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a Division of Research, Department of Obstetrics and Gynecology, Medical College of Ohio, Toledo, Ohio 43614
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The molecules and mechanisms involved in signal transduction pathways leading to sperm capacitation and acrosomal exocytosis are not clearly understood at present [8]. Results from our laboratory previously demonstrated that the protein phosphorylation at tyrosine residues of predominantly three sets of proteins of 95-kDa, 51-kDa (fertilization antigen; FA-1 antigen), and 14–18-kDa molecular identities, respectively, plays an important role in human sperm capacitation/acrosome reaction and zona pellucida binding [9–11]. Capacitation and zona pellucida (ZP) exposure increase the degree of tyrosine phosphorylation per sperm cell and increase the number of sperm that are phosphorylated, and also induce a subcellular shift in protein phosphorylation pattern of human sperm. FA-1 antigen is a well-characterized glycoprotein present on the post-acrosomal, midpiece, and tail regions of spermatozoa of various mammalian species [12–14]. It has receptor/ligand activity for zona pellucida of the human oocyte [15]. In addition to phosphorylation and autophosphorylation of FA-1 during capacitation/acrosomal exocytosis and zona pellucida binding, FA-1 antibodies (monoclonal and polyclonal) inhibit sperm-zona binding and in vitro fertilization in several mammalian species including humans. Recently, cDNA encoding for FA-1 antigen has been cloned and sequenced [16]. Based upon the above findings, the present study was conducted to investigate the involvement of serine and threonine phosphorylation in human sperm capacitation. Specifically, the study focused on examining the following questions: 1) which proteins are phosphorylated at serine/threonine residues during capacitation; 2) does exposure to solubilized human zona pellucida proteins increase the intensity of serine and threonine phosphorylation of FA-1 antigen; 3) are the proteins that are phosphorylated at tyrosine residues also phosphorylated at serine and/or threonine residues; and 4) which subcellular site(s) of the sperm cell is involved in serine and threonine phosphorylation, and does this pattern change as capacitation proceeds. These studies were conducted using recently available monoclonal antibodies that specifically recognize phosphorylated forms of serine or threonine residues.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human sperm were obtained from healthy fertile men (n = 6) after 48–72 h of sexual abstinence. Ejaculated semen was liquefied for 1 h at 37°C and analyzed for volume, sperm concentration, percentage motility, and progressive motility. Seven samples with a concentration of > 50 x 106 sperm/ml, motility > 60%, and progressive motility > 3 (on a scale of 0–5) and having immune cells < 1% were selected for further processing. Sperm were washed twice with Ham's F-10 medium (modified with 25 mM NaHCO3 and 1.0 mM calcium lactate, and supplemented with 0.3% BSA); they were then subjected to the swim-up procedure in the same medium [17]. The swim-up sperm population was obtained by overlaying the washed sperm pellet with 1.0 ml of supplemented Ham's F-10 and incubating the tube in a 20°-angled rack at 37°C in 5% CO2 in air for 1 h. The supernatant containing the swim-up population was harvested by careful aspiration, analyzed for motility, concentration, and any contamination, and used in the subsequent procedures.
The fertile men used in the present study were donor controls from our andrology laboratory. Sperm from these men undergo capacitation within 5 h of incubation [17, 18]. Approximately 10–18% of sperm after 5 h of capacitation undergo spontaneous and 46–68% undergo induced acrosome reaction after treatment (37°C, 1 h) with A23187 calcium ionophore (Sigma Chemical Co., St. Louis, MO) (10 µM final concentration). Also, sperm of these men show excellent sperm penetration of zona-free hamster oocytes.
Antibodies
Antibodies used in the present study to specifically detect phosphorylated serine and threonine residues of proteins were monoclonal antibodies (mAb) obtained from Sigma. These antibodies have been extensively used and have been reported to specifically bind to phosphoserine/phosphothreonine.
