of multiple organs [ 63 ] . Axin2 + cells induce mouse endometrial epithelial growth and regeneration, and Axin2 + cells exhibit bidirectional differentiation into GEs and LEs, as confirmed by lineage tracing [ 26 ] . Interestingly, the results of this study revealed that the AXIN2 gene is expressed both in SOX9-expressing epithelial cells and GE cells, suggesting that SOX9 + AXIN2 + cells have the potential to differentiate into GE, LE and CE cells. The results of this study revealed significant variations in the epithelial cell types of the endometria of Meishan pigs during both the proliferative and secretory phases. Notably, a substantial presence of GE cells and CE cells was observed during the secretory phase, indicating a strong correlation between these specific cell types and their biological functions within the endometrium during this period. MT3 and MGP were highly expressed in secretory glandular epithelial cells. MT3 specifically belongs to the metallothionein gene family and plays a pivotal role in maintaining zinc and copper homeostasis. MT3 induces angiogenesis in vitro, potentially regulating endometrial angiogenesis in vivo and the occurrence of miscarriage [ 64 ] . In the rat epididymis, the matrix Gla protein (MGP) plays an essential role in promoting calcium-dependent protein aggregation [ 65 ] . AQP3 is a transmembrane protein that plays a crucial role in facilitating water transport and is essential for the exchange of fluids between the maternal and foetal compartments, as well as the production of amniotic fluid [ 66 , 67 ] . Importantly, we identified a series of crucial genes and signalling pathways associated with Meishan pig uterine development at the single-cell level. These findings underscore the pivotal role of Meishan pig endometrial epithelial cells in facilitating embryo implantation and provide novel insights into gene expression patterns in the porcine endometrium across different stages of the estrous cycle. Conclusions Using scRNA-seq, we characterized distinct epithelial subpopulations in the uterine tissues of Meishan pigs throughout the estrous cycle. For the first time, we identified a novel subset of porcine endometrial epithelial progenitor cells called SOX9-expressing epithelial cells, which have the potential to differentiate into CE, GE, and LE cells. Moreover, we identified NUPR1 and HES1 as potential marker genes for endometrial SOX9-expressing epithelial cells in Meishan pigs. In summary, our study provides a high-resolution molecular and cellular characterization of the endometrial transformation in Meishan pigs throughout the estrous cycle, offering insights into this essential physiological process. Abbreviations AQP3 Aquaporin 3 CADPS2 Calcium Dependent Secretion Activator 2 CDH1 Cadherin 1 CDHR3 Cadherin Related Family Member 3 CETN2 Centrin2 CROCC Ciliary Rootlet Coiled-Coil, Rootletin CYP26A1 Cytochrome P450 26A1 EPCAM Epithelial Cell Adhesion Molecule FOXJ1 Forkhead Box J1 KRT18 Keratin 18 KRT8 Keratin 8 H2AJ H2A Histone Family Member J HES1 Hes Family BHLH Transcription Factor 1 HPGD 15-hydroxyprostaglandin dehydrogenase LPAR3 Lysophosphatidic Acid Receptor 3 MMP7 Matrix Metallopeptidase 7 MGP Matrix Gla Protein NMU Neuromedin U NUPR1 Nuclear Protein 1 PCM1 Pericentriolar Material 1 LPAR3 Lysophosphatidic Acid Receptor 3 PRDX5 Peroxiredoxin 5 PHGDH/3-PGDH 3-Phosphoglycerate dehydrogenase ST6GALNAC1 ST6 N-Acetylgalactosaminide Alpha-2,6-Sialyltransferase 1 SOX9 SRY-Box Transcription Factor 9 SLPI Secretory Leukocyte Peptidase Inhibitor SNTN Sentan-cilium apical structural protein SAT1 Spermidine/Spermine N1-Acetyltransferase 1 SPEF2 Sperm Flagellar 2 TPPP3 Tubulin Polymerization Promoting Protein Family Member 3 TP73 Tumor Protein P73 TSPO translocator protein 18 kDa Declarations Acknowledgements We would like to express our gratitude to Kunshan Meishan Pig Breeding Co. Ltd, China. Authors’ Contributions Conceptualization, R. H.; formal analysis, N. J.; investigation, N.J., W. X., C.L., Q.Z., J.M., Q.L., W.C. and X.X.; resources, C. Y. and B.X., writing—original draft preparation, N.J.; writing—review and editing, L.H. and R. H.; supervision, P.L., L.H. and Q.Z.; All authors have read and agreed to the published version of the manuscript. Funding This study was supported by the National Key Research and Development Program grant 2021YFD1301101, the Fundamental Research Funds for the Central Universities (KYT2023002), and the Project of Jiangsu Agricultural (Pig) Industry Technology Syprogenitor grants JATS (2023) 186. Availability of data and materials The single-cell RNA sequencing data used in this research have been deposited in the Genome Sequence Archive (GSA) at the Beijing Institute of Genomics (BIG), Chinese Academy of Sciences. The accession number for this dataset is CRA016749. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4582781","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":316335527,"identity":"d0fb19af-eaf0-46ea-893a-c5745d348cc5","order_by":0,"name":"Nengjing Jiang","email":"","orcid":"","institution":"Institute of Swine Science (Key Laboratory of Pig Genetic Resources Evaluation and Utilization, Ministry of Agriculture and Rural Affairs (Nanjing)), Nanjing Agricultural 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University","correspondingAuthor":true,"prefix":"","firstName":"Ruihua","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2024-06-14 14:46:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4582781/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4582781/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59520891,"identity":"a54477f0-9177-4c0f-8a79-5f5fe930394e","added_by":"auto","created_at":"2024-07-02 19:20:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4600265,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle-cell profiling of a Meishan pig uterus. \u003c/strong\u003e(A) Experimental workflow for single-cell RNA sequencing of the uterine horns of Meishan pigs. (B)\u003cstrong\u003e \u003c/strong\u003eEndometrial morphologies of a Meishan pig during the proliferative and secretory phases (HE staining). (C) Schematic illustration of a pig uterus showing the different layers and the morphological changes observed throughout the estrous cycle with respect to tissue sampling. (D) UMAP projections of scRNA-seq data from the uteri of two Meishan pigs. (E) Histogram showing the percentages of all cell types in uterine tissue from Meishan pigs. (F) A dot plot map displaying the marker genes for each cell type.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4582781/v1/9c75566b234cfff0e557b87b.jpg"},{"id":59520615,"identity":"96e34798-b827-42a5-b334-effa2ef06923","added_by":"auto","created_at":"2024-07-02 19:12:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6273073,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of endometrial epithelial cells.\u003c/strong\u003e(A) UMAP visualization of subclustered and subsampled epithelial cell populations. (B) The percentages of 4 types of epithelial cells present in the proliferative and secretory phases. (C) Dot plot illustrating the log2-transformed expression levels of genes distinctive to each subset of epithelial cells. (D) UMAP visualization maps of marker genes specific to subpopulations of endometrial epithelial cells.\u003c/p\u003e\n\u003cp\u003e(E) The immunofluorescence image shows a magnified view of the endometrial epithelium with a high density of SOX9 expression. 4',6-Diamidino-2-phenyl-indole (DAPI) was used as a nuclear counterstain. The lengths of the scale bars were 500 µm and 200 µm.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4582781/v1/302dd7533583dde999f729f1.jpg"},{"id":59520608,"identity":"657a5875-bc86-4196-928d-0d15de759c7c","added_by":"auto","created_at":"2024-07-02 19:12:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4526825,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of the biological functions of SOX9 expression in epithelial cells.