Single-Cell RNA-Sequencing Analysis of the Infundibulum, Ampulla, and Isthmus to Characterize Cell Populations in the Post-Ovulation Bovine Oviduct

Á. López-Valiñas¹, C. Mahé², E. Baumann³, A. I. Dyroff¹, C. Almiñana¹, M. Saint-Dizier², S. Bauersachs¹

1. Institute of Veterinary Anatomy, Vetsuisse Faculty, University of Zurich, Lindau (ZH), Switzerland.

2. INRAE, CNRS, Université de Tours, PRC, Nouzilly, France

3. Haverford College, Haverford, PA, USA

Abstract Text:

The oviduct (also called the Fallopian tube) plays an important role in transport and maturation of gametes, fertilization, and early embryonic development. It is divided into three functional parts: infundibulum, ampulla, and isthmus, with different roles in the early reproductive events. Ciliated and secretory epithelial cells are lining the lumen of each compartment to support these events. A deep characterization of the cellular heterogeneity of the oviduct and the gene expression profile of the three compartments will increase our understanding of their role in providing an optimal environment for reproductive success. Therefore, this study aimed to analyze the cellular diversity and gene expression profiles within infundibulum, ampulla, and isthmus of the bovine oviduct during the post-ovulatory phase using single cell RNA sequencing (scRNA-seq).

Oviducts were collected from five cyclic Swiss Brown cows at the early post-ovulatory stage. The infundibulum, ampulla, and isthmus were separated and single cells isolated by enzymatic digestion. After cell viability assessment, single cell capture, and library preparation were performed using the Illumina Single Cell 3' RNA Prep kit. The scRNA-seq libraries were sequenced on one 10B flow cell on the Illumina NovaSeq X Plus instrument (Functional Genomics Center Zurich). Raw sequencing reads were aligned to the Bos taurus genome (ARS-UCD2.0) using STAR. Data preprocessing and clustering were conducted using Seurat, retaining cells with 200-3,000 expressed genes and < 20% of reads from mitochondrial genes. Uniform Manifold Approximation and Projection (UMAP) was applied to visualize cellular heterogeneity across all samples. The most relevant markers per cluster were identified using “PrepSCTFindMarkers” function, followed by annotation based on gene expression profiles and literature.

Preliminary results revealed 75,877 high-quality single cells: 13,640 from the infundibulum, 33,962 from the ampulla, and 28,275 from the isthmus. UMAP clustering revealed 32 distinct cell populations, including 10 subclusters of secretory cells, 8 of multi-ciliated cells, and 5 subclusters of cells transitioning between secretory and multi-ciliated cells. Other cell types included cells with neuronal markers, endothelial cells, fibroblasts, and immune cells. Immune cell populations consisted of 7 subclusters: 4 of T cells, 2 of macrophages, and 1 of B cells. Secretory cells were the most abundant cell type across all parts, with the lowest proportion in the infundibulum (37.2 %) and comparable levels in ampulla (51.7%) and isthmus (47.3%). Multi-ciliated cells were the second most abundant cell population (25.7% in the infundibulum and 24.9% in the ampulla), except in the isthmus (9.8%), where transitioning epithelial cells (20.9%) and T cells (13.9%) were more prevalent. Cells with neuronal markers were exclusively detected in the ampulla, while fibroblasts and endothelial cells were detected in both the infundibulum and isthmus. Macrophages were detected in all regions, with the highest abundance in the infundibulum, whereas T cells were more prevalent in the infundibulum and isthmus than in ampulla. Notably, distinctive gene expression profiles were identified in the isthmus and ampulla for secretory and multi-ciliated epithelial cells, respectively.

This study outlines a comprehensive view of the cellular diversity of the oviductal compartments for first time, and its implications in providing an optimal milieu for the success of the early reproductive events. The study provides also with the first single-cell transcriptome atlas of the bovine oviduct, expanding our understanding of the oviduct biology in bovine, which can be extrapolated to other mammals.

Supported by SNSF (#205507) and ANR (#ANR-21-CE20-0042-01).