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A flow cytometric assay for global estimation of tyrosine phosphorylation associated with capacitation of spermatozoa from two marsupial species, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula)

¹ Cooperative Research Center for Conservation and Management of Marsupials and ² Fluorescence Research Group, Department of Biological Sciences, Macquarie University, NSW 2109, Australia and ³ Discipline of Biological Sciences, Center for Life Sciences, University of Newcastle, Australia

Correspondence should be addressed to K S Sidhu who is now at Human Stem Cell Group, Diabetes Transplant Unit, Prince of Wales Hospital and University of New South Wales, Randwick NSW 2031 Australia; Email: k.sidhu{at}unsw.edu.au

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The phosphorylation of tyrosine residues in cellular proteins is a major signal transduction event during sperm capacitation. In this study protein phosphorylation was monitored using a fluorescein isothiocyanate (FITC)-labeled antiphosphotyrosine monoclonal antibody and a flow cytometric procedure optimized for sperm. Using this technique, the correlation between tyrosine phosphorylation and sperm capacitation was examined in two marsupial species, the brushtail possum and the tammar wallaby and compared with that of ram spermatozoa. The levels of tyrosine phosphorylation in sperm from all three species were increased by the addition of cyclic AMP (cAMP) and vandate, a phosphotyrosine phosphatase inhibitor and were decreased by the addition of the phosphotyrosine kinase inhibitor, staurosporine. Oviductal conditioned media (CM) induced a progressive increase in tyrosine phosphorylation in both marsupial species and also induced morphological transition from a streamlined to a ‘T’-shape configuration in brushtail possum spermatozoa but not in tammar wallaby spermatozoa. Transition to the ‘T’-shape orientation associated with capacitation in marsupial spermatozoa was observed by 2 h of incubation in both species when tyrosine phosphorylation was increased by higher levels of cAMP i.e. 5 mM dibutyryl cAMP plus 3 mM pentoxyphylline. Thus the tyrosine phosphorylation trigger with CM may differ in these two marsupial species. Ram sperm tyrosine phosphorylation could be increased by addition of lower levels of cAMP (1 mM). These results support the finding that tyrosine phosphorylation is associated with sperm capacitation in marsupials. Similar results were obtained by using SDS PAGE/Western blot analysis of tyrosine phosphorylation in the brushtail possum spermatozoa. The specificity, efficiency and sensitivity of the procedure described here make it applicable for routine assessment of capacitation in large numbers of samples and in other species.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Mammalian spermatozoa acquire fertilization ability during their sojourn in the female reproductive tract by undergoing complex but not yet fully understood biochemical, physiological and molecular alterations that are collectively known as capacitation. In many domestic mammalian species including humans this phenomenon can be reproduced in vitro in defined media (for reviews see Sidhu & Guraya 1989, Sidhu, 1991, Yanagimachi, 1994). In Australian marsupials, however, in vitro capacitation cannot be achieved in defined media unless sperm are cocultured with oviductal cells and/or their secretions (Sidhu et al. 1998, 1999a,b; Mate et al. 2000).

Understanding the mechanism(s) of sperm capacitation has been the subject of many recent investigations. One of the major problems confronting gamete biologists is the lack of any definitive assay for sperm capacitation apart from in vitro fertilization. Hyperactive sperm motility, onset of the acrosome reaction (Yanagimachi, 1994) and changes in chlortetracycline (CTC) staining patterns have been used to monitor capacitation in some species (Maxwell & Johnson 1997, Dinkins & Brackett 2000, Parker et al. 2000, Rathi et al. 2001) but are not useful assays for monitoring the process in spermatozoa from Australian marsupials. There are no overt morphological changes associated with capacitation in eutherian spermatozoa, however there is a distinct reorientation of the sperm head (from streamlined to ‘T’-shape) during capacitation in marsupials (Bedford & Breed 1994, Molinia et al. 1998, Jungnickel et al. 1999, Sidhu et al. 1998, 1999a,b). Although the transition of marsupial sperm to the ‘T’-shape orientation is a useful indicator it is not definitive, as not all ‘T’-shape spermatozoa are capable of fertilization (Bedford & Breed 1994, Mate et al. 2000, Sidhu et al. 2003).

