Vascular Endothelial Growth Factor A (VEGFA) Isoform Loss Reduces Ovarian Size, Estrogen Concentrations, mRNA Abundance of Genes Critical to Follicular Development and Oocyte Growth Resulting in Subfertility.

Vascular Endothelial Growth Factor A (VEGFA) pro-angiogenic isoforms stimulate while anti-angiogenic isoforms inhibit progression of follicles to later stages of development. Furthermore, inappropriate regulation of these isoforms may alter the primordial follicle pool. Therefore, we hypothesized that loss of all VEGFA isoforms in granulosa cells would affect ovarian reserve and follicular development. To test this hypothesis we evaluated phenotypic and molecular markers of ovarian development with VEGFA loss in granulosa cells. Initially, we used a pDMRT-1 cre mouse crossed to a floxed VEGFA mouse to determine potential phenotypes in female granulosa and subpopulations of germ cell VEGFA (VEGFA-DGranG) loss. We demonstrated previously that these aged VEGFA-DGranG (~250 days, n=10) females displayed a 21% decrease (P < 0.05) in the combined weight of the ovaries and oviducts as well as a 45% reduction in serum estradiol concentrations (P < 0.05) compared to controls (n=11). Recently, we determined that the VEGFA-DGranG females had 44% fewer CLs than controls indicating they are having fewer ovulations. Furthermore, mRNA abundance for Foxo3a was 43% higher with the Bcl-2 to Bax ratio 96% greater in VEGFA-DGranG than controls suggesting that follicular development may be arrested or impaired. Steroidogenic enzyme gene expression and GDF-9 mRNA abundance were not different. However, time from mating to parturition was increased by 12 days in these females compared to controls. To determine the effect of VEGFA isoform loss at an earlier age and more specifically to granulosa cells without effects on germ cells, we crossed pAmhr2-cre mice with floxed VEGFA mice to create a granulosa cell (plus reproductive tract; VEGFA-GRT) knockout. The ovaries from VEGFA-GRT (n=5) and control (n=5) females were collected at approximately 80 days of age. Ovarian area of VEGFA-GRT was reduced by 95% (P < 0.01) when compared to controls. The estrogen concentrations in the VEGFA-GRT mice were numerically decreased by 26% at 80 days but did not differ significantly. To elucidate potential differences in mRNA abundance of genes involved in follicular development, qPCR analysis showed a trend (P = 0.08) for increased Fox03a and GDF-9 in younger VEGFA-GRT females from controls. Taken together, these results demonstrate that loss of VEGFA isoforms results in smaller ovaries with altered gene expression that may be due to abnormal follicular development or a reduced ovarian follicle pool. Consistent with this hypothesis, the trend for increased GDF-9 in younger VEGFA-GRT females may be a compensatory mechanism to maintain proper follicular development in the absence of VEGFA isoforms whereas the Foxo3a increase suggests suppression of primordial follicle activation likely resulting in the decreased ovarian size. Furthermore, the aged females appear to have increased symptoms of subfertility resulting in reduced number of ovulations and increased time to successful pregnancy and parturition. Thus, VEGFA isoform loss in granulosa cells appears to compromise follicular development and fertility in females. This research was supported in part by NIH/NICHD HD051979. (poster)