Proteomic analysis of mare follicular fluid during late follicle development

Follicular fluid accumulates into the antrum of follicle from the early stage of follicle development. Studies on its components may contribute to a better understanding of the mechanisms underlying follicular development and oocyte quality. With this objective, we performed a proteomic analysis of mare follicular fluid. First, we hypothesized that proteins in follicular fluid may differ from those in the serum, and also may change during follicle development. Second, we used four different approaches of Immunodepletion and one enrichment method, in order to overcome the masking effect of high-abundance proteins present in the follicular fluid, and to identify those present in lower abundance. Finally, we compared our results with previous studies performed in mono-ovulant (human) and poly-ovulant (porcine and canine) species in an attempt to identify common and/or species-specific proteins.

Follicular fluid samples were collected from ovaries at three different stages of follicle development (early dominant, late dominant and preovulatory). Blood samples were also collected at each time. The proteomic analysis was carried out on crude, depleted and enriched follicular fluid by 2D-PAGE, 1D-PAGE and mass spectrometry.

Total of 459 protein spots were visualized by 2D-PAGE of crude mare follicular fluid, with no difference among the three physiological stages. Thirty proteins were observed as differentially expressed between serum and follicular fluid. Enrichment method was found to be the most powerful method for detection and identification of low-abundance proteins from follicular fluid. Actually, we were able to identify 18 proteins in the crude follicular fluid, and as many as 113 in the enriched follicular fluid. Inhibins and a few other proteins involved in reproduction could only be identified after enrichment of follicular fluid, demonstrating the power of the method used. The comparison of proteins found in mare follicular fluid with proteins previously identified in human, porcine and canine follicular fluids, led to the identification of 12 common proteins and of several species-specific proteins.

This study provides the first description of mare follicular fluid proteome during the late follicle development stages. We identified several proteins from crude, depleted and enriched follicular fluid. Our results demonstrate that the enrichment method, combined with 2D-PAGE and mass spectrometry, can be successfully used to visualize and further identify the low-abundance proteins in the follicular fluid.