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From the Population Council, Center for Biomedical Research, New York, New York.
| Correspondence to: Dolores D. Mruk, Ph.D., Population Council, Center for Biomedical Research, 1230 York Avenue, New York, NY 10021. |
| Received for publication July 31, 2002; accepted for publication December 28, 2002. |
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Abstract |
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2-macroglobulin (a protease inhibitor
produced by Sertoli cells) or aprotinin (a serine protease inhibitor) into an
in vitro germ cell adhesion assay facilitated the attachment of fluorescently
labeled germ cells onto the Sertoli cell epithelium when compared to control,
which suggests that the assembly of adherens junctions may involve protease
inhibitors.
Key words: Protease inhibitor, Sertoli cell, germ cell, tight junction, adherens junction
In a previous report, which studied the assembly of adherens junctions between Sertoli and germ cells using an in vitro coculture system, it was shown that adhesion of germ cells onto the Sertoli cell epithelium is associated with an induction of proteases and protease inhibitors (Mruk et al, 1997). Another study showed that chloroquine, a protease inhibitor, could facilitate the assembly and maintenance of the Sertoli cell tight junction barrier (Okanlawon and Dym, 1996). Taken collectively, these results apparently suggest that proteases and protease inhibitors participate in junction dynamics. We herein describe the purification and partial characterization of a putative Sertoli cell protease inhibitor, known as tissue inhibitor of metalloproteases-1 (TIMP-1), from Sertoli cell conditioned medium (SCCM). The purification of TIMP-1 (Mr 31–29 kDa) is an extension of our earlier report on TIMP-2 (Mr 22 kDa) (Grima et al, 1996), because the retention times of both TIMPs overlapped in the preparative anion-exchange and C8 reverse-phase high-performance liquid chromatography (HPLC) steps. Anti-protease activity was also a characteristic of both of these proteins when [125I]-collagen was used as a substrate in a protease assay (Grima et al, 1996). On this note, we have used TIMP-1 as a target molecule to determine whether any changes in its expression could be detected during Sertoli-Sertoli and Sertoli–germ cell junction assembly in vitro, similar to our previous study (Mruk et al, 1997). Because many of the results presented in this report rely heavily on changes in the TIMP-1 mRNA level as detected by reverse transcription-polymerase chain reaction (RT-PCR), we have used an in vitro germ cell adhesion assay to demonstrate that protease inhibitors participate in junction dynamics by facilitating the binding of germ cells onto Sertoli cells.
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Materials and Methods |
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Preparation of Testicular Cell Cultures
Sertoli Cell Cultures—
Primary Sertoli cell cultures were prepared from 20-day-old rats by
sequential enzymatic treatments as described elsewhere
(Cheng et al, 1986) with
modifications as described below, which consistently yielded Sertoli cells
with a purity greater than 95%. In brief, minced tubules were resuspended in
serum-free Ham F12 nutrient mixture and Dulbecco modified eagle's medium
(F12/DMEM, 1: 1, [vol/vol]) (Sigma, St Louis, Mo) containing 1 mg/mL trypsin
(Type I, Sigma) and 20 µg/mL DNase (Sigma) and incubated for 30 minutes at
37°C. Thereafter, tubules were pelleted by centrifugation for 3 minutes at
800 x g and subjected to treatment with 1 M glycine and 2 mM
EDTA (pH 7.4) at 22°C containing 0.01% soybean trypsin inhibitor (STI,
[wt/vol], Type IIS; Sigma) and 20 µg/mL DNase for 10 minutes at 22°C to
lyse residual Leydig cells. The tubules were successively washed, resuspended
gently in 0.5 mg/mL collagenase (Type 1; Sigma) containing 0.005% STI (wt/vol)
and 5 µg/mL DNase, and incubated for 5 minutes at 37°C. This was
followed by resuspension in 1 mg/mL collagenase that contained 0.005% STI
wt/vol and 5 µg/mL DNase and incubation for 30 min at 37°C. After cells
were successively washed, they were resuspended in 1 mg/mL hyaluronidase that
contained 0.005% STI wt/vol and 5 µg/mL DNase and incubated for 30 minutes
at 37°C. Thereafter, cells were washed extensively and resuspended in
F12/DMEM supplemented with gentamicin (20 mg/L), sodium bicarbonate (1.2
gm/L), 15 mM HEPES, bovine insulin (10 µg/mL), human transferrin (5
µg/mL), bacitracin (5 µg/mL), and epidermal growth factor (2.5 ng/mL).
