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From the Institute for Reproductive Medicine, School of Veterinary Medicine Hannover, Hannover, Germany.
| Correspondence to: Dr Anna M. Petrunkina, Institute for Reproductive Medicine, School of Veterinary Medicine Hannover, Bünteweg 15, 30559 Hannover, Germany (e-mail: anna.petrounkina{at}tiho-hannover.de). |
| Received for publication September 13, 2002; accepted for publication December 5, 2002. |
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Abstract |
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Key words: Kinetics, hyperactivation, membrane destabilization, sperm function, phosphorylation
The time course of capacitation in vitro varies among species, individuals, and subpopulations of spermatozoa within one ejaculate (Harrison, 1996), among epididymal and ejaculated spermatozoa, and, furthermore, it depends on the composition of the capacitation medium (Yanagimachi, 1994). The composition of media for capacitating canine spermatozoa has been a matter of discussion for several years. The commonly used medium is canine capacitation medium (CCM; Mahi and Yanagimachi, 1978), although capacitation could also be completed in other media (Guérin et al, 1999; Hewitt and England, 1999). The time course of capacitation in canine sperm was reported to be similar to values observed in other species; capacitation is completed within 3–7 hours of incubation (Mahi and Yanagimachi, 1976, 1978), but capacitation-related changes appear to occur within a shorter incubation period (Shimazu et al, 1992, Kawakami et al, 1993, Guérin et al, 1999; Rota et al, 1999). The mechanisms of action that promotes capacitation and hyperactivation are poorly understood at the molecular level, however, some molecular events of significance to the initiation of capacitation have been identified. It is known that the changes finally revealed by CTC are preceded by earlier events: bovine serum albumin (BSA)-mediated cholesterol efflux, changes in lipid architecture of the membrane, increased membrane fluidity, modulations in intracellular ion concentrations, hyperpolarization of the sperm plasma membrane and changes in its lectin-binding properties, and increased protein tyrosine phosphorylation (Ashworth et al, 1995; Frazer 1995; Visconti and Kopf, 1998; Visconti et al, 1999). These molecular events are required for the subsequent induction of hyperactivation and the AR.
The majority of earlier events is induced by the bicarbonate ion (Harrison et al, 1996 and references therein; Gadella and Harrison, 2000). Bicarbonate activates adenylate cyclase, leading to increases in intracellular cyclic adenosine monophosphate (cAMP) levels. Cyclic AMP affects various cellular functions, presumably by signaling through specific protein phosphorylation (Harrison, 1996). Tyrosine phosphorylation of the membrane proteins occurs during the first stages of capacitation and is apparently regulated by a cAMP-dependent protein kinase pathway. The time course of protein tyrosine phosphorylation, changes in localization of phosphorylated proteins on the membrane, and some underlying molecular mechanisms have been characterized for other species (ie, mice, cows, hamsters, pigs) (Visconti et al, 1995a,b; Galantino-Homer et al, 1997; Flesch et al, 1999; Kulanand and Shivaji, 2001; Petrunkina et al, 2001a), but little is known about these signaling events during canine sperm capacitation. In mice, the first changes in protein tyrosine phosphorylation could be observed after 45 minutes of capacitation time, whereas capacitation was completed some hours later (Visconti et al, 1995a,b).
Because above referred canine capacitation systems appeared to induce capacitation too rapidly, we aimed to apply a capacitation system that facilitates hyperactivation on one hand, and does not induce accelerated capacitation and cell death on the other. By achieving capacitation in such a system, we aimed to record the changes in tyrosine phosphorylation of dog sperm membrane proteins and relate them to hyperactivation. As apart of physiological studies on the capacitation process in dog sperm, a major goal of this study was to determine a precise kinetic description of the sequence of capacitation-related events and determine a strictly temporary relationship directly between dynamic aspects of functional changes. Using mathematical models for approximation of observed changes as an adequate analytical strategy (Petrunkina et al, 2001a) and a set of various parameters (CTC staining for recording the capacitation state, propidium iodide [PI] for monitoring cell death, changes in cytosolic calcium content and motility patterns, and tyrosine phosphorylation patterns) we will give supporting mathematical evidence that 1) there is a specific order in the appearance of protein tyrosine phosphorylation, and 2) that different stages of specific tyrosine phosphorylation signals are functionally related to different stages/complexes of positive destabilizing events.
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Materials and Methods |
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Media In preliminary experiments, the CCM (Mahi and Yanagimachi, 1978) and modified canine medium (mCCM; CCM with reduced bicarbonate (25 mM) replaced by NaCl to maintain osmolality) were used.
