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RESEARCH |
Laboratorio de Estudios en Reproducción (LER), Buenos Aires, Argentina, 1 Departamento de Química Biológica, IQUIFIB (CONICET-UBA), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina and 2 CONRAD, Department of Obstetrics and Gynecology, The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, 601 Colley Ave., Norfolk, Virginia 23507, USA
Correspondence should be addressed to G F Doncel; Email: DoncelGF{at}evms.edu
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Abstract |
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Introduction |
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Tyrosine phosphorylation in spermatozoa is regulated by a complex series of molecular events involving membrane cholesterol efflux and hyperpolarization, increase in intracellular NaHCO3, Ca2 + and cAMP, and activation of phosphokinases (Visconti & Kopf 1998, Visconti et al. 2002). Following capacitation-associated cholesterol efflux and its consequent increase in membrane fluidity, sperm show a rise in cAMP produced by a NaHCO3-dependent soluble adenyl cyclase bound to the cyto-skeletal fraction in mature sperm, which precedes an upregulation of sperm motion parameters, hypermotility, and tyrosine phosphorylation (Visconti et al. 1995b, Leclerc et al. 1996, Bajpai & Doncel 2003, Luconi et al. 2005). Tyrosine (protein tyrosine kinases; PTK) and cAMP-dependent (protein kinase A; PKA) kinases are involved in these processes as their inhibition in human spermatozoa decreases tyrosine phosphorylation and motility concomitantly (Bajpai & Doncel 2003, Bajpai et al. 2003).
Studying sperm subpopulations from normozoospermic human semen samples, we also demonstrated that Percoll-isolated low-quality spermatozoa showed an impairment in the development of tyrosine phosphorylation during capacitation (Buffone et al. 2004). Such deficiency, however, was overcome with permeable activators of PKA, which led us to postulate that the main defect in those spermatozoa was localized upstream of PKA, possibly at the plasma membrane. Changes in plasma membrane composition have been associated with both capacitation (Davis 1981, Cross 1998) and sperm pathology (Calamera et al. 2003). Considering these findings as well as preliminary data gathered from samples with abnormal motility, we hypothesized that such combined alteration may be the underlying cause of certain sperm pathologies, especially those displaying low motility (Yunes et al. 2003).
In order to verify this hypothesis and to expand the characterization of such molecular defects in pathological sperm, we have studied the capacitation-associated changes of protein tyrosine phosphorylation and membrane fluidity in spermatozoa of asthenozoospermic, normozoospermic, and proven-fertile human subjects. Herein, we describe the results of this investigation.
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Materials and Methods |
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Sperm samples were classified as normozoospermic (sperm concentration >20 x 106 spermatozoa/ml, percentage of motile cells >50%, percentage of viable spermatozoa 
80%, and percentage of normal forms 14%) or asthenozoospermic (sperm concentration >20 x 106 spermatozoa/ml, percentage of motile cells <40%, percentage of viable spermatozoa 80%, and percentage of normal forms 14%). We also studied samples from seven sperm donors of proven fertility.
Motility parameters and sperm hyperactivation
Aliquots of each sperm suspension were loaded into a 20 µm deep disposable chamber (Microcell; Conception Technologies, San Diego, CA, USA) pre-warmed at 37 °C. Computer-assisted sperm motion analysis was performed using a Hamilton Thorne digital image analyzer (HTR-IVOS v 10.8 s; Hamilton Thorne Research) before and after a 6h capacitating incubation. At least 300 spermatozoa and five fields were assessed.
Eight motion parameters were assessed in this study: (1) motility (%); (2) average path velocity (VAP, µm/s); (3) track speed or curvilinear velocity (VCL, µm/s); (4) progressive or straight-line velocity (VSL, µm/s); (5) straightness (STR, %); (6) beat cross frequency (BCF, Hz); (7) linearity (LIN, %); (8) 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 cut off, 5 µm/s; medium VAP cut off, 25 µm/s; head size – non-motile, 12 pixels; head intensity – non-motile, 130 Units(U) ; static head size, 0.68–2.57 pixels; static head intensity, 0.31–1.21 U; static elongation, 23–100%. The playback function was used to accurately identify motile cells. Hyperactivated motility (%) was defined as motility with starspin or high-amplitude trashing patterns and short trajectory distances (Burkman 1984). This percentage represents the portion of motile spermatozoa displaying hyperactivated movement. The criteria for detecting hyperactivated spermatozoa were: VCL >150 µm/s; ALH >7.0 µm; LIN <50% (Mortimer et al. 1998).