All enzymatic incubations at 37°C were done in an oscillating water bath
with a volume of 40 mL, and all washings were done by resuspending cells in
F12/DMEM and centrifuging for 3 minutes at 800 x g. For
high-density cell cultures where tight, anchoring, and gap junctions
assembled, isolated cells were plated on Matrigel Matrix (BD Biosciences,
Bedford, Mass) (diluted 1: 7 with F12/DMEM)–coated 12-well dishes at a
density of 0.5 x 106 cells/cm2 in F12/DMEM
supplemented with factors as described above. Cells were incubated at 35°C
in a humidified atmosphere of 95% air and 5% CO2 (vol/vol). To
obtain Sertoli cells with a purity greater than 95%, cultures were
hypotonically treated 48 hours after plating with 20 mM Tris (pH 7.4) at
22°C for 2.5 minutes to lyse contaminating germ cells
(Galdieri et al, 1981), followed immediately by 2 successive washes with lab stock F12/DMEM. For
low-density cell cultures where tight junctions did not form but cell-cell and
cell-substratum anchoring and gap junctions were present, isolated cells were
plated on Matrigel-free 100-mm dishes at a density of 5 x 104
cells/cm2 in F12/DMEM supplemented with factors as described above.
Media were replaced every 24 to 48 hours thereafter. In these cultures, time 0
(control) represents the time at which cells were plated. Total RNA was
subsequently extracted from these high- and low-density cultures by RNA
STAT-60 (Tel-test B Inc, Friendswood, Tex), as instructed by the manufacturer.
In experiments in which the steady-state TIMP-1 mRNA level was examined in
Sertoli cells isolated from aging testes, Sertoli cells were isolated as
described elsewhere with minor modifications
(Li et al, 2001), and cultured
for 5 days prior to their termination. Contaminating germ cells were removed
from 45- and 90-day-old Sertoli cells by consecutive hypotonic treatments on
days 2 and 3. In all experiments in which Matrigel was used, dishes were
coated 24 hours prior to the plating of Sertoli cells, to allow the Matrigel
to dry completely before plating cells.
Preparation of SCCM for the Purification of TIMP-1— Batches of approximately 10 L of SCCM routinely obtained from Sertoli cell cultures isolated from approximately 50 20-day-old rats were used for the purification of TIMP-1. Media were successively centrifuged at 800 and 45 000 x g in order to remove cellular debris. Samples were pooled, concentrated 100-fold at 4°C, and equilibrated against 20 mM Tris (pH 7.4) at 22°C using a Millipore (Bedford, Mass) Minitan tangential ultrafiltration unit equipped with 8 Minitan plates with a molecular weight cutoff of 10 kDa. The sample was then filtered through a 0.2µm filter unit and stored at –20°C until HPLC.
Assessing the Assembly of the Sertoli Cell Tight Junction Permeability Barrier In Vitro— The assembly of the Sertoli cell tight junction barrier was assessed by 2 different criteria. In brief, Sertoli cells were plated on Matrigel-coated bicameral units (Millicell-HA [mixed esters of cellulose] 0.45 µm membrane pore size, 12 mm diameter) (Millipore) at a density of 1.2 x 106 cells/cm2. First, transepithelial electrical resistance (TER) across the Sertoli cell epithelium was quantified using a Millicell electrical resistance system (Millipore), as previously described from this laboratory (Grima et al, 1998; Chung and Cheng, 2001; Lui et al, 2001). Second, the restricted influx of fluorescein isothiocyanate (FITC)–dextran (4.4 and 35.6 kDa, Sigma) from the apical to the basal compartment of bicameral units was monitored by cytofluorometry. Media (500 µL) with and without 50 µM FITC-dextran was added into apical and basal compartments, respectively. This was followed by incubation at 35°C for either 6 or 18 hours, at which time media was collected, centrifuged briefly at 800 x g to remove cellular debris, 100 µl aliquoted into 96-well plates, and fluorescence quantified by a Tecan GEN-ios cytofluorometer (Salzburg, Austria) at 530 nmEX/590 nmEM.
Germ Cell Cultures—
For Sertoli–germ cell coculture experiments, germ cells were isolated
from adult rat testes by a mechanical procedure without the use of trypsin as
described elsewhere (Aravindan et al,
1996,
1997), because trypsinization
has been shown to affect the functional properties of these cells. For germ
cells isolated from 90-day-old testes, the ratio of spermatogonia:
preleptotene spermatocytes: primary spermatocytes: round spermatids (elongate
spermatids and spermatozoa were removed by 
3 successive passages through
glass wool) in these cell preparations was 16.7: 8.3: 9.7: 65.3, as determined
by DNA flow cytometry in an independent series of experiments
(Clermont and Perey, 1965;
Aravindan et al, 1996). Somatic
cell contamination was virtually negligible when assessed by various criteria
as detailed elsewhere (Aravindan et al,
1996;
1997; Lee et al, 2002). Germ
cells were used within 1 hour of their isolation for Sertoli–germ cell
cocultures. In experiments in which the TIMP-1 mRNA level was examined in germ
cells isolated from testes of aging rats, germ cells were isolated as detailed
elsewhere and terminated immediately using RNA STAT-60.