For capacitation treatment of canine sperm, a complete Tyrode bicarbonate medium (Harrison et al, 1993) was used, consisting of 96 mmol/L NaCl, 3.1 mmol/L KCl, 5 mmol/L glucose, 0.4 mmol/L MgSO4, 15 mmol/L NaHCO3, 2 mmol/L CaCl2, 0.3 mmol/L NaH2PO4, 1 mmol/L sodium pyruvate, 21.6 mmol/L sodium lactate, 3 mg/mL BSA, 20 µg/mL of phenol red, and 20 mmol/L HEPES pH 7.6. Before use, it was equilibrated at 39°C for 1 hour in a humidified atmosphere containing 5% CO2 (pH 7.4, 300 mOsm/kg). Sperm loaded with fluo-3-AM were diluted in a HEPES-buffered saline (HBS) consisting of 137 mmol/L NaCl, 10 mmol/L glucose, 2.5 mmol/L KOH, 20 mmol/L HEPES, 1 mg/mL polyvinyl alcohol, and 1 mg/mL polyvinyl pyrrolidone (pH 7.4, 300 mOsm/kg; see Harrison et al, 1993). As washing medium, sucrose wash medium (SWM) was used, consisting of 200 mmol/L sucrose, 10 mmol/L NaCl, 10 mmol/L glucose, 2.5 mmol/L KOH, 20 mmol/L HEPES, 0.5 mg/mL polyvinyl alcohol, and 0.5 mg/mL polyvinyl pyrrolidone (Harrison et al, 1993). After preparation, all media were passed through a 0.2-µm single-use filter unit (Minisart Sartorius, Germany).
Sperm Preparation for the Basic Experiments Ejaculates were collected from 6 healthy fertile beagle dogs of the Institute's colony (aged 2–6 years) and used within 1 hour after collection. The samples were collected by digital manipulation in fractions (presecretion, sperm-rich fraction, sperm-poor fraction, and sperm-free fraction). The sperm-rich fraction was used for experiments. Sperm quality estimation including evaluation of sperm concentration, sperm motility, and assessment of morphological sperm alterations were performed as previously described (Petrunkina et al, 2001b) according to a classification described by Krause (1966). All ejaculates were of sufficient quality as judged by conventional spermatological parameters (Günzel-Apel et al, 1994). After collection and quality estimation, the semen was diluted in HBS to a concentration of about 1–1.5 x 108 cells/mL. Aliquots (4 mL) were washed through a two-step gradient of 35% and 70% iso-osmotic Percoll-saline (Harrison et al, 1993). After removal of the supernatant layers, the loose sperm pellet was resuspended in residual 70% Percoll (about 1 mL).
Experimental Design
Preliminary Experiments—
Sperm samples were incubated in CCM and mCCM for up to 6 hours. CTC
patterns evaluated as described below and membrane integrity evaluated by PI
were determined at 3, 60, 120, 180, 240, and 360 minutes and compared.
Furthermore, the CTC patterns and membrane integrity were compared for sperm
suspensions incubated in modified CCM and Tyrode medium at the same sampling
points.
Capacitational Changes (Experimental Series I)—
A total of 4 ejaculates from 4 different beagle dogs was used for this set
of experiments. Sperm were incubated in bicarbonate Tyrode medium at a
concentration of 
0.5 x 108 cells/mL under 5%
CO2 conditions at 39°C for 360 minutes. Evaluation of the
capacitation state of the sperm population was performed essentially as
previously described (Frazer et al
1995, Wang et al,
1995) and CTC patterns were evaluated at each sampling point (3,
30, 60, 90, 120, 180, and 360 minutes). Briefly, 20 µL of the incubated
sperm suspension was added to the equal volume of the freshly prepared
staining solution (130 mM NaCl, 20 mM Tris-HCl, 5 mM cysteine, and 0.515 mg/mL
chlortetracycline). At least 200 sperm were counted. Sperm motility (total
motility, straight line velocity [VSL], average path velocity [VAP], and
curvilinear velocity [VCL]) was recorded and evaluated as described below.
Membrane Integrity Changes(Experimental Series II)— A total of 6 ejaculates from 6 different dogs was used for this set of experiments. The membrane integrity was evaluated using PI staining (Harrison and Vickers, 1990). Percoll-washed sperm were added to the bicarbonate Tyrode medium containing 10 µL of the PI stock solution (concentration 0.5 mg/mL) and incubated as described above (sperm concentration about 5 x 107 sperm/mL, final concentration of PI, 5 µg/mL). The PI fluorescence was observed using a 546-nm excitation filter. At least 200 sperm were counted. Sperm motility was recorded as described below.
