Full text of DOI:10.1093/humrep/17.11.2919

Human Reproduction Vol.17, No.11 pp. 2919–2923, 2002
Quantitative differences in matrix metalloproteinase
(
MMP)-2, but not in MMP-9, tissue inhibitor of
metalloproteinase (TIMP)-1 or TIMP-2, in seminal plasma
of normozoospermic and azoospermic patients
E.Baumgart, S.V.Lenk, S.A.Loening and K.Jung1
Department of Urology, University Hospital Charit e´ , Humboldt University, Schumannstr. 20/21, D –10117 Berlin, Germany
To whom correspondence should be addressed. E-mail: klaus.jung@charite.de
BACKGROUND: Matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases
(TIMPs), have been detected in reproductive tissues and seminal plasma. The purpose of this study was to quantify
MMP-2, MMP-9, TIMP-1 and TIMP-2 in human seminal plasma and to evaluate their association with sperm.
METHODS: Seminal plasma was analysed using ELISA assays for all four analytes in 12 normozoospermic and
1
2 azoospermic patients and then for MMP-2 only in another 114 men with azoospermia (n ⍧ 16), after vasectomy
6
6
(n ⍧ 20) and with sperm counts within the following ranges: 0.3–19⍥10 /ml (n ⍧ 20), 20–23⍥10 /ml (n ⍧ 11),
6
6
6
6
49–57⍥10 /ml (n ⍧ 12), 96–110⍥10 /ml (n ⍧ 12), 139–161⍥10 /ml (n ⍧ 12) and 215–346⍥10 /ml (n ⍧ 11).
Additional zymographic analyses using SDS–PAGE were performed. RESULTS: All investigated MMPs and TIMPs
were detected. MMP-9, TIMP-1 and TIMP-2 were not significantly different in normozoospermia and azoospermia.
Only the MMP-2 concentration was significantly decreased in azoospermic compared with normozoospermic
patients (mean ⍨ SD: 650.6 ⍨ 288.9 versus 1677 ⍨ 910.4 ng/ml respectively; P ⍧ 0.0002) and significantly
correlated with the number of sperm (r ⍧ 0.54; P < 0.0001). CONCLUSION: MMP-2 in seminal plasma was
strongly correlated to the sperm count in a linear fashion. Its origin and potential function remain to be elucidated.
Key words: male fertility/MMP/seminal plasma/TIMP/zymography
Introduction
increased expression in the endometrium of the uterine wall
Salamonsen and Woolley, 1996; Xu et al., 2000) is believed
to contribute to the sloughing process. MMP-2 expression and
activity increase at the site of the rupturing follicle in the
ovary (Russell et al., 1995). Human trophoblasts utilize
MMP-2 and MMP-9 in the invasion of the uterine stroma
(
Matrix metalloproteinases (MMPs) form a family of structur-
ally-related, zinc-dependent proteases which are capable of
restructuring tissue components by proteolytic degradation
of extracellular matrix and basement membrane components
(Birkedal-Hansen, 1995). It is for this reason that they are
(Hulboy et al., 1997).
considered important in physiological growth and tissue
remodelling and also in cellular involution and apoptosis.
However, their involvement in multiple pathological processes,
such as tissue destruction in rheumatoid arthritis, neoplastic
growth, metastasis and neoangiogenesis, brings them into the
focus of new diagnostic and therapeutic strategies (Chambers
and Matrisian, 1997).
To date five subgroups, the collagenases, gelatinases, strome-
lysins, matrilysins and membrane type MMPs are known.
Common substrates of the gelatinases MMP-2 and MMP-9
are type I, IV, V, VII and X collagens, elastin, fibronectin and
tumour necrosis factor-α. Their activity is co-regulated and
inhibited by the tissue inhibitors of metalloproteinases (TIMP)
However, very little is known about the expression and
function of MMPs and their inhibitors in the male reproductive
tract. MMP-2 as well as TIMP-1 and -2 (Ulisse et al., 1994)
have been detected in rat Sertoli cell cultures. TIMP-2 extracted
from bovine seminal plasma binding to sperm was described
and its influence on bull fertility was postulated (McCauley
et al., 2001). A significant portion of MMPs such as MMP-2
and MMP-9 seem to come from accessory sex glands such as
the prostate and the seminal vesicles, as demonstrated by split
ejaculate analysis (Yin et al., 1990). This is supported by the
findings of two other groups who detected MMP-2 gelatinolytic
activity in prostatic secretions of benign hyperplastic tissue
(Lokeshwar et al., 1993; Wilson et al., 1993).
