Inserm U329, Hopital Debrousse, 29 rue sur Bouvier, 69322 Lyon Cedex 05, France
*Author for correspondence (e-mail: lemagueresse{at}lyon151.inserm.fr)
Accepted March 15, 2001
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SUMMARY |
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Key words: Metalloprotease, Testis, FSH, Sertoli cell, Germ cell
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INTRODUCTION |
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The testis is an organ in which a series of radical remodeling events occurs during development. It is composed of two main compartments: the interstitium, containing the steroidogenic Leydig cells, and the seminiferous tubules, composed of germ cells, Sertoli cells and peritubular cells. Sertoli cells play key roles in spermatogenesis: they are targets for follicle-stimulating hormone (FSH) and testosterone, the hormones responsible for the initiation and maintenance of spermatogenesis, and they form the tubule and provide structural and nutritional support for the developing germ cells (Ritzen et al., 1981; Skinner, 1991; Jégou, 1993; Parvinen, 1993; Sharpe, 1994; Griswold, 1995). Interestingly, the seminiferous tubule undergoes extensive restructuring in a cyclic fashion. As germ cells develop, they progress towards the tubular lumen without perturbing the structural integrity of the tubule. To accomplish this, germ cells, which have established intricate relationships with Sertoli cells, have to pass the blood-testis barrier. This barrier is formed at the initiation of puberty by a tight-junctional complex facing adjacent Sertoli cells. Once germ cells have passed the barrier, they enter the meiotic process and continue their migration towards the tubular lumen while differentiating into sperm cells. The last step in spermatogenesis is spermiation, or extrusion of sperm cells into the lumen, accompanied by Sertoli cell phagocytosis of the residual bodies (shed cytoplasts) (Russell, 1980). To permit these movements while maintaining integrity of the tubule, there must be a fine tuning of the different proteases and antiproteases present in the seminiferous tubules (Fritz et al., 1993). Previous reports have consistently suggested that serine and cysteine proteases could play a role in these restructuring events (Fritz et al., 1993; Mruk et al., 1997; Le Magueresse-Battistoni et al., 1998; Sigillo et al., 1999; Wong et al., 2000). Although several MMPs are known to be secreted by Sertoli cells, including MMP-2 and its inhibitor TIMP-2 (Fritz et al., 1993; Hoeben et al., 1996), no study to date has provided evidence of their involvement.
These observations prompted us to examine the in vivo and in vitro occurrence of MT1-MMP in the testis. The time course of the appearance of MT1-MMP transcript and protein, its cellular localization, the spatiotemporal distribution of TIMP-2 and MMP-2, and the predicted role of MT1-MMP strongly suggest an involvement of MT1-MMP in proMMP-2 activation within the testis throughout development. As the seminiferous tubule is the site of extensive remodeling, we next focused our study on the control of MMP-2 expression and activation in Sertoli cells. Using 20-day-old rat cultured Sertoli cells, we demonstrated that proMMP-2 activation is under both systemic (FSH-mediated) and local (exerted by germ cells) control.
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MATERIALS AND METHODS |
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Spermatogenic cells were isolated from 60- to 90-day-old rat testes by trypsinization (Meistrich et al., 1981) or a mechanical procedure (Aravindan et al., 1996). The resulting crude germ cell population (containing germ cells from all developmental steps but no somatic cells) was submitted to a centrifugal elutriation using a Beckman JE-6 rotor as described previously (Meistrich et al., 1981). Two fractions were harvested, the pachytene spermatocyte fraction (enrichment of 80-85%; contaminated primarily by early spermatids) and the early spermatid fraction (steps 1-8; enrichment 75-80% with primary contamination by both spermatocytes and elongated spermatids). The purity of cell types was assessed as previously described on air-dried smears stained with periodic acid Schiff (PAS) and hematoxylin (Meistrich et al., 1981; Le Magueresse et al., 1986), and by flow cytometry (Le Magueresse and Jégou, 1988). After collection, the different cell populations were either processed for RNA or protein analysis, or used for co-culture experiments. For culture studies, 2.5x106 germ cells (crude suspension or elutriated cells) were added to the 5-day-old cultured Sertoli cells (dish area 20 cm2) for 48 hours.
RNA extraction
Total RNA was isolated using acid-guanidium thiocyanate-phenol-chloroform extraction in a single step procedure as reported previously (Chomczynski et al., 1987). The polyadenylated (poly(A)+) RNA was isolated using µMACS mRNA Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany).