Tyrosine Phosphorylation (Experimental Series III)— Spermrich fractions from 5 different dogs were used for this experimental series (a total of 14 ejaculates). Multiple samples from the same animal were tested in this assay (2–4 ejaculates). Sperm were capacitated in Tyrode medium as described above, and air-dried smears were prepared at each sampling point (3, 30, 60, 90, 180, 240, and 360 minutes). The preparations were fixed in methanol for 10 minutes, the nonspecific binding was blocked by incubation overnight in 200 µL of blocking solution (50% goat serum and 0.1% Triton X-100 in phosphate-buffered saline [PBS]) at 37°C. After washing in wash buffer (0.1% Triton X-100 in PBS), 15 µL of the stock solution (50 µg/mL in antibody buffer consisting of 1% goat serum and 0.1% Triton X-100 in PBS) of the primary antibody mouse immunoglobulin G (IgG) antiphosphotyrosine (clone 4G10, Boehringer-Mannheim, Mannheim, Germany) were added. The preparations were incubated for 3 hours at 37°C in the wet chamber. After consecutive washing (3 x 10 minutes in wash buffer), 80 µL of the Cy3-conjugated anti-mouse IgG (from goat) solution (Jackson ImmunoResearch Laboratories, West Grove, PA; diluted 1:400 in antibody buffer) was added, and the preparations were incubated for 1 hour at 37°C. After final washing (3 x 10 minutes in wash buffer) the samples were dried and mounted in mounting medium (PBS/glycerin; 1:9). The Cy3-fluorescence was observed with fluorescence microscopy (excitation filter 546 nm) and at least 200 sperm were evaluated. The photographs were collected, saved, and edited using a microscope digital camera system (Olympus DP50; Olympus Optical Company GmbH, Hamburg, Germany) and Analysis 3.0 Software (Soft Imaging System GmbH, Münster, Germany).
To check the specificity of binding, two control series were performed for each of 5 experiments in this set. In the first series, the preparations were incubated only with the secondary antibody. In the second series, the primary antibody was preincubated with saturated ortho-phosphotyrosine solution for 1 hour at 37°C. The antityrosine/orthophosphotyrosine solution was added to the slides instead of the primary antibody.
Cytosolic Calcium Changes (Experimental Series III)—Sperm-rich fractions of ejaculates from 3 dogs processed and prepared as described above were used. Changes in cytosolic calcium content of the total cell population were recorded using the fluorescent probe fluo-3-AM (Molecular Probes, Leiden, The Netherlands) according to the method described by Harrison et al (1993). Cell suspensions (1.5 x 108 cells/mL, approximately 1 mL) were loaded with fluo-3-AM (final concentration 5 µM) in 15-mL conical plastic tubes (light-protected) at room temperature. After 10 minutes of incubation, 4 mL of HBS was added and the diluted suspension was incubated for an additional 20 minutes. This suspension was layered over 6 mL of sucrose wash medium, centrifuged as described by Harrison et al (1993), and then capacitated in Tyrode medium as described above. At each sampling point, the sperm sample was placed on a slide, covered with a coverslip, and observed with a Zeiss Axiovert 35 fluorescence microscope (Zeiss, Jena, Germany). The percentage of fluorescent cells (sperm with entire head and tail fluorescence) was evaluated by counting (at least 200 sperm).
Motility Changes— Four sets of semen samples (n = 4, n = 6, n = 6, and n = 3, respectively for 4 experimental series as described above) were used to characterize motility changes of dog spermatozoa during incubation under capacitating conditions and to relate these changes to other parameters.
The motility measurements were performed using a computerassisted motility analysis system (Minitüb, Tiefenbach, Germany). Samples were taken at each sampling point as described above. Eight microliters of sperm were placed in a Cell Chamber (MTM Mika Chamber, Minitüb) on a microscope. The tracks of at least 200 sperm were recorded and evaluated. On average, 4 images were evaluated in 32 frames per analysis at 20-millisecond intervals. The setting for canine sperm was chosen according to that described by Günzel-Apel et al (1993). The Cell Motion Analyser 2.0 software (Medical Technologies, Montreux, Switzerland) was used for evaluation. The percentage of motile sperm and track velocities (VAP, VSL, and VCL) were evaluated for experimental series I and IV, and total motility for series II. For experimental series III, the percentage of hyperactivated sperm (classification made according to the CMA threshold setting for sperm VCL, linearity, and lateral head displacement (LHD): VCL > 80 µm/s and linearity < 65%, and LHD > 6.5 µm), head beating frequency and LHD were also evaluated.
Data Analysis
For descriptive statistics, the means and SEM were calculated. Normality of
the distributions was tested; for analysis of variance, the general linear
model procedure was used to determine the effects of incubation time and
individual dog on the recorded parameters. The relationships between tyrosine
phosphorylation patterns and motility patterns during capacitation were
examined by Pearson correlation analysis. Regression analysis (linear
regression) and nonlinear modeling (functional approximation) were used to
describe the kinetic changes in terms of relationship between time of
coincubation and parameters were recorded. After unknown parameters of the
model function fitting the data were estimated, first derivatives were
obtained from these by differentiation. The functions and their first
derivatives were then compared pair-wise
(Petrunkina et al, 2001a).
