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Regular Article |
a MRC Reproductive Biology Unit, Centre for Reproductive Biology, Edinburgh EH3 9EW, United Kingdom
b School of Biological and Chemical Sciences, Centre for Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia
ABSTRACT
As mammalian spermatozoa migrate through the epididymis, they acquire functionality characterized by the potential to express coordinated movement and the competence to undergo capacitation. The mechanisms by which spermatozoa gain the ability to capacitate during epididymal transit are poorly understood. The purpose of this study was to investigate the impact of epididymal maturation on the signal transduction pathways regulating tyrosine phosphorylation, because this process is thought to be central to the attainment of a capacitated state and expression of hyperactivated motility. Western blot and immunocytochemical analyses demonstrated that epididymal maturation in vivo is associated with a progressive loss of phosphotyrosine residues from the sperm head. As cells pass from the caput to the cauda epididymis, tyrosine phosphorylation becomes confined to a narrow band at the posterior margin of the acrosomal vesicle. Epididymal maturation of rat spermatozoa was also associated with an acquired competence to respond to high levels of intracellular cAMP by phosphorylating tyrosine residues on the sperm tail. Immature caput spermatozoa were incapable of exhibiting this response, despite the apparent availability of cAMP and protein kinase A. These findings help to clarify the biochemical changes associated with the functional maturation of spermatozoa during epididymal transit.
sperm, sperm capacitation/acrosome reaction
INTRODUCTION
As mammalian spermatozoa migrate through the epididymis, they acquire the potential for fertilization, characterized by acquisition of the potential to express coordinated movement and the competence to undergo capacitation. The latter is a poorly defined process that is a prerequisite for fertilization. It was first described during the 1950s [1–4] and was defined as the period of time required for the final maturation of the spermatozoa that enabled them to successfully fertilize an oocyte [5]. Later, the ability of spermatozoa to undergo the acrosome reaction in response to contact with the zona pellucida was included in the definition [6–8].
The mechanisms by which epididymal maturation confers on mammalian spermatozoa the potential to capacitate is poorly understood. To address this problem, we have examined the impact of epididymal passage on the pattern of tyrosine phosphorylation exhibited by rat spermatozoa, because this signal transduction pathway is thought to be central to the attainment of a capacitated state [9–12] and the concomitant expression of hyperactivated motility [13, 14].
The regulation of tyrosine phosphorylation in mature mammalian spermatozoa is thought to be via a cAMP/protein kinase A (PKA) pathway that appears to be unique to this cell type. Thus, protein tyrosine phosphorylation in capacitating boar [15], mouse [10], and human spermatozoa has been found to be cAMP-dependent and inhibited by antagonists of this system [10, 16, 17]. Mahoney and Gwathmey [13] also demonstrated in Macaca fascicularis that tyrosine phosphorylation of sperm-tail proteins is an integral signaling pathway modulating some, but not all, of the motion characteristics associated with cAMP and caffeine-stimulated hyperactivation. A 55-kDa tyrosine phosphorylated protein in bovine spermatozoa was also found to be regulated by cAMP and associated with motility [18, 19].
Whereas tyrosine phosphorylation during capacitation has been observed in many species, to our knowledge, the impact of epididymal maturation on this signal transduction pathway has not yet been ascertained. Specifically, at which point in the cAMP-PKA-tyrosine phosphorylation pathway the epididymis controls the competence of mammalian spermatozoa to undergo capacitation is not known. The purpose of this study was to investigate the impact of epididymal maturation on the signal transduction pathways regulating tyrosine phosphorylation using the laboratory rat as an animal model.