\u003c/strong\u003e (A) Volcano plots depicting DEGs in SOX9-expressingepithelialcells. (B) The bar chart illustrates the top 30 enriched terms of SOX9-expressing epithelial cells obtained from gene ontology (GO) analysis.\u003c/p\u003e\n\u003cp\u003e(C) Bubble plots depicting the pivotal signalling pathways implicated in SOX9-expressing epithelial cells.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4582781/v1/eb2bf6b90b8dc1e6d5301e92.jpg"},{"id":59520892,"identity":"f79a9592-7cf2-4466-a533-bb8588baab28","added_by":"auto","created_at":"2024-07-02 19:20:30","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3873442,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of the biological functions of LE cells.\u003c/strong\u003e (A) Volcano plots depicting DEGs in LE cells. (B) Gene Ontology analysis of DEGs upregulated in LE cells. (C) Network interactions among target genes and signalling pathways.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4582781/v1/ca513cb4575891e832576f18.jpg"},{"id":59521390,"identity":"dcedb254-ef5a-4d01-9008-e0cc6f7fd83b","added_by":"auto","created_at":"2024-07-02 19:28:30","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5471483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of the biological functions of GE cells.\u003c/strong\u003e (A) UMAP plots depicting DEGs in GE cells. (B) Gene Ontology analysis of DEGs upregulated in GE cells. (C) Gene Ontology analysis of the \u003cem\u003eSPDEF\u003c/em\u003e gene. (D) Network interactions among target genes and signalling pathways.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4582781/v1/44a57a502fc851138d68aa19.jpg"},{"id":60132936,"identity":"f3902703-497c-4587-9bd1-fd85992b0b62","added_by":"auto","created_at":"2024-07-12 07:29:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":25429423,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4582781/v1/b32b00c6-6c35-47d1-af4f-ebd0eb60b23e.pdf"},{"id":59520611,"identity":"38a362b1-c179-429b-931e-f5d8c30d8772","added_by":"auto","created_at":"2024-07-02 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19:12:31","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":904340,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile11.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4582781/v1/d2c2370f89368b58efd38c90.xlsx"},{"id":59520622,"identity":"b480aa98-fd58-49df-8b9e-2308cf91536c","added_by":"auto","created_at":"2024-07-02 19:12:31","extension":"xlsx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":70811,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile12.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4582781/v1/9222feda5ad1e8f6391d220e.xlsx"},{"id":59520895,"identity":"880aa59e-a7e4-4c8e-badc-8dea2bb86f39","added_by":"auto","created_at":"2024-07-02 19:20:31","extension":"jpg","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":2220940,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4582781/v1/4f2d33237dc2e7a66edeb00a.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Single-Cell Analysis of the Endometrial Characteristics of Meishan Pigs Across the Estrous Cycle","fulltext":[{"header":"Background","content":"\u003cp\u003eThe Meishan breed of pigs, which originated in the Taihu Lake region in China, is one of the most globally renowned native pig breeds\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Renowned for its exceptional fertility, it can produce three to five more piglets per litter than traditional U.S. or European pig varieties\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The litter size of a pig is limited by the pig\u0026rsquo;s uterine capacity, which is influenced by factors such as the size of the uterus, dimensions of the placenta, and degree of vascularization\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Previous studies regarding the fecundity of Meishan sow pigs have indicated that their prolificacy stems from lower embryonic mortality than European sows with similar ovulation rates\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. The Meishan pig possesses a greater uterine capacity than does the Large White pig and sustains a greater number of implanted embryos throughout gestation\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Between gestation days 70 and 110, a twofold increase in blood vessel density in both the placenta and endometrium of Landrace pigs was observed. Conversely, the blood vessel densities in the placentae and endometria of Large White pigs remains relatively stable\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Meishan pigs have been exported into many countries, including the U.S. and Japan\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. It is regarded as an outstanding model animal for hybridization and the improvement of reproductive traits by genetic manipulation. The preservation of Meishan pigs is not only crucial for maintaining their exceptional traits but also offers valuable insights for improving the reproductive capabilities of other pig breeds and for developing new breeds. Therefore, investigating the physiological functions associated with reproductive traits in Meishan pigs is imperative.\u003c/p\u003e \u003cp\u003eThe uterus is a key component in the reproductive processes of female animals, and the endometrium is a pivotal site for embryo implantation\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Establishing a particular endometrial state is crucial for successful embryo implantation in female animals\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. The endometrium is most receptive to embryo implantation during the ovarian luteal phase following ovulation, this is the most favourable phase for successful implantation\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. During the luteal phase, the endometrial glands secrete cytokines, such as adhesins, which facilitate the settling of the embryo. These glands play a vital role in supporting pregnancy by providing necessary nutrients to the developing embryo\u003csup\u003e[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. The uterus is a complex organ with a heterogeneous anatomical structure. During the estrous cycle, the endometrium undergoes coordinated shedding, regeneration and differentiation, which are regulated by the hypothalamic-pituitary-ovarian axis. During the human menstrual cycle, endometrial epithelial cells undergo significant changes, including rapid cell differentiation and endometrial remodelling\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe uterus, a complex organ, is regulated by a diverse variety of cell types. Single-cell RNA sequencing (scRNA-seq) technology is widely used in heterogeneous tissues to identify various cell types and to construct cellular developmental trajectories at the single-cell level to determine the gene transcription profiles of cells\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Therefore, scRNA-seq is a valuable tool for examining the mechanisms that drive the dynamic alterations in endometrial tissue throughout the estrous cycle after sexual maturation. In 2021, scRNA-seq was employed to compare and analyse the cell types present in the human uterus during the proliferative and secretory phases, allowing for identification of a total of 14 distinct cell subsets within human uterine tissues\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. To date, several studies have conducted scRNA-seq on human\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e and mouse\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e uterine tissues to explore the gene transcription characteristics of endometrial epithelial cells during the estrous cycle. However, research on single-cell sequencing of porcine uterine tissues is scarce.