Mol. Hum. Reprod. Advance Access originally published online on October 18, 2006
Molecular Human Reproduction 2006 12(12):781-789; doi:10.1093/molehr/gal085
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Evidence for the involvement of proline-directed serine/threonine phosphorylation in sperm capacitation
1Center for Research in Contraceptive and Reproductive Health, Department of Cell Biology, University of Virginia, Charlottesville, VA and 2Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA, USA
3 To whom correspondence should be addressed at: Department of Veterinary and Animal Sciences, University of Massachusetts, 208 Paige Building, 161 Holdsworth Way, Amherst MA 01003, USA. E-mail: pvisconti{at}vasci.umass.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
To become fertilization competent, mammalian sperm undergo changes in the female reproductive tract termed capacitation. Capacitation correlates with an increase in tyrosine phosphorylation; however, less is known about the role of serine/threonine phosphorylation in this process. Proline-directed phosphorylation is one of the major regulatory phosphorylation events in many cellular processes such as cell proliferation and differentiation. Using mitotic phosphoprotein monoclonal-2 (MPM-2) antibody in this study, we observed that several mouse sperm proteins in the range of 70–250 kDa underwent increased serine/threonine–proline phosphorylation during capacitation. In contrast to the time course of tyrosine phosphorylation, proline-directed phosphorylation could be observed at shorter time points of sperm incubation, and it was found to be independent of NaHCO3 and adenosine 3'5'-cyclic monophosphate (cAMP). Similar to the regulation of the increase in tyrosine phosphorylation, cholesterol acceptors such as bovine serum albumin (BSA) or 2-hydroxypropyl-ß-cyclodextrin (2-OH-propyl-ß-CD) were essential for the regulation of proline-directed phosphorylation in mouse sperm. Furthermore, it was also found to be BSA dependent in human sperm. Among proline-directed kinases, extracellular signal-regulated kinase 1/2 (ERK1/2) is present in mammalian sperm; nevertheless, U0126 and PD098059, two inhibitors of the ERK pathway, did not block this phosphorylation in mouse sperm. In conclusion, capacitation is associated with an increase in proline-directed phosphorylation linked to cholesterol efflux in the sperm.
Key words: sperm/capacitation/phosphorylation/kinases/MPM-2
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
To become fertilization competent, mammalian sperm undergo a series of biochemical and physiological changes in the female reproductive tract collectively referred to as capacitation (Yanagimachi, 1994
). Capacitation can be studied in vitro in a defined medium containing energy sources (lactate and pyruvate), a protein source such as bovine serum albumin (BSA) and certain ions such as HCO3– and Ca2+. The process of capacitation was discovered in the 1950s (Austin, 1951; Chang, 1951); however, the signalling cascades involved in capacitation are still not fully understood. Based on studies in several species, it has been shown that capacitation is associated with an adenosine 3'5'-cyclic monophosphate (cAMP)-dependent increase in tyrosine phosphorylation (Visconti et al., 1995b; Leclerc et al., 1996; Galantino-Homer et al., 1997; Kulanand and Shivaji, 2001). Also, the role of protein kinase A (PKA), a serine/threonine kinase, in sperm capacitation has been well established. For example, null mutants of the testis-specific PKA catalytic subunit C2
are infertile and display capacitation-related defects such as a lack of the aforementioned increase in tyrosine phosphorylation (Nolan et al., 2004). Recently, antibodies against the phosphorylated form of the consensus target sequence for PKA have been commercially available, and using this antibody, the role of PKA-dependent phosphorylation has further been established in human sperm (O’Flaherty et al., 2004; Moseley et al., 2005) and boar sperm (Harrison, 2004). Other serine/threonine kinases, such as PKC (Breitbart et al., 1992; Kalina et al., 1995), Protein kinase B/v-akt murine thymoma viral oncogene (PKB/Akt) (Nauc et al., 2004), glycogen synthase kinase 3ß (GSK3ß) (Vijayaraghavan et al., 1996) and kinases from mitogen-activated PK (MAPK) pathway, such as MAPK/ERK kinase (MEK) (Thundathil et al., 2002; O’Flaherty et al., 2005) and extracellular signal-regulated kinase 1/2 (ERK1/2) (Luconi et al., 1998; de Lamirande and Gagnon, 2002), have been reported to be present in sperm.
The phosphorylation of proteins on serine or threonine residues that immediately precede a proline (phosphoserine/phosphothreonine–proline) plays an essential role in the regulation of cellular processes such as cell proliferation and differentiation. This specific serine/threonine phosphorylation, referred to as proline-directed phosphorylation, is known to be deregulated in conditions such as neurodegeneration in Alzheimer’s disease and cancer (Lu et al., 2002; Lu, 2004). Proline-directed kinases include cyclin-dependent protein kinases (CDK), MAPKs, GSK and Jun N-terminal kinase (JNK) (Lu et al., 2002). Serine/threonine phosphorylation is known to regulate protein function by bringing conformational changes in them. In addition, phosphorylated serine/threonine also function as binding motifs for recruiting proteins into signalling networks or placing enzymes within proximity to substrates (Pawson and Scott, 1997). Proline exists in two distinct forms cis and trans conformations, in relation to the adjacent phosphorylation site (serine/threonine), and their interconversion occurs spontaneously, albeit at a very slow rate. Upon phosphorylation of the adjacent serine/threonine, the isomerization is carried out very efficiently by a specific subgroup of peptidyl–prolyl isomerases (PPIases) from the Pin-1 family. The isomerization of cis and trans forms of proline can alter the local or global structure of the target proteins and thus regulate its function in biological processes. Pin-1 binds to the phosphoserine/phosphothreonine–proline motif of proteins, a site also known as mitotic phosphoprotein monoclonal-2 (MPM-2) antigen, because it is recognized by a phospho-specific mitosis marker antibody MPM-2 (Yaffe et al., 1997). The epitope for the MPM-2 antibody is phosphoserine/phosphothreonine–proline, and thus, Pin-1 and MPM-2 antibody share the same binding site. The MPM-2 antibody is a useful tool to study proline-directed phosphorylation in various cell systems. Two recent reports describe the use of the MPM-2 antibody in human sperm. In the first study, the antibody recognized two proteins (75 and 80 kDa) in the Triton-insoluble fraction of human sperm proteins (de Lamirande and Gagnon, 2002). These two proteins underwent capacitation-associated increase in proline-directed phosphorylation, and their phosphorylation was inhibited by the ERK pathway inhibitors namely U0126 and PD098059. The other report using the MPM-2 antibody is from a case study where the antibody localized the antigens in the sperm flagella (Porcu et al., 2003).
