1 Laboratorio de Estudios en Reproducción (LER), Buenos Aires, Argentina, 2 CONRAD Program, Department of Obstetrics and Gynecology, The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, 601 Colley Ave, Norfolk, VA 23507, USA and 3 Instituto de Biología y Medicina Experimental, CONICET-UBA, Buenos Aires, Argentina
4 To whom correspondence should be addressed. e-mail: DoncelGF{at}evms.edu
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Abstract |
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Key words: capacitation/human sperm/hyperactivation/Percoll gradient/protein tyrosine phosphorylation
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Introduction |
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Protein tyrosine phosphorylation is an important post-translation event involved at multiple levels of cellular regulation (Hunter, 1991). This type of protein modification has been associated with certain aspects of sperm physiology such as capacitation, acrosome reaction, and hyperactivation (Visconti and Kopf, 1998
).
In the mouse, protein tyrosine phosphorylation has been associated with sperm capacitation and fertilizing ability (Visconti et al., 1995a). This association appears to be a phenomenon common to many mammalian species, including humans (Carrera et al., 1996
; Vijayayraghavan et al., 1997
; Pukazhenthi et al., 1998
; Si and Okuno, 1999
; Yeung et al., 1999
; Beverley and Aitken, 2001
). The increase in protein tyrosine phosphorylation in humans is regulated by a cAMP-dependent pathway that involves protein kinase A (PKA) activation (Leclerc et al., 1996
). Furthermore, the in vitro dependence of sperm tyrosine phosphorylation on the albumin concentration of the incubation medium suggests a correlation between membrane cholesterol efflux and cAMP-induced protein tyrosine phosphorylation (Visconti et al., 2002
).
Protein tyrosine phosphorylation has been associated with the ability of normal sperm to capacitate and fertilize an egg. Its deficiency, on the other hand, has been postulated as a possible cause of sperm dysfunction, in particular asthenozoospermia (Yunes et al., 2003). Although sperm with varying degrees of quality possess different fertilizing potential, the relationship between the degree and incidence of tyrosine phosphorylation and the functional quality of human sperm subpopulations remains a matter of speculation.
The goal of the present work was to examine the relationship between sperm quality and level of protein tyrosine phosphorylation in different sperm subpopulations in an attempt to determine whether human sperm showing the poorest quality have an intrinsic deficiency in protein tyrosine phosphorylation. In the present study, human sperm were subjected to Percoll gradient centrifugation to separate different cell populations. Several parameters of sperm quality were measured (e.g. morphology, motility and hyperactivation) and compared with the degree and incidence of capacitation-related protein tyrosine phosphorylation in those cells. Additionally, sperm from each subpopulation were stimulated with activators of the PKA pathway, and their protein tyrosine phosphorylation response was assessed.
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Materials and methods |
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Samples were allowed to liquefy for 1 h at room temperature and then sperm concentration and motility were assessed using computer-assisted semen analysis (CASA; Hamilton Thorne IVOS V10.8s, Hamilton Thorne Research, USA). Sperm viability was assessed by light microscopy in the original semen samples as well as the isolated fractions at all incubation times using the Eosin Y assay (World Health Organization, 1999).
Percoll fractionation and incubation of sperm
Aliquots of semen (1 ml) were loaded onto a 45, 65 and 90% discontinuous Percoll (Sigma Chemical Co., USA) gradient. Density gradients were performed by layering 1 ml of each Percoll concentration into a 15 ml conical tube. The tube was then centrifuged at 400 g for 20 min. The resulting interfaces between the layers of 45 and 65% (L45), 65 and 90% (L65) and the 90% pellet (L90) were aspirated and transferred to separate tubes. Sperm suspensions were then diluted with Hams F10 medium containing 3 mg/ml bovine serum albumin (Ham/BSA) and centrifuged twice at 400 g for 10 min. An aliquot of each interface was used to assay sperm concentration, motility and morphology (Kruger et al., 1987).
Washed sperm were resuspended in 1 ml of Ham/BSA at a concentration adjusted to 10x106 sperm/ml. Other sperm parameters (motility and protein tyrosine phosphorylation) were determined immediately after washing (T0), or after incubation at 37°C, 5% CO2 for five (T5) and 18 h (T18).