\u003c/p\u003e \u003cp\u003eThe Meishan pig, a high-yielding local pig breed native to the Taihu Lake Basin, is known for its favourable intrauterine environment and high rate of embryo implantation. However, the characteristics and genetic mechanisms of endometrial development after sexual maturity in this pig breed remain largely unknown. In this study, we performed scRNA-seq on uterine tissues during the proliferative and secretory phases. Our goal was to identify the cell types present in the uteri of sexually mature Meishan pigs and to elucidate their growth and development patterns, as well as the transcriptional regulatory networks of endometrial epithelial cells throughout the estrous cycle.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and sample collection\u003c/h2\u003e \u003cp\u003eUteri were collected from two healthy 8-month-old Meishan pigs (n\u0026thinsp;=\u0026thinsp;2). The proliferative phase of the uterus was identified by the presence of ovarian follicles with no corpora lutea, while the secretory phase of the uterus was identified by the presence of four to six corpora lutea in the ovary. Approximately 2 cm of tissue was excised from one side of each uterine horn and divided into two pieces: one portion was immersed in 4% paraformaldehyde for histological examination, while the other segment was preserved in MACS Tissue Storage Solution (Miltenyi Biotec) for single-cell RNA sequencing. All the animals were provided by the Kunshan Meishan Pig Breeding Farm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSingle-cell dissociation\u003c/h2\u003e \u003cp\u003eThe collected uterine tissues were surgically removed and kept in MACS Tissue Storage Solution (Miltenyi Biotec) until processing. The tissue samples were processed as described below. Briefly, the samples were washed with phosphate-buffered saline (PBS), minced into small pieces (approximately 1 mm\u003csup\u003e3\u003c/sup\u003e) on ice and enzymatically digested with 2 mg/mL collagenase IV (Worthington), 1 mg/mL dispase II (Worthington) and 50 U/\u0026micro;L DNase I (Worthington) for 40 min at 37\u0026deg;C with agitation. After digestion, the samples were passed through a 70 \u0026micro;m cell strainer and centrifuged at 300 \u0026times; g for 5 min. After the supernatant was removed, the pelleted cells were suspended in 1\u0026times; red blood cell lysis buffer (Miltenyi Biotec) to lyse the red blood cells. After washing with PBS containing 0.04% BSA, the cell pellets were resuspended in PBS containing 0.04% BSA and filtered through a 40 \u0026micro;m cell strainer. Dissociated single cells were then stained with AO/PI for viability assessment using a Countstar Fluorescence Cell Analyser.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSingle-cell sequencing\u003c/h2\u003e \u003cp\u003eThe scRNA-seq libraries were generated using a 10x Genomics Chromium Controller Instrument and Chromium Single Cell 3\u0026rsquo; V3.1 Reagent Kits (10x Genomics, Pleasanton, CA). The process was as follows: 1) Cells were concentrated to approximately 1000 cells/\u0026micro;L and loaded into each channel to generate single-cell gel beads-in-emulsion (GEMs). 2) After the reverse transcription (RT) step, the GEMs were broken, and the resulting barcoded cDNA was purified and amplified. 3) The amplified barcoded cDNA was fragmented, subjected to A-tailing, and ligated with adaptors, followed by index PCR amplification. 4) The final libraries were quantified using a Qubit high-sensitivity DNA assay (Thermo Fisher Scientific), and the size distribution of each library was determined using a high-sensitivity DNA chip on a Bioanalyzer 2200 (Agilent). 5) All libraries were sequenced on an Illumina NovaSeq 6000 (Illumina, San Diego, CA) platform on a 150 bp paired-end run.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSingle-cell RNA statistical analysis\u003c/h2\u003e \u003cp\u003eThe scRNA-seq data analysis was performed by NovelBio Bio-Pharm Technology Co., Ltd., using the NovelBrain Cloud Analysis Platform (Shanghai, China). The analysis process included the following steps: 1) Quality control and filtering: The raw sequencing reads were processed with fastp using default parameters to filter the adaptor sequence and remove lower-quality reads to obtain clean data. 2) Alignment and quantification: The cleaned reads were then aligned to the pig genome (Sscrofa11.1 Ensemble: version 100) using CellRanger v6.1.1, which generated feature-barcode matrices. 3) Normalization: Down sampling analysis was performed across samples to normalize the data based on the number of mapped barcoded reads per cell for each sample. 4) Aggregation: The normalized data from individual samples were aggregated to form a comprehensive matrix. 5) Quality filtering: Cells with more than 200 expressed genes and a mitochondrial Unique Molecular Identifier (UMI) percentage of less than 20% were considered to have passed the cell quality filtering criteria. 6) Mitochondrial gene removal: Mitochondrial genes were subsequently removed from the expression table to focus on the nuclear transcriptome.\u003c/p\u003e \u003cp\u003eThe Seurat package (version: 4.0.3, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://satijalab.org/seurat/\u003c/span\u003e\u003cspan address=\"https://satijalab.org/seurat/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used for cell normalization and regression based on the expression table according to the UMI counts of each sample and percentage of mitochondria to obtain the scaled data. PCA was constructed based on the scaled data with the top 2000 highly variable genes, and the top 10 principal components were used for tSNE construction and UMAP construction. Utilising a graph-based clustering method, we acquired the unsupervised cell cluster results based on the top 10 principal components of the PCA, and we calculated the marker genes using the Find All Markers function with the Wilcoxon rank sum test algorithm under the following criteria: lnFC\u0026thinsp;\u0026gt;\u0026thinsp;0.25, \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and min.pct\u0026thinsp;\u0026gt;\u0026thinsp;0.1. To identify the cell types in detail, clusters of the same cell type were selected for re-tSNE analysis, graph-based clustering and marker analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePseudotime analysis\u003c/h2\u003e \u003cp\u003eWe applied single-cell trajectory analysis using Monocle2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cole-trapnell-lab.github.io/monocle-release\u003c/span\u003e\u003cspan address=\"http://cole-trapnell-lab.github.io/monocle-release\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with DDR-Tree and default parameters. Before Monocle analysis, we selected marker genes from the Seurat clustering results and the raw expression counts of the cells that passed filtering. The \"plot pseudotime heatmap\" function was utilized to generate a heatmap illustrating the signature genes and highly variable genes across pseudotime.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGene ontology (GO) and enrichment analysis\u003c/h2\u003e \u003cp\u003eDifferentially expressed genes (DEGs) across cluster subsets and designated cell types in the proliferative-phase and secretory-phase groups were determined using the Seurat package. The log2 (fold change [FC]) and false discovery rate (FDR) q values for DEGs were ranked after passing the following control thresholds: log2 (FC)\u0026thinsp;\u0026gt;\u0026thinsp;0.5 and q value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The marker genes of each cluster generally presented higher expression levels than did the other functional genes.