Mol. Hum. Reprod. Advance Access originally published online on July 28, 2005
Molecular Human Reproduction 2005 11(8):575-582; doi:10.1093/molehr/gah197
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Macrophage migration inhibitory factor in the human epididymis and semen
Centre de Recherche en Biologie de la Reproduction and Département d’Obstétrique-Gynécologie, Faculté de Médecine, Université Laval, Ste-Foy, Québec, Canada
1 Present address: Université Blaise Pascal, 24, Av des Landais, 63177 Aubière Cedex, France
2 To whom correspondence should be addressed at: Unité d’Ontogénie-Reproduction, Centre de Recherche, Centre Hospitalier de l’Université Laval, 2705 Boulevard, Laurier, Ste-Foy, Québec, Canada. E-mail: robert.sullivan{at}crchul.ulaval.ca
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Abstract |
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During epididymal transit, mammalian spermatozoa acquire new proteins involved in the acquisition of motility and of male gamete fertilising ability. We have previously shown that membranous vesicles called epididymosomes are involved in the transfer of epididymal-originating proteins to spermatozoa. The cytokine macrophage migration inhibitory factor (MIF) is one of these proteins but the role played by MIF in relation to epididymal sperm maturation still remains unclear. As this protein has already been shown to bear different functions depending on its location, we investigated its distribution along the epididymis and in different compartments of human semen. Northern and Western blot analysis as well as immunohistochemical studies show that MIF is expressed all along the epididymis with a higher level of transcript in the proximal segment. MIF is associated with two types of membranous vesicles, i.e. epididymosomes and prostasomes, the latter being prostate-originating membranous vesicles present in the semen. In semen, MIF is associated with spermatozoa, prostasomes as well as the soluble fraction. The amount of MIF in the seminal fluid varies from one individual to another but does not correlate with the amount of MIF associated with ejaculated spermatozoa. There is a negative correlation between the amount of sperm-associated MIF and the percentage of motility in different semen samples. Sperm separation using discontinuous Percoll gradient centrifugation shows a higher amount of MIF associated with poorly motile spermatozoa compared to highly motile spermatozoa present in the lower Percoll fraction. These results are discussed with regards to the possible involvement of MIF in sperm motility acquisition during the epididymal transit.
Key words: epididymis/macrophage migration inhibitory factor/sperm motility/spermatozoa
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Introduction |
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Macrophage migration inhibitory factor (MIF) was first described as a T-cell cytokine (Bloom and Bennett, 1966
). Since its discovery as a proinflammatory mediator, MIF has been shown to have a widespread distribution and, depending on its location, to play different functions. MIF can thus be considered as a moonlighting protein, e.g. playing different functions depending on its subcellular or tissue distribution or depending on the differentiation status of the cell expressing this protein (Jeffery, 1999). MIF has been shown to possess a tautomerase activity (Rosengren et al., 1997) and thiol-protein oxidoreduction properties (Kleemann et al., 1998). When secreted by Leydig cells, MIF modulates Sertoli cell inhibin production (Meinhardt et al., 1996; Meinhardt et al., 1998). In other physiological systems MIF modulates insulin secretion by pancreatic ß cells and regulates the glucocorticoid-mediated suppression of the immune response (Calandra et al., 1995). MIF is expressed in numerous tissues (Shimizu et al., 1996; Waeber et al., 1997; Yoshimoto et al., 1997) including male reproductive tract (Frenette et al., 1998; Eickhoff et al., 2001; Frenette et al., 2003).
