CHARACTERIZATION OF SEXUAL PHEROMONES IN APLYSIA

本提案中概述的实验将从化学上分离和 描述吸引海洋软体动物的信息素因素 对产卵同种进行交配诱导。 的因素 将从心房腺的酸性提取物中纯化， 器官分泌到输卵管，并从海水洗脱液最近 放置卵警戒线;将在T-迷宫中评估信息素活性， 交配实验 活跃因素的特点是： 成分和微序列或FAB/MS分析，和序列 合成用于HPLC共洗脱研究（以验证天然的 序列）和生物测定研究（以确认信息素活性）。 的 合成肽也可用于产生兔抗血清， 将提供一种免疫细胞化学方法来证明从 心房腺到鸡蛋警戒线;抗体特异性将是 通过免疫斑点试验、免疫细胞化学和预吸收法进行鉴定 问题研究 如果这种方法不成功，我们将结合使用 药理学和生物化学技术，以确定是否有 表征肽的替代组织来源;如果没有， 如果检测到，该肽可能起源于心房腺。 这一结论将通过检查在细胞中的肽释放来进一步检验。 体外（即，通过分析来自离体心房腺/大 高钾刺激前后两性导管的制备 人工海水，以及在暴露于袋式电池产品之前和之后）。 这些研究将证明心房腺是否是一个 性信息素的生理来源，以及信息素是否 这些因子是心房肌中表达的ELH相关基因的产物， 腺。 如果是的话，这些实验将提供一个初步的 一个可能感兴趣的模型系统的一个方面的表征 其中一个基因家族的不同子集的产物调节 不同的行为子集来产生协调的功能-- 袋细胞ELH基因的产物被分泌到血腔中， 诱导产卵，心房腺ELH相关基因的产物 沉积在卵圈上作为信息素吸引 其他的蜘蛛到卵层并诱导交配。 这些研究还将 有助于我们了解信息素和信息素的功能， 水生环境，通过鉴定第一肽信息素， 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.