\u003c/p\u003e \u003cp\u003eWe averaged gene expression across the specific clusters of each group after normalization by interrogation using the 10\u0026times; Genomics Loupe Cell Browser software. Volcano plots were generated with the X-axis displaying log2(FC)\u0026thinsp;\u0026gt;\u0026thinsp;0.26 and the y-axis showing a \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 using GraphPad Software. Kyoto Encyclopedia of Genes and KEGG enrichment analysis of cluster DEGs was performed with the cluster R package clusterProfiler. The results were visualized as dot plots using ggplot2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHistology and immunofluorescence staining\u003c/h2\u003e \u003cp\u003eFull-thickness uterine tissues from Meishan pigs were fixed in 4% paraformaldehyde (w/v) for a minimum of 24 h, dehydrated using an ethanol gradient, embedded in paraffin and then sectioned at a thickness of 4 \u0026micro;m. Then, 4 \u0026micro;m-thick paraffin sections were stained with haematoxylin and eosin. Immunostaining was carried out as follows: The 4 \u0026micro;m paraffin sections were rehydrated, subjected to antigen retrieval, rinsed three times with PBS and treated with blocking solution (1% BSA) for 1 h prior to incubation with primary antibodies at 4\u0026deg;C overnight. A primary antibody against SOX9 (Abcam, ab185966) was used. Goat anti-rabbit Alexa Fluor 546 (Invitrogen, A11035) and DAPI (Beyotime, China) secondary antibodies were used to label the primary antibodies and cell nuclei, respectively. All procedures were performed according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eA comprehensive transcriptional atlas of single cells in the full-thickness uteri of Meishan pigs\u003c/p\u003e\n\u003cp\u003eThe uteri of Meishan pigs, which comprise the endometria, myometria, and perimetria, were utilised for single-cell suspension preparation. To construct a cellular map of the Meishan pig uterus using scRNA-seq analysis, we followed a standard protocol to dissect the uterine tissues and isolate single cells from each experimental group and then conducted scRNA-seq using the 10\u0026times; Chromium Genomics pipeline. Subsequently, we applied filters based on the number of RNA molecules (n Count_ RNA \u0026lt; 60,000), number of expressed genes (n Feature_ RNA \u0026gt; 500), and mitochondrial content (percent. mt \u0026lt; 5) in each individual cell (Supplementary Fig S1A).\u003c/p\u003e\n\u003cp\u003eAfter performing computational quality control and RNA removal, we were able to obtain an average of 4,500-6,500 cells per sample. A total of 8,185 high-quality cells were subjected to uniform manifold approximation and projection (UMAP) analysis, leading to the identification of 7 major cell populations (Fig 1D). A histogram\u0026nbsp;illustrates the distribution of various cell types in full-thickness uterine tissue obtained from Meishan pigs (Fig 1E and Additional file\u0026nbsp;1).\u003c/p\u003e\n\u003cp\u003eEach of these 7 cell types in our dataset has a different specific marker gene: epithelial cells (\u003cem\u003eCDH1\u003c/em\u003e, \u003cem\u003eKRT18\u003c/em\u003e, \u003cem\u003eKRT8\u003c/em\u003e and \u003cem\u003eEPCAM\u003c/em\u003e); endothelial cells (\u003cem\u003ePECAM1\u003c/em\u003e, \u003cem\u003eVWF\u003c/em\u003e, \u003cem\u003eCD34\u003c/em\u003e, \u003cem\u003eSOX18\u003c/em\u003e, \u003cem\u003eCDH5\u003c/em\u003e and \u003cem\u003eCLDN5\u003c/em\u003e); fibroblasts (\u003cem\u003eCOL3A1\u003c/em\u003e, \u003cem\u003eHOXA10\u003c/em\u003e, \u003cem\u003eACTA2\u003c/em\u003e, \u003cem\u003eMYH11\u003c/em\u003e and \u003cem\u003eFN1\u003c/em\u003e); T-cells (\u003cem\u003eCD3D\u003c/em\u003e, \u003cem\u003eCD3E\u003c/em\u003e, \u003cem\u003eCD8A\u003c/em\u003e, \u003cem\u003eGZMB\u003c/em\u003e, \u003cem\u003eCD4\u003c/em\u003e and \u003cem\u003ePDCD1\u003c/em\u003e); macrophages (\u003cem\u003eC1QA\u003c/em\u003e, \u003cem\u003eC1QB\u003c/em\u003e and \u003cem\u003eC1QC\u003c/em\u003e); neutrophils (\u003cem\u003eCSF3R\u003c/em\u003e); and mast cells (\u003cem\u003eKIT\u003c/em\u003e and \u003cem\u003eMS4A2\u003c/em\u003e).\u0026nbsp;The top marker genes for each cell type are presented in a dot plot map\u0026nbsp;(Fig 1F). These clusters can be grouped into four main cellular categories: (1) epithelial; (2) endothelial; (3) fibroblast; and (4) immune (T cells, macrophages, neutrophils and mast cells).\u0026nbsp;Furthermore, we aimed to identify novel biomarkers specific to each cell type.\u0026nbsp;We identified genes that exhibited significantly greater expression levels in the cell type of interest than in other cell types using\u0026nbsp;the\u0026nbsp;Wilcoxon rank sum test (\u003cem\u003eP\u003c/em\u003e value \u0026lt;0.05) (Supplementary Fig S1C, Additional file 2).\u003c/p\u003e\n\u003cp\u003eEndometrial epithelial cells are composed of four cluster subsets based on gene heterogeneity\u003c/p\u003e\n\u003cp\u003eThe classification of epithelial cells resulted in the identification of distinct cell groups, with four subpopulations distinguishable based on marker gene expression (Fig 2).\u0026nbsp;(1) SOX9-expressing epithelium (\u003cem\u003eSOX9\u003c/em\u003e\u003csup\u003e[16]\u003c/sup\u003e), (2) ciliated epithelium (\u003cem\u003eTPPP3\u003c/em\u003e, \u003cem\u003eFOXJ1\u003c/em\u003e, and \u003cem\u003eTP73\u003c/em\u003e\u003csup\u003e[19]\u003c/sup\u003e), (3) glandular epithelium (GE) (\u003cem\u003eDLX5\u003c/em\u003e and\u003cem\u003e\u0026nbsp;DLX6\u003c/em\u003e\u003csup\u003e[20]\u003c/sup\u003e) and (4) luminal epithelium (LE) (\u003cem\u003eLPAR3\u003c/em\u003e\u003csup\u003e[21, 22]\u003c/sup\u003e) (Fig 2C and 2D).\u0026nbsp;During the proliferative phase, cell subpopulations predominantly consisted of SOX9-expressing epithelial cells (105, 87.5%), ciliated epithelial cells (9, 7.5%), and GE cells (6, 5%). In contrast, during the secretory phase of the endometrium, the cell subpopulations comprised SOX9-expressing epithelial cells (20, 3.13%), ciliated epithelial cells (337, 52.74%), GE cells (246, 38.5%) and LE cells (36, 5.63%) (Fig 2B, Additional file\u0026nbsp;3).\u003c/p\u003e\n\u003cp\u003eThe results of this experiment revealed a notably greater proportion of SOX9-expressing epithelial cells in the proliferative endometrium than in other cell types. Furthermore, immunofluorescence experiments revealed the specific expression of the SOX9 protein in the proliferative endometria of Meishan pigs, while it was not detected in the endometrium during the secretory phase (Fig 2E).\u003c/p\u003e\n\u003cp\u003eSOX9-expressing epithelial cells are pluripotent in the Meishan pig endometrium\u003c/p\u003e\n\u003cp\u003eThe results of the present study revealed significant changes in the abundance of SOX9-expressing epithelial cells within the endometria of Meishan pigs between the proliferative and secretory phases. A subpopulation of SOX9-expressing epithelial cells was identified as the progenitor cell population for the endometrial epithelia in Meishan pigs. Endometrial epithelial progenitor cells are a vital cell population capable of differentiating into specific cells to perform distinct biological functions\u003csup\u003e[23-25]\u003c/sup\u003e. The marker genes currently used to identify human endometrial epithelial progenitor cells include \u003cem\u003eAXIN2\u003c/em\u003e\u003csup\u003e[26]\u003c/sup\u003e, \u003cem\u003eCDH2\u003c/em\u003e\u003csup\u003e[27]\u003c/sup\u003e, \u003cem\u003eSSEA-1\u003c/em\u003e\u003csup\u003e[28]\u003c/sup\u003e, \u003cem\u003eLGR5\u003c/em\u003e\u003csup\u003e[29]\u003c/sup\u003e, and \u003cem\u003eSOX9\u003c/em\u003e\u003csup\u003e[24, 30]\u003c/sup\u003e. In this study, the expression of the \u003cem\u003eLGR5\u003c/em\u003e, \u003cem\u003eAXIN2\u003c/em\u003e, and \u003cem\u003eSOX9\u003c/em\u003e genes in Meishan pig endometrial epithelial stem cells was also analysed. Furthermore, our investigation revealed distinct upregulation of expression of the \u003cem\u003eNUPR1\u003c/em\u003e, \u003cem\u003eHES1\u003c/em\u003e, \u003cem\u003eIFI6\u003c/em\u003e, \u003cem\u003eSLPI\u003c/em\u003e, \u003cem\u003eMMP7\u003c/em\u003e, \u003cem\u003eMX1\u003c/em\u003e, \u003cem\u003eCD74\u003c/em\u003e, and \u003cem\u003eS100A2\u003c/em\u003e genes, specifically in SOX9-expressing epithelial cells of the Meishan pig endometria (Fig 3A, Additional file 4).