MATERIALS AND METHODS
Reagents
Reagents were obtained from BDH (Poole, Dorset, UK) unless otherwise specified. Pentoxifylline (PTX), N6, 2-O-dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP), genistein, SDS, Tris, Ponceau S concentrate, polyoxyethylene-sorbitan monolaurate/Tween-20 (T-20), O-phospho-L-tyrosine, and Fast Red substrate were all obtained from Sigma (Poole, Dorset, UK). Bovine serum albumin, glycine, and H89 were obtained from Calbiochem (Notts, UK). Complete mini tablets and 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS) were from Boehringer Mannheim GmbH (Mannheim, Germany). The following antibodies were obtained from Affiniti (Exeter, UK): PY20, a monoclonal antibody against phosphotyrosine; anti-mouse IgG; horse radish peroxidase (HRP) conjugate (raised in goat); anti-PKA (RI subunit used as an immunogen), anti-PKA RI
, anti-PKA RII, and anti-ß-catenin. The rabbit anti-mouse immunoglobulin (RAM) and mouse polyclonal alkaline phosphatase/anti-alkaline phosphatase antibody (APAAP) were both from Dako (Glostrup, Denmark), and the normal mouse serum was from Sapu (Carluke, Scotland, UK). The HRP-conjugated monoclonal antibody against phosphotyrosine was from UBI, TCS Biologicals (Bucks, UK), and the enhanced chemiluminescence (ECL) kit was from Amersham Life Sciences (Bucks, UK). The bicinchoninic acid method BCA protein assay kit was from Pierce (catalog no. 23225ZZ; Chester, UK). Methanol was obtained from Hayman Ltd. (Essex, UK). Ammonium persulfate, 2-mercaptoethanol, and N,N,N1-tetra-methylethylenediamine were from Biorad (Herts, UK). Seeblue molecular weight markers were obtained from Novex (Frankfurt, Germany), and Hyperfilm was obtained from Amersham. The 30% (w/v) acrylamide/bis-acrylamide stock solution (19:1 w/w) was from Anachem (Leeds, UK), and the filter paper was from Pharmacia Biotech (Herts, UK). The format A cAMP enzyme immunoassay kit, acetylated version, was obtained from Biomol (catalogue no. AK-200; Exeter, UK).
Extraction of Epididymal Spermatozoa
Epididymal spermatozoa were extracted from adult Han Wistar rats aged between 12 and 24 wk (Charles River Laboratory, Margate, UK). The rats were killed using carbon dioxide, followed by dislocation of the cervical vertebrae. The epididymides were removed from the animal and separated into the caput and caudal regions. To limit contamination, the tissue samples were carefully blotted on moist filter paper and carefully dissected free of blood clots and extraneous tissue before suspending each region into 10 ml of BWW [20] that had been prewarmed to 37°C. To reduce epididymal debris contamination, the caput and cauda were gently squeezed and punctured rather than minced. The spermatozoa were immediately placed into an incubator at 37°C and left for a short time to allow the spermatozoa to disperse throughout the media. Motility and density counts were then performed, and the cells were adjusted to a concentration of 10 x 106/ml BWW.
Extraction of Sperm Proteins
Spermatozoa were incubated at a concentration of 10 x 106/ml BWW for up to 3 h at 37°C, and sperm proteins were extracted at time periods of 0, 1, 2, and 3 h. Spermatozoa were also incubated under the same conditions for 3 h in the presence of the following reagents: PTX, dbcAMP, genistein, and H89. On completion of the incubation period, the motility was determined using a prewarmed, Thoma-ruled, 20-µm-deep hemocytometer (Weber, London, UK), and the cell suspensions were then centrifuged at 700 x g for 6 min using a MSE Mistral 3000E centrifuge (Sanyo Gallencamp, Leicester, UK). The supernatant was removed, and the samples were resuspended in approximately 4–6 ml of BWW without albumin (BWW-A) to give a sperm concentration of approximately 2 x 106 cells/ml. Following the second wash in BWW-A, the cells were resuspended at a concentration of 100 x 106/ml BWW-A containing protease inhibitors (one Complete mini tablet in 10 ml of BWW-A). Either 10% (w/v) CHAPS or 10% (w/v) SDS was added to the sperm suspension, so that the final concentration of CHAPS or SDS in the sperm suspensions was equal to 1%. Cells were treated with CHAPS to extract only surface and cytosolic proteins, whereas SDS was used to extract proteins of the fibrous sheath and cytoskeleton in addition. The sperm suspensions were briefly vortexed, and the CHAPS samples were placed on a rocker for 15 min at 25°C whereas the SDS suspensions were incubated for 1 h at 25°C. Following incubation, the samples were centrifuged for 6 min at 1000 x g, the supernatants retained, and the pellets discarded. Five microliters of each suspension were kept aside for use in a protein estimation assay, and the remaining supernatant was incubated at 100°C for 5 min with an equal amount of reduced sample buffer (0.375 M Tris [pH 8.85], 0.284 M 2-mercaptoethanol, 2% [w/v] SDS, 10% [w/v] sucrose, 0.05% [w/v] bromophenol blue). The samples were then stored at -20°C until they were required for use.
