|ABEDAL-MAJED, MOHAMED - University Of Nebraska|
|MANAMAGE, MANJULA - University Of Nebraska|
|VINTON, REBECCA - University Of Nebraska|
|Cushman, Robert - Bob|
|WOOD, JENNIFER - University Of Nebraska|
|CUPP, ANDREA - University Of Nebraska|
Submitted to: Society for the Study of Reproduction Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 6/2/2015
Publication Date: 6/2/2015
Citation: Abedal-Majed, M.A., Manamage, M.P., Vinton, R.J., Cushman, R.A., McNeel, A.K., Freetly, H.C., Wood, J.R., Cupp, A.S. 2015. Vascular Endothelial Growth Factor A 165 (VEGFA165), angiogenic isoform, promotes while VEGFA165b antagonizes VEGFA165 stimulated follicular progression in bovine ovarian cortical pieces cultured from pre-pubertal heifers [abstract]. Society for the Study of Reproduction Annual Meeting. p. 246.
Technical Abstract: Previously we have demonstrated in our lab that the VEGFA165 angiogenic isoform stimulates follicle progression in perinatal rat ovaries while inhibitors to VEGFA signal transduction pathways arrest follicular development. Thus, in the current study our objective was to determine if the anti-angiogenic VEGFA165b would antagonize the stimulatory effects of VEGFA165 angiogenic isoform and how this may affect the ability of follicles to progress in ovarian cortex from pre-pubertal heifers. Six pre-pubertal heifers were ovariectomized at 8-9 months and ovarian cortical strips were collected. Day 0 cortical pieces were taken before culture to measure the expression of angiogenic genes using Quantitative PCR (QPCR). There were no differences in any of genes performed: KDR, PECAM, VE-cadherin and NRP-1 suggesting no differences in vasculature prior to ovarian cortex culture. The cortical strips were cultured for 7 days in basic culture Waymouth’s MB 752/1 medium supplemented with antibiotics, ITS and BSA. This medium was then either supplemented with 50 ng/ml of angiogenic VEGFA165 (rhVEGF165; R&D Systems) alone or antiangiogenic VEGFA165b (rhVEGF165b; R&D Systems) alone or a combination of VEGFA165 and VEGFA 165b; or control (PBS). After the 7 days of culture, the cortical strips were fixed and embedded in paraffin and 4-5 serial sections of 5µ thickness were taken from each block. Slides of cortex sections were stained with hematoxylin and eosin (H&E) for follicle staging. Follicle stages were conducted using the following rubric: stage 0 (primordial follicle): An oocyte surrounded by a single layer of squamous pregranulosa cells, stage 1(transitional follicle): An oocyte surrounded by mostly squamous pregranulosa cells with some cuboidal cells; stage 2 (primary follicle): An oocyte surrounded by 1-1.5 layers of cuboidal granulosa cells; stage 3 (secondary follicle): An oocyte surrounded by 2 or more layers of cuboidal granulosa cells; stage 4 (Tertiary follicle: An oocyte surrounded by 2 or more layers of granulosa cells that contains a distinct antrum. Three different technicians counted 3 slides for each treatment and results were averaged per treatment. Neither VEGFA isoform treatment had an effect on numbers of primordial follicles (stage 0). However, there was an increase in the number of primary follicles (stage 2) in the VEGFA165 (0.55±0.02; P < 0.0001) treatment vs any other treatment: VEGFA165b (0.25±0.02), Combination (0.24 ±0.02), and PBS (0.20±0.02). Furthermore, there was also an increase in the number of secondary follicles (stage 3) that were in the bovine cortex treated with VEGFA165 (0.112±0.001; P < 0.0001) vs the other treatments: VEGFA165b (0.019±0.001), Combination (0.010 ±0.001), and PBS (0.037±0.001). Thus, we can conclude from these results that VEGFA isoform, VEGFA165, can stimulate follicle progression in bovine cortex which supports previous data in our lab. Interestingly, VEGFA165b alone or in combination with VEGFA165 arrests follicular progression suggesting that VEGFA165b can antagonize the actions of angiogenic isoform VEGFA165. Therefore, these bovine cortex cultures are an excellent model to understand the antagonistic mechanisms of VEGFA165b, the antiangiogenic isoform, on the ability of VEGFA165 to stimulate follicle progression. It is possible that VEGFA165b contributes to the block in follicle development in the ovary to maintain the ovarian reserve and guard against premature ovarian failure.