Preparation of spermatozoa
Sperm cells were separated from seminal plasma by dilution with Ham’s F10 medium (Gibco BRL, Grand Island, NY, USA) containing 3 mg/ml bovine serum albumin (BSA; Sigma, St Louis, MO, USA) (Ham/BSA) and centrifugation at 400 g for 5 min (three times). The pellet was resuspended in 1 ml Ham/BSA and an aliquot of it was used to assay sperm concentration and motility. This was considered the first incubation time (T0). Sperm concentration was adjusted to approximately 10 x 106 spermatozoa/ml and incubated for 6 h (T6) at 37 °C in 5% CO2. In some instances, sperm were separated from seminal plasma using Percoll-gradient centrifugation (500 g for 20 min) and subsequent washing with Ham/BSA. To increase intracellular levels of cyclic AMP (cAMP), sperm where incubated for 1 h with dibutyryl cAMP (dbcAMP; 1mM) and pentoxifylline (PTX; 1 mM).
Indirect immunofluorescence of spermatozoa
Immunofluorescence was employed to examine the sub-cellular localization of proteins phosphorylated in tyrosine residues as well as the incidence of tyrosine-phosphorylated sperm in a given population. Spermatozoa from the different groups were capacitated during various periods of time and washed twice with phosphate-buffered saline (PBS). Sperm concentration was adjusted to 5 x 106 cells/ml and 15 µl of the sperm suspension was spotted onto eight-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., Aurora, OH, USA), diluted 1:20 (50 µg/ml) in PBS–0.1% BSA, for 1.5 h at room temperature in a humidified chamber. After washing twice with PBS, slides were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (ICN Biomedicals Inc.) diluted 1:20 (50 µg/ml) in PBS–0.1% 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, Eugene, OR, USA). Spermatozoa were examined using a fluorescence microscope (Olympus BX40F, Melville, NY, USA). At least 200 cells were counted in different fields and the percentage of spermatozoa showing fluorescence in their tails was calculated. Negative controls were performed by blocking PY20 with ortho-D, L phosphotyrosine (Sigma).
Western blot analysis of sperm proteins
Proteins from spermatozoa were analyzed by SDS-PAGE and Western immunoblotting. Cells were washed twice with PBS and resuspended in Laemmli sample buffer (25 mM Tris, 0.5% SDS and 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 mM 2ß-mercaptoethanol and stored at –20 °C until use. Solubilized proteins (obtained from 2 x 106 spermatozoa per lane (<5 µg protein)) were separated on 7% polyacrylamide gels under denaturing conditions. Prestained molecular weight markers (Amersham Life Science Inc., Oakville, Ontario, Canada) were run in parallel. For Western blot analysis, proteins were electroblotted and transferred onto nitrocellulose (BioRad, Hercules, CA, USA) at 100 V at 4 °C for 2 h. To block non-specific binding sites, the membrane was first incubated with 2% dry skimmed milk in PBS–0.1% Tween 20 (blocking solution). Then it was incubated for 1 h with the monoclonal anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology, Lake Placid, NY, USA) diluted 1:5000 in blocking solution. After four washes with PBS–0.1% Tween 20, an anti-mouse peroxidase-conjugated IgG (Jackson Immuno-Research Laboratories Inc., West Grove, PA, USA) diluted 1:5000 in blocking solution was added. Following 1 h of incubation, the membrane was washed four times with PBS–0.1% Tween 20, and reactive bands were detected by enhanced chemiluminescence using the ECL kit (Amersham Life Science Inc.) according to the manufacturer’s 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 Institutes of Health, Bethesda, MD, USA).
Membrane fluidity
Sperm membrane fluidity was evaluated using the fluorescent probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan; Molecular Probes Inc., Eugene, OR, USA). Normozoospermic and asthenozoospermic spermatozoa were mixed with Laurdan at 1:1000 (probe:lipid ratio) and incubated for 15 min at 37 °C to allow the incorporation of the probe into the membrane. After incubation, membrane fluidity was evaluated by changes in Laurdan generalized polarization (GP) calculated as: GP = (I430 – I480)/(I430 + I480) where I430 and I480 are the fluorescence intensities at 430 nm and 480 nm respectively (
excitation: 350 nm) (Harris et al. 2002) measured in a Kontron SFM-25 spectrofluorometer with temperature control (Kontron Instruments SPA, Milan, Italy).
Statistical analysis
Results are expressed as means ± S.D. or S.E.M. Statistical differences between two groups were evaluated by Student’s t-test. Results obtained from different sperm groups at the same incubation time were compared by two-way ANOVA and Student–Newman–Keuls 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, San Diego, CA, USA).
