PDF(Pubmed)
Abstract：
Reproductive failure in dogs is often due to unknown causes, and correct diagnosis and treatment are not always achieved. This condition is associated with various congenital and acquired etiologies that develop inflammatory processes, causing an increase in the number of leukocytes within the female reproductive tract (FRT). An encounter between polymorphonuclear neutrophils (PMNs) and infectious agents or inflammation in the FRT could trigger neutrophil extracellular traps (NETs), which are associated with significantly decreased motility and damage to sperm functional parameters in other species, including humans. This study describes the interaction between canine PMNs and spermatozoa and characterizes the release of NETs, in addition to evaluating the consequences of these structures on canine sperm function. To identify and visualize NETs, May-Grünwald Giemsa staining and immunofluorescence for neutrophil elastase (NE) were performed on canine semen samples and sperm/PMN co-cultures. Sperm viability was assessed using SYBR/PI and acrosome integrity was assessed using PNA-FITC/PI by flow cytometry. The results demonstrate NETs release in native semen samples and PMN/sperm co-cultures. In addition, NETs negatively affect canine sperm function parameters. This is the first report on the ability of NETs to efficiently entrap canine spermatozoa, and to provide additional data on the adverse effects of NETs on male gametes. Therefore, NETs formation should be considered in future studies of canine reproductive failure, as these extracellular fibers and NET-derived pro-inflammatory capacities will impede proper oocyte fertilization and embryo implantation. These data will serve as a basis to explain certain reproductive failures of dogs and provide new information about triggers and molecules involved in adverse effects of NETosis for domestic pet animals.
摘要：
狗的生殖失败通常是由于未知的原因。正确的诊断和治疗并不总是能实现。这种情况与发展炎症过程的各种先天性和获得性病因有关，导致女性生殖道（FRT）内白细胞数量增加。在FRT中，多形核中性粒细胞（PMN）与感染因子或炎症之间的相遇可能会触发中性粒细胞胞外陷阱（NET），这与其他物种的精子活力和精子功能参数的显著下降有关，包括人类。这项研究描述了犬PMNs和精子之间的相互作用，并描述了NETs的释放，除了评估这些结构对犬精子功能的影响。为了识别和可视化NET，对犬精液样品和精子/PMN共培养物进行了May-GrünwaldGiemsa染色和中性粒细胞弹性蛋白酶（NE）的免疫荧光。使用SYBR/PI评估精子活力,并通过流式细胞术使用PNA-FITC/PI评估顶体完整性。结果显示天然精液样品和PMN/精子共培养物中的NETs释放。此外,NET对犬精子功能参数产生负面影响。这是关于NETs有效捕获犬精子能力的第一份报告，并提供有关NETs对雄性配子的不利影响的其他数据。因此,在未来的犬生殖衰竭研究中应考虑NETs的形成，因为这些细胞外纤维和NET衍生的促炎能力将阻碍正常的卵母细胞受精和胚胎植入。这些数据将作为解释狗的某些繁殖失败的基础，并提供有关NETosis对家养宠物的不利影响的触发因素和分子的新信息。
