GALANIN REGULATION OF PITUITARY REPRODUCTIVE HORMONES

大量的证据表明下丘脑神经肽甘丙肽在调节垂体生殖激素分泌中的作用。 甘丙肽神经元在两个不同的水平上调节垂体促黄体生成激素（LH）的分泌：1）。在下丘脑水平，甘丙肽通过刺激下丘脑释放因子促性腺激素释放激素（GnRH）的产生间接调节LH，和2）。在脑垂体水平，甘丙肽直接刺激促性腺激素分泌LH，并增强GnRH刺激的LH分泌。 甘丙肽对下丘脑促性腺激素释放激素（GnRH）分泌的影响已被较好地描述，但关于甘丙肽直接在脑垂体水平调节促黄体激素（LH）分泌的作用却知之甚少。本提案中概述的实验旨在表征垂体下丘脑释放因子促性腺激素释放激素（GnRH），以及2）。在脑垂体水平，甘丙肽直接刺激促性腺激素分泌LH，并增强GnRH刺激的LH分泌。 虽然甘丙肽对GnRH分泌的下丘脑作用相对较好地表征，但关于甘丙肽直接在脑垂体水平调节LH分泌的情况知之甚少。 本提案中概述的实验旨在以大鼠为动物模型，表征甘丙肽对雄性和雌性生殖激素分泌的垂体影响。 本建议的具体目标1是确定性腺类固醇是否改变促性腺细胞对甘丙肽刺激的敏感性。 将在一系列体内输注实验中检验该假设，这些实验检查1）完整、去势和去势+睾酮替代雄性大鼠;和2）4天发情周期中不同时间点的雌性大鼠对甘丙肽的LH分泌反应。 在雌性动物中进行的研究将专门探讨促性腺激素对甘丙肽刺激的敏感性是否在发情前期（即排卵前）内分泌环境中上调。 具体目标2的实验旨在表征在垂体前叶促性腺激素中表达的甘丙肽受体亚型。 将进行双标记原位杂交实验，以确定对GnRH有反应的垂体前叶细胞中是否存在甘丙肽2型受体（GalR 2）信使RNA（mRNA）表达，如果存在，则确定促性腺激素细胞与对甘丙肽有反应的其他垂体前叶细胞类型中GalR 2 mRNA表达的相对水平。 本提案的具体目标3是研究甘丙肽对垂体前叶生殖激素基因表达的影响。 将使用体外灌流系统来确定甘丙肽除了刺激LH分泌外，是否还对Lh β亚基的mRNA表达有影响。 此外，将评估甘丙肽对GnRH受体mRNA表达的影响。 这些研究的结果将提供有关甘丙肽在调节垂体促性腺激素LH合成和分泌中的作用的重要信息，这些事件对于维持男性和女性的生殖能力至关重要。此外，参与执行本提案中概述的实验将为本科生提供一个独特的机会，学习生殖生理学和分子生物学的基本技术，这些经验将为他们提供良好的服务，因为他们追求研究生学习。

A substantial body of evidence now exists which implicates a role for the hypothalamic neuropeptide galanin in the regulation of pituitary reproductive hormone secretion. Galanin neurons regulate pituitary lutenizing hormone (LH) secretion at two distinct levels: 1). at the level of the hypothalamus, where galanin indirectly regulates LH by stimulating the production of the hypothalamic releasing factor gonadotropin-releasing hormone (GnRH), and 2). at the level of the pituitary gland, where galanin directly stimulates the secretion of LH from gonadotropes and enhances GnRH-stimulated LH secretion. While the hypothalamic effects of galanin on GnRH secretion are relatively well-characterized, little is known about galanin regulation of LH secretion directly at the level of the pituitary gland. Experiments outlined in this proposal were designed to characterize the pituitathe hypothalamic releasing factor gonadotropin-releasing hormone (GnRH), and 2). at the level of the pituitary gland, where galanin directly stimulates the secretion of LH from gonadotropes and enhances GnRH-stimulated LH secretion. While the hypothalamic effects of galanin on GnRH secretion are relatively well-characterized, little is known about galanin regulation of LH secretion directly at the level of the pituitary gland. Experiments outlined in this proposal were designed to characterize the pituitary effects of galanin on reproductive hormone secretion in both males and females, using the rat as an animal model. Specific aim number 1 of this proposal in to determine whether gonadal steroids alter gonadotrope sensitivity to galanin stimulation. This hypothesis will be tested in a series of in vivo infusion experiments examining LH secretory responses to galanin in 1) intact, castrated, and castrated + testosterone-replaced male rats; and 2) female rats at different timepoints during the four day estrous cycle. Studies in females will specifically address whether gonadotrope sensitivity to galanin stimulation is upregulated within the context of the proestrous (i.e. preovulatory) endocrine milieu. Experiments of Specific aim number 2 are designed to characterize the galanin receptor subtype that is expressed in gonadotropes in the anterior pituitary gland. Double-label in situ hybridization experiments will be performed to determine if galanin type 2 receptor (GalR2) messenger RNA (mRNA) expression is present in anterior pituitary cells that respond to GnRH, and if so, to determine the relative levels of GalR2 mRNA expression in gonadotropes vs. other anterior pituitary cell types that respond to galanin. Specific aim number 3 of this proposal is to investigate the effects of galanin on reproductive hormone gene expression in the anterior pituitary gland. An in vitro perifusion system will be used to determine if galanin, in addition to stimulating the secretion of LH, also has an effect on the mRNA expression of the Lhbeta subunit. Furthermore, the effects of galanin of GnRH receptor mRNA expression will be assessed. Results from these studies will provide important information regarding the role of galanin in regulating LH synthesis and secretion from pituitary gonadotropes, events that are critically important for maintaining reproductive competence in both males and females. Additionally, participation in the execution of experiments outlined in this proposal will provide a unique opportunity for undergraduate students to learn fundamental techniques in reproductive physiology and molecular biology, experiences that will serve them well as they pursue their graduate studies.