\u003c/p\u003e\n\u003cp\u003eGO enrichment analysis revealed that SOX9-expressing epithelial cells primarily participate in biological processes associated with cilium assembly and movement, as well as those associated with molecular functions involving ATP binding and nucleotide binding (Fig 3B, Additional file 5). This finding suggests that CE cells may differentiate from SOX9-expressing endometrial epithelial stem cells. KEGG functional enrichment analysis revealed significant enrichment in signalling pathways regulating the pluripotency of stem cells, GnRH secretion, the FOXO signalling pathway, the oestrogen signalling pathway, the mTOR signalling pathway, PI3K/Akt signalling pathway, the WNT signalling pathway, TGF-\u0026beta; signalling pathway, and MAPK signalling pathway (Fig 3C, Additional file 6).\u003c/p\u003e\n\u003cp\u003eEndometrial luminal epithelial cells exhibit enriched expression of genes relevant to embryo implantation\u003c/p\u003e\n\u003cp\u003eIn the present study, it was observed that LE cells initiate their formation within the endometrium during the secretory phase. The LE of the endometrium plays a crucial role in embryo implantation. \u003cem\u003eLPAR3\u003c/em\u003e, \u003cem\u003eNMU\u003c/em\u003e, \u003cem\u003eDEFB1.1\u003c/em\u003e, \u003cem\u003eTSPO\u003c/em\u003e, \u003cem\u003eHPGD\u003c/em\u003e, \u003cem\u003ePHGDH\u003c/em\u003e, and \u003cem\u003eH2AJ\u003c/em\u003e were identified as genes with significantly elevated expression in LE cells (Fig 4A, Additional file 7). GO enrichment analysis revealed significant enrichment of genes involved in membrane, integral component of membrane, cytosol, cytoplasm, identical protein binding, oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor, NAD binding, extracellular region, identical protein binding, extracellular space and membrane oxidoreductase activities\u0026nbsp;(Fig\u0026nbsp;4B,\u0026nbsp;Additional file\u0026nbsp;8).\u003c/p\u003e\n\u003cp\u003eInteractions between specific genes and signalling pathways were observed in LE cells (Fig 4C, Additional file 9). Based on Fig 3C, it can be inferred that \u003cem\u003eLPAR3\u003c/em\u003e primarily functions in the Rap1 signalling pathway, the PI3K/Akt signalling pathway, the phospholipase D signalling pathway, and the neuroactive ligand‒receptor interaction pathway. The \u003cem\u003ePHGDH\u003c/em\u003e gene primarily functions in metabolic pathways, including amino acid biosynthesis, carbon metabolism, cysteine and methionine metabolism, and glycine, serine, and threonine metabolism.\u003c/p\u003e\n\u003cp\u003ePrevious studies have reported that the local balance of progesterone and estrogen signalling in the mouse uterus is disrupted in Lpar3-knockout mice during implantation, leading to a delay in embryo implantation\u003csup\u003e[22]\u003c/sup\u003e. Cytochrome P45026A1 (cyp26a1) is reportedly expressed in the mouse uterus prior to implantation, and inhibition of the activity of this protein reduces the number of implantation sites for mouse embryos\u003csup\u003e[31, 32]\u003c/sup\u003e. Embryo-derived 15-HPGD expression is necessary for maintaining pregnancy in mice, with 15-HPGD\u003csup\u003e-/-\u003c/sup\u003e mice experiencing early embryo loss during gestation\u003csup\u003e[33]\u003c/sup\u003e. H2AJ is a mammal-specific histone variant with cell type-specific expression that is particularly abundant in the LE cells of various glands, including the mammary, prostate, pancreatic, thyroid, gastric, and salivary glands\u003csup\u003e[34]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eBiological functions of endometrial glandular epithelial cells in Meishan pigs\u003c/p\u003e\n\u003cp\u003eIn the present study, many endometrial glandular epithelial cells were found to be produced during the endometrial secretory phase. The endometrial glands and their secretions are critical for blastocyst survival, implantation, and embryo and placental development during early pregnancy. A total of 2374 genes exhibited significantly and specifically high expression in GE cells (Additional file 10). In this study, we focused on eight genes with significantly elevated expression in GE cells, namely, \u003cem\u003eST6GALNAC1\u003c/em\u003e, \u003cem\u003eAKR1A1\u003c/em\u003e, \u003cem\u003eMT3\u003c/em\u003e, \u003cem\u003eMT1A\u003c/em\u003e, \u003cem\u003eSPDEF\u003c/em\u003e, \u003cem\u003eCREB3L4\u003c/em\u003e, \u003cem\u003eAQP3\u003c/em\u003e and \u003cem\u003eMGP\u003c/em\u003e (Fig 5A).\u0026nbsp;The GE cells were implicated in the biological processes of protein stabilization, endoplasmic reticulum unfolded protein response, and negative regulation of the apoptotic process. The molecular functions included metal ion binding, transferase activity, protein binding, and DNA binding\u0026nbsp;(Fig\u0026nbsp;5B,\u0026nbsp;Additional file 11).\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eSPDEF\u003c/em\u003e gene primarily participates in the biological process of regulating transcription via RNA polymerase II and exerts a positive regulatory effect on apoptosis. Its molecular functions include DNA-binding transcription factor activity and sequence-specific DNA binding (Fig 5C). The interplay between target genes and signalling pathways in this network is depicted in Fig 5D. The genes \u003cem\u003eST6GALNAC1\u003c/em\u003e and \u003cem\u003eAKR1A1\u003c/em\u003e primarily participate in metabolic pathways, encompassing mucin type O-glycan biosynthesis, glycolysis/gluconeogenesis, ascorbate and aldarate metabolism, and pentose and glucuronate interconversions. The metalloenzyme family genes \u003cem\u003eMT3\u003c/em\u003e and \u003cem\u003eMT1A\u003c/em\u003e are primarily involved in the mineral absorption pathway. CREB3L4 is a canonical transcription factor primarily involved in the regulation of diverse signalling pathways, including the PI3K/Akt signalling pathway, oestrogen signalling pathways, growth hormone synthesis and secretion, the AMPK signalling pathway, and the cAMP signalling pathway. In addition, \u003cem\u003eCREB3L4\u003c/em\u003e and \u003cem\u003eAQP3\u003c/em\u003e jointly participate in the regulation of the antidiuretic hormone-mediated water reabsorption signalling pathway (Additional file 12).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe secretory ciliated cells evolved from proliferative SOX9-expressing\u0026nbsp;epithelial cells according to pseudotime analysis\u003c/p\u003e\n\u003cp\u003eThe developmental trajectory of endometrial epithelial cells in Meishan\u0026nbsp;pigs\u0026nbsp;was\u0026nbsp;constructed to study the\u0026nbsp;transcriptional characteristics of endometrial epithelial cells across the estrous cycle (Fig\u0026nbsp;6).\u0026nbsp;These\u0026nbsp;results indicate that SOX9-expressing\u0026nbsp;epithelial cells initiate differentiation primarily during the early stage of the estrous cycle, which is associated with endometrial hyperplasia. Subsequently, significant production of LE, GE, and CE cells occurs during late differentiation when the endometrium enters its secretory stage (Fig 6A and 6B).\u0026nbsp;The expression patterns of characteristic genes in endometrial epithelial cells can be classified into four modules, each containing subsets that represent gradual transitions from SOX9-expressing\u0026nbsp;epithelial cells to\u0026nbsp;GEs\u0026nbsp;and\u0026nbsp;LEs. This pathway consists of a branch and two cell fates: SOX9-GE (cell fate\u0026nbsp;1;\u0026nbsp;generation of ciliated epithelia) and SOX9-LE (cell fate\u0026nbsp;2;\u0026nbsp;generation of luminal\u0026nbsp;and glandular\u0026nbsp;epithelial\u0026nbsp;cells)\u0026nbsp;(Fig 6C).