(353) Unraveling the Cellular and Molecular Dynamics of Bovine Corpus Luteum Using Single-Cell RNA Sequencing

The corpus luteum (CL) plays a crucial role in regulating the reproductive cycle, fertility, and pregnancy maintenance by producing progesterone. CL undergoes dynamic molecular and structural changes throughout its lifespan in response to luteotropic and luteolytic signals. Despite significant advances in CL biology, the precise cellular and molecular mechanisms governing CL maturation and regression remain incompletely understood. This study employs single-cell RNA sequencing to characterize the cellular heterogeneity and molecular changes occurring in the bovine CL during its mature and regressive stages.

We collected mature CL tissues and blood samples (n=3) on day 11 post-ovulation from heat-synchronized, ultrasound-tracked cows via transvaginal surgery. To induce luteolysis, a separate group of animals (n=3) received an intramuscular injection of a prostaglandin F2α (PGF2α) analogue on day 11, and regressive CL tissues and blood samples were collected 8 hours post-injection. Radioimmunoassay analysis confirmed a significant decline in plasma progesterone levels following PGF2α administration, indicating the initiation of CL regression. All procedures were conducted with the approval of the competent animal ethics authorities. CL tissues were enzymatically dissociated using collagenase, and single cells were isolated via Ficoll gradient centrifugation. Equal proportions of mature and regressive samples were pooled for each stage, and single-cell libraries were generated using 10X Genomics technology. Sequencing was performed on the NovaSeq X Plus platform, and data analysis was carried out using Cell Ranger and R-based bioinformatics tools. Integration of mature and regressive CL cell clusters was performed using the Seurat toolkit. Post quality control, we analyzed 14,919 cells from mature CL and 13,131 cells from regressive CL.

Analysis identified that cellular composition of the mature CL included 43% steroidogenic cells, 19% endothelial cells, 15% immune cells, and 23% fibroblasts. In contrast, the regressing CL exhibited a compositional shift with 35% steroidogenic cells, 14% endothelial cells, 32% immune cells, and 18% fibroblasts. Notably, the increased immune cell population in the regressing CL was further validated by flow cytometric analysis of CD45, a well-established immune cell surface marker. This indicate recruitment of specific immune cell subtypes might be essential for triggering the regression process. Gene expression analysis of large luteal cells revealed an upregulation of inflammatory and apoptotic markers, including SERPINA14 and IL33, while steroidogenic genes such as STAR were significantly downregulated. Additionally, ingenuity pathway analysis identified key pathways activated in large luteal cells during the regression, including pro-inflammatory signaling and cell death pathways.
This study provides novel insights into the cellular dynamics and molecular transitions that drive CL regression in bovine. By leveraging single-cell transcriptomics, we identified novel gene markers distinguishing different luteal cell types and characterized differentially recruited immune cell populations and pathways associated with luteal maintenance and regression. These findings contribute to a deeper understanding of CL biology, with potential applications in fertility management and therapeutic strategies for reproductive disorders in livestock. 

Funding: This study is funded via FBN core fund.