Several lines of evidence indicate that the control of mammalian sperm function at fertilization is through protein tyrosine phosphorylation (Tesarik et al. 1993, Visconti et al. 1995, Aitken et al. 1998, Flesch et al. 1999, Jha & Shivaji 2002). Capacitation in particular appears to be associated with tyrosine phosphorylation of sperm proteins by a cAMP-dependent protein kinase (PKA) in several eutherian species (e.g. Visconti et al. 1995, 1999, Naz, 1999, Pavlok et al. 2001, Tardif et al. 2001). In human spermatozoa at least two second messenger systems utilize amplifier enzymes located on the inner surface of the plasma membrane (de Jonge et al. 1991a,b; Doherty et al. 1995). The first enzyme, adenylate cyclase, allows cAMP production and activation of cAMP-dependent protein kinase (PKA). The second, phospholipase C, converts polyphosphoinositide phosphatidylinositol 4,5-biphosphate into inositol 1,4,5-triphosphate and 1,2-diacylyglycerol. The latter activates a phospholipid Ca²⁺-dependent protein kinase (PKC). The activation of PKC has already been reported during marsupial sperm acrosome reaction, mediated by arachidonic acid derived from phospholipase A2 action on phosphatidylcholine (Sistina & Rodger 1997). The levels of cAMP within spermatozoa result from a balance between reactions catalyzed by adenylate cyclase and phosphodiesterase. Similarly, the phosphorylation on tyrosine residues of proteins is the result of phosphotyrosine kinase (PTK) enzyme and its counteraction by phosphotryosine phosphatase (PTPase) enzyme. Formation of both cAMP and phosphorylation at tyrosine residues of protein can be up-regulated or down-regulated by using stimulators or inhibitors in permeabilized. We have analyzed these pathways in marsupial sperm by using specific inhibitors or stimulators of these enzymes to investigate if these can modify the equilibrium.

The measurement of protein tyrosine phosphorylation in spermatozoa under conditions that permit capacitation thus gives a quantitative estimation of this event. In general, there are two methods used for estimating tyrosine phosphorylation of spermatozoa: (i) incorporation of radioactive phosphate on tyrosine residues, followed by precipitation with anti-phosphotyrosine antibodies or phosphoamino-acid determination (Naz, 1999) and (ii) Western blot using anti-phosphotyrosine monoclonal antibodies (Kalab et al. 1998). These methods are time consuming, cannot be applied to large numbers of samples and do not provide information on sub-populations within a sample. In this study, we have optimized the use of anti-phosphotyrosine antibodies for monitoring tyrosine phosphorylation in spermatozoa by flow cytometry. The global levels of sperm tyrosine phosphorylation under different in vitro conditions that induce capacitation were detectable in permeabilized cells using fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies against tyrosine phosphoproteins. The cell-associated fluorescence could be quantitated on a flow cytometer. With this technique we could estimate the effects of oviduct conditioned media and other intracellular modulators i.e. cAMP and phosphodiesterase inhibitors on Australian marsupial sperm tyrosine phosphorylation vis-à-vis capacitation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Animals
Adult brushtail possums were from the Landcare Research Animal Facility at Lincoln, New Zealand. Possums were housed individually in cages as described previously (Sidhu et al. 1999a) and fed a diet of cereal pellets supplemented with fruit and vegetables. Tammar wallabies were from the Macquarie University Fauna Park, Sydney, Australia. Wallabies were housed in outside yards and fed a diet of cereal pellets. All animals had access to water available ad libitum. This research was approved by the Animal Ethics Committees at Macquarie University and Landcare Research.

