Ontogeny of Tyrosine Phosphorylation-Signaling Pathways During ...

BIOLOGY OF REPRODUCTION 75, 588–597 (2006) Published online before print 21 June 2006. DOI 10.1095/biolreprod.106.052712

Ontogeny of Tyrosine Phosphorylation-Signaling Pathways During Spermatogenesis and Epididymal Maturation in the Mouse1 Minjie Lin,3 Yun Hwa Lee,3 Weian Xu, Mark A. Baker, and R. John Aitken2 Centre for Reproductive Science and Australian Research Council (ARC) Centre of Excellence in Biotechnology and Development, School of Environmental and Life Science, University of Newcastle, Callaghan, New South Wales 2308, Australia

acrosome reaction, calcium, cAMP kinase, epididymis, kinases, sperm, sperm hyperactivation, sperm maturation, sperm motility, tyrosine phosphorylation

zoa in the female tract via a process known as capacitation [2, 3]. Capacitation involves a series of time-dependent biochemical changes that enable these cells to bind to the zona pellucida, exhibit acrosomal exocytosis, and initiate fusion with the oocyte [4]. An important correlate of this process is the activation of an unusual cAMP/protein kinase A (PKA)-dependent signaling pathway that ultimately leads to the stimulation of tyrosine phosphorylation on a number of key proteins [5, 6]. This pathway is so conserved in spermatozoa that it has been identified in all mammalian species studied to date, including marsupials [7–15, 16]. Furthermore, in many species (including mouse, rat, bull, and horse), the cAMP/PKA-dependent tyrosine phosphorylation pathway that drives capacitation has been shown to be redox regulated, via mechanisms that appear to involve reactive oxygen species (ROS) generation [6, 7, 14, 17, 18]. According to these studies, exposure to oxidative stimuli elevates intracellular cAMP and stimulates patterns of tyrosine phosphorylation associated with capacitation. In rat and human spermatozoa, this cAMP-dependent pathway is also known to be sensitive to calcium, with the presence of this cation suppressing phosphotyrosine expression via mechanisms that involve the availability of intracellular ATP [19, 20]. Although this unique redox-regulated cAMP-induced tyrosine phosphorylation cascade has been studied in depth during the postejaculatory events leading up to fertilization [7, 21, 22], little is known about the maturation of this pathway during spermatogenesis and epididymal maturation. To determine when and where the capacity for tyrosine phosphorylation is activated in the mouse, we systematically sought evidence of this activity at all stages of germ cell development and examined the relationships between phosphotyrosine expression and acquisition of functional competence.

INTRODUCTION

MATERIALS AND METHODS

On leaving the testis, spermatozoa are incapable of progressive movement or the cascade of cellular interactions that result in fertilization of the oocyte. These functional attributes are only acquired as spermatozoa undergo maturation in the epididymis [1]. Ejaculation is associated with immediate expression of the facility for movement; however, the capacity for fertilization requires continued maturation of the spermato-

Reagents

ABSTRACT The objectives of this study were to map the ontogeny of tyrosine phosphorylation signal transduction pathways during germ cell development and to determine their association with the differentiation of a functional gamete. Until testicular germ cells differentiate into spermatozoa, cAMP-induced tyrosine phosphorylation is not detectable. Entry of these cells into the epididymis is accompanied by sudden activation of the tyrosine phosphorylation pathway, initially in the principal piece of the cell and subsequently in the midpiece. In the caput and corpus epididymides, the potential to express this pathway is inhibited by the presence of calcium in the extracellular medium. However, calcium has no effect on the expression of this pathway in caudal epididymal sperm. The competence of these cells to phosphorylate the entire sperm tail, from the neck to the tail-end piece, is accompanied by a capacity to exhibit hyperactivated motility on stimulation with cAMP. A distinctly different pattern of tyrosine phosphorylation, involving the acrosomal domain of the sperm head, is invoked as spermatozoa enter the caput epididymis, and phosphorylation remains high until these cells enter the distal corpus and cauda. The proportion of cells exhibiting this form of tyrosine phosphorylation is not affected by extracellular calcium or cAMP but is negatively correlated (R2 ¼ 0.99) with their ability to acrosome-react. However, this relationship is not causative. Our findings indicate that the development of functional spermatozoa is accompanied by carefully orchestrated changes in tyrosine phosphorylation, controlled by independent regulatory mechanisms in distinct subcellular compartments of these highly specialized cells.

The following reagents were used in this study: BSA (Research Organics, Cleveland, OH); Hepes, penicillin, and streptomycin (Gibco BRL, Paisley, U.K.); Mowiol (Calbiochem, San Diego, CA); unconjugated and fluorescein isothiocyanate (FITC)-conjugated clone 4G10 antiphosphotyrosine antibodies (Upstate Biotechnology, Lake Placid, NY); goat anti-mouse antibody (Santa Cruz Biochemicals, Santa Cruz, CA); ammonium persulphate, 2-mercaptoethanol, and N,N,N1-tetra-methylethylenediamine (Temed; BioRad, Hertfordshire, U.K.); and nitrocellulose hybond super-C membrane and enhanced chemiluminescence (ECL) kit (Amersham International, Buckinghamshire, U.K.). All other reagents were from Sigma-Aldrich (St. Louis, MO), unless otherwise stated.

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Supported by the University of Newcastle and by the ARC Centre of Excellence in Biotechnology and Development. 2 Correspondence: FAX: 02 4921 6953; e-mail: [email protected] 3 These authors contributed equally to this work.

