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,
From the * Endocrine Research Unit, Carmel Medical
Center, Haifa, Israel; the Faculty of
Medicine, Technion, Haifa, Israel; and the IVF
Unit, Ha'emek Medical Center, Afula, Israel.
| Correspondence to: Dr Z. Kraiem, Endocrine Research Unit, Carmel Medical Center, 7 Michal St, Haifa 34362, Israel (e-mail: zkraiem{at}clalit.org.il). |
| Received for publication December 17, 2001; accepted for publication May 6, 2002. |
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Abstract |
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Key words: Fertilization, oligo-terato-asthenospermia, infertility
The above scenario involves sperm penetration of 2 barriers imposed by the egg, the zona pellucida and the egg plasma membrane, the detailed molecular mechanisms of which are still unclear. The necessity of breakdown of physical barriers in the above cascade of events suggests that matrix metalloproteinases (MMPs) might be involved in this task. The MMPs are a family of proteolytic enzymes that degrade protein components of the extracellular matrix and basement membrane (Salamonsen, 1996; Hulboy et al, 1997). These enzymes, together with their tissue inhibitors (TIMPs), are believed to play a key role in a number of physiological processes, among which are ovulation and implantation (Salamonsen, 1996; Hulboy et al, 1997). Most MMPs are secreted from the cell as inactive zymogens (pro-MMP). Activation requires the disruption of a Cys—Zn+2 (cysteine switch) interaction and the removal of a propeptide (Hulboy et al, 1997). In this study, we have examined the presence of MMP and TIMP in normal and abnormal human sperm samples to determine if MMPs and TIMP play a role in fertilization.
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Materials and Methods |
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The semen samples were processed by adding an equal volume of human tubal fluid medium (HTF; Irvine Scientific, Santa Ana, Calif) in a conical falcon tube. (HTF serves as the most common medium for sperm treatment in fertility clinics.) The semen samples were then centrifuged (800 x g) for 10 minutes. The supernatant was discarded, and the pellet was suspended in 1 mL HTF medium and centrifuged once again (800 x g, 10 minutes) to wash seminal plasma. Two hundred microliters of HTF medium was added to the pellet and resuspended. The resuspended sperm was deposited at the bottom of a tube containing HTF medium and incubated for 1 hour at 37°C and 5% CO2. The sperm was collected, and viability (examined by trypan blue) was found to be above 87%. Sperm concentration was adjusted to 1 x 106 viable sperm/0.1 mL HTF. Sperm suspensions were incubated overnight at room temperature (except for the MMP/TIMP release experiments, in which the sperm was incubated and the medium collected at 2-hour intervals) and then centrifuged (800 x g) for 10 minutes. The supernatant was stored at -20°C for gel zymography and Western blot analyses.
Gel Zymography
Essentially, the same method as that described by Kleiner and
Stetler-Stevenson (1994) was
used. Samples for analysis (10 µL of stored supernatant) were prepared by
dilution into a buffer (4x) consisting of 0.4 M Tris (pH 6.8), 5% sodium
dodecyl sulfate (SDS), 20% glycerol, and 0.02% bromophenol blue. The samples
were applied onto an 8% polyacrylamide gel (PAGE) containing 0.5% gelatin.
After 90 minutes of electrophoresis, the gel was incubated for 30 minutes at
room temperature in 30 mL of 2.5% Triton X-100 on a rotary shaker. The Triton
X-100 solution was decanted, replaced with 30 mL of enzyme buffer (50 mM Tris,
pH 7.5, 200 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35), and incubated
again for 30 minutes at room temperature on a rotary shaker. The solution was
decanted, replaced with fresh enzyme buffer, and incubated overnight at
37°C. The gel was then stained with 0.5% Coomassie blue G in 30% methanol
and 10% acetic acid for 10 minutes at room temperature on a rotary shaker and
thereafter washed with water until clean bands were visualized. Finally, the
gel was incubated for 30 minutes in 45% methanol and 5% glycerol prior to
drying overnight between sheets of cellophane. Areas of proteolytic activity
are visualized by the absence of staining in areas of the gel in which the
protein substrate (gelatin) has been degraded, and this was quantitated by
densitometric analysis of the zymograms with the Bio Imaging gel documentation
system (Dinco & Renium, Jerusalem, Israel) and TINA software (Raytest,
Staubenhardt, Germany). The percentage of gelatinolytic activity of a sample
was defined as the sample's arbitrary optical density value compared to the
optical density value of a control sample. No MMP activity was evident on gel
zymography when HTF medium was run alone.
