147 Proteomic insights into the maturing follicle: a study of growing equine follicles

Follicular fluid (FF) is an essential component of the follicular environment. It originates from blood and follicular somatic cells, granulosa, and theca cells, and it is in direct contact with the oocyte. The composition of FF changes during follicular development to support oocyte maturation, fertilization, granulosa cell differentiation, ovulation, and luteinization. Therefore, understanding the physiological changes in the FF can help replicate the in vivo microenvironment in vitro. This understanding contributes to advancing biotechnologies such as intracytoplasmic sperm injection (ICSI) and IVF. Our main objective was to characterize protein profile differences in the equine FF between different developmental stages. Cyclic mares (n = 18) were examined from July to August in the Northern Hemisphere. After ovulation, width and height of the six largest follicles were measured using ultrasound to assess follicular waves. Upon follicular growth identification, mares were examined daily until FF collection. Mares were divided into two groups according to follicle diameter and were euthanized before follicle deviation (growing group with follicle diameter of 15–20 mm; n = 9) and during estrus (preovulatory group with follicle diameter of 35 mm; n = 9). Ovaries were collected and kept on ice, and FF was aspirated, snap-frozen, and stored at −80°C. Proteomic analysis was performed on one sample per animal and analyzed in duplicate. The proteomic profile of the samples was obtained using liquid chromatography with tandem mass spectrometry on an UltiMate 3000 nano-LC System coupled to an Orbitrap Fusion tribrid mass spectrometer (Thermo Fisher Scientific). The data were obtained in positive mode with high resolution and sensitivity. Using Proteome Discoverer 2.4, data were processed for a semiquantitative analysis against the horse proteome database (UniProt ID UP000002281). Protein fold changes between groups were calculated based with median pairwise comparison. Differentially abundant proteins (DAPs) were identified when the log2 fold-change was ≥1.0 or ≤−1.0 and the adjusted P-value (FDR) ≤ 0.05. DAPs were assigned to functional classification analysis in PANTHER v18.0. Fifty-four DAPs were identified (50 upregulated and four downregulated) in the growing group compared with the preovulatory group. The functional classification of the upregulated proteins classified and distributed them mainly in biological regulation and cellular processes. During follicular growth, the following proteins were upregulated in the growing follicle: inhibitor of growth protein (ING2), kinesin family member 1A (KIF1A), NAD-dependent protein lipoamidase sirtuin-4 mitochondrial (SIRT4), and cyclin-dependent kinase 13 (CDK13). These proteins have been described as crucial for follicular growth, follicular cell energy balance, and embryo preimplantation development in mice. We concluded that apparent follicular-size-dependent protein changes in the FF may affect follicular function. An in-depth understanding of these changes can lead to improvements in the composition of media for oocytes collected from follicles of different sizes, thereby enhancing the success of ICSI and IVF in horses.