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Results |
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). After 6 h of capacitating incubation, asthenozoospermic samples showed a proportionally greater decrease in motility (31.9% drop) than the other two groups (normozoospermic, 15.8% and fertile donors, 13.2%). Viability of spermatozoa, on the other hand, was not significantly different among the study groups at any time of incubation.
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Mean values of ALH displacement were significantly (P < 0.05) lower for asthenozoospermic when compared with normozoospermic and fertile samples at T0. This difference was no longer statistically significant at T6. Furthermore, no statistical differences were observed for values of flagellar BCF, STR, and LIN of trajectory at any incubation time.
The studied groups revealed different abilities to develop hyperactivated motility when they were incubated under capacitating conditions for 6 h (Fig. 1A). Out of the total number of motile spermatozoa, the percentage of hyperactivated cells in asthenozoospermic samples was significantly lower than that observed in normozoospermic (P < 0.01) and fertile samples (P < 0.05).
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) was significantly lower in the asthenozoospermic group than in the normozoospermic and fertile donor groups (P < 0.05). These latter groups were not statistically different from one another. The difference between asthenozoospermic and normozoospermic samples was observed regardless of the method used to separate sperm from seminal plasma (data not shown).
Similar results were observed following the Western blot analysis of spermatozoa (Fig. 3). Major differences were observed in the level of tyrosine-phosphorylated proteins recovered from the three groups studied. The lowest signal was detected in proteins extracted from spermatozoa of asthenozoospermic patients (Fig. 3). Densitometric analysis of the major protein bands (range: 77–170 kDa) showed that at T6 normozoospermic samples revealed a 1.8-fold increase in the overall signal compared with T0. The average increase in signal for fertile samples was 1.9-fold. Asthenozoospermic samples, however, showed no increase in the signal intensity of most of their protein bands at T6, revealing an inability to respond to capacitating conditions with the enhanced tyrosine phosphorylation observed in normozoospermic and fertile samples.
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Baseline incidence of phosphotyrosine-immunoreactive spermatozoa after a capacitating incubation (T6) was significantly lower (P < 0.05) in the asthenozoospermic than in the normozoospermic group (Fig. 4). cAMP stimulation induced a statistically significant (P < 0.05) increase in the number of tyrosine-phosphorylated spermatozoa in the asthenozoospermic group, which was equal to or even greater in relative magnitude to that of spermatozoa from normozoospermic samples, suggesting that the main block to increasing tyrosine phosphorylation during capacitation resides upstream of the cAMP-dependent kinase(s).
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). This molecule spontaneously incorporates into membranes at the glycerol backbone level, distributing itself evenly among the different lipid domains (Parasassi & Gratton 1995). Laurdan responds to variations in the number of water molecules accessible to the probe, which in turn depends on both lipid packing and cholesterol content. This alteration in the polarity of Laurdan’s microenvironment is visualized as an alteration in its fluorescence excitation and emission spectra. Results are expressed as ratios of the probe’s polarization. The lower the ratio, the higher the fluidity of the membrane. Asthenozoospermic samples showed a 21% higher GP ratio (P < 0.0001) prior to their capacitation (T0) in comparison with normozoospermic samples (0.097 ± 0.002 and 0.117 ± 0.004 for normozoospermic and asthenozoospermic spermatozoa respectively), indicating lower membrane fluidity.
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Discussion |
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Semen samples from asthenozoospermic patients showed not only a low percentage of motile spermatozoa but sperm motion of poor quality, reflected by decreased VAP, VSL, VCL, and ALH. As mentioned, these spermatozoa also displayed a marked impairment in their capacity to develop hyperactivated motility. Although these defects may be the cause of the reported poor fertilizing ability of asthenozoospermic samples, the pathogenetic mechanisms leading to such alterations are, for the most part, unknown.
Several groups, including ours, have reported a close association between sperm protein tyrosine phosphorylation and motility and hyperactivation in different mammalian species (Leclerc et al. 1996, Nassar et al. 1999, Si & Okuno 1999, Luconi et al. 2001, Yunes et al. 2003, Buffone et al. 2004). Differences in the level of hyperactivated sperm, especially after capacitation, could originate from the impaired capacity of these cells to phosphorylate proteins in tyrosine residues which, in turn, could be linked to reduced membrane fluidity.
