Studies on substantially increased proteins in follicular fluid of bovine ovarian follicular cysts using 2-D PAGE and MALDI-TOF MS

Abstract

Background: The objective of this study was to identify substantially increased proteins in bovine cystic follicular fluid (FF) in order to clarify the pathology and etiology of bovine ovarian follicular cysts (BOFC).

Methods: Proteins in normal and cystic FF samples were subjected to two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and were compared using silver stained gel images with PDQuest image analysis software. Peptides from these increased spots were analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and were identified based on the NCBI database by a peptide mass fingerprinting method.

Results: Comparative proteomic analysis showed 8 increased protein spots present in cystic FF. MS analysis and database searching revealed that the increased proteins in cystic FF were bovine mitochondrial f1-atpase (BMFA), erythroid associated factor (EAF), methionine synthase (MeS), VEGF-receptor, glyceraldehydes 3-phosphate dehydrogenase (GAPDH), heat shock protein 70 (HSP70), beta-lactoglobulin (BLG) and succinate dehydrogenase Ip subunit (SD).

Conclusion: Our results suggest that these proteins are overexpressed in BOFC, and that they may play important roles in the pathogenesis of BOFC. Furthermore, these proteins in the FF could be useful biomarkers for BOFC.

Figure 1

Silver stained 2-D PAGE images of proteins from bovine ovarian follicular fluid. Sample type A; Non treatment, Sample B; Depleted of impurities (salt, lipids, detergent and nuclei acid), Sample C; Depleted of abundant proteins (albumin and immunoglobulin G) and impurities.

Figure 2

Light micrographs of bovine ovarian follicles stained with hematoxylin and eosin. [A] Normal follicle, approximately 10 mm in diameter with granulosa. [B] Cystic follicle, approximately 30 mm in diameter with theca interna, but without granulosa. G: granulosa layer, TI: theca interna, TE: theca externa. Bar = 50 μm...

Figure 3

Representative silver stained 2-DPAGE images of proteins from normal follicles [A] and cystic follicles [B1 & B2]. Protein samples from both types of follicles were depleted of both abundant proteins (albumin and IgG) and impurities. Expressions of protein spots on the images from cystic and normal FFs which were processed for 2-D PAGE were simultaneously compared, and the results compared using each image image. Panel C is an enlarged image of square areas in panel B1, which contained some additional protein spots of cystic follicles.