Novel Perireceptor Role for Harderian Gland in Vomeronasal System

在大多数脊椎动物中，信息素的化学交流是主要的信息传递方式。雌性吊带蛇(Thamnopits Sirtalis)性信息素是一种非极性、疏水性的饱和和单不饱和甲基酮混合物，不溶于水。这种信息素由犁鼻器(VNO)检测，该器官专门用于接收非挥发性化学信号，但信息素进入器官水环境的机制尚不清楚。初步数据表明，哈德氏腺(HG)的分泌物具有增溶和/或信息素结合的特性，该分泌物专门进入蛇的VNO。300多年来，头部HG的功能一直是众多来源猜测的主题。当然，它们相对较大的体型，它们的系统发育年龄，以及几乎所有四足类从水环境到空气/陆地环境的持续保护表明，它们可能在适应陆地生命的生理上发挥了重要作用。该项目将研究HG作为媒介的作用，为雌性性信息素提供进入雄性吊袜带蛇的VNO的途径。利用放射性标记的束带蛇性信息素，研究HG分泌物对雌虫性信息素的增溶作用，以及HG分泌物具有信息素结合蛋白的潜力。去除HG对求偶行为和猎物攻击的影响，以及季节和激素变化对HG组成的影响，也将被调查，这两种行为都需要一个正常的VN系统。这里描述的综合研究将使用一个非常有特点的系统：吊袜蛇的VN系统，提供非极性、疏水的信息素分子到达VNO的新机制的演示。如果涉及信息素结合蛋白，它将是第一个在非哺乳动物物种中发现的蛋白质。这项研究将为研究HG参与其他哺乳动物和非哺乳动物脊椎动物的VNO功能的受体周围事件提供一个令人信服的模型。此外，这项研究利用了物种在其自然栖息地表现出的两种明确的、可量化的行为，这不仅为洞察繁殖行为和取食的季节性变化提供了一个稳健的模型，而且还提出了调节这些季节性调节变化的生理机制。这项提议涉及一种综合的、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)脊椎动物共同的信息素交流的统一原则产生重要的见解。这项提议的更广泛影响包括通过指导研究生、本科生和面向高中生和小学生进行研究和教育方面的多学科培训。本科生是PI实验室不可或缺的一部分，他们协助畜牧业、野外工作和行为实验。这些本科生是这两个实验室发表的23篇论文的共同作者。PI有长期的指导承诺，有十几名高中生参加了少数族裔培训计划和英特尔/西屋电气比赛，另外九名高中生参加了NSF发起的科学与工程学徒计划。这些经验，再加上适当的课程工作，将为这些受训科学家体验野外和实验室工作的喜悦和挫折奠定坚实的基础。

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.