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 LAquila, 67100 LAquila, Italy
4 To whom correspondence should be addressed. E-mail: elio.ziparo{at}uniroma1.it
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Abstract |
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Key words: apoptosis/Fas system/matrilysin/spermatozoa
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Introduction |
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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.
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Materials and methods |
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Seminal samples and sperm preparation
The samples were collected by masturbation into sterile plastic jars, after 35 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 streptavidinphycoerythrin (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 412% 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 416% 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 TrisHCl (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., 2002). 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 KruskalWallis test was used to assess differences among groups. P < 0.05 was considered significant.
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Results |
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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 KruskalWallis 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 (KruskalWallis 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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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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Discussion |
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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., 1999), although such Fas overexpression was not significantly associated with TUNEL positivity (Sakkas et al., 2002
; McVicar et al., 2004
). 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., 2000; Shen et al., 2002
).
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., 1996), although a subsequent report on Fas and FasL null mutant mice failed to show prevention of apoptosis in male reproductive organs (Sugihara et al., 2001
), 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., 1999; Zhang et al., 2002
). 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., 2002
). 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, 2003
). 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., 1998
). 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). 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)
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) and in human (Rodgers et al., 1993)
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., 1997
), 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)
. 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., 1997
).
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.
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Acknowledgements |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bellgrau D, Gold D, Selawry H, Moore J, Franzusoff A and Duke RC (1995) A role for CD95 ligand in preventing graft rejection. Nature 377,630632.[CrossRef][ISI][Medline]
Buchman-Shaked O, Kraiem Z, Gonen Y and Goldman S (2002) Presence of matrix metalloproteinases and tissue inhibitor of matrix metalloproteinase in human sperm. J Androl 23,702708.
DAlessio A, Riccioli A, Lauretti P, Padula F, Muciaccia B, De Cesaris P, Filippini A, Nagata S and Ziparo E (2001) Testicular FasL is expressed by sperm cells. Proc Natl Acad Sci USA 98,33163321.
Denison FC, Grant VE, Calder AA and Kelly RW (1999) Seminal plasma components stimulate interleukin-8 and interleukin-10 release. Mol Hum Reprod 5,220226.
Dhein J, Walczak H, Baumier C, Debatin KM and Krammer PH (1995) Autocrine T-cell suicide mediated by Apo-1/Fas/CD95. Nature 373,438448.[CrossRef][ISI][Medline]
Evans CH, Lee TS and Flugelman AA (1995) Spermine-directed immunosuppression of cervical carcinoma cell sensitivity to a majority of lymphokine-activated killer lymphocyte cytotoxicity. Nat Immunol 14,157163.
Fiedler P, Schaetzlein CE and Eibel H (1998) Constitutive expression of FasL in thyrocytes. Science 279,2015a.[CrossRef]
Filippini A, Riccioli A, Padula F, Lauretti P, DAlessio A, De Cesaris P, Gandini L, Lenzi A and Ziparo E (2001) Control and impairment of immune privilege in the testis and in semen. Hum Reprod Update 7,444449.
Fisher GH, Rosenberg FJ, Straus SE, Dale JK, Middleton LA, Lin AY, Strober W, Lenardo MJ and Puck JM (1995) Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 81,935946.[CrossRef][ISI][Medline]
Francavilla S, DAbrizio P, Rucci N, Silvano G, Properzi G, Straface E, Cordeschi G, Necozione S, Gnessi L, Arizzi M et al. (2000) Fas and Fas ligand expression in fetal and adult human testis with normal or deranged spermatogenesis. J Clin Endocrinol Metab 85,26922700.
Fujisawa M and Ishikawa T (2003) Soluble forms of Fas and Fas ligand concentrations in the seminal plasma of infertile men with varicocele. J Urol 170,23632365.[CrossRef][ISI][Medline]
Gandini L, Lombardo F, Paoli D, Caponecchia L, Familiari G, Verlengia C, Dondero F and Lenzi A (2000) Study of apoptotic DNA fragmentation in human spermatozoa. Hum Reprod 15,830839.
Green DR and Ferguson TA (2001) The role of Fas ligand in immune privilege. Nat Rev Mol Cell Biol 2,917924.[CrossRef][ISI][Medline]
Hashimoto K, Kihira Y, Matuo Y and Usui T (1998) Expression of matrix metalloproteinase-7 and tissue inhibitor of metalloproteinase-1 in human prostate. J Urol 160,18721876.[CrossRef][ISI][Medline]
Hulboy DL, Rudolph LA and Matrisian LM (1997) Matrix metalloproteinases as mediators of reproductive function. Mol Hum Reprod 3,2745.[Abstract]
Kelly RW (1995) Immunosuppressive mechanisms in semen: implications for contraception. Hum Reprod 10,16861693.[Abstract]
Kimmel SG, Ohbatake M, Kushida M, Merguerian P, Clarke ID and Kim PC (2000) Murine xenogeneic immune responses to the human testis: a presumed immune-privileged tissue. Transplantation 69,10751084.[CrossRef][ISI][Medline]
Knox PG, Milner AE, Green NK, Eliopoulos AG and Young LS (2003) Inhibition of metalloproteinase cleavage enhances the cytotoxicity of Fas ligand. J Immunol 170,677685.
Lee J, Richburg JH, Younkin SC and Boekelheide K (1997) The Fas system is a key regulator of germ cell apoptosis in the testis. Endocrinology 138,20812088.