In this work, the regulation of proline-directed serine/threonine phosphorylation during mouse sperm capacitation was investigated using MPM-2 antibodies. MPM-2 western blots showed increased signal in a cohort of proteins in capacitated sperm, suggesting that during capacitation a subset of sperm proteins undergo phosphorylation of the serine/threonine–proline motif. In contrast to previous findings on the regulation of tyrosine phosphorylation, the increase in proline-directed phosphorylation was observed also in the absence of HCO3–; it was not regulated by a cAMP-dependent pathway and occurred within 15 min of the initiation of capacitation. However, similar to the increase in tyrosine phosphorylation, cholesterol acceptors such as BSA or ß-cyclodextrins (ß-CDs) were necessary for the increase in serine/threonine–proline phosphorylation; consistent with these data, the addition of cholesterol SO4– to the sperm incubation medium inhibited the increase in proline-directed phosphorylation. Using a specific antibody, we confirmed the presence of ERK1/2 in mouse sperm. However, U0126 and PD098059, two inhibitors of the ERK pathway, did not block the increase in proline-directed phosphorylation in mouse sperm, suggesting that contrary to human sperm, the ERK pathway is not involved in the phosphorylation of the MPM-2 antigens reported in this work.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Materials
Anti-phosphoserine/phosphothreonine–proline antibody (MPM-2) and anti-phosphotyrosine (anti-PY) antibody (clone 4G10) were purchased from Upstate Biotechnology (Lake Placid, NY, USA). Mouse purified immunoglobulin G1 (IgG1) isotype antibody was purchased from BD Pharmingen (San Jose, CA, USA). The anti-ß-tubulin monoclonal antibody (clone E7), developed by Dr Michael Klymkowsky, was obtained from the Developmental Studies Hybridoma Bank under the auspices of the National Institute of Child Health and Human Development (NICHD) and maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA, USA). Antibody against p44/42 MAPK was purchased from Cell Signaling Technology (Danvers, MA, USA). Peroxidase-conjugated anti-mouse IgG and rhodamine-conjugated anti-mouse IgG were from Jackson Immunoresearch Laboratories (West Grove, PA, USA). Peroxidase-linked anti-rabbit IgG, enhanced chemiluminescence (ECL) and ECLplus chemiluminescence detection kits were from Amersham Biosciences (Uppsala, Sweden). Polyvinylidene difluoride (PVDF) membrane for the blotting was purchased from Biorad (Hercules, CA, USA). Protease inhibitor cocktail was purchased from Roche Applied Science (Indianapolis, IN, USA). 3-Isobutyl-1-methylxanthine (IBMX) was obtained from Biomol Research Laboratories (Plymouth Meeting, PA, USA). Dibutyryl-cAMP (dbcAMP), cholesterol SO4–, 2-hydroxypropyl-ß-CD (2-OH-propyl-ß-CD) and other Ultrapure® and culture-tested reagents were obtained from Sigma (St Louis, MO, USA). ERK1/2 inhibitors PD098059 and U0126 were purchased from Promega (Madison, WI, USA) and Calbiochem (San Diego, CA, USA), respectively.
Preparation of mouse sperm
Caudal epididymal sperm were collected from CD1 retired breeder males (Charles River Labs) killed in accordance with Institutional Animal Care and Use Committees (IACUC) guidelines. Caudal epididymis from each animal was placed in 1 ml of modified Krebs–Ringer medium (Whitten’s HEPES-buffered medium) (WH) (Moore et al., 1994) containing 100 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM glucose, 1 mM pyruvic acid, 4.8 mM L(+)-lactic acid and hemicalcium salt in 20 mM HEPES, pH 7.3. This medium, prepared in the absence of BSA and NaHCO3, does not support capacitation. Sperm released into the media during a 10-min period were counted and collected by centrifugation at 800 x g for 10 min at room temperature. Sperm pellets were resuspended in WH (without BSA or NaHCO3), and then 1–2 x 106 sperm were incubated in 1 ml of the medium at 37°C for the indicated time period. Capacitating medium consisted of 5 mg/ml of BSA plus 10 mM NaHCO3 in WH. In all cases, pH was maintained at 7.3. In some experiments, 2-OH-propyl-ß-CD was used instead of BSA in capacitating medium. Sperm pellets were resuspended in appropriate buffer depending on the study. Sperm motility was checked in all the experiments, and the number of motile sperm was routinely >80%. The motility was also normal in the experiments where reagents such as CDs, cholesterol SO4– or ERK1/2 pathway inhibitors were used.
Preparation of human sperm
Semen samples were obtained from normozoospermic volunteers according to WHO standards (WHO, 1999), as previously described (Ficarro et al., 2003). After complete liquefaction at room temperature, mature sperm were purified by the swim-up method, and sperm presenting >90% motility were used. Sperm concentration was adjusted to 5 x 106 cells/ml, and up to 2 ml of aliquots was incubated under different conditions at 37°C in 5% CO2 overnight. Sperm were then concentrated by centrifugation at 6500 x g for 5 min and washed once with phosphate-buffered saline (PBS). Sperm pellets were resuspended in appropriate buffer depending on the experiment.
Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and western blots
Phosphorylated proteins were analysed in protein extracts from mouse or human sperm. Sperm pellets were washed in 1 ml of PBS containing 1 mM sodium orthovanadate, resuspended in Laemmli sample buffer (Laemmli, 1970) without 2-mercaptoethanol and boiled for 5 min. After centrifugation, the supernatants were saved, and 2-mercaptoethanol was added to a final concentration of 5%. Samples were boiled for 5 min and subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) using 8–10% mini-gels; protein extracts equivalent to 1–2 x 106 sperm were loaded per lane. In some cases, either mid-size gels or commercially available Criterion® 7.5% gels (Biorad, Hercules, CA, USA) were used, as indicated in figure legends. A dual-prestained molecular weight standard was used (Biorad, Hercules, CA, USA). Proteins were transferred to PVDF membranes and incubated in appropriate blocking solution for 1 h at room temperature. The immunodetection of tyrosine-phosphorylated proteins was carried out using anti-PY monoclonal antibody (1:10000), as previously described (Visconti et al., 1995a). For MPM-2 western analyses, 5% non-fat dry milk in Tris-buffered saline-T (TBS-T) (20 mM Tris, 137 mM NaCl and 0.05% Tween-20, pH 7.6) was used both as blocking solution and as secondary antibody diluent; MPM-2 antibody (1:1000) was diluted in 5% BSA/TBS-T. For the immunodetection of ERK1/2, anti-p44/42 MAPK (1:1000) was used; blocking and antibody dilutions were carried out using 5% non-fat dry milk in TBS-T; in this case, detection was done with ECLplus chemiluminescence reagents. Loading of equal amounts of protein per lane was checked by reprobing immunoblots with anti-ß-tubulin antibody (clone E7; 1:5000). After washes, protein detection was performed by incubating membranes with appropriate horseradish peroxidase-conjugated secondary antibodies (1:10000) followed by ECL kit (Amersham Biosciences), according to the manufacturer’s instructions.
Two-dimensional gel electrophoresis
For two-dimensional (2D) gel electrophoresis (PAGE), sperm pellets were resuspended in a modified Celis extraction/rehydration buffer [5 mM urea, 2 mM thiourea, 2% 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.2% ampholytes, pH 3–10, 50 mM dithiothreitol (DTT), 0.0002% Bromophenol blue, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 30 mM ß-glyceraldehyde and 2 mM EGTA] and protease inhibitors, vortexed for 2 min and kept on ice for 30 min. After centrifugation at 12000 x g for 5 min, extracted proteins (equivalent to 2.5 x 106 sperm per strip) were loaded passively onto Immobilized pH gradient (IPG) strips (pH 3–10) and incubated overnight at room temperature. Isoelectric focusing (IEF) was performed using Protean IEF Cell apparatus (BioRad, Richmond, CA, USA). After focusing, IPG strips were equilibrated in equilibration buffer (6 M urea, 2% SDS, 0.05 M Tris–HCl, 2% DTT and 20% glycerol) at room temperature for 10 min, followed by incubation on a second equilibration buffer in which DTT was replaced with 2.5% iodoacetamide, for 10 min at room temperature. Two-dimensional gel electrophoresis was performed on 10% SDS–PAGE mini-gels. Proteins were transferred onto PVDF membranes and analysed by western blotting, as described above. Proteins extracts, run in parallel gels, were silver stained, as previously described (Ficarro et al., 2003).
Indirect immunofluorescence localization
Mouse sperm were incubated in the medium containing BSA and NaHCO3 for 15 min. Following incubation, they were air-dried on slides and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. After fixation, the sperm were washed with PBS (four washes each for 5 min) and permeabilized with methanol for 7 min. Following permeabilization, the sperm were blocked with 10% normal goat serum in PBS for 30 min at room temperature. Sperm were then incubated either with MPM-2 antibody (1:250) diluted in PBS containing 1% normal goat serum or with normal mouse purified IgG1 isotype control antibody at the same concentration for 1 h at room temperature. After the primary antibody incubation, the sperm were washed with PBS (four washes) and incubated with rhodamine-conjugated anti-mouse IgG (1:200) diluted in PBS containing 1% normal goat serum for 1 h at room temperature. The secondary antibody treatment was followed by four washes in PBS, treatment with slow-fade light (Molecular Probes, Eugene, OR, USA) and observation by phase-contrast and epifluorescence microscopy using a Zeiss Axiophot microscope (magnification x60) (Carl Zeiss, Thornwood, NY, USA). Controls using secondary antibody alone were also used to check for antibody specificity.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Capacitation-associated serine/threonine–proline phosphorylation
To evaluate whether proline-directed phosphorylation is regulated during capacitation, we incubated mouse sperm in media that were either conducive (+BSA, +NaHCO3) or non-conducive (–BSA, –NaHCO3) to capacitation. After 15 and 90 min, sperm lysates were prepared and subjected to western blot analysis using the MPM-2 antibody. This antibody recognized a set of proteins that underwent increased proline-directed phosphorylation when incubated in the medium containing NaHCO3 and BSA (Figure 1A). In the absence of HCO3– and BSA, much less phosphorylation on serine–proline and/or threonine–proline was detected. The molecular masses of the major phosphorylated proteins were approximately 210, 96, 91 and 70 kDa. Interestingly, increase in proline-directed phosphorylation occurred rapidly (within 15 min) in contrast to the capacitation-associated tyrosine phosphorylation. As a control, the tyrosine phosphorylation was checked with anti-PY monoclonal antibody (clone 4G10) in a duplicate set of samples; as described before (Visconti et al., 1995a
), an increase in protein tyrosine phosphorylation was only observed when the sperm were incubated in medium supporting capacitation for 90 min (Figure 1B). The increase in proline-directed phosphorylation was also detected using MPM-2 western blots of sperm protein extracts separated using 2D gel electrophoresis (Figure 1C). Consistent with 1D western blot, capacitated sperm displayed a much higher level of proline-directed phosphorylation when compared with non-capacitated sperm. Interestingly, although proteins 
100 kDa showed very high MPM-2 staining, other proteins of lower molecular masses were also recognized by this monoclonal antibody in the 2D blot.