Sertoli–Germ Cell Cocultures— Sertoli cells isolated as described above were used for Sertoli–germ cell coculture experiments (Mruk et al, 1997). Sertoli cells were plated on Matrigel-coated 24-well dishes at a density of 0.5 x 106 cells/cm2. They were hypotonically treated 48 hours after their isolation. Thereafter, cells were cultured for an additional 3 days to allow for the formation of an epithelium with intact tight, anchoring, and gap junctions as described elsewhere (Grima et al, 1992; Mruk et al, 1997; Chung et al, 1999). These cells were then used for coculture experiments. In brief, isolated germ cells were cocultured with Sertoli cells using a Sertoli: germ cell ratio of 1: 1 in F12/DMEM supplemented with 6 mM sodium DL-lactate, 2 mM sodium pyruvate, insulin (10 µg/mL), transferrin (5 µg/mL), bacitracin (5 µg/mL), and epidermal growth factor (2.5 ng/mL). In these cocultures time 0 (control) represents the time at which germ cells were added onto the Sertoli cell epithelium, so that the relative ratio of Sertoli:germ cell RNA at this time point was 1:1. Thereafter, cocultures were terminated at specified time points by using RNA STAT-60. Control experiments consisted of Sertoli cells cultured under the same conditions as described above but without the addition of germ cells. The viability of germ cells cocultured with Sertoli cells was greater than 95% throughout the entire culture period, as judged by the trypan blue dye exclusion test (Phillips and Terryberry, 1957).
Purification of TIMP-1 from SCCM
Anion-Exchange HPLC—
Approximately 200 mg of SCCM protein obtained as described above was loaded
onto a preparative Mono Q (Pharmacia Biotech, Piscataway, NJ) anion-exchange
HPLC column (HR 16/10; 16 x 100 mm, internal diameter [id]). Bound
proteins were eluted using a linear gradient from 0% to 80% Solvent B (20 mM
Tris [pH 7.4] at 22°C containing 600 mM NaCl) at a flow rate of 4 mL/min
over a period of 90 minutes, as described elsewhere
(Grima et al, 1996). The
eluents were monitored by UV absorbance at 280 nm, and fractions of 4 mL were
collected. For this and all subsequent fractionation steps, an aliquot from
selected fractions was withdrawn for sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) run under reducing
conditions, and proteins were visualized by silver staining as detailed
elsewhere (Wray et al,
1981).
C8 Reverse-Phase HPLC— On the basis of TIMP-1's electrophoretic mobility on SDS-PAGE, fractions obtained from the preceding step were pooled, lyophilized, and resuspended in solvent A (5% acetonitrile [vol/vol], 95% water [vol/vol] containing 0.1% trifluoroacetic acid [vol/vol]) and loaded onto a Vydac (Hesperia, Calif) C8 reverse-phase HPLC column (4.6 x 250 mm, id). Bound proteins were eluted using a linear gradient from 5% to 80% solvent B (95% acetonitrile [vol/vol], 5% water [vol/vol] that contained 0.1% trifluoroacetic acid [vol/vol]) at a flow rate of 1 mL/min over a period of 60 minutes. The eluents were monitored by UV absorbance at 280 nm, and fractions of 1 mL were collected.
High-Performance Electrophoresis Chromatography (HPEC)— Fractions obtained from the preceding step containing TIMP-1 were pooled, lyophilized, and resuspended in 40 µL double-distilled water. This was combined with an equal volume of HPEC sample buffer (7.5 mM Tris [pH 6.8] at 22°C containing 0.5% SDS [wt/vol], 1.6% 2-mercaptoethanol [vol/vol], 15% glycerol [vol/vol], and 0.005% bromophenol blue [wt/vol]) and subsequently denatured. This sample was then loaded onto a 10% T (%T = total acrylamide concentration) gel (3.5 x 50 mm, id) in an Applied Biosystems 230A HPEC System (Foster City, Calif) using a Tris-phosphate buffer system and fractionated as described elsewhere (Leone et al, 1993; Saso et al, 1993).
