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The Population Council (G.R.A., D.M., C.Y.C.), Center for Biomedical Research, New York, New York 10021; and Department of Zoology (D.M., W.M.L.), The University of Hong Kong, Hong Kong
Address all correspondence and requests for reprints to: C. Yan Cheng, Ph.D., The Population Council, 1230 York Avenue, New York, New York 10021. E-mail: yan{at}popcbr.rockefeller.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2-macroglobulin (a Sertoli cell secretory product) also
in a dose-dependent manner. A study on the interactions between
different classes of protease inhibitors and the purified 41-kDa
protein revealed that it is a serine protease. At doses ranging between
0.5 and 50 ng/ml, 41-kDa-P induced a dose-dependent inhibition of
Sertoli cell secretory function using testin and clusterin as markers
without any apparent proteolytic activity. However, at doses greater
than 0.5 µg/ml, 41-kDa-P was found to cleave
[125I]-collagen and [125I]-testin at
physiological pH, indicating that this 41-kDa protein has dual
biological activities whose primary action is concentration dependent.
In view of the biological activities of this protease, it is postulated
that this protein may be involved in facilitating germ cell migration
in the epithelium. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-macroglobulin (6, 7),
TIMP-2 (8, 9), TIMP-1 (8), cystatin C (10); and cell
junction-associated proteins such as actin, vinculin (11), zonula
occludens 1 (ZO-1) (12), N-cadherin (13, 14), and testin (15, 16, 17). Recent studies on the expression of rat cathepsin L mRNA and its protein localization by immunohistochemical techniques in different stages of the spermatogenic cycle have shown that this cysteine protease is predominantly present in stages Vl–VII of the cycle preceding spermiation, suggesting that it may be involved in the degradation of cell adhesion molecules between mature spermatids and Sertoli cells, thereby facilitating the release of spermatids into the seminiferous tubular lumen (1, 18). Proteases are known to participate in tissue remodeling in the testis (for review, see 19 and in other organs such as in the ovary during ovulation (for review, see 20 and during tumorigenesis (for review, see 21 . Moreover, specific proteolysis of tight junctions has been postulated to be responsible for the translocation of spermatocytes across the blood-testis barrier (4, 22).
During the past decade, studies from several laboratories have
demonstrated that germ cells release or express several signaling
molecules that modulate Sertoli cell secretory functions such as: 1)
basic fibroblast growth factor (FGF) derived from pachytene
spermatocytes that stimulates Sertoli cell transferrin expression (23);
2) nerve growth factor (NGF) that was localized in germ cells of the
testis (24) has been shown to be a potent mitogen secreted by round
spermatids that may participate in germ cell proliferation during
spermatogenesis (25); and 3) germ cells are known to express
interferon- and -
(26), among others. However, in a painstaking
effort to purify a potent biological factor from germ cell-conditioned
medium (GCCM) that modulates Sertoli cell clusterin and testin
secretion, it was found to be the residual trypsin that was used
initially to isolate germ cells from the tubules rather than a putative
germ cell factor (27). The use of trypsin for the isolation of highly
purified germ cells for in vitro studies was an accepted
procedure used by many laboratories including ours (28, 29, 30). Other
studies have shown that trypsin can also induce changes on the adhesion
and binding properties of spermatogenic cells (31). In the current
study, we report the identification, purification, and characterization
of a factor from GCCM where germ cells were isolated from the rat
testis by a mechanical procedure without the use of trypsin (27). This
protein was found to possess dual biological actions that affect
Sertoli cell secretory function or cleaves multiple proteins depending
on its concentration in the in vitro assay.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GCCM preparation
GCCM was prepared by a nonenzymatic mechanical method as
previously described (27, 32) using testes from adult Sprague-Dawley
rats (250–300 g BW). The purity of germ cells isolated by this
procedure was assessed by DNA flow cytometry and direct microscopic
examination. Both approaches did not reveal any observable somatic cell
contaminations (27). Moreover, when total RNA (about 1 µg) extracted
from these germ cell preparations was used for RT-PCR to detect testin,
an authentic Sertoli and Leydig cell product, it failed to amplify the
testin complementary DNA (16, 27), suggesting the somatic cell
contamination is almost negligible. The recovered cells were
reconstituted in Ham’s F12/DMEM supplemented with 1.2 g/liter sodium
bicarbonate, 15 mM HEPES, gentamycin (20 mg/liter), 6
mM sodium DL-lactate, 2 mM sodium pyruvate, and
bacitracin (5 µg/ml, a protease inhibitor) and plated at a
concentration of 2.5 x 106 cells/ml in 75
cm2 culture flasks in a final volume of 15 ml. Cells were
incubated at 35 C in 95% air/5% CO2 (vol/vol) for 20
h. Under these conditions, the cell viability was greater than 90% at
the end of the 20-h culture period when examined by erythrosine red dye
exclusion test. The spent medium was carefully decanted and centrifuged
successively at 800 x g (1 h) and 5000 x
g (1 h) to remove residual germ cells. The supernatant was
stored at -20 C until used.
