Mol. Hum. Reprod. Advance Access originally published online on May 28, 2004
Molecular Human Reproduction 2004 10(8):605-611; doi:10.1093/molehr/gah075
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Influence of macrophage migration inhibitory factor (MIF) on the zinc content and redox state of protein-bound sulphydryl groups in rat sperm: indications for a new role of MIF in sperm maturation
1Department of Anatomy and Cell Biology, 2Department of Dermatology and Andrology, 3Institute for Plant Ecology, Justus Liebig University, D-35385 Giessen, 4Department of Anatomy and Cell Biology, Philipps University of Marburg, D-35037 Marburg, Germany and 5Department of Dermatology, Juntendo University, School of Medicine, Tokyo, Japan
6 To whom correspondence should be addressed at: Department of Anatomy and Cell Biology, Justus-Liebig-University of Giessen, Aulweg 123 D-35385 Giessen, Germany. Email: andreas.meinhardt{at}anatomie.med.uni-giessen.de
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The function of macrophage migration inhibitory factor (MIF) in sperm maturation was studied by investigating its role in the biochemical maturation of the outer dense fibres. Rat sperm obtained from the caput and cauda epididymis were stimulated overnight with either recombinant MIF or MIF-containing vesicles originating from epididymal fluid at various concentrations. The zinc content of both the sperm and the medium was determined by means of atomic absorption spectrometry. Incubation in both recombinant MIF and vesicular MIF resulted in a statistically significant decrease of the zinc content in stimulated caput sperm of
50%. In parallel, the conditioned media showed a clear increase in the concentration of this trace metal. The effect of MIF was less marked in cauda sperm. In addition, we demonstrated a statistically significant increase of detectable free thiol groups in the sperm mid- and principle piece in isolated rat sperm after stimulation with MIF at concentrations of 25 and 50 ng/ml. Our data suggest that MIF plays an important role in the maturation process of rat sperm during epididymal transit by inducing the elimination of zinc and affecting the amount of free sulphydryl groups in the sperm flagella. Key words: migration inhibitory factor/sperm/zinc
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Introduction |
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Macrophage migration inhibitory factor (MIF) is a multifunctional, mostly pro-inflammatory cytokine of broad target specificity (reviewed in: Calandra and Roger, 2003
; Lolis and Bucala, 2003). Distinguishable from other cytokines, MIF exhibits at least two catalytic activities, a tautomerase (Rosengren et al., 1996, 1997) and a thiol-protein oxidoreductase (TPOR) activity (Kleemann et al., 1998a,b). Shown to catalyse the reduction of insulin and 2-hydroxyethyldisulphide in a glutathione- and lipoamide-dependent manner (Kleemann et al., 1998b), MIF's TPOR activity has been found to be dependent on the presence of the cysteines in the motif Cys57-Ala-Leu-Cys60 (Kleemann et al., 1999; Nguyen et al., 2003a). This CXXC motif is a common feature of all TPOR enzymes and can be found in thioredoxin/adult T-cell leukaemia-derived factor and the disulphide bond proteins (e.g. protein disulphide isomerase and glutaredoxin), amongst others. Similar to the other TPOR, MIF catalyses the reduction of protein disulphides in a cysteine-based dithiol/disulphide reaction. It is this enzymatic action that is thought to be responsible for at least part of the immunological activity of MIF, as the substitution of either Cys 57 or Cys 60 by serine can result in the partial or complete inactivation of the immunological actions of the MIF protein (Bendrat et al., 1997; Swope et al., 1998; Hermanowski-Vosatka et al., 1999; Jung et al., 2001). The involvement of MIF in cellular redox processes is further substantiated by the findings that it is capable of binding to and regulating peroxiredoxin PAG (Jung et al., 2001) and able to inhibit oxidative stress-induced apoptosis (Nguyen et al., 2003b).
It is only recently that the binding of MIF to CD74, the cell surface form of MHC class II-associated invariant chain, was demonstrated (Leng et al., 2003). This is the first finding that MIF binds to a cell surface protein, but it does not explain how MIF targets such a broad spectrum of cells, as CD74 is found only on a limited number of cells, mainly leukocytes. Consequently, in the search to elucidate the molecular mechanism of MIF's actions, attention has also focused on the investigation of its enzymatic properties and to the identification of its natural substrates. However, thus far, neither has a natural substrate for the enzymatic activity of MIF been found nor has a conventional cytokine receptor been identified.