In one set of experiments, 1 mmol/l dibutyryl cyclic adenosine monophosphate (dbcAMP) and 1 mmol/l pentoxifylline (PTX) (Sigma) were added to the incubation medium.
Motility parameters and sperm hyperactivation
Aliquots of each sperm suspension were loaded into a 20 µm deep disposable chamber (Microcell; Conception Technologies, USA), pre-warmed at 37°C. Computer-assisted sperm motion analysis was performed using a Hamilton Thorne digital image analyzer (HTR-IVOS v 10.8s). At least 300 sperm and five fields were assessed.
Six motion parameters were assessed in this study: motility (%); track speed (VCL, µm/s); progressive velocity (VSL, µm/s); straightness (STR, %); beat cross frequency (BCF, Hz); and lateral head amplitude (ALH, µm). The settings used during the analysis were: frames acquired, 30; frame rate, 60 Hz; minimum contrast, 85; minimum cell size, 4 pixels; straightness threshold, 80%; low VAP threshold, 5 µm/s; medium VAP threshold, 25 µm/s; head sizenon-motile, 12 pixels; head intensitynon-motile, 130; static head size, 0.682.57; static head intensity, 0.311.21; static elongation, 23100. The playback function was used to accurately identify motile cells. Hyperactivated motility (HA, %) was defined as a motility with starspin or high-amplitude thrashing patterns and short trajectory distances (Burkman, 1984). The criterion for detecting hyperactivated sperm was: VCL >150 µm/s, ALH >7.0 µm, LIN <50% (Mortimer et al., 1998
).
Indirect immunofluorescence of sperm
Immunofluorescence was employed to examine the subcellular localization of proteins phosphorylated in tyrosine residues. Sperm from the different Percoll fractions were capacitated during various periods of time and washed twice with phosphate-buffered saline (PBS). Sperm concentration was adjusted to 5x106 cell/ml and 15 µl of the sperm suspension were spotted onto 8-well glass slides. Cells were air-dried on the slides, fixed and permeabilized with methanol for 30 min at room temperature. The slides were incubated with anti-phosphotyrosine antibody PY20 (ICN Biomedicals Inc., USA), diluted 1:20 (50 µg/ml) in PBS0.1% BSA, for 1.5 h at room temperature in a humidified chamber. After washing twice with PBS, slides were incubated with goat anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC; ICN Biomedicals Inc.), diluted 1:20 (50 µg/ml) in PBS0.1% BSA for 30 min at room temperature in a humidified chamber. Following the incubation, slides were washed with PBS three times, air-dried and mounted with Antifade® (Molecular Probes, USA). Sperm were examined using a fluorescence microscope Olympus BX40F (USA). At least 200 cells were counted in different fields and the percentage of sperm showing fluorescence in their tails was calculated. Negative controls were performed by blocking PY20 with o-D,L-phosphotyrosine (Sigma).
Western blot analysis of sperm proteins
Proteins from sperm were analysed by sodium dodecyl sulphate (SDS)polyacrylamide gel electrophoresis and western immunoblot analysis. Cells were washed twice with PBS and resuspended in Laemmli sample buffer (0.025 mol/l Tris, 0.5% SDS, 5% glycerol, pH 6.8) (Laemmli, 1970). Samples were centrifuged at 6000 g for 5 min. The supernatants were recovered and heated at 100°C for 5 min in the presence of 70 mmol/l 2-
mercaptoethanol and stored at 20°C until use. Solubilized proteins [obtained from 2x106 sperm per lane (
5 µg of protein)] were separated on 7% polyacrylamide gels under denaturing conditions. Prestained molecular weight markers (Amershan Life Science Inc., Canada) were run in parallel. For western blot analysis, proteins were electroblotted and transferred onto nitrocellulose (Bio Rad, USA) at 100 V, 4°C for 2 h. To block non-specific binding sites, the membrane was first incubated with 2% dry skimmed milk in PBS0.1% Tween 20 (blocking solution). After that it was incubated for 1 h with a monoclonal anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology, USA) diluted 1:5000 in blocking solution. After 4 washes with PBS0.1% Tween 20, an antimouse peroxidase-conjugated IgG (Jackson Immuno-Research Laboratories Inc., USA) diluted 1:5000 in blocking solution was added. Following 1 h of incubation, the membrane was washed four times with PBS0.1% Tween 20, and reactive bands were detected by enhanced chemiluminescence using the ECL kit (Amershan Life Science Inc., Canada) according to the manufacturers instructions. All incubations were performed at room temperature.
