The balance of proangiogenic and antiangiogenic VEGFA isoforms regulate follicle development

Abstract

Vascular endothelial growth factor A (VEGFA) has been extensively studied because of its role in follicular development and is a principal angiogenic factor essential for angiogenesis. Since vascularization of the theca layer increases as follicles progress in size through preantral and antral stages, VEGFA might influence follicle growth via the regulation of angiogenesis. However, VEGFA might also influence follicular development through nonangiogenic mechanisms, since its expression has been localized in nonvascular follicles and cells. Alternative mRNA splicing of eight exons from the VEGFA gene results in the formation of various VEGFA isoforms. Each isoform has unique properties and is identified by the number of amino acids within the mature protein. Proangiogenic isoforms (VEGFA_XXX) are encoded by exon 8a, whereas a sister set of isoforms (VEGFA_XXXB) with antiangiogenic properties is encoded by exon 8b. The antiangiogenic VEGFA_XXXB isoforms comprise the majority of VEGFA expressed in most tissues, whereas expression of the proangiogenic VEGFA isoforms is upregulated in tissues undergoing active angiogenesis. Although proangiogenic and antiangiogenic isoforms can now be distinguished from one another, many studies evaluating VEGFA in ovarian and follicular development up to now have not differentiated proangiogenic VEGFA from antiangiogenic VEGFA. Experiments from our laboratory indicate that proangiogenic VEGFA promotes follicle recruitment and early follicular development and antiangiogenic VEGFA inhibits these processes. The balance of proangiogenic versus antiangiogenic VEGFA isoforms is thus of importance during follicle development. Further studies are warranted to elucidate the way that this balance regulates follicular formation and progression.

Fig. 1

Alternate splicing of the human vascular endothelial growth factor A (VEGFA) gene results in different VEGFA isoforms. Each isoform is encoded by a specific set of exons and the resulting proteins are named by their unique number of amino acids. Exon 8a encodes the proangiogenic isoforms, whereas exon 8b encodes the antiangiogenic “B” isoforms

Fig. 2

Quantitative reverse transcription with the polymerase chain reaction was conducted to detect mRNA levels for Vegfa_165, Vegfa_165b, Vegfa_189 and Vegfa_189b in rat ovaries from embryonic day 13 (E13) through postnatal day 5 (P5) of ovarian development. Gapdh (D-glyceraldehyde-3-phosphate dehydrogenase) was used as an endogenous control to account for differences in starting material. These data are the result of at least three different pools of tissue from each age group. The mean normalized values obtained for E13 have been set at 1 and the values for the other developmental ages are presented as a fold-change from E13. Therefore, values greater than 1 indicate increased mRNA levels and values less than 1 indicate reduced mRNA levels in comparison with E13 (a, b). The primary morphologic stages that occur during development of the rat ovary from E13-P5 are presented in c

Fig. 3

One ovary from postnatal day 3/4 rat was cultured for 2 weeks with either recombinant VEGFA_165, antibodies to VEGFA_XXXB isoforms, an inhibitor of KDR and FLT1 (VEGFA receptors), or an inhibitor to NRP1. The paired ovary from each rat was cultured without treatment to serve as a control. The mean number of follicles at each stage of development was calculated as a percent of total follicles, and these percentages were compared between treated and control ovaries (↑ significant increas in the percent of follicles at each stage in treated ovaries in comparison with controls, ↓ significant decrease in the percent of follicles at each stage in treated ovaries in comparison with controls, - no difference in the percent of follicles at each stage in treated ovaries in comparison with controls). All developing follicles include early primary, primary, transitional, and secondary follicle stages

Fig. 4

Representation of the proposed role for VEGFA isoforms in follicle development. Proangiogenic isoforms appear to promote initial recruitment and development of ovarian follicles, whereas antiangiogenic isoforms appear to suppress these processes