Presence of membrane and soluble forms of Fas ligand and of matrilysin (MMP-7) activity in normal and abnormal human semen

A. Riccioli1, V.Dal Secco1, P.De Cesaris2, D. Starace1, L. Gandini3, A. Lenzi3, F. Dondero3, F. Padula1, A. Filippini1 and E. Ziparo1,4

1 Department of Histology and Medical Embryology, Istituto Pasteur-Fondazione Cenci Bolognetti, 3 Department of Medical Physiopathology, University of Rome ‘La Sapienza’, 00161 Rome and 2 Department of Experimental Medicine, University of L’Aquila, 67100 L’Aquila, Italy

4 To whom correspondence should be addressed. E-mail: elio.ziparo{at}uniroma1.it


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The aim of this study is to shed some light on the role of the Fas system in human semen, by investigating whether there is an association between the expression of the molecules regulating the Fas system [membrane-bound Fas ligand (mFasL), soluble Fas ligand (sFasL) and matrilysin, the metalloprotease cleaving mFasL to sFasL] and sperm parameters. METHODS: We investigated, by flow cytometric analysis, the presence of FasL on spermatozoa from normozoospermic and teratozoospermic subjects and, by western blot, the presence of sFasL and matrilysin in the seminal plasma of the same samples as well as on samples from azoospermic subjects. The enzymatic activity of matrilysin was examined by gel zymography. RESULTS: We observed that sperm cells expressed mFasL in 22% of normozoospermic men, whereas it was absent from spermatozoa from teratozoospermic patients. Higher levels of sFasL and augmented enzymatic activity of matrilysin were found in azoospermic samples. CONCLUSIONS: The presence of mFasL on sperm from normozoospermic men and its absence in pathological samples emphasize the role of the Fas system in human semen. Moreover, the presence of both sFasL and matrilysin in seminal plasma implies a fine regulation of the function of the Fas system and, consequently, of the apoptotic process in the human genital tract.

Key words: apoptosis/Fas system/matrilysin/spermatozoa


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The metalloproteinases (MMPs) form a family of structurally related enzymes capable of degrading specific components of the extracellular matrix (ECM) in a zinc-dependent manner at physiological pH. They are produced as proenzymes and secreted into the ECM where they are activated by proteolytic cleavage. The MMP family currently comprises 23 gene products in humans (Somerville et al., 2003Go). Matrilysin or MMP-7 is considered a member of the stromelysin subfamily, comprising MMPs able to degrade a broad range of substrates, such as fibronectin, laminin, elastin, collagen and proteoglycans. In addition to its activity on basement membrane and ECM components, matrilysin has been reported to cleave recombinant and membrane-bound Fas ligand (mFasL) to a functional soluble form (sFasL) (Powell et al., 1999Go). The Fas system is involved in the control of immune system homeostasis by downregulating the immune response of activated T cells (Dhein et al., 1995Go) and a non-functional Fas system leads to autoimmune disease in mice (Watanabe-Fukunaga et al., 1992Go; Takahashi et al., 1994Go) and humans (Fisher et al., 1995)Go. Fas is abundantly expressed in various tissues, whereas FasL is more restricted and tightly regulated in its expression, so that the functionality of the Fas system often depends on FasL induction (for a review see Nagata, 1999)Go. Human sFasL is a 26–35 kDa glycoprotein and consists of the specific extracellular region of FasL which binds to Fas to induce apoptosis. The human active sFasL has been identified in the supernatant of activated human peripheral T cells (Tanaka et al., 1995Go). In contrast, recent studies demonstrated that blockade of FasL cleavage with a matrilysin inhibitor significantly enhances FasL-induced apoptosis, suggesting that sFasL may antagonize the effect of mFasL (Tanaka et al., 1995Go; Knox et al., 2003)Go. Therefore, the balance between two different forms of FasL (mFasL and sFasL) may be critical in the regulation of Fas-mediated apoptosis, and matrilysin activity may be important in this control system. The majority of the MMPs are produced primarily by mesenchymal cells in the stroma, whereas matrilysin expression is restricted to the epithelial component (Wilson et al., 1995)Go, and in particular is preferentially expressed by cells of glandular epithelium (Saarialho-Kere et al., 1995Go). Although expression of matrilysin has been associated primarily with neoplastic lesions and metastatic progression of tumours (Leeman et al., 2003Go), evidence for its expression in normal tissues is beginning to accumulate. In mouse, matrilysin mRNA shows a very restricted tissue pattern of localization: high constitutive levels of matrilysin are found in epithelial cells in the uterus; in the extratesticular ducts, namely the epithelial cells lining the efferent ducts; in mammary epithelial cells; in the crypts of the small intestine (Wilson et al., 1995)Go; and in the prostatic epithelial cells in both mouse and in human (Hashimoto et al., 1998Go; Zhang et al., 2002Go). This expression pattern of matrilysin, restricted almost exclusively to reproductive tissues, prompted us to investigate the presence of matrilysin in human semen which might function as a regulator of the Fas system in the reproductive tracts. We have previously detected FasL protein on the epididymal sperm cell surface in mouse and in rat (D’Alessio et al., 2001Go; Filippini et al., 2001Go) and proposed a possible new function for FasL expressed on murine spermatozoa: a self-defence mechanism of male gametes against lymphocytes present in the female genital tract (Riccioli et al., 2003Go). In the mouse, the testis represents the main source of constitutive FasL in the body (Suda et al., 1993Go) and it has been proposed that the Fas system is involved both in maintaining the immune-privileged nature of the testis (Bellgrau et al., 1995Go) and in the regulation of physiological testicular germ cell apoptosis in mouse and rat (Lee et al., 1997Go; Nair and Shaha, 2003Go). As for human reproduction, the expression and function of Fas and FasL are a matter of debate. It has been reported that xenografts of human testes in mice are rejected and that, unlike in rodents, in human testis FasL mRNA is not expressed while Fas is highly expressed. Consequently, the concept of immune privilege, as defined by prolonged survival after transplantation and its relationship to FasL expression, cannot be extended to human testis (Kimmel et al., 2000Go). Conversely, other authors found that, like in rodent testis, FasL is highly expressed in human testis, whereas Fas is confined to Leydig cells and to sporadic degenerating spermatocytes (Francavilla et al., 2000Go). The discrepancy between these results is probably due to the use of inappropriate antibodies to detect FasL (Restifo, 2000Go; Green and Ferguson, 2001Go).

