Graduate Research Assistant University of Missouri Columbia, Missouri, United States
Abstract Authors: Isabella Sellmer Ramos1, Monica O. Caldeira1, Amanda L. Patterson1,2 and Matthew C. Lucy1
1Division of Animal Sciences, University of Missouri, Columbia, Missouri, USA
2Department of Obstetrics, Gynecology and Women’s Health, University of Missouri, Columbia, Missouri, USA
Abstract Text: The endometrial glandular epithelium (GE) plays a critical role in fertility of mammalian species. In a recent study (Sellmer Ramos et al. 2024; Biol. Reprod. 110:1-4), we observed that the earliest phase of glandular development (adenogenesis) in the neonatal bovine uterus was characterized by the patch-like expression of FOXA2 within the luminal epithelium (LE; postnatal day 0, PND0) that was sustained until PND 28. FOXA2 is a pioneer transcription factor, expressed specifically in the GE of the adult, and required for adenogenesis. The unexpected results led us to further investigate the mechanisms that lead to the spatially-constrained induction of FOXA2 within the LE that precede adenogenesis. Spatial transcriptomics was performed on uterine cross-sections to evaluate gene expression within calves at distinct stages of adenogenesis including stage 0 (absence of glandular epithelium, and little to no expression of FOXA2 within the LE, n=2) and developmental stage 2 (presence of GE and islands of FOXA2+ cells within the LE, n=2) for PND0 (n=2 stage 0, n=1 stage 2) and PND7 (n=1 stage 2). Uterine cross sections were collected, embedded in O.C.T., frozen and stored in a -80 ºC. Tissue blocks were sectioned and placed in a Visium (10X Genomics) slide containing 4 capture areas and sequenced in an Illumina NovaFlowSeq 6000 (sequencing depth of 50,000 read pairs per 50 mm spot). Each cross-section was H&E stained and subsequently imaged. Images and raw FASTQ files were processed using the Space Ranger pipeline and aligned to the Bos taurus genome. Raw counts were normalized using SCTransform in Seurat v.5.0 (R Statistical Software v.4.3.1). Normalized data were merged and integrated using Harmony v.1.2 and submitted to the standard Seurat clustering pipeline prior to downstream analysis. Differential gene expression analysis was performed using a Wilcoxon rank sum test (FindAllMarkers) and clusters were annotated using the PanglaoDB database and manually confirmed based on spatial localization. We found 3 epithelial clusters present in both developmental stages using the FindSubCluster function: early-stage LE (EarlyLE), GE (FOXA2+) and late-stage LE (LateLE). Differential expression analysis revealed distinct markers for each cell type. EarlyLE was characterized by expression of IGFBP1, CLDN7, GHRH, PGA5, SAA3 and FXYD4. When compared with LateLE, the EarlyLE cluster had greater SOX17, MMP7, OVOL1, FUT6, WNK1, SPON2, DKKL1 and LGR4. The GE cluster was characterized by FOXA2, ALDH1A1, TUBA1D, IHH, SLC2A5 and FOXJ1. When compared with LE clusters, the GE cluster had greater AXIN2, WNT5B, PTCH2, PTCH1, PENK, CXCL12, FZD2 and CDH11. The LateLE cluster was characterized by KLRJ1, SBSPON, CSF1, GSTT2 and RIMS2. When compared with EarlyLE, the LateLE had increased expression of CDH1, JUN, TFF3, GGT5, IGFBP4, TGFBI, MKI67. Importantly, at developmental stage 0, UMAP plots revealed that all epithelial populations clustered in close proximity, with fewer unique mapping identity (UMI) spots representing LateLE and GE clusters. At developmental stage 2, these cell populations clustered separately, indicating a potential timeline for determination and specification of epithelial phenotypes. We characterized the EarlyLE phenotype across developmental time points using the FindConservedMarkers function, and found that markers OVOL2, KLF5, ITGB4, ITGA6, SOX17, SPDEF, OVOL1 and TERT were conserved, suggesting that a progenitor-like epithelial population is maintained within the endometrium. These findings shed light on potential markers that might drive adenogenesis and highlight distinct epithelial populations in the neonatal bovine uterus. Supported by the NICHD award R01HD092254.