(Woessner, 1994).
MMPs have been linked extensively with female reproduc-
tive function (Hulboy et al., 1997). During menstruation, their
Parallel to our investigations, the occurrence of MMP-2 and
MMP-9 activity in human seminal plasma has been recently
©
European Society of Human Reproduction and Embryology
2919

E.Baumgart et al.
reported (Shimokawa et al., 2002). However, no quantitative
data have been described until now using robust assays such
as ELISAs.
The purpose of this study was: (i) to quantitate MMP-2 and
MMP-9, as well as their inhibitors TIMP-1 and TIMP-2, in
human seminal plasma; and (ii) to examine the relationship
between their concentrations and the sperm count and other
characteristics of sperm.
parameter method for calculation of concentrations (Mikrowin soft-
ware, version 3.0; Mikrotek Laborsysteme, Overath, Germany).
Zymography for MMP-2 and -9 was performed in sodium dodecyl
sulphate polyacrylamide gel electrophoresis (SDS–PAGE; 8%) con-
taining 0.5% gelatin on the Midget Electrophoresis Unit 2050 (LKB,
Uppsala, Sweden). Samples of seminal plasma and sperm extracts
were mixed with non-reducing sample buffer and subjected to
electrophoretic analysis without boiling. Purified human proMMP-2
and proMMP-9 (Chemicon Inc., Temecula, USA) were included in
each gel run as standards. After electrophoresis, gels were soaked for
2
h in 2.5% Triton-X 100 solution with four washing steps. The gels
were then incubated for 18 h at 37°C in buffer containing 50 mmol/l
Material and methods
Tris–HCl, pH 7.6, 150 mmol/l NaCl, 10 mmol/l CaCl , 0.2% Brij-
2
Study subjects and specimens
3
5 and 0.02% NaN . After incubation, the gels were stained with
3
Samples of seminal plasma were prepared from ejaculates collected
for semen analysis from 138 men who attended the infertility clinic
0.2% Coomassie Blue and destained until clear proteolytic bands
appeared. Gels were scanned using a flatbed scanner (Scanmaker 4;
Microtek Lab, Redondo Beach, USA). Studies with inhibitors in the
incubation solution (10 mmol/l EDTA or 1 mmol/l o-phenanthroline)
verified the specificity of the method. Preincubation of samples with
1 mmol/l p-amino-phenyl-mercuric acetate (18 h, 37 °C) as an in-
vitro MMP activator was used to differentiate between latent and
active forms of MMPs (Crabbe et al., 1993). Controls were performed
without activator but with a corresponding volume of buffer solution
instead of p-amino-phenylmercuric acetate.
(median age 33 years, range 25–50). This included 12 men with
azoospermia (seven idiopathic, five obstructive) and normozoospermia
for the initial evaluation of MMP-2, MMP-9, TIMP-1 and TIMP-2.
A further 114 patients were then analysed for a possible correlation
between MMP-2 and sperm count: 16 patients with azoospermia
(10 idiopathic; six obstructive), 20 patients with azoospermia after
vasectomy, and 78 patients with increasing sperm counts within
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6
the following ranges: 0.3–19ϫ10 /ml (n ϭ 20), 20–23ϫ10 /ml
6
6
(
n ϭ 11), 49–57ϫ10 /ml (n ϭ 12), 96–110ϫ10 /ml (n ϭ 12),
39–161ϫ10 /ml (n ϭ 12), 215–346ϫ10 /ml (n ϭ 11). There were
6
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1
Statistical analysis
no exclusion criteria such as smoking or alcohol consumption. Ethical
approval was obtained from the Ethics Commission at the Charite´
University Hospital.