\u003c/p\u003e\n\u003cp\u003ePseudotime analysis of endometrial epithelial cells revealed four distinct gene expression modules.\u0026nbsp;Module 1 exhibited a distinct set of marker genes in ciliated epithelial cells, including \u003cem\u003eTP73\u003c/em\u003e, \u003cem\u003eTPPP3\u003c/em\u003e,\u0026nbsp;and \u003cem\u003eFOXJ1\u003c/em\u003e.\u0026nbsp;Genes with high expression levels in module 1 included\u0026nbsp;\u003cem\u003eHMGCS1\u003c/em\u003e, \u003cem\u003ePCM1\u003c/em\u003e, \u003cem\u003eCFAP54\u003c/em\u003e, \u003cem\u003eCFAP44\u003c/em\u003e, \u003cem\u003eCFAP45\u003c/em\u003e,\u003cem\u003e\u0026nbsp;CFAP52\u003c/em\u003e,\u003cem\u003e\u0026nbsp;DNAH5\u003c/em\u003e, \u003cem\u003eCIBAR2\u003c/em\u003e, \u003cem\u003eRSPH4A\u003c/em\u003e, \u003cem\u003eMNS1\u003c/em\u003e, \u003cem\u003eSPEF2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;SNTN\u003c/em\u003e,\u003cem\u003e\u0026nbsp;DYDC2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;CAPS2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;LRRC71\u003c/em\u003e,\u003cem\u003e\u0026nbsp;LRRC10B\u003c/em\u003e,\u003cem\u003e\u0026nbsp;LRRC46\u003c/em\u003e,\u003cem\u003e\u0026nbsp;ODAD2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;CAPS2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;CIBAR2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eFAM183A\u003c/em\u003e, \u003cem\u003eFAM216B\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eCETN2\u003c/em\u003e,\u0026nbsp;\u003cem\u003eC20orf85\u003c/em\u003e,\u003cem\u003e\u0026nbsp;MORN5\u003c/em\u003e,\u003cem\u003e\u0026nbsp;RIBC2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u0026nbsp;\u003cem\u003eSP\u003c/em\u003e\u003cem\u003eACA9\u003c/em\u003e. PCM1 binds to CROCC, and this interaction is critical not only for the accumulation of centriolar satellites near centrosomes and basal bodies but also for cilia formation\u003csup\u003e[35]\u003c/sup\u003e. \u003cem\u003eRSPH4A\u003c/em\u003e was found to be associated with primary ciliary dyskinesia\u003csup\u003e[36]\u003c/sup\u003e.\u0026nbsp;MNS1 and DNAH5 have been implicated in the assembly of axonemes in murine respiratory epithelial cells\u003csup\u003e[37]\u003c/sup\u003e.\u0026nbsp;MNS1 is essential for spermiogenesis and motile ciliary functions in mice\u003csup\u003e[38]\u003c/sup\u003e.\u0026nbsp;SPEF2 is essential for the assembly of sperm flagella in humans\u003csup\u003e[39, 40]\u003c/sup\u003e.\u0026nbsp;The sentinel-cilium apical structural protein (SNTN) is specifically expressed in multiciliated cells of the nasal epithelium\u003csup\u003e[41]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eModule 2 exhibited high expression levels of\u0026nbsp;\u003cem\u003eST6GALNAC1\u003c/em\u003e,\u0026nbsp;\u003cem\u003eCADPS2\u003c/em\u003e,\u0026nbsp;\u003cem\u003eMT3\u003c/em\u003e,\u003cem\u003e\u0026nbsp;MGP\u003c/em\u003e,\u0026nbsp;\u003cem\u003eHPGD\u003c/em\u003e,\u0026nbsp;\u003cem\u003eCYP26A1\u003c/em\u003e,\u0026nbsp;\u003cem\u003eNMU\u003c/em\u003e,\u0026nbsp;\u003cem\u003eMT1A\u003c/em\u003e,\u0026nbsp;\u003cem\u003eTSPO\u003c/em\u003e and\u0026nbsp;\u003cem\u003eAQP3\u003c/em\u003e, which were specifically highly expressed in GE and LE\u0026nbsp;cells.\u0026nbsp;Additionally, several other genes, such as \u003cem\u003eNBL1\u003c/em\u003e, \u003cem\u003eAKR1A1\u003c/em\u003e, \u003cem\u003eCCL28\u003c/em\u003e, \u003cem\u003eSAT1\u003c/em\u003e, \u003cem\u003ePLA2G7\u003c/em\u003e and \u003cem\u003eMRPS36\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e exhibited significant and specific expression within this module.\u0026nbsp;Spermine N\u0026rsquo;-adenosyltransferase (SAT1) converts spermine into N1-acetylspermine,\u0026nbsp;and\u0026nbsp;polyamine production is crucial for follicular development, ovulation, and reproductive\u0026nbsp;processes\u0026nbsp;in mammals, as well as for the establishment and maintenance of gestation\u003csup\u003e[42]\u003c/sup\u003e.\u0026nbsp;Module 3 exhibited high expression levels of\u0026nbsp;\u003cem\u003eUBB\u003c/em\u003e,\u0026nbsp;\u003cem\u003eRPL8\u003c/em\u003e, \u003cem\u003eRPL\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e\u003cem\u003e7\u003c/em\u003e, \u003cem\u003eRPS3A\u003c/em\u003e, \u003cem\u003eRPS1\u003c/em\u003e\u003cem\u003e2\u003c/em\u003e and \u003cem\u003eRPS19\u003c/em\u003e.\u0026nbsp;Finally, module 4 displayed high expression levels of\u0026nbsp;\u003cem\u003eSOX9\u003c/em\u003e, \u003cem\u003eMMP7\u003c/em\u003e,\u003cem\u003e\u0026nbsp;HES1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;NUPR1\u003c/em\u003e and \u003cem\u003eSLPI\u003c/em\u003e, which were highly expressed specifically in SOX9-expressing\u0026nbsp;epithelial cells\u0026nbsp;(Fig 6C).\u003c/p\u003e\n\u003cp\u003eThe expression patterns of the representative genes were consistent along the pseudotime axis, as illustrated in Fig\u0026nbsp;6D. As\u0026nbsp;shown\u0026nbsp;in the\u0026nbsp;figure, the expression levels of \u003cem\u003eMMP7\u003c/em\u003e and \u003cem\u003eNURP1\u003c/em\u003e were high during the early stage (proliferative stage), gradually decreased,\u0026nbsp;and then remained stable after reaching their lowest expression levels in the middle stage (secretory stage). The initial expression levels of \u003cem\u003eAQP3\u003c/em\u003e, \u003cem\u003eMGP\u003c/em\u003e, \u003cem\u003eNUM\u003c/em\u003e and \u003cem\u003eTPSO\u003c/em\u003e were\u0026nbsp;low but gradually increased during proliferation before reaching their lowest expression levels during secretion and remaining stable thereafter.\u0026nbsp;Moreover,\u0026nbsp;the expression levels of \u003cem\u003eFOXJ1\u003c/em\u003e and \u003cem\u003eTPPP3\u003c/em\u003e remained consistently low throughout the proliferative phase but gradually increased during the secretory phase (Fig 6D).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious studies have predominantly focused on gene transcription in endometrial tissue using bulk RNA-seq.\u0026nbsp;However, it is crucial to consider the heterogeneity and cellular interactions among distinct cell subpopulations within complex tissues and organs. The use of scRNA-seq technology has become a primary method for exploring the diversity and complexity of RNA transcripts within individual cells. This technique enables the elucidation of cellular compositions and functions in heterogeneous organs or structurally intricate tissues\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. In this study, we conducted detailed cellular mapping of the Meishan pig uterus, encompassing the most comprehensive tissue characterization to date.