Amount of mRNA and localization of vascular endothelial growth factor and its receptors in the ovarian follicle during estrous cycle of water buffalo (Bubalus bubalis)

The objective of the present study was to characterize the temporal patterns of gene expression for vascular endothelial growth factors (VEGF) and VEGF receptors during ovarian follicular growth, development and maturation in buffalo (Bubalus bubalis). Follicles were classified into four groups according to size and the concentration of estradiol-17β (E2) in follicular fluid (FF): Group I (small), 4–6 mm diameter, E2 > 0.5 ng/ml of FF; Group II (medium), 7–9 mm, E2 = 0.5–5 ng/ml; Group III (large), 10–13 mm, E2 = 5–40 ng/ml; Group IV(pre-ovulatory), >13 mm, E2 > 180 ng/ml). The mRNAs for FSH receptor (FSHR), LH receptor (LHR) and aromatase (CYP19A1) in theca interna and granulosa layers were also determined, further defining the maturational state of each group. The relative expression of VEGF isoforms (120, 164, and 188 amino acid forms), as determined by quantitative real-time PCR (qRT-PCR), increased during follicular development in both the granulosa (P < 0.05) and theca layers. Relative amounts of VEGF receptors (VEGFR-1 and VEGFR-2) were least in granulosa cell (GC) and theca interna cell (TI) layers of Gp-I follicles. The amount of VEGFR-2 transcripts increased in the granulosa layer throughout development, reaching a maximum in Gp-IV follicles (P < 0.05). The relative amount of VEGF isoforms and receptors in follicle lysates, as determined by western blotting, increased throughout follicular maturation to maximum amounts in pre-ovulatory follicles. Immunohistochemistry revealed a clear localization of VEGF isoforms and receptors in both steroidogenic cell types (GC and TI) and of VEGF receptors in the vascular endothelial cells of the thecal blood vessels. The most intense immunofluorescence was evident in pre-ovulatory follicles compared to other smaller follicles. These data provide evidence that the VEGF may contribute to the extensive capillary proliferation associated with the increase in size, selection, and maturation of the pre-ovulatory follicle. This may facilitate follicle maturation by enhancing the supply of nutrients, hormones, and other essential blood-borne signals to the follicle. VEGF may also promote maturation of follicles through recently recognized, non-angiogenic mechanisms.