Retrieval, analysis and classification of the ejaculates were per-
formed according to World Health Organization recommendations
Data were analysed using the statistical softwares SPSS 10.0 for
Windows (SPSS, Chicago, USA) and GraphPad Prism 3.02 (GraphPad
Software, San Diego, USA). Student’s t-test, analysis of variance,
linear regressions and correlation analysis using the correlation
coefficient according to Pearson (r) were performed. P Ͻ 0.05 was
considered significant.
(World Health Organization, 1993). Briefly, samples were obtained
by masturbation into a sterile plastic tube after 4–5 days of abstinence.
Following physical examination of the ejaculate (pH, volume, consist-
ency, motility, morphology etc.), samples were centrifuged at 3000 g
and the seminal plasma was stored at –80°C until analysis.
To investigate whether the release of MMPs from sperm was
dependent on time after sampling, semen specimens from five patients
were divided into five aliquots of 0.3 ml, stored at room temperature
Results
In a first step, seminal plasma samples of men with normozoo-
spermia (group 1; mean sperm count 52 Ϯ 2.2ϫ10 /ml) and
azoospermia (group 2) were analysed using ELISAs for
MMP-2, MMP-9, TIMP-1 and TIMP-2. Only MMP-2
6
(23°C) and centrifuged after 1, 2, 4, 8 and 24 h after sampling. The
(Figure 1) was significantly different (P ϭ 0.0002) in normo-
supernatants were immediately frozen at –80°C for subsequent use.
To characterize the MMP-2 and -9 patterns in sperm, pelleted
sperm were suspended in 0.25% Triton X-100 solution after the final
centrifugation and an ultrasonic disintegration was performed. Prior
to this, the pelleted sperm were washed three times with 5 ml
phosphate-buffered saline to avoid any influence of seminal plasma
remnants. The homogenate was then centrifuged (13 000 g for 15 min)
and the supernatant was used for further investigations.
zoospermic subjects (mean Ϯ SD, 1677 Ϯ 910.4 ng/ml)
compared with azoospermic subjects (650.6 Ϯ 288.9 ng/ml).
However, with regard to the aetiology of the azoospermia
(idiopathic versus obstructive) there appeared to be no
differences for any of the investigated proteins. The ratio of
MMP-9 either to TIMP-1 or TIMP-2 did not show any
significant differences between the groups. No significant
correlation was found between MMP-2 and TIMP-2 in the
normozoospermic group (r ϭ –0.46, not significant).
Assays
The quantification of MMP-2, MMP-9, TIMP-1 and TIMP-2 was
performed using ELISAs (ELISAs for MMP-2 and MMP-9 from
Oncogene, Boston, USA; TIMP-1 and TIMP-2 from Amersham Int.,
Little Chalfont, UK).
All measurements were performed in duplicate according to the
manufacturer’s instructions. Briefly, seminal plasma in a 1:5 to 1:51
dilution was placed into microwells equipped with biotinylated
monoclonal anti-human MMP-2 antibody and incubated for 2 h. After
washing, horseradish peroxidase was added to catalyse the conversion
of chromogenic tetramethylbenzidine into a blue solution. Following
the addition of 2.5 mol/l sulphuric acid as a stopping reagent, the
absorbance was determined using the microplate reader Anthos
HTIII (Anthos Labtec Instruments, Salzburg, Austria) with the four-
As a consequence of these results, only MMP-2 was
investigated in more detail. Samples of a vasectomy group
with azoospermia and of subjects with sperm counts of
6
6
6
0
1
.3–19ϫ10 /ml, 20–23ϫ10 /ml, 49–57ϫ10 /ml, 96–110ϫ
0 /ml, 139–161ϫ10 /ml and 215–346ϫ10 /ml were analysed
6
6
6
(Figure 2). Scatter plots in Figure 2 show that the MMP-2
concentration in seminal plasma increased along with rising
number of sperm in the ejaculate. Furthermore, there was no
significant difference between the vasectomy and azoospermic
groups (759.1 Ϯ 335.3 versus 650.6 Ϯ 288.9 ng/ml, Figure 2).
