REGULATION OF GAMETE USE AND NEURAL PATHWAYS IN REPRODUCTION

项目摘要 成功地产生健康的受精卵涉及到几十个基因的活动， 女性这些基因介导了两性之间在分子，细胞， 神经和整个机体水平。在目前的资助期内，我们确定了高水平的自然资源， 雌性神经系统中调节精子优先顺序的基因发生变异。在这 更新申请，我们提出的目标1一组实验，问同种精子是否 优先顺序是由相同的基因介导的，这些基因被选择用于种内精子 竞争与来自错误物种的雄性交配可能会产生精子选择性反应， 相同的机制，作为那些调解之间的同类男性的偏好，或者可能有 质的差异。这个问题将通过精子优先的基因测试直接解决， 转录组比较，在物种内和物种间交配。我们将定量RNA的转移， 雄性果蝇，绘制这种转移中涉及变异的基因，并将RNA转移与精子竞争联系起来 结果。在目标2中，我们询问高度保守的章鱼胺（OA）神经信号传导是否以及如何 途径被调节以在雄性X雌性精子竞争相互作用中发挥作用。我们展示与 初步结果表明，在女性中直接操作OA会影响精子的差异保留， 第一个交配的雄性，这就提出了一个问题，即两性如何利用这一途径来影响彼此的行为。 生殖结果。我们将直接测试这个问题，通过遗传操纵OA信号， 精子优先的测试，并通过检查OA信号的变化，及其与精子的关系 竞争结果，在自然近交参考系。在目标3中，我们探索了外泌体在以下方面的作用： 调节男性和女性的互动外泌体是脂质结合的囊泡，其携带蛋白质和小分子蛋白质。 RNA分子。他们定义了一种新的细胞间通讯机制，其作用多种多样， 生育和转移。外泌体在精液中转移到女性并影响生殖 哺乳动物精子成熟的现象。果蝇精液中的类似囊泡与苍蝇融合 精子，并可能与女性生殖道，影响女性交配后的反应。有可能 敲除果蝇中的精液外泌体传递，并跟踪这些外泌体的存在、转移和融合。 带有荧光标记的外来体。我们建议使用这些操作来确定外泌体是否 在男性和女性之间传递影响精子使用的信号。我们研究外来体变异的工作 生产和转移将需要应用GWAS方法来鉴定外泌体中的新遗传因子 生物学我们将要研究的许多过程都显示出高度的进化保守性，这意味着， 我们的研究结果将扩大我们对生殖过程中雄性x雌性相互作用的理解， 与可能涉及伴侣之间遗传不相容的特发性人类不育症病例相关。

PROJECT SUMMARY Successful production of healthy zygotes involves the activity of dozens of genes in both males and females. These genes mediate a complex set of interactions between the sexes at the molecular, cellular, neurological and whole-organism levels. In the current funding period, we identified high levels of naturally occurring variation in genes that act in the female's nervous system to modulate sperm precedence. In this renewal application, we propose as Aim 1 a set of experiments that ask whether conspecific sperm precedence is mediated by the same genes that are selected upon for within-species sperm competition. Mating with males from the wrong species might produce a sperm-selective response through the same mechanisms as the ones that mediate preference among conspecific males, or there may be qualitative differences. This question will be addressed directly by genetic tests of sperm precedence, and by transcriptome comparisons, in within- and between-species matings. We will quantify the transfer of RNAs by male Drosophila, map genes involved in variation in this transfer, and relate RNA transfer to sperm competition outcomes. In Aim 2, we ask whether and how the highly conserved octopamine (OA) neural-signaling pathway is modulated to play a role in male x female sperm-competitive interactions. We show with preliminary results that direct manipulation of OA in females influences differential retention of sperm from the first male to mate, raising the question of how the sexes use this pathway to influence each others' reproductive outcome. We will test this question directly, by genetic manipulation of OA signaling followed by tests of sperm precedence, and by examining variation of OA signaling, and its relationship to sperm competition outcomes, in natural inbred reference lines. In Aim 3 we explore the role of exosomes in mediating male x female interactions. Exosomes are lipid-bound vesicles that carry both proteins and small RNA molecules. They define a new cell-cell communication mechanism with roles in processes as diverse as fertility and metastasis. Exosomes are transferred to females in seminal fluid and impact reproductive phenomena such as sperm maturation in mammals. Similar vesicles in Drosophila seminal fluid fuse with fly sperm, and possibly with the female reproductive tract, affecting female post-mating responses. It is possible to knock out seminal exosome transmission in flies, and to follow the presence, transfer and fusion of these exosomes with a fluorescent marker. We propose using these manipulations to determine whether exosomes transfer signals between males and females that impact sperm use. Our work to examine variation in exosome production and transfer will entail application of GWAS methods to identify new genetic factors in exosome biology. Many of the processes that we will study show high levels of evolutionary conservation, implying that our results will expand our understanding of male x female interactions in reproduction that may have relevance to cases of idiopathic human infertility that may involve genetic incompatibility between the partners.