Reverse-transcriptase polymerase chain reaction
3 µg total RNA were reverse-transcribed in 10 µl of reaction mixture consisting of 0.2 mM dNTPs (Sigma), 10 U ml-1 of MMLV reverse transcriptase (Gibco BRL), 0.01 M DTT (Gibco BRL) and 5 µM random hexamers (Sigma) in 20 mM Tris-HCl buffer (Gibco BRL). Reverse transcription (RT) was carried out for 60 min at 37°C and 100°C for 5 min. 10 µl of water was added per sample at the end of the RT. Primers for MMP-2 were: sense (5'-CTATTCTGTCAGCACTTTGG-3') and antisense (5'-CAGACTTTGGTTCTCCAACTT-3'), with an expected product of 309 bp (Wells et al., 1996). Primers for MT1-MMP were: sense (5'-GCTCATTCATGGGTAGCGAT-3') and antisense (5'-AGTAAAGCACTCGCTTGGGT-3'), with an expected product of 329 bp. Primers for TIMP-2 were: sense (5'-ATCAGAGCCAAAGCAGGTGAGCG-3') and antisense (5'-GGTAATGTGCATCTTGCCATCTCC-3'), with an expected product of 243 bp. Primers for GAPDH were: sense (5'-TCCCACCACCCTGTTGCTGTA-3') and antisense (5'-ACCACAGTCCATGCCATCAC-3') with an expected product of 455 bp. All primers were purchased from Oligoexpress (Montreuil, France).
PCR was carried out using 2 µl of the RT reaction, 1 µM of the designed specific primers in the presence of Taq polymerase (0.01 U µl-1, Eurobio, France), dNTPs (100 µM; Sigma), MgCl2 (1.5 mM; Eurobio) and 50 mM Tris-HCl (Eurobio) in a final volume of 20 µl. The polymerase chain reaction amplification was performed using a Mastercycler gradient (Eppendorf). The optimal temperature of annealing was of 55°C for MMP-2, 59°C for MT1-MMP and GAPDH, and 70°C for TIMP-2. Briefly, after a first denaturation at 94°C for 4 minutes, samples were submitted to 35 cycles (GAPDH, 30 cycles) of denaturation (94°C, 30 seconds), annealing (optimal temperature, 30 seconds) and elongation (72°C, 60 seconds). Final elongation was performed at 72°C for 5 minutes. Negative controls contained water instead of cDNA. Amplified cDNAs were visualized in a 1.5% agarose gel stained with ethidium bromide. A DNA ladder (Gene Ruler, Fermentas, St Leon-Rot, Germany) was loaded on each gel. PCR with no RT reactions gave no band, eliminating the possibility of a genomic DNA contamination in the RNA preparations (not shown).
Northern blot analysis
The probes used for hybridization were the PCR products for MMP-2 (309 bp), MT1-MMP (329 bp) and GAPDH (455 bp), a 700 bp HindIII-XhoI fragment of human TIMP-2 complementary DNA (cDNA) (D. R. Edwards, Calgary University, Canada), an 1100 bp BamHI-EcoRI fragment of rat 18S cDNA (A. Ferguson, University of Texas, USA). Probes were labeled using the RTS RadPrime DNA Labeling System (Life Technologies, Gaithersburg, UK). Northern blot analysis was performed as previously described (Le Magueresse-Battistoni et al., 1994), using either total RNA (10-20 µg as measured by absorbance at 260 nm) or poly(A)+ (3 µg). A 0.28 kb to 9.5 kb Promega RNA ladder was used to determine the size of the transcripts.
Western blot analysis
Primary antibodies used in this study were a rabbit anti-MMP-2 (1:500), a rabbit anti-TIMP-2 (1:1000) (Chemicon International, Temecula, CA) and a rabbit anti-MT1-MMP (1:1000) (Sigma). SDS-PAGE and western blotting were carried out using (1) tenfold concentrated cell culture media (Centripep, cut-off at 10 kDa; Amicon, Beverly, MA, USA) for the analysis of TIMP-2, (2) a immunoprecipitated proteins from concentrated cell culture media (MAGmol protein A MicroBeads, Miltenyi Biotec) for the analysis of MMP-2 or (3) cell lysates prepared using PBS containing 1% NP-40 and 5 mM EDTA for the analysis of MT1-MMP (Okada et al., 1997).