Monoclonal anti-phosphoserine antibody (mouse IgG1 isotype) (#P3430; clone PSR-45) was raised by immunizing mice against phosphoserine conjugated to keyhole limpet hemocyanin (KLH); it specifically reacts with phosphoserine and does not react with nonphosphorylated serine or with phosphorylated or nonphosphorylated threonine and tyrosine, AMP, and ATP.
Monoclonal anti-phosphothreonine antibody (mouse IgG2b isotype) (#P3555; clone PTR-8) was raised by immunizing mice against phosphothreonine conjugated to KLH. This antibody specifically reacts with phosphothreonine and does not react with nonphosphorylated threonine or with phosphorylated or nonphosphorylated serine and tyrosine, AMP, and ATP.
The affinity-purified monoclonal antibody of IgG2b subclass (#56329) obtained from Organon-Teknika Corporation (Durham, NC) was used as control Ig.
Immunoprecipitation Procedure
Human sperm capacitated at various times (0–5 h) (37°C, 5% CO2 in air) [18] were washed twice in PBS (pH 7.4) containing 1 mM PMSF and 1 mM orthovandate. Washed sperm pellet was solubilized overnight at 4°C in lysis buffer (20 mM Hepes, pH 7.5, 1.5 mM MgCl2, 10% glycerol, 0.05% Nonidet P-40, 10 µM PMSF, 2 mM sodium orthovandate, and 10 µg/ml leupeptin). The lysate was centrifuged, and the supernatant (sperm extract) was used in the immunoprecipitation procedure, as described in detail elsewhere [19, 20]. Briefly, sperm extract containing ~80–150 µg protein/100 µl, as determined by the Micro BCA protein assay reagent kit (Pierce Chemical Co., Rockford, IL), was mixed with anti-phosphoserine mAb/anti-threonine in mAb/control myeloma Ig (10 µg/100 µl sperm suspension) in modified RIPA buffer (50 mM NaCl, 10 mM Tris, 1 mM EDTA, 1 mM PMSF, and 0.1 M sodium orthovandate). The reaction mixture was incubated overnight at 4°C; then 50 µl of protein A/G agarose beads (Oncogene Science, Inc., Uniondale, NY) was added to the reaction mixture, and incubation was continued for an additional 1.5 h. The reaction mixture was centrifuged (10 000 rpm) for 5 min and the supernatant discarded. The pellet was washed (three times) with RIPA buffer containing 0.1% Triton X-100, boiled for 10 min in SDS sample buffer (nonreduced), and centrifuged; the supernatant was subjected to analysis in SDS-PAGE (5–15% gradient slab gel) [21]. After PAGE, the gels were stained with silver stain (Bio-Rad, Richmond, CA).
Western Blot Procedure
Western blot procedure was performed using the swim-up population that was capacitated (0–3 h) with or without solubilized preparation of human zona pellucida (HZP, 5–7 µg protein/200 µl of sperm suspension), as described above. The human oocytes were obtained from female partners of infertile couples who attended the Infertility Service for the in vitro fertilization-embryo transfer procedure, as approved by the Institutional Review Board [22]. Those infertile couples whose infertility was attributed to the male partner were selected for this study. These women were of midreproductive age (21–35 yr old) and did not have any ovarian abnormality. Informed consent was obtained for study participation and ova collection. Ova were excess leftover mature eggs, cryopreserved in 3.5 M propanediol containing 0.27 sucrose and 5% BSA in PBS until used. These eggs had never been exposed to sperm and could have otherwise been discarded. The eggs were rapidly thawed, washed in Ham's F-10 medium, and freed of adhering granulosa cells by incubation with 0.1% hyaluronidase (Sigma) in PBS for 5–10 min to disperse the cumulus cells. The eggs were washed three times in PBS to remove adhering cumulus cells. Zonae were heat solubilized in sodium carbonate buffer (0.001 M, pH 9.0) at 60°C for 1 h as described elsewhere [22]. The solubilized human zona protein (HZP) was dialyzed against PBS for 48 h at 4°C before use.