In the male reproductive tract, MIF has been found in both the testis and the epididymis. In the testis, it is expressed by the Leydig cells and is involved in the paracrine regulation of testicular function (Meinhardt et al., 1996, 2000). MIF has also been found to be produced in a regional specific manner by the epithelial cells of the epididymis, with maximal expression in the caput (Eickhoff et al., 2001; Frenette et al., 2002). In addition, MIF was localized in membranous vesicles secreted by the epithelial cells of the epididymis, which are in close contact with epididymal sperm. Therefore, a role for MIF in post-testicular sperm maturation has been hypothesized (Eickhoff et al., 2001). In the epididymis, many secretory proteins are expressed in a regional-specific pattern and it is assumed that they interact with the sperm surface in a sequential manner. This includes proteins which are transported to the sperm by membranous vesicles released by the epithelial cells of the epididymis (Kirchoff and Hale, 1996; Frenette et al., 2002, 2003).
Despite the locomotive apparatus in the sperm tail being formed during spermatogenesis, testicular sperm are unable to move in a coordinated manner. This developmental change, essential for the ultimate function of sperm, occurs during their transit through the epididymis. It is during spermatogenesis that considerable amounts of zinc are incorporated into the outer dense fibres (ODF), a structural component which extends along 60% of the length of the principal piece of the sperm flagellum (Baccetti et al., 1973; Serres et al., 1983). In ejaculated sperm >93% of the zinc is located in the flagellum (Henkel et al., 1999) and it has been demonstrated that in humans the flagellar zinc content is negatively correlated with motility (Henkel et al., 1999, 2003b). Zinc has been shown to bind to the sulphydryl groups of the ODF proteins, which are unusually rich in cysteine, and through the formation of zinc–mercaptide complexes prevents the premature oxidation of these groups to disulphide bridges (Baccetti et al., 1976; Henkel et al., 2003b). During the passage of the sperm through the epididymis, zinc is eliminated again from sperm by an unknown mechanism and subsequently reabsorbed by the epithelial cells of the epididymis (Calvin and Bleau, 1974; Henkel et al., 2003a,b). Concurrent to the removal of zinc, and due to the now unrestrained oxidation processes, there is a progressive increase in the degree of disulphide bridges between proteins within the ODF. This results in the greater structural stability of the maturing epididymal sperm (Calvin and Bedford, 1971), which in turn provides protection against any breakage caused by shear forces during ejaculation (Baltz et al., 1990) and imparts the counterforce essential for an efficient tail beat and motility (Lindemann and Kanous, 1995). As a consequence, the flagella attain full functional competence.
The considerable amounts of MIF found in the caput region of the epididymis, together with its known catalytic properties as a thiol-protein oxidoreductase, led us to postulate that MIF may be one of the factors influencing the thiol status of sperm as they progress through the epididymis, and that MIF may therefore play an important role in the attainment of motility and ultimately the ability of sperm to fertilize an oocyte. In the course of our study we also investigate a candidate natural substrate for the enzymatic activity of MIF and obtain further insight into a possible molecular mechanism underlying MIF's actions.
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Reagents and animals
Male adult Wistar rats (290–310 g body weight) were purchased from Charles River (Germany). For organ removal, animals were anaesthetized and killed by CO2 asphyxiation. The local animal ethics committee (RP Giessen) approved the investigations. In order to determine the thiol status of the rat sperm flagella, a 5 mmol/l solution of N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM) was made in acetone at 4°C to prevent autofluorescence occurring at pH >8.0. DACM binds specifically and quantitatively to protein-bound free sulphydryl groups. Sperm were isolated in human tubal fluid (HTF) medium containing 25 mmol/l NaHCO3, 101.6 mmol/l NaCl, 4.69 mmol/l KCl, 2.04 mmol/l CaCl2, 0.2 mmol/l MgSO4, 0.37 mmol/l KH2PO4, 2.78 mmol/l glucose, 0.33 mmol/l Na pyruvate, 21.4 mmol/l Na lactate, 20 mmol/l HEPES, 0.005 mg/ml Phenol Red, 0.06 mg/ml penicillin, 0.05 mg/ml streptomycin sulphate (Quinn et al., 1985
). Prior to use, osmolarity of the HTF medium was adjusted to 280 mOsmol/kg and bovine serum albumin (1% w/v) was added (HTF–BSA). To avoid possible contamination with zinc, all chemicals were of the highest possible quality (supra pure). The water was purchased from Merck (p.A. grade, Germany). All solutions were prepared in sterile polyethylene tubes and zinc-free water was used. Recombinant MIF and the polyclonal anti-MIF antibody were generously provided by Dr Bacher (Marburg, Germany).
