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To study the mechanism leading to elevated semen cytokines in men with spinal cord injury (SCI) and to understand if inflammasome pathways are involved in this process. To investigate inflammasome components and end-product cytokines in semen of SCI and control subjects.
Major university medical center.
Men with and without SCI (n = 28 per group).
Main Outcome Measure(s)
Seminal plasma concentrations of caspase-1, interleukin (IL) 1β, and IL-18 were quantified by ELISA. Caspase-1 in sperm fractions and apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) in seminal plasma and sperm fractions were identified by Western blot. Localization of proteins in sperm was accomplished by immunocytochemistry.
ASC, caspase-1, IL-1β, and IL-18 concentrations were elevated in the seminal plasma of SCI subjects compared with control subjects. ASC and caspase-1 were elevated in sperm cells of SCI subjects. Immunocytochemistry revealed that ASC was located in the acrosome, equatorial segment, and midpiece, and caspase-1 in the midpiece.
This study provides the first evidence of ASC in human semen and demonstrates the involvement of inflammasome proteins in semen of men with SCI. These findings suggest an immunologic basis for abnormal semen quality in men with SCI.
). We hypothesized that this effect was due to elevated semen concentrations of specific inflammatory cytokines (interleukin [IL] 1β, IL-6, and tumor necrosis factor α), the neutralization of which led to improved sperm motility (
). The most common inflammasomes consist of a nucleotide oligomerization domain–like receptor (NLR), the adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1. The activation of the inflammasome results in cleavage of procaspase-1 into active caspase-1, which then cleaves pro–IL-1β and pro–IL-18 into their active forms. IL-1β and IL-18 are potent proinflammatory cytokines that play a major role in innate immunity and the body’s response to tissue injury, including lymphocyte activation, recruitment of other inflammatory cells and their products and cytokines, and induction of secondary inflammatory cytokines and other cellular products in T cells and natural killer cells (
). The inflammasome complex itself may be activated by a wide variety of causes, such as bacteria, fungi, yeasts, viruses and many of their products, cell wall components, toxins, nucleic acids, foreign compounds, such as asbestos, particles, such as urates, and other things, such as lipopolysaccharides and beta-amyloid.
As a first step toward determining if the inflammasome contributes to elevated cytokines in semen of men with SCI, the present study investigated whether components of the inflammasome were present in the semen of men with SCI as well as in noninjured healthy men as control subjects.
Materials and methods
Semen samples from 28 men with SCI and 28 age-matched control subjects were examined in this study. Control subjects were healthy volunteers who were specifically recruited for this study. All control subjects were noninjured normospermic men with no known history of infertility. The mean age (±SEM) of SCI subjects was 35.0 ± 1.6 years (range 21.0–55.0 years) and 31.0 ± 1.6 years (range 20.0–50.0 years) for the control subjects. All SCI subjects were past the period of spinal shock (i.e., ≥12 months after injury). Their level of injury ranged from C4 to T8. All SCI and control subjects were in good general health and were participants in the Male Fertility Research Program of the Miami Project to Cure Paralysis, University of Miami Miller School of Medicine. The study was approved by the University of Miami Institutional Review Board, and informed consent was obtained from each of the subjects.
Semen was obtained from SCI subjects using the standard methods of penile vibratory stimulation (PVS) or electroejaculation (EEJ) as previously described (
). Only antegrade semen specimens were used in this study. Non-SCI control subjects collected their semen by masturbation. Semen was analyzed for sperm concentration and sperm motility according to World Health Organization criteria (
Each semen sample was divided into two portions: one for gradient separation of sperm, and the other for recovering seminal plasma. Seminal plasma was obtained by centrifugation of an aliquot of semen at 1,000g for 15 minutes at room temperature. The seminal plasma (i.e., the supernatant) was collected and stored at −80°C.
