Extrusion of Neutrophil Extracellular Traps (NETs) Negatively Impacts Canine Sperm Functions: Implications in Reproductive Failure

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.

Keywords: PMNs; canine; neutrophil extracellular traps (NETs); sperm functionality; spermatozoon.

Figure 1

Characterization of dog semen samples. The chart in (A) shows the score determined for the presence of PMNs in each sperm sample, with 1 being scarce, 2 moderate, and 3 abundant. The table in (B) shows the results of the quality parameters of the semen sample. The images in (C) are representative of fluorescence microscopy and light microscopy observed with the 20× and 100× objective, respectively, showing PMNs infiltration and NET production (scale bars = 20 μm).

Figure 2

Representative immunofluorescence images show PMNs releasing NETs, and entrapped sperm present in dog semen samples by immunofluorescence. (A,B,C.3) MERGE indicates the co-location of both channels (DAPI and Alexa Fluor™ 488). (C.1) DAPI marker with blue fluorescence indicates nuclear sperm DNA and PMNs. (C.2) Alexa Fluor™ 488 marker with green fluorescence indicates NE from released NETs. (D–F) contains representative images of the May–Grünwald Giemsa stain, where the orange arrows indicate PMNs present in the samples and the blue arrows indicate sperm (scale bars = 20 μm).

Figure 3

Representative immunofluorescence images of dog PMN/sperm co-cultures. (A–C) show the colocalization of the DAPI and Sytox Orange markers and different phenotypes of NETs. Orange arrows indicate PMNs, blue arrows indicate sperm, white arrows indicate aggNETs, yellow arrows indicate sprNETs, and red arrows indicate diffNETs ((A,C), scale bar = 50 μm, (B), scale bars = 20 μm).

Figure 4

Representative images of the different NET phenotypes (aggNETs, sprNETs, and diffNETs) from PMNs stimulated with dog sperm. (A,D,G) The white arrow in (G) indicates the structure of aggNETs. In (B,E,H), the red arrows in (H) indicate diffNETs, and in (C,F,I), the yellow arrow in (I) indicates sprNETs. DNA can be observed with orange fluorescence in (A–C) (Sytox© Orange), and the presence of NE with green fluorescence (Alexa Fluor 488) in (D–I) shows co-location of both channels of extracellular structures attributable to NETs (scale bars = 20 μm).

Figure 5

Representative image of nuclear area expansion (NAE) of PMNs isolated from peripheral blood exposed to dog sperm. (A) NAE−based quantification of sperm-activated PMNs at 15 and 120 min of exposure with their respective controls. The letters (B–E) represent scatter plots of PMNs nuclear segmentation. p < 0.01, **, p < 0.0001, ***.

Figure 6

Effect of different concentrations of NET components on the cell membrane integrity of dog sperm. (A) Myeloperoxidase (MPO); (B) Cathepsin G (Cat G); (C) Histone 2 A (H2A), (D) Neutrophil elastase (NE), and (E) Cathelicidin (LL-37). The results are shown as mean ± standard deviation. The asterisks on the bar indicate differences in the three biological replicates compared to the control for MPO p < 0.02, *** (A); Cat-G p < 0.0001 *** (B), NE p < 0.05, * (D).

Figure 7

Effect of different concentrations of NET components on the state of the acrosome in dog sperm. (A) Myeloperoxidase (MPO); (B) Cathepsin G (Cat G); (C) Histone 2A (H2A), (D) Neutrophil elastase (NE), and (E) Cathelicidin (LL-37). The results are shown as mean ± standard deviation. The asterisks on the bar indicate differences in the three biological replicates compared to the control. For MPO p < 0.02, * (A); Cat-G p < 0.01 ** (B).