|
To investigate the localization of proteins phosphorylated at serine and/or threonine residues followed by proline, we conducted indirect immunofluorescence using MPM-2 monoclonal antibody in sperm incubated in complete medium for 15 min (Figure 2). Most of the MPM-2 signal localized to the sperm principal piece; however, a lighter signal was also observed in the sperm anterior head (Figure 2A). Normal mouse purified IgG1 isotype control used at the same concentration as MPM-2 antibody (Figure 2B), and the secondary antibody alone (data not shown) confirmed the specificity of MPM-2 antibody. Altogether, these data suggest that an early change in proline-directed phosphorylation occurs in conditions that support capacitation.
|
Regulation of proline-directed phosphorylation in mouse sperm
BSA and NaHCO3 are essential components of mouse sperm capacitation media and for the capacitation-associated increase in tyrosine phosphorylation (Visconti et al., 1995a). To investigate the independent action of these compounds on the increase in proline-directed phosphorylation, we incubated mouse sperm for 90 min in media that lacked either BSA or HCO3–, extracted in sample buffer and analysed by western blot using MPM-2 or anti-PY monoclonal antibodies (Figure 3). Under these conditions, the increase in proline-directed phosphorylation was regulated by BSA (Figure 3A and B, lanes 2 and 4) but not by HCO3– (Figure 3A and B, lanes 3 and 4), and similar results were obtained at 15 min (data not shown). As tyrosine phosphorylation is regulated by a cAMP/PKA pathway, it was important to determine whether the phosphorylated epitope recognized by the MPM-2 antibody was also regulated by this pathway. Thus, this experiment was also conducted in the absence or presence of a permeable cAMP analogue (dbcAMP) and a general inhibitor of phosphodiesterases (IBMX) (Figure 3). As expected, the addition of cAMP agonists did increase tyrosine phosphorylation in sperm incubated in the absence of either HCO3– or BSA (Figure 3D); however, no increase in proline-directed phosphorylation was observed either at 15 min (data not shown) or at 90 min (Figure 3B) of sperm incubation in the presence of the cAMP agonists under any of the conditions tested, suggesting that this type of phosphorylation is cAMP independent.
|
Regulation of the serine/threonine–proline phosphorylation by BSA in mouse sperm
To further confirm that proline-directed phosphorylation during capacitation is regulated by BSA, we studied the effect of various concentrations of BSA on this phosphorylation. The concentration of BSA needed to detect an increase in proline-directed phosphorylation was 1 mg/ml (Figure 4A) similar to the BSA concentration needed for mouse sperm capacitation and for the capacitation-associated increase in tyrosine phosphorylation (Visconti et al., 1995a). It has been proposed that the role of BSA in capacitation is associated with the ability of this protein to act as a sink for plasma membrane cholesterol (Visconti et al., 1999a,b). Therefore, the finding that BSA is also needed for increased proline-directed phosphorylation suggests that this type of phosphorylation occurs downstream to the cholesterol efflux. To investigate this possibility, we carried out two experiments. First, we assayed whether another cholesterol-binding compound such as 2-OH-propyl-ß-CD was able to replace BSA; second, we tested whether a cholesterol homologue such as cholesterol SO4– was able to inhibit the increase in proline-directed phosphorylation in sperm incubated in complete capacitation medium. In the first experiment, mouse sperm were incubated in the absence of BSA and in the presence of different concentrations of 2-OH-propyl-ß-CD. After 15 min incubation, a concentration-dependent increase in MPM-2 signal was detected (Figure 4B). As loading control, the same blots used for the BSA and 2-OH-propyl-ß-CD concentration curves were analysed using an anti-ß-tubulin monoclonal antibody (Figure 4A and B, lower panels).
|
For the second experiment, mouse sperm were incubated in complete capacitation medium in the absence or presence of 16 µM cholesterol SO4–. As non-capacitated negative control, the sperm were also incubated in the absence of BSA and HCO3–. As shown in previous experiments, sperm incubated in complete medium underwent an increase in proline-directed phosphorylation (Figure 4C). This increase was blocked in the presence of cholesterol SO4– (Figure 4C, lane 3). As loading control, the same blots were analysed using an anti-ß-tubulin monoclonal antibody (Figure 4A–C). Altogether, the effects of BSA, CD and cholesterol SO4– suggest that serine/threonine–proline phosphorylation is a BSA-dependent process, and it occurs downstream to cholesterol efflux.
As mentioned in the Introduction, at least two proline-directed kinases have been described in mammalian sperm, ERK1/2 and GSK3ß. Using western blot analysis, the presence of ERK1/2 in mouse sperm was confirmed (Figure 5A). In human sperm, proline-directed phosphorylation appears to be inhibited by ERK pathway inhibitors (de Lamirande and Gagnon, 2002). To analyse whether ERK1/2 was involved in the regulation of the BSA-dependent increase in proline-directed phosphorylation, we incubated mouse sperm in the presence of two inhibitors of the ERK pathway, U0126 and PD098059. In this experiment, sperm were collected in non-capacitated medium in the presence or absence of different concentrations of the inhibitors. After pre-incubation for 15 min, the sperm were diluted in complete medium so that the final concentration of NaHCO3 and BSA was kept constant (20 mM and 5 mg/ml, respectively); the ERK pathway inhibitors were present in the media throughout the period of experiment, at a final concentration indicated in Figure 5. The increase in proline-directed phosphorylation was evaluated by western blots with MPM-2 antibody. Results obtained after 15 min incubation are shown in Figure 5; similar results were obtained after 90 min incubation in the presence of the inhibitors (data not shown). Under these conditions, the ERK pathway inhibitors did not block proline-directed phosphorylation (Figure 5B), suggesting that ERK is not involved in the phosphorylation of the MPM-2 antigens. As in the other experiments, loading of controls was conducted in the same membrane by western blot with anti-ß-tubulin antibody.