Medium Biggers, Whitten, and Whittingham (BWW) medium [23] consisted of 95 mM NaCl, 44 lM sodium lactate, 25 mM NaHCO3, 20 mM Hepes, 5.6 mM Dglucose, 4.6 mM KCl, 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 0.27 mM sodium pyruvate, 0.3% (w/v) BSA, penicillin (5 U/ml), and streptomycin (5 lg/ml) [pH 7.4]. BWW-calcium consisted of normal BWW except that CaCl2 was substituted with 1.7 mM NaCl. This buffer contained 2.0 6 0.5 lM

Received: 29 March 2006. First decision: 28 April 2006. Accepted: 14 June 2006. Ó 2006 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

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Ca2 þ, as measured by atomic adsorption spectrometry. BWW/polyvinyl alcohol (PVA) was prepared by substituting BSA with PVA (1 mg/ml). In specific experiments, spermatozoa were treated with 5 mM dibutyryl cAMP (dbcAMP) and 3 mM pentoxifylline (PTX) in BWW/PVA to stimulate high levels of tyrosine phosphorylation. For experiments involving the biological assessment of acrosomal exocytosis, these concentrations were reduced to 1 mM dbcAMP and to 1 mM PTX to reduce the risk of nonspecific effects due to plasma membrane perturbation by these hydrophobic reagents.

Sperm Preparation Sexually mature male Swiss mice (8–14 wk old, bred in the university animal facility) were killed by CO2 asphyxiation, as approved by the institutional animal ethics committee. The reproductive tracts were removed and cleared of fat and connective tissue, and the testes, epididymides, and vas deferens were dissected out. The epididymides were further divided into caput (zones 1–3), corpus (zones 4a and 4b), and caudal (zones 5a and 5b) regions (Fig. 1) [24]. The tissue was transferred to small Petri dishes, each containing 0.5 ml of BWW buffer surrounded by water-saturated mineral oil that had been prewarmed to 378C. Each piece of tissue was repeatedly punctured with a 26gauge needle and then left for a further 15 min to allow outward diffusion of the spermatozoa. The concentration of spermatozoa was ultimately determined using a Neubauer hemocytometer. The cells were centrifuged (200 3 g, 2 min), and the final concentration was adjusted to 1 3 106 cells/ml before incubation at 378C under an atmosphere of 5% CO2 and 95% air.

Preparation of Germ and Testicular Cells The testes were dissected free of fatty tissue and placed into approximately 5 ml of BWW medium at 378C. The tunica albuginea was discarded, and the seminiferous tubules were transferred to 5 ml of fresh BWW medium containing collagenase (0.5 mg/ml) at 378C. The sample was left to rotate on a laboratory wheel for 15 min, following which the tubule mass was centrifuged (600 3 g, 5 min), the supernatant was removed, and 5 ml of BWW containing 0.25% trypsin was added to the pellet, followed by rotation for a further 15 min. BSA (1%) was then added to the suspension to inactivate the trypsin. The tubules were gently pipetted to release the cells from within the ducts. The sample was subsequently filtered through a 70-lm cell strainer (Becton Dickinson) to remove unwanted material, and the filtrate was passed over a BSA gradient to separate the germ cells, as previously described [25]. Arachis hypogaea lectin (peanut agglutinin [PNA]) was used to stain the developing acrosome and to facilitate the discrimination of pachytene spermatocytes and spermatids [25]. In addition to these populations of isolated germ cells, studies were also conducted on an immortalized spermatogonial cell line (GC1).

FIG. 1. Regional differentiation of the mouse epididymis and vas deferens (VD). The epididymis is divided into seven zones according to the classification by Takano [24]. Zones 1–3 comprise the caput epididymis, zones 4a and 4b the corpus epididymis, and zones 5a and 5b the caudal epididymis. ECL kit (Amersham International, Sydney, Australia) according to the manufacturer’s instructions.

Stripping Nitrocellulose Membranes To confirm equal loading of protein, blots that had been probed for phosphotyrosine proteins were stripped and reprobed with an antibody against a-tubulin. For this procedure, approximately 30 ml of stripping buffer (consisting of 2% [w/v] SDS, 62.5 mM Tris, pH 6.7, and 100 mM 2mercaptoethanol) was added to the membrane for 1 h with constant shaking at 608C. The membrane was then washed (three times for 10 min in TBS), blocked, and probed with the primary antibody as described.

Extraction of Protein After incubation, cells were centrifuged (500 3 g, 3 min) and washed in 1 ml of BWW lacking BSA before being solubilized (2% [w/v] SDS, 0.375 M Tris, pH 6.8, and 10% sucrose) and heated to 1008C for 5 min. Following centrifugation at 20 000 3 g for 10 min, the supernatant was retained and boiled in SDS sample buffer containing 2% (v/v) 2-mercaptoethanol. Samples were then stored at 208C until used. To ensure that equal amounts of protein were loaded into the gels, protein estimations were performed on each sample using a bicinchoninic acid kit (Pierce Chemical, Rockford, IL) according to the manufacturer’s instructions. A minimum of 1 lg of total protein was loaded per lane.

