Department of Urology, University Hospital Charité, Humboldt University, Schumannstr. 20/21, D 10117 Berlin, Germany
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Abstract |
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Key words: male fertility/MMP/seminal plasma/TIMP/zymography
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Introduction |
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To date five subgroups, the collagenases, gelatinases, stromelysins, matrilysins and membrane type MMPs are known. Common substrates of the gelatinases MMP-2 and MMP-9 are type I, IV, V, VII and X collagens, elastin, fibronectin and tumour necrosis factor-. Their activity is co-regulated and inhibited by the tissue inhibitors of metalloproteinases (TIMP) (Woessner, 1994
).
MMPs have been linked extensively with female reproductive function (Hulboy et al., 1997). During menstruation, their increased expression in the endometrium of the uterine wall (Salamonsen and Woolley, 1996
; Xu et al., 2000
) is believed to contribute to the sloughing process. MMP-2 expression and activity increase at the site of the rupturing follicle in the ovary (Russell et al., 1995
). Human trophoblasts utilize MMP-2 and MMP-9 in the invasion of the uterine stroma (Hulboy et al., 1997
).
However, very little is known about the expression and function of MMPs and their inhibitors in the male reproductive tract. MMP-2 as well as TIMP-1 and -2 (Ulisse et al., 1994) have been detected in rat Sertoli cell cultures. TIMP-2 extracted from bovine seminal plasma binding to sperm was described and its influence on bull fertility was postulated (McCauley et al., 2001
). A significant portion of MMPs such as MMP-2 and MMP-9 seem to come from accessory sex glands such as the prostate and the seminal vesicles, as demonstrated by split ejaculate analysis (Yin et al., 1990
). This is supported by the findings of two other groups who detected MMP-2 gelatinolytic activity in prostatic secretions of benign hyperplastic tissue (Lokeshwar et al., 1993
; Wilson et al., 1993
).
Parallel to our investigations, the occurrence of MMP-2 and MMP-9 activity in human seminal plasma has been recently reported (Shimokawa et al., 2002). However, no quantitative data have been described until now using robust assays such as ELISAs.
The purpose of this study was: (i) to quantitate MMP-2 and MMP-9, as well as their inhibitors TIMP-1 and TIMP-2, in human seminal plasma; and (ii) to examine the relationship between their concentrations and the sperm count and other characteristics of sperm.
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Material and methods |
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Retrieval, analysis and classification of the ejaculates were performed according to World Health Organization recommendations (World Health Organization, 1993). Briefly, samples were obtained by masturbation into a sterile plastic tube after 45 days of abstinence. Following physical examination of the ejaculate (pH, volume, consistency, motility, morphology etc.), samples were centrifuged at 3000 g and the seminal plasma was stored at 80°C until analysis.
To investigate whether the release of MMPs from sperm was dependent on time after sampling, semen specimens from five patients were divided into five aliquots of 0.3 ml, stored at room temperature (23°C) and centrifuged after 1, 2, 4, 8 and 24 h after sampling. The supernatants were immediately frozen at 80°C for subsequent use.
To characterize the MMP-2 and -9 patterns in sperm, pelleted sperm were suspended in 0.25% Triton X-100 solution after the final centrifugation and an ultrasonic disintegration was performed. Prior to this, the pelleted sperm were washed three times with 5 ml phosphate-buffered saline to avoid any influence of seminal plasma remnants. The homogenate was then centrifuged (13 000 g for 15 min) and the supernatant was used for further investigations.
Assays
The quantification of MMP-2, MMP-9, TIMP-1 and TIMP-2 was performed using ELISAs (ELISAs for MMP-2 and MMP-9 from Oncogene, Boston, USA; TIMP-1 and TIMP-2 from Amersham Int., Little Chalfont, UK).
All measurements were performed in duplicate according to the manufacturers instructions. Briefly, seminal plasma in a 1:5 to 1:51 dilution was placed into microwells equipped with biotinylated monoclonal anti-human MMP-2 antibody and incubated for 2 h. After washing, horseradish peroxidase was added to catalyse the conversion of chromogenic tetramethylbenzidine into a blue solution. Following the addition of 2.5 mol/l sulphuric acid as a stopping reagent, the absorbance was determined using the microplate reader Anthos HTIII (Anthos Labtec Instruments, Salzburg, Austria) with the four-parameter method for calculation of concentrations (Mikrowin software, version 3.0; Mikrotek Laborsysteme, Overath, Germany).
Zymography for MMP-2 and -9 was performed in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE; 8%) containing 0.5% gelatin on the Midget Electrophoresis Unit 2050 (LKB, Uppsala, Sweden). Samples of seminal plasma and sperm extracts were mixed with non-reducing sample buffer and subjected to electrophoretic analysis without boiling. Purified human proMMP-2 and proMMP-9 (Chemicon Inc., Temecula, USA) were included in each gel run as standards. After electrophoresis, gels were soaked for 2 h in 2.5% Triton-X 100 solution with four washing steps. The gels were then incubated for 18 h at 37°C in buffer containing 50 mmol/l TrisHCl, pH 7.6, 150 mmol/l NaCl, 10 mmol/l CaCl2, 0.2% Brij-35 and 0.02% NaN3. After incubation, the gels were stained with 0.2% Coomassie Blue and destained until clear proteolytic bands appeared. Gels were scanned using a flatbed scanner (Scanmaker 4; Microtek Lab, Redondo Beach, USA). Studies with inhibitors in the incubation solution (10 mmol/l EDTA or 1 mmol/l o-phenanthroline) verified the specificity of the method. Preincubation of samples with 1 mmol/l p-amino-phenyl-mercuric acetate (18 h, 37 °C) as an in-vitro MMP activator was used to differentiate between latent and active forms of MMPs (Crabbe et al., 1993). Controls were performed without activator but with a corresponding volume of buffer solution instead of p-amino-phenylmercuric acetate.
