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From the * Urology Research Laboratory, Royal
Victoria Hospital and Faculty of Medicine, McGill University, Montréal,
Québec, Canada; and the Center for
Research in Contraception and Reproductive Health, Department of Cell Biology,
University of Virginia, Charlottesville, Virginia.
| Correspondence to: Dr Claude Gagnon, Urology Research Laboratories, Room H6.47, Royal Victoria Hospital, 687 Ave des Pins ouest, Montréal, Québec, Canada H3A 1A1 (e-mail: claude.gagnon{at}muhc.mcgill.ca). |
| Received for publication February 28, 2002; accepted for publication May 7, 2002. |
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Abstract |
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Key words: Phosphodiesterases, cyclic adenosine monophosphate, calcium ionophore A23187, signal transduction
Intracellular levels of cAMP are regulated by both the adenylyl cyclase (AC) that synthesizes cAMP from adenosine triphosphate (ATP) and the cAMP phosphodiesterases (PDEs) that degrade cAMP to 5'-AMP. The presence of calmodulin-dependent PDE, cyclic guanosine monophosphate (cGMP)-inhibited PDE, and cAMP-specific PDE, but not cGMP-specific PDE, has been reported in human spermatozoa (Fisch et al, 1998; Lefièvre et al, 2000, in press). Mammalian spermatozoa contain a unique form of soluble AC (sAC), an enzyme with no transmembrane domain structurally related to that of AC found in procaryotes (Buck et al, 1999).
In mammalian cells, the principal action of cAMP is mediated through the activation of the cAMP-dependent protein kinase A (PKA). PKA is a tetrameric enzyme containing 2 regulatory (R) subunits bound to 2 catalytic (C) subunits, which can form either a type I or type II holoenzyme, depending on the particular subclass of R subunit (RI or RII) present. The activation of PKA occurs when 4 molecules of cAMP bind to the R subunits, 2 to each R subunit, causing the dissociation of 2 free and active C subunits from the 2 R subunits. Most cells are equipped with A-kinase-anchoring proteins (AKAPs), which bind PKA, via its R subunits, to the cytoskeleton or to subcellular organelles in close proximity to target proteins (Feliciello et al, 2001). In mammalian spermatozoa, the cellular localization of R subunits of PKA to specific locations (RI and RII to the acrosomal cap and flagellum) (Pariset and Weinman, 1994; Vijayaraghavan et al, 1997; Visconti et al, 1997) might suggest that these cells contain other AKAPs besides those presently known (AKAP82, AKAP110, AKAP 220, etc) (Feliciello et al, 2001), so that targeted phosphorylation can occur at the time of capacitation or acrosome reaction, in specific compartments.
Inhibitors of PKA blocked sperm capacitation and the associated increase in protein tyrosine phosphorylation (Visconti et al, 1995; Leclerc et al, 1996) as well as prevented the acrosome reaction (De Jonge et al, 1991), suggesting that PKA takes part in these events. Moreover, PKA activity progressively increased in epididymal mouse spermatozoa during capacitation, and RI and C subunits of PKA were found in the head, midpiece, and principal piece of mouse spermatozoa (Visconti et al, 1997). One of the roles of PKA during capacitation appears to be the indirect regulation of protein tyrosine phosphorylation (Visconti et al, 1995; Leclerc et al, 1996). On the other hand, the role of PKA in the acrosome reaction could be to activate a voltage-dependent calcium channel (Spungin and Breitbart, 1996).
Since capacitation and acrosome reaction are regulated by cAMP and PKA, the aim of the present study was to evaluate a possible relationship between human sperm intracellular levels of cAMP and PDE and PKA activities during capacitation induced by fetal cord serum ultrafiltrate (FCSu) and acrosome reaction induced by calcium ionophore A23187.
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Materials and Methods |
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-32P] ATP were purchased from
Amersham Pharmacia Biotech (Baie d'Urfé, PQ, Canada; Piscataway, NJ).