Although numerous studies have been carried out in the testis, so far there are no data at all about the presence and the role of Fas system in the human genital tract and there is no evidence demonstrating the presence of FasL protein on the human sperm cell surface in either fertile or infertile males. On the basis of our data on rodents, in order to elucidate the possible role of the Fas system in the male genital tract, we investigated the presence of mFasL on human freshly prepared spermatozoa and of sFasL and matrilysin in the seminal plasma from donors with normal and abnormal sperm parameters.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients
We selected 71 male subjects who collected seminal fluid at the Laboratory of Seminology and Immunology of Reproduction of the Department of Medical Pathophysiology, University of Rome ‘La Sapienza’. Samples were subdivided into four groups. Group A were 41 normozoospermic subjects, aged 32.7 ± 7.9 years, attending the clinic for andrological control for a pre-marital check-up or to submit ejaculate for the first time for an examination of the couple’s infertility. Group B were 14 teratozoospermic subjects aged 35.4 ± 8.6 years, suffering from primary infertility for at least 2 years and affected by various andrological pathologies (varicocele, genital inflammation and testicular trauma). Group C were 13 azoospermic subjects, aged 34.2 ± 4.4 years, of whom 10 were affected by secretory azoospermia (volume 3.5 ± 1.8 ml; pH 7.5 ± 0.1) and three had excretory azoospermia (volume 0.6 ± 0.2 ml; pH 6.5 ± 0.1). Group D were three subjects, aged 34.0 ± 8.9 years, with a sperm concentration range of 0.1–3.0 x 106/ml, affected by seminal vesicle dysfunction as demonstrated by very low volume (0.8 ± 0.1 ml), pH (6.7 ± 0.3) and fructose concentration in seminal plasma (22.6 ± 3.5). None of the subjects in the four groups had been medically or surgically treated in the 3 months prior to the study. Groups C and D were selected to study whether sFas ligand was correlated to the presence of sperm and to evaluate sFasL origin (testicular, prostatic or vesicular).

Seminal samples and sperm preparation
The samples were collected by masturbation into sterile plastic jars, after 3–5 days of sexual abstinence. They were allowed to liquefy for 30 min at room temperature (22ºC) and were then evaluated according to the World Health Organization guidelines [World Health Organization, 1992 (for morphology reference values), 1999]. The variables taken into consideration were: ejaculate volume (ml), pH, sperm concentration (n x 106/ml), total sperm count (n x 106), forward motility (%) and morphology (% atypical forms).