Western Blot Analysis
Western blot analysis was performed as described in our previous study
(Goldman et al, 1997). Sperm
suspensions (30 µL of stored supernatant) after SDS-PAGE were blotted onto
0.45-µm nitrocellulose membranes (Schleicher & Schuel, Dassel,
Germany). Nonspecific binding sites were blocked by incubating the
nitrocellulose membranes overnight with 20% nonfat milk and Tris-buffered
saline containing 0.01% Tween-20. The membranes were then washed twice with
Tris-buffered saline containing 0.5% Tween-20 and incubated for 1 hour with
mouse anti-human TIMP-1 (1:100) or MMP-2 (1:100) and with rabbit anti-human
MMP-9 (1:2500) monoclonal antibodies (Oncogene Science, Cambridge, Mass) in
10% nonfat milk and Tris-buffered saline containing 0.01% Tween-20. The
membranes were subsequently washed with Tris-buffered saline containing 0.5%
Tween-20 and incubated for 1 hour with horseradish peroxidase—conjugated
anti-rabbit (1:5000) secondary antibody (Jackson ImmunoResearch, West Grove,
Pa) in 10% nonfat milk and Tris-buffered saline containing 0.01% Tween-20. The
membranes were washed 3 times with Tris-buffered saline containing 0.5%
Tween-20 and then detected by enhanced chemiluminescence (Amersham Int,
Piscataway, NJ) and quantitated by densitometry as specified above.
Immunofluorescence
Fresh sperm suspensions were twice poured slowly on the side of a cold
glass tube on ice and then washed twice by centrifugation and resuspension
with Dulbecco phosphate-buffered saline (DPBS) at 400 x g for
10 minutes. The samples were fixed for 10 minutes in 2% paraformaldehyde and
washed by centrifugation and resuspension with DPBS at 400 x g
for 10 minutes. Remaining steps were carried out at ambient temperature. The
samples were incubated for 30 minutes in blocking solution (DPBS plus 50 mg/mL
bovine serum albumin). Polyclonal rabbit anti-human MMP-9 (1:100) (Oncogene)
or monoclonal mouse anti-human MMP-2 (1:50) (R&D Systems, Abingdon, United
Kingdom) was added to sperm samples at a concentration of 1.0 µg/mL (which
had in preliminary experiments been determined to allow adequate
immunostaining of sperm cells). The samples were incubated for 60 minutes,
washed twice, and resuspended in 1 mL DPBS. Fluorescein
isothiocyanate—conjugated goat anti-rabbit or donkey anti-mouse
immunoglobulin G (Jackson) was added as a second antibody to the samples (1:50
dilution in DPBS), which were then incubated for an additional 60 minutes.
After secondary antibody incubation, the cell suspensions were washed twice
with DPBS. Sperm cell samples were placed on glass microscope slides with
coverslips and a fluoroguard anti-fade reagent (Fluoromount G, Southern
Biotechnology, Birmingham, Ala). The localization of MMP-2 and pro-MMP-9 in
the sperm was visually demonstrated by a Bio-Rad MRC 1024 Laser Scanning
Confocal Microscope (Bio-Rad House, Hertfordshire, United Kingdom) linked to a
Nikon Diaphot 300 microscope (Nikon Corp, Tokyo, Japan) at 1000x and
interfaced to a Compaq Prosignia 300 (Compaq Computer Corp, Houston, Tex).