Iodination of collagen
Collagen obtained commercially was purified to apparent
homogeneity by reversed-phase HPLC using a Vydac C4 (4.6 x 250
mm, id) column. About 600 µg of collagen was suspended in 200 µl of
the solvent A [5% acetonitrile (ACN)/95% water (vol/vol), containing
0.1% trifluoroacetic acid (TFA, vol/vol)], and loaded onto the C4
column. Proteins were eluted using a linear gradient of 20–80%
solvent B [95% ACN/5% water, containing 0.1% TFA] at a flow rate
of 1 ml/min over a period of 45 min. Fractions of 0.5 ml each were
collected, and an aliquot from each fraction was resolved by SDS-PAGE
onto 5% T SDS-polyacrylamide gels under reducing
conditions. For iodination, approximately 5 µg of purified collagen
recovered by lyophilization was resuspended in iodination buffer (50
mM sodium acetate, 0.15 M NaCl, 0.05%
NaN3, pH 5.0, at 22 C) and radiolabeled using
Na-[125I] by Iodogen (33). Radiolabeled collagen was
separated from the free isotope by Sephadex G-100 chromatography using
the iodination buffer as the elution buffer.
Detection of 41-kDa-P by a protease assay
To detect protease activity in GCCM, a collagen thin film assay
was used as previously described (34). Briefly,
[125I]-collagen, about 30,000 cpm, suspended in 25 µl
of the iodination buffer was coated onto the bottom of 96-multiwell
flat-bottom cell culture plates by incubating the plates at 37 C for
3–6 h under humidified conditions. Unattached
[125I]-collagen was removed by immersing the plates in a
water tray for 30 min before air drying at room temperature overnight.
About ten such 96-well plates were routinely prepared at a time that
remained stable for approximately 4–6 weeks. A standard curve was
generated in each protease assay by incubating the
[125I]-collagen coated well with 0.1, 0.5, 1, and 5 µg
of trypsin in triplicate by suspending the protease or samples in a
protease buffer (50 mM Tris, 0.2 M NaCl, 1
mM CaCl2, 0.02% NaN3, pH 7.4, at
22 C) in a final reaction volume of 200 µl/well. Total hydrolysis was
estimated by using 20 µg of trypsin. Nonspecific hydrolysis was
assessed by incubating 200 µl of protease buffer alone in the
[125I]-collagen coated well. The specificity of protease
induced hydrolysis was also assessed by incubating the above described
concentrations of trypsin in the presence of 20 µg of soybean trypsin
inhibitor (STI). The amount of [125I]-collagen released
into the supernatant was quantified after 3 h of incubation by
spectrophotometry using a Packard -counter (Model Cobra II) by
aliquoting 150 µl of the sample for radioactivity determination.
Hydrolysis of [125I]-collagen by 41-kDa-P in the
presence of protease inhibitors
To determine the protease class of 41-kDa-P, multiple protease
inhibitors at various concentrations were used as follows: ABSF (a
serine protease inhibitor), sodium chloromercuribenzoate (a cysteine
protease inhibitor), pepstatin (an aspartyl protease inhibitor), EDTA
(a metalloprotease inhibitor), and 1,10-phenanthroline (a
metalloprotease inhibitor). ABSF, pepstatin, and 1,10-phenanthroline
were dissolved in ethanol and appropriate controls were run using
ethanol without protease inhibitors. EDTA and sodium
chloromercuribenzoate were dissolved in PBS (10 mM sodium
phosphate, 0.15 M NaCl, pH 8.0, at 22 C). Protease assay
was performed as described above except that appropriate amounts of
each protease inhibitor was added to triplicate wells before the
addition of purified 41-kDa-P.
