In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation

Rupture of the ovarian follicle releases the oocyte at ovulation, a timed event that is critical for fertilization. It is not understood how the protease activity required for rupture is directed with precise timing and localization to the outer surface, or apex, of the follicle. We hypothesized that vasoconstriction at the apex is essential for rupture. The diameter and blood flow of individual vessels and the thickness of the apical follicle wall were examined over time to expected ovulation using intravital multiphoton microscopy. Vasoconstriction of apical vessels occurred within hours preceding follicle rupture in wild-type mice, but vasoconstriction and rupture were absent in Amhr2^cre/+SmoM2 mice in which follicle vessels lack the normal association with vascular smooth muscle. Vasoconstriction is not simply a response to reduced thickness of the follicle wall; vasoconstriction persisted in wild-type mice when thinning of the follicle wall was prevented by infusion of protease inhibitors into the ovarian bursa. Ovulation was inhibited by preventing the periovulatory rise in the expression of the vasoconstrictor endothelin 2 by follicle cells of wild-type mice. In these mice, infusion of vasoconstrictors (either endothelin 2 or angiotensin 2) into the bursa restored the vasoconstriction of apical vessels and ovulation. Additionally, infusion of endothelin receptor antagonists into the bursa of wild-type mice prevented vasoconstriction and follicle rupture. Processing tissue to allow imaging at increased depth through the follicle and transabdominal ultrasonography in vivo showed that decreased blood flow is restricted to the apex. These results demonstrate that vasoconstriction at the apex of the follicle is essential for ovulation.During ovulation in typically mono-ovulatory species such as humans, as well as in poly-ovulatory species such as rodents, the oocyte is released from the preovulatory follicle by extrusion through a rupture site on the outer surface, or apex, of the follicle, which protrudes from the surface of the ovary (1). Precise timing and accurate spatial localization of rupture at the apex are essential to allow capture of the oocyte by a hormonally primed oviduct where fertilization occurs, but the mechanisms involved are not yet understood. The rupture site breaches multiple layers of cells and their associated extracellular matrix and basement membranes (2). These include the single layer of epithelial cells that covers the surface of the ovary, the basement membrane that supports it, and the multiple cell layers comprising the wall of the preovulatory follicle. The outer wall of the ovarian follicle contains androgen-secreting theca cells and extensive vasculature. This vasculature consists of an inner and an outer plexus of capillaries with associated arterioles and venules that supply nutrients to the entire follicle (3–5). Underlying the theca and separated from it by a basement membrane is the avascular granulosa cell layer that serves as the major source of estrogen. The oocyte resides in the center of the follicle surrounded by multiple layers of specialized granulosa cells known as “cumulus cells.” In a mature preovulatory follicle, formation of a fluid-filled antral cavity separates the oocyte–cumulus complex from the mural granulosa cells that form the wall of the follicle except at a region known as the “stalk,” which connects the oocyte–cumulus complex to the antral granulosa cells of the follicle wall. At ovulation the oocyte is released from the follicle in association with attached cumulus cells.The preovulatory release of surge levels of luteinizing hormone (LH) from the anterior pituitary acts on receptors in the follicle to trigger events critical for the rupture and remodeling of the follicle and differentiation of granulosa and theca cells into progesterone-producing cells of the corpus luteum. The cumulus cells are induced to secrete a mucoelastic extracellular matrix which causes loosening of contacts between granulosa cells and between granulosa cells and the oocyte, a process known as “cumulus expansion,” which is essential for ovulation (1). Expression of proteases belonging to several major families, including the matrix metalloproteinase, plasminogen activator/plasmin, and ADAMTS (a disintegrin and metalloproteinase with thrombospondin-like motifs) families, increases. Simultaneously, follicle cells express protease inhibitors such as tissue inhibitors of metalloproteinases (TIMPs 1–4) and plasminogen activator inhibitors (PAI 1–3) (6, 7). The increase in protease activity is essential for rupture of the follicle and for the breakdown of the basement membrane separating theca and granulosa cells to allow the ingrowth of blood vessels to establish the corpus luteum. The mechanisms that regulate the balance of protease and protease inhibitor activity in the follicle to allow precise rupture at the apex while protecting most of the follicle structure from protease activity are not understood (1, 6, 7).We postulated that vasoconstriction of vessels within the theca at the apex of the follicle is required to promote follicle rupture. Our first approach was to examine mice with conditional expression of a dominant active allele of smoothened (SMO), the transmembrane protein that relays signaling by the hedgehog (HH) pathway. In these Amhr2^cre/+SmoM2 mice, preovulatory follicles develop normally in many respects, including changes in the expression of critical genes in response to the preovulatory LH surge (8, 9). However, follicles fail to rupture, and oocytes remain trapped as the follicles luteinize. The major ovarian phenotype in these mice is a pronounced deficiency of vascular smooth muscle (VSM) surrounding vessels in the theca cell layer, whereas other vessels that are present throughout the stroma of the ovary have normal maturation with VSM. Because VSM is required for vasoconstriction, the mice provided a model to test whether failure of vasoconstriction contributes to anovulation. In additional experiments with wild-type mice, we blocked the increase in the expression of endothelin 2 (Edn2) by granulosa cells that normally occurs within hours before follicle rupture (10, 11). Because EDN2 is a potent vasoconstrictor, this approach allowed us to test the effect on follicle rupture of inhibiting vasoconstriction versus treatment with exogenous compounds to restore vasoconstriction. In addition, treatment of wild-type mice with EDN2 receptor antagonists was used to test the role of EDN2 in vasoconstriction and rupture. Vasoconstriction and changes in the follicle wall were monitored repeatedly relative to the time of ovulation using intravital multiphoton microscopy.