Vascular Endothelial Growth Factor A 164b (Vegfa164b) Inhibitory Isoform is Up-regulated in Bovine Granulosa Cells Prior to the LH Surge May Be a Marker of Follicle Status.

Abstract

The Vegfa gene has 8 different exons that can be alternatively spliced to form several different angiogenic isoforms: Vegfa120, Vegfa164, Vegfa188, Vegfa205. Recently, human VEGFA inhibitory (b) isoforms were discovered that contain exon 8b instead of 8a (present in angiogenic isoforms). Furthermore, for every angiogenic isoform there is a similar size inhibitory or b isoform (ie. Vegfa120b, Vegfa164b, etc) that blocks its actions. Our laboratory has determined that VEGFA inhibitory isoforms regulate rat follicle progression in vitro. Thus, the objective of the current study was to amplify and sequence the bovine Vegfa164b isoform and determine its expression in relation to Vegfa164 prior to the LH surge in bovine granulosa cell aspirates. Furthermore, differences in these isoforms were determined in estrogenic verses non-estrogenic follicles. Cows were administered two injections of prostaglandin F2alpha (PG; 25 mg/ head) 14 days apart and the largest follicle was aspirated at 12, 18, 24, 30, 36 and 48 hr after the second injection of PG (n= 10 at each aspiration). Estrogen (E2) and progesterone (P4) assays were conducted on follicular fluid to determine whether the follicle was estrogenic (E2/P4 > 1) or non-estrogenic (E2/P4 < 1). Granulosa cells were extracted for RNA and quantitative real-time PCR (QRT-PCR) was conducted using reverse primers specific for: 1) Vegfa164 (exon 8a); 2) Vegfa164b (exon 8b); and 3) both Vegfa164 and Vegfa164b (reverse primer in exon 7; to compare with previous data). The Vegfa164b inhibitory isoform was amplified through RT-PCR and demonstrated to be 97% homologous with its human Vegfa165b counterpart. Furthermore, the predicted bovine protein sequence contained one amino acid less than the human. Prior to the LH surge, Vegfa164b in estrogenic follicles was greater at 18 hr (P=0.06) after the second injection of PG; however, there were no differences in amount of Vegfa164. The reverse primer that amplified both Vegfa164 and 164b (exon 7) had peak expression at 18 hr (P < 0.08) in estrogenic follicles similar to the results from the Vegfa164b primer (exon 8b). To determine if there were differences in isoforms based on follicle status, the area under the curve for Vegfa164 and 164b was determined for granulosa cells from estrogenic and non-estrogenic follicles aspirated at 12, 18, 24 and 48 hr after PG. In estrogenic follicles there was 2 fold greater Vegfa164b and 1.6 fold greater Vegfa164 isoforms than non-estrogenic follicles. However, in the reverse primers detecting both isoforms there was only 1.24 fold greater Vegfa164a&b isoform detected in estrogenic versus non-estrogenic follicles. Thus, inhibitory Vegfa164b isoform but not angiogenic Vegfa164 isoform is up-regulated in the granulosa cell prior to the LH surge and may be involved in follicle development or oocyte maturation. Furthermore, amount of Vegfa164b isoform in bovine granulosa cells may be utilized in the future to detect oocyte quality and developmental competence due to estrogenic or non-estrogenic status of the follicle.