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

 描述(申请人提供)：需要检验的总体假设是，排卵时卵泡壁的破裂完全取决于顶端血管的收缩；这种依赖性是由于需要减少血清中丰富的蛋白水解酶抑制物的输送，从而允许顶壁的最终局部变薄。建议的实验将通过使用我们开发的用于对小鼠卵巢进行活体成像的新的多光子显微镜(MPM)程序来促进。MPM是理想的，因为它是微创的，可以用来成像光学厚度的组织。在注射罗丹明标记的葡聚糖后，可以看到排卵前卵泡的血管，并且可以确定直径和通过单个血管的血流的时间变化。可以同时进行的测量包括血管密度、顶端卵泡壁的厚度、卵泡的形状、卵母细胞的位置和荧光标记的强度。此外，还可以在向法氏囊腔内注入药物的过程中进行成像，以询问假说的各个方面。第一个具体目的是通过靶向排卵前颗粒细胞产生的血管收缩因子-内皮素-2(EDN_2)来确定是否需要对卵泡顶端血管的血管收缩来实现蛋白酶活性、卵泡壁变薄和破裂。这将通过检查阻止颗粒细胞产生内源性EDN_2的效果，然后确定用外源性EDN_2替代的效果来实现。一个补充的方法将是使用EDN受体拮抗剂来抑制内源性产生的EDN2的作用。第二个特定目标将测试血清蛋白水解酶抑制物是否在维持卵泡壁的蛋白酶和抗蛋白酶活性之间的平衡中起关键作用，以及这种平衡是否因血管收缩而改变，从而有利于组织降解导致卵泡破裂。第一个实验将测试卵泡顶端的血管收缩和随后的管壁变薄之间的潜在因果关系。我们将确定外源性血清蛋白水解酶抑制剂是否能阻止卵泡破裂，而不影响通常发生在排卵前的血管收缩。这将提供证据表明，排卵前卵泡顶端血流减少不仅仅是对卵泡壁变薄的补偿反应；相反，血管收缩与顶端蛋白酶活性增加之间可能存在因果关系。第二个实验将确定在卵泡破裂前血流减少前后卵泡内血清蛋白水解酶抑制物浓度的局部变化。这些研究将填补我们对卵泡破裂所需步骤的准确顺序的理解的一个重大空白。了解排卵的机制将促进新的避孕药和不孕症治疗的发展。拟议的研究还将建立MPM程序，用于研究体内排卵的生理学，并促进该技术在其他器官中的推广使用。

 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.