Purification of Sperm with Discontinuous-Gradient Separation
Semen specimens were processed with the use of a discontinuous gradient (Allgrad; Life Global) as follows: 2 mL Allgrad 90% was placed in a 15-mL centrifuge tube then another 2 mL Allgrad 45% was added. The liquefied semen (0.5–2.0 mL) was carefully added to the top of the 45% layer and centrifuged at 300g for 20 minutes. The resulting pellet was recovered, washed, and resuspended in 0.5 mL of Allgrad Wash. This fraction was referred to as the pure sperm fraction (F1). The supernatant from gradient was collected, washed with Allgrad wash, centrifuged at 500g, and the resulting pellet resuspended in 0.5 mL Allgrad Wash. This fraction was referred to as the mixed cell fraction (F2). An aliquot of the pure sperm fraction (F1) was stained with ortho-toluidine reagents and examined microscopically for leukocytes (
). Specimens containing traces of leukocytes were labeled F1WBC+ and those free of leukocytes were labeled F1WBC−. Sperm cells from all three fractions (F1WBC+, F1WBC−, and F2) were centrifuged to a pellet, washed with phosphate-buffered saline solution (PBS), and resuspended in PTN50 lysis buffer (50 mmol/L sodium phosphate buffer, pH 7.4, containing 50 mmol/L NaCl and 1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, and 1% protease inhibitor cocktail; Sigma-Aldrich). Specimens were then subjected to freezing and thawing twice in dry ice. Protein was recovered by centrifugation at 5,500g for 5 minutes; protein concentration was determined with the use of bicinchoninic acid protein assay reagents (Thermo Scientific).
Western Blot Analysis
Seminal plasma samples were retrieved from storage at −80°C and thawed at room temperature. Seminal plasma (1 μL) from each subject was mixed with loading buffer (to a final concentration of 50 mmol/L Tris-HCl, pH 7.0, containing 2% sodium dodecyl sulfate [SDS], 10% glycerol, 5% β-mercaptoethanol, and 0.002% bromphenol blue), heated at 95°C for 10 minutes, and subjected to SDS-polyacrylamide gel electrophoresis (Mini-protein GTX 4%–20%; Biorad). An ASC-positive sample was included in each gel to serve as a positive standard. The gel was blotted to an immune-blot polyvinylidene difluoride membrane (Biorad). The membrane was blocked with 0.4% I-Block (Applied Biosystem) in PBS (pH 7.4) containing 0.1% Tween-20 and incubated with anti-ASC antibody (polyclonal; Protein Tech Group) followed by goat anti–rabbit IgG horseradish peroxidase (HRP)–conjugated secondary antibody (Cell Signaling). The blot was treated with Lumiglo chemiluminescent substrate (Cell Signaling) and exposed to x-ray film. Quantification of band density was performed with the National Institutes of Health ImageJ software, and the intensity of the ASC-specific band from each specimen was normalized to the intensity of the ASC-positive standard on the same gel and presented as relative intensity.
Sperm cell extracts were mixed with loading buffer and analyzed with the use of anti-ASC, anti–caspase-1 (monoclonal; Imgenex), and anti-GAPDH (polyclonal; Sigma) as primary antibodies and followed by HRP-conjugated secondary antibody. The intensity of the band of interest was normalized to that of GAPDH and presented as the relative intensity. Precision Plus Protein Standards (Biorad) were used as the protein molecular weight markers.
An aliquot of gradient-purified sperm was resuspended in Allgrad Wash and incubated with Mitotracker Green (Molecular Probes; Invitrogen) at a final concentration of 1 μmol/L at room temperature for 90 minutes. Sperm smears were prepared. Slides were fixed with 2% paraformaldehyde for 20 minutes and washed with PBS and incubated with permeabilization/blocking solution (PBS containing 5% normal goat serum and 0.2% Triton X-100) at 25°C for 30 minutes. Slides were incubated with primary antibody diluted in PBS containing 5% goat serum and 0.1% Triton X-100 at room temperature for 1 hour and then stored overnight at 4°C. Primary antibodies used were polyclonal rabbit anti-ASC (Anaspec) at 1:40 dilution, or anti–caspase-1 (1:100 dilution; Imgenex). Secondary antibodies were Alexa Fluor 594 goat anti–rabbit (or –mouse) IgG (Molecular Probes; Invitrogen) at 1:200 dilution. Slides were covered with mounting medium Prolong Anti-fade with 4′,6-diamidino-2-phenylindole (Molecular Probes; Invitrogen) and coverslips. Images were taken with an Olympus Fluoview FV1000 laser scanning confocal microscope. Rabbit or mouse IgG was used to replace primary antibodies to serve as negative control samples.
Caspase-1, IL-1β, and IL-18 were measured in seminal plasma samples by ELISA as described by the manufacturer (R&D Systems). Seminal plasma samples were retrieved from storage at −80°C and thawed at room temperature. The samples were then placed in microtitration plates precoated with a monoclonal antibody specific for IL-1β, IL-18, or caspase-1. Each sample was assayed in duplicate.