Protein Assay
To ensure equal loading of protein onto an SDS gel for SDS-PAGE and Western blot analysis, protein estimations were performed on every sample using the bicinchoninic acid method BCA protein assay kit. A standard curve ranging from 50 to 250 µg of protein/ml was prepared using 50 µl of each standard and 1 ml of the supplied solution AB. For the sperm protein extracts, 45 µl of dH2O and 1 ml of solution AB were added to 5 µl of each protein extract. The standards and extracts were then placed in a water bath heated to 60°C for between 15 and 30 min, depending on the intensity of color change. A calibration curve using the standards was calculated, and the protein concentration of the extracts was measured at a wavelength of 562 nm using a Shimadzu (Milton Keynes, UK) UV-VIS scanning spectrophotometer (UV-2101PC). The volume of each extract to be loaded onto the gel and adjusted to equal the amount of protein found in the sample with the lowest concentration of protein, with the minimum amount loaded per lane being 50 µg of protein.
SDS-PAGE and Western Blot Protocol
The SDS-PAGE was carried out on the sperm protein extracts using 7.5% SDS-PAGE gels. Following electrophoresis at 30-mA constant current, the proteins were transferred overnight onto nitrocellulose hybond super-C membrane (Amersham) at 25-V constant voltage in wet-blot buffer (0.1 M Tris, 0.767 M glycine, and 20% [v/v] methanol). Ponceau S concentrate was added to the membrane and incubated at 25°C, then rinsed off with dH2O to check if the proteins had transferred successfully from the gel to the membrane. The membrane was blocked for 1 h in Tris-buffered saline (TBS; 20 mM Tris and 150 mM NaCl [pH 7.6]) with 3% (w/v) BSA at 25°C. The membrane was then probed with either PY20, a monoclonal antibody against phosphotyrosine, or an HRP-conjugated monoclonal antibody against phosphotyrosine, both at a 1:1000 (v/v) dilution in TBS with 0.1% (v/v) T-20 and 1% (w/v) BSA for 2 h at 25°C. This was followed by repeated washes in TBS/0.1% T-20 (once for 15 min and three times for 5 min) and then incubation with anti-mouse IgG, HRP conjugate (raised in goat), at a concentration of 1:6000 (v/v) in TBS/0.1% T-20/1% (w/v) BSA for 1 h. Incubation with the secondary antibody was not required for membranes probed with the HRP-conjugated monoclonal antibody against phosphotyrosine. The washes were repeated, and detection of proteins was performed using the ECL kit.
In addition to probing proteins for phosphotyrosine, primary antibodies for detecting PKA were also used as part of this study. These included anti-PKA (RI subunit used as an immunogen), anti-PKA RI, and anti-PKA RII, which were all used at a 1:1000 (v/v) dilution for 2 h at 25°C. The washing procedure and secondary antibody used were identical to the protocol employed with PY20. To confirm antibody specificity, some samples were either probed with a monoclonal antibody against ß-catenin (instead of an antibody against phosphotyrosine) or probed with a monoclonal antibody against phosphotyrosine that had previously been incubated with 20 mM O-phospho-L-tyrosine and rotated for 60 min at 25°C. The primary antibody was also omitted in some experiments to determine secondary antibody specificity. All of these controls demonstrated that the Western blot protocols used in this study were specific for the target antigens (data not shown).
Immunocytochemistry
Following various treatments, sperm suspensions were diluted in PBS to a concentration of 2.5 x 106/ml and dried onto Hendley slides (Hendley Ltd., Essex, UK). Slides were either fixed in 1% (w/v) paraformaldehyde for 10 min or 100% methanol/3% H2O2 for 30 sec. No statistical difference was found in the percentage of stained cells or localization of staining when the two methods of fixing were compared (data not shown). The slides were probed with PY20 at a concentration of 1:10 (v/v) in TBS, followed by incubation with RAM at a concentration of 1:25 (v/v) with a 1:10 (v/v) solution of normal rabbit serum in TBS. This was followed by a 1:50 (v/v) dilution of the antibody APAAP complex in TBS, followed in turn by incubation with Fast Red substrate. The cells were counterstained with hematoxylin and mounted in Aquamount (BDH). The slides were observed using an Olympus (Hamburg, Germany) AX70 microscope, and a minimum of 200 cells on each slide were counted for staining. Duplicate slides were prepared for use as negative controls whereby PY20 was replaced with normal mouse serum or only the RAM and APAAP antibodies were added.
