Novel Perireceptor Role for Harderian Gland in Vomeronasal System

信息素的化学通讯是大多数脊椎动物信息传递的主要方式。雌性袜带蛇（Thamnophis sirtalis）的性信息素是一种饱和和单不饱和甲基酮的非极性疏水混合物，不溶于水溶液。这种信息素是由犁鼻器官（VNO）检测到的，这是专门用于接收非挥发性化学信号的，但信息素进入该器官水环境的机制尚不清楚。初步数据表明，哈德氏腺（HG）的分泌物，专门进入蛇的VNO，具有溶解和/或信息素结合的特性。300多年来，头侧HG的功能一直是众多猜测的主题。当然，它们相对较大的体型，它们的系统发育年龄，以及在几乎所有四足动物群体中从水环境到空气/陆地环境的持续保存，都表明它们可能在适应陆地生活的生理过程中发挥了重要作用。本项目将研究HG作为中介在提供雌性性信息素进入雄性吊带蛇的VNO中的作用。利用放射性标记的袜带蛇性信息素，研究HG分泌对雌性性信息素的溶解作用以及HG分泌具有信息素结合蛋白的潜力。去除汞柱对求偶行为和猎物攻击的影响，这两个都需要一个功能性的VN系统，也将被研究，以及季节和激素变化对VN组成的影响。这里描述的综合研究将展示一种新的机制，通过这种机制，非极性、疏水信息素分子到达VNO，使用一个非常有特征的系统：吊带蛇的VN系统。如果涉及信息素结合蛋白，这将是首次在非哺乳动物物种中发现。本研究将为其他哺乳动物和非哺乳动物脊椎动物HG参与介导VNO功能的周围受体事件提供一个令人信服的模型。此外，本研究利用了物种在其自然栖息地中表现出的两种明确的、可量化的行为，不仅为深入了解繁殖行为和摄食的季节性变化提供了一个强大的模型，而且还提出了一种调节这些季节性调节变化的生理机制。这一建议涉及一个综合的、多学科的方法，包括实地和实验室研究，这将对许多（如果不是大多数的话）脊椎动物共同的信息素交流的统一原则产生重要的见解。该提案的更广泛影响包括通过指导研究生、本科生和扩展到高中和小学学生，在研究和教育方面进行多学科培训。本科生是PI实验室的重要组成部分，协助进行畜牧业、野外工作和行为实验。这些本科生在这两个实验室发表了23篇论文。PI有长期的指导承诺，有十几名高中生参加了少数民族培训项目和英特尔/西屋公司的竞赛，另有九名高中生参加了国家科学基金会发起的科学与工程学徒项目。这些经验，加上适当的课程作业，将为这些在培训中的科学家提供一个坚实的基础，使他们能够体验实地和实验室工作的喜悦和挫折。

Chemical communication by pheromones is the predominant mode of information transfer in most vertebrates. The female garter snake (Thamnophis sirtalis) sex pheromone, a nonpolar, hydrophobic blend of saturated and monounsaturated methyl ketones, is insoluble in aqueous solutions. This pheromone is detected by the vomeronasal organ (VNO), which is specialized for the reception of nonvolatile chemical cues, but the mechanism by which the pheromone gains access to the aqueous environment of the organ is unknown. Preliminary data indicate that the Harderian glands' (HG) secretions, which duct exclusively into the VNO in snakes, contain solubilizing and/or pheromone-binding properties. For over 300 years, the function of the cephalic HG has been the subject of speculation from numerous sources. Certainly their relatively large size, their phylogenetic age, and persistent conservation in almost all tetrapod groups as they emerged from an aqueous to an air/land environment suggest that they probably play an important role in the physiological adaptation to terrestrial life. This project will examine the role of the HG as a mediator in providing access for the female sex pheromone to the VNO of male garter snakes. Using radiolabeled garter snake sex pheromone, studies will be conducted to determine the solubilizing effect of HG secretion on the female sex pheromone and the potential of the HG secretion to possess pheromone-binding proteins. The effects of HG removal on courtship behavior and prey attack, both known to require a functional VN system, will also be investigated, as will the effects of seasonal and hormonal variation on the composition of the HG. The integrative research described here will provide demonstrations of a novel mechanism by which nonpolar, hydrophobic pheromone molecules reach the VNO, using a very well characterized system: the VN system of garter snakes. If a pheromone-binding protein is involved, it will be the first identified in a non-mammalian species. This research will provide a compelling model for the examination of HG involvement in perireceptor events mediating VNO function of other mammalian and non-mammalian vertebrates. In addition, this study takes advantage of two unambiguous, quantifiable behaviors exhibited by a species in its natural habitat that not only offers a robust model for insights into seasonal shifts in reproductive behavior and feeding, but also proposes a physiological mechanism mediating these seasonally regulated changes. This proposal involves an integrative, multidisciplinary approach including field and lab studies that will yield significant insights into the unifying principles of pheromonal communication common to many, if not most, vertebrates. The broader impacts of this proposal include multidisciplinary training in research and education through the mentoring of graduate students, undergraduates and outreach to high school and grade school students. Undergraduates are an integral part of the PI's laboratories, assisting in animal husbandry, field work and behavior experiments. These undergraduates have been co-authors on 23 published papers from the two laboratories. The PI's have a long-term commitment to mentoring, with more than a dozen high school students involved in minority training programs and Intel/Westinghouse competitions, and an additional nine high school students having worked in an NSF-initiated Apprenticeships in Science and Engineering program. These experiences, coupled with appropriate course work, will provide a firm foundation for these scientists-in-training to experience the joys and frustration of field and laboratory work.