Purification of 41-kDa-P from GCCM
GCCM, in batches of 5–15L, was concentrated and equilibrated in
20 mM Tris, pH 7.4, at 4 C using a Millipore Minitan
tangential ultrafiltration unit (Millipore Corp., Milford, MA) equipped
with eight Minitan plates with an Mr cut-off at 10,000.
Anion-exchange HPLC.
The concentrated and equilibrated
sample obtained by ultrafiltration was loaded onto a Mono Q (Pharmacia,
Uppsala, Sweden) anion exchange preparative column (HR 16/10, 16
x 100 mm, id; particle size, 10 µm) as described (35) at a flow rate
of 4 ml/min. Bound proteins were eluted using a linear gradient of
0–80% solvent B (20 mM Tris, pH 7.4, at 22 C, containing
600 mM NaCl) over a 90-min period. Eluents were monitored
by UV absorbance at 280 nm. An aliquot from each fraction was assayed
for protease activity and for protein visualization by SDS-PAGE.
Vydac C4 reversed-phase HPLC.
Following the preparative
anion-exchange HPLC step, fractions exhibiting protease activity were
pooled, lyophilized, reconstituted in solvent A [5% ACN/95% water
containing 0.1% TFA] and loaded onto a Vydac C4 preparative HPLC
column (22 x 250 mm, id; particle size, 10 µm) at a flow rate
of 4 ml/min. Bound proteins were eluted using a linear gradient of
20–80% solvent B [95% ACN/5% water, containing 0.1% TFA] over a
period of 90 min at a flow rate of 4 ml/min. Eluents were monitored by
UV absorbance at 280 nm. Fractions of 4 ml each were collected,
lyophilized, resuspended in sterile water, and an aliquot from each
fraction was assayed for protease activity and for protein
visualization by SDS-PAGE. For all subsequent steps, the compositions
of solvents A and B were the same as in this Vydac C4 step unless
specified otherwise.
Vydac C8 reversed-phase HPLC.
The fractions from the Vydac
C4 step that exhibited protease activity were pooled, lyophilized,
reconstituted in solvent A and loaded onto a Vydac C8 reversed-phase
HPLC column (4.6 x 250 mm, i.d.). Bound proteins were eluted
using a linear gradient of 20–80% solvent B over a period of 45 min
at a flow rate of 1 ml/min. Eluted proteins were monitored by UV
absorbance at 280 nm. Fractions were lyophilized, resuspended in
sterile water, and an aliquot from each fraction was assayed for
protease activity and for protein visualization by SDS-PAGE.
Vydac C18 reversed-phase HPLC.
The active fractions
containing protease activity obtained from the Vydac C8 step were
pooled, lyophilized, and fractionated further using a Vydac C18
reversed-phase column (4.6 x 250 mm, id). The lyophilized
material was reconstituted in 200 µl of solvent A and loaded onto the
column at a flow rate of 1 ml/min. Bound proteins were eluted using a
linear gradient of 10–70% solvent B over a period of 45 min at a flow
rate of 1 ml/min, and fractions of 0.5 ml each were collected. An
aliquot from each fraction was withdrawn, dried under nitrogen, and
assayed for protease activity, and proteins visualized by SDS-PAGE.
High performance electrophoresis chromatography (HPEC).
The
fractions obtained from the Vydac C18 HPLC step that exhibited protease
activity were pooled, lyophilized and resuspended in 50 µl of HPEC
sample buffer (0.125 M Tris-PO4, pH 6.8 at 22
C, containing 10% glycerol and 0.002% bromophenol blue, wt/vol). The
sample was loaded onto a 7.5% T polyacrylamide gel of
3.5 x 50 mm (id) in an Applied Biosystems (Foster City, CA) 230A
HPEC system as described (36). Eluents were monitored by UV absorbance
at 280 nm, and fractions of about 270 µl each were collected. The
flow rate of the elution buffer (75 mM
Tris-PO4, pH 7.5, at 22 C) was about 27 µl/min. An
aliquot from each fraction was assayed for protease activity, and
proteins were visualized by SDS-PAGE. Because the active fractions
collected from this HPEC step still contained a few contaminants, they
were pooled, concentrated to about 50 µl using an Amicon (Danvers,
MA) Microcon-3 ultrafiltration unit, suspended in 40 µl of the HPEC
sample buffer, and further fractionated by a 12.5% T HPEC
polyacrylamide gel (3.5 x 50 mm, id) using procedures as described
above.
