1 Department of Dermatology and Allergy, 3 Institute for Medical Statistics and Epidemiology, Technical University Munich, and 2 Department of Anatomy, University of Munich, Germany
4 To whom correspondence should be addressed at: Department of Dermatology and Allergy, Technical University Munich, Biedersteiner Str. 29, D-80802 Munich, Germany. Email: weidinger{at}lrz.tum.de
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Abstract |
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Key words: ERK1/2/human spermatozoa/mast cell/motility/PAR-2/tryptase
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Introduction |
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The importance of MCsperm interactions for reproduction is indeed indicated by some clinical studies showing improved semen parameters and pregnancy rates using MC blockers such as ketotifen (Schill et al., 1986; Yamamoto et al., 1995
; Hibi et al., 2001
). Whether the reduced release of tryptase as an expected result of MC blocker treatment is the cause, remains to be shown. However, tryptase is thought to be the major MC product in all human MCs, including those found in the male and female genital tract, e.g. the uterus and Fallopian tubes (Yamanaka et al., 2000
; Sivridis et al., 2001
; see Weidinger et al., 2003
for additional details). Our previous studies also showed that both seminal plasma and follicular fluid contain tryptase (Weidinger et al., 2003
). Furthermore, with respect to the male genital organs, tryptase-containing MCs, which are present in testes of healthy men, are dramatically increased and activated in patients with spermatogenic arrest and Sertoli-cell-only-syndrome (Maseki et al., 1981
; Meineke et al., 2000
). Thus, based on these studies, interactions between tryptase and spermatozoa during their migration to the oocyte may be hypothesized.
In contrast to PAR-1 (see for example Vu et al., 1991), little is known about the intracellular signalling of PAR-2. In rat cardiomyocytes, activation of PAR-2 was shown to promote inositol trisphosphate accumulation, stimulate the mitogen-activated protein kinases (MAPK) ERK1/2 (extracellular signal regulated kinases 1/2) and elevate intracellular calcium concentration (Sabri et al., 2000
). Activation of PAR-2 has been shown further to cause tyrosine phosphorylation of cellular proteins in a pertussis toxin (PTX)-sensitive fashion and to increase early gene transcription through the activation of the c-fos promoter, also involving G proteins insensitive to PTX (Yu Z et al., 1997
). In human bronchial epithelial cells, activation of PAR-2 appears to stimulate a signalling cascade involving Ras, MAPK kinase (MEK) and ERK (Page et al., 2003
). Importantly, ERK1/2 in human spermatozoa have been suggested to contribute to the acquisition of sperm motility (Lu et al., 1999).
Since virtually nothing is known about the cellular pathways of PAR-2 signalling in human spermatozoa, we aimed to investigate second messenger systems involved in tryptase action, including the role of Ca2+, PTX-sensitive pathways and the MAPK ERK1/2.
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Materials and methods |
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Sperm preparation
Fresh semen was collected from healthy donors (n=30) who had no history of diseases related to infertility and who had given informed consent. Semen samples were allowed to liquefy at room temperature for 30 min; then they were analysed for standard semen parameters according to the World Health Organization (1999), including determination of pH, volume, total sperm count, sperm density, motility, morphology (Shorr stain), vitality (eosin Y stain) and peroxidase-positive leukocytes. Only ejaculates with <1 x 106 white blood cells/ml and motility (WHO a + b) > 50% or linear progressive motility (WHO a) >25% were used for the experiments. For experiments, samples were washed (500 g, 10 min) and resuspended in 300 µl of HTFM, and sperm concentration was adjusted to 12 x 107/ml.
Sperm motility (WHO a/b/c) was assessed by computer-assisted semen analysis (CASA) using the StroembergMika cell motion analyser (Version 4.4, Mika Medical GmbH, Rosenheim, Germany). Briefly, 5 µl aliquots of sperm suspensions were transferred into disposable counting chambers (10 µm depth). Measurements of motility parameters were performed at 36°C. The specimens were observed by a high resolution CCD-video camera and a microscope Optiphot-2 (Nikon Co, Tokyo, Japan). A minimum of 200 spermatozoa from at least four different fields was analysed, and motility of spermatozoa (% WHO a/b/c) was determined in each specimen. The parameter settings and thresholds were as detailed by Krause (1995). Sperm vitality was assessed using eosin staining. Motility results reported in the present study represent WHO a/b.