Seminal samples were diluted 1:2 with 0.5 mmol/l phosphate-buffered saline (PBS)-EDTA and centrifuged for 10 min at 600 g; the pellet was re-suspended with the same solution and centrifuged again as above. The final pellet was re-suspended in 1 ml of PBS-10% glycerol at a concentration of 4 x 106/ml, frozen and stored at –20°C until analysis. A 1 ml aliquot of the seminal samples was centrifuged at 500 g for 10 min; the supernatant was then further centrifuged at 10 000 g for 10 min. The plasma thus obtained was stored at –20°C.

Flow cytometric analysis
For detection of FasL expression on the surface of human spermatozoa, we used the biotin-conjugated mouse IgG1 anti-human FasL monoclonal antibody (clone NOK-1 Pharmingen; Becton Dickinson, San Josè, CA). Specific monoclonal antibody or the appropriate isotypic control monoclonal antibody were used at 20 µl per test (1 x 106 cells) for 30 min on ice. Cells were then washed twice with PBS + 1% bovine seum albumin (BSA) and incubated with streptavidin–phycoerythrin (PE) conjugate (Sav-PE; Becton Dickinson) for 30 min on ice and, after two washes, analysed with a Coulter Epics XL flow cytometer (Beckman Coulter, CA). Cells were gated using forward versus side scatter to exclude dead cells and debris. Fluorescence of 104 cells/sample was acquired in logarithmic mode for visual inspection of the distributions and in linear mode for quantitating the expression of the relevant molecules by calculating the mean fluorescence intensity.

Western blot analysis
The protein concentration of each seminal plasma sample was determined by using the micro BCA method (Pierce, Rockford, IL). Equal amounts of proteins (70 µg) or 1 µl of seminal plasma were electrophoresed on a NuPAGE Novex 4–12% Bis-Tris polyacrylamide gel (Invitrogen, Carlsbad, CA) using the XCell SureLockTM Mini-Cell apparatus (Invitrogen) and then transferred onto nitrocellulose. Non-specific binding sites were blocked by incubating the nitrocellulose membranes for 1 h with 5% non-fat dry milk (Biorad, Hercules, CA) in Tris-buffered saline containing 0.1% Tween-20. The membranes were then incubated with purified mouse IgG1 anti-human FasL monoclonal antibody (clone G247-4 Pharmingen; Becton Dickinson) or with polyclonal rabbit IgG anti-human matrilysin (Oncogene Research Products, Cambridge, MA) overnight at 4ºC. The membranes were subsequently washed three times for 15 min with Tris-buffered saline containing 0.1% Tween and incubated for 1 h with the secondary goat anti-mouse horseradish peroxidase (HRP)-conjugated antibody (Biorad) and donkey anti-rabbit HRP antibody (Amersham Biosciences, UK) for the two primary antibodies, respectively. After incubating with the secondary antibody, the membranes were washed three times for 15 min with Tris-buffered saline containing 0.1% Tween, and finally immunostained bands were detected by a chemiluminescence system (ECL Advance kit, Amersham Biosciences).

Human malignant prostatic cell line LNCaP, used for matrilysin positive control, was kindly provided by Dr D.Farini (University of Rome ‘Tor Vergata‘). Recombinant human sFasL protein used as a positive control was purchased from Upstate Biotechnology (Charlottesville, VA).

Casein zymography
Novex 4–16% zymogram blue casein gels (Invitrogen) were used to detect matrilysin enzymatic activity levels. An equal volume for each seminal plasma sample (1 µl/lane) was loaded and the gels were run in Tris/glycine SDS running buffer under non-denaturating conditions. The gels were washed twice in 2.5% (v/v) Triton X-100 for 30 min at room temperature to remove SDS. Zymograms were subsequently developed by incubation for 16 h at 37ºC in zymogram developing buffer [0.2 mol/l NaCl, 5 mmol/l CaCl2, 1% Triton X-100 and 0.02% NaN3 in 50 mmol/l Tris–HCl (pH 7.4)]. Enzymatic activity was visualized as a clear band against a blue background of stained casein. Serum-free conditioned medium from the human malignant prostatic cell line LNCaP was used as a positive control for matrilysin activity (Zhang et al., 2002Go). The matrilysin protein in the conditioned medium was activated by incubation with 1 mmol/l organic mercuride p-aminophenylmercuric acetate (APMA) (Sigma Aldrich, St Louis, MO) at 37ºC for 30 min. before electrophoresis.