Measurement of Intracellular cAMP Generation in Spermatozoa
Cyclic AMP was extracted from cells pelleted by centrifugation for 6 min at 600 x g to remove the BWW, followed by incubation with 0.1 M HCl (250 µl, 10 x 106 cells) for 10 min at 25°C. The samples were centrifuged again for 6 min at 600 x g, and the supernatants were removed and kept for use in the assay. Intracellular cAMP concentrations of spermatozoa incubated with various treatments were measured using the format A cAMP enzyme immunoassay kit, acetylated version, in accordance with the manufacturer's guidelines. The Assay Zap program from BioSoft (Cambridge, UK) was used to calculate the intracellular cAMP concentration of the samples.
Statistics
All experiments were replicated at least three times, and the statistical significance of any differences observed was determined by analysis of variance using the Statview 2 program (Abacus Concepts Inc., Berkeley, CA). Post-hoc testing of differences between group means was accomplished using the Fisher protected least significant difference test, with the significance level set at P < 0.05.
RESULTS
Tyrosine Phosphorylation of Rat Spermatozoa Isolated from the Caput and Cauda Epididymis
Considering the importance of tyrosine kinase activity in sperm function [9, 10, 21, 22], it was of interest to determine the patterns of tyrosine phosphorylation of epididymal rat spermatozoa, cultured for various time intervals, in a simple, defined culture medium capable of supporting the capacitation of functionally mature cells.
Rat spermatozoa were incubated in BWW at 37°C for up to 3 h. Protein was extracted from the spermatozoa at the beginning of the incubation and at 1, 2, and 3 h thereafter. Western blot analyses of sperm-surface proteins extracted with 1% CHAPS revealed high levels of phosphotyrosine expression in caput epididymal spermatozoa, particularly in a cohort of proteins of approximately 50–64 kDa (Fig. 1A). The level of phosphorylation of these proteins did not alter to a great extent over the 3-h incubation period.
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In contrast, Western blot analysis of proteins extracted from caudal spermatozoa under the same conditions revealed low levels of tyrosine phosphorylation in proteins of approximately 60 and 73 kDa (Fig. 1A). Tyrosine phosphorylation of these caudal sperm proteins also decreased over the 3-h period, with maximum intensity being observed immediately following extraction from the epididymis.
This trend was also observed with the more severe extraction conditions imposed by SDS (1%), revealing the presence of additional, heavily phosphorylated proteins in caput cells, with molecular weights ranging between 91 and 127 kDa (Fig. 1B). As with the CHAPS extraction protocol, the overall level of tyrosine phosphorylation in caudal spermatozoa was markedly reduced in SDS-extracted cells compared to the caput population, with phosphorylation being restricted to proteins of 41, 52, and 127 kDa (Fig. 1B).
Immunolocalization of Tyrosine Phosphorylated Proteins
Immunolocalization of tyrosine phosphorylated proteins in caput spermatozoa demonstrated heavy labeling over the entire acrosomal domain of the sperm head (Figs. 2, A–D, 3A, and 4A). Approximately 100% of the caput sperm population exhibited this pattern of phosphorylation, and this did not change significantly over time (Figs. 2, A–D, 3A, and 4A). In contrast, protein phosphorylation of caudal epididymal cells was clearly confined to the posterior margin of the acrosome in between 70% and 80% of the sperm population (Figs. 2, E–H, 3B, and 4B), although this staining was occasionally accompanied by labeling at the apex of the sperm head, in close proximity to the perforatorium (Fig. 2E). The percentage of caudal cells exhibiting phosphorylation at the posterior acrosomal margin remained relatively constant over time (Fig. 4B). Tyrosine phosphorylation of the entire acrosomal domain was observed in a small population (10%–20%) of caudal cells at the beginning of the incubation period (Fig. 4A). However, the percentage of cells exhibiting this pattern of staining declined significantly over the incubation period, to represent less than 5% of the sperm population after 3 h. The decline in this subpopulation of labeled caudal epididymal cells could account for the time-dependent decline in tyrosine phosphorylation levels seen in the Western blot analysis (Fig. 1A).
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Regulation of Tyrosine Phosphorylation in Rat Spermatozoa by cAMP
Tyrosine phosphorylation in spermatozoa has been studied in many species, including human [11, 12, 23, 24], murine [9, 10], bovine [18, 19], and sea urchin [25], and it is central to attainment of the capacitated state [9, 10]. Because cAMP plays a pivotal role in the regulation of tyrosine phosphorylation [10, 13, 15, 17–19, 25], it was of interest to investigate the status of this signal transduction pathway during epididymal maturation in the rat.