Following its differentiation in the testis the mammalian spermatozoon travels along the epididymis. It is during this journey along this single convoluted tubule that the male gamete will acquire its fertilising ability. Under androgen control, the epithelial cells bordering the epididymal lumen secrete proteins that interact with the sperm surface (Cooper, 1998). Some of these proteins are added to the spermatozoa and are essential for the production of fully functional gametes (Sullivan, 1999; Cuasnicu et al., 2002). We have previously shown that small membranous vesicles called epididymosomes are secreted in an apocrine manner by the epididymal principal cells (Legare et al., 1999b; Frenette and Sullivan, 2001; Frenette et al., 2002; Sullivan et al., 2003). Many proteins are associated with the epididymosomes and some of them are transferred to different subcellular compartments of the maturing spermatozoa (Frenette et al., 2002). MIF is one of the proteins transferred to spermatozoa during the epididymal transit (Frenette et al., 2003). Interestingly, it has been shown that MIF associated with epididymosomes is transferred to the dense fibres of the sperm flagellum (Eickhoff et al., 2001). These structures are known to be involved in sperm flagellar beating and acquire rigidity during the epididymal transit because of the formation of disulphide bonds that progressively increase during sperm maturation. MIF has also been shown to be present in large quantities in human semen, probably originating from the prostatic secretion containing membranous vesicles similar to epididymosomes and described as prostasomes (Frenette et al., 1998; Utleg et al., 2003). Considering the potential role of MIF in the modulation of sperm flagellar beating and motility, we investigated the distribution of MIF along the human epididymis as well as in semen samples.
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Materials and methods |
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Biological material
The use of human tissue and body fluids was approved by our institutional ethic committee. Human epididymides were obtained through our local organ transplantation program. Donors were of 20–49 years with no known medical pathologies that could affect reproductive function. Tissues were collected under optimal conditions while artificial circulation was maintained to preserve organs assigned for transplantation. Epididymides were immediately set on ice and transported to the laboratory and dissected in caput, corpus and cauda epididymidis as previously described (Legare et al., 1999a
). Tissues were fixed in freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS) and embedded in OCT (optimal cutting temperature: Canemco supplies, Canada) for immunohistochemical studies or snap frozen in liquid nitrogen and kept at –80°C for subsequent RNA or protein extractions. Paraffin blocks of human testicular tissues fixed with 4% paraformaldehyde were processed for immunohistochemistry. Sperm samples were obtained from our clinical andrology laboratory. Spermogram values, sperm count, percentage of total and progressive motility and normal morphology were determined using a Hamilton Thorn Computer Assisted Sperm Analysis (CASA) system. After liquefaction at room temperature, semen samples were diluted with two volumes of TN buffer (30 mM Tris, 130 mM NaCl, pH 7.5) and pelleted at 1200 g for 10 min. Spermatozoa were washed two times in TN and proteins extracted with 1% sodium dodecyl sulphate (SDS).
Western blotting
Epididymal tissues were homogenized in 1% SDS in water and centrifuged at 16 000 g for 20 min. Epididymal proteins in supernatants were precipitated with methanol/chloroform and resuspended in sample buffer for sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) (Laemmli, 1970). Protein concentrations were evaluated by amido black staining on dot-blots (Chapdelaine et al., 2001). Electrophoretic patterns were transferred onto nitrocellulose membrane, saturated with PBS containing 0.1% Tween and 5% skim milk. The membrane was incubated with a goat antiserum directed against MIF (R&D Systems, Minneapolis, MN, USA) diluted 1 µg/ml. After washing, the membrane was incubated in a peroxidase conjugated rabbit anti-goat immunoglobulin G (IgG) diluted 1/10 000. The immune complexes were revealed using a peroxidase chemiluminescent substrate (Amersham, Buckinghamshire, UK).
Northern blot analysis of MIF mRNA
Five micrograms of total RNA extracted from human lung was reverse-transcribed using superscript II (InVitrogen, Burlington, Ontario, Canada). 5'-CTCTCCGAGCTCACCCAGCAG-3' and 5'-CGCGTTCATGTCGTAATAGTT-3' primers were used to amplify a 255 bp region of MIF mRNA. The reaction conditions were as follows: initial denaturation at 95°C for 5 min followed by 30 cycles of denaturation at 94°C, annealing at 55°C and extension at 72°C for 1 min. The PCR product was purified on QIAquick PCR purification kit, cloned in pGEM-T and sequenced using our institution core facilities. The MIF cDNA probe was random-prime labelled using Ready Prime II kit (Amersham).
