VEGFA164_B mRNA Abundance Has a Positive Relationship to AMH, BCL2 and the Ratio of E2:A4 in Mural Granulosa Cells of Estrogen Active and Inactive Follicles Prior to Ovulation.

Abstract

Previously, we reported differential expression of Vascular Endothelial Growth Factor A (VEGFA) isoforms in somatic cells from bovine females of differing fertility. Thus, we sought to determine how expression of VEGFA pro and antiangiogenic isoforms were regulated after synchronization protocols that stimulated normal or persistent follicle development. Furthermore, we examined relationships between VEGFA isoforms, steroid hormones and genes that regulate follicular development in mural granulosa cells and cumulus oocyte complexes (COC). Our hypothesis was that dominant estrogen active (EA) follicles would have greater mRNA abundance of VEGFA angiogenic isoforms and genes that stimulate follicle development; while estrogen inactive (EI) follicles would contain greater mRNA expression of VEGFA antiangiogenic isoforms. Thirty-five composite beef cows [75% MARC III (1/4 Angus, 1/4 Hereford, 1/4 Pinzgauer, 1/4 Red Poll) and 25% Red Angus] were treated with either 1) a modified Co-Synch protocol (CIDR) or 2) injected with prostaglandin and offered Melengesterol Acetate (MGA) for 14 days. Dominant follicle aspirations were performed 18, 36, and 60 hours after the last PGF2a injection utilizing transvaginal ultrasound guided ovum pickup. Follicular fluid steroid hormone concentrations were determined via RIA (E2 and P4) or ELISA (A4). Follicles were then classified as EA (E2:P4 > 1) or as EI (E2:P4 = 1). There was no effect of time on gene expression or steroid hormone levels, thus animals were grouped based on treatment and follicle E2-activity. CIDR and MGA EI follicles had a greater (P = 0.02) concentrations of progesterone compared to CIDR EA follicles. Although A4 concentration was similar among groups, CIDR EA follicles had greater E2:A4 ratio compared to EI follicles suggesting reduced efficiency in conversion of A4 to E2.To determine if the reduced E2:A4 ratio in EI follicles alters the mRNA abundance of VEGFA isoforms in the COC or mural granulosa cells quantitative, real-time RT-PCR was performed. There was no difference in VEGFA_164 gene expression among groups. However, abundance of VEGFA_164B mRNA was 7-fold and 2-fold greater (P < 0.01) in the mural granulosa cells of MGA EI and CIDR EI, respectively, compared to CIDR EA follicles. Furthermore, there was a trend (P = 0.06) for an increased ratio of VEGFA_164/ VEGFA_164B in mural granulosa of CIDR EA follicles. Follicles classified as MGA EI had greater (7-fold; P = 0.02) mRNA abundance of AMH compared to CIDR EA follicles in granulosa cells. Furthermore, BCL2 mRNA expression tended to be increased (P = 0.07) in MGA EI follicles. In contrast to the mural granulosa cells, VEGFA_164B mRNA abundance was not different in COC. In mural granulosa cells, there was a positive correlation between VEGFA_164B and BCL2; VEGFA_164B and AMH; VEGFA_164B and the ratio of E2:P4; and the ratio of VEGFA_164/VEGFA_164B and BCL2.Taken together these data suggest a tendency for increased levels of anti-apoptotic factors and reduced efficiency of conversion of A4 to E2 in granulosa cells of EI follicles. Furthermore, there is a positive correlation between the antiangiogenic VEGFA isoform and BCL2 and AMH which play a role in regulating follicle development. USDA is an equal opportunity provider and employer.