Protein Microsequencing
Approximately 15–20 µg of purified TIMP-1 was resolved on a 12.5%
T SDS–polyacrylamide gel run under reducing conditions. This
was followed by electrophoretic transfer onto a polyvinylidene difluoride
membrane (Applied Biosystems) using a buffer system consisting of 10 mM
3-(cyclohexylamino)-1-propanesulfonic acid (pH 11.0) at 22°C containing
10% methanol (vol/vol). Thereafter, the blot was stained with Coomassie blue
R-250 (0.1% Coomassie blue R-250 [wt/vol], 50% methanol [vol/vol], and 10%
acetic acid [vol/vol]) and sequenced as described elsewhere
(Cheng et al, 1988; Leone et al, 1993;
Saso et al, 1993; Mruk and
Cheng, 1999). Phenylthiohydantoin (PTH)–amino acids were identified and
quantified by HPLC using a Brownlee PTH-C18 (2.1 x 220 mm, id) column
(Applied Biosystems) in an Applied Biosystems 473A pulsed-liquid phase protein
sequencer. The repetitive yield was approximately 96%. Protein sequencing was
repeated at least twice using 2 different batches of purified protein, and
identical results were obtained in both instances.
TIMP-1 Anti-Protease Assay
To quantify the anti-protease activity of TIMP-1, a
[125I]-collagen film assay
(Johnson-Wint, 1980) was used,
with minor modifications (Grima et al,
1996; Aravindan et al,
1997; Mruk et al,
1997). In brief, collagen was iodinated using Iodogen and
[125I]-Na (Amersham Pharmacia Biotech, Arlington Heights, Ill), and
approximately 20 000 cpm of [125I]-collagen resuspended in buffer A
(0.05 M sodium acetate [pH 5.5] at 22°C containing 0.15 M NaCl and 1 mM
CaCl2) was used for coating 96-well, flat-bottom plates (Corning)
as described elsewhere (Grima et al,
1996; Mruk et al,
1997). TIMP-1 anti-protease activity was monitored by its ability
to inhibit collagenase (a metalloprotease) from cleaving
[125I]-collagen, releasing iodinated fragments into the
supernatant, which were subsequently collected for the determination of
radioactivity by spectrometry using a 
-counter (Packard Cobra II) with
98% counting efficiency. To quantify the anti-protease activity of TIMP-1, it
was necessary to add protease inhibitor (TIMP-1 or STI, Sigma) into
[125I]-collagen–coated wells prior to the addition of
collagenase. Each well contained the appropriate amount of collagenase,
TIMP-1, or STI to a total volume of 200 µL. The assay plate was then
incubated at 35°C for 2 hours in a humidified atmosphere to allow
hydrolysis of [125I]-collagen. Total counts were determined by
incubating wells with 10 µg of collagenase. A nonspecific control was
performed by using protease buffer (50 mM Tris [pH 7.4] at 22°C,
containing 0.15 M NaCl, 1 mM CaCl2, and 0.02% NaN3)
alone without the addition of collagenase or TIMP-1. Each data point had
triplicate wells, and each experiment was repeated at least 3 times using
different batches of highly purified TIMP-1.
RNA Extraction and RT-PCR
Total RNA was extracted from cells using RNA STAT-60, according to the
manufacturer's instructions. RT-PCR was performed essentially as described
elsewhere (Mruk and Cheng, 1999). In brief, 2 µg of total RNA was reverse
transcribed into cDNAs using 0.3 µg of oligo (dT)15 and an M-MLV
reverse transcriptase kit (Promega, Madison, Wisc) in a final reaction volume
of 25 µL. From this reaction product, 3 µL served as a template for PCR
in combination with 0.3 µg each of the TIMP-1 sense and antisense primers.
The primers used for the amplification of TIMP-1
(Okada et al, 1994) and S16
(Chan et al, 1990) were as
follows: 5'-TGGTTATAAGGGCTAAAT-3' (TIMP-1, sense, nucleotides 137
to 154), 5'-GCCCGCGATGAGAAACTC-3' (TIMP-1, antisense, nucleotides
331 to 348), 5'-TCCGCTGCAGTCCGTTCAAGTCTT-3' (S16, sense,
nucleotides 15 to 38), and 5'-GCCAAACTTCTTGGATTCGCAGCG-3' (S16,
antisense, nucleotides 376 to 399). Coamplification with S16 was included to
ensure that equal amounts of RNA were reverse transcribed and amplified in
each reaction tube. The cycling parameters for the PCR reaction were as
follows: denaturation at 94°C for 1 minute, annealing at 58°C for 2
minutes, and extension at 72°C for 3 minutes, using a Perkin Elmer GeneAmp
2400 PCR system. Twenty-five cycles were performed. The cycles were followed
by an extension period at 72°C for 15 minutes. Thereafter, aliquots of 10
µL were withdrawn from each of the PCR reaction tubes and resolved onto 5%
T polyacrylamide gels in TBE buffer (45 mM Tris, 45 mM boric acid,
and 1 mM EDTA [pH 8.0] at 22°C). In some instances, 0.2 µg of the
TIMP-1 antisense primer was 5' end-labeled with
[32P]-ATP (specific activity, 6000 Ci/mmol; Amersham Pharmacia
Biotech) by using T4 polynucleotide kinase (Promega). The S16 antisense primer
was also 5' end-labeled for coamplification, as described above. Under
these conditions, the amplifications of TIMP-1 and S16 were both in the linear
range, as verified in a series of preliminary experiments when different
concentrations of TIMP-1 and S16 sense and antisense primers were combined
with testis, Sertoli, or germ cell cDNA. Thereafter, aliquots of 10 µL were
withdrawn from each of the PCR reaction tubes in cycles 18, 20, 22, 25, 27,
and 30 for gel analysis. PCR products were either visualized by ethidium
bromide staining or autoradiography using Kodak X-ray films (Rochester,
NY).
