CHARACTERIZATION OF SEXUAL PHEROMONES IN APLYSIA

这项提案中概述的实验将在化学上分离和 描述吸引海洋软体动物的信息素 海兔对产卵同种生物产生反应并诱导它们交配。影响因素 将从心房的酸性提取物中提纯，心房是一种外分泌 分泌到输卵管的器官，以及最近从海水洗脱液中分泌的器官 储存的卵警戒线；信息素活性将在T-迷宫和 交配实验。积极的因素将表现为 成分和微序列或FAB/MS分析，以及序列 合成用于高效液相色谱洗脱研究(以验证天然的 序列)和生物测定研究(以确认信息素活性)。这个 合成肽还将用于制备兔抗血清，该抗血清 将提供一种免疫细胞化学方法来演示从 将房腺放在卵子的警戒线上；抗体的特异性 免疫斑点分析、免疫细胞化学和预吸收 学习。如果此方法不成功，我们将使用 药理和生化技术来确定是否有 特征性多肽的替代组织来源；如果没有 检测到，该肽很可能起源于心房腺。 这一结论将通过检查多肽释放来进一步验证 体外(即，通过分析来自分离的心房腺体/大的 高钾刺激前后两性导管的制备 人工海水，以及暴露于袋状电池产品前后)。 这些研究将证明心房腺是否是一种 性信息素的生理来源，以及信息素是否 因子是在心房中表达的ELH相关基因的产物 腺体。如果是这样的话，这些实验将提供一个初步的 潜在有趣模型系统的一个方面的刻画 其中一个基因家族不同子集的产物调节 不同的行为子集来产生协调的功能-- BAG细胞ELH基因产物分泌到血腔中以 诱导产卵，以及心房腺ELH相关基因的产物 沉积在卵警戒线上，作为信息素吸引 其他海兔进入产蛋期并诱导交配。这些研究还将 有助于我们理解信息素和信息素的功能 在水生环境中，通过鉴定第一个肽信息素(S) 软体动物。

The experiments outlined in this proposal will isolate and chemically characterize the pheromonal factors that attract the marine mollusc Aplysia to egg-laying conspecifics and induce them to mate. The factors will be purified from acidic extracts of the atrial gland, an exocrine organ secreting into the oviduct, and from seawater eluates of recently deposited egg cordons; pheromonal activity will be assessed in T-maze and mating experiments. The active factors will be characterized by compositional and microsequence or FAB/MS analyses, and the sequences synthesized for use in HPLC coelution studies (to verify the native sequence) and in bioassay studies (to confirm pheromonal activity). The synthetic peptide will also be used to generate rabbit antisera, which will provide an immunocytochemical approach to demonstrating release from the atrial gland onto the egg cordon; antibody specificity will be characterized by immunodot assay, immunocytochemistry and preabsorption studies. If this approach is unsuccessful, we will use a combination of pharmacological and biochemical techniques to determine whether there are alternative tissue sources of the characterized peptide; if none are detected, the peptide is likely to have originated in the atrial gland. This conclusion will be further tested by examining peptide release in vitro (i.e., by analyzing perfusates from an isolated atrial gland/large hermaphroditic duct preparation before and after stimulation with high K artificial seawater, and before and after exposure to bag cell products). These studies will demonstrate whether the atrial gland is a physiological source of sexual pheromones, and whether the pheromonal factors are products of the ELH-related genes expressed in the atrial gland. If they are, these experiments will provide an initial characterization of one facet of a potentially interesting model system in which products of different subsets of a family of genes regulate different subsets of behavior to produce a coordinated function -- products of the bag-cell ELH gene being secreted into the hemocoel to induce egg laying, and products of the atrial gland ELH-related genes being deposited onto the egg cordon to act as pheromones that attract other Aplysia to the egg layer and induce mating. The studies will also contribute to our understanding of pheromones and pheromonal function in an aquatic environment, by identifying the first peptide pheromone(s) in molluscs.