Bioassay of 41-kDa-P
The ability of purified 41-kDa-P to modulate Sertoli cell
secretory function was bioassayed using primary cultures of Sertoli
cells isolated from 20-day-old Sprague-Dawley rats (28). Sertoli cells
cultured in F12/DMEM supplemented with insulin (10 µg/ml), human
transferrin (5 µg/ml), epidermal growth factor (2.5 ng/ml), and
bacitracin (5 µg/ml) were plated in 24-well culture plates at a
concentration of 2 x 105 cells in 500 µl suspension
and incubated at 35 C in a humidified atmosphere of 5%
CO2/95% air. After 48 h, Sertoli cells were subjected
to a 2.5-min hypotonic treatment (20 mM Tris, pH 7.4, at 22
C) to lyze contaminating germ cells followed by two additional washes.
Twenty-four hours later, aliquots of HPLC/HPEC fractions ranging from
5–50 µl from different purification steps were incubated with these
cells for an additional 24 h. The spent media were collected for
specific RIAs. In some experiments, the cell numbers before and after
the 24 h incubation with purified 41-kDa-P were assessed by a
Coulter counter. In all cases, cell numbers and viability as monitored
by trypan blue dye exclusion test did not change more than 5%.
Effect of different pHs on the proteolytic activity of 41-kDa-P
To examine the optimal pH range required for 41-kDa-P to exert
its protease activity, different doses of purified 41-kDa-P were
reconstituted in buffers of different pHs similar to the standard
protease buffer described above. For pHs at 2.5–5.5, 50 mM
Tris was substituted with 50 mM sodium acetate. The pH was
adjusted using acetic acid, whereas the other components remained the
same (0.2 M NaCl, 1 mM CaCl2, 0.02
NaN3). In assays screening for the effect of
metalloprotease inhibitors on 41-kDa-P, all metallic cations were
omitted in the protease buffer. Aliquots of 0.3 ng-1.5 µg of the
highly purified 41-kDa-P in duplicate were suspended in each of these
buffers in a total volume of 200 µl, transferred to
[125I]-collagen coated wells, and incubated for 3 h
at 37 C. Thereafter, 150 µl of the supernatant was collected for
radioactivity determination.
Proteolysis of [125I]-testin by 41-kDa-P
The ability of 41-kDa-P to cleave testin in vitro was
tested under different experimental conditions by incubating
[125I]-testin (about 50,000 cpm) at 37 C in the absence
or presence of different doses of purified 41-kDa-P in F12/DMEM,
protease buffer, Sertoli cell-conditioned medium (SCCM), or RIA buffer
(10 mM sodium phosphate, 0.15 M NaCl, pH 7.4,
at 22 C containing 0.5% BSA, wt/vol). The rationale for performing
these experiments was to assess whether the previously noted inhibition
of Sertoli cell testin secretion by fractions of purified 41-kDa-P in
the in vitro bioassay was an authentic biological inhibitory
activity or an artefact of proteolysis. About 50,000 cpm of
[125I]-testin was incubated in the absence or presence of
0.01, 0.5, 1, and 1.5 µg of purified 41-kDa-P with either F12/DMEM,
SCCM, or RIA buffer in a final incubation volume of 75 µl in
corresponding buffer, and incubated at 37 C for 20 h. We also
studied the effect of various doses of 2-macroglobulin,
a Sertoli-cell secreted protease inhibitor (6), on the proteolytic
activity of 41-kDa-P. Hydrolysis of [125I]-testin by
41-kDa-P was assessed by SDS-PAGE.