Ten micrograms of total RNA extracted from human caput, corpus, and cauda epididymidis were separated on a 1% agarose gel containing MOPS and formaldehyde and transferred onto a nylon membrane. Prehybridization was performed in Express Hyb (Clontech, Palo Alto, CA, USA) and hybridization was performed in the same solution containing 3 x 106 dpm/ml MIF-labelled probe. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to probe the same membrane and used as a ubiquitously expressed gene.
Immunohistochemistry
Cryosections of paraformaldehyde-fixed epididymal tissues embedded in OCT were immersed in 3% H2O2 prepared in methanol for 10 min to neutralize endogenous peroxidases. The sections were blocked with 5% rabbit preimmune serum, incubated for 90 min with a goat anti-human MIF at 3 µg/ml, followed by incubation with a biotinylated rabbit anti-goat IgG antiserum. Both first and second antibodies were diluted in 0.2% bovine serum albumin (BSA) in PBS. The immune complexes were revealed with avidin-peroxidase preformed complex (Vectastain ABC reagent, Vector, Burlingame, CA, USA) and the sections were counterstained with Harris’s hematoxylin. Preimmune goat serum diluted 1/2500 in 0.2% BSA in PBS was used as a negative control.
MIF in epididymosomes and prostasomes
The association of MIF with membranous vesicles present along the male reproductive tract was investigated by preparing epididymosomes and prostasomes from fluid collected in the vas deferens or from semen samples, respectively. Fluid was collected from the scrotal portion of the vas deferens during surgical vasectomy reversal, and epididymosomes were prepared as previously described (Frenette et al., 1998; Frenette and Sullivan, 2001). Samples were centrifuged two times at 3000 g for 10 min to eliminate spermatozoa and other cellular constituents. The supernatant was ultracentrifuged at 120 000 g for 2 h, and the pellet was resuspended in TN buffer and ultracentrifuged a second time under the same conditions.
Semen samples were processed in a similar way to prepare prostasome pellets. Following the first ultracentrifugation, the pellet was resuspended in TN buffer and chromatographed on a Sephacryl S-500 HR (Pharmacia, Baie d’Urfée, Québec, Canada). The prostasome-containing fractions corresponding to the void volume of the column were pooled and ultracentrifuged again at 120 000 g for 2 h.
The pellets containing epididymosomes or prostasomes were submitted to Western blot analysis to detect MIF using conditions similar to the ones described above.
To further document the association of MIF with prostasomes prepared from semen samples, the prostasome suspension obtained following Sephacryl S-500 HR column fractioning was mixed with isotonic Percoll to obtain a final 55% Percoll concentration. The density gradient was formed by centrifugation at 30 000 g for 30 min using a Beckman 50.2 Ti rotor. Fractions were collected along the density gradient and submitted to Western blot analysis using the anti-MIF antiserum. Density marker beads (Sigma, Oakville, Ontario, Canada) were centrifuged in parallel to establish the density gradient.
Discontinuous Percoll gradient centrifugation of ejaculated spermatozoa
Ejaculated spermatozoa were submitted to discontinuous Percoll gradient centrifugation to determine whether MIF was preferentially associated with a subpopulation of spermatozoa. Percoll solutions (85% and 60%) were prepared by mixing different volumes of Ham F-10 medium with an isotonic Percoll solution. One millilitre semen sample from each donor was layered on the top of two successive 1.5 ml layers of 60 and 85% Percoll solutions and submitted to centrifugation at 500 g for 20 min. Spermatozoa were collected in the pellet under the 85% Percoll layer and at the 60/85% Percoll interface. After motility evaluation, spermatozoa were washed three times with TN and proteins were extracted with 1% SDS. MIF associated with spermatozoa was analyzed by Western blotting and quantitated by densitometry. Quantities of MIF associated with dense spermatozoa (under 85% Percoll) was expressed as a percentage of MIF associated with the same number of spermatozoa recovered at the 60/85% Percoll interface: MIF on dense sperm (>85%)/MIF on less dense sperm (60/85%).