Phosphocellulose paper, Whatman P81, was purchased from Whatman Incorporated
(Clifton, NJ). Other chemicals used were at least of reagent grade. The
specific antiserum against cAMP (CV27) was obtained through the National
Hormone and Peptide Program, National Institute of Diabetes and Digestive and
Kidney Diseases, and Dr A.F. Parlow (Torrance, Calif). Fetal cord blood was collected at the birthing center of the Royal Victoria Hospital (Montréal, PQ, Canada). An informed consent was obtained from the patients, and the ethics board of the Royal Victoria Hospital approved the present study. Fetal cord blood was centrifuged (1000 x g, 30 minutes, 4°C), and serum samples were pooled and frozen (-20°C) until used. FCSu's were prepared from at least 15 individual samples using Amicon YM3 membranes (exclusion limit: 3 kd; Oakville, ON, Canada) (de Lamirande and Gagnon, 1993). IBMX and A23187 were dissolved in dimethylsulfoxide (DMSO) and then diluted so that the concentration of DMSO did not exceed 0.1% (vol/vol), an amount that did not influence sperm motility, capacitation, or acrosome reaction. Other chemicals were dissolved in distilled water.
Sperm Preparation and Treatments
Semen samples were obtained by masturbation from healthy volunteers after 3
days of sexual abstinence. After liquefaction, semen was layered on top of a
gradient composed of 2-mL fractions of 20%-40%-65% Percoll and 0.1 mL of 95%
Percoll made isotonic and buffered in HEPES balanced saline (HBS: 115 mM NaCl,
4 mM KCl, 0.5 mM MgCl2, 14 mM fructose, and 25 mM HEPES, pH 8). The
samples were centrifuged for 30 minutes at 2300 x g, and
spermatozoa at the 65%-95% interface and within the 95% Percoll fraction were
recovered, pooled, and diluted to 500 x 106 cells/mL with 95%
Percoll solution. Percoll-washed spermatozoa were analyzed by video-microscopy
using the CellSoft computer-assisted digital image analysis system (Cryo
Resources, Montgomery, NY) to measure sperm concentration and motility, and
only samples in which progressive sperm motility was at least 70% were
used.
Spermatozoa were further diluted in Biggers, Whitten, and Whittingham medium (BWW; pH 8) (Biggers et al, 1971) devoid of bicarbonate and albumin, containing 1.2 mM CaCl2 and supplemented with 25 mM HEPES, pH 8 (Lefièvre et al, 2000). For capacitation studies, spermatozoa were incubated for up to 3 hours at 37°C in BWW alone (noncapacitating condition; control) or supplemented with 7.5% (vol/vol) FCSu (capacitating condition). The acrosomal status of spermatozoa was evaluated after permeabilization with ethanol using FITC-conjugated P sativum agglutinin as described by Cross et al (1986). At least 200 spermatozoa were evaluated for the acrosome reaction in each sample. The levels of spontaneous acrosome reaction in spermatozoa incubated with BWW alone or supplemented with FCSu for 3.5 hours at 37°C were 7.5% plus or minus 1.6% and 8.9% plus or minus 1.2%, respectively. When challenged with A23187 (1 µM, in the absence of BSA for an additional 15 minutes at 37°C) (de Lamirande et al, 1998), the levels of acrosome reaction were 8.9% plus or minus 1.2% for control spermatozoa (BWW alone) and 17.5% plus or minus 0.7% for capacitated spermatozoa (treated with FCSu). Moreover, sperm motility was monitored during the 3 hours of incubation. The percentage of motility was not affected by the different treatments, and hyperactivation was observed in spermatozoa incubated with FCSu.
PDE Assay
PDE activity was measured in spermatozoa (duplicates) collected at various
times of incubation using the modified 2-stage radio-isotopic method of
Sonnenburg et al (1998).