Intracellular cAMP levels were raised in rat spermatozoa by the addition of the phosphodiesterase-inhibitor PTX in conjunction with the membrane permeant analogue of cAMP, dbcAMP. Rat spermatozoa were incubated for 3 h at 37°C in BWW with various concentrations of PTX and dbcAMP, and their proteins were extracted with 1% SDS. In both caput and caudal epididymal spermatozoa, tyrosine phosphorylation was significantly up-regulated following treatment with these two reagents, although these effects were much more marked in caudal cells (Fig. 5, A and B). Treatment of caudal spermatozoa with both PTX and dbcAMP induced tyrosine phosphorylation in a new set of proteins, with molecular weights of approximately 58, 62, 79, 98, 115, 125, 153, and 205 kDa. Up-regulation of phosphorylation was also observed in proteins of approximately 52, 64, and 84 kDa. Increased phosphorylation was particularly evident with a combination of 3 mM PTX and 5 mM dbcAMP (Fig. 5B). Tyrosine phosphorylation was also induced in caput spermatozoa under the same conditions, although the effect observed was not as significant as that found with the caudal cells (Fig. 5A). An increase in tyrosine phosphorylation of proteins of approximately 28–33, 60, 84, and 120 kDa was observed in caput spermatozoa treated with PTX and dbcAMP, whereas phosphorylation of a new protein of approximately 215 kDa was induced. These effects were not as clearly dose dependent as those observed in caudal cells.
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A similar picture emerged when, instead of using SDS as a solubilization agent, cytosolic and membrane proteins were extracted with CHAPS. Elevation of intracellular cAMP by the combination of dbcAMP and PTX specifically enhanced the phosphorylation of three high-molecular-weight proteins of approximately 98, 115, and 125 kDa in caudal, but not in caput, cells extracted with CHAPS (Fig. 6). In contrast, elevated intracellular cAMP induced the phosphorylation of a group of proteins in the range of 50–64 kDa in caput, but not in caudal, cells (Fig. 6). That PTX had to accompany dbcAMP to induce high levels of tyrosine phosphorylation in both caput and caudal cells suggests the presence of highly active, intracellular phosphodiesterases at all stages of epididymal maturation (Fig. 6).
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Immunocytochemistry demonstrated that in caudal cells only, stimulation by dbcAMP in the presence of PTX leads to the phosphorylation of a new set of proteins located in the tail of up to 80% of the sperm population (Figs. 7, C and D, and 8). The percentage of each sperm population demonstrating this positive tail stain varied significantly between treatments (Fig. 8). Populations of caudal spermatozoa treated with PTX or dbcAMP alone contained a significantly smaller population of cells with tyrosine phosphorylated tails than those treated with dbcAMP combined with PTX, in accordance with the results of the Western blot analysis (Figs. 7 and 8). However, in caput spermatozoa treated under the same conditions, tyrosine phosphorylated proteins remained restricted to the acrosomal domain in the face of cAMP stimulation, with less than 3% of cells demonstrating any phosphorylation on the sperm tail (Fig. 7, A and B).
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Tyrosine kinase involvement in the cAMP-mediated effects on phosphotyrosine expression in the sperm tail was indicated by the ability of genistein to significantly suppress this response (Fig. 9, A and B).
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Tyrosine Phosphorylation In Vivo
To determine whether the tyrosine phosphorylation patterns observed with rat spermatozoa were present in vivo or the result of cells being incubated in BWW, spermatozoa were dissected into BWW containing 10 µM genistein, a known tyrosine kinase inhibitor [23]. Under these conditions, spontaneous tyrosine phosphorylation as a consequence of the spermatozoa's release into BWW should have been inhibited. The results of this analysis indicated that genistein had no effect on the percentage of cells stained in the entire acrosomal domain or in the posterior acrosomal margin in caput and caudal spermatozoa, respectively (Fig. 9, C and D). This was not because of any deficiency on the part of genistein as a tyrosine kinase inhibitor under these conditions, because this reagent clearly suppressed the PTX- and dbcAMP-induced tyrosine phosphorylation of tail proteins in caudal spermatozoa (Fig. 9B). This result is consistent with the notion that the distinct patterns of phosphorylation observed in caput and caudal epididymal spermatozoa in vitro were already extant in vivo. This conclusion was confirmed by the immunocytochemical studies in which sperm cells were not released into BWW but were smeared directly from the epididymis onto slides. The results of this analysis clearly demonstrated that in vivo, spermatozoa recovered directly from the caput and cauda epididymides were phosphorylated over the entire acrosomal region and posterior acrosomal margin, respectively, just as observed with the cells incubated in BWW (Fig. 10). This result was also confirmed when histopathological sections of the epididymis were stained with an antiphosphotyrosine antibody (data not shown).