In Vitro Germ Cell Adhesion Assay
Sertoli cells (0.5 x 106 cells/cm2, 20 days
old) isolated as previously described were plated on Matrigel-coated 24-well
dishes and cultured for 5 days. Thereafter, isolated germ cells (90 days old)
were fluorescently labeled with
1,1'-dioctadecyl-6,6'-di(4-sulfophenyl)-3,3,3',3'-tetramethylindocarbocyanine
[SP-DiIC18(3)] (Molecular Probes, Eugene, Ore).
SP-DiIC18(3) is a lipophilic, sulfonated carbocyanine derivative
that is soluble in aqueous buffer (ie, does not precipitate during cell
labeling), binds only to the membranes of viable germ cells, and is stable for
the entire culture period used in this study. In brief, germ cells were
labeled by incubating with 1–2 µM of SP-DiIC18(3)/1
x 106 cells/1 mL media for 5 minutes at 35°C. The stock
solution of SP-DiIC18(3)(20 µM) was prepared in DMSO (Sigma).
This was followed by an additional incubation for 15 minutes at 4°C. These
fluorescently labeled germ cells were then successively washed in F12/DMEM to
remove unbound dye, and added onto the Sertoli cell epithelium using a
Sertoli: germ cell ratio of 1: 1 in the absence (control) or presence of
2-macroglobulin (50 µg/mL; 2-MG,
purified from SCCM as described elsewhere
[Cheng et al, 1990]), aprotinin
(100 µg/mL; Calbiochem, La Jolla, Calif), or a monospecific
anti-2-MG antibody (1: 1000 dilution, decomplemented at
56°C for 30 minutes before use) as characterized elsewhere
(Cheng et al, 1990;
Stahler et al, 1991). At
specified time points, cocultures were terminated by successively washing each
well with 1 mL of lab stock F12/DMEM to remove germ cells that did not adhere
onto the Sertoli cell epithelium, and fluorescence was quantified by
cytofluorometry. The viability of germ cells was not affected during the
labeling procedure, because the final concentration of DMSO was below 10%.
Poor cell viability, after all, would prevent the binding of germ cells onto
the Sertoli cell epithelium. In addition, the incubation of germ cells at the
lower temperature did not affect cell viability. Instead, it slowed down
endocytosis, which prevented the dye from accumulating in cytosolic vesicles.
This allowed for efficient labeling of the plasma membrane. Although this
assay quantitatively estimates the adhesion of germ cells onto the Sertoli
cell epithelium, it also assesses the assembly of adherens junctions between
these cells, because cell attachment is a prerequisite for the assembly of
adherens junctions, which are known to form within 24 to 48 hours
(Enders and Millette, 1988;
Cameron and Muffly, 1991). It
must also be noted that we had initially attempted to perform this study using
TIMP-1. However, the yield of TIMP-1 isolated from SCCM was too low (we could
only obtain 10 µg of highly purified TIMP-1 from 5 L of SCCM vs 1 mg of
2-MG from the same amount of SCCM) and this in vitro assay
required at least 1 mg of highly purified protein. Therefore, we opted to use
2-MG and aprotinin instead. Each data point had triplicate
wells, and each experiment was repeated at least 3 times using different
batches of fluorescently labeled germ cells.
General Methods
SDS-PAGE was performed as described elsewhere
(Laemmli, 1970;
Cheng et al, 1985). The
resolving gel consisted of 12.5% T (%T = gm/100 ml
acrylamide + bis-acrylamide) and 2.6% C
N,N'-methylene-bis-acrylamide as cross-linker [%C =
cross-linker concentration; 100 x methylenebis(acrylamide)/[acrylamide +
methylenebis(acrylamide)]. The stacking gel consisted of 8.5% T and
15% C N,N-diallytartardiamide (DATD, cross-linker). All sample
aliquots, unless noted otherwise, were denatured and reduced in SDS sample
buffer (0.125 M Tris [pH 6.8] at 22°C containing 1% SDS [wt/vol], 20%
glycerol [vol/vol], and 1.6% 2-mercaptoethanol [vol/vol]) at 100°C for 10
minutes. For subunit structural analysis of TIMP-1, approximately 0.25 µg
of purified TIMP-1 obtained from the HPEC purification step was resolved by
SDS-PAGE under reducing and nonreducing conditions. For light microscopy,
Sertoli cells (0.5 x 106 cells/cm2) cultured on
Matrigel-coated bicameral units as described elsewhere, were processed by
excising membranes on which cells were cultured, submerging membranes in
Bouin's fixative for 5 minutes, and briefly rinsing in PBS (pH 7.4).