NH2-Terminal protein sequencing
Highly purified 41-kDa-P (about 0.1 nmol) was resolved by
SDS-PAGE onto 12.5% T SDS-polyacrylamide gels, transferred
onto a polyvinylidene difluoride (PVDF) membrane using a buffer system
consisting of 10 mM CAPS/10% methanol (vol/vol), pH 11, at
22 C. 41-kDa-P electroblotted onto the PVDF membrane was stained by
Coomassie blue R-250 as described (36) and sequenced as detailed
elsewhere (35, 36, 37). In one experiment, purified 41-kDa-P (about 0.1
nmol) obtained from the HPEC step was coated onto a BioBrene coated
cartridge and sequenced directly. Phenylthiohydantoin (PTH)-amino acids
were identified and quantified by HPLC using a Brownlee PTH-C18 (id,
2.1 x 220 mm; particle size, 5 µm) column. The repetitive yield
was about 96%. Protein sequencing was repeated five times using
different batches of highly purified 41-kDa-P.
Testin RIA
Purified rat testin (about 5 µg) was radioiodinated using
[125I]-Bolton-Hunter reagent (38). Testin RIA was
performed as previously described (35). The interassay and intraassay
coefficients of variation were 11% and 8%, respectively. The minimal
detectable dose was 0.06 ng/assay tube and 50% displacement was at 1.3
ng.
Clusterin RIA
Clusterin RIA was performed as detailed elsewhere (39). The
interassay and intraassay coefficients of variation were 14% and 10%,
respectively. The minimal detectable dose was at 0.12 ng/assay tube and
the 50% displacement was at 14 ng.
General methods
Analytical PAGE was performed according to the procedures of
Laemmli (40). The resolving gel consisted of 12.5% T (total
acrylamide concentration) and 2.6% cross-linker using
methylene-bisacrylamide (% CBis), with a stacking gel of
5% T and 15% N,N-diallyltartardiamide (%
CDATA). Protein estimation was performed by Coomassie blue
dye binding assay using BSA as a standard (41). Transferrin RIA was
performed as detailed elsewhere (28).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), a preparative C4 column
(Fig. 1B), an analytical C8 column (Fig. 1C), an analytical C18 column
(Fig. 1D), and two consecutive HPEC
steps using 7.5% (Fig. 1F) and 12.5% T (Fig. 1H)
polyacrylamide gels under nonreducing conditions. In each of the
purification steps, 41-kDa-P was assessed by the
[125I]-collagen film assay. Each protease assay was
accompanied by a calibration curve to ensure assay reliability using
increasing doses of trypsin with and without STI (Fig. 1A, inset). The inset shown in Fig. 1A demonstrated
that trypsin induced dose-dependent proteolysis of
[125I]-collagen that could be inhibited by STI. Using
this protease assay, it was noted that protease activity in GCCM eluted
in fractions 20–38 from the preparative Mono Q HPLC step under protein
peaks 6–10 (Fig. 1A). The active fractions were then fractionated by
sequential C4 (Fig. 1B), C8 (Fig. 1C), and C18 (Fig. 1D) reversed-phase
HPLC columns as detailed in Materials and Methods. When an
aliquot of 20 µl of sample from selected fractions between 5 and 95
from the C18 reversed-phase HPLC step (Fig. 1D) was resolved by
SDS-PAGE and the proteins visualized by silver staining (Fig. 1E), the
proteolytically active fractions in 48–52 derived from the C18 HPLC
step (Fig. 1D) still contained multiple contaminating proteins
(fractions 48–52 in Fig. 1E). These proteolytically active fractions
were pooled, concentrated, equilibrated, and further purified by HPEC
using a 7.5% T polyacrylamide gel. Protease activity was
found in fractions 26–38 (Fig. 1F) which, apart from a few
contaminating bands, corresponded to a heterogeneous band of 41 kDa
under reducing conditions that could not be readily silver stained
(Fig. 1G). To verify that this 41-kDa protein is indeed the protease,
it was further purified by HPEC using a 12.5% T
polyacrylamide gel (Fig. 1H). When the proteolytically active fraction
was resolved by SDS-PAGE under nonreducing and reducing conditions, a
protein band of 44 kDa and 41 kDa, respectively, was noted when stained
by Coomassie blue R-250 (Fig. 2A). Silver staining was
not used for the reason given above (see Fig. 1G); however, when this
gel was visualized with silver staining, no other contaminant was
detected (data not shown). An increase in electrophoretic mobility
under reducing conditions vs. nonreducing conditions, 41
vs. 44 kDa, can be explained by complete denaturation of the
polypeptide chain under reducing conditions that cause linearization of
the polypeptide chain and thus a better electrophoretic mobility.