SDS-PAGE and Western Blot SDS-PAGE was conducted on 1 lg of solubilized protein using 7.5% or 10% polyacrylamide gels at 10 mA constant current per gel. The proteins were then transferred onto nitrocellulose hybond super-C membrane (Amersham International, Sydney, Australia) at 350 mA constant current for 1 h. The membrane was blocked for 1 h at room temperature with Tris-buffered saline (TBS; 0.02 M Tris, pH 7.6, and 0.15 M NaCl) containing 3% (w/v) BSA. The membrane was then incubated for 2 h at room temperature in a 1:4000 dilution of a monoclonal antiphosphotyrosine (clone 4G10) or anti-a tubulin (clone B5-1-2) antibody in TBS containing 1% (w/v) BSA and 0.1% (v/v) Tween. After incubation, the membrane was washed four times for 5 min with TBS containing 0.01% Tween 20 and was then incubated for 1 h at room temperature with goat anti-mouse immunoglobulin G (IgG) horseradish peroxidase conjugate, at a concentration of 1:3000 in TBS containing 1% (w/v) BSA and 0.1% (v/v) Tween 20. The membrane was again washed as already described, and then the phosphorylated proteins were detected using an

Immunocytochemistry Extracted spermatozoa were incubated with 5 mM dbcAMP and 3 mM PTX in BWW/PVA buffer at 378C for 90 min in an incubator with 5% CO2 and 100% humidity. Following incubation, cells were fixed with 4% paraformaldehyde for 30 min at room temperature. After three washes in PBS (pH 7.4), cells were airdried onto poly-L-lysine-coated slides and then permeabilized with 0.2% Triton X-100 in PBS for 10 min. Following a PBS rinse, cells were blocked with 10% goat serum in 1% BSA in PBS at 378C for 90 min and were then incubated with 1:100 antiphosphotyrosine antibody in PBS for 60 min at 378C or overnight at 48C. After three washes in PBS, cells were incubated with 1:100 goat antimouse IgG-FITC antibody in 1% BSA in PBS for 60 min at 378C. After three PBS washes, coverslips were mounted on 5 ll of antifade reagent (13% Mowiol 4–88, 33% glycerol, 66 mM Tris [pH 8.5], and 2.5% 1,4 diazobcyclo[2.2.2]octane), and cells were examined and photographed using a Carl Zeiss MC 200 Chip microscope camera on an Axiovert S100 inverted phase-contrast microscope (Zeiss, Jena, Germany). Fluorescent images were captured through a Zeiss No. 9 (FITC) filter system with blue excitation of 450–490 nm.

Assessment of Acrosome Reaction After incubation with dbcAMP (1 mM) and PTX (1 mM), spermatozoa were treated with an equivalent volume of 1.25 lM A23187 in dimethyl sulfoxide, or vehicle alone, for 30 min at 378C. To assess sperm viability, the latter were diluted 1:10 with prewarmed hypoosmotic swelling medium, which contained 0.735% (w/v) sodium citrate and 1.351% (w/v) fructose, and were incubated for 60 min at 378C. Cells were centrifuged and fixed in ice-cold 100% methanol for 5 min and then placed on a glass slide and air-dried. After rehydration in PBS for 30 min, cells were treated with rhodamine-conjugated

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LIN ET AL. velocity cutoffs, 10.0 lm/sec and 25 lm/sec, respectively. For the assessment of hyperactivation, a 20-ll droplet of sperm suspension at 1 3 106/ml was placed onto a coverslip and viewed under darkfield illumination. The percentage of hyperactivated cells was assessed on at least 100 cells after 45 min capacitation in the presence of dbcAMP (5 mM) and PTX (3 mM), and the importance of tyrosine phosphorylation was determined by the concomitant addition of 10 lM herbimycin A.

Statistical Analysis All experiments were replicated with samples from at least three different mice, and the statistical significance of differences between group means was determined using ANOVA; Fisher protected least significant difference was used to test the statistical significance of differences between group means. Where appropriate, data are given as mean 6 SEM. The numbers of experiments are indicated in the figure legends. Percentage data were subjected to arcsine transformation before performing ANOVA. Differences between groups with P , 0.05 were considered statistically significant.

RESULTS Nonactivation of cAMP-Dependent Pathway in Testicular Germ Cells FIG. 2. Tyrosine phosphorylation in precursor germ cells. A) Pachytene spermatocytes (PS) and round spermatids (RS) do not respond to NADPH or dbcAMP/PTX as assessed by Western blot analysis. B) Reprobing of the membrane with a-tubulin demonstrates equal protein loading of the gel.

PNA in PBS (0.5 mg/ml) for 20 min in a humid chamber at room temperature. Following three washes with PBS, cells were mounted with 5 ll of antifade medium as already described, and the acrosomal status of viable spermatozoa was assessed by epifluorescence microscope using the No. 15 (rhodamine) filter set. Double-labeling experiments involving the simultaneous assessment of tyrosine phosphorylation and acrosomal status were scored using a Zeiss LSM 510 scanning confocal microscope.

Zona Pellucida-Induced Sperm Acrosome Reaction Assay Oocytes were collected, and their capacity to bind spermatozoa was preserved at 48C in a medium consisting of 1.5 mM MgCl2, 0.1% (w/v) dextran, 0.01 M Hepes buffer, and 0.1% (w/v) PVA. Ova were subsequently recovered from the storage medium, washed three times in BWW/PVA, and warmed to 378C. Spermatozoa from the proximal corpus (zone 4a) were isolated as described and incubated for 1 h in BWW/PVA supplemented with dbcAMP (1 mM) and PTX (1 mM). Preincubated sperm were deposited under water-saturated mineral oil at 378C, and 10 oocytes were added. Following 30min incubation, the oocytes were removed, washed three times in BWW/PVA with a micropipette to remove loosely adherent spermatozoa, and fixed immediately with 4% paraformaldehyde. In addition, an aliquot of the freeswimming population of capacitated spermatozoa, as well as an aliquot of control spermatozoa that had not been treated with dbcAMP and PTX, was removed and fixed with 4% paraformaldehyde. Fixed cells were washed in PBS before being air-dried onto poly-L-lysine-coated slides. After permeabilization with 0.2% Triton X-100 for 10 min, the slides were rinsed and blocked with 10% goat serum in 3% BSA for 30 min before being stained with a 1:64 dilution of FITC-conjugated antiphosphotyrosine (clone PT66) at 378C for 1 h. After three washes, the cells were counterstained with tetramethyl rhodamine isothiocyanate-conjugated PNA (0.2 mg/ml) at 378C for 20 min, washed, and mounted in antifade medium. Slides were viewed using epifluorescence and confocal microscopy as already described.

