GRANULOSA CELL-CONTROL OF NA+/H+ ANTIPORT AND K+ CHANNEL

细胞外K+和Na+的缺乏导致 促性腺激素和生长因子的异常影响类固醇激素合成和 卵巢颗粒细胞的其他生物合成功能。这些 观测结果被解释为反映了这些离子的重要性。 颗粒细胞的功能。然而，人们对此知之甚少。 这些离子进入或离开内分泌的调节和途径 单元格的类型。 本提案的目的是检验所提出的假设。 根据我们的初步数据，Na+/H+逆向转运体是EGF的介体 在颗粒细胞中的作用。我们还将检验这一假设 电兴奋、K+、Cl-通道和膜电位发挥作用 在黄体生成素诱导的孕酮产生和生长中的密切作用 因子-(EGF、IGF-1、TGFa和TGFb)影响类固醇合成和其他 颗粒细胞的生物合成功能。具体目标是：(1) 确定Na+/H+逆向转运体介导的细胞质碱化是否 生长因子激活的颗粒细胞中的强制性事件，以及 使用对pH敏感的荧光色素进行卵泡发育。(2)确定 未成熟颗粒细胞长期(3-4天)暴露对卵巢的影响 生长因子对体外细胞Na~+/H~+逆向转运蛋白活性的影响 文化模式。(3)研究促黄体生成素和生长因子的调节(直接或 信使介导的卵巢颗粒细胞K~+和Cl~-通道 应用膜片钳技术观察不同成熟阶段的卵泡。 (4)观察长期(3-4天)暴露对幼虫的影响 颗粒细胞对黄体生成素和生长因子的发生及活性的影响 K+和Cl-通道。 这些研究将通过使用膜片钳技术和 家鸡颗粒细胞的荧光分光光度测定 在卵泡发育的不同阶段。与众不同的 鸡卵巢的解剖特征与强大的膜片钳结合 以及光谱荧光技术使详细研究 卵泡发育成熟过程中颗粒细胞离子通量的调节 卵巢排卵前卵泡。我们的结果将解释离子是如何 酶催化重要过程所需的微环境 (如腺苷环化酶和胆固醇侧链裂解酶) 控制住了。这些研究还将提供有关如何 促性腺激素和生长因子调节颗粒细胞分化 在卵泡成熟期间。此外，他们还将提供有关 生长因子在颗粒细胞中的作用机制。

The absence of K+ and Na+ in extracellular compartment leads to the aberration of gonadotropin- and growth factor-influenced steroidogenic and other biosynthetic functions of ovarian granulosa cells. These observations have been interpreted to reflect the importance of these ions in granulosa cell function. However, very little is known about the regulation and pathways by which these ions enter or leave this endocrine type of cell. The objective of the present proposal is to test the hypothesis suggested by our preliminary data, that the Na+/H+ antiporter is a mediator of EGF action in granulosa cells. We will also test the hypothesis that electrical excitation, K+ and Cl- channels and membrane potential play an intimate role in the LH-induced production of progesterone, and in growth factor-(EGF, IGF-1, TGFa and TGFb) influenced steroidogenesis and other biosynthetic functions in granulosa cells. The specific aims are: (1) Determine if the Na+/H+ antiporter-mediated cytoplasmic alkalinization is an obligatory event in growth factor-activated granulosa cells, and on follicular development using pH sensitive flourochrome. (2) Determine the effect of long term (3-4 days) exposure of immature granulosa cells to growth factor on the Na+/H+ antiporter activity using an in vitro cell culture mode. (3) Study the LH and growth factor regulation (direct or messenger-mediated) of K+ and Cl- channels in granulosa cells isolated from follicles at different stages of maturation using patch clamp techniques. (4) Examine the influence of long term (3-4 days) exposure of immature granulosa cells to LH and growth factor on the occurrence and activity of K+ and Cl- channels. The studies will be accomplished by using patch clamp techniques and spectrofluorometry on granulosa cells isolated from domestic hen (Gallus domesticus) at different stages of follicular development. The distinctive anatomical features of the hen ovary coupled with the powerful patch clamp and spectroflourometric techniques make possible a detailed study of the regulation of ion fluxes in granulosa cells during the maturation of ovarian preovulatory follicles. Our results will explain how the ionic microenvironment necessary for important processes catalysed by enzymes (such as adenylate cyclase and cholesterol side chain cleavage enzyme) are controlled. These studies will also provide information on how gonadotropins and growth factors regulate granulosa cell differentiation during follicular maturation. In addition, they will give knowledge about the mechanism of action growth factors in granulosa cells.