Thereafter, membranes were embedded in gelatin (10% [wt/vol]; Type B from
bovine skin, 75 bloom, Sigma), 8-µm sections cut perpendicular to the plane
of the membrane, sections stained with hematoxylin, and mounted for microscopy
using an Olympus BX-40 system microscope (Olympus America Inc, Melville, NY).
After germ cells were fluorescently labeled, they were plated on 6-well
dishes. Cells were then terminated 3 and 24 hours after plating by rinsing
wells extensively with F12/DMEM, mounted with VECTASHIELD (Vector Laboratories
Inc, Burlingame, Calif), and examined microscopically using fluorescent
optics. All images were captured with an Olympus Micro-Publisher Cooled
Imaging digital camera with FireWire connection. The authenticity of the
212-bp TIMP-1 PCR product was verified by direct nucleotide sequencing using
Sequenase (Amersham Pharmacia Biotech), as described elsewhere
(Cheng et al, 1988).
Densitometric scanning of all autoradiograms was performed using SigmaGel
software package (version 1.0; SPSS Inc, Chicago, Ill). Statistical analysis
was performed by either Student's t-test (all data points were
compared against the control) or ANOVA (all data points were compared against
each other) using SigmaStat (version 2.0; SPSS Inc). All experiments performed
in the present study had triplicate data points, and each experiment was
repeated 2 to 5 times.
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Results |
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Anti-Protease Activity of TIMP-1
The biological activity of highly purified TIMP-1 was monitored by its
ability to block the action of collagenase from cleaving
[125I]-collagen. As shown in the Table, 10 µg collagenase (total
counts, positive control) actively cleaved [125I]-collagen,
releasing iodinated fragments into the supernatant, which were subsequently
collected for the quantitation of radioactivity. Although STI, a serine
protease inhibitor, had no effect in blocking collagenase activity (Table),
TIMP-1 effectively arrested the activity of collagenase (Table). These results
illustrate that TIMP-1 is a biologically active polypeptide possessing
anti-protease activity that can block metalloproteases, such as
collagenase.
Developmental Expression of TIMP-1 in the Testis
We proceeded to determine whether any changes in TIMP-1 expression could be
detected in developing testes, during which there is an increase in the number
of germ cells and Sertoli–germ cell interactions. It was found that the
steady-state TIMP-1 mRNA level increased fourfold by 60 days of age, followed
by a significant decrease in expression from 90 to 120 days of age
(Figure 2A and B).
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Developmental Expression of TIMP-1 in Sertoli and Germ Cells
To assess whether the increase in testicular TIMP-1 expression reflects
changes in the Sertoli: germ cell ratio during development, we investigated
the steady-state TIMP-1 mRNA level in Sertoli and germ cells during
maturation. When RNA was extracted from Sertoli cells isolated from testes at
20, 45, and 90 days of age for semiquantitative RT-PCR, the TIMP-1
steady-state mRNA level was shown to increase during aging
(Figure 3A and B). This
increase in TIMP-1 expression is, however, in sharp contrast to the trend that
was detected in germ cells (Figure 3C and
D). Figure 3C and D
illustrate the presence of TIMP-1 mRNA in 10-, 15-, and 20-day-old germ cells
(ie, spermatogonia and primary spermatocytes). This was not the result of
somatic cell contamination when aliquots of RNA from these samples were
processed for RT-PCR using primer pairs specific for testin, a Sertoli cell
product; 3ß-hydrosteroid dehydrogenase, a Leydig cell product; and
fibronectin, a peritubular myoid cell product, as described elsewhere (Lee et
al, 2002). TIMP-1 expression was undetectable in germ cells isolated from
testes at 45, 60, 90, and 120 days of age
(Figure 3C and D).
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Changes in the TIMP-1 Steady-State mRNA Level During Sertoli Cell
Junction Assembly
Using semiquantitative RT-PCR, the TIMP-1 steady-state mRNA level increased
transiently but significantly (Figure 4A
and B) when Sertoli cells were cultured at high density (0.5
x 106 cells/cm2), as shown in
Figure 4C, to induce the
formation an epithelium with intact tight, anchoring, and gap junctions.
