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。常见的治疗方法 对于人类不孕症，如体外受精和卵胞浆内单精子注射，费用昂贵，并增加了 各种问题的风险。更多关于输卵管如何与精子、卵丘-卵母细胞复合体相互作用的知识 (COC)，发育中的胚胎可能会提高生育力，减少对治疗的需要或导致 改进的治疗方法(即试管受精的改进)。输卵管是精子的储存库，在精液沉积和 在受精前。与输卵管结合可维持精子活力并抑制精子活力。精子被释放出来以便于移动 上输卵管(壶腹)使卵母细胞受精。在这个精子-输卵管相互作用的模型中有很多空白，但我们的 研究已经开始填补其中的一些空白。我们使用了一种糖链方法来筛选数百种多糖，并发现 对猪精子有亲和力的葡聚糖有两个基序之一，硫酸化的Lewis X三糖或支化的6-糖- 唾液酸化的复合多糖。我们还确定了精子膜上这两种多糖的两个候选受体， PKDREJ和ADAM5，它们与糖链结合未知。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，如果这些多糖被固定在珠子或显微镜载玻片上，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寿命一样。最后，我们发现COCS 分泌孕酮，通过诱导精子过度激活释放精子信号，从而使精子能够移动 向受精地点进发。这一更新的具体目标将提供对精子如何 绑定输卵管，绑定如何延长精子寿命，以及这些结果如何转化为改善试管受精。目标1： 阻断精子中PKDREJ和ADAM5各蛋白及基因突变以确定其功能 一头猪。来自这些基因突变的猪的精子已经被生产出来。这些精子中的每一个都有缺陷 将对蛋白质进行检测，以确定它们结合输卵管细胞的能力和生育能力是否受到影响。目标2.目标 确定精子与多糖结合是否会减少氧化磷酸化和延长柠檬酸循环 精子寿命。与固定化多糖结合的精子将被检查，以确定特定的代谢变化 以及导致这些变化的细胞内信号。目的3.确定输卵管多糖是否选择 用于储存和体外受精的优质精子。我们将检查通过多糖黏附选择的精子是否具有 改进的特征本身，并通过以下方式产生更类似于体内产生的胚胎的胚胎 胚胎转录本和甲基组的综合分析。这些具体目标的完成将提供 解决精子如何与输卵管结合的重要关键是储存在输卵管中，并在 COC。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 the 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 or lead to the development of improved therapies (i.e. improvements in IVF). 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 but our studies have begun to fill some of these gaps. We have used a glycomic approach to screen hundreds of glycans and found that glycans with affinity for porcine sperm have either of two motifs, sulfated Lewis X trisaccharide or branched 6- sialylated complex glycans. We also identified two candidate receptors for both glycans on the sperm membrane, PKDREJ and ADAM5, that were not known to bind glycans. 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 or microscope slides, they can extend sperm lifespan, much like binding to oviduct cells prolongs the lifespan of sperm. Finally, we found that COCs secrete progesterone that signals sperm release from the lower oviduct by inducing hyperactivation so sperm can move toward the site of fertilization. The Specific Aims of this renewal will provide a mechanistic understanding of how sperm bind the oviduct, how binding prolongs sperm lifespan, and how these results may be translated to improve IVF. 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 have mutations in these genes have been produced. Sperm that are deficient in each of these proteins will be examined to determine if their ability to bind oviduct cells and their fertility are affected. Aim 2. To determine if sperm binding to glycans diminishes oxidative phosphorylation and the citric acid cycle to lengthen sperm lifespan. Sperm bound to immobilized glycans will be examined to ascertain the specific metabolic changes that are induced and the intracellular signaling that leads to these changes. Aim 3. To determine if oviduct glycans select superior sperm for storage and in vitro fertilization. We will examine whether sperm selected by glycan adhesion have improved characteristics themselves and also produce embryos that more closely resemble in vivo-produced embryos by comprehensive analysis of embryo transcriptomes and methylomes. The completion of these Specific Aims will provide important keys to resolving how sperm bind to the oviduct, are stored in the oviduct, and are released in response to the COC. This fundamental information could be used to develop simpler, safer, and less costly assisted reproductive technologies.