Effect of seminal plasma on MIF content in spermatozoa
If MIF association to spermatozoa negatively correlates with motility, we can expect that there is a dissociation of MIF following ejaculation. One way to investigate this possibility is to coincubate cauda epididymal spermatozoa with seminal plasma. For obvious reasons, these experiments are very difficult to perform using human biological material. Knowing that MIF is also present in bovine epididymis (Frenette et al., 2003), we have performed these experiments using this species. Epididymides were obtained from the slaughterhouse, and cauda epididymal spermatozoa were collected as previously described (Frenette and Sullivan, 2001). After washing, spermatozoa were resuspended in PBS to which 0.5 volume of seminal plasma was added. In control experiments, 150 mM NaCl or fetal bovine serum (FBS) (InVitrogen) was added instead of seminal plasma. Seminal plasma was prepared by centrifugation of fresh semen obtained from our local Center of Artificial Insemination (Alliance Bovitech, St-Hyacinthe, Québec, Canada) and kept at –80°C until used. Following 75 min at 37°C, spermatozoa were washed by centrifugation, proteins were extracted with 1% SDS and MIF content was determined by densitometric determination of Western blots probed with an anti-bovine MIF antiserum provided by Dr. M.Nishibori (Okayama University, Japan) (Frenette et al., 2003).
The quantity of MIF associated with cauda epididymal spermatozoa was determined in four independent control experiments. Each experiment was performed using a distinct pool of cauda epididymal spermatozoa. For each SDS–PAGE performed, the mean of MIF quantities was normalized at 100. For each control experiment, six to eight determinations were made and the standard deviation was calculated. The effect of plasma seminal coincubation on MIF quantities associated to spermatozoa was measured in parallel with control experiments. The effect of each of the seven different seminal plasma was determined three to seven times. T-test was used to compare individually the mean of MIF determination performed for each seminal plasma to each set of control experiments. Significance was set at P < 0.01.
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MIF mRNA and protein expression in the human epididymis
MIF mRNA was detectable all along the human epididymis. It was strongly expressed in the caput epididymidis and then gradually decreased in the corpus and cauda segments. The levels of MIF mRNA were in the order of four-fold higher in the proximal epididymidis compared to the distal segment (Figure 1A and B). Like the mRNA, MIF protein was detectable all along the human epididymis. The level of MIF detectable by Western blots performed on epididymal tissue homogenates was much more constant from one epididymal segment to the other when compared to the coding transcript (Figure 1C). Immunohistochemistry performed on testis sections showed that MIF is strongly detected in cells of the interstitial compartment. In some seminal tubule sections, a few spermatocytes were lightly stained (Figure 2A). MIF was strongly detectable in the principal cells of the epididymis. The cellular distribution and intensity were comparable all along the epididymis. MIF was distributed uniformly in the cytoplasm of principal cells with a slightly higher signal at the basal and apical region of the epididymal epithelium. The interstitial tissue did not show staining higher than background. MIF detection was specific as shown by the absence of staining when preimmune serum was used as a negative control (Figure 2B–D).