Graphpad Prism 5.0 was used for statistical analysis of the data, including calculation of means, standard errors of means, medians, interquartile intervals, P values, correlation, and linear regressions. The results were expressed as mean ± SEM for parametric variables (subject’s age) and analyzed by Student t test. Data with unpaired nonparametric variables (motility and sperm concentration) were expressed as median (interquartile range) and analyzed by Mann-Whitney test.
Sperm Motility Is Lower in SCI Subjects Compared with Control Subjects
The median (interquartile range) sperm concentration in the SCI group (93 [55–134] million/mL) was not statistically significant compared with the control group (98 [75–135] million/mL; P=.5294). In contrast, the median sperm motility in the SCI group was significantly lower than the control group (35% [23%–47%] vs. 61% [54%–71%]; P<.0001).
Inflammatory Cytokines IL-1β and IL-18 Are Elevated in Seminal Plasma of SCI Subjects
To determine the levels of inflammatory cytokines in seminal plasma, we measured IL-1β and IL-18 concentrations in SCI and control subjects with the use of ELISA. Seminal plasma concentrations of IL-1β and IL-18 were significantly higher in SCI versus control subjects (97.9 ± 21.5 vs. 14.9 ± 4.4 pg/mL [P<.001] and 2,129.0 ± 805.8 vs. 68.7 ± 20.5 pg/mL [P<.01], respectively; Supplemental Fig. 1, available online at www.fertstert.org).
Inflammasome Proteins Caspase-1 and ASC Are Elevated in Seminal Plasma of SCI Subjects
To establish whether inflammasome proteins were present in seminal plasma and whether men with SCI had altered levels of these proteins, we performed analysis of seminal plasma of SCI and control subjects. Caspase-1 concentrations were significantly elevated in the seminal plasma of SCI subjects (1,104.0 ± 204.6 pg/mL) compared with control subjects (183.8 ± 43.6 pg/mL; P<.0001; Fig. 1A). In addition, by using Western blot analysis, levels of the inflammasome adaptor protein ASC were also found to be significantly elevated in seminal plasma samples from SCI versus control subjects (Fig. 1B). The mean ± SEM relative intensity of ASC was 1.045 ± 0.159 in SCI subjects and 0.115 ± 0.033 in control subjects. The difference was statistically significant (P<.0001).
Caspase-1 Is Elevated in Sperm Fractions of SCI Subjects
The elevated ASC and/or caspase-1 levels in the seminal plasma of SCI subjects may arise from leukocytes that are often present in the semen of men with SCI. We therefore fractionated semen with the use of discontinuous-gradient separation to remove leukocytes from the semen. Cell extracts were prepared from sperm fractions and analyzed by Western blot analysis for levels of procaspase-1 and caspase-1. Increased levels of procaspase-1 (45 kD) were found in purified sperm fractions (F1) of SCI subjects (Fig. 2). After microscopic examination, leukocytes were not detected in 15 of 28 F1 fractions from SCI subjects and 27 of 28 F1 fractions from control subjects. The levels of procaspase-1 in these F1WBC− fractions are shown in Figure 2. The mean ± SEM relative intensity was 0.626 ± 0.201 in SCI subjects and 0.026 ± 0.005 in control subjects (P<.001; Fig. 2, bottom left). A similar elevation in procaspase-1 was present in mixed cell fractions (F2) of SCI subjects (mean relative intensity 0.746 ± 0.082) versus control subjects (0.216 ± 0.061; P<.0001; Fig. 2, bottom center). However, F2 fractions also contained increased levels of active caspase-1 (26 kD) in SCI subjects (mean relative intensity 0.530 ± 0.069) versus control subjects (0.132 ± 0.063; P<.0001; Fig. 2, bottom right).
ASC Is Expressed in Sperm Fractions
Next, we analyzed whether sperm also produced ASC after SCI. As shown in Figure 3, ASC levels were significantly elevated after SCI. The relative intensity of ASC was quantified (Fig. 3A). In the F1WBC− fraction, the mean ± SEM relative intensity of ASC for the SCI group was 0.256 ± 0.067 and 0.025 ± 0.007 for the control group (Fig. 3A). The intensity of ASC was lower in F1 fractions versus F2 fractions of the same subjects, indicating the contribution of leukocytes to ASC expression. In F2 fractions, the mean ± SEM for the SCI group was 0.755 ± 0.046 versus 0.199 ± 0.045 for the control group (P<.0001; Fig. 3B).