Statistical analysis
Spearman rank (r) correlation test was used to evaluate the relationship between the sperm concentration and sFasL/matrilysin densitometric values. The non-parametric Kruskal–Wallis test was used to assess differences among groups. P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The mean and SD of age and sperm parameters of the two groups of patients analysed for FasL expression on spermatozoa are reported in Table I. We investigated by flow cytometric analysis the presence of mFasL on spermatozoa from both normozoospermic (group A) and teratozoospermic (group B) subjects. All samples of group B were negative, whereas 22% of samples from group A (nine out of 41) expressed mFasL (Figure 1A). From flow cytometric profiles of the positive samples, it appears that mFasL is homogeneously expressed in the whole sperm cell population and the intensity of FasL expression is ~2-fold more than the isotype control value, as indicated by the values of mean fluorescence shown in Figure 1B.


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Table I. Patient age and characteristics of seminal parameters (mean ± SD)

 


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Figure 1. Cell surface expression of FasL on human spermatozoa. (A) Percentage of patients expressing FasL on spermatozoa measured by flow cytometric analysis. Group A represents 41 normozoospermic subjects and group B represents 14 teratozoospermic subjects. (B) Flow cytometric profiles representative of the positive samples from group A. The mFasL is homogeneously expressed in the whole sperm cell population and the intensity of FasL expression is ~2-fold more than the isotype control value, as indicated by the values of mean fluorescence. Cells were incubated with anti-human FasL mAb NOK-1 (open histograms) or with isotype control mAb (grey histograms).

 

Since the freshly ejaculated semen samples are allowed to liquefy at room temperature for at least 30 min, it is reasonable that, during this incubation, the mFasL could be converted to its soluble form by the activity of the specific metalloproteinase matrilysin. To verify this hypothesis, we investigated by western blot the presence of both sFasL and matrilysin in the seminal plasma of the groups A, B, azoospermic samples (group C) and in samples from subjects affected by seminal vesicle dysfunction (group D). We observed bands both for sFasL (25 kDa) and matrilysin (28 and 19 kDa) in all the samples investigated. Results from a representative experiment are shown in Figure 2A. Quantification of the bands was performed by densitometric analysis shown in Figure 2B. The average differences of sFasL are significant among the four groups considering Kruskal–Wallis one-way ANOVA, H = 9.105, P = 0.035, and sFasL levels clearly increase in groups C and D compared with groups A and B. Conversely, Figure 2B shows that matrilysin was detected at higher levels in groups A and B, but the differences were not significant (Kruskal–Wallis test: H = 5.6, P = 0.061). We also observed high levels of sFasL and matrilysin in three azoospermic patients with ejaculatory duct obstruction (i.e. the second lane of group C in Figure 2A). Moreover, our data indicate no difference in sFasL and matrilysin concentration between groups A and B in relation to different sperm number. In fact, when a correlation index was calculated to compare seminal sFasL and matrilysin concentration with the sperm concentration of the patients, no significant correlation was observed (sFasL/sperm concentration r = 0.336; P > 0.05 and matrilysin/sperm concentration r = 0.144; P > 0.05).



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Figure 2. Western blot analysis of seminal plasma samples with different sperm parameters. (A) An equal volume of seminal plasma (1 µl/lane) was loaded for each sample. Each sample was loaded at least three times. Data from seminal plasma samples representative of group A, group B, group C (13 azoospermic subjects) and group D (three subjects having a lack of seminal vesicle secretion) are shown. The second lane from the left of group C shows a representative sample from a patient with obstructed ejaculatory ducts. Recombinant sFasL protein (1 ng) of 35 kDa was loaded for the determination of the specificity of anti-FasL antibody. Protein extracted from serum-free conditioned medium of cell line LNCaP (50 µg) was used as a positive control for matrilysin; only the active form (19 kDa) is present. (B) Densitometric analysis of the results shown in (A).

