Accumulation, Storage, and Release of Sperm in the Oviduct

项目摘要 生育依赖于成功的受精和早期发育，这些过程发生在输卵管中。普普通通 人类不孕不育的治疗方法，如体外受精和胞浆内单精子注射，费用昂贵，而且 增加各种问题的风险。更多关于输卵管如何与精子、卵丘-卵母细胞相互作用的知识 复合体(COC)和发育中的胚胎可能会提高生育力，减少治疗的需要。输卵管作为 精液沉积之后和受精前的精子储存库。与输卵管的结合维持精子活力和 抑制运动性。精子被释放到输卵管上部(壶腹)，使卵母细胞受精。有很多差距。 在这个精子-输卵管相互作用的模型中。我们的研究已经开始填补其中的一些空白。我们使用了一种血糖调节剂 对377个多糖进行筛选，发现所有对猪精子有亲和力的多糖都具有两个基序中的一个， 硫酸化的Lewis X三糖或支化的6-唾液酸化的复合多糖。我们已经确定了两个候选受体 精子膜上的两种多糖：PKDREJ和ADAM5。值得注意的是，小鼠精子缺乏PKDREJ和其他 亚当斯不会积聚在子宫-输卵管交界处以外，但目前尚不清楚这是否由于 在输卵管中的结合和滞留。值得注意的是，如果这些葡聚糖固定在珠子上，它们可以延长精子 寿命，就像与输卵管细胞结合可以延长精子的寿命一样。最后，我们发现卵丘-卵母细胞 复合体(COCs)分泌分子，发出精子从输卵管下部释放的信号，从而使精子能够向 受精。这些特定的目标将提供对精子如何结合输卵管的机械理解，如何结合 延长精子寿命以及COC如何将精子从输卵管中释放出来。目标1：确定 在猪的精子中，通过阻断每个蛋白质和突变每个基因，PKDREJ和ADAM5。来自猪的精子 这些蛋白质中每一种的缺陷都将被检查以确定它们结合输卵管细胞的能力和它们的生育能力 是受影响的。目的2.确定精子与固定的输卵管多糖结合是否抑制ROS的产生， 保存三磷酸腺苷，稳定质膜，延长精子寿命和生育力。精子被绑定到 将检查固定化的葡聚糖，以确定粘附力引起的变化。第二组实验将 通过添加清除ROS和稳定血浆的试剂来确定哪些变化足以延长寿命 薄膜。目的3.确定卵丘-卵母细胞复合体信号如何从输卵管细胞释放精子和 多聚糖。我们将检查黄体酮、前列腺素或可能由COCs分泌的蛋白质是否可能 诱导猪精子从输卵管细胞和固定化多糖中释放。这些具体目标的完成将 阐明精子如何与输卵管结合，储存在输卵管中，并对卵丘-卵母细胞做出反应而释放 很复杂。这些基本信息可以用来开发更简单、更安全、成本更低的辅助生殖 技术。

Project Summary Fertility depends on successful fertilization and early development, processes that occur in the oviduct. Common therapies for human infertility, such as in vitro fertilization and intracytoplasmic sperm injection, are expensive and increase risks of a variety of problems. More knowledge of how the oviduct interacts with sperm, the cumulus-oocyte complex (COC) and the developing embryo may improve fertility and reduce the need for therapies. The oviduct serves as a reservoir for sperm, after semen deposition and before fertilization. Binding to the oviduct maintains sperm viability and suppresses motility. Sperm are released to move to the upper oviduct (ampulla) to fertilize oocytes. There are many gaps in this model of sperm-oviduct interaction. Our studies have begun to fill some of these gaps. We have used a glycomic approach to screen 377 glycans and found that all glycans with affinity for porcine sperm have either of two motifs, sulfated Lewis X trisaccharide or branched 6-sialylated complex glycans. We have identified two candidate receptors for both glycans on the sperm membrane, PKDREJ and ADAM5. Notably, mouse sperm deficient in PKDREJ and other ADAMs do not accumulate beyond the utero-tubal junction, but it is not known if this is due to a problem in binding and retention in the oviduct. Remarkably, if these glycans are immobilized on beads, they can extend sperm lifespan, much like binding to oviduct cells prolongs the lifespan of sperm. Finally, we found that cumulus-oocyte complexes (COCs) secrete molecules that signal sperm release from the lower oviduct so they can move toward the site of fertilization. The Specific Aims will provide a mechanistic understanding of how sperm bind the oviduct, how binding prolongs sperm lifespan and how sperm are released from the oviduct by the COC. Aim 1: To determine the function of PKDREJ and ADAM5 in sperm by blocking each protein and mutating each gene in swine. Sperm from pigs that are deficient in each of these proteins will be examined to determine if their ability to bind oviduct cells and their fertility is affected. Aim 2. To determine if sperm binding to immobilized oviduct glycans suppresses ROS production, conserves ATP, and stabilizes the plasma membrane, which extends sperm lifespan and fertility. Sperm bound to immobilized glycans will be examined to ascertain what changes adhesion induces. The second group of experiments will determine which changes are sufficient to prolong lifespan by adding agents that scavenge ROS and stabilize the plasma membrane. Aim 3. To determine how the cumulus-oocyte complex signals release of sperm from oviduct cells and glycans. We will examine whether progesterone, prostaglandins, or proteins that may be secreted from COCs might induce porcine sperm release from oviduct cells and immobilized glycans. The completion of these Specific Aims will elucidate how sperm bind to the oviduct, are stored in the oviduct and are released in response to the cumulus-oocyte complex. This fundamental information could be used to develop simpler, safer and less costly assisted reproductive technologies.