Analysis of Sperm Motility Following the induction of capacitation with dbcAMP and PTX, spermatozoa were transferred to two prewarmed Makler slide chambers and were placed in the 378C sample holder of a Hamilton-Thorne sperm motility analyzer (IVOS version 10.1, Beverly, MA). The movement of approximately 300 spermatozoa from each zone of the epididymis and vas deferens was assessed using the following settings: 30 frames analyzed at 50 Hz; minimum contrast, 25; magnification 31.96; minimum cell size, 3 pixels; nonmotile head size, 5 pixels; straightness threshold, 75%; and low and medium average path

To investigate the existence of a cAMP-mediated tyrosine phosphorylation pathway in testicular germ cells, pachytene spermatocytes and round spermatids were purified and then stimulated with NADPH and dbcAMP/PTX, and the resultant Western blots were probed with an antiphosphotyrosine antibody. Both cell types failed to increase their level of tyrosine phosphorylation on addition of either agonist compared with the vehicle controls (Fig. 2). This was true whether the cells were incubated in the presence (Fig. 2A, lanes 1–6) or absence (Fig. 2A, lanes 7–12) of extracellular Ca2 þ. In similar fashion, immortalized type A spermatogonia (GC1 cells) were found to be incapable of generating a tyrosine phosphorylation response to NADPH or dbcAMP/PTX compared with the vehicle control (data not shown). Furthermore, the phosphorylation status of these cells was not significantly affected by the presence or absence of extracellular Ca2 þ (data not shown). To confirm these Western blot analyses, immunohistochemistry was performed on pachytene spermatocytes and round spermatids. Again, no significant increase in the fluorescence level was detected in this study (data not shown) irrespective of whether these cells were treated with NADPH or dbcAMP/ PTX. Therefore, these results indicate that precursor germ cells, including immortalized spermatogonia, spermatocytes, and round spermatids, cannot exhibit the cAMP-dependent tyrosine phosphorylation signal transduction pathway that is characteristic of the fully differentiated male gamete. cAMP-Dependent Pathway in Testicular and Epididymal Spermatozoa In contrast, the analyses shown in Figure 3 demonstrate that spermatozoa from the cauda epididymis could respond to dbcAMP/PTX stimulation with a dramatic increase in tyrosine phosphorylation in the presence (Fig. 3, lane 9) and absence (Fig. 3, lane 12) of extracellular Ca2þ. According to the Western blot analysis, NADPH did not elicit a similar increase in tyrosine phosphorylation in these cell populations, regardless of the Ca2þ status of the medium (Fig. 3, lanes 8 and 11). Immature cells from the caput epididymis were also unable to generate a detectable response to NADPH (Fig. 3, lanes 2 and 5) but could respond to cAMP/PTX, provided that Ca2þ was omitted from the medium (Fig. 3, lane 6). Findings from these studies suggest that, somewhere between the round spermatid stage of development and the entry of the spermatozoa into the caput epididymis, a capacity to exhibit the cAMP-driven


tyrosine phosphorylation cascade is acquired. Attempts to investigate the existence of this pathway in testicular spermatozoa were hampered by difficulties in obtaining purified populations of testicular spermatozoa for protein extraction. To overcome this problem, we adopted an immunocytochemical approach to resolve the ontogeny of this tyrosine phosphorylation pathway during the functional differentiation of spermatozoa. The results of these immunofluorescence analyses revealed a dramatic effect of the stage of sperm maturation on the ontogeny of cAMP-induced tyrosine phosphorylation. For this analysis, the three cellular domains that are competent to exhibit phosphotyrosine expression (principal piece, midpiece. and acrosome) are considered separately. Principal piece. An extremely small (1%–3%) proportion of testicular spermatozoa treated with cAMP/PTX exhibited strong tyrosine phosphorylation on the principal piece of the tail in the presence or absence of calcium (Fig. 4, A and B). This phosphorylation pattern stopped abruptly at the annulus, so that no phosphorylation was observed on the midpiece of these highly immature testicular cells (Fig. 5). In the caput region of the epididymis (zones 1–3), the spontaneous level of tyrosine phosphorylation in the principal piece was low (,10%) but increased dramatically on exposure to cAMP/PTX, with expression in approximately 50% of distal caput spermatozoa (zone 3) in the presence of extracellular calcium and in approximately 80% of cells in the absence of this cation (Fig. 4, A and B). After spermatozoa had passed through the corpus epididymis (beyond zone 4a), the spontaneous levels of principal piece phosphorylation increased progressively to reach maximal levels in the vas deferens, at which point 48.9% 6 11.2% and 61.1% 6 6.7% of spermatozoa were positive in the presence and absence of calcium, respectively (Figs. 4, A and B, and 5). The percentage of sperm responding to dbcAMP/PTX remained at approximately 50% through the corpus epididymis (zones 4a and 4b) and increased to more than 80% in the cauda and vas deferens in the presence of calcium (Fig. 5A). However, in the absence of calcium, the principal piece of more than 80% of the spermatozoa was competent to respond to cAMP from the distal caput onward (Fig. 4B). Therefore, the signal transduction pathway that mediates cAMP-dependent tyrosine phosphorylation in the principal piece was clearly established in a vast majority of spermatozoa by the time they had reached the caput epididymis (Figs. 4B and 5). However, the presence of extracellular calcium had obfuscated its existence in about one third of the spermatozoa [20]. The suppressive effect of extracellular calcium was no longer visible in zone 5 and beyond.