\u003c/p\u003e \u003cp\u003eSome studies have identified the transcription factor SOX9 as being crucial for various biological processes, including uterine gland development\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e, cartilage differentiation\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e, hair growth\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e, and testis determination\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. Interestingly, our study revealed obvious SOX9 protein localization in the basal layer of the Meishan pig endometrium. Hence, the \u003cem\u003eSOX9\u003c/em\u003e gene was identified as a potential marker of endometrial epithelial progenitor cells in Meishan pigs. Moreover, the results of this study revealed that \u003cem\u003eNUPR1\u003c/em\u003e and \u003cem\u003eHES1\u003c/em\u003e may serve as marker genes for identifying endometrial SOX9-expressing epithelial cells. \u003cem\u003eNUPR1\u003c/em\u003e serves as a transcriptional regulator that plays a decisive role in regulating the differentiation of intestinal epithelial cells\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eHes1\u003c/em\u003e, a major target gene in the Notch signalling pathway, regulates the fate and differentiation of various cell types in many developmental systems. \u003cem\u003eHes1\u003c/em\u003e regulates the development of the mouse cornea and the homeostatic functions of corneal epithelial stem/progenitor cells\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, in addition to identifying marker genes for porcine endometrial epithelial stem cells, several candidate marker genes potentially associated with adenoepithelial and ciliated epithelial cells were also identified. \u003cem\u003eSPDEF\u003c/em\u003e was specifically highly expressed in GE cells, which renders it a potential candidate marker gene for GE cells. Researchers have discovered that expression of the \u003cem\u003eSpdef\u003c/em\u003e gene regulates the differentiation of both goblet cells and antral mucous gland cells\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. In \u003cem\u003eSpdef\u003c/em\u003e-knockout mice, there are reduced numbers of goblet cells in the intestinal\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e, conjunctival\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e, and tracheobronchial mucosa, as well as impaired terminal differentiation. Similarly, expression of multiple key genes affecting cilia differentiation were identified in ciliated cells. It was indicated that the protein encoded by \u003cem\u003eC20orf85\u003c/em\u003e is associated with cilia functionality\u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e. CFAP45 regulates adenine nucleotide homeostasis to facilitate mammalian ciliary and flagellar motility, thereby contributing to the maintenance of normal sperm function in asthenospermia patients\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. Researchers have discovered that CDHR3 is a transmembrane protein whose expression is increased specifically in the ciliated cells of lung bronchioles\u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e. The colocalization of expression of the \u003cem\u003eFAM183A\u003c/em\u003e gene, specifically with the \u003cem\u003eFOXJ1\u003c/em\u003e gene, was observed in human endometrial ciliated epithelial cells\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. A study by Kwon revealed that the \u003cem\u003eRIBC2\u003c/em\u003e gene plays a significant role in regulating ciliary motility in airway epithelial cells\u003csup\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e. A study conducted by Gui et al. revealed that the microtubule-binding protein SPACA9 can form both spirals and striations within the microtubules of human cilia\u003csup\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn both humans and mice, endometrial epithelial stem cells have been shown to possess the capacity to differentiate into LE and GE cells. Hapangama et al.\u003csup\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e reported that SSEA1 and nuclear SOX9 (nSOX9), which serve as indicators of basal epithelial cells, exhibited certain glandular characteristics in vitro. Jin et al.\u003csup\u003e[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e found that endometrial epithelial progenitor cells exhibit bidirectional differentiation capabilities, leading to the formation of both GE and LE cells in mice. Remarkably, this study provides a groundbreaking finding that SOX9-expressing epithelial cells in Meishan pigs may also exhibit the potential to differentiate into ciliated cells.\u003c/p\u003e \u003cp\u003eSpecifically, the \u003cem\u003eMMP7\u003c/em\u003e gene was highly expressed in SOX9-expressing epithelial cells according to the heatmap of gene expression analysis in the proposed time series. Endometrial expression of \u003cem\u003eMMP7\u003c/em\u003e occurs during menstrual breakdown and subsequent estrogen-mediated growth but not during the secretory phase\u003csup\u003e[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eMMP7\u003c/em\u003e expression regulates the remodelling of the human endometrium to facilitate blastocyst implantation\u003csup\u003e[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eAxin2\u003c/em\u003e is a well-known target gene of the WNT pathway that is expressed in the stem cells of multiple organs\u003csup\u003e[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/sup\u003e. Axin2\u003csup\u003e+\u003c/sup\u003e cells induce mouse endometrial epithelial growth and regeneration, and Axin2\u003csup\u003e+\u003c/sup\u003e cells exhibit bidirectional differentiation into GEs and LEs, as confirmed by lineage tracing\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Interestingly, the results of this study revealed that the \u003cem\u003eAXIN2\u003c/em\u003e gene is expressed both in SOX9-expressing epithelial cells and GE cells, suggesting that SOX9\u003csup\u003e+\u003c/sup\u003e AXIN2\u003csup\u003e+\u003c/sup\u003e cells have the potential to differentiate into GE, LE and CE cells.\u003c/p\u003e \u003cp\u003eThe results of this study revealed significant variations in the epithelial cell types of the endometria of Meishan pigs during both the proliferative and secretory phases. Notably, a substantial presence of GE cells and CE cells was observed during the secretory phase, indicating a strong correlation between these specific cell types and their biological functions within the endometrium during this period. \u003cem\u003eMT3\u003c/em\u003e and \u003cem\u003eMGP\u003c/em\u003e were highly expressed in secretory glandular epithelial cells. MT3 specifically belongs to the metallothionein gene family and plays a pivotal role in maintaining zinc and copper homeostasis. MT3 induces angiogenesis in vitro, potentially regulating endometrial angiogenesis in vivo and the occurrence of miscarriage\u003csup\u003e[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/sup\u003e. In the rat epididymis, the matrix Gla protein (MGP) plays an essential role in promoting calcium-dependent protein aggregation\u003csup\u003e[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]\u003c/sup\u003e. AQP3 is a transmembrane protein that plays a crucial role in facilitating water transport and is essential for the exchange of fluids between the maternal and foetal compartments, as well as the production of amniotic fluid\u003csup\u003e[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eImportantly, we identified a series of crucial genes and signalling pathways associated with Meishan pig uterine development at the single-cell level. These findings underscore the pivotal role of Meishan pig endometrial epithelial cells in facilitating embryo implantation and provide novel insights into gene expression patterns in the porcine endometrium across different stages of the estrous cycle.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eUsing scRNA-seq, we characterized distinct epithelial subpopulations in the uterine tissues of Meishan pigs throughout the estrous cycle. For the first time, we identified a novel subset of porcine endometrial epithelial progenitor cells called SOX9-expressing epithelial cells, which have the potential to differentiate into CE, GE, and LE cells. Moreover, we identified \u003cem\u003eNUPR1\u003c/em\u003e and \u003cem\u003eHES1\u003c/em\u003e as potential marker genes for endometrial SOX9-expressing epithelial cells in Meishan pigs. In summary, our study provides a high-resolution molecular and cellular characterization of the endometrial transformation in Meishan pigs throughout the estrous cycle, offering insights into this essential physiological process.