Insulin reduction assay
The enzymatic redox activity of rec MIF was measured as previously described by Kleemann et al. (1998b)) based on the insulin reduction assay of Chandler and Varandani (1975) and Holmgren (1979). Briefly, this enzymatic assay is based on the reduction of insulin and subsequent insolubilization of the insulin ß-chain. The time-dependent increase in turbidity is then measured spectrophotometrically at 650 nm. The reaction was started by adding 177.5 µl recombinant (r)MIF dissolved in 10 mmol/l Tris, 50 mmol/l NaCl, pH 7.4 or a control solution (containing buffer alone), and 22.5 µl 200 mmol/l reduced glutathione (GSH) to 700 µl ice-cold reaction mixture containing 1 mg/ml insulin, 100 mmol/l sodium phosphate buffer (pH 7.2) and 2 mmol/l EDTA. MIF-catalysed insulin reduction was measured against the control solution (containing GSH) in the same experiment.
Isolation of epididymal sperm from rat
Epididymides were collected from rats and the epididymal duct fluid containing sperm was obtained separately from the caput and cauda region. The ducts of the respective epididymal segments were carefully cut open, minced with scissors and then rinsed in HTF–BSA. Epididymal sperm were separated from the epididymal secretion by centrifugation at 600 g for 10 min at room temperature. After washing, the resulting pellet was resuspended in HTF–BSA and the sperm counted in a haemocytometer.
Isolation of vesicles from epididymal fluid
Vesicles were isolated according to the method described by Fornes et al. (1991). Briefly, epididymides from five male adult Wistar rats were removed, and epididymal duct fluid was obtained separately from the caput and cauda region by rinsing the respective epididymal segments in ice-cold sucrose buffer (250 mmol/l sucrose, 10 mmol/l Tris–Cl, 1 mmol/l EDTA, and 1 mmol/l Pefabloc SC inhibitor; pH 7.4). All successive steps were carried out at 4°C. Sperm were removed by centrifugation at 600 g for 10 min. The supernatant was centrifuged at 2000 g for 20 min to precipitate tissue fragments and cellular debris. The resulting supernatant was ultracentrifuged at 100 000 g for 30 min to isolate vesicles from epididymal duct fluid. The pellet was then resuspended and centrifuged a second time at 100 000 g for 30 min. Microsomes may represent a considerable contamination of the isolated vesicle fraction. Finally, the vesicles were suspended in 800 µl of sucrose buffer and the protein content of the samples was analysed according to the colorimetric method described by Bradford (1976). Finally, the MIF content of the isolated vesicles was estimated by western blot analysis, by comparison with defined quantities of bioactive rMIF using a polyclonal rabbit anti-MIF antibody.
Western blot analysis
Vesicles isolated from epididymal fluid were separated on a 10% Tricine–sodium dodecyl sulphate polyacrylamide gel and subsequently transferred from the gel to a 0.2 µm nitrocellulose membrane (Schleicher & Schuell, Germany). Increasing concentrations of rMIF were run in separate lanes for estimation of MIF concentration in the vesicles. MIF was detected using a polyclonal rabbit antibody (1:400) raised against rMIF (data not shown).
Stimulation of rat sperm
In six independent experiments, epididymal rat sperm obtained separately from the caput and cauda were suspended in HTF–BSA containing either rMIF or a suspension of MIF-containing vesicles which were freshly isolated from epididymal fluid. rMIF was added at concentrations of 10, 25, 50 and 75 ng/ml. Alternatively, suspensions of epididymal membranous vesicles containing MIF at the identical concentrations (as determined by western blot) as those for the rMIF were added to the sperm. HTF–BSA without rMIF or vesicle suspension served as negative control. Aliquots containing 5 x 106 sperm in each treatment group (each experiment was conducted in two sets of triplicates) were added to 6-well culture dishes (Falcon, France) and incubated overnight at 32°C. Overnight incubation was chosen in consideration of the relatively slow onset of MIF–TPOR activity and the fact that sperm cells are in contact with the factor for 2 weeks during epididymal passage. The next day, the sperm were separated by centrifugation at 400 g for 10 min, then washed in HTF–BSA, and finally the resulting pellet was resuspended in fresh HTF–BSA. Sperm and conditioned media were stored at –20°C prior to determination of the zinc concentration. For the specific staining of free sulphydryl groups with DACM, sperm were used immediately after incubation.