Human sperm capacitated with or without HZP for various times were solubilized in lysis buffer. The sperm lysate at each time point was divided into two aliquots; one aliquot was immunoprecipitated using protein A/G agarose beads with anti-FA-1 monoclonal antibody [10, 18, 19] and the other with control myeloma Ig, as described above. After the immunoprecipitation procedure, the beads were centrifuged and washed three times with RIPA buffer. The washed beads were boiled for 10 min in SDS sample buffer (nonreduced) and centrifuged, and the supernatant was subjected to SDS-PAGE. Proteins resolved in SDS gels were transferred electrophoretically (100 mA for 18 h at room temperature) onto nitrocellulose membranes [23]. The membranes were blocked with 3% BSA, and the proteins were immunodetected by incubating first with anti-phosphoserine mAb or anti-phosphothreonine mAb and then with alkaline phosphatase-conjugated anti-mouse antibodies. This was followed by washing and then incubation with a substrate mixture of nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate [19, 20]. Finally, the membranes were washed in distilled water, dried, and photographed. The Image analysis system (Center for Information Technology, NIH, Bethesda, MD) was used to quantify the changes in intensity of various bands.
In Vitro Kinase Assay
To investigate phosphorylation of the proteins that were phosphorylated at serine/threonine as well as tyrosine residues during capacitation, the cell-free in vitro kinase assay was performed. The assay was performed both on the beads as well as in the solution. The human sperm cells (5 x 106 to 10 x 106 sperm/ml) were capacitated for 3–5 h and washed three times with PBS, and the membrane proteins were solubilized overnight at 4°C in 200 µl of lysis buffer. The lysate was centrifuged at 10 000 revs/min in an Eppendorf (Hamburg, Germany) microfuge for 10 min at 4°C, and the supernatant was used for further analysis. For assay on the beads, the extract prepared in lysis buffer was incubated (2 h, 37°C) with anti-phosphoserine mAb/anti-phosphothreonine mAb/control myeloma Ig bound onto A/G agarose beads; unbound proteins were washed off, and the beads were used for the kinase reaction. The reaction was carried out for 15 min at 4°C in a total volume of 40 µl of buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 5 mM manganese chloride, 1 mM magnesium chloride, and 100 µM ATP [9, 10, 18, 20]). The reaction was stopped by adding nonreduced SDS sample loading buffer and centrifuged (10 000 rev/min, 10 min); the supernatant was resolved in SDS-PAGE and transferred to nitrocellulose paper for Western blot analysis.
For the in vitro kinase assay performed in the solution, no antibody was added to the reaction mixture, and the reaction was carried out as described above using sperm extracts capacitated for various times. After the assay, the reaction mixture was divided into three aliquots that were immunoprecipitated with anti-phosphoserine mAb, anti-phosphothreonine mAb, or control myeloma Ig, respectively, coupled onto A/G agarose beads. The beads were washed three times, boiled in SDS sample loading buffer, and centrifuged; the proteins in the supernatant were resolved in SDS-PAGE and transferred to nitrocellulose membrane for Western blot analysis. The Image analysis system was used to measure the changes in band intensity, as described above.
To examine whether the proteins that were phosphorylated at the serine and threonine residues in the in vitro kinase assay were also autophosphorylated at the tyrosine residues, the Western blot procedure was performed. The in vitro kinase reaction products from the assay performed on beads/in solution and resolved on SDS-PAGE were transferred to nitrocellulose membrane and probed with the PY-20 monoclonal antibody (20 µg/10 ml incubation buffer). The reacted antigens were localized as described above. The PY-20 monoclonal antibody (cat #p11120; IgG2b) used in this experiment was purchased from Signal Transduction Laboratories (Lexington, KY). This antibody reacts specifically with phosphotyrosine residues and does not cross-react with phosphoserine or phosphothreonine residues [24].