Briefly, the standard incubation mixture (pH 7.5) containing 20 mM Tris-HCl,
20 mM imidazole, 3 mM MgCl2, 15 mM magnesium acetate, 0.2 mg/mL
BSA, 0.1% Triton X-100, 5 µg/mL leupeptin, 5 µg/mL pepstatin, 10
µg/mL aprotinin, 200 µM PMSF, and 1 µM cAMP was added to the sperm
suspension (50 x 106 cells/mL, final concentration). The
reaction was started by the addition of 50 000 counts/min of [8-3H]
cAMP. The reaction was stopped after 10 minutes at 30°C by heat
denaturation at 100°C for 1 minute, and the resulting 5'-AMP was
converted to adenosine with 2.5 mg/mL of C atrox venom for an
additional 5 minutes at 30°C. The reaction products were separated by
anion-exchange chromatography on Dowex 1X8, and the amount of unbound
[3H] adenosine was quantified by liquid scintillation counting.
cAMP Measurement
Sperm samples (2.5 x 106 cells; duplicates) were
centrifuged at 3000 x g for 5 minutes, resuspended in 0.5 mL of
90% (vol/vol) ice-cold ethanol, and mixed by vortexing. After 30 minutes at
-20°C and 30 minutes at 4°C, samples were centrifuged at 19 000
x g, supernatants were collected, and the ethanol was
evaporated in a SpeedVac concentrator. Dried pellets were stored at -20°C
until used. Intracellular cAMP was measured by radioimmunoassay using
[125I] cAMP as described previously
(Lefièvre et al,
2000).
PKA Assay
PKA activity was measured as described by Visconti et al
(1997) using Kemptide as a
substrate. Briefly, spermatozoa were adjusted to a final concentration of 20
x 106 cells/mL, incubated at 37°C under the different
conditions as described in the "Sperm Preparation and Treatments"
section, and snap frozen in liquid nitrogen at appropriate time points. Frozen
sperm samples (triplicates) were supplemented with 2x assay cocktail
(final concentrations: 100 µM Kemptide, 1 µCi [-32P]
ATP, 40 µM ATP, 1% [vol/vol] Triton X-100, 1 mg/mL BSA, 10 mM
MgCl2, 40 mM ß-glycerophosphate, 5 mM p-nitrophenyl
phosphate, 25 mM HEPES [pH 7.4], 10 µM aprotinin, 10 µM leupeptin, and
100 µM vanadate). Another triplicate of the same sperm samples was measured
in conditions that maximize PKA activity (addition of 1 mM dbcAMP + 100 µM
IBMX to the assay mixture) to eliminate the possibility that differences in
PKA activity observed would be due to different amounts of enzyme or
spermatozoa in the PKA assays. The samples were then incubated for 15 minutes
at 37°C, and the reaction was stopped by the addition of trichloroacetic
acid (10% final concentration). Samples were cooled and centrifuged at room
temperature for 3 minutes at 10 000 x g. Thirty microliters of
the supernatant were spotted onto phosphocellulose papers, which were then
washed in 5 mM phosphoric acid and dried. The radioactivity on the
phosphocellulose paper was quantified by liquid scintillation counting.
Subcellular Localization
Spermatozoa were washed twice with HBS (5 minutes at 1000 x
g). Pellets were resuspended at a concentration of 50 x
106 cells/mL in a hypotonic buffer (50 mM Tris-HCl [pH 7.5], 2 mM
EDTA, 1 mM dithiothreitol, 3 mg/mL BSA, 5 µg/mL leupeptin, 5 µg/mL
pepstatin, 10 µg/mL aprotinin, and 200 µM PMSF). Subcellular
fractionation was performed as described by Lefièvre et al
(2000). Briefly, the
suspension was homogenized in a glass Dounce homogenizer and centrifuged at
100 000 x g at 4°C for 30 minutes. The supernatant was
recovered (cytosol fraction), and the pellet was resuspended in hypotonic
buffer (see above) containing 0.1% Triton X-100, rehomogenized, incubated at
4°C for 10 minutes, and centrifuged at 19 000 x g for 10
minutes at 4°C. The Triton X-100 soluble fraction (membrane fraction) was
recovered, and the pellet (particulate fraction) was resuspended in the
hypotonic buffer as described above. Aliquots from the initial homogenization
(total PDE activity) and the different fractions were assayed for PDE activity
as described earlier.
Data Analysis
Statistical differences between treatments were measured by the Fisher
Protected least significant difference test following a 2-tailed analysis of
variance with multiple paired treatment. A difference was considered
statistically significant at P less than or equal to .05.
