105 Proteome profiling of equine follicular fluid before, during, and after selection of the dominant follicle

Understanding the signalling coordination among the follicular fluid milieu and follicle cells is essential for a better comprehension of the selection mechanism and development of the dominant ovulatory follicle. Follicular fluid is a vital microenvironment for the growing oocyte and follicle; its unique and evolving biochemical composition makes it an ideal reservoir for potential biomarkers’ discovery to facilitate the improvement of assisted reproductive technologies. Herein, we performed a proteome profiling and comparison of follicular fluid collected in vivo (n = 17 mares) at different developmental stages. A new follicular wave was induced with ablation of all follicles ≥6 mm during the mid-cycle, and follicle growth was tracked daily using ultrasonography. Follicular fluid was aspirated when follicles reached 18–20 mm (Predeviation), 22–25 mm (Deviation = “selection”), 26–29 mm (Postdeviation), 30–35 mm (Preovulatory), or >35 mm (Impending signs of ovulation). After being harvested, follicular fluid samples were immediately centrifuged and stored at −80°C until proteomic shotgun analysis through nanoLC-MS/MS and protein identification. A total of 294 unique proteins were identified (P-value and false discovery rate (FDR) < 0.01), corresponding to 65 common proteins, and 124, 142, 167, 132, and 142 proteins were present in the Predeviation, Deviation, Postdeviation, Preovulatory, and Impending ovulation samples, respectively. Proteins belonged to the classical biological categorisations (cellular component (CC), molecular function (MF), and biological processes (BP)), with the number of gene ontologies being significantly and differentially enriched (P and FDR < 0.01) in the CC (Spherical high-density lipoprotein particle and Extracellular region), in the MF (Oxygen binding and Phosphatidylcholine binding), and in the BP (Positive regulation of cholesterol and acute phase response) during follicle development. The complement and coagulation cascades were the main significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways during follicle development, with potential key roles of 43 differentially expressed proteins associated with follicle selection, dominance, and ovulation. This study highlights the existence of core proteins for folliculogenesis, although many others may have critical roles in regulating follicle development.