To quantify changes in protein tyrosine phosphorylation, rectangular boxes were drawn around bands on scanned digital images of ECL contact photographs of western blots, and adjusted optical densities for each lane were obtained using ImageJ software 1.30 V (National Institute of Health, USA).
Statistical analysis
Results are expressed as mean ± SD. Statistical differences between two groups were evaluated by Students t-test. Results obtained on different sperm fractions at the same incubation time were compared by two-way analysis of variance (ANOVA) and StudentNewmanKeuls test. All tests were two-tailed with a statistical significance assessed at the P < 0.05 level. Statistical analysis was performed using the Graphpad InStat program (GraphPad software, USA).
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Results |
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Although sperm showed phosphotyrosine immunolabelling on both head and tail, the signal associated with tail proteins was stronger and more consistent than that of the head; therefore, tail labelling was used to consider a spermatozoon as positive (Figure 6).
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Effect of cAMP and pentoxifylline on protein tyrosine phosphorylation in Percoll-separated sperm subsets
Sperm tyrosine phosphorylation has been shown to be stimulated by cAMP analogues and/or phosphodiesterase inhibitors via direct protein kinase A activation which occurs downstream from action at the plasma membrane (Visconti et al., 1995b; Leclerc et al., 1996
). To test whether sperm from the L45 subpopulation could be forced to overcome their protein tyrosine phosphorylation deficiency, we designed an experiment where each subpopulation of sperm was incubated with pentoxifylline and dbcAMP.
Sperm recovered from the three layers were incubated for 6 h with pentoxifylline in combination with dbcAMP to increase their endogenous levels of cAMP and induce tyrosine phosphorylation. Untreated control samples showed higher percentages of tyrosine-phosphorylated sperm than those previously observed. This difference may be due to a longer incubation time as well as the smaller sample size employed in this study. The pentoxifylline/dbcAMP experiments (n = 4) showed an increase in the incidence of phosphotyrosine-immunoreactive sperm in all three fractions (P < 0.05) (Figure 9). Although the incidence of phosphotyrosine-immunoreactive sperm was lower in L45 than in L90 (P < 0.05), compared to the untreated sample, protein kinase A stimulation induced a statistically significant increase in the number of tyrosine-phosphorylated sperm from the L45 fraction, which was equal to or even greater in relative magnitude than that of sperm from L65 or L90 fractions.
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Discussion |
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Studying enriched populations of highly and poorly motile human sperm separated by the swim-up technique, Turner et al. (1999) found that the percentages of tyrosine-phosphorylated cells were not statistically significantly different between these two subpopulations. Furthermore, they found no association between the tyrosine phosphorylation status of the major fibrous sheath protein of human sperm, hAKAP82, and significant differences in motility. Although these results appear to be in disagreement with those presented in our report, differences in the composition of the sperm subpopulations studied and in the ingredients of the medium used for incubation, as well as the length of the capacitating challenge may explain, at least in part, such apparent discrepancy. Turner and co-workers used a swim-up protocol to separate sperm, incubated the cells for 90 min in HTF + 3% BSA, and focused their analysis on the phosphorylation and processing of two tail proteins, pro-hAKAP82 and hAKAP82. In contrast, we used a discontinuous three-layer Percoll gradient which separated sperm subpopulations more precisely, incubated sperm under capacitating conditions for up to 18 h, and based our assessment on the incidence and intensity of all phosphotyrosine proteins. These conditions may have widened the differences between the sperm subsets, making the tyrosine phosphorylation deficiency of poor quality sperm more evident.