Various reports have described an active participation of the sperm plasma membrane in the process of capacitation, mainly through loss of cholesterol (Davis 1981, Cross 1998, Visconti et al. 1999a). Cholesterol efflux produces an increase in membrane fluidity which directly or indirectly impacts protein function, such as that of surface ion channels and enzymes. Changes in ion fluxes across the membrane, mainly HCO3–, Na +, K +, and Ca2 +, have been implicated in initiating sperm capacitation as well as tyrosine phosphorylation (Yanagimachi 1994, Visconti & Kopf 1998). More specifically, an increase in intracellular HCO3– stimulates soluble adenyl cyclase which rapidly increases cAMP, protein tyrosine phosphorylation, sperm motility, and acrosome reaction (Visconti et al. 1999b, Gadella & Harrison 2000, Luconi et al. 2005). The endpoint of these capacitation-associated changes, at least those related to motility and hyperactivation, appears to be an increased binding of the regulatory subunits of PKA, especially typeII, to tyrosine-phosphorylated forms of A-kinase anchoring proteins 3 and 4. Interruption of this binding leads to decreased sperm motility (Vijayaraghavan et al. 1997, Luconi et al. 2004).
Sperm tyrosine phosphorylation can be stimulated by cAMP analogs and/or phosphodiesterase inhibitors via direct PKA activation, which occurs downstream of the plasma membrane (Visconti et al. 1995b, Leclerc et al. 1996, Nassar et al. 1999, Bajpai & Doncel 2003, Buffone et al. 2004). In order to evaluate the participation of a plasma membrane defect in the inability of asthenozoospermic sperm to undergo protein tyrosine phosphorylation, we designed an experiment in which spermatozoa were treated with dbcAMP and pentoxyfilline to increase their endogenous levels of cAMP. Results showed that defective tyrosine phosphorylation of asthenozoospermic spermatozoa could be overcome when those stimulators were added to the incubation medium, indicating that signal transduction mechanisms downstream of cAMP were not significantly affected in those cells.
These findings suggested that the deficiency in tyrosine phosphorylation observed in asthenozoospermic spermatozoa and, possibly, their dysfunctional motility could be associated with an alteration in the dynamics of the plasma membrane. Removal of cholesterol and increased membrane fluidity is a key step in the initiation of capacitation (Cross 1998). It decreases the cholesterol/phospholipid ratio (Davis 1981), allowing for redistribution of membrane lipids and proteins (Cross & Overstreet 1987, Gadella et al. 1999). Capacitation can be inhibited by adding cholesterol or cholesterol analogs to the capacitating medium (Visconti et al. 1999a), and can be stimulated by cholesterol acceptors such as ß-cyclodextrins (Osheroff et al. 1999). Changes in membrane dynamics have been associated with tyrosine phosphorylation as well as sperm function and fertilizing ability (Gadella et al. 1999, Flesch et al. 2001).
In order to verify if asthenozoospermic sperm have a membrane dynamics defect that could be associated with their tyrosine phosphorylation and motility deficiencies, we have measured changes in the membrane fluidity of the sperm during a capacitating incubation, studying changes in Laurdan fluorescence (Ambrosini et al. 2001). Spermatozoa from the asthenozoospermic group revealed significantly less fluid membranes at the beginning of the incubation (T0). Capacitation increased the membrane fluidity of these cells, but never to the level achieved by normozoospermic spermatozoa.
Such difference in membrane fluidity could be due to the increased susceptibility of these spermatozoa to suffer peroxidative damage (Calamera et al. 2003), as the generation of membrane lipid hydroperoxides has been associated with membrane fluidity reduction (Aitken et al. 1993, 1994, Windsor et al. 1993). This susceptibility of asthenozoospermic sperm could be explained, in part, by their membrane composition, which is responsible for their reported higher oxidation coefficient (Calamera et al. 2003). Sperm membranes of asthenozoospermic samples contain high levels of polyunsaturated fatty acids, making them more prone to attack by reactive oxygen species. Since oxidizing conditions are normal during sperm capacitation and have been linked to signal transduction and tyrosine phosphorylation (Aitken et al. 1994, 1998), the predisposition of the asthenozoospermic samples to oxidative damage may be the origin of their membrane dysfunction, resulting in tyrosine phosphorylation deficiency and alteration of motility.
The biological and clinical significance of the above-described findings resides in the association between a common sperm pathology such as asthenozoospermia and a defective molecular mechanism, closely related with the acquisition of the sperm’s fertilizing ability. 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.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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D.Y. Liu, G.N. Clarke, and H.W.G. Baker Hyper-osmotic condition enhances protein tyrosine phosphorylation and zona pellucida binding capacity of human sperm Hum. Reprod., March 1, 2006; 21(3): 745 - 752. [Abstract] [Full Text] [PDF]
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