 

Finally, in order to assess the functional significance of matrilysin expression, we tested the enzyme activity by using gel zymography. All samples displayed an intense band of casein-degrading activity, corresponding for promatrilysin to a mol. wt of 28 kDa; however, a more intense specific band is present in group C, in which was evident also the 19 kDa caseinolytic activity attributable to the activated form of matrilysin (Figure 3). Despite the fact that, by western blot, equal densitometric values of matrilysin have been measured in group C compared with other samples (Figure 2), a higher enzymatic activity was detected in this group by gel zymography (Figure 3). This result may account for the higher values of sFasL detected in azoospermic seminal plasma compared with the other samples (Figure 2).



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Figure 3. Gel zymography of representative normal and abnormal human seminal plasma samples as in Figure 2. The respective pro-matrilysin and proteolytically activated form with mol. wts of 28 and 19 kDa are indicated. Protein extracted from serum-free conditioned medium of cell line LNCaP was used as a positive control for matrilysin activity. The zymograph shown is representative of at least three loadings for each sample.

 


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Human seminal plasma has powerful immunosuppressive properties, containing high concentrations of prostaglandins (Kelly, 1995)Go, soluble p55 tumour necrosis factor-{alpha} (TNF-{alpha}) receptor (Liabakk et al., 1993Go), transforming growth factor-{beta} (TGF-{beta}) (Nocera and Chu, 1993Go) and spermine (Evans et al., 1995Go). These immunosuppressive factors are thought to confer a survival advantage to spermatozoa within the hostile female genital tract. In contrast, a profound pro-inflammatory leukocytosis has been observed post-coitum in animals and humans (Thompson et al., 1992Go; Denison et al., 1999)Go and the factor or factors which initiate the leukocytosis are unknown. Studies performed on mice have revealed the presence of FasL on epididymal spermatozoa, suggesting that this system plays a self-protective role of male gametes against immune attacks along the male and female genital tract (D’Alessio et al., 2001Go; Riccioli et al., 2003Go). In order to investigate the possible role of the Fas system in the physiopathology of human male fertility, we studied the expression of FasL on the surface of human sperm cells and its regulation.

We have detected the presence of mFasL only in spermatozoa from normozoospermic men and not in sperm from subjects with severe infertility (group B). This result might provide evidence for a role for the Fas system in human semen as a protection mechanism of the gametes against anti-sperm activated lymphocytes present in both male and female genital tracts. Our findings are consistent with the notion that FasL expression could represent one of the factors improving sperm survival. The mFasL positivity only for a partial number of sperm samples may be due to the fact that, during semen preparation, samples are kept for 30 min at room temperature to allow the liquefaction process. The reduced mFasL posititivity on sperm (22%) might be the consequence of an enhanced matrilysin-driven cleavage of mFasL during this time lapse. However, liquefaction is a process normally occurring in the female genital tract. As a result, the experimental procedure employed simply mimics a physiological condition. Moreover, the evidence that mFasL is expressed in 22% of the normal samples leads us to suppose the possible presence of alternative protective mechanisms for immunoprotection of spermatozoa along the male and female genital tracts.

The Fas system is also considered to be crucial in tissue homeostasis for both growth control and elimination of abnormal cells. Other authors have examined human sperm cells for the presence of membrane-bound Fas. They showed that men with oligoteratozoospermia had overall higher Fas expression on spermatozoa than men with normal sperm parameters (Sakkas et al., 1999Go), although such Fas overexpression was not significantly associated with TUNEL positivity (Sakkas et al., 2002Go; McVicar et al., 2004Go). These authors proposed that the presence of Fas-positive spermatozoa in the ejaculate is indicative of an ‘abortive apoptosis’ having taken place, whereby the normal apoptotic mechanism would have misfunctioned. Our data might explain their findings, as the absence of FasL on the spermatozoa of teratozoospermic men could represent the missing trigger for the fratricide elimination via apoptosis of Fas-overexpressing sperm, whereby these cells expressing Fas and destined to undergo apoptosis might escape the clearance mechanism. The survival of defective gametes contributes to poor sperm quality. In conclusion, our data suggest that the quality control system for spermatozoa selection could function not only during spermatogenesis, but also along the male genital tract.

Therefore, our present and previous data suggest that the FasL expressed on spermatozoa might perform both functions proposed: control of sperm quality and escape from immune surveillance. However, a redundancy of apoptosis-triggering mechanisms seems to exist, since we and others observed that the percentage of apoptotic spermatozoa is significantly higher in infertile patients than in normozoospermic subjects (Gandini et al., 2000Go; Shen et al., 2002Go).