Direct N-terminal sequence analysis of purified 41-kDa-P from at least
five batches of samples using protein either from the HPEC step (Fig. 1H) or 41-kDa-P electroblotted onto PVDF membrane revealed an identical
partial N-terminal sequence of NH2-KYEFYEIXLL (Fig. 2B),
suggesting that the protein has been purified to apparent homogeneity.
Comparison of this partial N-terminal sequence with existing database
at GenBank, PIR, and BLAST revealed that 41-kDa-P is a unique protein.
Also, it did not display significant homology to any existing sequences
in the protease database. Using the purification scheme described
above, we routinely obtained 300–500 µg of purified 41-kDa-P from
each 12–15L batch of GCCM. A total of five separate sets of
experiments were carried out, and a similar result was obtained in each
set of experiments.
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) and clusterin (Fig. 3B) dose dependently, whose
inhibitory effect became visible at a dose of about 0.25 ng/500
µl/dish with an ED50 at 0.5–1.5 ng/500 µl/dish. A
similar observation was achieved using two different batches of
purified 41-kDa-P. The observed biological effect of 41-kDa-P on
Sertoli cell testin and clusterin secretion does not appear to be the
result of nonspecific proteolysis. Because in the bioassay using
primary Sertoli cell cultures, 5 µg of bacitracin/ml was included in
the F12/DMEM that acted as a protease inhibitor. Furthermore, Sertoli
cells secreted about 1.5–2 µg 2-macroglobulin/ml
medium in vitro under the present culture conditions when
quantified by RIA as previously described (42). In view of the data
reported in Fig. 5 (see below) where 1:1 molar ratio of
2-macroglobulin:41-kDa-P is effective in limiting the
proteolytic activity of 41-kDa-P, the amount of
2-macroglobulin released by Sertoli cells (1.5–2
µg/ml) should be sufficient to inhibit the proteolytic activity of
41-kDa-P because the effective biological dose of 41-kDa-P was at
0.5–50 ng/ml (Fig. 3, A and B).
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, A and B) was effective in modulating Sertoli cell
secretory function, we next sought to examine whether it possessed
proteolytic activity at these concentrations. We also assessed the
optimal pH required for its activity. At the indicated pH values
between 2.5 and 5.5, 41-kDa-P did not exhibit any significant
proteolytic activity at doses between 0.3 ng and 1.5 µg/200
µl/assay well (=1.5 ng/ml–7.5 µg/ml) (Fig. 4). At
pH 7.4, however, 41-kDa-P cleaved [125I]-collagen dose
dependently, and the proteolytic activity was detectable only when its
concentration was at about 0.1 µg/200 µl/assay well (=0.5 µg/ml)
(Fig. 4). More important, this proteolytic activity was not inhibited
by STI indicating 41-kDa-P is different from trypsin (Fig. 4).
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) (for review, see
58 , we examined their effects on the proteolytic activity of
41-kDa-P using the [125I]-collagen film assay (Table 1).
It was found that 41-kDa-P can be inhibited by ABSF but not by the
other inhibitors suggesting it is a member of the serine protease
family (Table 1).
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1–5), and serum-free F12/DMEM (Fig. 5, lanes 6–10) 41-kDa-P was found
to cleave [125I]-testin dose dependently yielding
complete hydrolysis of testin (Fig. 5; lanes 3–5 vs. 1–2
and lanes 8–10 vs. 6–7). Apparently,
[125I]-testin was protected from being proteolyzed by
41-kDa-P in the presence of an excessive amount (0.375 mg per assay
tube/75 µl) of BSA (Fig. 5, lanes 11–15) or SCCM (Fig. 5, lanes
16–20). We speculate that the inhibitory effect of SCCM on 41-kDa-P
proteolytic activity may be the result of multiple protease inhibitors
present in this medium because it is known that Sertoli cells secrete
both 2-macroglobulin and TIMP-2 when cultured in
vitro (6, 7, 8, 9). Under these culture conditions, Sertoli cells
secrete as many as 1.5–2 µg of 2-macrogloublin/ml
medium (42). We, therefore, sought to examine the effect of
2-macroglobulin on the proteolytic action of 41-kDa-P.
