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

体内多光子显微镜揭示排卵时卵泡破裂的机制

• 批准号: 8807067
• 负责人: SUSAN MARY QUIRK
• 金额: $ 7.75万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-03 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8807067
• 关键词: Accounting; Affect; Animal Model; Anti-Inflammatory Agents; Anti-inflammatory; Apical; Blood flow; Caliber; Cells; Characteristics; Complex; Contraceptive Agents; Contraceptive methods; Dependence; Development; Dextrans; Echinomycin; Endothelin-2; Equilibrium; Event; Hypoxia Inducible Factor; Image; Immune Sera; In Vitro; Incidence; Individual; Indomethacin; Infertility; Infusion procedures; Injection of therapeutic agent; Investigation; Knowledge; Label; Leukocytes; Life; Measurement; Measures; Mediating; Methodology; Microscopy; Movement; Mus; Oocytes; Organ; Oryctolagus cuniculus; Outcome; Ovary; Ovulation; Ovulation Inhibition; Pathway interactions; Peptide Hydrolases; Perfusion; Pharmaceutical Preparations; Phosphodiesterase Inhibitors; Physiological; Physiology; Play; Positioning Attribute; Procedures; Production; Prostaglandin Production; Prostaglandins; Protease Inhibitor; Protocols documentation; Rat-1; Rattus; Reagent; Relative (related person); Reproductive Health; Research; Research Methodology; Research Project Grants; Rhodamine; Rodent; Role; Rupture; Serum; Shapes; Stimulus; Structure; Syndrome; Technology; Testing; Thick; Time; Tissues; Vasoconstrictor Agents; Woman; base; density; granulosa cell; in vivo; in vivo imaging; inhibitor/antagonist; light microscopy; minimally invasive; novel; ovulation time; public health relevance; receptor; research and development; research study; response; vasoconstriction

 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.

 描述（由申请方提供）：待检验的总体假设是排卵时卵泡壁破裂完全依赖于顶端血管的血管收缩;这种依赖性是由于需要减少血清中大量存在的蛋白酶抑制剂的递送，从而使顶端壁最终局部变薄。拟议的实验将促进使用一种新的多光子显微镜（MPM）的程序，我们已经开发的小鼠卵巢在体内成像。MPM是理想的，因为它是微创的，可用于成像光学厚组织。向小鼠注射罗丹明标记的右旋糖酐后，排卵前卵泡的血管可见，并且可以确定单个血管的直径和血流的时间变化。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，成像可以在将药剂输注到囊腔期间进行，以询问假设的各个方面。第一个具体目标是通过靶向排卵前颗粒细胞产生的血管收缩剂内皮素2（EDN 2）来确定卵泡顶端血管的血管收缩是否是蛋白酶活性、卵泡壁变薄和破裂所需的。这将通过检查阻断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。一种补充方法是使用EDN受体拮抗剂抑制内源性产生的EDN 2的作用。第二个具体目标将测试血清蛋白酶抑制剂是否在维持卵泡壁中蛋白酶和抗蛋白酶活性之间的平衡中起关键作用，以及这种平衡是否被血管收缩改变，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壁变薄之间的潜在因果关系。我们将确定外源性血清蛋白酶抑制剂是否阻止卵泡破裂而不影响排卵前正常发生的血管收缩。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，血管收缩和顶端蛋白酶活性增加之间可能存在因果关系。第二个实验将确定卵泡破裂前血流量减少前后卵泡中血清蛋白酶抑制剂浓度的局部变化。这些研究将填补我们对卵泡破裂所需步骤的精确顺序的理解中的一个主要空白。了解排卵机制将促进新的避孕药和不孕症治疗方法的开发。拟议的研究还将建立MPM程序，用于体内排卵生理学的研究，并促进该技术在其他器官中的应用。