Unless otherwise stated, all materials were obtained from Sigma Chemical Company (St Louis, Mo).
Statistical Analysis
Statistical analysis of the data was carried out by the Student's
t test when comparing 2 groups, and analysis of variance was used
when comparing more than 2 groups. A P value less than .05 was
considered significant.
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Results |
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MMP and TIMP in Normal and Abnormal Sperm Samples
Four major bands of gelatin-degrading activity, corresponding to the
molecular weights of 92, 72, 62, and 28 kd, were evident on gel zymography of
normal sperm samples (Figure 1,
lanes 2, 4, and 6). No gelating-degrading activity was evident on gel
zymography when HTF medium was run alone. Inhibition of the bands was obtained
by incubation with 1, 10-phenanthroline (data not shown), as expected for
metalloproteinases. The molecular weights of 92, 72, and 62 kd are consistent
with those of pro-MMP-9, pro-MMP-2, and MMP-2, respectively. The presence of
MMP-2 and pro-MMP-9 was confirmed by Western blot analysis
(Figure 2A and B). It is to be
noted that an additional band of 28 kd was observed with the MMP-2 monoclonal
antibody in the Western blot (Figure
2A). The 28-kd band was not MMP-7 or fertilin, as tested by
Western blot (data not shown).
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The bands obtained by gel zymography were quantified by densitometric scanning. The results, described in Figure 3A, showed that the dominant MMP in normal sperm samples was the 72-kd band with relatively lower band intensities at 92, 62, and 28 kd (P < .05, P < .05, and P < .01, respectively). Western blot analysis demonstrated the presence of TIMP-1 in normal sperm samples (Figure 4).
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Three major bands of gelatin-degrading activity, corresponding to the molecular weights of 92, 72, and 62 kd, were evident on gel zymography of abnormal sperm samples (Figure 1, lanes 3, 5, and 7). A very faint band of 28 kd could also be seen. The molecular weights of 92, 72, and 62 kd are consistent with those of pro-MMP-9, pro-MMP-2, and MMP-2, respectively. The presence of pro-MMP-9 and MMP-2 was confirmed by Western blot analysis (Figure 2A and B). The bands obtained by gel zymography were quantified by densitometric scanning. The results, described in Figure 3A, showed no significant differences between the band intensities at 92, 72, and 62 kd; however, a significantly less intense band at 28 kd was shown (P < .001). Western blot analysis demonstrated the presence of TIMP-1 in abnormal sperm samples (Figure 4).
MMP and TIMP Expression in Normal vs Abnormal Sperm Samples
A comparison of normal and abnormal sperm samples revealed that in normal
samples, 28-kd activity was 4.5-fold higher (P < .001) and that
92-kd activity was significantly lower (P < .05) than in abnormal
samples (Figure 3B). No
significant difference was found when bands between 72 and 62 kd
(Figure 3A) and the TIMP-1 band
(Figure 4) were compared with
normal and abnormal sperm samples using either the gel zymography or Western
blot technique (Western blot data not shown).
Time Course of MMP and TIMP Release
The time course of MMP and TIMP-1 release is shown in
Figure 5. Pro-MMP-9, pro-MMP-2,
MMP-2, and TIMP-1 reached maximal values at 2 hours and then dropped sharply
to reach very low (pro-MMP-9) or even undetectable values after a 10-hour
incubation. On the other hand, the 28-kd band was maximally active at 4 hours
and kept 40% of this activity after 10 hours of incubation.
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MMP-2 and MMP-9 Localization
MMP-2 was detected by immunofluorescence in normal
(Figure 6A and B) and abnormal
sperm samples (Figure 6C and D)
in the acrosome region and in the midpiece.