It was found that 2-macroglobulin isolated from SCCM is
a potent inhibitor of 41-kDa-P (Fig. 5, lanes 21–25) with a
dose-dependent inhibitory effect (Fig. 5, lanes 26–30). These analyses
revealed that 41-kDa-P is likely a germ cell product whose proteolytic
activity can be blocked by a Sertoli cell secreted protease
inhibitor. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-macroglobulin in the spent media of the bioassay is
about 1.5–2 µg/ml that is sufficient to limit the action of 41-kDa-P
because it is present only at a concentration between 0.5 and 50 ng/ml
in the bioassay given the fact that 2-macroglobulin is
capable to limit the action of 41-kDa-P at a molar ratio of 1:1 with
41-kDa-P (Fig. 5). It is likely that 2-macroglobulin is
the factor in SCCM that inhibits 41-kDa-P because it was shown to be a
putative Sertoli cell secretory product (6). The maximum concentration
of 41-kDa-P used in the bioassay at 50 ng/ml was found neither to
affect Sertoli cell viability nor reduce cell number as demonstrated by
trypan blue dye exclusion test, measurement of lactic dehydrogenase
level, and cell number determination by a Coulter counter. In addition,
it is noteworthy to mention that trypsin at a concentration of up to 1
µg/ml did not affect Sertoli cells detrimentally (27).
The fact that 41-kDa-P, a gonadal peptide, has dual biological
actions is not without precedence. TIMP-1, a protease inhibitor
released by Sertoli cells (8), has recently been shown to be also a
potent stimulator of Leydig cell steroidogenic function (43). This
stimulation is maximal when TIMP-1 forms a molecular aggregate with
cathepsin L (43), a cysteine protease actively synthesized and secreted
by Sertoli cells (1, 2). Besides, activin, originally purified from
follicular fluid (44, 45) and found to be expressed in Leydig cells
(46), has also been shown to exhibit multiple biological actions. On
one hand, activin stimulates the synthesis (47) and release (48) of FSH
from the pituitary. On the other hand, activin acts as a mitogen by
stimulating mammalian organogenesis (49) and spermatogonial
proliferation in vitro (50). Furthermore, activin can
regulate the mRNA expression of follistatin in granulosa cells (51).
Similarly, 2-macroglobulin is a protease inhibitor in
the testis but is also a binding protein of various growth factors such
as TGF-ß (52, 53) and activin (54) and is also known to stimulate the
growth of EC and RME cells in vitro (55, 56).
Throughout the purification of 41-kDa-P, this protein was found difficult to be visualized by silver staining, possibly due to extensive glycosylation, unless the oligosaccharides were oxidized by periodic acid treatment (data not shown). However, 41-kDa-P can be readily visualized by Coomassie blue staining. The heterogeneity of this protein was also visible by SDS-PAGE, which displayed a rather heterogeneous band on polyacrylamide gel; however, direct protein sequencing of discrete segments within this heterogenous band electroblotted onto PVDF membrane consistently yielded the same N-terminal sequence.
Spermatogenesis is a precisely controlled and timed process that consists of multiple biochemical and molecular events and are inextricably linked to the kinetics of germ cell migration and turnover of intertesticular cell junctions. The demonstration of dual activities of 41-kDa-P, in particular its effect on testin, suggest a likely role for 41-kDa-P in junctional restructuring during spermatogenesis. Testin has been implicated to be a dynamic component of intertesticular cell junctions (15, 16, 17, 57). The testin mRNA expression appears to be dependent upon the status of tissue restructuring in a given organ (17, 57). Moreover, the expression of testin is the highest in the gonad possibly due to the continual tissue restructuring as a result of germ cell or follicle development. Even though testin can be cleaved by trypsin at Gln-94 from the N-terminus, it is resistant to further tryptic digest in vitro unless it is exposed to low pH for an extended period of time (17). 41-kDa-P, on the other hand, is capable of inducing complete proteolysis of testin at physiological pH suggesting this molecule may be important in mediating the action of testin in the epithelium. Even though further investigation is necessary to define the function of this 41-kDa protein, it appears this protein may play a critical role in Sertoli cell-germ cell interactions in the epithelium during spermatogenesis.
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2 Current address: Olson Biochemistry Laboratories, ASC-136, South
Dakota State University, Brookings, South Dakota 57007.
Received December 3, 1996.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-macroglobulin.
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2-macroglobulin and clusterin secretion by peritubular
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Biol Chem 257:14093–14101This article has been cited by other articles:
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