|
Proline-directed phosphorylation in human sperm
Capacitation-associated changes such as cholesterol efflux, PKA activation and tyrosine phosphorylation have been found to be conserved in sperm from various species. In human sperm, an increased signal in MPM-2 has been reported in western blots (de Lamirande and Gagnon, 2002). To investigate whether proline-directed phosphorylation is dependent on BSA present in the capacitation medium, we collected human sperm by the swim-up method and incubated overnight either in the absence or in the presence of different concentrations of BSA. Taking into consideration the time course of human sperm capacitation (Zarintash and Cross, 1996), human sperm were incubated for 18 h under these conditions before extraction with sample buffer. Protein extracts were then analysed by western blots using MPM-2 monoclonal antibody. Under these conditions, similar to mouse sperm, BSA was necessary for the increase in proline-directed phosphorylation in a concentration-dependent manner (Figure 6). As in previous figures, loading of controls was conducted in the same membrane by western blot with anti-ß-tubulin antibody.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Post-translational modifications through serine/threonine or tyrosine phosphorylation by PKs, and/or the dephosphorylation of these residues by phosphoprotein phosphatases, play a major role in many cellular processes including the transduction of extracellular signals, intracellular transport and cell cycle progression (Manning et al., 2002
). With the exception of PKA, the kinases involved in the regulation of sperm function are not well defined. Other kinases, described in mature mammalian sperm using antibodies, include PKC (Rotem et al., 1990), GSK3ß (Vijayaraghavan et al., 1996), casein kinase II (Chaudhry et al., 1991a,b), ERK1/2 (Luconi et al., 1998) and at least one member of the testis-specific serine kinase (Tssk) family (Hao et al., 2004). However, very little is known about the role of the proteins undergoing phosphorylation during capacitation. Capacitation-associated changes in protein tyrosine phosphorylation have been demonstrated in multiple species including the mouse (Visconti et al., 1995a), bovine (Galantino-Homer et al., 1997), human (Leclerc et al., 1996; Osheroff et al., 1999), pig (Kalab et al., 1998) and hamster (Visconti et al., 1999c; Kulanand and Shivaji, 2001). In addition, it has been demonstrated that capacitation-associated increase in protein tyrosine phosphorylation is downstream of a cAMP/PKA pathway in mouse sperm (Visconti et al., 1995b) and sperm of other species (Leclerc et al., 1996; Galantino-Homer et al., 1997; Kalab et al., 1998). In mice, this pathway appears to be tightly controlled by components in the capacitation medium; for example, in the absence of BSA, Ca2+ or HCO3–, neither the capacitation nor the increase in tyrosine phosphorylation is observed.
Furthermore, concentrations of these compounds needed for tyrosine phosphorylation are associated with those required for capacitation (Visconti et al., 1995a). The role of PKA in the regulation of capacitation has been highlighted by two recent publications. First, sperm from mice that lack soluble adenylyl cyclase (sAC), the HCO3–-regulated sAC, do not display changes in their tyrosine phosphorylation pattern after incubation in a capacitation-supporting media (Esposito et al., 2004; Hess et al., 2005); second, sperm from knockout mice lacking C2, the testis-specific PKA catalytic subunit, are not able to move actively and are unable to undergo increase in tyrosine phosphorylation as observed in the wild-type mouse (Nolan et al., 2004). These knockout mice confirm results obtained using cAMP agonists and antagonists on the capacitation-associated increase in tyrosine phosphorylation.
The essential role of PKA in sperm implies that serine/threonine phosphorylation plays a role in the regulation of sperm capacitation. However, less is known regarding the regulation of serine and threonine phosphorylation in sperm. One reason for this lack of information is that antibodies against isolated phosphoserine and phosphothreonine residues do not display the quality and sensitivity obtained with anti-PY antibodies. This limitation can be addressed using antibodies against specific phosphorylated motifs; in sperm, phospho-specific antibodies have been used to evaluate PKA substrates (Harrison, 2004; O’Flaherty et al., 2004; Moseley et al., 2005), ERK phosphorylation (Thundathil et al., 2002; O’Flaherty et al., 2005) and AKT phosphorylation (Nauc et al., 2004).
Another very well-characterized phospho-specific antibody is MPM-2. This monoclonal antibody can recognize phosphorylated residues of serine or threonine that are immediately followed by proline. Two articles reported the use of this monoclonal antibody in human sperm. One of them is a case report suggesting that this antibody can be used to detect infertile sperm in a clinical setting (Porcu et al., 2003). The second one, by de Lamirande and Gagnon (2002) used this antibody to investigate the ERK1/2 pathway in human sperm. In our study, MPM-2 antibody was used to study whether proline-directed phosphorylation is associated with mouse sperm capacitation. MPM-2 antibodies recognized a series of proteins in sperm incubated under capacitating conditions, whereas non-capacitated sperm showed a significantly reduced signal, suggesting that capacitation is associated with an increase in proline-directed phosphorylation. When compared with the increase in protein tyrosine phosphorylation, the increase in proline-directed phosphorylation presented several differences. First, it can be observed at 15 versus 60–90 min in the case of tyrosine phosphorylation. Second, it does not require HCO3– in the sperm incubation medium. Third, the treatment of sperm with cAMP agonists did not increase this type of phosphorylation. On the contrary, similar to the increase in protein tyrosine phosphorylation, the increase in proline-directed phosphorylation appears to require BSA in the incubation medium both for mouse and for human sperm. Moreover, the increase in proline-directed phosphorylation required between 0.3 and 1 mg/ml of BSA similar to the concentration needed for both capacitation and the capacitation-associated increase in protein tyrosine phosphorylation (Visconti et al., 1995a).