Proteins (exactly 40 µg), measured by BCA protein assay (Pierce/Interchim, Montluçon, France), were separated by electrophoresis performed on polyacrylamide gels (7.5% for MMP-2 and MT1-MMP; 15% for TIMP-2) under reducing conditions. The proteins were then electrophoretically transferred to a polyvinylidine difluoride (PVDF) membrane (Biorad). After treatment with a blocking solution (5% bovine serum albumin (BSA) in Tris-buffered saline (TBS)) for 3 hours, the membrane was incubated overnight with the primary antibody at 4°C. The PVDF membrane was washed and incubated with peroxidase-conjugated goat anti-rabbit IgG antibody (Covalab, Lyon, France). After washing in TBS containing 0.1% Tween-20, proteins were detected using a chemiluminescent detection system, ECL+ (Amersham Pharmacia Biotech).
Gelatin zymography
Cell culture media were concentrated tenfold using Centripep (Amicon, Beverly, MA, USA). 40 µg of proteins were electrophoresed at 4°C on 10% polyacrylamide gels containing 1 mg ml-1 gelatin (Sigma) in the absence of any reducing agent. Following electrophoresis, SDS is removed from the gel by exchange in Triton X-100 (two washes of 30 minutes at room temperature in 2.5% Triton X-100 followed by two washes of 20 minutes in distilled water). The gel is subsequently incubated at 37°C for 48 hours in 100 mM Tris-base, pH 7.6 containing 15 mM CaCl2. In these conditions, gelatinases present in the samples renatured and autoactivated. White zones of lysis indicating gelatin degrading activity were revealed by staining with Coomassie Brillant Blue R-250.
Immunohistochemistry
Testes were fixed in AFA (acetic acid/formaldehyde/alcohol) or in Bouins liquid. Tissues were then dehydrated and embedded in paraffin. Sections (4 µm thick) were applied to 3-aminopropyl trietoxysilane-coated slides. After deparaffinization and rehydration, nonspecific binding of antibodies was blocked with phosphate-buffered saline (PBS) containing 1% BSA for 2 hours. Endogenous peroxidases were blocked with H2O2 for 10 minutes at room temperature. Sections were then incubated with the rabbit anti-MT1-MMP antibody (used in western-blotting studies) for 1 hour at room temperature. Anti-MT1-MMP antibody was diluted (1:200) using DAKO antibody diluent (Dako, Trappes, France). The diluent alone was used to prepare negative controls. Such diluent is particularly useful for reducing background staining while maintaining specific reactivity. After rinsing with 0.05 M Tris-HCl, pH 7.6, containing 0.15 M NaCl, sections were incubated with goat anti-rabbit immunoglobulin conjugated to peroxidase (Envision+, Dako, Trappes, France) for 30 minutes at room temperature and the immunohistochemical reaction was carried out using diaminobenzidine (DAB). Sections were next stained with PAS, counterstained with Harris hematoxylin and mounted. Sections were observed under an Axioplan microscope (Zeiss).
Data analysis
All experiments were repeated at least three times using independent cell preparations and triplicate dishes; a representative experiment from each series is presented here. The band densities obtained in northern blotting analyses were determined by scanning densitometric analysis (Alcatel TITN Answare, Massy, France). The amount of RNA in each lane of each northern blot was internally standardized within a blot by assessing the amount of GAPDH or 18S mRNA per lane. The significance of the results was determined by Students t test when comparing data from two groups. Differences are accepted as significant at P<0.05.
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RESULTS |
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Role of FSH in MMP-2 activation in cultured Sertoli cells
Functional demonstration of the impact of FSH on MMP-2 was provided by gelatin zymography. This technique allows discrimination between active and inactive gelatinases, as they migrate differently. Two groups of gelatinolytic bands were detected in Sertoli cell culture media (Fig. 9A). One group migrated at or near 92 and 84 kDa and the second group migrated as a triplet around 72 kDa, 66 kDa and 62 kDa. Based on these sizes and on previous data from the testis (Hoeben et al., 1996) and in other systems (Birkedahl-Hansen, 1995; Stanton et al., 1998; Hernandez-Barrantes et al., 2000; Wang et al., 2000), these bands are likely to correspond to MMP-9 (the pro- and active forms) and MMP-2 (the pro-, intermediate and active forms), respectively. The temporal production of MMP-2 was next examined in culture in the presence or absence of FSH (Fig. 9B). In the absence of FSH, production of the 72 kDa proMMP-2 declined with the duration of culture, the intermediate 66 kDa band stayed constant in intensity and the active 62 kDa form first appeared at 48 hours and increased in intensity at 72 hours of culture. Addition of FSH (50 ng ml-1) resulted in an enhancement of the three MMP-2 lytic bands after 72 hours of treatment. Note that after 48 hours of treatment with FSH, a slight but reproducible (n=3) increase of the 62 kDa band could be observed. The lytic bands corresponding to MMP-9 were also increased by FSH after 72 hours of treatment.