Leeman MF, Curran S and Murray GI (2003) New insights into the roles of matrix metalloproteinases in colorectal cancer development and progression. J Pathol 201,528534.[CrossRef][ISI][Medline]
Liabakk NB, Lien E, Sundan A, Sunde A, Austgulen R and Espevik T (1993) High concentrations of the soluble p55 tumour necrosis factor receptor in human seminal plasma. Hum Reprod 8,18371842.[Abstract]
McVicar CM, McClure N, Williamson K, Dalzell LH and Lewis SE (2004) Incidence of Fas positivity and deoxyribonucleic acid double-stranded breaks in human ejaculated sperm. Fertil Steril 81 Suppl 1,767774.
Nagata S (1999) Fas ligand-induced apoptosis. Annu Rev Genet 33,2955.
Nair R and Shaha C (2003) Diethylstilbestrol induces rat spermatogenic cell apoptosis in vivo through increased expression of spermatogenic cell Fas/FasL system. J Biol Chem 278,64706481.
Nocera M and Chu TM (1993) Transforming growth factor beta as an immunosuppressive protein in human seminal plasma. Am J Reprod Immunol 30,18.[ISI][Medline]
Powell WC, Fingleton B, Wilson CL, Boothby M and Matrisian LM (1999) The metalloproteinase matrilysin proteolytically generates active soluble Fas ligand and potentiates epithelial cell apoptosis. Curr Biol 9,14411447.[CrossRef][ISI][Medline]
Restifo NP (2000) Not so Fas: re-evaluating the mechanisms of immune privilege and tumor escape. Nat Med 6,493495.[CrossRef][ISI][Medline]
Riccioli A, Salvati L, DAlessio A, Starace D, Giampietri C, De Cesaris P, Filippini A and Ziparo E (2003) The Fas system in the seminiferous epithelium and its possible extra-testicular role. Andrologia 35,6470.[CrossRef][ISI][Medline]
Rodgers WH, Osteen KG, Matrisian LM, Navre M, Giudice LC and Gorstein F (1993) Expression and localization of matrilysin, a matrix metalloproteinase, in human endometrium during the reproductive cycle. Am J Obstet Gynecol 168,253260.[ISI][Medline]
Rudolph-Owen LA, Hulboy DL, Wilson CL, Mudgett J and Matrisian LM (1997) Coordinate expression of matrix metalloproteinase family members in the uterus of normal, matrilysin-deficient, and stromelysin-1-deficient mice. Endocrinology 138,49024911.
Saarialho-Kere UK, Crouch EC and Parks WC (1995) Matrix metalloproteinase matrilysin is constitutively expressed in adult human exocrine epithelium. J Invest Dermatol 105,190196.[CrossRef][ISI][Medline]
Sakkas D, Mariethoz E and St.John JC (1999) Abnormal sperm parameters in humans are indicative of an abortive apoptotic mechanism linked to the Fas-mediated pathway. Exp Cell Res 251,350355.[CrossRef][ISI][Medline]
Sakkas D, Moffatt O, Manicardi GC, Mariethoz E, Tarozzi N and Bizzaro D (2002) Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis. Biol Reprod 66,10611067.
Shen HM, Dai J, Chia SE, Lim A and Ong CN (2002) Detection of apoptotic alterations in sperm in subfertile patients and their correlations with sperm quality. Hum Reprod 17,12661273.
Somerville RP, Oblander SA and Apte SS (2003) Matrix metalloproteinases: old dogs with new tricks. Genome Biol 4,216.[CrossRef][Medline]
Suda T, Takahashi T, Goldstein P and Nagata S (1993) Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 75,11691178.[CrossRef][ISI][Medline]
Sugihara A, Yamada N, Tsujimura T, Iwasaki T, Yamashita K, Takagi Y, Tsuji M and Terada N (2001) Castration induces apoptosis in the male accessory sex organs of Fas-deficient lpr and Fas ligand-deficient gld mutant mice. In Vivo 15,385390.[ISI][Medline]
Suzuki A, Matsuzawa A and Iguchi T (1996) Down regulation of Bcl-2 is the first step on Fas-mediated apoptosis of male reproductive tract. Oncogene 13,3137.[ISI][Medline]
Takahashi T, Tanaka M, Brannan CI, Jenkins NA, Copeland NG, Suda T and Nagata S (1994) Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell 76,969976.[CrossRef][ISI][Medline]
Tanaka M, Suda T, Takahashi T and Nagata S (1995) Expression of the functional soluble form of human Fas ligand in activated lynphocytes. EMBO J 14,1129.[Abstract]
Thompson LA, Barratt CL, Bolton AE and Cooke ID (1992) The leukocytic reaction of the human uterine cervix. Am J Reprod Immunol 28,8589.[ISI][Medline]
Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA and Nagata S (1992) Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356,314.[CrossRef][ISI][Medline]
Werb Z (1997) ECM and cell surface proteolysis: regulating cellular ecology. Cell 91,439442.
World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and SemenCervical Mucus Interactions, 3rd edn. Cambridge University Press, Cambridge.
World Health Organization (1999) WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction, 4th edn. Cambridge University Press, Cambridge.
Wilson CL, Heppner KJ, Rudolph LA and Matrisian LM (1995) The metalloproteinase matrilysin is preferentially expressed by epithelial cells in a tissue-restricted pattern in the mouse. Mol Biol Cell 6,851869.[Abstract]
Wilson CL, Heppner KJ, Labosky PA, Hogan BL and Matrisian LM (1997) Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrilysin. Proc Natl Acad Sci USA 94,14021407.
Zhang J, Jung K, Lein M, Kristiansen G, Rudolph B, Hauptmann S, Schnorr D, Loening SA and Lichtinghagen R (2002) Differential expression of matrix metalloproteinases and their tissue inhibitors in human primary cultured prostatic cells and malignant prostate cell lines. Prostate 50,3845.[CrossRef][ISI][Medline]
Submitted on December 22, 2004; resubmitted on April 26, 2005; accepted on May 19, 2005.
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