Role of calcium in tryptase action on spermatozoa
To examine whether tryptase effects depend on extracellular Ca2+, phosphate-buffered saline (PBS) alone and PBS supplemented with a defined Ca2+ concentration of 2 mmol/l were used instead of HTFM. Pilot studies performed in PBS ruled out that during the observation period of 30 min PBS negatively affected motility of spermatozoa (data not shown). After 10 and 30 min, sperm motility was determined by CASA.
Intracellular Ca2+ levels in human spermatozoa were measured to investigate possible effects of the PAR-2 activator tryptase (120 mIU/ml), and for control purposes also with the PAR-1 and PAR-2 activator trypsin (0.44 µmol/l; PAA, Cölbe, Germany). To this end, human ejaculated spermatozoa from various donors were pooled, washed with HTFM and subjected to centrifugation at 400 g for 10 min. After resuspending the pellet in 1 ml of extracellular buffer (ECB) containing 140 mmol/l NaCl, 3 mmol/l KCl, 1 mmol/l CaCl2, 10 mmol/l HEPES, 10 mmol/l glucose, pH 7.4, cells were loaded with 5 µmol/l fluo 4-AM (Molecular Probes, Eugene, OR) for 30 min at 37°C, centrifuged for 10 min at 400 g and resuspended in 1 ml of ECB. All centrifugation steps were carried out at room temperature. Approximately 2 x 107 loaded and non-loaded (control) spermatoza were transferred into the wells of a 24-well plate in a total volume of 1 ml of ECB per well. Vitality and motility of the cells were checked with an Axiovert 135 microscope (Zeiss, Jena, Germany). Intracellular Ca2+ concentrations, [Ca2+]i, were monitored at 32°C by means of a fluorescence plate reader (BMG Labtechnologies GmbH, Offenburg, Germany). To this end, fluo-4 was excited at 485 nm and fluorescence was detected at 520 nm. Autofluorescence of unloaded spermatozoa was subtracted from all fluorescence values. The corrected fluorescence intensities were then normalized for each well. Stimulants were applied using the built-in pump system of the plate reader. In order to test for capability of our experimental set-up, we raised [Ca2+]i by application of the Ca2+ ionophore 4-bromo-A23187 (4 µmol/l; from a stock solution of 10 mmol/l in DMSO; EMD Biosciences, Inc., San Diego, CA).
Effects of tryptase on MAPK ERK1/2 signalling in spermatozoa
In order to examine involvement of the ERK1/2 MAPK pathway in tryptase action on sperm motility, washed spermatozoa were incubated with 105 mol/l PD98059 diluted in HTFM for 10 min before 120 mIU/ml tryptase was added. Sperm motility was determined after 10 and 30 min using CASA. Untreated spermatozoa and samples treated with tryptase only were measured as controls. Immunoblotting experiments were performed to investigate ERK1/2 phosphorylation. Therefore, spermatozoa were incubated for 5, 10, 20 and 30 min in the presence or absence (0 min) of tryptase alone, tryptase plus 105 mol/l PD98059 or equivalent concentrations of DMSO in controls. Immunoblots were performed as described (Frungieri et al., 2002) by using a monoclonal mouse anti-human phospho-ERK 1/2 antibody (1:500; Cell Signaling Technology, New England Biolabs GmbH, Frankfurt am Main, Germany), and a polyclonal rabbit anti-human ERK 1/2 antiserum (1:1000; Cell Signalling Technology).
Effect of pertussis toxin (PTX) on tryptase action on sperm motility
Since in some cells PAR-2 were shown to act on a PTX-sensitive pathway, the effects of PTX on the impairment of sperm motility by tryptase were investigated. Therefore, washed spermatozoa were incubated with 100 ng/ml PTX diluted in HTFM for 10 min before tryptase was added. Sperm motility was determined after 10 and 30 min. Untreated spermatozoa and samples treated with tryptase only were measured as controls.
Statistical analyses
Continuous variables are summarized as mean±SD. For graphical visualization, only upper limits are drawn to indicate the limits of uncertainty. The Friedman test was applied to test the significance between the experimental scenarios. In case of significance, the post hoc MannWhitney U-test was performed. All tests are two-tailed, and P-values <5% indicate statistical significance. Analyses were performed using SPSS version 11.5 for Windows.
