The mechanism of follicle rupture at ovulation revealed by multiphoton microscopy in vivo

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 DESCRIPTION (provided by applicant): The overall hypothesis to be tested is that rupture of the follicle wall at ovulation is absolutely dependent upon vasoconstriction of vessels at the apex; this dependence is due to a requirement to decrease the delivery of protease inhibitors that are present in abundance in serum and thereby allow the final localized thinning of the apical wall. The proposed experiments will be facilitated by use of a novel multiphoton microscopy (MPM) procedure we have developed for imaging the mouse ovary in vivo. MPM is ideal because it is minimally-invasive and can be used to image optically thick tissues. Vessels of preovulatory follicles are visible after injection of mice with rhodamine-labeled dextran and temporal changes in diameter and blood flow through individual vessels can be determined. Measurements that can be made simultaneously include vessel density, the thickness of the apical follicle wall, the shape of the follicle, the position of the oocyte, and the intensity of fluorescent markers. In addition, imaging can be performed during infusion of agents into the bursal cavity to interrogate aspects of the hypothesis. The first specific aim is to determine whether vasoconstriction of apical vessels of the follicle is required for protease activity, thinnng of the follicle wall and rupture by targeting a vasoconstrictor produced by granulosa cells before ovulation, endothelin 2 (EDN2). This will be accomplished by examining the effect of blocking the production of endogenous EDN2 by granulosa cells and then determining the effect of replacement with exogenous EDN2. A complementary approach will be to inhibit the action of endogenously produced EDN2 using EDN receptor antagonists. The second specific aim will test if serum protease inhibitors play a crucial role in maintaining a balance between protease and anti-protease activity in the follicle wall and if this balance is altered by vasoconstriction o favor tissue degradation leading to follicle rupture. The first experiment will test a potential cause and effect relationship between vasoconstriction at the follicle apex and subsequent thinning of the wall. We will determine if exogenously supplied serum protease inhibitors block follicle rupture without affecting the vasoconstriction that normally occurs just prior to ovulatio. This would provide evidence that the decrease in blood flow at the apex of the follicle before ovulation is not simply a compensatory response to thinning of the follicle wall; instead, there may be a cause and effect relationship between vasoconstriction and increased protease activity at the apex. The second experiment will identify localized changes in the concentration of serum protease inhibitors in the follicle before and after the decrease in blood flow preceding follicle rupture. These studies will fill a major gap in our understanding of the precise sequence of steps required for follicle rupture. Understanding the mechanism of ovulation will promote development of new contraceptives and treatments for infertility. The proposed studies will also establish the MPM procedure for investigation of the physiology of ovulation in vivo and promote extension of this technology for use in other organs.