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FIG. 3. Response of caput and caudal epididymal sperm preparations to NADPH and dbcAMP/PTX as assessed by Western blot analysis. Caput spermatozoa did not respond to NADPH or to dbcAMP/PTX in the presence of calcium, although a significant response to dbcAMP/PTX was observed in the absence of the cation. Caudal cells exhibited a profound response to dbcAMP/PTX in the presence and absence of calcium.

Midpiece. The phosphorylation of the sperm midpiece followed the same general trend as that observed in the principal piece (Fig. 5 and Fig. 6, A and B). Therefore, spontaneous phosphorylation of the midpiece was virtually undetectable in testicular and caput epididymal spermatozoa but increased gradually during epididymal maturation to become maximally expressed in the vas deferens. At this point, 24.5% 6 2.5% of cells were positive in the presence of extracellular calcium and 38.5% 6 17.5% in the absence of this cation. When considered in conjunction with Figure 4, this means that, following incubation in BWW medium, approximately half of the mature caudal epididymal spermatozoa exhibiting tyrosine phosphorylated principal pieces also possessed a tyrosine-phosphorylated midpiece. Analysis of the cellular response to dbcAMP/PTX again revealed that this pathway was essentially established by the time spermatozoa had completed their passage through the caput epididymis, with 98.0% 6 1.9% of cells in zone 3 staining positively in the midpiece following exposure to cAMP/PTX in the absence of calcium (Fig. 6B). However, this competence was obscured in about two thirds of the cells from the distal caput and proximal corpus (zones 3 and 4a) if the extracellular medium contained

FIG. 4. Immunocytochemical analysis of tyrosine phosphorylation in the principal piece of the sperm tail at different stages of epididymal maturation in the presence or absence of stimulation with dbcAMP/PTX. A) In the presence of calcium. B) In the absence of calcium. T indicates testes; 1– 5b, epididymal zones; and VD, vas deferens. Two-way ANOVA indicated significant differences associated with treatment (P , 0.001) and region of sperm recovery (P , 0.001).

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FIG. 5. Phase-contrast (left column) and immunofluorescence (right column) micrographs of mouse spermatozoa showing dynamic changes in the subcellular locations of phosphotyrosine expression after dbcAMP/PTX stimulation. In testicular spermatozoa (A and B), weak phosphotyrosine expression is seen only on the principal piece of a small number of cells. In zones 1 and 2 of the proximal caput region, tyrosine phosphorylation is observed in the acrosome and principal piece, with little or no expression in the midpiece (C and D). In zones 3, 4a, and 4b, phosphotyrosine expression is still observed in the sperm head and principal piece, but at this point in epididymal maturation the spermatozoa are also capable of exhibiting a strong tyrosine phosphorylation response in the midpiece (E and F). Most spermatozoa recovered from the cauda epididymal zones 5a and 5b (G and H) and vas deferens (I and J) respond to cAMP with intense phosphorylation of the principal piece and midpiece of the sperm tail but exhibit complete dephosphorylation of the acrosome. Bar ¼ 5 lM.

added calcium (Fig. 6A). The percentage of cells within which this suppression was observed decreased with increasing maturity [20, 26, 27]. Acrosome. An intense phosphotyrosine signal was recorded in the acrosomal region of approximately 30% of testicular spermatozoa but increased to represent 70%–80% of spermatozoa recovered from zones 1–3 of the caput epididymis (Fig. 5 and Fig. 7, A and B). Acrosomal phosphotyrosine expression was also high (approximately 70%–80%) in the proximal corpus region of the epididymis but fell dramatically in the distal corpus to represent 20%–30% of the sperm population (Fig. 7, A and B). In the cauda epididymis and vas deferens, the proportion of spermatozoa exhibiting this pattern of tyrosine phosphorylation fell still further to represent 5%–10% of such cells (Fig. 5 and Fig. 7, A and B). This acrosomal pattern of FIG. 6. Immunocytochemical analysis of tyrosine phosphorylation in the midpiece of the sperm tail at different stages of epididymal maturation in the presence or absence of stimulation with dbcAMP/PTX. A) In the presence of calcium. B) In the absence of calcium. T indicates testes; 1–5b, epididymal zones; and VD, vas deferens. Two-way ANOVA indicated significant differences associated with treatment (P , 0.001) and region of sperm recovery (P , 0.001).

tyrosine phosphorylation was not significantly affected by exposure to dbcAMP/PTX or by the level of calcium in the extracellular medium (Fig. 7, A and B). This progressive decline in acrosomal phosphotyrosine expression during epididymal maturation was also observed in cells freshly recovered from the epididymis in the absence of any incubation in BWW or dbcAMP/PTX (data not shown). In summary, these immunocytochemical data indicate that the cAMP–dependent tyrosine phosphorylation pathway is predominantly expressed by male germ cells following their entry into the epididymis. The capacity of maturing spermatozoa to phosphorylate targets in the principal piece of the cell was acquired slightly in advance of the midpiece. By the time these cells had reached the distal caput (zone 3), most of the cells were able to tyrosine phosphorylate the entire flagellum


593 FIG. 7. Immunocytochemical analysis of tyrosine phosphorylation in the acrosomal region of the sperm head at different stages of epididymal maturation in the presence or absence of stimulation with dbcAMP/PTX. A) In the presence of calcium. B) In the absence of calcium. T indicates testes; 1– 5b, epididymal zones; and VD, vas deferens. Two-way ANOVA indicated significant difference associated with region of sperm recovery (P , 0.001) but not with dbcAMP/ PTX treatment.