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MIF in human epididymosomes and prostasomes
Epididymosomes and prostasomes were prepared by a series of centrifugations of epididymal fluid and seminal plasma, respectively. The electrophoretic patterns of proteins associated with these membranous vesicles showed major differences depending on their origin, e.g. epididymal fluid or seminal plasma. The major protein band located between 65 and 97 kDa standards in the epididymosome electrophoretic pattern had not been identified yet but comparison with human serum albumin revealed a distinct migration pattern (data not shown) excluding the possibility of blood contamination occurring during epididymal fluid aspiration performed during assisted reproduction technology procedures (Figure 3A). Corresponding Western blots revealed that MIF was associated with both epididymosomes and prostasomes. The amount of immunodetectable MIF was much higher in prostasomes when compared to the MIF quantities associated with epididymosomes (Figure 3B). MIF in the seminal plasma was found to be associated with prostasomes but was also found in the soluble fraction prepared from seminal plasma obtained from different men. MIF was roughly distributed in equal quantities between prostasomes and the soluble fraction of the seminal plasma. This distribution was similar between semen samples obtained from different men (Figure 4). In different animal species, prostasomes have been shown to have a buoyant density of 1.05. The association of MIF with prostasomes was thus investigated by submitting these membranous vesicles to a continuous gradient centrifugation. Following centrifugation, Western blots of each Percoll fraction revealed that MIF was associated with the 1.05 density fraction (Figure 5).
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MIF associated with spermatozoa
Western blot analysis of human semen components showed that MIF was found as a soluble component and a constituent of both prostasomes and spermatozoa (Figures 4 and 6). The amount of MIF associated with a constant number of spermatozoa showed a great variability from one man to the other. The amount of MIF in seminal plasma was also characterized by an interindividual variability (Figure 6). There was no correlation between the amount of MIF associated with spermatozoa and the amount of MIF detectable in the seminal plasma from the same individual (Figure 6).
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The amount of MIF associated with a constant number of spermatozoa showed a reverse correlation with the percentage of motility in the semen sample (Figure 7). A similar reverse correlation was also observed between MIF quantities and percentage of spermatozoa with normal morphology or with progressive motility (data not shown). Different subpopulations of spermatozoa from different semen samples were prepared by Percoll discontinuous gradient centrifugation. MIF was detectable in highly motile (>85% Percoll density) and less motile (60/85% Percoll density) spermatozoa. However, in a given semen sample, the amount of MIF associated with a constant number of spermatozoa was much less in the highly motile population of spermatozoa (denser) when compared to the normal morphology spermatozoa with lower motility (less dense). An average of 32% less MIF was detectable in the >85% Percoll density sperm population (Figure 8).
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For obvious reasons, the effect of seminal plasma on cauda epididymal spermatozoa cannot be performed using human material. Bovine biological material was thus used to evaluate the effect of seminal plasma on the amount of MIF associated with spermatozoa. In four control experiments performed to evaluate the quantity of MIF associated with spermatozoa, the mean, SD and number of determinations were 100, 4.4, 8; 100, 6.13, 6; 100, 7.74, 8 and 100, 5.35, 8 for a total 100, 5.71, 30. The quantity of MIF associated with cauda epididymal spermatozoa incubated with seminal plasma was decreased when compared to the same population of sperm incubated with control isotonic NaCl solution. The ability of seminal plasma from seven different bulls to remove MIF from spermatozoa was evaluated in triplicate. The amount of MIF removed from spermatozoa varied from one seminal plasma sample to the other; from 5 to 35% with a mean of 17%. When compared to control experiments, the decrease in MIF content caused by each of seven seminal plasma was highly significant at P < 0.01 (Figure 9). When cauda epididymal spermatozoa were incubated with FBS, MIF amount associated with sperm cells remained the same (data not shown).
