Effect of diet on ability of Vascular Endothelial Growth Factor A (VEGFA) isoforms to alter follicular progression in bovine ovarian cortical cultures

Authors: ABEDAL-MAJED, MOHAMED - University Of Nebraska; CUPP, ANDREA - University Of Nebraska; WOOD, JENNIFER - University Of Nebraska; Cushman, Robert - Bob; McNeel, Anthony; KURZ, SCOTT - University Of Nebraska; MAGAMAGE, MANJULA - Sabaragamuwa University Of Sri Lanka; VINTON, REBECCA - University Of Nebraska; Freetly, Harvey

Submitted to: Society for the Study of Reproduction Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 3/2/2016
Publication Date: 7/16/2016
Citation: Abedal-Majed, M.A., Cupp, A.S., Wood, J.R., Cushman, R.A., McNeel, A.K., Kurz, S.G., Magamage, M., Vinton, R., Freetly, H.C. 2016. Effect of diet on ability of Vascular Endothelial Growth Factor A (VEGFA) isoforms to alter follicular progression in bovine ovarian cortical cultures [abstract]. Society for the Study of Reproduction Annual Meeting. Abstract #239 (Scientific Program p. 181). Available: http://www.ssr.org/sites/ssr.org/files/uploads/attachments/node/320/2016_ssr_abstracts.pdf

Interpretive Summary:

Technical Abstract: The objective of this study was to determine the effect of changes in diet on ability of VEGFA isoforms to alter follicle progression in bovine ovarian cortex cultures. Our hypothesis was that diet would affect the magnitude of VEGFA isoform actions on follicular development. Heifers (n = 30) received the Control diet (30% alfalfa hay, 69.8% corn silage, 0.2% salt) before 6 heifers were ovariectomized at 8 months of age (collection 1). The remaining 24 heifers were divided into 2 treatment groups. Controls were offered 228 Kcal ME/BW Kg **0.75 for 84 days. Stair-Step heifers received 157 Kcal ME/BW Kg **0.75 for 84 d. Six heifers were ovariectomized from each group (collection 2). Following the 84-d restriction, heifers in the Stair-Step received 277 Kcal ME/BW Kg **0.75 over a 63 day period while control heifers remained on the restricted diet (collection 3). At each ovariectomy, ovarian cortical strips and RNA were collected. Ovary cross sections were fixed, embedded in paraffin, and 5 sections of 6µm thickness were taken from each heifer, stained with hematoxylin and eosin (H&E), and the area of the cortex and medulla were measured. The cortical strips were cultured for 7 days in Waymouth’s MB 752/1 medium supplemented with antibiotics, ITS and BSA. Cultures were supplemented with 50 ng/ml of angiogenic VEGFA165 (rhVEGF165; R&D Systems) alone, antiangiogenic VEGFA165b (rhVEGF165b; R&D Systems) alone, a combination of VEGFA165 and VEGFA165b; or PBS (control). After 7 days of culture, the cortical strips were fixed and embedded in paraffin and 4-5 sections of 5µm thickness were taken from each block. Slides of cortex sections were stained with hematoxylin and eosin (H&E) for follicle staging. Follicle stages were analyzed by three different technicians with procedures previously published. No differences were detected via QPCR for Kdr, Pecam, VE-cadherin and Nrp-1 mRNA in collection 1 or 2. In collection 3, VE-cadherin and Nrp-1 mRNA were increased in Stair-Steps (P < 0.05). There were no interactions of in vitro VEGFA isoform treatments with in vivo diets at any collection time. However, VEGFA165 increased follicular progression to later stages of development and VEGFA165b inhibited the action of VEGFA165. In histological sections, Stair-Step heifers had less primordial follicles (P = 0.01) at collection 2 while at collection 3, Stair-Step heifers had more primordial follicles (P = 0.001) than Control heifers. There was a greater percent of the ovarian area occupied by cortex at collection 2 in Stair-Step heifers and a decreased percent of the ovarian area occupied by cortex at collection 3 in Stair-Step heifers (P < 0.01). Thus, we can conclude from these results that VEGFA isoform, VEGFA165, can stimulate follicle progression independent of diet. Interestingly, VEGFA165b inhibited VEGFA165-dependent stimulation of follicular progression, suggesting that VEGFA165b can antagonize the actions of angiogenic isoform VEGFA165. These bovine cortical cultures are an excellent model to understand the mechanisms of VEGFA isoforms; especially since previous diet does not impair or affect their actions on follicular progression in bovine cortical cultures.