Collection of sperm
Adult male possums were killed at Landcare Research by using a mask with CO₂:O₂ (21:11/min) followed by an intracardial injection of sodium barbiturate (125 mg/kg). Testes and epididymides were removed and sperm were recovered by back-flushing the vas deferens with 0.5–1.0 ml EMEM. Sperm samples from several males were pooled and pellet-frozen according to the method of Molinia & Rodger (1996) and were transported to Macquarie University for use after thawing at 37 °C. Sperm samples from tammar wallabies were collected by electro-ejaculation according to the method of Rodger & White (1978). Sperm samples from rams (frozen and fresh) were a gift from Dr Chis Maxwell’s Laboratory at the University of Sydney, Australia.

In vitro capacitation of sperm using oviduct cultures and a co-culture system
Oviduct explants cultures and conditioned media (CM) from oviduct explants were prepared from pregnant mare’s serum gonadotropin/luteinizing hormone-primed females as described previously (Sidhu et al. 1999a). Briefly, 3–4 mm² pieces of oviduct tissue were prepared from oviducts that were slit open longitudinally keeping the epithelium, stroma and musculature associations intact so as to simulate in vivo-like conditions. These explants were washed twice with PBS and cultured in EMEM, pH 7.4 containing polyvinyl alcohol (PVA) (1 mg/ml), 100 IU penicillin/ml, 100 µg streptomycin/ml and growth factors (10 ng epidermal growth factor/ml, 5 µg insulin/ml, 5 ng transferrin/ml, 50 IU selenium/ml) (Sigma Chemical Co., St Louis, MO, USA). Oviduct explants (5 mg tissue per 250 µl medium) were cultured in 35-mm petri dishes at 36 °C under 5% CO₂ in humidified air. After 4 h of culture, the medium was replaced and the explants were cultured for another 20 h. Explant-conditioned medium (CM) was collected after centrifugation at 4000 g for 15 min at 4 °C and supernatants (CM) were preserved at - 20 °C.

CM was used to induce sperm capacitation as described previously (Sidhu et al. 1999a). Highly motile sperm samples were obtained after swim up in EMEM medium plus PVA from 4–5 frozen–thawed epididymal sperm pellets in the case of possums, and from ejaculated wallaby semen. Spermatozoa were incubated in CM (diluted 1:1) at a final concentration of 2 x 10⁶/ml at 36°C in 5% CO₂ and humidified air in 1.5 ml plastic tubes. Ram spermatozoa were incubated under in vitro conditions that induce capacitation as described by Gillan et al. (1997).

The percentage of motile sperm, progressive motility and percentage ‘T’-shape (sperm head–tail orientation) were assessed subjectively using a phase contrast inverted microscope at 0, 2 and 4 h. The onset of ‘T’-shape orientation in sperm is an indicator of sperm capacitation in marsupials (Sidhu et al. 1998, 1999b). Sperm viability was also assessed using a Fertilight Sperm Viability Kit according to the instructions of the supplier (Molecular Probes Inc., Eugene, OR, USA) and as described previously (Sidhu et al. 1999a).

Flow cytometric analysis
Sperm aliquots (40 x 10⁶) were taken at the times indicated above and fixed in ice-cold 1% formaldehyde in PBS, pH 7.2 for 30 min at 4 °C. After centrifugation (1000 x g, 3 min), spermatozoa were permeabilized in 0.05% saponin (Sigma Chemical Co.) for 10 min at room temperature. Permeabilization of spermatozoa is a critical step in staining, as the kinase enzyme is most likely localized on the inside of sperm plasma membrane. Permeabilization had no apparent effect on sperm morphology detectable by phase contrast microscopy. Non-specific binding sites were blocked by incubating in 0.1% BSA and 0.1% Tween 20 in PBS for 30 min at room temperature. Tyrosine phosphoproteins were localized using a monoclonal antibody (mAb) (10 µg/ml) against human tyrosine phosphoprotein conjugated with FITC (clone PT-66-FITC, Sigma Chemical Co.) in PBS containing 0.1% BSA for 30 min at room temperature. A non-specific FITC-conjugated mAb, MOPC-21 (Sigma Chemical Co.) was used as a control for non-specific binding. After two washes in PBS, labeled sperm were resuspended in PBS containing 0.1% BSA for flow cytometric analysis and examination by fluorescence microscopy.