Figure 4A and B show that the
induction in the TIMP-1 mRNA level was restricted to days 1 and 2 during the
assembly of the Sertoli cell tight junction permeability barrier when TER
across the epithelium was measured in cells cultured at high density (1.2
x 106 cells/cm2)
(Figure 4D). The tight junction
permeability barrier also assembled in Sertoli cells cultured at a much lower
cell density (0.5 x 106 cells/cm2,
Chung et al, 2001), the
density used for experiments shown in
Figure 4A and B. When Sertoli
cells were cultured at low density (5 x 104
cells/cm2), tight junctions failed to assemble because of the lack
of proximity between cells (Figure
4D). After tight junction assembly was completed by day 2, TIMP-1
expression decreased to a barely detectable level
(Figure 4A and B). The
formation of the tight junction barrier in Sertoli cells cultured at high
density (1.2 x 106 cells/cm2) was confirmed when
the restricted influx of FITC-dextran (4.4 kDa,
Figure 4E; and 35.6 kDa,
Figure 4F) from the apical to
the basal compartment was quantified by cytofluorometry. After tight junction
assembly, diffusion of FITC-dextran into the basal compartment of bicameral
units became restricted. An increase in TIMP-1 expression was also detected
when Sertoli cells were cultured at a substantially lower density (5 x
104 cells/cm2) in which cell-cell and cell-substratum
anchoring, and gap, but not tight, junctions were capable of forming
(Figure 4G and H).
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Changes in the TIMP-1 Steady-State mRNA Level During
Sertoli–Germ Cell Adherens Junction Assembly
Because testicular TIMP-1 expression was up-regulated during development
(Figure 2A and B), and this was
not likely the result of a change in the Sertoli: germ cell ratio
(Figure 3 A through D), we
examined whether the increase in the TIMP-1 mRNA level can be attributed to
Sertoli–germ cell interactions. To investigate this, we cocultured germ
cells with Sertoli cells using a Sertoli: germ cell ratio of 1: 1. Prior to
adding germ cells, Sertoli cells (0.5 x 106
cells/cm2) were cultured for 5 days to allow for the establishment
of tight, anchoring, and gap junctions. When germ cells were added, it was
found that the steady-state TIMP-1 level increased three- to sixfold during
the adhesion of germ cells onto the Sertoli cell epithelium
(Figure 5A and B). When
adherens junctions had already assembled between Sertoli and germ cells by day
2, TIMP-1 expression returned to its basal level
(Figure 5A and B). We did not
detect changes in TIMP-1 expression when Sertoli cells were cultured under the
same conditions as described above but without the addition of germ cells
(control, data not shown).
|
Protease Inhibitors Can Facilitate the Adhesion of Germ Cells Onto
Sertoli Cells
Results presented in this report suggest the participation of TIMP-1 in
cell junction assembly. However, the precise role of protease inhibitors in
junction dynamics has not yet been defined. To determine whether protease
inhibitors could affect the adhesion (a prerequisite of junction assembly) of
germ cells onto Sertoli cells, we used an in vitro cytofluorometric adhesion
assay. The inclusion of 2-MG (50 µg/mL) facilitated the
adhesion of germ cells onto Sertoli cells by 24 hours compared with controls
(Figure 6A), whereas the
presence of anti-2-MG antibody perturbed germ cell adhesion
(Figure 6A). The effects of
2-MG on germ cell adhesion onto the Sertoli cell epithelium
could be reproduced by using aprotinin, a serine protease inhibitor not
produced by Sertoli or germ cells (Figure
6A). Figure 6B
shows fluorescently labeled germ cells, consisting largely of spermatogonia,
spermatocytes, and round spermatids, cultured on 6-well dishes 3 and 24 hours
after labeling, which illustrates that germ cells remained labeled during the
coculture period.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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To determine whether changes in the TIMP-1 mRNA level correlated with the events of junction assembly and/or disassembly, such as during the formation of the blood-testis barrier or the first wave of spermiation, we relied on semiquantitative RT-PCR. The increase in testicular TIMP-1 expression from 40 to 60 days of age is suggestive of its participation in the events of spermiation. However, the increase in testicular TIMP-1 expression cannot be ascribed to germ cells, because we failed to detect TIMP-1 mRNA in germ cells isolated from rats at 45 to 120 days of age, which is similar to a previously published report by Gronning et al (2000). Subsequently, the increase in testicular TIMP-1 expression was justified when the Sertoli cell TIMP-1 mRNA level was found to increase from 45 to 90 days of age. The increase in testicular TIMP-1 may also be assigned to Sertoli–germ cell interactions or to peritubular myoid, but not Leydig cells, because they were also found to express TIMP-1 (Gronning et al, 2000). Taking these results collectively, it is possible that an elevated level of Sertoli cell TIMP-1 may be required to neutralize endogenous MMP activity immediately after spermiation. As such, TIMP-1 may function by promoting junction assembly and, in turn, maintaining the integrity of the epithelium. It is also likely that other TIMP family members participate in these events as well.
