NEUROENDOCRINE BASES OF REPRODUCTIVE BEHAVIOR

（改编自研究者摘要）：卵巢激素 雌二醇（E）和孕酮（P）作用于下丘脑和视前区 刺激排卵前垂体释放 促性腺激素和协调生殖交配的表达 行为，即女性通过以下动作的前凸 去甲肾上腺素（NE）。拟议研究的目标是检查 雌二醇和孕酮调节信号的分子机制 HPOA中α 1-和β-肾上腺素受体的转导， 这些都是生殖行为的表现。具体目标1将测试 雌二醇升高α 1B-肾上腺素受体的假说， 表达ER的HPOA神经元群体。免疫细胞化学方法 将被用来确定：1）是否E增加α 1B-肾上腺素受体 HPOA中也表达ER的区域中的蛋白质; 2）A1 B是否 - 肾上腺素受体和ER共定位于这些神经元中的一些或全部中;并且 3)表达α 1B肾上腺素受体的下丘脑神经元是否投射 中脑中央灰质（MCG）。具体目标2将测试 假设在E-primed雌性的HPOA中，P将转换α 1 从磷脂酶C激活到钙离子的肾上腺素受体信号传导 一氧化氮（NO）/可溶性鸟苷酸环化酶的依赖性激活 通路支持这一假设的证据来自以下观察： α 1肾上腺素能激活NO合成影响排卵前 E+P中LH的释放和生殖行为的表达 处理的雌性大鼠被NO合成酶抑制剂抑制， 可溶性鸟苷酸环化酶的抑制剂。具体目标3将测试 假设E增加一种或多种蛋白激酶表达 C（PKC）同工酶在HPOA。PKC是一种主要的下游介质， α 1-肾上腺素能信号转导;因此，诱导PKC可以 进一步放大HPOA中的α 1-肾上腺素能信号。实验将 利用PKC催化活性以及佛波酯结合的测定， 和蛋白质印迹以鉴定单独的同工酶。具体目标4将 测试假设，E调节分子参与肾上腺素能 受体-G蛋白偶联。分子生物学和免疫学 方法将用于确定E对：1）mRNA和蛋白质的影响 b-肾上腺素能受体激酶1和2（b-ARK 1和b-ARK 2）水平和2） b-抑制蛋白1和b-抑制蛋白2的mRNA和蛋白水平。这些是 重要的问题，因为b-ARKs和b-arrestins阻碍了 β-肾上腺素能、α 2-肾上腺素能和μ-阿片受体与G 蛋白质，他们发现E治疗降低了所有蛋白质的功能， HPOA中的这些受体中的三种可测量地下调 受体。

(Adapted from the investigator's abstract): The ovarian hormones estradiol (E) and progesterone (P) act in the hypothalamus and preoptic area (HPOA) to stimulate the preovulatory release of pituitary gonadotropins and coordinate the expression of reproductive mating behavior, namely lordosis by the female through the action of norepinephrine (NE). The goal of the proposed research is to examine the molecular mechanisms by which estradiol and progesterone modulate signal transduction on alpha1-and beta-adrenoceptors in the HPOA and to relate these to the expression of reproductive behavior. Specific Aim 1 will test the hypothesis that estradiol elevates alpha1B-adrenoceptors in populations of HPOA neurons that express ER. Immunocytochemical approaches will be employed to determine: 1) whether E increases alpha1B-adrenoceptor protein in regions of the HPOA that also express ER; 2) whether A1B - adrenoceptors and ER are colocalized in some or all of these neurons; and 3) whether hypothalamic neurons expressing alpha1B-adrenoceptors project to the midbrain central gray (MCG). Specific aim 2 will test the hypothesis that in the HPOA of E-primed females P will switch alpha1 adrenoceptor signaling from activation of phospholipase C to calcium- dependent activation of the nitric oxide (NO)/soluble guanylyl cyclase pathway. Support for this hypothesis is derived from the observation that alpha1-adrenergic activation of NO synthesis influences the preovulatory release of LH and because expression of reproductive behavior in E+P treated female rats is inhibited by NO synthetase inhibitors and inhibitors of soluble guanylyl cyclase. Specific aim 3 will test the hypothesis that E increases the expression of one or more protein kinase C (PKC) isoenzymes in the HPOA. PKC is a major downstream mediator of alpha1-adrenergic signal transduction; hence, induction of PKC could further amplify alpha1-adrenergic signaling in the HPOA. Experiments will utilize assays of PKC catalytic activity as well as phorbol ester binding and Western Blots to identify the separate isoenzymes. Specific Aim 4 will test the hypothesis that E regulates molecules involved in adrenergic receptor-G protein coupling. Molecular biological and immunological methods will be used to determine the effects of E on: 1) mRNA and protein levels of b-adrenergic receptor kinase 1 and 2 (b-ARK1, and b-ARK2) and 2) the mRNA and protein levels of b-arrestin1 and b-arrestin2. These are significant questions because b-ARKs and b-arrestins impede the interactions of b-adrenergic, a2-adrenergic and u-opioid receptors with G proteins, and they find that E treatment decreases the function of all three of these receptors in the HPOA with measurably downregulating the receptors.