Stained samples were analyzed using a BD FACS-Calibur fitted with a fluorescence-activated cell sorting module (Becton Dickinson, Sydney, Australia) equipped with a 15 mW argon laser emitting light at 488 nm. Sheath fluid was Osmosol (LabAids Pty. Ltd, Sydney, Australia). The cytometer was equipped with forward-angle light scatter (FSC; <15°), side-angle light scatter (SSC; >15°), and three fluorescence detectors: FL1 515–565 nm, FL2 565–605 nm, and FL3 >605 nm. The green fluorescence due to FITC was detected in FL1. Logarithmic amplification was used throughout, and fluorescence acquisition was gated by light scatter parameters. The detection threshold was adjusted in FL1 to eliminate particles emitting green fluorescence significantly below that of spermatozoa. Compensation was set so as to remove FL2 fluorescence from the FL1 channel and FL1 from the FL2 channel. The typical flow rate was 1000 cells/s for immunofluorescence measurement. At least 10 000 cells were analyzed for each sample and data were stored in a histogram mode on a log scale.

Activation/inhibition of tyrosine phosphorylation in sperm
In order to determine the pathways of tyrosine phosphorylation activation in sperm by CM, dibutyryl cAMP (dbcAMP – a cAMP analog) and inhibitors of phosphodiesterase (3-isobutyl-1-methylxanthine (IBMX), caffeine, pentoxyphylline) and the tyrosine phosphorylation pathway (staurosporine, phosphotyrosine) were used prior to flow cytometric analysis. Media containing varying concentrations of dbcAMP, IBMX, caffeine, and pentoxyphylline alone or in combination were made up as 100 x concentrated stocks. Concentrations of dbcAMP (1 mM), caffeine (2 mM) or IBMX (0.1 mM) that were effective in ram spermatozoa had no effect on tyrosine phosphorylation of marsupial spermatozoa (data not shown). These preliminary experiments demonstrated that marsupial sperm require higher levels of cAMP for activation of the tyrosine phosphorylation pathway than eutherian sperm. In subsequent experiments, 5 mM dbcAMP + 3 mM pentoxyphylline were used to optimally activate tyrosine phosphorylation in marsupial spermatozoa.

SDS PAGE and Western blotting
In order to validate this novel flow cytometric assay for sperm tyrosine phosphorylation, SDS PAGE/Western blotting assay was also run for comparison using only the brushtail possum spermatozoa with similar treatments.

The spermatozoa were incubated in a total volume of 150 µl in Eppendorf tubes at a final concentration of 2.4 million sperm/ml in different sets of four tubes each. Each treatment group comprised 5 mM dbcAMP + 3 mM pentoxyphylline at 0 h and 2 h and in the presence of the phosphotyrosine kinase inhibitors staurosporine (400 nM) and genistein (400 nM) respectively. At each time point, spermatozoa were washed through 500 µl of 1 mM orthovandate at 8000 x g for 7 min and pellets were extracted by boiling for 7 min in 50 µl 2% w/v SDS, 0.375 M Tris, pH 6.8, 3% w/v mercaptoethanol, 1 mM orthovandate plus a cocktail of protease inhibitors (Sigma Chemical Co.). The supernatants were then stored at -20°C until required. In order to ensure that equal amounts of protein were loaded into the gels, protein estimations were performed on each sample using a bicinchoninic acid (BCA) kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. A minimum of 1 mg total protein was loaded per lane. In order to confirm equal loading of protein, blots that had been probed for phosphotyrosine proteins were stripped and reprobed with an antibody against -tubulin (Aitken et al. 1998). For this procedure, approximately 30 ml stripping buffer, consisting of 2% (w/v) SDS, 62.5 mM Tris pH 6.7 and 100 mM 2-mercaptoethanol, were added to the membrane for 1 h with constant shaking at 60°C. The membrane was then washed for 3 x 10 min in Tris-buffered saline (TBS; 0.02 M Tris pH 7.6, 0.15 M NaCl), blocked and probed with the primary antibody.