Linear regression analysis utilizing the individual MMP-2
concentrations showed a linear relationship between MMP-2
2920

MMPs and TIMPs in human seminal plasma
be assumed that the MMP-2 concentrations measured were
not a result of a secretion by inflammatory cells.
To further characterize the MMP pattern in seminal plasma,
samples were assessed with SDS–PAGE (Figure 3). Figure 3A
shows a major band at 72 kDa representing proMMP-2 and a
faint band at 92 kDa that is equivalent to proMMP-9. The
MMP-2 and MMP-9 patterns are similar in seminal plasma
and sperm (Figure 3B). The minor band at 62 kDa corresponds
to the active MMP-2 (Figure 3A) confirmed by in-vitro
experiments with samples preincubated with 1 mmol/l
p-amino-phenylmercuric acetate (Figure 3C). In comparison
with control samples without the treatment with p-amino-
phenylmercuric acetate, samples preincubated with the in-vitro
MMP activator showed the typically changed zymographic
pattern: an increased band of the active form at 62 kDa and a
decreased band of the proMMP-2 at 72 kDa (Figure 3C). A
similar change in the MMP-9 pattern was observed (Figure 3C).
In order to investigate whether the differences of MMP-2
concentration in seminal plasma in normozoospermic versus
azoospermic patients resulted from an increased release from
the sperm, samples from five patients with differing sperm
6
counts (2.5–247.5ϫ10 /ml) were divided into five aliquots of
0.3 ml and centrifuged after liquefaction at 1, 2, 4, 8 and 24 h.
MMP-2 concentrations were related to the values measured at
the starting point 1 h after liquefaction. They remained stable
for the first 2 h and decreased gradually in an almost linear
fashion (Figure 4). After 8 h, significant decreases were
observed (P Ͻ 0.05).
Figure 1. MMP-2, MMP-9, TIMP-1 and TIMP-2 concentrations in
the seminal plasma of normozoospermic versus azoospermic
individuals. Normo. ϭ normozoospermia; azoo. ϭ azoospermia.
Horizontal bars in the scatter plots indicate mean values.
Discussion
To date, only scarce knowledge exists about the expression
and function of MMPs and TIMPs in the male reproductive
system. It is generally believed that the gelatinases MMP-2
and -9 are involved in remodelling processes of structural
proteins (Woessner, 1994; Hulboy et al., 1997). Gelatinolytic
activity coinciding with differentiation stages has been found
in maturing prostate and seminal vesicles in a rat model
(Wilson et al., 1992) and also during prostate involution
following castration (Wilson et al., 1991). In rat Sertoli cell
cultures, MMP-2 was detected and was suspected to contribute
to remodelling of the basement membrane during development
of the seminiferous tubules and in the release of differentiating
germ cells from the basal lamina (Sang et al., 1990). Since
MMPs and TIMPs are ubiquitary, it seems only logical that
they should also be present in seminal plasma.
Figure 2. MMP-2 concentrations in seminal plasma prepared from
ejaculates with increasing sperm counts. Scatterplots (with mean
values as horizontal bars) represent groups within the depicted
6
ranges of sperm counts ϫ10 /ml. For further details see Text.
Utilizing ELISAs for MMP-2, MMP-9, TIMP-1 and
TIMP-2 as well as SDS–PAGE, these proteins were identified in
all measured specimens. After we had finished our experiments,
another group (Shimokawa et al., 2002) published similar
results. They described several gelatinolytic proteins with
molecular weights of 72, 67, 92 and 84 kDa in seminal plasma
with gelatin zymography without giving quantitative data.
Using Western blot techniques, proMMP-2 (72 kDa) and
proMMP-9 (92 kDa), as well as their active forms MMP-2
and -9, were identified. The authors discussed a possible
levels and sperm counts (r ϭ 0.54, P Ͻ 0.0001). However,
no correlation was found between MMP-2 levels and sperm
parameters such as motility or morphology.
To discern any possible influence of leukocytospermia on
MMP-2 levels in the experimental groups, a correlation analysis
was performed for granulocyte counts and associated MMP-2
concentrations. It showed no significant correlation between
these two parameters (r ϭ 0.007, P ϭ 0.96). Therefore, it can
2921

E.Baumgart et al.