Serum albumin is considered one of the main components of in vitro capacitation media and is believed to function as a sink for cholesterol from the sperm plasma membrane (Go and Wolf, 1985; Langlais and Roberts, 1985; Suzuki and Yanagimachi, 1989; Cross, 1996, 1998). Although serum albumin may have other roles during capacitation (Espinosa et al., 2000), its ability to facilitate cholesterol efflux is required for capacitation. For example, capacitation is inhibited by the addition of cholesterol and/or cholesterol analogues to the capacitation medium (Visconti et al., 1999b). Furthermore, serum albumin can be substituted in in vitro capacitation media with cholesterol-binding compounds such as high-density lipoproteins (HDLs) (Therien et al., 1997; Visconti et al., 1999b) and ß-CDs (Choi and Toyoda, 1998; Cross, 1999; Osheroff et al., 1999; Visconti et al., 1999a) to induce capacitation. Similar to the increase in protein tyrosine phosphorylation, experiments presented in this article suggest that cholesterol efflux is required for the increase in proline-directed phosphorylation. First, 2-OH-propyl-ß-CD was able to induce proline-directed phosphorylation in the absence of BSA; second, proline-directed phosphorylation was inhibited by cholesterol SO4– in complete capacitation medium.
Understanding how cholesterol efflux couples to the regulation of signal transduction pathways intrinsic to capacitation remains rudimentary at present. It can be hypothesized that similar to other cell types, in sperm, cholesterol may be concentrated in lipid rafts and that cholesterol efflux is related to changes in these membrane microdomains. Supporting this hypothesis, caveolin has been recently detected in the plasma membrane overlying the acrosomal region and the flagellum of mouse and guinea-pig sperm (Travis et al., 2000; Treviño et al., 2001), suggesting the presence of a special type of lipid raft, called caveolae, in these cells. Furthermore, other recent studies have shown that the incubation of sperm in capacitation-supporting medium induces lateral redistribution of raft marker proteins such as the Glycosylphoshatidylinositol (GPI)-anchored CD59 and caveolin within the sperm head plasma membrane (Cross, 2004; Shadan et al., 2004). Although no particular sperm functional change has yet been ascribed to alterations in lipid raft structure and distribution, one may expect that such changes, resulting from cholesterol removal, will have profound effects on subsequent signalling events. In somatic cells, the disruption of lipid rafts by cholesterol-binding compounds results in the activation of several signalling pathways. It can be speculated that similar activation occurs in sperm during capacitation (Sleight et al., 2005); in this respect, changes in proline-directed phosphorylation might play a role in regulatory pathways downstream to cholesterol efflux. Interestingly, although MPM-2 antigens appear to localize both to the anterior head and to the sperm flagellum, most signal is detected in the principal piece of the sperm, suggesting that proline-directed phosphorylation might play a role in the regulation of sperm motility.
As shown by de Lamirande and Gagnon (2002), western blots using MPM-2 antibodies revealed a series of proteins in human sperm. In this work, we present evidence that proline-directed phosphorylation in human sperm is also regulated by BSA concentration. However, western blots shown in Figure 6 revealed more proteins than the ones reported by de Lamirande and Gagnon (2002). This is likely due to the use of total sperm extracts instead of the Triton-insoluble protein extracts used by these authors. Alternatively, these differences could be due to the amount of proteins loaded per lane, exposure time and/or secondary antibody concentration. Although in human sperm the phosphorylation of these MPM-2 antigens is inhibited by ERK pathway inhibitors (de Lamirande and Gagnon, 2002), proline-directed phosphorylation does not appear to be regulated by this pathway in mouse sperm. ERK1/2 is present in mouse sperm; however, two different ERK pathway inhibitors, U0126 and PD098059, were not able to block proline-directed phosphorylation in mouse sperm. The different effect of these inhibitors in mouse and human sperm could indicate that different pathways are activated during capacitation in different species. Other proline-directed kinases present in mammalian sperm could be responsible for this regulation in the mouse; one of them is GSK3ß, a kinase shown to be important in the regulation of sperm motility (Vijayaraghavan et al., 1996). Further studies will be necessary to determine the role of this kinase and other proline-directed kinases in mouse sperm capacitation and the overall relevance of proline-directed phosphorylation to mouse sperm capacitation.
|
Acknowledgements |
|---|
This study was supported by the Andrew W. Mellon Foundation, NIH HD38082 and HD44044 (to PEV), NIH U5429099 (to JCH), grants from Schering AG (to JCH) and post-doctoral fellowship (to KNJ) from the Fogarty International Center grant D43 TW/HD 00654 (to JCH).
|
Notes |
|---|
* The authors equally contributed to this work.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Austin CR (1951) Observations on the penetration of the sperm in the mammalian egg. Aust J Sci Res (B) 4,581–596.
Breitbart H, Lax J, Rotem R and Naor Z (1992) Role of protein kinase C in the acrosome reaction of mammalian spermatozoa. Biochem J 281,473–476.
Chang MC (1951) Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature 168,697–698.[Medline]
Chaudhry PS, Nanez R and Casillas ER (1991a) Purification and characterization of polyamine-stimulated protein kinase (casein kinase II) from bovine spermatozoa. Arch Biochem Biophys 288,337–342.[CrossRef][ISI][Medline]
Chaudhry PS, Newcomer PA and Casillas ER (1991b) Casein kinase I in bovine sperm: purification and characterization. Biochem Biophys Res Commun 179,592–598.[CrossRef][ISI][Medline]
Choi YH and Toyoda Y (1998) Cyclodextrin removes cholesterol from mouse sperm and induces capacitation in a protein-free medium. Biol Reprod 59,1328–1333.
Cross NL (1996) Effect of cholesterol and other sterols on human sperm acrosomal responsiveness. Mol Reprod Dev 45,212–217.[CrossRef][ISI][Medline]
Cross NL (1998) Role of cholesterol in sperm capacitation. Biol Reprod 59,7–11.