SDS-PAGE was conducted on 1 µg solubilized sperm proteins using 7.5 or 10% polyacrylamide gels at 20 mA constant current per gel according to the method of Aitken et al. (1998). The proteins were then transferred onto a 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 TBS containing 3% (w/v) BSA. The membrane was then incubated for 2 h at room temperature in a 1:4000 dilution of a monoclonal anti-phosphotyrosine (clone 4G10) or anti--tubulin (clone B-5-1-2) 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 then incubated for 1 h at room temperature with goat anti-mouse immunoglobulin G 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 described above and then the phosphorylated proteins were detected using an enhanced chemiluminescence (ECL) kit (Amersham International) according to the manufacturer’s instructions.

Statistical analysis
The log scale data from the flow cytometric analyses were transformed to linear scale as per instructions on a BD FACS-Calibur machine and the mean linear values of the treatment groups were compared using the Student’s t-test (SigmaStat for Windows, Jandel Scientific, San Rafael, CA, USA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Specificity of the assay
Increasing the levels of cAMP in marsupial sperm using dbcAMP (5 mM) and the phosphodiesterase inhibitor, pentoxyphylline (3 mM), significantly (P < 0.01) increased phosphorylation at tyrosine residues of sperm proteins in both the tammar wallaby and the brushtail possum as shown by this assay (Figs 1, 2). Staurosporine (400 nM), a PTK inhibitor added to permeabilized sperm prior to incubation with antibody, significantly (P < 0.01) decreased the phosphorylation at tyrosine residues of sperm proteins in both the tammar wallaby and the brushtail possum (Figs 1, 2). Addition of 10 mM phosphotyrosine significantly (P < 0.01) decreased the level of cell-associated fluorescence. In both the marsupial species, relatively high levels of cAMP (i.e. 5 mM dbcAMP plus 3 mM pentoxyphylline) were required for induction of tyrosine phosphorylation. In ram spermatozoa, however, tyrosine phosphorylation occurred in the presence of lower concentrations of cAMP (1 mM) and caffeine (2 mM) (Fig. 3).

View larger version (30K): [in this window] [in a new window]   Figure 1 A flow cytometric analysis of the effects of 5 mM cAMP (A) + 3 mM pentoxyphylline (P), the phosphotyrosine kinase inhibitor (PKI) staurosporine (400 nM), and phosphotyrosine (PT) (10 mM) on phosphotyrosine levels in the tammar wallaby spermatozoa at different incubation times (0, 2, 4 h). Spermatozoa were incubated in the presence of cAMP and pentoxyphylline and aliquots taken for flow cytometric analyses as described under Materials and Methods. Staurosporine was added at the start of incubation and phosphotyrosine was added during the blocking step. The original relative fluorescence intensities on the X-axis are shown on a log scale. The sperm counts are on the Y-axis. The mean linear values for fluorescent intensities on an arbitrary scale (0–1200) are represented at the top of each peak. The experiments were repeated four times on different samples with similar results. The mean linear values with different superscripts differ significantly (P < 0.01).

