Spatiotemporal analysis of the protein expression of angiogenic factors and their related receptors during folliculogenesis in rats with and without hormonal treatment

This study investigated the protein expression and cellular localization of ANGPT1, ANGPT2, and their receptor TEK, as well as vascular endothelial growth factor A (VEGFA) and its receptor KDR (VEGFR2) during folliculogenesis. To obtain follicles at different stages for immunochemistry and western analyses, we used prepubertal untreated, diethylstilbestrol- and equine chorionic gonadotropin-treated rats. To confirm that these hormonal treatments reflect physiological change, we used non-treated adult rats. No expression of ANGPT1 was observed in granulosa cells (Gc) from immature hormone-treated and non-treated rats at any follicular stage. By contrast, ANGPT1 expression in theca cells (Tc) increased with follicular maturation. ANGPT2 protein was either absent or weakly expressed in Gc at all follicular stages. In Tc, minimal expression of ANGPT2 protein was detected in the preantral follicle (PF), whereas it was stronger in the early antral follicle (EAF) and preovulatory follicle (POF). TEK staining was absent in Gc but was intense in Tc at every follicular stage. Staining for VEGFA was either absent or weakly present in Gc and Tc in PF and EAF, although in POF it was stronger in Gc and Tc. Staining for KDR was absent in Gc and very low in Tc from PF. Gc and Tc of EAF showed positive staining for KDR and in POF the staining was stronger. These results were confirmed by western immunoblot. A similar pattern of expression of these proteins was observed in cycling rats. In conclusion, we observed that the protein expression of ANGPT1, ANGPT2, VEGFA and their receptors increased during follicular development in rats.