Mechanisms involved in follicular angiogenesis.

Angiogenesis is a critical component of follicular function: maintenance of the theca vascular bed and follicular health are strictly related. Non-growing primordial follicles and slow-growing preantral follicles do not present a vascular supply but, as the antrum develops, the thecal layer acquires a vascular sheath consisting of two capillary networks located in the theca interna and externa, respectively. Angiogenesis is accompanied by vasodilatation; both thecal capillary vasodilatation and angiogenic process basically support the gradual increase of ovarian blood flow during follicle growth, thus assuring adequate levels of oxygen, nutrients and hormone precursors. Even though angiogenesis takes place in theca layer, granulosa cells exert an important role, by producing several angiogenic factors that act in the theca; among these, the most important one is VEGF. The VEGF family is composed by at least six members (VEGF A-F); in addition, five molecular forms of VEGF-A are produced in humans, as a result of alternative splicing of the gene transcript. VEGF effects are almost exclusively exerted via two receptors, VEGFR-2 (involved in mediating endothelial cell proliferation and survival and vascular permeability) and VEGFR-1 (which might play an inhibitory role by sequestering VEGF). VEGF is a potent migration-stimulating and mitogenic factor for endothelial cells; it modulates granulosa cell differentiation in the initial phases of antral follicle development, enhances proliferation in large follicles, plays a role in structural maintenance and increases vessel permeability. In the periovulatory interval, VEGF levels markedly increase in follicular fluid and the peptide is easily detectable in granulosa-derived luteinized cells. In the follicle, VEGF production is regulated differently depending on follicle size. VEGF mRNA and protein in the primate ovary are expressed in the theca layer of antral follicles and in the granulosa cells nearest the oocyte in preovulatory follicles but not in granulosa cells of primordial and preantral follicles. In bovine and porcine follicles VEGF is weakly expressed during early follicular development and becomes more pronounced in granulosa and theca cells along with follicle development. Expression of VEGF mRNA and/or protein in granulosa layer of several species is upregulated by LH and hCG, which also increase VEGFR-1 and –2 expression. The injection of VEGF gene fragments to eCG-treated gilts increases the population of large follicles and the vascular density in the theca interna; in addition, it increases VEGF levels in follicular fluid, its expression in granulosa cells and VEGFR-1 mRNA expression in theca layer. The permeabilizing action of VEGF is thought to be essential also for antrum formation; in large follicles, VEGF seems to slowly diffuse out and to create an angiogenic gradient that attracts blood vessels toward the granulosa. Medium follicles with high VEGF levels in follicular fluid and extensive vascularization accumulate high E2 concentrations: these follicles are supposed to ovulate or at least to undergo the endocrine scenario of the preovulatory phase. As ovulation approaches, VEGF production ceases, probably to create the best conditions for follicular rupture. In swine and bovine (but not in humans), VEGF production is stimulated by hypoxia. As the follicle develops, O2 tension in follicular fluid progressively decreases thus stimulating angiogenic activity via an increase in VEGF production; O2 sensing is possibly mediated by reactive oxygen species (ROS). The stimulatory effect of granulosa cells cultured in hypoxic conditions is inhibited by a VEGF Trap (a potent VEGF receptor blocker), which has been demonstrated to inhibit the stimulatory effect of granulosa cells on endothelial cell proliferation; in addition, it which reduces follicular angiogenesis, recruitment and growth of antral follicles and VEGF receptors. VEGF production is also inhi...