International Research Fellowship Program: Unraveling Genetic and Nongenetic Causes of Sperm Precedence in Stalk-eyed Flies

0913116 birge2009年美国复苏和再投资法案（公法111-5）为该奖项提供资金。国际研究奖学金项目使美国科学家和工程师能够在国外进行9至24个月的研究。该计划的奖励为联合研究提供了机会，并利用独特或互补的设施、专业知识和国外的实验条件。该奖项将支持由Leanna M. Birge博士与英国伦敦大学学院的Andrew Pomiankowski博士以及美国马里兰大学的Gerald Wilkinson博士共同开展的为期24个月的研究。雌性多次交配为射精之间的竞争创造了一个舞台。作为回应，雄性会采用各种机制来影响受精的成功，比如丧失能力。然而，丧失能力作为精子优先机制的重要性是有争议的，因为尚不清楚雄性是如何伤害竞争对手的。他射精时不会伤害到自己。为了评估丧失能力的效用并识别其诱因，需要一个系统，在这个系统中，竞争者之间的遗传差异？射精可以被识别。柄眼蝇提供了这样一个系统。在白胞藓中，产生无偏性别比的雄性精子使产生由X染色体减数分裂驱动的雌性偏性别比的雄性精子丧失能力。在本项目中，我们使用该系统进行了两项互补研究：失能性的稳健性测试和与失能性相关的蛋白质的分子表征。为了评估丧失能力的强度，使用伦敦大学学院制定的人口和方案来控制成人饮食，并在马里兰大学开发的体外精子活力测定中对驱动和非驱动雄性进行竞争。对活精子/死精子计数进行方差分析。先验地，由于饮食应激对非驱动雄性造成蛋白质组成的改变，驱动雄性有望克服低蛋白饮食治疗中的丧失能力。该项目的分子部分在马里兰大学进行，重点是确定与失能相关的射精蛋白谱的变化。这项工作的一部分包括从睾丸和男性附属腺体中构建表达序列标签（EST）文库。串联质谱对三种不同组合的精囊/副腺样品以及内部标准进行鉴定蛋白质谱。串联质谱分析结果与EST文库进行比较，并对不同处理之间蛋白质组成和表达的差异进行量化，以确定候选失能基因。拟议工作的更广泛影响包括博士后和马里兰大学本科生的高级培训。此外，该项目还促进了博士后与英国和美国两名高级研究员之间的国际合作。这项研究的结果有可能为了解精子发育和功能障碍提供见解，这对了解男性不育的原因具有生物医学意义。最后，大量的DNA序列信息存储在在线存储库中，从而增强了其他研究者进行比较基因组研究的能力。

0913116BirgeThis award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).The International Research Fellowship Program enables U.S. scientists and engineers to conduct nine to twenty-four months of research abroad. The program's awards provide opportunities for joint research, and the use of unique or complementary facilities, expertise and experimental conditions abroad.This award will support a twenty-four-month research fellowship by Dr. Leanna M. Birge to work with Dr. Andrew Pomiankowski at University College London in the United Kingdom and with Dr. Gerald Wilkinson at the University of Maryland in the U.S.Female multiple mating creates an arena for competition to occur among ejaculates. In response, males employ a variety of mechanisms to bias their fertilization success, such as incapacitation. However, the importance of incapacitation as a mechanism for sperm precedence is controversial because it is unclear how a male can damage a rival?s ejaculate without also damaging his own. To evaluate the utility of incapacitation and identify its agents, a system is needed in which genetic differences between competitors? ejaculates can be identified. Stalk-eyed flies provide such a system. In Cyrtodiopsis whitei, sperm from males that produce unbiased sex ratios incapacitate sperm from males that produce female-biased sex ratios caused by X chromosome meiotic drive. In this project, we conduct two complementary studies using this system: a test of the robust nature of incapacitation and a molecular characterization of proteins associated with incapacitation. To assess the strength of incapacitation, adult diet is manipulated using a population and protocols developed at University College London and drive and non-drive males are competed in an in vitro sperm viability assay developed at University of Maryland. Analysis of variance is performed across treatments on live/dead sperm counts. A priori, drive males are expected to overcome incapacitation in the low protein diet treatment due to changes in protein composition caused by dietary stress to non-drive males. The molecular portion of the project is conducted at the University of Maryland and focuses on identifying changes in ejaculatory protein profiles associated with incapacitation. A portion of this work includes the construction of expressed sequence tag (EST) libraries from testes and male accessory glands. Tandem mass spectroscopy is performed on three different combinations of seminal vesicle/accessory gland samples as well as an internal standard to identify protein profiles. Tandem mass spectroscopy results are compared to EST libraries and differences between treatments in protein composition and expression are quantified to identify candidate incapacitation genes.Broader impacts of the proposed work include advanced training for the postdoctoral fellow and University of Maryland undergraduate students. In addition, the project fosters international collaboration between the postdoctoral fellow and two senior investigators in the UK and US. Results from this study have the potential to provide insight into sperm development and dysfunction, which have biomedical relevance for understanding causes of male infertility. Finally, a large amount of DNA sequence information is deposited in online repositories, thereby enhancing the ability of other investigators to conduct comparative genomic studies.