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

0913116 Birge该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。国际研究奖学金计划使美国科学家和工程师能够在国外进行9到24个月的研究。 该计划的奖项提供了联合研究的机会，以及使用独特或互补的设施，专业知识和国外的实验条件。Birge与英国伦敦大学学院的Andrew Pomiankowski博士和美国马里兰州大学的Gerald威尔金森博士合作。女性多次交配为射精之间的竞争创造了一个竞技场。作为回应，男性采用各种机制来偏见他们的受精成功，如失能。 然而，失能作为精子优先机制的重要性是有争议的，因为还不清楚雄性如何损害竞争对手？他射精时也不会伤害自己。 为了评估失能的效用，并确定其代理人，需要一个系统，其中竞争对手之间的遗传差异？精液可以被识别。 有柄眼的苍蝇就提供了这样一个系统。在Cyrtodiopsis whitei中，来自产生无偏倚性比的雄性的精子使来自产生由X染色体减数分裂驱动引起的雌性偏倚性比的雄性的精子丧失能力。 在这个项目中，我们使用这个系统进行两个互补的研究：一个测试的强大的失能性和与失能相关的蛋白质的分子表征。 为了评估失能的强度，使用伦敦大学学院开发的群体和方案来操纵成人饮食，并在马里兰州大学开发的体外精子活力测定中对驾驶和非驾驶男性进行竞争。 对各处理组的活/死精子计数进行方差分析。 先验地，驱力雄性动物预期克服低蛋白饮食治疗中的失能，这是由于饮食应激对非驱力雄性动物造成的蛋白质组成变化。 该项目的分子部分在马里兰州大学进行，重点是确定与失能相关的射精蛋白质谱的变化。 这项工作的一部分，包括表达序列标签（EST）文库的建设，从睾丸和男性附腺。 对精囊/附属腺样品的三种不同组合以及内标物进行串联质谱法以鉴定蛋白质谱。 将串联质谱结果与EST文库进行比较，并对蛋白质组成和表达的处理之间的差异进行量化，以鉴定候选失能基因。拟议工作的更广泛影响包括博士后研究员和马里兰州大学本科生的高级培训。 此外，该项目还促进了博士后研究员与英国和美国两名高级研究人员之间的国际合作。 这项研究的结果有可能提供深入了解精子发育和功能障碍，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序列信息被储存在在线知识库中，从而提高了其他研究人员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.