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DISCUSSION |
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To confirm and extend these findings, we used isolated testicular cells (somatic and germ cells) and northern and western blotting. We confirmed the previous finding (Sato et al., 1994) of a single 4.5 kb transcript and the protein migrating as a doublet of 63-60 kDa. An additional band of 45 kDa was detected in germ cell extracts only. Such band might correspond to the 44 kDa processed MT1-MMP lacking the N-terminal portion of the molecule and the entire catalytic domain (Stanton et al., 1998; Maquoi et al., 2000; Hernandez-Barrantes et al., 2000). We indeed used an antibody directed against the hinge domain located in the C-terminal part of the molecule. However, the reason why Sertoli cells and peritubular cells did not exhibit the processed band is currently unknown.
Collectively, our data are consistent with the idea that MT1-MMP could be one of the proteases involved in the extensive remodeling that occurs during the prepubertal period and in the adult rat testis at specific stages of the seminiferous cycle. MT1-MMP has been described as an interstitial collagenase (Ohuchi et al., 1997). However, this protease is known for its ability to activate proMMP-2 (Sato et al., 1994) in the presence of TIMP-2 (Caterina et al., 2000; Wang et al., 2000). As we observed that MMP-2 and TIMP-2 were transcribed together with MT1-MMP throughout testicular development and in somatic cells, we speculated that one role for MT1-MMP in testes might be to activate proMMP-2 in the presence of TIMP-2. To substantiate our hypothesis further, use was made of a model of cultured Sertoli cells treated with FSH or co-cultured with germ cells to mimic an immature (Griswold, 1995) or a mature (Le Magueresse and Jégou, 1988; Han et al., 1993; Wong et al., 2000) developmental period, respectively.
We first determined the kinetics of expression of the three partners (i.e. MMP-2, TIMP-2 and MT1-MMP) in Sertoli cells treated with FSH for up to 72 hours or not treated. Our data showed that MT1-MMP gene expression was high and constitutive (no fluctuation with time nor in the presence of FSH), whereas TIMP-2 protein levels increased with time and were significantly enhanced by FSH. Therefore the ratio of TIMP-2 to MT1-MMP increased as a function of time during the culture period analyzed (72 hours). Interestingly, in FSH-treated cultures at 48 hours and in control cultures at 72 hours, TIMP-2 antigen levels were more or less the same. A careful examination of the zymograms at these time points revealed the appearance of a distinct band at 62 kDa, corresponding to the active form of MMP-2. This 62 kDa lytic band increased further at 72 hours in FSH-treated cells, as did TIMP-2 antigen levels. These data are consistent with the literature documenting that proMMP-2 activation is highly sensitive to the stoichiometric ratios of TIMP-2 to MT1-MMP (Strongin et al., 1995; Shofuda et al., 1998). Whether TIMP-2 becomes inhibitory at a particular high concentration in the testis, as shown elsewhere (Hernandez-Barrantes et al., 2000; Wang et al., 2000), remains to be determined. Finally, we demonstrated that FSH not only promoted activation of proMMP-2 but also augmented MMP-2 expression at the mRNA and protein levels.
In a second part of this study, we examined germ cell control of Sertoli cell proMMP-2 activation. Spermatogenesis is known to depend on intricate and intimate relationships between Sertoli cells and germ cells (Jégou, 1993; Le Magueresse-Battistoni, 1993; Parvinen, 1993; Sharpe, 1994). We thus predicted that MMP-2 of Sertoli cell origin would aid the progressive movement of germ cells towards the tubular lumen during their differentiation into sperm cells. In turn, germ cells would control the rate and extent of proMMP-2 activation. Our prediction was also based on the finding of a processed MT1-MMP form in germ cells. Indeed, this form has been previously associated with binding to TIMP-2 and proMMP-2 activation (Hernandez-Barrantes et al., 2000). We thus examined whether germ cells that do not express MMP-2 (Fritz et al., 1993; this study) expressed TIMP-2. We found, in contrast to a previous RT-PCR study (Grima et al., 1996) that germ cells contained TIMP-2 transcripts and produced the 21 kDa TIMP-2 protein. At present, we cannot explain this discrepancy with the previous report.