Indirect Immunofluorescence Technique (IFT)
To examine the subcellular localization of the proteins phosphorylated at serine and threonine residues, IFT was performed. The sperm were capacitated for various times and washed twice with PBS; sperm concentration was adjusted to 1 x 106/ml, and 20 µl of the sperm suspension was applied into wells of immunofluorescence slides as described elsewhere [19, 20]. The slides were then air dried, fixed in methanol for 30 min, and air dried again. The slides were then incubated for 1.5 h with anti-phosphoserine mAb, anti-phosphothreonine mAb, or control myeloma Ig at room temperature in a humidified chamber. The slides were washed with PBS (three times) and incubated for 1 h with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse Ig (1:20 dilution in PBS-BSA). The slides were then washed with PBS again, and a drop of mounting medium (PBS containing 90% glycerol, 0.1% sodium azide, and 10 mg/ml of 1,4-diazabicyclo(2,2,2)octane) was applied into each well; slides were covered with a coverslip and examined using a fluorescence microscope. At least 200 sperm were counted in different fields, and the percentage of sperm showing fluorescence in various regions was calculated. The experiment was repeated 3–5 times on various days using sperm from 3–6 donors, and the percentage of sperm showing various fluorescence patterns was calculated for each time point. The values were expressed as mean ± SEM; the significance of differences was examined using one-way ANOVA. A p value of < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Anti-phosphoserine mAb specifically recognized protein bands predominantly belonging to four molecular regions, as compared to the control myeloma Ig in the immunoprecipitation procedure (Fig. 1). Three bands reacted in the 43–55-kDa region, one band in the 94 ± 2-kDa region, and one band each in the 110-kDa and 190-kDa regions. In addition, another faint band was seen in each of the 18-kDa and 35-kDa molecular regions. The intensity of the three bands in the 43–55-kDa region increased by 2.7- to 3.8-fold as capacitation proceeded from 1 h to 3 h, and then started decreasing at 5 h. The bands in the 18-kDa, 35-kDa, and 94 ± 2-kDa regions were visible only in samples capacitated for 1 h and 3 h, not in those capacitated for 0 h and 5 h. The intensity of protein bands in the 110-kDa and 190-kDa regions did not change much up to 3 h and decreased by 1.4- to 1.8-fold at the 5-h capacitation period.
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Anti-phosphothreonine mAb specifically recognized protein bands in six molecular regions similar to those recognized by the anti-phosphoserine mAb, except that the 18-kDa, 35-kDa, and 94 ± 2-kDa protein bands were sharper and thicker (Fig. 1). Also, an additional thicker band was observed in the 110-kDa molecular region. The intensity of the three bands in the 43–55-kDa region increased by 1.9- to 3.1-fold as capacitation proceeded from 0 h to 1–3 h, and then started decreasing at 5 h. The 18-kDa and 34-kDa bands were visible only in the samples capacitated for 1 h and 3 h, not in samples capacitated for 0 h and 5 h. The intensity of the bands in the 94 ± 2-kDa, 110-kDa, and 190-kDa regions did not change much for up to 5 h of capacitation.
Some nonspecific bands were observed with the control myeloma Ig that were also detected with the anti-phosphoserine and anti-phosphothreonine mAbs (Fig. 1).
