Drosophila Genes Causing Male X Female Interactions in Reproduction

描述（由申请人提供）：精子和卵子的特定组合能否成功形成受精卵的决定因素涉及无数的分子、细胞、神经和整个生物体的行为属性。我们过去的工作主要集中在多次交配的雌性果蝇精子成功差异的现象学上，对影响精子竞争的基因的筛选已经阐明了这一过程的许多方面。我们继续努力的第一个目标是确定女性在生殖道中表达的基因在精子成功中的作用。为了剖析这些基因在精子使用中的功能作用，我们将应用高度靶向的RNAi敲除，并评估这些基因在女性生殖道中表达缺失的影响。使用在女性生殖道中表达的GAL4驱动因子，可以很容易地实现这一点。我们将采用明智的标准来选择敲除候选基因，我们对具有神经功能的女性基因表达特别感兴趣。雌性反应基因的敲除将在一系列精子竞争测试中进行测试，测试对象是一组16个已知近交序列的雄性和精液蛋白零，以试图量化雄性与雌性的相互作用以及测试特定的相互作用。我们的第二个目标是量化雄性在影响雌性交配后转录组反应的能力方面的遗传差异，以及这些表达变化在精子竞争中的作用。雌性将与来自果蝇遗传参考小组的雄性交配（基因组序列和基因表达数据已经可用），生殖道将被解剖并进行rna测序。分析将提供详细的图片，哪些基因对交配有转录反应，哪些基因对不同的男性基因型有不同的反应。这些基因将依次在雌性体内被敲除，以观察它们是否在精子竞争中发挥作用。因为我们研究的基因变异涉及基本的生殖功能，我们的结果将扩大对特发性人类不孕症病例的理解基础，特别是那些没有性别特异性原因的不孕症，因此可能反映了伴侣之间的遗传不相容。

DESCRIPTION (provided by applicant): The determinants of which particular combinations of sperm and egg are successful in forming a zygote involve a myriad of molecular, cellular, neurological and whole-organism behavioral attributes. Our past work has focused on the phenomenology of differential success of the sperm in multiply mated female Drosophila, and screens for genes that impact this sperm competition have illuminated many aspects of the process. The first aim of our continuing effort is to determine the role(s) in sperm success of genes expressed by the female in her reproductive tract. To dissect the functional role of these genes in sperm use, we will apply highly targeted RNAi knockdown and assess the impact of loss of expression of these genes in the female reproductive tract. This is readily accomplished with the UAS-GAL4 system, using GAL4 drivers that express in the female reproductive tract. Judicious criteria will be applied to select candidates for knockdown and we are especially interested in the set of genes expressed in females that have neurological function. Knockdowns of female response genes will be tested in a battery of sperm competition tests against a set of 16 inbred sequence-known males and seminal protein nulls to try to quantify male x female interaction as well as testing specific interactions. Our second aim is to quantify genetic differences among males in their ability to influence post-mating transcriptome responses in females, and the roles of those expression changes on sperm competition. Females will be mated with males drawn from the Drosophila Genetic Reference Panel (for which both genome sequence and gene expression data are already available) and reproductive tracts will be dissected and subjected to RNA-sequencing. Analysis will provide a detailed picture of which genes respond transcriptionally to mating, and which genes respond differentially to different male genotypes. These genes will in turn be knocked down in females to see if they play a role in sperm competition. Because the genes whose variation we examine involve basic reproductive functions, our results will expand the foundation for understanding cases of idiopathic human infertility, specifically those in which no sex-specific cause for the infertility can be identified and may thus reflect genetic incompatibility between the partners.