View larger version (34K): [in this window] [in a new window]   Figure 2 A flow cytometric analysis of the effects of 5 mM cAMP (A) + 3 mM pentoxyphylline (P), phosphotyrosine kinase inhibitor (PKI) staurosporine (400 nM), and phosphotyrosine (PT) (10 mM) on phosphotyrosine levels in the brushtail possum spermatozoa at different incubation times (0, 2 h). Spermatozoa were incubated in the presence of cAMP and pentoxyphylline and aliquots were taken for flow cytometric analyses as described under Materials and Methods. Staurosporine was added at the start of incubation and phosphotyrosine was added during the blocking step. The original relative fluorescence intensities on the X-axis are shown on a log scale. The sperm counts are on the Y-axis. The mean linear values for fluorescent intensities on an arbitrary scale (0–1200) are represented at the top of each peak. The experiments were repeated four times on different samples with similar results. The mean linear values with different superscripts differ significantly (P < 0.01).

View larger version (38K): [in this window] [in a new window]   Figure 3 A flow cytometric analysis of the effects of 1 mM cAMP (A) plus 2 mM caffeine (C), the phosphotyrosine kinase inhibitor (PKI) staurosporine (400 nM), and phosphotyrosine (PT) (10 mM) on phosphotyrosine levels in the ram spermatozoa at different incubation times (0, 2, 4 h). Spermatozoa were incubated in the presence of A + C and aliquots were taken for flow cytometric analyses as described under Materials and Methods. The pathway for phosphorylation was assessed by using an inhibitor of phosphotyrosine kinase enzyme, staurosporine (400 nM), at the start of incubation and phosphotyrosine was added during the blocking step to saturate the system. The original relative fluorescence intensities on the X-axis are shown on a log scale. The sperm counts are on the Y-axis. The mean linear values for fluorescent intensities on an arbitrary scale (0–1200) are represented at the top of each peak. The experiments were repeated four times on different samples with similar results. The mean linear values with different superscripts differ significantly (P < 0.01).

Sperm capacitation and tyrosine phosphorylation
Effects of CM
Incubating marsupial spermatozoa (tammar wallaby and brushtail possum) in oviduct CM (1:1) maintained higher sperm percentage motility, progressive motility and percentage viability than in controls, and in the possum, a significantly higher percentage of motile spermatozoa were transformed to a ‘T’-shape orientation by 2-h incubation as reported earlier (Sidhu et al. 1998, 1999a). Aliquots of sperm taken from CM showed an increase in phosphorylation at tyrosine residues of sperm proteins at 2 h in both the tammar wallaby and the brushtail possum (Figs 4, 5). In the brushtail possum, the increase in tyrosine phosphorylation caused by oviduct CM was correlated with the onset of ‘T’-shape orientation in sperm at 2 h of incubation. In the tammar wallaby sperm the increase in tyrosine phosphorylation was not correlated with transformation of sperm to ‘T’-shape orientation. Preincubation of brushtail possum spermatozoa with staurosporine or phosphotyrosine (inhibitors of phosphotyrosine kinase) prior to exposure to CM maintained tyrosine phosphorylation at basal levels (Figs 4, 5) and prevented transformation to the ‘T’-shape orientation.

View larger version (31K): [in this window] [in a new window]   Figure 4 A flow cytometric analysis of the effects of oviduct CM on phosphotyrosine levels in the tammar wallaby spermatozoa at different incubation times (0, 2 h). Spermatozoa were incubated in the presence of CM (1:1) and aliquots taken for flow cytometric analyses as described under Materials and Methods. The pathway of CM-induced phosphorylation was assessed by using an inhibitor of phosphotyrosine kinase enzyme, staurosporine (400 nM), at the start of incubation and phosphotyrosine was added during the blocking step to saturate the system. The original relative fluorescence intensities on the X-axis are shown on a log scale. The sperm counts are on the Y-axis. The mean linear values for fluorescent intensities on an arbitrary scale (0–1200) are represented at the top of each peak. The experiments were repeated four times on different samples with similar results. The mean linear values with different superscripts differ significantly (P < 0.01).