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Intracellular cAMP Concentration in Rat Spermatozoa
Given the importance of cAMP in regulating tyrosine phosphorylation, it was of interest to determine whether spermatozoa from the separate regions of the epididymis differed in their capacity to generate this second messenger. Direct measurement of cAMP levels revealed no significant difference in intracellular cAMP concentration between caput and caudal spermatozoa, although levels tended to be higher in caput cells (Fig. 11). Additionally, when cells were stimulated with 3 mM PTX, intracellular cAMP levels were increased in both caput and caudal spermatozoa, although this increase was not statistically significant. Such results could explain why the addition of dbcAMP to PTX was required to raise intracellular cAMP levels to the point at which optimal levels of tyrosine phosphorylation were observed (Figs. 6 and 8). These results also emphasize that the distinct phosphorylation patterns observed in caput and caudal epididymal spermatozoa, both in vivo and in vitro, are unlikely to result from differences in intracellular cAMP alone.
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cAMP-Dependent PKA and Tyrosine Phosphorylation
Because caput and caudal spermatozoa were clearly capable of generating equal levels of cAMP, it was important to determine whether the factor inhibiting cAMP action in immature caput epididymal sperm cells could be downstream of cAMP generation. Protein kinase A catalyzes transfer of the terminal phosphate group from ATP to specific serines or threonines of selected proteins [26]. In the inactive state, PKA consists of a complex of two regulatory subunits, which bind cAMP, and two catalytic subunits. The binding of cAMP alters the conformation of the regulatory subunits, causing them to dissociate from the catalytic units, which are consequently activated to phosphorylate specific substrate protein molecules [26]. Four distinct regulatory subunits (RI, RIß, RII, and RIIß) define the type I and type II classes of cAMP-dependent protein kinases.
Proteins were extracted from rat spermatozoa with 1% SDS following incubation with various treatments for 3 h at 37°C. Following SDS-PAGE, Western blot analysis was carried out using three separate PKA antibodies: anti-PKA (RI subunit used as an immunogen), anti-PKA RI, and anti-PKA RII. When sperm proteins were probed with the anti-PKA antibody, equal amounts of PKA were found in both caput and caudal spermatozoa (Fig. 12C). Additionally, no difference was found in the intracellular presence of the PKA RI and RII subunits in caput and caudal epididymal spermatozoa (Fig. 12, A and B). However, in both caput and caudal spermatozoa, there appeared to be a greater amount of type II PKA compared to type I (Fig. 12, A and B).
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To confirm PKA involvement in the cAMP-mediated induction of tyrosine phosphorylation in the rat, spermatozoa were incubated with the PKA-inhibitor H89 (10 µM) for 3 h at 37°C, with and without 3 mM PTX and 5 mM dbcAMP. The H89 completely inhibited the PTX- and dbcAMP-associated inducement of tyrosine phosphorylation in rat spermatozoa (Fig. 12D).
DISCUSSION
The results presented here highlight how differences in protein tyrosine phosphorylation of rat spermatozoa reflect their maturational status. Thus, phosphotyrosine expression by rat spermatozoa markedly decreased as they matured in the epididymis, in complete contrast to the situation in the mouse [9]. In the rat, spermatozoa recovered from the caput region of the epididymis exhibited extensive phosphorylation of several proteins, exhibiting relative molecular masses of 50–64 and 91–127 kDa following extraction with CHAPS and SDS, respectively. Immunocytochemical analysis clearly revealed that these phosphorylated proteins were located in the entire acrosomal domain of the sperm head. In complete contrast, Visconti et al. [9] found that mouse caput epididymal spermatozoa exhibited low levels of tyrosine phosphorylation, with the only phosphorylated band being the p95/116 hexokinase. Those authors also demonstrated that subsequent incubation of mouse caput epididymal spermatozoa for prolonged periods of time in vitro did not influence the tyrosine phosphorylation status of these cells. Similarly, when rat caput epididymal spermatozoa were incubated for up to 3 h in vitro, no change in phosphotyrosine expression was observed.