Statistical analysis
Data were analysed using the statistical softwares SPSS 10.0 for Windows (SPSS, Chicago, USA) and GraphPad Prism 3.02 (GraphPad Software, San Diego, USA). Students t-test, analysis of variance, linear regressions and correlation analysis using the correlation coefficient according to Pearson (r) were performed. P < 0.05 was considered significant.
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Results |
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To further characterize the MMP pattern in seminal plasma, samples were assessed with SDSPAGE (Figure 3). Figure 3A
shows a major band at 72 kDa representing proMMP-2 and a faint band at 92 kDa that is equivalent to proMMP-9. The MMP-2 and MMP-9 patterns are similar in seminal plasma and sperm (Figure 3B
). The minor band at 62 kDa corresponds to the active MMP-2 (Figure 3A
) confirmed by in-vitro experiments with samples preincubated with 1 mmol/l p-amino-phenylmercuric acetate (Figure 3C
). In comparison with control samples without the treatment with p-amino-phenylmercuric acetate, samples preincubated with the in-vitro MMP activator showed the typically changed zymographic pattern: an increased band of the active form at 62 kDa and a decreased band of the proMMP-2 at 72 kDa (Figure 3C
). A similar change in the MMP-9 pattern was observed (Figure 3C
).
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Discussion |
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Utilizing ELISAs for MMP-2, MMP-9, TIMP-1 and TIMP-2 as well as SDSPAGE, these proteins were identified in all measured specimens. After we had finished our experiments, another group (Shimokawa et al., 2002) published similar results. They described several gelatinolytic proteins with molecular weights of 72, 67, 92 and 84 kDa in seminal plasma with gelatin zymography without giving quantitative data. Using Western blot techniques, proMMP-2 (72 kDa) and proMMP-9 (92 kDa), as well as their active forms MMP-2 and -9, were identified. The authors discussed a possible function of MMPs in liquefaction of the ejaculate. Our results not only confirm the presence of the two MMPs and TIMPs in seminal plasma, but also show quantitative data for the first time. In addition, we could demonstrate a strong correlation between the sperm count and MMP-2, but not MMP-9, TIMP-1 or TIMP-2. There were significantly higher concentrations of MMP-2 in normozoospermic compared with azoospermic plasma. No difference within the azoospermic group with regard to the aetiology, whether obstructive or idiopathic, could be found.
In the present study, the analysis of seminal plasma originating from ejaculates with increasing sperm counts produced evidence of a link between sperm count and MMP-2. This is further supported by the fact that there were no significant differences in MMP-2 levels between azoospermic and vasectomy samples. Since MMP-2 is also secreted by accessory sex glands, the non-linear relationship in the sperm count range of 49110x106/ml could be due to a changed baseline secretion by these glands. However, it remains unclear exactly where MMP-2 is expressed. It has been shown that MMP-2 is secreted by Sertoli cells and seems to be involved in the release of differentiating germ cells from the basal lamina in the seminiferous tubules (Sang et al., 1990). It therefore seems possible that it is also released into the tubular lumen this way.
TIMPs represent natural inhibitors to MMPs (Woessner, 1994). They bind in a 1:1 ratio to the haemopexin domain of MMPs and interfere with their activation (DeClerck et al., 1993
). TIMP-1 is specific for MMP-9 and TIMP-2 for MMP-2 (Goldberg et al., 1989
). In bovine seminal fluid, a 24 kDa heparin-binding protein (HBP-24) was recently described (McCauley et al., 2001
). This protein bound to sperm membranes was identified as TIMP-2 and was suggested to have a role in bull fertility. The authors suspected an interaction with MMP-2 either expressed in the seminal plasma or directly on sperm (McCauley et al., 2001
).
However, in somatic cells one way of MMP-2 activation is through formation of a trimolecular complex between MMP-14, TIMP-2 and proMMP-2 on the cell surface (Strongin et al., 1995). TIMP-2, which usually acts as an inhibitor to MMP-2, interestingly is also required for its activation (Wang et al., 2000
) and could be derived from the seminal plasma. Activated MMP-2 is subsequently released into the extracellular matrix. Although not yet reported, the same activation process could theoretically be present on sperm, and was responsible for the detection of MMP-2 in the present study. This would explain the correlation between sperm count and MMP-2 concentration.
MMP-2 concentrations in seminal plasma decreased with time, being significantly lower from 8 h onwards following ejaculation (Figure 4). The decrease was somewhat linear and could be explained by the degradation of MMP-2. This would, however, exclude any prolonged release of MMP-2 by the sperm themselves unless it is outweighed by degradation processes.
In summary, in this work we have reported on the concentrations of MMP-2 and MMP-9 and their inhibitors TIMP-1 and TIMP-2 in human seminal plasma. MMP-2 was strongly correlated to the sperm count in a linear fashion. Its origin and function remain to be elucidated.
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Acknowledgements |
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Notes |
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References |
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Submitted on March 22, 2002; resubmitted on June 13, 2002; accepted on July 23, 2002.