Effect of Capacitation and Exposure to Human Zona Pellucida on Serine/Threonine Phosphorylation of FA-1 Antigen
This set of experiments was performed to examine whether or not the FA-1 antigen, which has been shown to undergo tyrosine phosphorylation during capacitation and after zona pellucida exposure, is phosphorylated at serine/threonine residues and, if so, whether the exposure to HZP increases the intensity of phosphorylation. The results shown in Figure 2 demonstrate that the FA-1 antigen was phosphorylated at both serine (Fig. 2A) and threonine residues (Fig. 2B), and exposure to HZP increased the intensity of phosphorylation at both of these residues. There was no phosphorylation in noncapacitated (0 h) sperm. The phosphorylation was observed as early as 30 min of capacitation and further increased by 1.6-fold (1 h) and 3.2-fold (3 h). At each time point, exposure to HZP (lane c) further increased (1.5- to 2.8-fold) the intensity of serine/threonine phosphorylation compared to that in sperm capacitated without HZP (lane a). These protein bands were specific for phosphorylation, since they were immunoprecipitated only with the FA-1 mAb (lanes a and c) and not with the same amount of isotype-specific control myeloma Ig (lane b).
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Proteins Phosphorylated at Serine/Threonine as Well as Tyrosine Residues
In the in vitro kinase assay carried out on beads (Fig. 3A), proteins belonging to two molecular regions, namely 110 ± 10 kDa (1–2 proteins) and 210 ± 10 kDa (1 protein), were phosphorylated at the serine and tyrosine residues. There was a 1.2- to 1.7-fold increase in intensity of these bands as capacitation proceeded from 0 h to 3–5 h. In the assay carried out in the solution (Fig. 3B), proteins belonging to five molecular regions, namely 20 ± 4 kDa (1 weak protein band), 43–55 kDa (2–3 proteins), 80 ± 4 kDa (1 protein), 110 ± 10 kDa (1–2 proteins), and 210 ± 10 kDa (1 protein), were phosphorylated at the serine and tyrosine residues. The intensity of these bands did not vary much over time as the capacitation proceeded.
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In the assay carried out on the beads, proteins belonging to five molecular regions, namely 43–55 kDa (1–2 proteins), 94 ± 2 kDa (2 proteins), 110 ± 10 kDa (2 proteins), and 210 ± 10 kDa (1 protein), were phosphorylated at both the threonine and tyrosine residues (Fig. 3A). The intensity of these bands increased by 1.8- to 3.1-fold as capacitation proceeded, with maximal intensity observed at 3 h. When the assay was carried out in the solution (Fig. 3B), similar proteins were phosphorylated, except that proteins belonging to two additional molecular regions, namely 20 ± 4 (1 protein) and 80 ± 4 kDa (1–2 proteins), were also phosphorylated. Again, the intensity of these proteins increased by 2.1- to 3.8-fold as capacitation proceeded, with maximal intensity of most of these bands observed at 3 h.
Subcellular Patterns of Serine/Threonine Phosphorylation
In the indirect immunofluorescence studies performed on methanol-fixed human sperm capacitated for various times (0 h, 1 h, and 3–5 h), anti-phosphoserine mAb reacted weakly with the acrosomal and tail regions of sperm at 0 h of incubation (Fig. 4b). As capacitation proceeded, the intensity and percentage of sperm showing fluorescence increased (% sperm showing fluorescence, mean ± SEM: 0 h, 15 ± 6; 1 h, 45 ± 9; 3–5 h, 68 ± 8; p < 0.05 at each time point) (Fig. 4). The maximum intensity of fluorescence was observed at 3 h and began to decrease on further incubation. Along with an increase in intensity and percentage of sperm showing fluorescence, there was a subcellular shift in the sperm regions binding to anti-phosphoserine mAb, from the acrosomal region (0 h) to postacrosomal and tail regions (1 h) and then to postacrosomal, midpiece, and tail regions (3 h), as capacitation proceeded (Fig. 4).