Figure 3. Gelatin zymography of (A) seminal plasma samples, (B) protein extracts of sperm and (C) seminal plasma samples and protein
extracts of sperm after preincubation with and without 1 mmol/l p-amino-phenylmercuric acetate. (A) Lane S contained standards of MMP-2
(
72 kDa) and MMP-9 (92 kDa). Lanes 1–3: typical digestion patterns of seminal plasma of an azoospermic, oligozoospermic and
normozoospermic patient respectively. Equal volumes were loaded. (B) Lane S contained standards as mentioned; lane 1 presents a digestion
pattern of the protein extract of sperm. (C) Lane S ϭ standard; lanes 1,3: seminal plasma sample and spermatozoal protein extract after
preincubation without 1 mmol/l p-amino- phenylmercuric acetate; lanes 2,4: seminal plasma sample and spermatozoal protein extract after
preincubation with 1 mmol/l p-amino-phenylmercuric acetate. For further details see Materials and methods.
time. In addition, we could demonstrate a strong correlation
between the sperm count and MMP-2, but not MMP-9,
TIMP-1 or TIMP-2. There were significantly higher concentra-
tions of MMP-2 in normozoospermic compared with azoo-
spermic plasma. No difference within the azoospermic group
with regard to the aetiology, whether obstructive or idiopathic,
could be found.
In the present study, the analysis of seminal plasma
originating from ejaculates with increasing sperm counts
produced evidence of a link between sperm count and
MMP-2. This is further supported by the fact that there
were no significant differences in MMP-2 levels between
azoospermic and vasectomy samples. Since MMP-2 is also
secreted by accessory sex glands, the non-linear relationship
6
in the sperm count range of 49–110ϫ10 /ml could be due
Figure 4. Changes of MMP-2 concentration in seminal plasma with
to a changed baseline secretion by these glands. However,
time after ejaculation (ejaculates were stored at room temperature
it remains unclear exactly where MMP-2 is expressed. It
has been shown that MMP-2 is secreted by Sertoli cells
and seems to be involved in the release of differentiating
germ cells from the basal lamina in the seminiferous tubules
and centrifuged after 1, 2, 4, 8 and 24 h). Percentual changes
compared with the initial MMP-2 values 1 h after ejaculation are
displayed (percentage means Ϯ SD). Significant decreases were
seen from 8 h onwards (*P Ͻ 0.05).
(Sang et al., 1990). It therefore seems possible that it is
function of MMPs in liquefaction of the ejaculate. Our results
not only confirm the presence of the two MMPs and TIMPs
in seminal plasma, but also show quantitative data for the first
also released into the tubular lumen this way.
TIMPs represent natural inhibitors to MMPs (Woessner,
1994). They bind in a 1:1 ratio to the haemopexin domain of
2922

MMPs and TIMPs in human seminal plasma
DeClerck, Y.A., Yean, T.D., Lee, Y., Tomich, J.M. and Langley, K.E.
MMPs and interfere with their activation (DeClerck et al.,
993). TIMP-1 is specific for MMP-9 and TIMP-2 for
MMP-2 (Goldberg et al., 1989). In bovine seminal fluid, a
4 kDa heparin-binding protein (HBP-24) was recently
described (McCauley et al., 2001). This protein bound to
sperm membranes was identified as TIMP-2 and was suggested
to have a role in bull fertility. The authors suspected an
interaction with MMP-2 either expressed in the seminal plasma
or directly on sperm (McCauley et al., 2001).
However, in somatic cells one way of MMP-2 activation is
through formation of a trimolecular complex between
MMP-14, TIMP-2 and proMMP-2 on the cell surface (Strongin
et al., 1995). TIMP-2, which usually acts as an inhibitor to
MMP-2, interestingly is also required for its activation (Wang
et al., 2000) and could be derived from the seminal plasma.
Activated MMP-2 is subsequently released into the extracellu-
lar matrix. Although not yet reported, the same activation
process could theoretically be present on sperm, and was
responsible for the detection of MMP-2 in the present study.
This would explain the correlation between sperm count and
MMP-2 concentration.