DACM staining of free sulphydryl groups
The staining method for the utilized protein-bound free sulphydryl groups, other than some slight modifications, followed the protocol developed by Ogawa et al. (1979). Briefly, suspended sperm were treated with 1% Triton X-100 in phosphate-buffered saline (PBS) for 15 min and then incubated with 0.01 mmol/l DACM in 5 mmol/l Tris–acetate buffer, 0.85% NaCl (TAS buffer, pH 6.8) for 3 min at room temperature. After incubation, sperm were centrifuged at 600 g for 10 min, washed in cold TAS buffer and smeared on glass slides. Fluorescence intensity was quantified using a PTI Photomultiplier in combination with a cuvette system (Delta Scan Illumination System; Photon Technologies International, USA). Cells were excited with a wavelength of 400 nm and the intensity of emission at a wavelength of 480 nm was recorded. For documentation, DACM-labelled sperm were photographed using a Zeiss LSM 410 inverted laser scanning microscope (Zeiss, Germany).
Determination of zinc concentrations by means of atomic absorption spectrometry
Since zinc is a ubiquitous element, special care had to be taken when handling the samples to avoid contaminations. Therefore, for all procedures only chemicals of highest purity, ‘supra pure’ or ‘pro analysi’, were used. For determination of zinc, samples were thawed at room temperature. Subsequently, sperm samples were dissolved with 0.1 mol/l KOH (supra pure; Merck) and diluted 1:50 000 with 1% nitric acid (supra pure; Merck) and incubated for 30 min. Media samples were only diluted with 1% nitric acid and incubated for 30 min. Samples were measured with an atomic absorption spectrometer (Perkin–Elmer M 2100; Perkin–Elmer, Germany) using a graphite furnace (Perkin–Elmer GRK/PP 2100) under argon at a wavelength of 213.9 nm. For each measurement, 5 µl of the diluted sample was used. For preparation of a calibration curve, a zinc standard solution (0, 1, 3, 5 µg/l; Merck) was diluted in 1% nitric acid. The data were recorded as ng zinc/106 sperm and ng zinc/ml respectively. Due to the variation of zinc concentration from experiment to experiment and for better comparison of the effects of the different treatments, a conversion of the original data into ‘percentage zinc to control’ was performed.
Statistical analysis
Mann–Whitney rank sum test was used for comparing differences between experimental groups. All statistical analyses were performed with the Sigma Stat software (USA). All values stated are mean±SD (n=6 experiments per group) unless otherwise indicated.P<0.05 was considered significantly significant.
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MIF TPOR activity
TPOR activity of rMIF was obtained by comparing control incubations with those supplemented with rMIF. MIF, in a glutathione-dependent manner, was found significantly to accelerate the rate of precipitation of insulin and to reduce the lag-time that is observed prior to insolubilization (data not shown). Onset of TPOR enzyme activity was recorded after
30 min.
Effect of MIF on zinc release in sperm
Control experiments of freshly isolated sperm, and sperm cells which were harvested after overnight incubation in HTF–BSA only, revealed a decrease in intracellular zinc concentration of 40% from 75 250 to 32 500 ng/106 sperm of the original in caput sperm and 60% from 36 800 to 21 350 ng/106 sperm in cauda sperm. Compared to untreated control samples, a statistically significant further decrease in zinc concentration was found in epididymal sperm isolated from caput and cauda origin which were incubated in HTF–BSA containing either rMIF or isolated epididymal vesicles. In general, similar effects on intracellular zinc liberation were observed when caput and cauda sperm were incubated with MIF-containing vesicles or rMIF (Figures 1A and 2A Figures 1A and 2A). In addition, the effect of both treatments was much more pronounced in caput sperm than in sperm from the cauda epididymis. The maximal decrease of the zinc content (–55%) compared to untreated control concentrations was determined for caput sperm cells which were incubated with MIF-containing membranous vesicles (75 ng/ml MIF; Figure 1A). Treatment of cauda sperm with vesicular MIF resulted in a much smaller reduction of zinc (Figure 1A). In comparison to vesicular MIF, rMIF caused a marginally less prominent liberation of zinc from caput sperm (–51% at 50 ng/ml rMIF; –47% at 10 ng/ml MIF). The data are summarized in Table I.
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Concomitantly with zinc liberation from sperm, the concentrations of the trace element were elevated in the conditioned culture media (Figures 1B and 2BFigures 1B and 2B). In agreement with the stronger zinc-eliminating effect of MIF on caput sperm, the concentrations of zinc in the culture supernatant of these cells showed the highest increase. The main rise (to 160%, absolute values: 119–194 ng/ml) in culture medium were observed at rMIF concentrations of 10 ng/ml (Figure 2B), which paralled the maximum zinc liberation from caput sperm (Figure 2A).