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Discussion |
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As previously reported in rat (Eickhoff et al., 2001
) and bovine (Frenette et al., 2003), MIF is detected in the human epididymis both at the transcriptional and protein level. Whereas the level of immunodetectable MIF along the epididymis is relatively constant, the mRNA level is higher in the caput epididymidis and then progressively decreases in the more distal portions. Thus, once secreted MIF is uniformly distributed in the epididymal fluid. In human testis, MIF is not immunodetectable in Sertoli cells or in spermatid suggesting that MIF to which spermatozoa are exposed in the excurrent duct is mainly secreted by the epididymis. MIF has been shown to be secreted by the epididymal epithelium in an apocrine manner (Eickhoff et al., 2001; Flieger et al., 2003; Nickel, 2003). This is based on the fact that its mRNA lacks a signal peptide and that this protein is associated with intraluminal membranous vesicles called epididymosomes (Jones, 1998; Saez et al., 2003). The situation appears to be the same in humans as MIF is immunodetectable in the cytoplasm of principal cells all along the epididymis. The association with epididymosomes may protect MIF from degradation and favours uniform concentration within the epididymal fluid. Many proteins are associated with epididymosomes and some of them are selectively transferred to spermatozoa during sperm epididymal transit (Frenette and Sullivan, 2001; Frenette et al., 2002). These proteins have been hypothesized to be major players in sperm maturation, i.e. acquisition of fertilising ability and modulation of sperm motility parameters (Yanagimachi et al., 1985; Sullivan et al., 2003). MIF may be one of these proteins.
The quantity of immunodetectable MIF in epididymosomes and in sperm protein extracts is relatively low when compared to the quantity detected in semen samples. In semen, MIF is associated with spermatozoa, prostasomes and is also found as a soluble constituent. As epididymosomes are a minor constituent of membranous vesicles present in total semen (Frenette et al., 2002), MIF in sperm free semen samples probably originates from prostatic secretion (Frenette et al., 1998). Thus, the function of MIF in semen may be distinct from its role during epididymal maturation. In fact, this cytokine has been shown to play different functions in different biological fluids(Bloom and Bennett, 1966; Calandra et al., 1995; Meinhardt et al., 1996; Rosengren et al., 1997; Kleemann et al., 1998). MIF being a major constituent of human seminal plasma, it may play a role in the female reproductive tract. MIF being well known as a proinflammatory cytokine (Bloom and Bennett, 1966), this protein may be involved in the control of vaginal immune response against spermatozoa (Frenette et al., 1998). MIF in the seminal plasma is associated with prostasomes as well as with the soluble fraction. The association of MIF with prostasomes is clearly demonstrated by its distribution following prostasome centrifugation on a Percoll density gradient. MIF distribution corresponds to a density of 1.05 which is a biophysical characteristic of prostasomes (Ronquist et al., 1978; Frenette et al., 2003). This indicates that MIF is secreted by the prostate in the classical way and a nonclassical apocrine mode (Aumuller et al., 1999; Hermo and Jacks, 2002; Nickel, 2003; Utleg et al., 2003; Ronquist and Nilsson, 2004). The biological significance of these two forms of MIF in the seminal fluid remains to be determined.
The quantity of MIF varies by a ten-fold factor in different seminal plasma samples (Frenette et al., 1998). The quantity of sperm-associated MIF also shows interindividual variability but does not correlate with the quantities of MIF present in the seminal plasma. Considering this absence of correlation and the fact that spermatozoa are in contact for a short period of time with the accessory gland secretions, it is reasonable to postulate that MIF associated with spermatozoa at the level of the outer dense fibres is probably transferred to spermatozoa during the epididymal transit(Eickhoff et al., 2001). There is a strong negative correlation between sperm motility and the quantity of MIF associated with a constant number of spermatozoa. Considering that sperm motility parameters are modulated during epididymal transit (Yeung and Cooper, 2002), the epididymosomes origin of sperm-associated MIF can be postulated.
In rat epididymal fluid, MIF have been shown to be associated with epididymosomes that interact with spermatozoa during epididymal transit. Contemporary, MIF accumulates in spermatozoa in association with the outer dense fibres of the mid and principal piece of the flagellum(Eickhoff et al., 2001). Eickhoff et al. (2001) clearly showed that MIF is associated with sperm midpiece and principal piece. This subcellular distribution of MIF correlates with localization of outer dense fibres in spermatozoa. These cytoskeletal components are known to be rich in cysteines and an increase in disulphide bonds in the flagellar cytoskeletal elements and in nuclear protamines occurs during epididymal transit (Calvin and Bedford, 1971). MIF is a trimer formed by identical peptides each having three cysteines. Interestingly, structural analysis reveals that the cysteines are present as free thiols (Sun et al., 1996). It has recently been shown that MIF decreases the zinc content of rat epididymal spermatozoa. In the epididymis, zinc binds to sulphydryl groups of outer dense fibres of maturing spermatozoa preventing premature disulphide bond formation (Eickhoff et al., 2004). By acting on zinc content, MIF can thus modulate motility patterns acquired during the epididymal transit (Yeung et al., 1993) (Figure 10). Interestingly, zinc content negatively correlates with sperm motility in human ejaculates (Henkel et al., 1999).