On the other hand, we were able to detect TIMP-1 expression in 10-, 15-, and 20-day-old germ cells, consisting of spermatogonia and primary spermatocytes. The presence of TIMP-1 mRNA in germ cells was not due to somatic cell contamination because, in a series of preliminary experiments by RT-PCR, we did not amplify testin (a Sertoli cell product; Cheng et al, 1989; Zong et al, 1994), 3ß-hydrosteroid dehydrogenase (a Leydig cell product; Zwain et al, 1991), and fibronectin (a peritubular myoid cell product; Tung et al, 1984), as described elsewhere (Lee et al, 2002). Although we do not know the immediate reason for the presence of TIMP-1 mRNA in 10-to 20-day-old germ cells, an elevated level of TIMP-1 may need to be restricted to the basal compartment of the epithelium to facilitate the assembly of the blood-testis barrier, which takes place at 15 to 18 days of age in the rat. Despite the fact that germ cells per se do not contribute directly to the assembly of the Sertoli cell tight junction, it is very likely that these cells participate indirectly via a yet-to-be-identified mechanism. Second, we failed to detect an increase in testicular TIMP-1 expression from 10 to 20 days of age, probably because other events, unrelated to the assembly of the blood-testis barrier, are taking place, thereby masking the up-regulation in TIMP-1 mRNA. Third, these germ cells, which reside in the basal compartment adjacent to the basal lamina, may also regulate the remodeling of the extracellular matrix. Last, TIMP-1 expression was not detected in 45- to 120-day-old germ cells, which suggests that secondary spermatocytes and round spermatids (elongate spermatids and spermatozoa were removed from all of our germ cell isolations) do not synthesize this protease inhibitor.
More important, when Sertoli cells were cultured at high density, TIMP-1
expression was found to increase approximately threefold during junction
assembly, followed by a significant decline from days 3 to 5 after junctions
assembled. At present, it is difficult to target changes in the TIMP-1 mRNA
level to the assembly of a single junction type, because the assembly of
tight, anchoring, and gap junctions in epithelial cells is interdependent. We
also do not know whether similar changes in expression could be detected with
other protease inhibitors, such as 2-MG, cystatin C, or
plasminogen activator inhibitor. However, we do anticipate that an array of
protease inhibitors functions during junction assembly with a majority of the
inhibitors being up-regulated, although some may be down-regulated.
Similar changes in TIMP-1 expression were detected when germ cells were cocultured with Sertoli cells, co-inciding with the adhesion of germ cells onto the Sertoli cell epithelium. The increase in TIMP-1 expression as early as 5 minutes suggests that this protease inhibitor may be required to limit endogenous MMP activity at the site of Sertoli–germ cell interaction. This study did not determine, however, whether a higher level of TIMP–anti-protease activity can be detected in apical chambers of bicameral units. In control cultures, we failed to detect changes in TIMP-1 expression when Sertoli cells were cultured alone without the addition of germ cells.
The results presented in this study demonstrate that the inclusion of
protease inhibitors can facilitate the adhesion of fluorescently-labeled germ
cells onto the Sertoli cell epithelium, whereas anti-2-MG
antibody was able to perturb adhesion when compared with control cocultures.
We speculate that the inclusion of TIMP-1 into this in vitro adhesion assay
would also improve germ cell binding. Ideally, an experiment should be
performed in which inhibitors that block serine, cysteine, aspartic, and
metalloprotease activity are added into this in vitro assay and the adhesion
of germ cells assessed; commercially available protease inhibitors, however,
are toxic to cells. To circumvent this technical difficulty, we are presently
attempting to localize proteases and protease inhibitors by fluorescent
microscopy to the site of Sertoli–germ cell interaction using
cross-sections of Sertoli–germ cell co-cultures that have been cultured
on bicameral units. This experiment should demonstrate the importance of these
molecules in junction assembly.
It is also possible that other yet-to-be-defined factors, such as
cytokines, are functioning in this in vitro system, in addition to proteases
and protease inhibitors. For instance, 2-MG can bind growth
factors, such as TGF-ß3, rendering TGF-ß3 inactive
(O'Connor-McCourt and Wakefield,
1987; Huang et al,
1988). In the testis, TGF-ß3 can regulate Sertoli cell tight
junction dynamics, because its presence can perturb the tight junction
permeability barrier in vitro by modulating occludin and ZO-1 levels
(Lui et al, 2001). On the
other hand, basic fibroblast growth factor ß and TGF-ß3 can affect
proteases, such as plasminogen activator, a serine protease, in Sertoli cells
in vitro (Jaillard et al,
1987; Nargolwalla et al,
1990). Taken collectively, the results presented in the present
study suggest the participation of protease inhibitors in cell junction
dynamics.
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Footnotes |
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