NEUROENDOCRINE BASES OF REPRODUCTIVE BEHAVIOR

(改编自研究人员的摘要)：卵巢激素 雌二醇(E)和孕酮(P)作用于下丘脑和视前区 促排卵前垂体释放面积(HPOA) 促性腺激素与生殖交配的协调表达 行为，即女性通过以下行为而产生的前凸 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α1和β肾上腺素能受体的转导及其关系 这些都是生殖行为的表现。特定目标1将测试 雌激素升高肾上腺皮质激素α1B受体的假设 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)E是否增加α1B肾上腺素受体 HPOA区也表达ER的蛋白质；2)A1B- 肾上腺素能受体和内质网共存于部分或全部神经元； 3)表达α1B-肾上腺素能受体的下丘脑神经元是否投射 至中脑中央灰质(MCG)。《特定目标2》将测试 在E启动的雌性HPOA中，P将切换Alpha1的假设 从磷脂酶C激活到钙离子的肾上腺素受体信号转导 一氧化氮(NO)/可溶性鸟苷酸环化酶的依赖激活 路径。对这一假设的支持来自于观察到的 α1肾上腺素能激活NO合成对排卵前期的影响 黄体生成素的释放与E+P生殖行为的表达 治疗的雌性大鼠被一氧化氮合酶抑制剂和 可溶性鸟苷酸环化酶抑制剂。《特定目标3》将测试 E可增加一种或多种蛋白激酶表达的假说 HPOA中的C(PKC)同工酶。PKC是一个主要的下游调停者 α1-肾上腺素能信号转导；因此，PKC的诱导可以 进一步放大HPOA中的α1-肾上腺素能信号。实验将会 利用PKC催化活性和佛波酯结合的测定 和蛋白质印迹来鉴定分离的同工酶。具体目标4将 检验E调节肾上腺素能相关分子的假说 受体-G蛋白偶联。分子生物学和免疫学 将使用多种方法来确定E对：1)信使核糖核酸和蛋白质的影响 B-肾上腺素能受体激酶1和2(b-ARK1和b-ARK2)和2的水平 B-arrestin1和b-arrestin2的mRNA和蛋白水平。这些是 重大问题，因为b型方舟和b型逮捕阻碍了 B-肾上腺素能、a2-肾上腺素能和u-阿片受体与G的相互作用 蛋白质，他们发现E治疗降低了所有人的功能 HPOA中的三种受体可明显下调 感受器。

(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.