MMP-2 concentrations in seminal plasma decreased with
time, being significantly lower from 8 h onwards following
ejaculation (Figure 4). The decrease was somewhat linear and
could be explained by the degradation of MMP-2. This would,
however, exclude any prolonged release of MMP-2 by the
sperm themselves unless it is outweighed by degradation
processes.
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1
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Hulboy, D.L., Rudolph, L.A. and Matrisian, L.M. (1997) Matrix
metalloproteinases as mediators of reproductive function. Mol. Hum.
Reprod., 3, 27–45.
Lokeshwar, B.L., Selzer, M.G., Block, N.L. and Gunja-Smith, Z. (1993)
Secretion of matrix metalloproteinases and their inhibitors (tissue inhibitor
of metalloproteinases) by human prostate in explant cultures: reduced tissue
inhibitor of metalloproteinase secretion by malignant tissues. Cancer Res.,
2
53, 4493–4498.
McCauley, T.C., Zhang, H.M., Bellin, M.E. and Ax, R.L. (2001) Identification
of a heparin-binding protein in bovine seminal fluid as tissue inhibitor of
metalloproteinases-2. Mol. Reprod. Dev., 58, 336–341.
Russell, D.L., Salamonsen, L.A. and Findlay, J.K. (1995) Immunization
against the N-terminal peptide of the inhibin alpha 43-subunit (alpha N)
disrupts tissue remodeling and the increase in matrix metalloproteinase-2
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Sang, Q.X., Dym, M. and Byers, S.W. (1990) Secreted metalloproteinases in
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Shimokawa Ki, K., Katayama, M., Matsuda, Y., Takahashi, H., Hara, I.,
Sato, H. and Kaneko, S. (2002) Matrix metalloproteinase (MMP)-2 and
MMP-9 activities in human seminal plasma. Mol. Hum. Reprod., 8, 32–36.
Strongin, A.Y., Collier, I., Bannikov, G., Marmer, B.L., Grant, G.A. and
Goldberg, G.I. (1995) Mechanism of cell surface activation of 72-kDa
type IV collagenase. Isolation of the activated form of the membrane
metalloprotease. J. Biol. Chem., 270, 5331–5338.
Ulisse, S., Farina, A.R., Piersanti, D., Tiberio, A., Cappabianca, L., D’Orazi,
G., Jannini, E.A., Malykh, O., Stetler-Stevenson, W.G., D’Armiento, M.
et al. (1994) Follicle-stimulating hormone increases the expression of tissue
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Metalloproteinase activities expressed during development and maturation
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Wilson, M.J., Norris, H., Kapoor, D., Woodson, M., Limas, C. and Sinha,
A.A. (1993) Gelatinolytic and caseinolytic proteinase activities in human
prostatic secretions. J. Urol., 149, 653–658.
In summary, in this work we have reported on the concentra-
tions of MMP-2 and MMP-9 and their inhibitors TIMP-1 and
TIMP-2 in human seminal plasma. MMP-2 was strongly
correlated to the sperm count in a linear fashion. Its origin
and function remain to be elucidated.
Acknowledgements
The authors would like to thank Gisela Maicher, Silke Klotzek, Ines
Baumert and Sabine Becker for their help with the experimental set-
up. This work was supported in part by grants from the Research
fund of the Charit e´ Hospital of the Humboldt University.
Woessner, J.F. Jr (1994) The family of matrix metalloproteinases. Ann. NY
Acad. Sci., 732, 11–21.
Xu, P., Wang, Y.L., Zhu, S.J., Luo, S.Y., Piao, Y.S. and Zhuang, L.Z. (2000)
Expression of matrix metalloproteinase-2, -9, and -14, tissue inhibitors of
metalloproteinase-1, and matrix proteins in human placenta during the first
trimester. Biol. Reprod., 62, 988–994.
Yin, H.Z., Vogel, M.M., Schneider, M., Ercole, C., Zhang, G., Sinha, A.A.
and Wilson, M.J. (1990) Gelatinolytic proteinase activities in human seminal
plasma. J. Reprod. Fertil., 88, 491–501.
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