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Weak pro-MMP-9 labeling was found mostly in the midpiece and flagella but was almost undetectable in the acrosome region in normal sperm samples (Figure 6E and F). In sharp contrast, abnormal sperm samples showed strong MMP-9 labeling in the acrosome region, midpiece, and flagella (Figure 6G and H).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Gel zymography showed 92-, 72-, 62-, and 28-kd molecular-weight bands exhibiting gelatin-degrading activity. The 92-, 72-, and 62-kd bands with gelatinolytic activity are consistent with pro-MMP-9, pro-MMP-2, and MMP-2, respectively.
Since Western blot analysis with a monoclonal antibody against MMP-2 showed—apart from the expected MMP-2 band—a lower-molecular-weight 28-kd band, as was also observed in gel zymography, this latter band could be a degradation product of MMP-2. Proteolytic processing of MMPs generating lower-molecular-weight products has been frequently reported (see, for example, Okada et al, 1988; Bjarnson and Fox, 1995; Shipley et al, 1996; Stanton et al, 1998). Moreover, unlike normal sperm samples in which a 28-kd band with gelatinolytic activity was clearly seen on gel zymography as well as Western blot analysis, in abnormal sperm samples, an enzymatically active 28-kd band was barely detected in gel zymography but was clearly observed by Western blot. This may indicate that such a 28-kd protein is found in both normal and abnormal sperm samples but is active only in normal sperm. It may therefore be speculated that a lack of 28-kd activation may be characteristic of certain types of male infertility. Of course, experimental verification is necessary to test the possibility of MMP-2 cleavage generating an active/inactive 28-kd product.
Comparison analysis showed no significant difference in 72-kd MMP, 62-kd MMP, and TIMP-1 between normal and abnormal human sperm samples. In contrast, a higher 28-kd MMP activity and a lower 92-kd MMP activity in normal relative to abnormal sperm samples were detected. The relevance to male infertility, if any, of MMP and TIMP found in sperm samples is presently unknown.
The time-course experiments showed that pro-MMP-9, pro-MMP-2, MMP-2, and TIMP-1 reached maximal release after 2 hours, while the 28-kd protein reached maximal activity after 4 hours. It may therefore be possible, that regarding the 28-kd protein, release is being stimulated by one of the MMPs or TIMP-1 or that, as mentioned earlier, the 28-kd product is an MMP degradation product. MMP-9 and the 28-kd protein, in contrast to pro-MMP-2 and MMP-2, remained visible on gel zymography in the time-course experiment even after 10 hours. This may be the result of a gradual release of pro-MMP-9 and 28-kd protein. A possible explanation for the quick release of MMPs into the medium is that MMPs are bound to the sperm from accessory sex gland secretions upon ejaculation. This process is analogous to the situation in mice-ejaculated spermatozoa, which have urokinase associated on the cell surface (Huarte et al, 1987). The mouse spermatozoa pick up urokinase from the vas deferens and seminal vesicle secretion. However, the repeated washing steps of the ejaculates, together with the immunofluorescence experiments demonstrating the presence of MMPs in different regions of the sperm, indicate that the above explanation is a remote one.
The time-course experiments together with the immunofluorescence assays suggest that most of the MMPs were secreted within a short time after sperm was incubated with the capacitation medium (HTF). A fast protein release from the sperm, as observed with MMP-2, can take place if the MMP is localized in the acrosome region. The immunofluorescence assays support this hypothesis, since MMP-2 in normal and abnormal sperm samples and pro-MMP-9 mostly in abnormal sperm samples were found localized in the acrosome region.
In conclusion, the necessity of breakdown of physical barriers in the fertilization process suggests that MMPs are involved in this task. This is the first report of MMP activity in normal and abnormal human sperm samples and of TIMP presence in sperm samples. The data indicate a different MMP profile between normal and abnormal sperm samples, with a higher 28-kd MMP activity and a lower 92-kd MMP activity in normal relative to abnormal samples.
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Footnotes |
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