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The sperm cytoplasm is small resulting in a low ATP synthesis by the glycolytic pathway. Even though sperm forward motility is acquired during the epididymal transit, sperm motility has to be suppressed in the excurrent duct to maintain sperm energy. Epididymal proteins with sperm immobilising properties (Usselman and Cone, 1983; Usselman et al., 1985), acidification of intraluminal fluid by H+ATPase (Breton et al., 1996) and modulation of energy sources produced by the polyol pathway along the epididymis (Frenette et al., 2004) are different mechanisms proposed to control epididymal spermatozoa motility. The reverse correlation between MIF quantities associated with spermatozoa and motility suggest that this protein may be involved in repression of sperm motility in the epididymis. When submitted to Percoll gradient centrifugation, morphologically normal spermatozoa distributed themselves in two bands with different buoyant densities. We have previously hypothesized that the ‘dense’ spermatozoa were more mature when compared with the less dense spermatozoa. This was based on the fact that the proportion in number of ‘dense’ spermatozoa increases along the epididymis and that these spermatozoa are characterized by proteins known to be acquired during the epididymal transit (Sullivan and Robitaille, 1989; Sullivan et al., 1989). According to this hypothesis, we can deduce that the association of MIF with a subpopulation of spermatozoa is part of the sperm epididymal maturation processes that are involved in the maintenance of low motility along the excurrent duct. At ejaculation, MIF would then have to be removed from spermatozoa to favour forward motility and sperm functionality within the female genital tract. As demonstrated using bovine biological material, seminal plasma decreases the amount of MIF associated with cauda epididymal spermatozoa. Considering the relatively high MIF concentration in seminal plasma, this removing process is not driven by a diffusion process (Frenette et al., 1998) (Figure 10). Modifications of fibrous sheath of rabbit spermatozoa have also been shown to occur at ejaculation (Kim et al., 1997).
Moonlighting proteins are proteins that possess different functions that vary with subcellular localization, differentiation status or tissue distribution (Jeffery, 1999). In mammalian spermatozoa, the selenoprotein phospholipids hydroperoxide glutathione peroxidase (PHGPx) is an elegant example of a moonlighting protein. In spermatids, this protein is a soluble peroxidase whereas in differentiated spermatozoa it is a structural protein on the tail midpiece (Ursini et al., 1999). MIF may also be a moonlighting protein inhibiting sperm motility in the epididymis and playing another function in seminal plasma, possibly modulation of the female immune response.
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Acknowledgements |
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We thank Dr. Michel Thabet for providing us with vas deferens fluids recovered during surgical vasectomy reversal, Dr. Pierre Leclerc for gift of histological sections of human testis, Dr. M. Nishibori from Department pharmacology, Okayama University for generous gift of anti-bovine MIF antiserum, Mrs. Diane Dorval from the andrology laboratory of our institution for semen samples processing and information regarding spermogram parameters, The Centre d’Insémination Artificielle du Québec (CIAQ), St-Hyacinthe, Québec, Canada, for generous gift of fresh bovine semen samples and Dr. Pierre Leclerc for stimulating discussion. This work was supported by ‘Canadian Institutes for Health Research’ and ‘Natural Sciences en Engineering Research Council of Canada’ grants to R.S.
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Submitted on May 2, 2005; accepted on June 3, 2005.
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