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Results |
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Subcellular Localization of PDE Activity During Sperm
Capacitation
To determine whether PDE activity was redistributed during capacitation,
sperm cytosol, membrane, and particulate fractions were prepared
(Figure 2). As previously
observed (Lefièvre et al,
2000), for the PDE hydrolysis of cAMP, 45% was recovered in the
cytosol, 20% in the membrane, and 15% in the particulate fractions
(Figure 2). PDE activity
recovered from the different sperm fractions was similar in noncapacitating
(Figure 2A) and capacitating
(Figure 2B) spermatozoa and did
not vary over the course of the incubation.
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Sperm PDE Activity, Intracellular Levels of cAMP, and PKA Activity
During Acrosome Reaction Induced by A23187
Spermatozoa were incubated for 3.5 hours in capacitating or noncapacitating
conditions and then with A23187 as the acrosome reaction inducer.
Intracellular cAMP levels and PDE and PKA activity were assayed 15 minutes
later (Figure 3). Sperm PDE
activity did not change during treatment with A23187 and was similar in
acrosome reacting and nonacrosome reacting spermatozoa
(Figure 3A). Levels of sperm
intracellular cAMP and PKA activity were higher in capacitated spermatozoa
treated with A23187 (induction of the acrosome reaction) than in
noncapacitated or nonacrosome reacting spermatozoa
(Figure 3B and C). The maximal
PKA activity (addition of dbcAMP and IBMX to the assay mixture) in
noncapacitating and capacitating spermatozoa was the same
(Figure 3D), indicating that
the differences observed in Figure
3C were really due to an activation of PKA during the acrosome
reaction and not to a difference in the amount of enzymes or spermatozoa in
PKA assay.
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Discussion |
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The intracellular concentration of cAMP was reported to increase in bull and human (Parrish et al, 1994; Parinaud and Milhet, 1996) or decrease in mouse and guinea pig (Hyne and Garbers, 1979; Stein and Fraser, 1984) during sperm capacitation. Our data indicate that the levels of cAMP in capacitating and noncapacitating spermatozoa were similar (Figure 1B) and corresponded to those previously reported for human spermatozoa (ranges from 20 to 40 pmol/108 spermatozoa) (Parinaud and Milhet, 1996; Calogero et al, 1998). Surprisingly, there was a progressive and important decrease in sperm cAMP over the 3-hour incubation period (Figure 1B).
Sperm AC, sAC activity is not regulated by forskolin and guanine nucleotides (Buck et al, 1999), but rather, is activated by bicarbonate anions (Okamura et al, 1985; Rojas et al, 1992; Chen et al, 2000) in a pH-independent manner (Chen et al, 2000). The presence of bicarbonate appears essential for the capacitation of epididymal but not of ejaculated spermatozoa, because the latter are in the presence of high concentrations (25 mM) of this anion in the seminal plasma of human, bull, rat, mouse, and dog (Okamura et al, 1985). One of the functions of bicarbonate in spermatozoa is to stimulate sAC within minutes of its addition (Okamura et al, 1985; Rojas et al, 1992; Chen et al, 2000). Therefore, the exposure of spermatozoa to bicarbonate that takes place by contact to seminal plasma during the 30 to 60 minutes following ejaculation (liquefaction time) appears sufficient to support sperm functions (Okamura et al, 1986). The low level of bicarbonate in the incubation medium used in this study (2 mM; from FCSu) compared to those used by other groups (25 mM) could then explain the progressive decrease of cAMP concentration observed (Figure 1B). Previous studies from our laboratory demonstrate that human sperm capacitation and the associated increase in protein tyrosine phosphorylation and hyperactivation can be induced in media devoid of bicarbonate but supplemented with FCSu (de Lamirande and Gagnon, 1993; Leclerc et al, 1996; de Lamirande et al, 1997). Leclerc et al (1996) also observed that there was no further increase in the level of FCSu-induced capacitation after the addition of bicarbonate to this medium. Therefore, these results indicate that in human spermatozoa, low levels of bicarbonate and cAMP are sufficient to induce capacitation.