from the neck to the tail-end piece (Fig. 6). However the inability of immature spermatozoa to control their intracellular calcium homeostasis meant that it was not until the cauda epididymis that a majority of spermatozoa could induce phosphorylation of the entire flagellum in culture medium containing 1.7 mM calcium. As the competence to phosphorylate the flagellum was being acquired in the epididymis, the ability to phosphorylate the acrosome was being lost via mechanisms that were entirely independent of calcium homeostasis and cAMP stimulation. To understand the physiological significance of these highly ordered, dynamic patterns of tyrosine phosphorylation, we next examined the relationships between these activities and the functional competence of spermatozoa recovered from different regions of the epididymis. Sperm Motility and Hyperactivation To investigate the relationship between activation of the cAMP-dependent tyrosine phosphorylation pathway and the development of sperm motility, we stimulated spermatozoa obtained from different regions of epididymis with dbcAMP/ PTX in the presence of physiological calcium levels and then used a Hamilton-Thorne CASA system to measure their

FIG. 8. CASA analysis of sperm movement for cells isolated from different regions of the male reproductive tract and treated with dbcAMP/ PTX. VSL indicates straight line velocity; VAP, average path velocity; VCL, curvilinear velocity; T, testes; 1–5b, epididymal zones; and VD, vas deferens. Two-way ANOVA indicated significant difference associated with region of sperm recovery (P , 0.001).

movement characteristics. Our data revealed that spermatozoa obtained from the testis and initial segment of the caput epididymis (zone 1) had no capacity to exhibit movement in the presence of cAMP (Fig. 8) in concert with their lack of tyrosine phosphorylation on the sperm tail (Figs. 4A and 6A). The onset of tyrosine phosphorylation in zones 2 and 3 of the caput epididymis was correlated with the initiation of flagellar activity (Figs. 5A and 7A). However, it is unlikely that the two events were causally linked, because full expression of coordinated movement was evident in spermatozoa recovered from zone 4a (proximal corpus) onward, while the competence to phosphorylate the entire flagellum was still developing. A much closer association was observed between tyrosine phosphorylation of the flagellum and hyperactivated movement (Fig. 9). Hyperactivation was only observed after spermatozoa had entered the corpus epididymis (zones 4a and 4b) and rose to reach a peak in cells recovered from the cauda epididymis and vas deferens when most cells treated with dbcAMP/PTX exhibited phosphorylation of the entire tail. The fact that percentage hyperactivation could be suppressed by 90% with tyrosine kinase inhibitors such as herbimycin in the absence of any change in overall sperm motility (Fig. 9) indicated that tyrosine phosphorylation of the sperm flagellum was critical for expression of this pattern of movement.

FIG. 9. Analysis of hyperactivated movement for spermatozoa removed from different regions of the male reproductive tract and treated with dbcAMP/PTX. The capacity for hyperactivated movement progressively increased once spermatozoa had reached the corpus epididymis (zone 4a). T indicates testes; 1–5b, epididymal zones; and VD, vas deferens. Two-way ANOVA indicated significant difference associated with region of sperm recovery (P , 0.001). Inset shows the suppressive effect of herbimycin (10 lM) on the expression of hyperactivated motility at different stages of sperm maturation.

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FIG. 10. Analysis of acrosome reaction rates for spermatozoa removed from different regions of the male reproductive tract and treated with dbcAMP/PTX with or without A23187. VD indicates vas deferens; 1–5b, epididymal zones. Two-way ANOVA indicated significant differences associated with region of sperm recovery (P , 0.001) and treatment (P , 0.001). *P , 0.05 and **P , 0.01 for differences compared with BWW control within a particular region of sperm recovery.

Tyrosine Dephosphorylation in the Acrosomal Region and the Acrosome Reaction To investigate the effect of phosphotyrosine expression of the sperm head (Fig. 7) on acrosomal exocytosis, spermatozoa at different stages of maturation were examined for their ability to acrosome-react in the presence and absence of an ionophoreinduced calcium influx. The results of this analysis revealed highly significant effects (P , 0.001) for treatment and region of sperm recovery by ANOVA. When tyrosine phosphorylation levels in the sperm head were high (testicular spermatozoa and sperm from zones 3–4a), spermatozoa did not exhibit a spontaneous acrosome reaction, nor could one be induced by raising the intracellular levels of cAMP (Fig. 10). Addition of A23187 also failed to induce acrosomal exocytosis in these immature cells except in zone 4a, where a low but significant (P , 0.05) increase in acrosome reaction rate was observed. In zone 4b, the spontaneous acrosome reaction rate slightly increased, but this response could not be augmented with cAMP or A23187. By contrast, spermatozoa from the cauda epididymis and vas deferens, which exhibited low levels of tyrosine phosphorylation in the acrosome (Fig. 7), exhibited high spontaneous levels of acrosomal exocytosis that responded to cAMP (P , 0.01) and A23187 (P , 0.05) stimulation in the cauda (zone 5a; Fig. 10). By the time spermatozoa had reached the vas deferens, the spontaneous level of acrosomal exocytosis was already so high that treatment with neither cAMP nor A23187 could further augment the response (Fig. 10). When these data were pooled and assessed by linear regression analysis, it was clear that a highly significant correlation existed (R2 ¼ 0.99; P , 0.001) between the percentage of cells competent to undergo the acrosome reaction and the percentage of cells exhibiting dephosphorylated acrosomes (Fig. 11). In light of these results, it was important to determine whether this close association between the ability of spermatozoa to acrosome-react and tyrosine dephosphorylation was causative (i.e., whether dephosphorylation of the acrosomal domain is a prerequisite for acrosomal exocytosis). To address this issue, we took spermatozoa from region 4a, at which point around 20% of the spermatozoa did not possess tyrosine phosphorylated acrosomes (Fig. 7) and an equivalent proportion of cells had gained the ability to acrosome-react on exposure to ionophore (Fig. 10). We sought to determine whether the spermatozoa that had gained the ability to acrosome-react in region 4a were the same spermatozoa that