As regards the expression of FasL in the male genital tract, it has been analysed in animal models only in pathological conditions. The involvement of the Fas pathway in epididymal apoptotic cell death after androgen withdrawal is controversial. An initial study indicated that Fas signalling is involved in apoptosis of male reproductive organs, specifically the prostate and epididymis, after orchidectomy (Suzuki et al., 1996Go), although a subsequent report on Fas and FasL null mutant mice failed to show prevention of apoptosis in male reproductive organs (Sugihara et al., 2001Go), indicating that the Fas system is not essential in mediating apoptosis in these tissues after orchidectomy.

To better understand the role of the Fas system in human semen, we examined some molecules controlling FasL function, namely sFasL and the enzyme involved in its production, matrilysin. We detected both molecules in all the seminal plasma samples analysed but found no significant difference between normozoospermic and teratozoospermic samples and no correlation between sperm concentration and amount of sFasL or matrilysin. Higher levels of sFasL and augmented enzymatic activity of matrilysin were found in azoospermic samples, indicating that the sFasL found in seminal plasma is not derived from shedding from the sperm membrane. Therefore, in order to assess the origin of sFasL and matrilysin contained in seminal plasma, we analysed samples from azoospermic patients with and without ejaculatory duct obstruction and patients characterized by seminal plasma with no vesicle secretion, and we found a high concentration of both molecules in all samples. These results confirm that most of the sFasL and matrilysin in seminal plasma does not originate only from the testis or the seminal vesicles but also from the prostate, which has been described as a source of both molecules (Powell et al., 1999Go; Zhang et al., 2002Go). Accordingly, a recent study reported MMP activity in conditioned medium from human spermatozoa assayed by gel zymography and that the molecular weight of the bands with gelatinolytic activity was consistent with MMP-9 and MMP-2, whereas the presence of matrilysin was excluded (Buchman-Shaked et al., 2002Go). Our data disagree with those of other researchers who reported that the sFasL concentration in human seminal plasma was under the limit of detection using an enzyme immunoassay (EIA) (Fujisawa and Ishikawa, 2003Go). We can explain these divergent results as due to the use of different antibodies. We are confident that the data we report are valid since they were obtained with reliable antibodies (Fiedler et al., 1998Go). In fact, western blot analysis permits visualization of the molecular weight of the protein, and the band size we detect is the expected one. Moreover, the specificity of the anti-FasL antibody was determined by recognizing a recombinant sFasL protein.

Moreover, we show the first evidence for the presence of functional matrilysin in human semen. This is an important finding since this isoform is distinct from the other known MMPs as regards structure, tissue expression and function (Werb, 1997)Go. Matrilysin lacks the haemopexin domain thought to be important in interactions with TIMPs (specific tissue inhibitors of MMPs) and is thus believed to be less sensitive to the inhibitory action of these proteins (Baragi et al., 1994)Go and therefore likely to undergo specific and as yet unknown control mechanisms.

Although the expression pattern of matrilysin both in mouse (Wilson et al., 1995)Go and in human (Rodgers et al., 1993)Go tissues suggests that it is particularly involved in the remodelling associated with reproductive processes, including menstruation, trophoblast invasion and involution of the post-partum uterus (Hulboy et al., 1997Go), mice with a null mutation in the gene encoding matrilysin have no obvious reproductive defects, proceeding normally through the estrous cycle, pregnancy and uterine involution (Wilson et al., 1997)Go. Since agents that affect reproduction are key targets for natural selection, it follows that there may be considerable redundancy in the contribution of MMP family members to reproductive processes. In fact, interestingly, the expression of other MMPs, stromelysin 1 and stromelysin 2, is dramatically upregulated in the stroma of post-partum involuting uterus in the matrilysin nullizygous mice (Rudolph-Owen et al., 1997Go).

In conclusion, we provide some insights for a possible role for the Fas system in the physiopathology of human semen; further investigations will be required to assess correlations between dysfunctions in FasL expression on sperm cells or deregulation of sFasL/matrilysin production and infertility or subfertility sine causa.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We wish to thank Professor Fioretta Palombi for critically reading the manuscript. This work was supported by grants from M.I.U.R./COFIN 2003 to E.Z. and F.D.


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 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on December 22, 2004; resubmitted on April 26, 2005; accepted on May 19, 2005.





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