Tyrosine phosphorylation in rat spermatozoa recovered from the cauda epididymis was greatly reduced relative to caput cells, regardless of the extraction conditions used to recover the proteins. Similarly, Visconti et al. [9] found that initial levels of tyrosine phosphorylation in caudal mouse spermatozoa were relatively low and confined to the p95/116 hexokinase, which Kalab et al. [27] demonstrated to be the major phosphotyrosine-containing protein in mouse sperm membranes. However, when caudal rat and mouse spermatozoa were subsequently incubated in vitro, quite different patterns of tyrosine phosphorylation were observed.
In the mouse, tyrosine phosphorylation of a subset of proteins in the range of 40–120 kDa increased gradually over time, with maximum levels being achieved after 90-min incubation [9]. However, in the rat, the overall levels of phosphotyrosine expression became reduced during a 3-h incubation period in vitro. These differences may reflect the contrast between these species in ability of the spermatozoa to capacitate in vitro. Whereas capacitation of rat spermatozoa in vitro is extremely difficult, mouse spermatozoa readily engage in this process, attaining a full state of capacitation within 90 min, as assessed by the ability of the spermatozoa to undergo a zona pellucida-induced acrosome reaction [6, 8, 28] as well as their ability to fertilize zona pellucida-intact eggs [28, 29].
Immunocytochemical analyses revealed striking differences between caput and caudal epididymal spermatozoa regarding localization of the phosphotyrosine residues. Whereas extensive staining of the acrosomal region was observed in virtually all caput spermatozoa, a majority of caudal cells exhibited a narrow band of tyrosine phosphorylation at the posterior acrosomal margin of the sperm head. Evidently, epididymal maturation in the rat is characterized by a progressive loss of phosphotyrosine residues from proteins associated with the acrosome. The caudal cell population also contained a small percentage of spermatozoa exhibiting an extensive, caput-like staining of the acrosome, but the cells exhibiting this phosphorylation pattern reduced from 10% to 20% to less than 5% over a 3-h incubation period. These results suggest that the caudal sperm population is heterogeneous and includes some immature cells that are competent to complete the maturation process when incubated in a simple, defined medium in vitro. Such late maturation may reflect the competence of the cells to regulate their intracellular calcium levels (see below).
These differences in phosphotyrosine expression between caput and caudal epididymal spermatozoa appeared to reflect the normal in vivo situation and were not a consequence of sperm dilution in BWW and subsequent incubation in vitro. Thus, when the tyrosine kinase-inhibitor genistein was incorporated into the media to suppress de novo phosphorylations, the characteristic differences in tyrosine phosphorylation between caput and caudal epididymal spermatozoa were still observed. In contrast genistein was effective in suppressing the de novo phosphorylations induced in caudal cells by a combination of PTX and dbcAMP. Confirmation that the observed differences in tyrosine phosphorylation between caput and caudal epididymal spermatozoa also exist in vivo came from immunocytochemical analyses of spermatozoa sampled directly from the epididymis without being diluted in BWW.
The purpose of these changes in tyrosine phosphorylation are unknown, but they could be related to the control of acrosomal exocytosis. Caput epididymal spermatozoa are incapable of undergoing the acrosome reaction, even when they are exposed to high concentrations of the divalent cation ionophore A23187. As spermatozoa mature, they acquire a capacity to acrosome react in response to a calcium stimulus in concert with the progressive dephosphorylation of proteins in the sperm head. These phosphoproteins might negatively regulate sperm function by suppressing the membrane-fusion events associated with the acrosome reaction. To address this hypothesis, ultrastructural data are clearly required to determine the precise localization of the phosphotyrosine residues on the sperm head and their fate during induction of the acrosome reaction.
In concordance with findings in many other species, including the sea urchin [25], cynomologous monkey [13], human [16, 17], bovine [18, 19], boar [15], and mouse [10], we found that tyrosine phosphorylation in rat spermatozoa was also regulated by a cAMP-dependent signal transduction pathway. A particularly detailed and lucid study of this area has been reported by Visconti et al. [10], who found that active cAMP analogues could induce phosphorylation of capacitation-associated proteins in caudal mouse spermatozoa. When caudal mouse spermatozoa were incubated in media devoid of bicarbonate, calcium chloride, or BSA, they did not exhibit the capacitation-associated increases in protein tyrosine phosphorylation. However, such inhibition could be completely overcome by the addition of biologically active cAMP analogues. As in all species examined to date, we found that many new proteins were phosphorylated in caudal rat spermatozoa, in a dose-dependent manner, following addition of the active cAMP analogue dbcAMP and the phosphodiesterase-inhibitor PTX. Tyrosine phosphorylation was most pronounced when PTX and dbcAMP were used in combination, suggesting that these cells possess a particularly powerful phosphodiesterase. Such activity may contribute significantly to the low levels of spontaneous tyrosine phosphorylation observed in caudal epididymal rat spermatozoa and the difficulties encountered in capacitating these cells in vitro.