Sperm capacitation and associated events (such as hyperactivation and protein tyrosine phosphorylation) are regulated by cAMP and PKA (Visconti et al, 1995; Leclerc et al, 1996). In mouse, PKA activity increased in a time-dependent manner in capacitating epididymal spermatozoa (Visconti et al, 1997). In the present study, we observed that PKA activity was higher in capacitating than in noncapacitating human spermatozoa throughout the 3 hours of incubation (Figure 1C). This observation and the absence of a parallel between the changes in intracellular cAMP levels and PKA activity could suggest that even low levels of cAMP are sufficient to activate PKA and/or that cAMP concentrations of the whole cells do not necessarily reflect the levels present in the different compartments of the spermatozoa (discussed below).
The acrosome reaction induced with A23187 was associated with an increase in intracellular cAMP levels and PKA activity (Figure 3B and C). However, sperm PDE activity was not modified by the treatment of spermatozoa with A23187 (Figure 3A), emphasizing again the probable role of AC as the main regulator of intracellular cAMP levels in spermatozoa. A23187 triggers the influx of calcium, an activator of sperm AC (Rojas et al, 1992), which could possibly explain the increase in cAMP levels observed.
Sperm PDE activity was not modified by capacitation or acrosome reaction (Figures 1,2,3). We could hypothesize that this is due to the presence of already activated forms of PDE1, such as PDE1A (calcium- and calmodulin-dependent PDE), in human spermatozoa (Lefièvre et al, in press). We reported that PDE activity in whole-sperm extract or after partial purification by anion-exchange chromatography was not stimulated by the addition of calcium or calcium plus calmodulin and that the binding between human sperm PDE1A and calmodulin is stronger than that observed in other cell types, since strong chelators (ethyleneglycoltetraacetic acid [EGTA] + EDTA) were not able to dissociate this complex (Lefièvre et al, in press). These results also suggest that net cAMP concentration is probably under the control of AC, since PDE activity is constant during sperm capacitation and acrosome reaction.
Enzyme diffusion is not an efficient method for targeting intracellular signals via their specific enzymes. It is becoming clear that signaling events occur in spatially discrete compartments, in specific regions of most types of cells, and in spermatozoa (Travis and Kopf, 2002) through the use of molecular anchors, scaffolds, or adaptor proteins (Pawson and Scott, 1997). AKAPs were first identified for tethering PKA to specific cellular locations (Feliciello et al, 2001). In spermatozoa, AKAP82, AKAP110, and AKAP220 were identified (Feliciello et al, 2001), and immunolocalization identified C as well as RI and RII subunits of PKA in the flagellum and the head of spermatozoa from different species (Pariset and Weinman, 1994; Vijayaraghavan et al, 1997; Visconti et al, 1997). Moreover, it is now recognized that some AKAPs have the ability to bind and anchor signaling enzymes other than PKA, such as PKC, calcineurin (Feliciello et al, 2001), and even PDE4 (Dodge et al, 2001). Therefore, AKAPs provide a precise control of signal transduction complexes in discrete regions and cellular compartments of the cells, facilitating the propagation of second messenger signals by activating specific pools of protein kinases.
In spermatozoa, the generation of cAMP also appears to be compartmentalized. sAC associates with organelles (Travis and Kopf, 2002) so that the production of cAMP is localized precisely at the points where it is needed and does not stimulate other nearby pathways. Furthermore, we recently observed that PDE1A was localized in the equatorial segment and flagellum and that PDE3A was localized in the flagellum (Lefièvre et al, in press) of human spermatozoa. The presence of AKAPs, and possibly of other anchoring/scaffolding systems, as well as the specific localization of enzymes such as AC and PDEs in spermatozoa, emphasizes the importance of the local control that these very compartmentalized cells have over the enzymatic system composed of cAMP/PKA, AC, and PDEs. It is then possible that the intracellular levels of cAMP measured in whole human spermatozoa (Figures 1 and 3) are not representative of the cAMP present in precise compartments and involved in specific aspects of sperm functions. Visconti et al (1997) previously suggested that sperm PKA activity does not always correlate with increases in cAMP concentrations and, likewise, that increases in cAMP levels do not always correlate directly with PKA activation. Furthermore, cAMP can induce events that are independent of PKA, such as the regulation of the cyclic nucleotide gated ion channel or guanine exchanging factors (Skalhegg and Tasken, 2000), and PKA can be activated independently of cAMP—for example, by proteolysis of the R subunit as observed in Aplysia sensory neurons (Greenberg et al, 1987; Hedge et al, 1993) or by activation of the C subunit caused by degradation of the inhibitor of kappa B protein as observed in rabbit lung (Zhong et al, 1997).