Effect of MIF on protein-bound sulphydryl groups in sperm
Treatment of freshly isolated rat epididymal sperm with DACM, an agent which specifically and quantitatively reacts with protein-bound sulphydryl groups to form a highly fluorescent product, resulted in intense labelling of the sperm midpiece. Confocal microscopy revealed that the signal intensity decreased towards the more distal parts of the sperm tail, whereas the sperm head was completely negative (Figure 3). Slightly, but not significantly, higher levels of free sulphydryl groups in untreated cauda sperm were measured in comparison to caput sperm using a fluorescence-ratio-image system for quantification (Figure 4). Based on the similar effects of rMIF- and MIF-containing vesicles on sperm zinc elimination, only rMIF was used for the following investigations in the redox state of protein-bound sulphydryl groups in sperm. rMIF caused a statistically significant increase of detectable thiol groups both in caput and cauda sperm. In caput sperm, a significant rise (40%) of free sulphydryl groups was obvious when stimulated with 50 ng/ml rMIF, whereas the lower concentration (25 ng/ml) was only marginally effective (8% increase; Figure 4). In contrast, both rMIF concentrations caused a significant increase of free protein-bound sulphydryl groups in cauda sperm (55 and 40% respectively; Figure 4), where the maximum value (55%, 25 ng/ml MIF) markedly exceeded the value determined in caput sperm (40%; Figure 4).
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In the rat and bovine testis, MIF has been shown to be secreted by the epididymis as a constituent of small apical blebs [epididymosomes (Eickhoff et al., 2001
; Frenette et al., 2003)]. Epididymosomes are released from the apical plasma membrane of the epithelial cells of the epididymis and contain vesicular structures that fragment in the luminal milieu. Proteins secreted in this manner are often found to be glycosyl phosphatidylinositol (GPI)-linked, anchored to the membrane of the epididymosomes, and their sequences are characterized by the absence of a signal peptide (Legare et al., 1999; Frenette et al., 2003). The latter is the case for aldolase reductase and for MIF (Eickhoff et al., 2001; Frenette et al., 2003). Considering that some of these proteins have been identified on the sperm surface during epididymal transit, it is likely that the apocrine secretion of these vesicles may play a role in sperm maturation. MIF has been shown to be associated with the cortex of sperm outer dense fibres (ODF), suggesting that MIF may play a role in the attainment of motility by sperm during their transit. This premise becomes more likely, as the proteins of the ODF are unusually rich in cysteines.
During spermatogenesis, zinc is incorporated into the ODF of testicular sperm to prevent premature oxidation of the cysteine residues by the formation of zinc–mercaptide complexes. Release of this protective zinc is therefore a prerequisite for the formation of disulphide bonds in the ODF of epididymal sperm. Coincidentally, previous studies on human semen from infertile patients and healthy donors revealed repeatedly a negative correlation between the zinc concentration in the flagella and the capacity for sperm motility and velocity (Henkel et al., 1999). Recently, a significant increase in the percentage of progressively motile human sperm, accompanied by a significant increase of straight line velocity, was observed after incubation with extracellular zinc chelators (Wroblewski et al., 2003). In vivo, comparable changes in the motility pattern take place during epididymal sperm maturation. In the cauda epididymidis, sperm show only slow, sluggish and non-linear movement. After maturation, however, they are progressively motile with a high straight line velocity (Yeung et al., 1993).
In this study, the zinc values that we found are higher than those published by Kaminska et al. (1987). Apart from methodological aspects of using flame atomic absorption spectrophotometer (Kaminska et al., 1987) or the graphite furnace atomic absorption spectrophotometer (this study), which is 100–1000-fold more sensitive, the high range of the zinc values has to be mentioned. In our previous work on human sperm (Henkel et al., 1999, 2003b), we found ranges of 8.5–121.3 and 1.1–334.7 ng zinc/106 sperm respectively. In addition, considering that rat ODF are 30 times bigger than human ODF and that we used epididymal sperm, which contain much higher amounts of zinc than ejaculated sperm, the measured zinc content for epididymal rat sperm corresponds with the expected values. Finally, while Kaminska et al. (1987) only washed the sperm with physiological saline solution (of which the purity is unknown), we incubated them overnight in a culture medium composed of ingredients of the highest purity available. Even the water used as solvent was of analytical grade with extremely low zinc content. Despite these precautions (zinc is a ubiquitous element), however, some contamination of the medium cannot completely be ruled out, which in turn could result in an increased measured zinc concentration.