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Anti-phosphothreonine mAb reacted with the equatorial regions of sperm at 0 h of incubation (Fig. 5a). As capacitation proceeded, the intensity and percentage of sperm showing fluorescence increased (% sperm showing fluorescence, mean ± SEM: 0 h, 12 ± 5; 1 h, 42 ± 11; 3 h, 62 ± 6; 5 h, 81 ± 9; p < 0.05 at each time point). Along with an increase in intensity and percentage of sperm showing fluorescence, there was a subcellular shift in the sperm regions binding to anti-phosphothreonine mAb. As the time of capacitation lengthened, sperm were showing fluorescence in the midpiece and tail regions, in addition to the fluorescence in the equatorial region observed at 0 h of incubation; and at 5 h capacitation, ~80% of sperm were showing fluorescence in the equatorial as well as midpiece and tail regions (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The protein bands detected with the anti-phosphoserine/anti-phosphothreonine mAb were specific since 1) the same amount of control myeloma Ig of the same class/isotype specificity did not react with these bands and 2) these mAbs specifically recognize only the phosphorylated forms of their specific residue (serine/threonine) and also do not react with phosphorylated/nonphosphorylated tyrosine, AMA, and ATP [25, 26]. Immunoadsorption (4°C, overnight) of the mAb with phosphoserine/phosphothreonine residues (1:10, antibody:antigen ratio, w:w) completely abolished their immunoreactivity with the specific sperm proteins as tested in the ELISA and the immunoprecipitation procedure (data not shown).
In the 43–55-kDa molecular region, three proteins were phosphorylated at serine/threonine residues. Our data from the second set of experiments using anti-FA-1 mAb indicate that one of these three proteins is a well-characterized sperm glycoprotein, designated FA-1 antigen, that has been previously shown to be phosphorylated at the tyrosine residues [9–11]. The findings further indicate that besides phosphorylation at tyrosine residues, FA-1 also undergoes phosphorylation at the serine and threonine residues. The phosphorylation starts as early as 30 min, and the intensity of phosphorylation increases as capacitation proceeds, which is further enhanced after exposure to human zona pellucida. These results are in agreement with the previous studies [9–11] showing an increase in tyrosine phosphorylation of FA-1 antigen after capacitation and zona pellucida exposure.
Of the eight proteins phosphorylated at serine/threonine residues in six molecular regions, 4–8 proteins belonging to similar molecular regions, namely 20 ± 4 kDa, 43–55 kDa, 94 ± 2 kDa, and 110 ± 10 kDa, as well as in 80 ± 4 and 210 ± 10 kDa regions, were phosphorylated at the serine/tyrosine and/or threonine/tyrosine residues in the in vitro kinase assay. There was a difference in phosphorylation pattern when the kinase assay was carried out on the beads versus in the solution. These types of differences have also been observed in other studies [9–11, 20]. It is possible that there are some conformationally dependent differences in the availability of sites for phosphorylation performed in solution versus solid support. It is equally possible that the requirement for the kinase reaction of proteins varies under different conditions. The degree of phosphorylation depended upon the physiological state of the sperm population and increased as capacitation proceeded, with intensity reaching maximum after 1 h and decreasing after the 5-h capacitation period. The quantitative increase in phosphorylation may be due to better conformation and/or enhanced activation state of these proteins/enzymes, through addition/deletion of subunits/domains in response to second messengers, as capacitation proceeds. In the in vitro kinase assay, the phosphorylation observed might be due to autophosphorylation through the kinase domain of a membrane protein or be mediated through the endogenous kinases that relocate from the cytosolic compartment, become membrane bound after activation, and are solubilized along with the other membrane proteins.