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAQP3 \u0026nbsp; \u0026nbsp;Aquaporin 3\u003c/p\u003e\n\u003cp\u003eCADPS2 \u0026nbsp; Calcium Dependent Secretion Activator 2\u003c/p\u003e\n\u003cp\u003eCDH1 \u0026nbsp; \u0026nbsp;Cadherin 1\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCDHR3 \u0026nbsp; Cadherin Related Family Member 3\u003c/p\u003e\n\u003cp\u003eCETN2 \u0026nbsp; \u0026nbsp;Centrin2\u003c/p\u003e\n\u003cp\u003eCROCC\u0026nbsp; \u0026nbsp;Ciliary Rootlet Coiled-Coil, Rootletin\u003c/p\u003e\n\u003cp\u003eCYP26A1 \u0026nbsp; Cytochrome P450 26A1\u003c/p\u003e\n\u003cp\u003eEPCAM \u0026nbsp; Epithelial Cell Adhesion Molecule\u003c/p\u003e\n\u003cp\u003eFOXJ1 \u0026nbsp; \u0026nbsp;Forkhead Box J1\u003c/p\u003e\n\u003cp\u003eKRT18 \u0026nbsp; \u0026nbsp;Keratin 18\u003c/p\u003e\n\u003cp\u003eKRT8 \u0026nbsp; \u0026nbsp; Keratin 8\u003c/p\u003e\n\u003cp\u003eH2AJ \u0026nbsp; \u0026nbsp;H2A Histone Family Member J\u003c/p\u003e\n\u003cp\u003eHES1 \u0026nbsp; \u0026nbsp; Hes Family BHLH Transcription Factor 1\u003c/p\u003e\n\u003cp\u003eHPGD \u0026nbsp;15-hydroxyprostaglandin dehydrogenase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLPAR3 \u0026nbsp; \u0026nbsp; Lysophosphatidic Acid Receptor 3\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMMP7 \u0026nbsp; \u0026nbsp;Matrix Metallopeptidase 7\u003c/p\u003e\n\u003cp\u003eMGP \u0026nbsp; \u0026nbsp; \u0026nbsp;Matrix Gla Protein\u003c/p\u003e\n\u003cp\u003eNMU \u0026nbsp; \u0026nbsp; \u0026nbsp;Neuromedin U\u003c/p\u003e\n\u003cp\u003eNUPR1 \u0026nbsp; \u0026nbsp;Nuclear Protein 1\u003c/p\u003e\n\u003cp\u003ePCM1\u0026nbsp; \u0026nbsp; \u0026nbsp; Pericentriolar Material 1\u003c/p\u003e\n\u003cp\u003eLPAR3 \u0026nbsp; \u0026nbsp; Lysophosphatidic Acid Receptor 3\u003c/p\u003e\n\u003cp\u003ePRDX5 \u0026nbsp; \u0026nbsp;Peroxiredoxin 5\u003c/p\u003e\n\u003cp\u003ePHGDH/3-PGDH \u0026nbsp; 3-Phosphoglycerate dehydrogenase\u003c/p\u003e\n\u003cp\u003eST6GALNAC1 \u0026nbsp; \u0026nbsp;ST6 N-Acetylgalactosaminide Alpha-2,6-Sialyltransferase 1\u003c/p\u003e\n\u003cp\u003eSOX9 \u0026nbsp; \u0026nbsp; SRY-Box Transcription Factor 9\u003c/p\u003e\n\u003cp\u003eSLPI \u0026nbsp; \u0026nbsp; \u0026nbsp; Secretory Leukocyte Peptidase Inhibitor\u003c/p\u003e\n\u003cp\u003eSNTN \u0026nbsp; \u0026nbsp; Sentan-cilium apical structural protein \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSAT1 \u0026nbsp; \u0026nbsp; \u0026nbsp;Spermidine/Spermine N1-Acetyltransferase 1\u003c/p\u003e\n\u003cp\u003eSPEF2\u0026nbsp; \u0026nbsp; \u0026nbsp;Sperm Flagellar 2\u003c/p\u003e\n\u003cp\u003eTPPP3 \u0026nbsp; \u0026nbsp; Tubulin Polymerization Promoting Protein Family Member 3 \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTP73 \u0026nbsp; \u0026nbsp; \u0026nbsp;Tumor Protein P73\u003c/p\u003e\n\u003cp\u003eTSPO \u0026nbsp; \u0026nbsp; translocator protein 18 kDa\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our gratitude to Kunshan Meishan Pig Breeding Co. Ltd,\u0026nbsp;China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, R. H.; formal analysis, N. J.; investigation, N.J., W. X., C.L., Q.Z., J.M., Q.L., W.C. and X.X.; resources, C. Y. and B.X., writing\u0026mdash;original draft preparation, N.J.; writing\u0026mdash;review and editing, L.H. and R. H.; supervision, P.L., L.H. and Q.Z.; All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Key Research and Development Program grant 2021YFD1301101, the Fundamental Research Funds for the Central Universities (KYT2023002), and the Project of Jiangsu Agricultural (Pig) Industry Technology Syprogenitor grants JATS (2023) 186.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe single-cell RNA sequencing data used in this research have been deposited in the Genome Sequence Archive (GSA) at the Beijing Institute of Genomics (BIG), Chinese Academy of Sciences. The accession number for this dataset is CRA016749.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal trials were performed following Guidelines for the Care and Use of Laboratory Animals prepared by the Institutional Animal Welfare and Ethics Committee of Nanjing Agricultural University, Nanjing, China [Certification No: SYXK (Su) 2017-0007].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLiu C, Li P, Zhou W, et al. 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Mol Med, 2023, 29: 1-14.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Meishan pig, endometrium, endometrial epithelial progenitor cells, single-cell transcriptomic sequencing","lastPublishedDoi":"10.21203/rs.3.rs-4582781/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4582781/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The Meishan pig, native to China, is renowned for its superior reproductive capabilities, including a high ovulation rate, substantial uterine capacity, and an impressively high rate of embryo implantation. The endometrium plays a pivotal role in facilitating embryo implantation and sustaining pregnancy. It is regulated by ovarian hormones and uterine prostaglandins and undergoes a complex series of coordinated processes across the estrous cycle, including proliferation, differentiation, shedding, and regeneration. A detailed examination of the intricate sow endometrial gene expression patterns during this cycle can yield valuable insights into creating ideal conditions for successful embryo implantation and early embryonic development. To gain a comprehensive understanding of the Meishan pig endometrial biological functions across the estrous cycle, we specifically used uterine tissues in the proliferative and secretory phases for single-cell transcriptomic sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe comprehensive transcriptional profile of uterine cells was elucidated throughout the estrous cycle in Meishan pigs. We identified 7 distinct cell types within the primary cell categories, with 4 subpopulations specifically discerned among the endometrial epithelial cells. Considerable variability was observed in the types and quantities of epithelial cell subpopulations spanning the proliferative and secretory phases of the estrous cycle. Significantly, SOX9-expressing epithelial cells were characterised as potential endometrial epithelial stem cells in Meishan pigs. \u003cem\u003eNURP1\u003c/em\u003e and \u003cem\u003eHES1\u003c/em\u003ewere identified as potential marker genes for these stem cells. Pseudotime analysis indicated that these SOX9-expressing epithelial cells can differentiate into glandular epithelial (GE) or luminal epithelial (LE) cells. We also observed that SOX9-expressing epithelial cells may differentiate into ciliated epithelial (CE) cells. There was a marked increase in the number of GE and CE cells during the secretory phase compared to the proliferative phase. GE cells are vital for processes such as glycolysis, amino acid biosynthesis, and N-glycan biosynthesis, all of which are crucial for supplying essential nutrients required for embryo implantation and early stages of embryonic development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eWe reveal the integrated transcriptional profile of uterine cells in sexually mature Meishan pigs and delineate the gene expression patterns within the uterine horns throughout the estrous cycle. These findings provide potential new diagnostic indicators for determining the estrous cycle in sows.\u003c/p\u003e","manuscriptTitle":"Single-Cell Analysis of the Endometrial Characteristics of Meishan Pigs Across the Estrous Cycle","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-02 19:12:25","doi":"10.21203/rs.3.rs-4582781/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"010fd039-2459-4849-8afa-68cf92a50987","owner":[],"postedDate":"July 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-12T07:21:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-02 19:12:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4582781","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4582781","identity":"rs-4582781","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}