FIG. 11. Correlation between acrosome reaction rates and the percentage of cells with dephosphorylated acrosomes for spermatozoa removed from different regions of the male reproductive tract and treated with dbcAMP/PTX with or without A23187. R2 ¼ 0.99.

had dephosphorylated their acrosomes. For this objective, we examined the phosphorylation status of region 4a spermatozoa that had acrosome-reacted on the zona surface using a doublestaining technique that allowed us to simultaneously assess the tyrosine phosphorylation status of these cells and their ability to acrosome-react (Fig. 12). In the absence of stimulation (Fig. 12, open bars), a majority of these free-swimming spermatozoa (.80%) had intact acrosomes and exhibited tyrosine phosphorylation over the sperm head. Even when capacitation was stimulated with dbcAMP/PTX, most of the free-swimming spermatozoa (approximately 65%) were acrosome intact and tyrosine phosphorylated (Fig. 12, hatched bars). However, under these circumstances, approximately 20% of the spermatozoa had acrosome-reacted, and about two thirds of these cells still exhibited phosphotyrosine residues over the sperm head. When the subpopulation of these capacitated spermatozoa that had bound to the zona pellucida were examined, approximately 50% had acrosome-reacted, and more than half of these cells still exhibited tyrosine phosphorylation in the acrosomal domain (Fig. 12, arrow). We conclude from these studies that tyrosine dephosphorylation is not an essential prerequisite for the acrosome reaction to occur. DISCUSSION This study demonstrates for the first time (to our knowledge) that the unusual cAMP-dependent tyrosine phosphorylation-signaling cascade found in murine spermatozoa is acquired during the final stages of male germ cell differentiation. In particular, it is only when male germ cells have differentiated into spermatozoa and entered the epididymis that they are able to respond to cAMP with an increase in the tyrosine phosphorylation of proteins on the principal piece of the flagellum (Fig. 4B). Further maturation of the spermatozoa permits the induction of a cAMP-dependent tyrosine phosphorylation response in the midpiece area, via mechanisms that are essentially established by the time the spermatozoa have reached the distal caput (Fig. 6B). As a consequence of these maturation events, most of the spermatozoa entering the corpus epididymis (zone 4a) have the potential to exhibit a cAMP tyrosine phosphorylation response that runs the entire length of the tail from the neck to the tail-end piece. However, in the presence of 1.7 mM extracellular calcium, only approximately 30% of these cells can express this potential. The size of the


sperm population that can exhibit this pattern of tyrosine phosphorylation is biologically important because it correlates with the ability of spermatozoa to exhibit sperm-zona binding [28, 29] and hyperactivation [30]. When spermatozoa are not pharmacologically driven with PTX and dbcAMP, this state is achieved by about 25% of spermatozoa recovered from the vas deferens in calcium-containing medium and by about 40% of cells in the absence of this cation (Fig. 6). Our observation that Ca2þ is a powerful negative regulator of the tyrosine phosphorylation-signaling pathway in immature cells from the caput and corpus epididymis is consistent with studies on human, porcine, bovine, and murine spermatozoa [21, 28, 31–34]. These studies demonstrated that omission of calcium from the incubation medium results in an increase in the tyrosine phosphorylation patterns associated with sperm capacitation. It had been envisaged that calcium might have a direct action on the kinase/phosphatase system regulating tyrosine phosphorylation [31] or might act indirectly via regulation of calcium-dependent channels in the sperm plasma membrane [20, 32]. However, both of these explanations appear to be incorrect. When caput epididymal spermatozoa are placed in medium containing millimolar amounts of free ionic calcium, their internal calcium levels rise dramatically, presumably because the mechanisms that regulate the intracellular concentration of this cation have not yet matured [27]. Such high calcium levels place substantial strain on the availability of ATP, which is consumed by the futile activation of energy-dependent mechanisms to remove this cation from the cytoplasm [21]. Phosphotyrosine expression is subsequently suppressed because intracellular ATP levels fall below the Km value required to drive the cAMP-dependent tyrosine kinase [21]. This explanation applies particularly to spermatozoa from the corpus epididymis, which are capable of phosphorylating the entire flagellum in response to cAMP (Figs. 4 and 6) but are prevented from doing so by the presence of extracellular calcium. By the time spermatozoa have arrived in the cauda epididymis, calcium no longer has such a significant inhibitory effect on the response to cAMP, presumably because by this time the mechanisms responsible for internal calcium homeostasis have been fully established. This is why intracellular calcium levels are significantly lower in caudal compared with caput spermatozoa when extracellular calcium is in millimolar range [21]. The nature of the mechanisms that establish intracellular calcium homeostasis in caudal epididymal spermatozoa has not yet been elucidated. Calcium adenosine triphosphatases, sodium-calcium exchangers, and mitochondrial uniporters have been proposed (and occasionally refuted) in various studies [35–37]. The ability of cAMP to stimulate flagellar tyrosine phosphorylation in calcium-containing medium increased progressively as spermatozoa moved beyond the distal caput (Figs. 4 and 6). This increase was associated with the stimulation of progressive motility and the induction of sperm hyperactivation. Clearly, tyrosine phosphorylation is not the only factor involved in the stimulation of these various forms of movement [37]. Nevertheless, if the cAMP-dependent tyrosine phosphorylation pathway is suppressed, hyperactivated movement cannot occur in human spermatozoa [38] or in murine spermatozoa, as demonstrated in the present study. Identification of the kinases and target proteins involved in this response will be instructive, and, in this context, the recent demonstration that hamster sperm capacitation is associated with the phosphorylation of a phospholipid hydroperoxide glutathione peroxidase is notable [39]. This protein is a major constituent of the mitochondrial capsule, and it is tempting to suggest that the phosphorylation of such targets is associated