Both PTX and dbcAMP also induced an increase in protein tyrosine phosphorylation in spermatozoa retrieved from the caput epididymis. However, this stimulatory effect was insignificant compared with the dramatic stimulation of phosphotyrosine expression seen in caudal sperm. Moreover, it involved a completely different group of proteins, located in a completely different region of the cell. The stimulation of caudal spermatozoa with PTX and dbcAMP induced phosphorylation of a new set of proteins in the sperm tail, whereas caput spermatozoa treated under the same conditions only exhibited phosphotyrosine expression in the acrosomal region of the sperm head. The reasons behind the failure of caput epididymal spermatozoa to exhibit cAMP-dependent phosphorylation of tyrosine residues in the sperm tail are currently unknown. Recent, unpublished results from our laboratory suggest that high intracellular calcium levels are responsible for the lack of cAMP-mediated phosphorylation in caput epididymal spermatozoa. These findings could reflect an immaturity of the Ca2+/Mg2+-ATPases in the sperm plasma membrane of caput epididymal spermatozoa, resulting in high intracellular calcium levels and a consequential elevation of calcium-dependent phosphatase activity. Studies are currently underway to determine whether the lack of cAMP-dependent phosphorylation in caput cells is, as hypothesized, a consequence of elevated calcium-dependent phosphatase activity. Of course, other explanations are possible, including differences in tyrosine kinase activity and the modifying impact of epididymal secretions.
In terms of the cAMP/PKA pathway, no significant differences were found between caput and caudal epididymal spermatozoa in the availability of intracellular cAMP or PKA. Western blot analyses indicated that both caput and caudal epididymal spermatozoa contained the PKA RI, RI, and RII subunits, with the latter predominating over RI. This is in accordance with the results of previous studies, indicating that in rat and bovine spermatozoa, the regulatory subunit (RII) of a type II cAMP-dependent protein kinase is tightly associated with the sperm flagellum [30–32]. Lieberman et al. [33] also demonstrated that a type II cAMP-dependent protein kinase was located at the outer mitochondrial membrane of bovine sperm. They suggested that it may play a role in the direct, cAMP-mediated stimulation of mitochondrial respiration during sperm activation. Pariset and Weinman [34] found two isoforms of the regulatory subunit RII of cAMP-dependent protein kinase in human sperm, one of which was localized to the microtubules and the other detected among the cytoplasmic proteins. Additionally, Macleod et al. [35] localized RII and its binding proteins to the fibrous sheath of rat sperm flagellum. Clearly, the PKA in mammalian spermatozoa is actively engaged in regulating the tyrosine phosphorylation status of these cells, as indicated by the ability of the PKA-inhibitor H89 to suppress tyrosine phosphorylation in both caput and caudal epididymal spermatozoa [10, 16]. If the cAMP/PKA pathway is functional at all stages of epididymal maturation, it suggests that the differences between caput and caudal spermatozoa observed in this study may result from alterations in protein phosphatase activity. Studies are currently underway to examine this possibility.
In conclusion, the results presented here indicate that epididymal maturation of rat spermatozoa is associated with a progressive loss of tyrosine phosphorylated proteins from the acrosomal domain of the sperm head. Epididymal maturation is also associated with an acquired competence to respond to high levels of intracellular cAMP by phosphorylating tyrosine residues on the sperm tail. These findings provide important clues regarding the mechanisms by which spermatozoa acquire the competence for movement and capacitation as they transit the epididymis and become programmed for fertilization.
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ACKNOWLEDGMENTS
We are grateful to fellow lab members and the staff of the animal unit at the MRC Reproductive Biology Unit, Edinburgh, for their technical advice. In addition, we would like to thank Dr. D.R. Newall from GlaxoWellcome for his advice and support.
FOOTNOTES
First decision: 3 October 2000.
1 Supported by a grant from GlaxoWellcome Research and Development and the Medical Research Council of the United Kingdom. 
2 Correspondence: R. John Aitken, School of Biological and Chemical Sciences, University of Newcastle, Callaghan, NSW 2308, Australia. FAX: 61 2 49 21 6923; jaitken{at}mail.newcastle.edu.au
Accepted: January 8, 2001.
Received: August 30, 2000.
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