The observed differences in the functional quality and tyrosine phosphorylation of the sperm subpopulations evaluated in our study could result from defects at the sperm plasma membrane originated during spermatogenesis or epididymal maturation. Sperm subsets isolated by a similar gradient have been reported to present differences in their sperm membrane lipid composition, particularly in their content of docosahexaenoic acid and cholesterol (Ollero et al., 2000). Since cholesterol efflux has been implicated in triggering capacitation (Cross, 1998
) and tyrosine phosphorylation (Osheroff et al., 1999
), an incompletely or abnormally differentiated sperm membrane with high content of docosahexaenoic acid and cholesterol could be the cause for inadequate tyrosine phosphorylation of sperm proteins. The phosphorylation deficiency might also be due to incomplete or abnormal differentiation of the signal transduction systems, which could be part of a more general sperm abnormality, including the membrane and other sperm structures (Huszar and Vigue, 1993
; Aitken et al., 1998
; Gil-Guzman et al., 2001
).
Sperm tyrosine phosphorylation has been shown to be stimulated by cAMP analogues and/or phosphodiesterase inhibitors via a direct protein kinase A activation which occurs downstream of the plasma membrane (Visconti et al., 1995b; Leclerc et al., 1996
). In the present study, the effect of such compounds upon protein tyrosine phosphorylation was analysed on sperm subpopulations of different quality isolated through Percoll-gradient centrifugation. Results showed that the defective tyrosine phosphorylation of L45 sperm could be overcome when those stimulators were added to the incubation medium, indicating that signal transduction mechanisms downstream of PKA were not significantly affected in these cells. This stimulation recruited a number of newly phosphorylated sperm in L45, which represented a 100% increase over the basal (untreated) values. Moreover, the percentage of tyrosine-phosphorylated sperm after stimulation reached the level of untreated L90 cells. These findings suggest that the deficiency in tyrosine phosphorylation observed in the poor quality sperm from L45 would be associated with defects in the composition and/or dynamics of the plasma membrane. Alternatively, such deficiency could also be associated with alterations of other enzymes located upstream of PKA, e.g. soluble adenyl cyclase.
The role of adenyl cyclase, bicarbonate and cholesterol acceptors in capacitationinduction has been well demonstrated in sperm from several mammalian species (Harrison, 1996; Visconti and Kopf, 1998
; Gadella and Harrison, 2000
). In porcine sperm, bicarbonate induces a change in membrane architecture consisting of phospholipid scrambling and lateral re-distribution and apical concentration of cholesterol, phenomena that facilitate cholesterol removal by acceptor molecules such as albumin (Flesch et al., 2001
). Only a subpopulation of ejaculated sperm responds to bicarbonate, and such a property appears to be related to the degree of epididymal maturation.
We propose that the low level of protein tyrosine phosphorylation observed in poor quality sperm recovered from the 45% layer may be related to their membrane lipid composition, particularly to high cholesterol content, which would impair the ability of these sperm to respond to capacitation-inducing stimuli with adequate membrane architectural changes and related signal transduction. The biological and clinical significance of the above-described findings lies in the association between structural and functional parameters of human sperm quality and a relatively new biochemical marker of capacitation, an indispensable process in human fertilization. This association represents a step toward the elucidation of defective molecular mechanisms that may be the real cause of the reduced fertilizing capacity of pathological sperm.
In conclusion, data presented above show that human sperm subpopulations isolated from normal semen samples through Percoll gradient centrifugation display clear differences in their ability to undergo capacitation-associated protein tyrosine phosphorylation. The subpopulation bearing the poorest quality shows an intrinsic impairment of tyrosine phosphorylation; a defect that may be related to incomplete or abnormal differentiation of these cells, and in particular, to a specific defect of their plasma membrane which contains high levels of cholesterol. This deficiency would impair a normal development of the capacitation process, ultimately compromising the acquisition of optimal fertilizing capacity by the sperm.
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Acknowledgement |
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Submitted on May 23, 2003; resubmitted on September 25, 2003; accepted on September 25, 2003.