248 EXPRESSION AND LOCALIZATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 2 IN EQUINE FOLLICLES

Abstract

Ovarian follicles undergo pronounced morphological changes, alternating between periods of growth and regression. The equine follicle will grow to an average of 45 mm in diameter at ovulation, and during the phase of growth, there is an increase in blood supply to the follicle. Vascular endothelial growth factor (VEGF) is a cytokine that interacts with tyrosine kinase receptors to stimulate angiogenesis, endothelial cell proliferation and vascular permeability. The aim of the study was to evaluate the expression and localization of VEGF and the VEGF-receptor 2 (VEGF-R2) in equine follicles. Ovaries were collected from a slaughterhouse. Granulosa cells from follicles were pooled regardless of the size of the follicles. Western blots were performed using protein extracted from granulosa cells and follicular fluid. Blots were probed with rabbit anti-human VEGF and rabbit anti-mouse VEGF-R2 antibodies and visualized with chemiluminescence. Total RNA was extracted from granulosa cells and integrity of the RNA samples was tested by the amplification of β-actin. Complementary DNA was synthesized by reverse transcription, followed by polymerase chain reaction amplification of cDNA encoding with bovine primer sequences for VEGF and VEGF-R2. The PCR product was resolved on 1% agarose gel and the resulting VEGF and VEGF-R2 bands were sequenced. Immunostaining for VEGF and VEGF-R2 was performed on fixed, paraffin-embedded sections of follicle wall from follicles larger than 30 mm. Western blot analysis of granulosa cell lysates revealed 22 kDa bands for VEGF, and 210 kDa bands for VEGF-R2. VEGF protein was present in follicular fluid, whereas VEGF-R2 was not detectable. RT-PCR experiments revealed the presence of VEGF and VEGF-R2 mRNA in isolated granulosa cells. Sequencing demonstrated 93% and 99% homology to known sequences of equine VEGF and VEGF-R2, respectively. Immunofluorescence experiments performed on dissected equine follicles localized VEGF to the granulosa cell layer and sporadically to the theca cell layer. VEGF-R2 co-localized with VEGF in the granulosa cells, and was relatively absent in the theca layer. The present study detected novel expression patterns for VEGF and VEGF-R2 in equine ovarian follicles. The results of these experiments suggest an extra-vascular role for the VEGF family in follicle development. Future studies will be directed at studying the genomic and proteonomic profiles of follicles during the selection of the dominant follicle in mares.