As zinc release and thiol redox reactions in the sperm midpiece are intricately linked to each other, both parameters were assessed to determine whether the TPOR enzymatic activity of MIF could contribute to the redox processes occurring in the ODF during epididymal sperm maturation. As it was unclear from preliminary experiments whether MIF could directly interact with the sperm after disintegration of the epididymosomes or whether an intact membranous vesicle would be essential to deliver MIF to the sperm, we applied both rMIF and freshly isolated MIF containing epididymosomes in our assays. In vitro experiments performed with epididymal rat sperm obtained from opposite regions of the rat epididymis (caput and cauda) resulted in a significant decrease of the zinc concentration in MIF-stimulated sperm. For caput sperm the effect was considerably stronger than for cauda sperm, a finding that may be attributed to the fact that the zinc content of cauda sperm cells is already much lower than in caput sperm and consequently less accessible to the actions of MIF. Interestingly, the effects of both rMIF- and MIF-containing vesicles were similar—a finding that suggests that MIF functions either indirectly by binding to mediating partners on the plasma membrane of sperm (such as potentially CD74) or by direct uptake into the cell. MIF uptake and subsequent interaction with cytosolic proteins has already been shown with Jab1 in HeLa cells and PAG in kidney fibroblasts (Kleemann et al., 2000; Jung et al., 2001). However, whether this is also the mechanism by which MIF influences sperm maturation remains to be elucidated. At the very least, we have demonstrated the immunological presence of MIF in rat sperm ODF (Eickhoff et al., 2001).
To investigate whether the zinc-eliminating effect of MIF was associated with a TPOR reaction, we performed experiments to measure the amount of free sulphydryl groups in isolated sperm. In untreated epididymal sperm, a prominent staining of sulphydryl groups in the midpiece and principle piece of the sperm tail was demonstrated using DACM. These data confirm previous biochemical analyses which identified an unusually high amount of cysteines in the ODF proteins, which are located in the sperm midpiece and principal piece (Bedford and Calvin, 1974; Calvin et al., 1975). No fluorescent staining of the sperm head was observed, indicating that the oxidation of the sulphydryl groups of the protamines for chromatin condensation must have taken place before the sperm reached the epididymis. Quantification of the fluorescent intensity of DACM-treated sperm stimulated with rMIF revealed a significant increase of detectable free sulphydryl groups in both caput and cauda sperm. The most active concentrations of rMIF were different among the assays performed. Optimal stimulation of cauda sperm was obtained with 25 ng/ml. In contrast, caput sperm showed a significant increase only after stimulation at a concentration of 50 ng/ml. This coincides with the in vivo observation that the luminal fluid of the caput epididymis contains much higher concentrations of MIF than the more distal parts (Eickhoff et al., 2001). In terms of mechanism, the elevated amounts of free sulphydryls after MIF stimulation can be the consequence of both zinc elimination from the zinc–mercaptide complex, which in turn allows the free sulphydryl groups to be accessed by DACM, or alternatively a direct TPOR activity of the cytokine.
As cysteine residues of the ODF are normally oxidized during sperm passage through the epididymis (Bedford and Calvin, 1974; Calvin et al., 1975), we initially expected a decrease of DACM-labelled sulphydryl groups in unstimulated cauda sperm compared to caput sperm. However, a small increase of free thiol groups was recorded. This observation could imply that the elevated amounts of free sulphydryl groups measured in this study are not due to a direct oxidizing effect of MIF. We rather suppose an indirect effect of the cytokine causing or supporting the elimination of zinc from the zinc–mercaptide complexes in the ODF, which in turn allows the free sulphydryl groups to be accessed by the fluorescent reagent DACM. The oxidation of the SH groups to disulphide groups would then be executed by another mechanism that is still unknown.
In conclusion, our data implicate a new role for MIF in the maturation process of rat sperm during epididymal transit by inducing or supporting the elimination of zinc from zinc–mercaptide complexes and therefore affecting the number of free SH groups in the sperm flagella.
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Acknowledgements |
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The authors wish to thank Prof. J.Bernhagen for his help with the MIF enzymatic assay and Dr Con Mallidis and Mrs S.Henkel for critically reading the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Me 1323/2-4 and We 2344/4-1).
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Submitted on April 6, 2004; resubmitted on May 10, 2004; accepted on May 12, 2004.
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