Cross NL (1999) Effect of methyl-beta-cyclodextrin on the acrosomal responsiveness of human sperm. Mol Reprod Dev 53,92–98.[CrossRef][ISI][Medline]
Cross NL (2004) Reorganization of lipid rafts during capacitation of human sperm. Biol Reprod 71,1367–1373.
Espinosa F, Lopez-Gonzalez I, Munoz-Garay C, Felix R, De la Vega-Beltran JL, Kopf GS, Visconti PE and Darszon A (2000) Dual regulation of the T-type Ca (2+) current by serum albumin and beta-estradiol in mammalian spermatogenic cells. FEBS Lett 475,251–256.[CrossRef][ISI][Medline]
Esposito G, Jaiswal BS, Xie F, Krajnc-Franken MA, Robben TJ, Strik AM, Kuil C, Philipsen RL, van Duin M, Conti M et al. (2004) Mice deficient for soluble adenylyl cyclase are infertile because of a severe sperm-motility defect. Proc Natl Acad Sci USA 101,2993–2998.
Ficarro S, Chertihin O, Westbrook VA, White F, Jayes F, Kalab P, Marto JA, Shabanowitz J, Herr JC, Hunt D et al. (2003) Phosphoproteome analysis of human sperm. Evidence of tyrosine phosphorylation of AKAP 3 and valosin containing protein/P97 during capacitation. J Biol Chem 278,11579–11589.
Galantino-Homer HL, Visconti PE and Kopf GS (1997) Regulation of protein tyrosine phosphorylation during bovine sperm capacitation by a cyclic adenosine 3'5'-monophosphate-dependent pathway. Biol Reprod 56,707–719.[Abstract]
Go KJ and Wolf DP (1985) Albumin-mediated changes in sperm sterol content during capacitation. Biol Reprod 32,145–153.[Abstract]
Hao Z, Jha KN, Kim YH, Vemuganti S, Westbrook VA, Chertihin O, Markgraf K, Flickinger CJ, Coppola M, Herr JC et al. (2004) Expression analysis of the human testis-specific serine/threonine kinase (TSSK) homologues. A TSSK member is present in the equatorial segment of human sperm. Mol Hum Reprod 10,433–444.
Harrison RA (2004) Rapid PKA-catalysed phosphorylation of boar sperm proteins induced by the capacitating agent bicarbonate. Mol Reprod Dev 67,337–352.[CrossRef][ISI][Medline]
Hess KC, Jones BH, Marquez B, Chen Y, Ord TS, Kamenetsky M, Miyamoto C, Zippin JH, Kopf GS, Suarez SS et al. (2005) The "soluble" adenylyl cyclase in sperm mediates multiple signaling events required for fertilization. Dev Cell 9,249–259.[CrossRef][ISI][Medline]
Kalab P, Peknicova J, Geussova G and Moos J (1998) Regulation of protein tyrosine phosphorylation in boar sperm through a cAMP-dependent pathway. Mol Reprod Dev 51,304–314.[CrossRef][ISI][Medline]
Kalina M, Socher R, Rotem R and Naor Z (1995) Ultrastructural localization of protein kinase C in human sperm. J Histochem Cytochem 43,439–445.[Abstract]
Kulanand J and Shivaji S (2001) Capacitation-associated changes in protein tyrosine phosphorylation, hyperactivation and acrosome reaction in hamster spermatozoa. Andrologia 33,95–104.[CrossRef][ISI][Medline]
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680–685.[CrossRef][Medline]
de Lamirande E and Gagnon C (2002) The extracellular signal-regulated kinase (ERK) pathway is involved in human sperm function and modulated by the superoxide anion. Mol Hum Reprod 8,124–135.
Langlais J and Roberts JD (1985) A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa. Gamete Res 12,183–224.
Leclerc P, de Lamirande E and Gagnon C (1996) Cyclic adenosine 3',5' monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. Biol Reprod 55,684–692.[Abstract]
Lu KP (2004) Pinning down cell signaling, cancer and Alzheimer’s disease. Trends Biochem Sci 29,200–209.[CrossRef][ISI][Medline]
Lu KP, Liou YC and Zhou XZ (2002) Pinning down proline-directed phosphorylation signaling. Trends Cell Biol 12,164–172.[CrossRef][ISI][Medline]
Luconi M, Barni T, Vannelli GB, Krausz C, Marra F, Benedetti PA, Evangelista V, Francavilla S, Properzi G, Forti G et al. (1998) Extracellular signal-regulated kinases modulate capacitation of human spermatozoa. Biol Reprod 58,1476–1489.
Manning G, Plowman GD, Hunter T and Sudarsanam S (2002) Evolution of protein kinase signaling from yeast to man. Trends Biochem Sci 27,514–520.[CrossRef][ISI][Medline]
Moore GD, Ayabe T, Visconti PE, Schultz RM and Kopf GS (1994) Roles of heterotrimeric and monomeric G proteins in sperm-induced activation of mouse eggs. Development 120,3313–3323.[Abstract]
Moseley FL, Jha KN, Bjorndahl L, Brewis IA, Publicover SJ, Barratt CL and Lefievre L (2005) Protein tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction are enhanced in IVF media: an effect that is not associated with an increase in protein kinase A activation. Mol Hum Reprod 11,523–529.
Nauc V, De Lamirande E, Leclerc P and Gagnon C (2004) Inhibitors of phosphoinositide 3-kinase, LY294002 and wortmannin, affect sperm capacitation and associated phosphorylation of proteins differently: Ca2+-dependent divergences. J Androl 25,573–585.
Nolan MA, Babcock DF, Wennemuth G, Brown W, Burton KA and McKnight GS (2004) Sperm-specific protein kinase A catalytic subunit Calpha2 orchestrates cAMP signaling for male fertility. Proc Natl Acad Sci USA 101,13483–13488.