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FIG. 12. Analysis of the ability of spermatozoa from the proximal corpus (zone 4a) to undergo the acrosome reaction using a double-staining technique. A) Staining with PNA alone to illustrate position of the acrosome as visualized using rhodamine filter. B) Same image examined through an FITC filter set showing staining pattern obtained with antiphosphotyrosine antibody. Note that one of the spermatozoa has become dephosphorylated (arrow). C) Spermatozoa primed with dbcAMP (1 mM) and PTX (1 mM) bound to the zona pellucida; approximately half of these bound cells underwent the acrosome reaction. Black arrow indicates acrosome-reacted spermatozoa recovered from the zona surface that still express phosphotyrosine residues over the sperm head. Control ¼ free-swimming spermatozoa not treated with dbcAMP/PTX; pY ¼ cells exhibiting tyrosine phosphorylation on the sperm head.

with an increase in mitochondrial function that, in turn, contributes to the induction of coordinated flagellar activity and the onset of hyperactivation. If tyrosine phosphorylation of mitochondrial proteins is essential for the expression of normal sperm function, it is probably not just a simple consequence of enhanced energy production by these organelles. The complete lack of coordinated sperm motility in mice experiencing targeted disruption of a key enzyme in glycolysis (glyceraldehyde 3phosphate dehydrogenase-S), despite unchanged levels of mitochondrial oxygen consumption [40], tells us that sperm movement is not dependent on oxidative phosphorylation. Most of the energy requirements for sperm motility are evidently met by glycolysis, the enzymes for which are tightly associated with the fibrous sheath and not confined to the sperm midpiece. Similarly, the finding that testis-specific cytochrome c-null mice produce functional spermatozoa [41] suggests that mitochondrial energy production is not essential for the support of sperm motility or the phosphorylation events that underpin this activity. Rather, the role of sperm mitochondria in regulating the biological activity of these cells must depend on other potential functions performed by these organelles such as the regulation of cytoplasmic calcium or the maintenance of axonemal stability. The factors responsible for activating this cAMP-dependent tyrosine phosphorylation cascade in the sperm midpiece are poorly understood. The only clue that we have at present is that the induction of tyrosine phosphorylation in this specific location is dependent on the metabolism of glucose through the

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hexose monophosphate shunt (HMS) [42, 43]. The enzyme responsible for regulating HMS activity, glucose-6-phosphate dehydrogenase, is present in the midpiece of murine spermatozoa. Removal of glucose from the incubation medium delays the onset of tyrosine phosphorylation in this region of the cell, while the presence of this sugar stimulates the HMS to generate NADPH. The latter is, in turn, held to stimulate redox activity in mammalian spermatozoa via a poorly characterized NADPH oxidoreductase activity that promotes tyrosine phosphorylation through a combination of increased intracellular cAMP availability and decreased tyrosine phosphatase activity [22]. The case for ROS involvement in cAMP generation and tyrosine phosphorylation has now been made for human [7, 22], rat [14], mouse [6, 44], bovine [17], and equine [18] spermatozoa. If the control of ROS generation in the sperm midpiece is involved in activating this tyrosine phosphorylation cascade, it raises important questions concerning the mechanisms by which the epididymis affects HMS activity and the redox status of NADP þ/NADPH in maturing spermatozoa. Although the tyrosine phosphorylation associated with the sperm tail is functionally important in the context of hyperactivated movement, tyrosine phosphorylation of the sperm head did not appear to be causally associated with the ability of epididymal spermatozoa to acrosome-react. The loss of phosphotyrosine residues from the sperm head was highly correlated with the ability of these cells to acrosome-react because these processes are independent measures of sperm maturation. The purpose of the acrosomal pattern of tyrosine phosphorylation is unknown, to date, and cannot be addressed until we understand the mechanisms by which tyrosine phosphorylation in the sperm head is controlled. All we know at present is that these control mechanisms involve neither cAMP nor calcium. In conclusion, the results of this study show for the first time that the signaling pathway leading to cAMP-mediated tyrosine phosphorylation in the flagellum of murine spermatozoa is only present when germ cells differentiate into spermatozoa. The potential to express this pathway is established in the caput epididymis but is suppressed by the presence of extracellular calcium. By the time spermatozoa have reached the cauda epididymis, this suppressive effect of calcium is no longer observed. The ability of spermatozoa to express this cAMPdependent phosphorylation pattern in the presence of extracellular calcium is positively correlated with their capacity to exhibit hyperactivated movement. Conversely, phosphotyrosine expression in the acrosome is not modulated by calcium or cAMP and does not appear to be causally involved in the regulation of acrosomal exocytosis. Further elucidation of the mechanisms by which these independent tyrosine phosphorylation pathways are differentially established and activated in distinct subcellular compartments will shed light on the maturation process and may have implications for future developments in contraception and in infertility. ACKNOWLEDGMENTS We thank Belinda M. Attard, Daniel G. Blackmore, and Donna Buckingham for their assistance with various aspects of this research project.

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