Morphological and TUNEL Studies
The first objective was to analyze the effects of the inhibition of VEGFA on follicular development and apoptosis in the rat ovary. To inhibit VEGFA, a soluble truncated form of the fms-like tyrosine kinase (FLT) fused to IgG (VEGF Trap R1/Fc Chimera: Trap) was administered for different time periods. The time course effects of Trap on follicle growth are depicted in Figure 1. Rats were treated with intrabursal injections of 0.5 ig of Trap, and the ovaries were removed 12, 24, and 48 h later and then processed and analyzed as described above.
Histological ovarian slides were stained with hematoxylin-eosin to determine the number of different follicle stages (Fig. 1A). Injection of 0.5 ig of Trap per ovary did not change the number of PFs or EAFs (Fig. 1B; a and b); however, it significantly decreased the number of POFs when compared to the control group 48 h after surgery (Fig. 1B; c) (control: 14.08% 6 1.49%; Trap: 8.54% 6 1.72%, P < 0.01, n = 6), and it significantly increased the number of atretic follicles (Fig. 1B; d) (control: 10.46% 6 0.54%; Trap: 16.38% 6 1.27%, P < 0.05, n = 6).
FIG. 1. Time course of VEGFA inhibition (Trap treatment) on follicle growth in ovaries from gonadotropin-stimulated rats. A) Representative fields of X100 ovarian sections. Control ovary (a);Trap-treated ovary (b). EAFs, Early antral follicles; AtF, atretic follicles;POFs, periovulatory follicles; GC, granulosa cells;TC, theca cells. B) Preantral follicles (a);early antral follicles (b);peri-ovulatory follicles (c) (control: 14.08% 6 1.49%; Trap: 8.54% 6 1.72%). d) Atretic follicles (control: 10.46% 6 0.54%; Trap: 16.38% 6 1.27%). Prepubertal rats were injected with 0.5 ig of Trap under the bursa of one ovary. The contralateral ovary served as a control and was injected with vehicle. Subsequently, 25 IU of eCG were administered. The ovaries were removed at 12, 24, or 48 h after injection and prepared for histology. *P < 0.05;**P < 0.01;n = 6.