GALANIN REGULATION OF PITUITARY REPRODUCTIVE HORMONES

目前已有大量证据表明下丘脑神经肽甘丙氨酸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； 2)在垂体水平，甘丙肽直接刺激促性腺激素分泌LH，增强GnRH刺激的LH分泌。虽然甘丙肽对下丘脑GnRH分泌的影响已经比较明确，但对甘丙肽直接在垂体水平上调节LH分泌的作用知之甚少。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）， 2)在垂体水平上，甘丙氨酸直接刺激促性腺激素的LH分泌，并增强GnRH刺激的LH分泌。虽然甘丙肽对下丘脑GnRH分泌的影响已经比较明确，但对甘丙肽直接在垂体水平上调节LH分泌的作用知之甚少。本提案中概述的实验旨在描述甘丙肽对雄性和雌性生殖激素分泌的垂体作用，以大鼠为动物模型。本提案的第一个具体目的是确定性腺激素是否会改变促性腺激素对丙氨酸刺激的敏感性。这一假设将在一系列体内输注实验中得到验证：1)完整、去势和去势+睾酮替代的雄性大鼠体内LH分泌对丙氨酸的反应；2)雌性大鼠在4天发情周期的不同时间点。对女性的研究将专门探讨促性腺激素对丙氨酸刺激的敏感性是否在发情（即排卵前）内分泌环境中上调。特异性靶数2的实验旨在表征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，以确定在对GnRH有反应的垂体前叶细胞中是否存在gal丙氨酸2型受体（GalR2）信使RNA （mRNA）表达，如果存在，则确定促性腺激素与其他对gal丙氨酸有反应的垂体前叶细胞类型中GalR2 mRNA表达的相对水平。本提案的第三个具体目的是研究甘丙肽对垂体前叶生殖激素基因表达的影响。体外灌注系统将用于确定丙氨酸除了刺激LH分泌外，是否也对Lhbeta亚基mRNA表达有影响。此外，还将评估丙氨酸对GnRH受体mRNA表达的影响。这些研究的结果将提供关于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，参与本提案中概述的实验执行将为本科生提供学习生殖生理学和分子生物学基础技术的独特机会，这些经验将为他们继续研究生学习提供良好的服务。

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.