O’Flaherty C, de Lamirande E and Gagnon C (2004) Phosphorylation of the arginine-X-X-(serine/threonine) motif in human sperm proteins during capacitation: modulation and protein kinase A dependency. Mol Hum Reprod 10,355–363.
O’Flaherty C, de Lamirande E and Gagnon C (2005) Reactive oxygen species and protein kinases modulate the level of phospho-MEK-like proteins during human sperm capacitation. Biol Reprod 73,94–105.
Osheroff JE, Visconti PE, Valenzuela JP, Travis AJ, Alvarez J and Kopf GS (1999) Regulation of human sperm capacitation by a cholesterol efflux-stimulated signal transduction pathway leading to protein kinase A-mediated up-regulation of protein tyrosine phosphorylation. Mol Hum Reprod 5,1017–1026.
Pawson T and Scott JD (1997) Signaling through scaffold, anchoring, and adaptor proteins. Science 278,2075–2080.
Porcu G, Mercier G, Boyer P, Achard V, Banet J, Vasserot M, Melone C, Saias-Magnan J, D’Ercole C, Chau C et al. (2003) Pregnancies after ICSI using sperm with abnormal head-tail junction from two brothers: case report. Hum Reprod 18,562–567.
Rotem R, Paz GF, Homonnai ZT, Kalina M and Naor Z (1990) Protein kinase C is present in human sperm: possible role in flagellar motility. Proc Natl Acad Sci USA 87,7305–7308.
Shadan S, James PS, Howes EA and Jones R (2004) Cholesterol efflux alters lipid raft stability and distribution during capacitation of boar spermatozoa. Biol Reprod 71,253–265.
Sleight SB, Miranda PV, Plaskett NW, Maier B, Lysiak J, Scrable H, Herr JC and Visconti PE (2005) Isolation and proteomic analysis of mouse sperm detergent-resistant membrane fractions: evidence for dissociation of lipid rafts during capacitation. Biol Reprod 73,721–729.
Suzuki F and Yanagimachi R (1989) Changes in the distribution of intramembranous particles and filipin-reactive membrane sterols during in vitro capacitation of golden hamster spermatozoa. Gamete Res 23,335–347.[CrossRef][ISI][Medline]
Therien I, Soubeyrand S and Manjunath P (1997) Major proteins of bovine seminal plasma modulate sperm capacitation by high-density lipoprotein. Biol Reprod 57,1080–1088.[Abstract]
Thundathil J, de Lamirande E and Gagnon C (2002) Different signal transduction pathways are involved during human sperm capacitation induced by biological and pharmacological agents. Mol Hum Reprod 8,811–816.
Travis AJ, Vargas LA, Merdiushev T, Moss SB, Hunnicutt GR and Kopf GS (2000) Expression and localization of caveolin-1 in mouse and guinea pig spermatozoa. Mol Biol Cell 11,121a.
Treviño CL, Serrano CJ, Beltran C, Felix R and Darszon A (2001) Identification of mouse trp homologs and lipid rafts from spermatogenic cells and sperm. FEBS Lett 509,119–125.[CrossRef][ISI][Medline]
Vijayaraghavan S, Stephens DT, Trautman K, Smith GD, Khatra B, da Cruz e Silva EF and Greengard P (1996) Sperm motility development in the epididymis is associated with decreased glycogen synthase kinase-3 and protein phosphatase 1 activity. Biol Reprod 54,709–718.[Abstract]
Visconti PE, Bailey JL, Moore GD, Pan D, Olds-Clarke P and Kopf GS (1995a) Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. Development 121,1129–1137.[Abstract]
Visconti PE, Moore GD, Bailey JL, Leclerc P, Connors SA, Pan D, Olds-Clarke P and Kopf GS (1995b) Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Development 121,1139–1150.[Abstract]
Visconti PE, Galantino-Homer H, Ning X, Moore GD, Valenzuela JP, Jorgez CJ, Alvarez JG and Kopf GS (1999a) Cholesterol efflux-mediated signal transduction in mammalian sperm. beta-cyclodextrins initiate transmembrane signaling leading to an increase in protein tyrosine phosphorylation and capacitation. J Biol Chem 274,3235–3242.
Visconti PE, Ning X, Fornes MW, Alvarez JG, Stein P, Connors SA and Kopf GS (1999b) Cholesterol efflux-mediated signal transduction in mammalian sperm: cholesterol release signals an increase in protein tyrosine phosphorylation during mouse sperm capacitation. Dev Biol 214,429–443.[CrossRef][ISI][Medline]
Visconti PE, Stewart-Savage J, Blasco A, Battaglia L, Miranda P, Kopf GS and Tezon JG (1999c) Roles of bicarbonate, cAMP, and protein tyrosine phosphorylation on capacitation and the spontaneous acrosome reaction of hamster sperm. Biol Reprod 61,76–84.
WHO (1999) WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Cambridge, UK: Cambridge University Press.
Yaffe MB, Schutkowski M, Shen M, Zhou XZ, Stukenberg PT, Rahfeld JU, Xu J, Kuang J, Kirschner MW, Fischer G et al. (1997) Sequence-specific and phosphorylation-dependent proline isomerization: a potential mitotic regulatory mechanism. Science 278,1957–1960.
Yanagimachi R (1994) Mammalian fertilization. In Knobil E and Neill JD (eds) The Physiology of Reproduction. Raven Press, New York, pp. 189–317.
Zarintash RJ and Cross NL (1996) Unesterified cholesterol content of human sperm regulates the response of the acrosome to the agonist, progesterone. Biol Reprod 55,19–24.[Abstract]
Submitted on August 23, 2006; resubmitted on September 11, 2006; accepted on September 18, 2006.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Z. Zhang, X. Shen, B. H Jones, B. Xu, J. C Herr, and J. F Strauss III Phosphorylation of Mouse Sperm Axoneme Central Apparatus Protein SPAG16L by a |