Localization of Inflammasome Proteins in Sperm Cells
To visualize inflammasome proteins in sperm cells, we performed immunohistochemistry followed by confocal microscopy. As shown in Figure 4, caspase-1 staining was present in the midpiece section of sperm cells. A different pattern of staining was observed for ASC than for caspase-1. ASC was found in more than one location in the sperm cell: the acrosome, the equatorial segment region, and the midpiece. ASC was positively identified in specimens from SCI subjects but not from control subjects. Thus, inflammasome proteins are present in sperm and are localized in discrete areas of the sperm cell.
Linear regression analysis showed a positive correlation between the levels of ASC and caspase-1 in the seminal plasma (n = 56; r2 = 0.49; P<.0001; Supplemental Fig. 2A [available online at www.fertstert.org]) and between the levels of ASC and procaspase-1 in the F1WBC− fractions (n = 42; r2 = 0.44; P<.0001; Supplemental Fig. 2B) and F2 fractions (n = 56; r2 = 0.29; P<.0001; Supplemental Fig. 2C). There was a negative correlation between sperm motility and the levels of ASC in the seminal plasma (n = 56; r2 = 0.32; P<.0001; Supplemental Fig. 2D).
The semen of most men with SCI contains normal sperm concentrations but abnormally low sperm motility. Elevated concentrations of inflammatory cytokines in the semen are detected in this unusual semen profile (
). Whether the inflammasome signaling mechanism is active in semen of men with SCI, however, had not been previously tested. The present study shows, for the first time, that the inflammasome components ASC and caspase-1 are present in semen of men with SCI and that increased levels of these proteins are correlated with increased expression of the proinflammatory cytokines IL-1β and IL-18.
Our results show that ASC and caspase-1 levels in semen of SCI subjects are significantly elevated compared with control subjects, with levels 9.1- and 6-fold higher for ASC and caspase-1, respectively. Additionally, concentrations of IL-1β and IL-18 were 6.6- and 31.0-fold higher, respectively, in SCI versus control subjects. One of the most pronounced abnormalities in men with SCI is leukocytospermia. In the examination of sperm samples almost free of leukocytes, high levels of ASC expression were found in 53.3% of the samples, whereas only one control subject (3.7%) showed slightly elevated ASC expression. Moreover, the average relative intensity of ASC in this group was 10-fold higher in SCI versus control subjects. Similarly, in this group, elevated expression of procaspase-1 was observed in 60% of the SCI subjects versus none of the control subjects. In the F2 fraction (the fraction of mixed sperm and leukocytes), ASC expression was 3.8-fold higher and procaspase-1 expression 3.5-fold higher in SCI versus control subjects. These results suggest that high levels of ASC and caspase-1 activity in the semen of men with SCI arises from both leukocytes and sperm.
Immunocytochemical analysis confirmed the expression of both ASC and caspase-1 in sperm. ASC-positive signals were found mainly in the midpiece, with no positive signals found in purified sperm fractions of control subjects. Caspase-1 signals were observed in the midpiece section of sperm cells. A positive association was found between the levels of ASC and caspase-1 in the seminal plasma, and a negative association was found between these proteins and sperm motility.
The findings of our study suggest a role of the inflammasome complex in abnormal semen quality in men with SCI. The detailed composition of the inflammasome has not been elucidated, nor have specific “triggers” to the activation of the inflammasome been described. A further study is underway to determine if antiinflammasome therapies, such as antibody neutralization of ASC, might present a new approach to improve sperm motility and consequently improve fertility in this patient population.
In conclusion, this study provides the first evidence of ASC in human sperm. This study further demonstrates the involvement of inflammasome proteins caspase-1 and ASC in semen of men with SCI. Increased levels of inflammasome complex proteins and end-product cytokines were found in semen of men with SCI versus non-SCI healthy men. These findings provide further evidence to suggest an activation of innate immunity for abnormal semen quality in men with SCI.
All of the authors receive grant support from the Craig H. Neilsen Foundation . X.Z. has nothing else to disclose. E.I. has nothing else to disclose. J.P.d.R.V. has patents pending on regulation of innate immune responses. G.L. has nothing else to disclose. T.C.A. has nothing else to disclose. W.D.D. has received National Institutes of Health and Department of Defense grants. R.W.K. has patents pending and has received a National Institutes of Health grant. C.M.L. has nothing to disclose. N.L.B. receives travel support from the Craig H. Neilsen Foundation.
Supported by the Craig H. Neilsen Foundation (grant no. 124464 ).