REGULATION OF GAMETE USE AND NEURAL PATHWAYS IN REPRODUCTION

项目总结 健康受精卵的成功生产涉及到数十个基因的激活，男性和 女性。这些基因在分子、细胞、 神经学和整个生物体的水平。在目前的资助期内，我们确定了高水平的自然 雌性神经系统中调节精子优先顺序的基因发生变异。在这 作为目标1，我们提出了一组实验，询问同种精子是否 优先顺序是由物种内精子所选择的相同基因介导的 竞争。与来自错误物种的雄性交配可能会通过 与调节同种雄性偏爱的机制相同，或者可能存在 质的差异。这个问题将通过精子优先的基因测试直接解决，并通过 在种内和种间配对中的转录组比较。我们将通过以下方式量化RNA的转移 雄性果蝇，定位参与这种转移变异的基因，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)神经信号是否以及如何 途径被调控，在男性和女性精子-竞争性相互作用中发挥作用。我们用来展示 初步结果表明，在女性中直接操作骨性关节炎会影响精子的不同保留。 第一个交配的男性，提出了一个问题，即两性如何利用这一途径相互影响 生殖结果。我们将通过对OA信号的遗传操作来直接测试这个问题，然后 对精子优先顺序的测试，以及通过检查OA信号的变异及其与精子的关系 比赛结果，在自然近亲繁殖参照系中。在目标3中，我们探讨了外切体在 调节男性和女性之间的互动。外切体是脂类结合的囊泡，携带蛋白质和小分子 核糖核酸分子。它们定义了一种新的细胞-细胞通信机制，在流程中扮演的角色多种多样 生育和转移。外体在精液中传递给雌性并影响生殖 哺乳动物的精子成熟等现象。果蝇精液中类似的囊泡与苍蝇融合 精子，并可能与女性生殖道一起，影响女性交配后的反应。这是可能的 敲除果蝇精液外胚体的传递，并跟踪这些外胚体的存在、转移和融合 用荧光标记标记的外显体。我们建议使用这些操作来确定外切体是否 在男性和女性之间传递影响精子使用的信号。我们研究外切体变异的工作 生产和转移将需要应用GWAS方法来识别外切体中的新遗传因素 生物学。我们将研究的许多过程显示出高度的进化守恒，这意味着 我们的结果将扩大我们对男性和女性在生殖过程中的相互作用的理解 与可能涉及配偶之间基因不相容的特发性人类不孕不育病例的相关性。

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.