View larger version (33K): [in this window] [in a new window]   Figure 5 A flow cytometric analysis of the effects of oviduct CM on phosphotyrosine levels in the brushtail possum spermatozoa at different incubation times (0, 2 h). Spermatozoa were incubated in the presence of CM (1:1) and aliquots taken for flow cytometric analyses as described under Materials and Methods. The pathway of CM-induced phosphorylation was assessed by using an inhibitor of phosphotyrosine kinase enzyme, staurosporine (400 nM), at the start of incubation and phosphotyrosine (PT) was added during the blocking step to saturate the system. The original relative fluorescence intensities on the X-axis are shown on a log scale. The sperm counts are on the Y-axis. The mean linear values for fluorescent intensities on an arbitrary scale (0–1200) are represented at the top of each peak. The experiments were repeated four times on different samples with similar results. The mean linear values with different superscripts differ significantly (P < 0.01).

A comparison of the flow cytometric assay with the SDS PAGE/Western blot assay for estimating tyrosine phosphorylation using the brushtail possum sperm
In order to validate the flow cytometric assay for sperm tyrosine phosphorylation, an SDS PAGE/Western blot assay was also run for comparison using the brushtail possum sperm. Cyclic AMP/pentoxyphylline stimulated tyrosine phosphorylation at 2 h of incubation and the same was inhibited by using two phosphorylation inhibitors, staurosporine and genistein (Fig. 6). Staurosporine appeared to be more potent than genistein in inhibiting phosphorylation, as was also observed in the flow cytometric assay.

View larger version (17K): [in this window] [in a new window]   Figure 6 Western blot analysis of tyrosine phosphorylation in the brushtail possum spermatozoa. Effects of cAMP (5 mM) + pentoxyphylline (3 mM) at 0 h (lane 1), 2 h (lane 2), plus staurosporine (400 nM) (lane 3), and plus genistein (400 nM) (lane 4).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

In the brushtail possum there was a correlation between the onset of ‘T’-shape orientation (capacitation) and tyrosine phosphorylation during incubation in oviductal CM. Our data suggest that this CM-induced transformation of possum spermatozoa to the ‘T’-shape orientation may be directly related to tyrosine phosphorylation, as the addition of the phosphotyrosine kinase inhibitor, staurosporine, prevented both the increase in tyrosine phosphorylation and ‘T’-shape morphology. Although the onset of ‘T’-shape orientation was not observed in the tammar wallaby during incubation in oviductal CM, there was an increase in tyrosine phosphorylation with cAMP and time. Interestingly, the addition of stimulators of phosphotyrosine phosphorylation pathways (i.e. dbcAMP and pentoxyphylline) did induce ‘T’-shape orientation (capacitation) in the tammar wallaby within 2 h of incubation indicating that CM alone is not sufficient or is slower to induce capacitation and head re-orientation in this species.

In summary, the flow cytometric technique described provides rapid and reliable estimates of global cellular phosphotyrosine levels in spermatozoa. The use of intact cells in this process makes it a very attractive technique for studying the effects of various modulators of tyrosine phosphorylation under in vitro conditions. Estimating tyrosine phosphorylation using flow cytometry also provides an opportunity to quantitate sub-populations within samples. It is particularly relevant for the study of sperm capacitation as in any given sample there are sub-populations of spermatozoa at various stages of capacitation that could be estimated by this technique. The only limitation of this technique is that it gives only the global estimates of tyrosine phosphorylation in sperm, and phospho-residues cannot be localized to specific sperm proteins. However, the rapidity of this technique makes it attractive for physiological study of sperm capacitation under different conditions.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The Australian Government Cooperative Research Center (CRC) Program funded this study. The supply of frozen brushtail possum sperm pellets from Landcare Research, Lincoln, New Zealand by Dr Frank Molinia, and frozen ram sperm pellets from Sydney University, Australia by Dr Chis Maxwell are thankfully acknowledged.

	Footnotes

 
Received 4 August 2003
First decision 16 September 2003
Accepted 7 October 2003

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