In summary, we report for the first time that there is an increase in PKA activity during human sperm capacitation and that this increase does not correlate with cAMP levels. On the other hand, the increase in PKA was associated with an increase in intracellular levels of cAMP during the acrosome reaction induced by A23187. Taken altogether, these data could suggest that the levels of bicarbonate and cAMP needed for human sperm PKA activation are low and/or that the local concentration of cAMP in sperm compartments may be different.
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Acknowledgments |
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Footnotes |
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 K. Bedu-Addo, L. Lefievre, F.L.C. Moseley, C.L.R. Barratt, and S.J. Publicover Bicarbonate and bovine serum albumin reversibly 'switch' capacitation-induced events in human spermatozoa Mol. Hum. Reprod., September 1, 2005; 11(9): 683 - 691. [Abstract] [Full Text] [PDF]
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F.L.C. Moseley, K.N. Jha, L. Bjorndahl, I.A. Brewis, S.J. Publicover, C.L.R. Barratt, and L. Lefievre Protein tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction are enhanced in IVF media: an effect that is not associated with an increase in protein kinase A activation Mol. Hum. Reprod., July 1, 2005; 11(7): 523 - 529. [Abstract] [Full Text] [PDF]
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C. O'Flaherty, E. de Lamirande, and C. Gagnon Reactive Oxygen Species and Protein Kinases Modulate the Level of Phospho-MEK-Like Proteins During Human Sperm Capacitation Biol Reprod, July 1, 2005; 73(1): 94 - 105. [Abstract] [Full Text] [PDF]
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H. Breitbart, G. Cohen, and S. Rubinstein Role of actin cytoskeleton in mammalian sperm capacitation and the acrosome reaction Reproduction, March 1, 2005; 129(3): 263 - 268. [Abstract] [Full Text] [PDF]
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L. Liguori, E. de Lamirande, A. Minelli, and C. Gagnon Various protein kinases regulate human sperm acrosome reaction and the associated phosphorylation of Tyr residues and of the Thr-Glu-Tyr motif Mol. Hum. Reprod., March 1, 2005; 11(3): 211 - 221. [Abstract] [Full Text] [PDF]
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M. Luconi, I. Porazzi, P. Ferruzzi, S. Marchiani, G. Forti, and E. Baldi Tyrosine Phosphorylation of the A Kinase Anchoring Protein 3 (AKAP3) and Soluble Adenylate Cyclase Are Involved in the Increase of Human Sperm Motility by Bicarbonate Biol Reprod, January 1, 2005; 72(1): 22 - 32. [Abstract] [Full Text] [PDF]
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 L. Liguori, M. G. Rambotti, I. Bellezza, and A. Minelli Electron Microscopic Cytochemistry of Adenylyl Cyclase Activity in Mouse Spermatozoa J. Histochem. Cytochem., June 1, 2004; 52(6): 833 - 836. [Abstract] [Full Text] [PDF]
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C. O'Flaherty, E. de Lamirande, and C. Gagnon Phosphorylation of the Arginine-X-X-(Serine/Threonine) motif in human sperm proteins during capacitation: modulation and protein kinase A dependency Mol. Hum. Reprod., May 1, 2004; 10(5): 355 - 363. [Abstract] [Full Text] [PDF]
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K. Mitra and S. Shivaji Novel Tyrosine-Phosphorylated Post-Pyruvate Metabolic Enzyme, Dihydrolipoamide Dehydrogenase, Involved in Capacitation of Hamster Spermatozoa Biol Reprod, April 1, 2004; 70(4): 887 - 899. [Abstract] [Full Text] [PDF]
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