60 Single-cell transcriptome analysis of fetal and adult bovine ovaries reveals developmental progression in cell population composition and function

The ovary supports female germline development from the time primordial germ cells (PGCs) arrive at the forming gonad during fetal development through the ovulation of mature oocytes in an adult. The objective of this analysis was to compare the transcriptomic landscape of the bovine fetal ovary soon after PGC colonisation with the adult ovary that had given rise to pregnancy. Fetal and adult bovine ovarian samples were collected and processed for single-cell RNA-sequencing (scRNA-seq) in two independent experiments. Fetal ovaries were dissected from two bovine fetuses at ∼50 days of pregnancy (4 cm crown-rump length), and adult ovarian cortex samples were dissected from two nulliparous heifers at 49 and 52 days of pregnancy. After dissociation and viability assessment, cell suspensions were processed for individual barcoding and cDNA synthesis before undergoing scRNA-seq using the 10× Genomics Chromium controller and Illumina HiSEqn 4000 platform. Alignment of reads to the bovine genome and initial analyses were performed using the CellRanger pipeline, followed by quality control, sample normalisation and integration, and secondary analyses using the Seurat package of R. An elbow plot of the merged dataset was used to determine fit, and clusters were visualised by uniform manifold approximation and projection dimensionality reduction. A total of 46,174 (16,442 fetal and 29,732 adult) cells were plotted and cluster identity was assigned using the 10 most differentially expressed genes and conserved marker genes. Eleven cell clusters were identified as stromal (3 clusters), endothelial (2 clusters), epithelial, granulosa, theca, PGCs, and lymphoid and myeloid immune cells, with fluctuations in relative proportions by life stage. While PGCs (OCT4, NANOS3) were captured in the fetal sample, oocytes had been size-excluded from sequencing and no cells resembling PGCs were identified in the adult ovary. A distinct theca cell (CYP11A1, STAR) cluster was only detectable in the adult ovary, whereas cells from both stages contributed to the granulosa cell (GC) (AMH, INHA) cluster. Network analysis of upregulated genes by age within the GC cluster (FDR ≤ 0.05, FC ≥ 1.5) highlighted enrichment of cholesterol and steroid metabolism and hormone secretion in adult compared to fetal GCs. In addition, epithelial cells (KRT19, MSLN) in the adult ovary had enhanced markers of immune response and cellular adhesion. Of the three stromal cell (DCN, COL1A2/3A1) clusters, fetal cells comprised 81% of one cluster (named “Str1”), while adult cells represented 74 ± 1% of the other two clusters (named “Str2–3”). Ingenuity pathway analysis of upregulated genes in Str1 versus Str2–3 indicated enrichment of processes, including post-transcriptional and translational modification, protein folding, and cell cycle. In conclusion, these data represent two stages during the developmental trajectory of the bovine ovary and reflect the shift in roles that the ovary plays in gamete development, hormone synthesis, and tissue remodelling based on life stage.

This research was supported by USDA NRSP8 and GEEAP NNF.