Tissues were then incubated for 30 min with a peroxidase-conjugated anti-digoxygenin monoclonal antibody, and apoptotic cells were visualized as positively immunostained structures after reaction with diaminobenzidine. Negative controls included TdT omission. Sections were counterstained with hematoxylin. The number of apoptotic cells was determined by counting labeled cells from follicles in 400X microscopic fields (three sections per ovary; five ovaries) and expressed as the apoptotic cell mean per field.
The individual ovarian follicles were dissected from the ovary under a stereoscopic microscope as previously described. In brief, healthy antral follicles (300^-50 im in diameter) from eight ovaries per group were frozen and used for biochemical assays. From each ovary, a pool of isolated follicles was frozen, and the results obtained from each pool were considered a single datum.
To evaluate changes in general structure, the ovaries were removed and immediately fixed in 4% neutral-buffered formalin for 12 h and then embedded in paraffin. Four-micrometer step sections were mounted at 50-im intervals onto microscope slides to prevent counting the same follicle twice, according to the method described by Woodruff et al.. One set of slides was stained with hematoxylin-eosin in order to count the number of different stages of follicles per ovary section, and the others were immunostained by the TUNEL technique.
Rats then received either 0.1 or 0.5 ig of Trap in 5 il of PBS with 0.1% BSA under the bursa of one ovary (Trap ovary). The contralateral ovary was injected with 5 il of vehicle (PBS with 0.1% BSA, control ovary). Taking into account that the VEGF Trap protein is a chimera that contains the Fc region of human immunoglobulin G (IgG), an additional control was designed in which one ovary was injected with 0.65 ig of human IgG, and the contralateral ovary was injected with vehicle. No significant differences were found in the number of follicles at any stage between the human IgG and the vehicle-injected ovary.
Materials and Reagents
Recombinant mouse-soluble VEGF receptor 1/Fc Chimera (Trap) (R&D Systems, Inc., Minneapolis, MN) was dissolved in PBS buffer with 0.1% BSA. The eCG (Novormon) was provided by Syntex S.A. (Buenos Aires, Argentina). Polyclonal primary antibodies for BAX (N-20), BCL2 (N-19), BCL2L1 (S-18), TNFRSF6 (FL-335), and FASLG (Q-20) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); actin B antibody was obtained from Novus Biologicals (ab-6276; Littleton, CO). Anti-rabbit and anti-goat secondary antibodies conjugated with horseradish peroxidase were purchased from Sigma-Aldrich. All other chemicals were of reagent grade and were obtained from standard commercial sources.
In fact, the presence of VEGFA and its receptor in human and rodent ovaries has been reported at both protein and mRNA levels, in granulosa cells as well as in theca cells. We suggest that apoptotic cell death in granulosa cells of those follicles selected for ovulation can be prevented by the paracrine/ autocrine actions of VEGFA. VEGFA is a cytoprotective agent for endothelial cells, which protects these cells from apoptosis and induces the expression of antiapoptotic proteins in human endothelial and mouse ovarian cells.
The molecular mechanism of apoptosis is a matter of an active debate. It is known that ovarian apoptosis takes place to eliminate follicular cells in atretic follicles. FSH and LH are the primary survival factors for ovarian follicles; the antiapoptotic effects of these gonadotropins are probably mediated by the production of ovarian growth factors. It has been demonstrated that various growth factors and cytokines (IGF1, EGF, TGFA, FGF2, FGF7, and interleukin 1B) prevent apoptosis in antral follicles.
Expression of VEGFA in ovarian follicles depends on follicular size. In bovine and porcine follicles, VEGFA is weakly expressed during early ovarian follicular development and becomes more pronounced in granulosa and theca cells, along with dominant follicle development. Similar results were found in the rat ovary, where, in addition, some secondary follicles showed extremely strong VEGFA immunoreactivity in the zona pellucida.
Angiogenesis is a process of vascular growth that is mainly limited to the reproductive system in healthy adult animals. The development of new blood vessels in the ovary is essential to guarantee the necessary supply of nutrients and hormones to promote follicular growth and corpus luteum formation.
Preantral follicles (PFs) have no vascular supply of their own, but rather, they depend on vessels in the surrounding stroma.