GAP JUNCTIONS AND LUTEAL FUNCTION

本项目的长期目标是研究黄体细胞的作用 语料库生长、分化和退化过程中的相互作用 Lutea（CL）。 拟议研究的具体目标是 研究：1）接触依赖性细胞间通讯的个体发生 在体外通过评估三个阶段的黄体细胞类型之间， 发情周期（对应于快速生长、分化和 回归;实验1）、2）促黄体生成素（LH）的体外作用 和前列腺素F/2 α（PGF）对接触依赖性细胞间 通讯和第二信使系统的黄体细胞（实验。1和2）， 3)第二信使系统对接触依赖性 体外黄体细胞间通讯（Exp.（3）、（4） 细胞间隙连接通讯与 黄体细胞产生孕酮（实验1和3），5）的存在 黄体细胞和组织中的间隙连接蛋白（Exp. 1和4）和6） 促黄体生成素和前列腺素F对黄体细胞间通讯的影响 细胞（实验4）、黄体生长、分化和退化期。 CL在整个过程中表现出动态的结构和功能变化 动情周期的过程。 黄体包含几种细胞类型 它们相互作用以维持正常的组织功能。 这些细胞可以 通过旁分泌或内分泌途径或通过缝隙连接相互作用 渠道 结构和功能的缝隙连接已被证明 在几个物种的黄体组织，并可能发挥关键作用， 黄体细胞的增殖、分化和退化。 在 CL的添加、生长、分化和退化受到调节 全身和局部因素，包括促黄体生成激素和 前列腺素F/2 α。 这些全身和局部调节剂影响黄体功能， 细胞第二信使（即，cAMP、蛋白激酶C和/或钙）， 已知其调节间隙连接。 然而，接触的作用- 协调黄体生长的依赖性（间隙连接）相互作用， 分化和退化尚未得到很好的定义。 为了研究接触依赖性相互作用，我们将利用绵羊CL，其 形态和功能已经被广泛研究。 拟议 实验中，交互式激光细胞仪将用于检查接触- 黄体细胞类型之间的依赖性细胞间通讯 存在或不存在黄体功能调节剂。 交互式激光器 细胞计量术也将用于测量细胞内的 cAMP、蛋白激酶C或钙离子在单个活细胞中的存在 或缺乏黄体功能调节剂。 此外，差距的存在 将在黄体细胞培养物和黄体细胞中评价连接蛋白。 组织中 培养基中的孕酮浓度将用作 黄体细胞分化的功能，也作为一个估计， 缝隙连接通讯与孕酮的关系 分泌物 培养的黄体细胞DNA和蛋白质含量以及 类固醇生成能力的免疫组织化学标记物将用于 评价细胞群体动力学并确定最终的细胞数量 在培养 阐明了调节 细胞间的相互作用将提供对协调的深入了解， 黄体生长、分化和退化过程中的细胞过程。

The Long-Term Goal of this project is to study the role of luteal cell interactions during growth, differentiation and regression of corpora lutea (CL). The Specific Objectives of the proposed research are to study: 1) the ontogeny of contact-dependent intercellular communication between luteal cell types in vitro by evaluating three stages of the estrous cycle (corresponding to rapid growth, differentiation and regression; Exp. 1), 2) the in vitro effects of luteinizing hormone (LH) and prostaglandin F/2alpha (PGF) on contact-dependent intercellular communication and second messenger systems of luteal cells (Exp. 1 and 2), 3) the effects of second messenger systems on contact-dependent intercellular communication between luteal cells in vitro (Exp. 3), 4) the relationship of gap junctional intercellular communication with progesterone production by luteal cells (Exp. 1 and 3), 5) the presence of gap junction proteins in luteal cells and tissues (Exp. 1 and 4), and 6) the in vivo effects of LH and PGF on intercellular communication of luteal cells (Exp. 4), during luteal growth, differentiation and regression. The CL exhibits dynamic structural and functional changes during the course of the estrous cycle. Corpora lutea contain several cell types that interact to maintain normal tissue function. These cells may interact by paracrine or endocrine pathways or through gap junctional channels. Structural and functional gap junctions have been demonstrated in luteal tissues of several species, and probably play a critical role in proliferation, differentiation and regression of luteal cells. In addition, growth, differentiation and regression of the CL are regulated by systemic and local factors, including luteinizing hormone and prostaglandin F/2alpha. These systemic and local regulators affect luteal cell second messengers (i.e., cAMP, protein kinase C and/or calcium), which are known to modulate gap junctions. However, the role of contact- dependent (gap junctional) interactions in coordination of luteal growth, differentiation and regression has not been well defined. To study contact-dependent interactions, we will utilize ovine CL, whose morphology and function have been studied extensively. In the proposed experiments, interactive laser cytometry will be used to examine contact- dependent intercellular communication between luteal cell types in the presence or absence of regulators of luteal function. Interactive laser cytometry also will be used to measure intracellular concentrations of cAMP, protein kinase C or calcium in individual live cells in the presence or absence of regulators of luteal function. Moreover, presence of gap junction proteins will be evaluated in luteal cell cultures and in luteal tissues. Progesterone concentrations in media will be used as an index of luteal cell differentiated function, and also as an estimate of the relationship between gap junctional communication and progesterone secretion. DNA and protein contents of cultured luteal cells as well as immunohistochemical markers of steroidogenic capacity will be used to evaluate cell population dynamics and to determine cell numbers at the end of culture. Elucidation of contact-dependent mechanisms that regulate cellular interactions will provide insight into the coordination of cellular processes during luteal growth, differentiation and regression.