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 < 60,000), number of expressed genes (n Feature_ 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 SPACA9. 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, RPL17, RPS3A, RPS12 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).