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Results |
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Discussion |
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PARs are a family of transmembrane-spanning domain G-protein-coupled receptors consisting of four members named PAR-1, PAR-2, PAR-3 and PAR-4 (Dery et al., 1998). The physiological activator of PAR-1, PAR-3 and PAR-4 is thrombin, whereas PAR-2 is activated by trypsin-like enzymes and the MC protease tryptase, but not by thrombin (MacFarlane et al., 2001
).
Signalling mechanisms after activation of PARs are partially known. Concerning PAR-2 signalling, MAPK may be involved, as shown in rat aortic smooth muscle cells and bovine pulmonary arterial fibroblasts (Belham et al., 1996). In line with these results, we found that activation of PAR-2 on human spermatozoa by tryptase induces phosphorylation of ERK1/2. This increase in phosphorylation of ERK1/2 activity seems to be related to the decrease in sperm motility. This can be concluded because tryptase-mediated inhibition of sperm motility and phosphorylation of ERK1/2 were completely blocked in the presence of the MAPK inhibitor PD98059.
Preliminary experiments showed no effects on progesterone and calcium ionophore A23187-induced human sperm acrosome reaction, when the spermatozoa had been capacitated in the presence of tryptase (data not shown). Therefore, an association with altered sperm capacitation (Luconi et al., 1998; de Lamirande et al., 2001
) seems unlikely.
Studies of signal transduction events following activation of PAR-1, and to a lesser extent PAR-2, have so far been conducted in several other cell types, including enterocytes, keratinocytes and transfected cells (Dery et al., 1998). Depending on the receptor and the cell type, two major pathways were identified: a PTX-insensitive, Ras-independent pathway leading to inositol trisphosphate formation (Hawes et al., 1995
; Hershenson et al., 1995
), and a PTX-sensitive, Ras-dependent pathway linked to cytosolic tyrosine kinases including Src (Alblas et al., 1993
; Winitz et al., 1993
). In our present study, pre-treatment of spermatozoa with PTX failed to inhibit the decrease of motility caused by tryptase, indicating a PTX-insensitive signalling pathway in human spermatozoa.
Recently, epidermal growth factor receptor (EGFR) transactivation by tyrosine phosphorylation of EGFR family members in a PTX-insensitive pathway has been suggested as a major mechanism of PAR-2 signalling, whereby ERK1/2 are stimulated after activation of PAR-1 and PAR-2 (Sabri et al., 2002; Darmoul et al., 2004
). EGFR is present on ejaculated human spermatozoa and shows an increased expression in infertile patients (Damjanov et al., 1993
; Foresta and Varotto, 1994
). More extensive studies are needed to clarify the detailed molecular mechanisms of the specific regulation of sperm motility by the tryptasePAR-2 interaction and a possible involvement of EGFR.
Activation of PAR-1 (Jeng et al., 2004) and of PAR-2 has been shown to mobilize intracellular Ca2+ and to stimulate Ca2+ influx from extracellular fluid in kidney and intestinal epithelial cells (Bohm et al., 1996
). However, in our present study, neither tryptase (PAR-2 agonist) nor trypsin (a PAR-1 and PAR-2 agonist used for additional control purposes) influenced intracellular Ca2+ levels, and equally strong inhibitory effects of tryptase on sperm motility were observed in the presence and absence of extracellular calcium. These observations indicate that tryptase action of PAR-2 on spermatozoa is not mediated by extracellular or intracellular Ca2+.
Tryptase is a major MC product, but its possible role in reproduction is far from being understood. Nevertheless, several studies showed the involvement of MCs in the pathophysiology of reproduction at different levels (Schill et al., 1986; Yamamoto et al., 1995
; Hibi et al., 2001
; Meineke et al., 2000
; Frungieri et al., 2002
). Our previous (Weidinger et al., 2003
) and present findings clearly show that the major MC product tryptase, via PAR-2 and ERK1/2 MAPK, significantly blocks human sperm motility. We propose that further insights into the mechanisms of tryptase-induced PAR-2 signalling in human spermatozoa may foster novel approaches for the treatment of infertility or contraceptive strategies.
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Acknowledgements |
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References |
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Submitted on June 28, 2004; resubmitted on September 23, 2004; accepted on October 22, 2004.
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