Phosphorylation of structural and regulatory proteins is a major intracellular control mechanism in eukaryotes [2–5]. The phosphorylation state of a protein is a dynamic process controlled by both protein kinases and protein phosphatases [6, 7]. Those phosphotransferases with a protein alcohol group as acceptor are protein-serine/threonine kinases, and those with a protein phenolic group as acceptor are protein-tyrosine kinases [3]. The total number of distinct kinase domain sequences available now is over 400 [6], and they have similar sequences in several domains. The protein-serine/threonine family is more diverse than the protein-tyrosine kinase family [3]. Many of the kinases and phosphatases are specific for serine, threonine, or tyrosine residues, and others have dual specificity [3]. Several of the serine/threonine/tyrosine phosphoregulating enzymes, such as cyclic nucleotide-, diacylglycerol-, and calcium/calmodulin-regulated protein kinases [27–29], cdc2 family of proteins [30], c-ras proteins [31], and several tyrosine kinase proteins such as epidermal growth factor receptor [32] and c-Abl protein [20], have been found to be present in human sperm. The findings of the present study indicate that besides residue-specific kinases, the human sperm cell also has dual-specificity kinases that cause phosphorylation at more than one residue (serine/tyrosine or threonine/tyrosine) of proteins. Previous studies from our [9–11] and other laboratories [25, 33, 34] have demonstrated that among the protein(s) that are tyrosine phosphorylated/autophosphorylated during capacitation of human sperm, those belonging to the 94 ± 2-kDa molecular region are among the major proteins along with FA-1 antigen. Recently, the 94 ± 2-kDa set of proteins has drawn considerable attention. The present data indicate that besides phosphorylation at tyrosine residues, this set of proteins also undergoes phosphorylation/autophosphorylation at the serine and threonine residues.
Immunofluorescence results on fixed human sperm revealed that capacitation increases the intensity of serine and threonine phosphorylation per sperm cell as well as the number of sperm cells that are phosphorylated. Interestingly with these changes, there was also a shift in the site of serine/threonine-specific fluorescence from the acrosomal to the post-acrosomal regions (in case of serine phosphorylation) and from the equatorial to the equatorial, midpiece, and tail regions (in case of threonine phosphorylation). Results from our laboratory previously showed a shift in subcellular localization of various proteins of human sperm after tyrosine phosphorylation [9]. Using other systems, there are studies indicating a shift in subcellular localization after phosphorylation. In human epidermoid carcinoma A431 cells, it has been shown that the binding of epidermal growth factor to its receptor triggers redistribution of phospholipase C-r1 from a predominantly cytosolic localization to membrane-bound activity following phosphorylation at tyrosine residues [35]. The subcellular shift of proteins after phosphorylation during capacitation may have physiological significance in consolidating certain phosphorylated proteins to specific sperm regions involved in acrosomal exocytosis and zona pellucida recognition, binding, and penetration.
In conclusion, our data indicate that there are at least 8–9 proteins (major or minor) belonging to six molecular regions, namely 18 kDa, 35 kDa, 43–55 kDa, 94 ± 2 kDa, 110 kDa, and 190 kDa, that undergo serine and threonine phosphorylation, which increases as capacitation proceeds. One of these proteins in the 43–55-kDa region is the FA-1 antigen, which has been shown to be tyrosine phosphorylated during capacitation. The present findings indicate that this protein is also phosphorylated at serine and threonine residues. The intensity of phosphorylation is further enhanced after exposure to human zona pellucida. Of these proteins, 4–8 proteins belonging to four similar molecular regions, namely 20 ± 2-kDa, 43–55-kDa, 94 ± 2-kDa, and 110 ± 10-kDa, and in the 80 ± 4- and 210 ± 10-kDa region, showed dual phosphorylation at the serine/tyrosine and/or threonine/tyrosine residues. Capacitation increased the intensity of serine/threonine phosphorylation per sperm cell, increased the number of sperm cells that were phosphorylated, and induced a shift in subcellular localization of serine/threonine-specific fluorescence. These findings indicate that the serine/threonine phosphorylation/autophosphorylation of a specific set of proteins is involved and may have a physiological role in sperm capacitation/acrosomal exocytosis and zona pellucida binding, and its derangement may cause defective sperm function leading to male infertility.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Rajesh K. Naz, Division of Research, Health Education Building, Rm 211, Medical College of Ohio, 3055 Arlington Avenue, Toledo, OH 43614-5806. FAX: 419 383 4473; rnaz{at}mco.edu
Accepted: February 1, 1999.
Received: September 30, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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