When Sertoli cells and germ cells were co-cultured, we detected a dramatic increase in the accumulation of the MMP-2 antigen. The enhancement in MMP-2 production was even more evident with a crude germ cell suspension than with either pachytene spermatocytes or early spermatids. This probably resulted from the fact that the crude fraction contains elongated spermatids, which we found strongly immunoreactive for MT1-MMP. Using gelatin zymography, we extended these data and showed that germ cells could also enhance the intensity of the 62 kDa lytic band, corresponding to the active form of MMP-2. The second gelatinase (probably MMP-9) was also activated by germ cells.
The fact that MMP-2 levels declined for 72 hours from the time of plating was consistent with the time-dependent decrease in the number of contaminating germ cells in the Sertoli cell cultures and with the enhancement in MMP-2 production in co-culture. By contrast, we demonstrated that germ cells expressed TIMP-2 and MT1-MMP, and that TIMP-2 levels increased with time in culture whereas MT1-MMP levels did not change. We also observed that the germ cell specific MT1-MMP form was not present in the fresh Sertoli cell preparation, although it was contaminated with 10-15% germ cells. It appears therefore that the contaminating germ cells present in the fresh preparations of Sertoli cells were either not sufficient in number to reproduce the co-culture effects or that the contaminating spermatogonia and early spermatocytes do not influence the Sertoli cell MMP-2 machinery as more developed germ cells do. The possibility of isolating highly enriched populations of spermatogonia for co-culture with Sertoli cells should clarify the point.
Whether germ cell effects require cell-cell contact or whether they can occur with germ cell conditioned media will be addressed in future studies. Another issue to be resolved is identification of the possible intraepithelial substrates for MMPs. MMPs are known to cleave not only interstitial collagens and basement membrane components but also non-matrix components, with significant biological ramifications (Nagase and Woessner, 1999). Indeed, MMPs, or a cascade of proteolytic events activated by MMPs, can lead to cleavage and release of a variety of active molecules from cell surfaces, including bFGF. This is secreted by germ cells (Han et al., 1993) and is a potent activator of MMPs. Additionally, the junctional proteins that compose the blood-testis barrier (Byers et al., 1993) are potential targets for MMPs, as shown in other systems for integrin (Von Bredow et al., 1997), occludin (Wachtel et al., 1999) and cadherin (Steinhusen et al., 2001). Finally, it has been demonstrated that protease-sensitive elements hold spermatids and Sertoli cells together (Russell, 1980), but the nature of these protease-sensitive elements is at yet unknown.
In conclusion, we demonstrated that FSH and germ cells could enhance MMP-2 gene expression and levels of active MMP-2, with germ cells having a substantially greater effect than FSH under the conditions employed. Active MMP-2 might then be involved in the extensive remodeling that occurs within the seminiferous epithelium throughout development. MMP-2 is certainly not the sole protease involved. Mice with a single deficiency in various proteases and antiproteases including MMP-2 and TIMP-2 are overtly normal, viable and fertile. MT1-MMP-deficient mice, which displayed severe runting, wasting and increased mortality, can still reproduce (Carmeliet and Collen, 1998; Holmbeck et al., 1999). Serine- and cysteine- proteases are thought to be involved in the germ cell migratory route (Fritz et al., 1993; Mruk et al., 1997; Le Magueresse-Battistoni et al., 1998; Sigillo et al., 1999; Wong et al., 2000). Finally, four other MT-MMPs can activate proMMP-2 in a TIMP-2-sensitive fashion (Murphy et al., 1999; English et al., 2000). This suggests that the different proteases and antiproteases in the testis exert overlapping functions.
A challenge for the future will be to identify the full complement of proteases and their regulatory mechanisms. This will enable the design of additional studies to define precisely the role and relative importance of each in the complex steps of spermatogenesis. Then, the phenotypic effects in gene knockout experiments can be interpreted with knowledge of their integrated roles and potential for compensatory action.
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ACKNOWLEDGMENTS |
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