Accumulation, Storage, and Release of Sperm in the Oviduct

项目摘要 生育力取决于成功的受精和早期发育，这些过程发生在输卵管中。共同 用于人类不育症的疗法，例如体外受精和胞浆内精子注射，是昂贵的， 增加了各种问题的风险。更多关于输卵管如何与精子、卵丘-卵母细胞相互作用的知识 复合体（COC）和发育中的胚胎可以提高生育能力并减少对治疗的需求。输卵管作为 在精子沉积后和受精前储存精子。与输卵管结合可维持精子活力， 抑制运动。精子被释放到输卵管上部（壶腹）使卵母细胞受精。存在不少差距 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。我们使用了一种糖分析剂 筛选377种聚糖的方法发现所有对猪精子具有亲和力的聚糖都具有两种基序之一， 硫酸化刘易斯X三糖或支链6-唾液酸化复合聚糖。值得注意的是，如果这些聚糖被固定 在珠子或显微镜载玻片上，它们可以延长精子的寿命，就像与输卵管细胞结合一样， 精子我们已经确定了精子膜上两种聚糖的两种候选受体，PKDREJ和ADAM 5。 值得注意的是，PKDREJ和其他亚当斯的小鼠精子不会在子宫输卵管连接处以外积累， 但不知道这是否是由于输卵管中的结合和保留问题。为了确定这些候选人 受体是精子与输卵管细胞结合和生育所必需的，我们已经生产出在这两个方面都有插入缺失的猪。 PKDREJ和ADAM 5。由NIH资助的国家猪研究和资源中心生产这些动物 在COVID-19大流行开始后， 由于NSRRC实验室和屠宰场关闭而推迟。我们终于在2021年9月/10月获得了动物。我们 接受了无成本扩展研究动物，但不幸的是，所有基因编辑的ADAM 5动物都是雌性， 需要额外的育种来产生纯合的基因编辑的雄性。从最初的RO 1中获得的资金现在 因此我们无法完成PKDREJ编辑的雄性的研究。这一行政补充的目标是 以保存这些独特的遗传资源供进一步研究。我最近提交了一份更新原始RO 1提案的申请， 包括对这些基因编辑动物的研究。具体目的是冷冻保存男性精液，胚胎 来自雌性的体细胞和来自在ADAMA 5和PKDREJ基因中具有插入缺失的所有动物的体细胞。这种保存下来的基因 材料可用于人工授精、胚胎移植和体细胞核移植以产生 用于RO 1更新研究的动物。

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. 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. 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. To determine if these candidate receptors are required for sperm binding to oviduct cells and for fertility, we have produced swine with indels in both PKDREJ and ADAM5. Production of these animals by the NIH-funded National Swine Research and Resource Center was supported by an administrative supplement that was funded just after the COVID-19 pandemic started so production was delayed due to NSRRC lab and abattoir shut-downs. We finally obtained the animals in Sept/Oct of 2021. We received a No Cost Extension to study the animals but unfortunately, all the gene-edited ADAM5 animals were female so additional breeding is required to produce homozygous gene-edited males. Funding from the original RO1 is now depleted so we are unable to complete the studies of PKDREJ-edited males. The goal of this administrative supplement is to preserve these unique genetic resources for further study. I recently submitted a renewal of the original RO1 proposal that will include studies of these gene-edited animals. The Specific Aim is to cryopreserve semen from males, embryos from females, and somatic cells from all animals with indels in ADAMA5 and PKDREJ genes. This preserved genetic material can be used for artificial insemination, for embryo transfer, and for somatic cell nuclear transfer to produce animals for study in the RO1 renewal.