Differences were considered to be significant if the calculated probability of
their occurring by chance was less than or equal to 5% (P 
.05).
Excel software and the SAS program package (SAS Institute Inc, Cary, NC) were used for calculations in the above-described test procedures.
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Results |
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Comparing mCCM and Tyrode medium, more capacitated (B) and AR cells were observed at the end of the incubation period in mCCM (B = 25% and AR = 21% vs B = 16% and AR = 10% in Tyrode). Similarly, more dead cells were observed in mCCM than in Tyrode medium (51.7% vs 24.8%).
The rates of changes in all media were compared directly using linear
regression. The rate of changes in pattern B (capacitated cells) in CCM was
0.056, in mCCM 0.06, and in Tyrode medium 0.038
(R2 = 0.87, 0.95, and 0.97, respectively). The rate of
changes in AR cells in CCM was 0.096, in mCCM 0.063, and in Tyrode
0.02 (R2 = 0.96, 0.92, and 0.61, respectively).
Time Course of Capacitation-Related Events During Incubation in
Tyrode Medium
Changes in CTC Patterns and Membrane Integrity—
Spermatozoa incubated in Tyrode medium underwent capacitation-induced
changes during a 360-minute incubation period. Significant changes (P
< .05) were observed in percentages of F (uncapacitated), B (capacitated),
and AR (acrosome-reacted) patterns after 90 minutes of incubation. The changes
in the proportions of cells expressing patterns B and AR progressed
continuously during capacitation treatment and reached 24.25% and 13%,
respectively, after 360 minutes of incubation. The temporal changes of cell
proportions corresponding to these patterns fitted the linear regression
models (P < .05; Fig.
1).
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The first significant changes in the percentage of dead cells were observed after 90 minutes of incubation. Cell death progressed during the entire incubation period as a linear function of time (P < .05; Fig. 2), reaching a level of 21.2% of dead cells after 360 minutes of incubation. Cell death progressed more slowly than capacitation: the rate of cell death was about half the rate of capacitation-induced changes (r = 0.04 and r = -0.08 are the rates of change in the percentage of dead cells and noncapacitated cells, respectively).
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Motility Changes Accompanying Changes in CTC Patterns and Membrane Integrity— Capacitation and cell death were accompanied by a decrease in total motility. Similar to the two former parameters, the first significant changes occurred after 90 minutes of incubation and progressed linearly, reaching 25%–48% of motile cells after 360 minutes of incubation (Fig. 2, linear decrease of motility in experimental series II, graph for the linear decrease of motility in experimental series I not shown). The decrease in motility progressed with the rate r = -0.19 and -0.11 for experimental series I and II, respectively.
Oscillations of VCL, VSL, and VAP were observed during the first 120 minutes of incubation, followed by a logarithmical decrease after this sampling point (Fig. 3). The oscillations of all velocities proceeded in the same phase with a half cycle length of about 30 minutes. The decrease after 30 minutes was significant for VAP and VSL (93.5 and 83.8 at 3 minutes vs 65.3 and 53.8 µm/s at 30 minutes, P < .05), followed by a tangential increase after 60 minutes (>35%, P < .07), and a second periodic decrease after 90 minutes for VAP, VCL (P < .05), and VSL (P < .07) to values similar to those found at 30 minutes. These oscillations and the following decrease were observed in all tested dogs in the same phase.
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Changes in Cytosolic Ca2+ Content— Changes in cytosolic calcium ion content during incubation were characterized by estimating the percentage of cells with the fluo-3-AM fluorescence in the entire head and tail region. The intensity of fluorescence of fluo-3-AM increases in the presence of Ca2+. The use of fluo-3-AM with its large fluorescence response may be a sensitive means for detecting destabilizing changes in membranes of spermatozoa (Harrison et al, 1993). The changes in population of sperm cells with high internal calcium ion concentration were represented by a curve with a biphasic profile. A first increase in the percentage of cells with high calcium content occurred after 60 minutes of incubation (P < .05). The second increase began after 240 minutes of incubation and progressed to 360 minutes, reaching a value of 45%, which was significantly different from time points t <240 minutes (Fig. 4). The first increase in Ca2+ was accompanied by a decrease in motility and trajectory velocities.
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Changes in Tyrosine Phosphorylation of Membrane Proteins
Localization of Tyrosine Phosphorylation on Sperm Membrane and
Classification of Patterns—
Phosphorylation of the sperm proteins occurred both in the tail and head.
Two patterns of tail fluorescence were observed: fluorescence in the midpiece
and fluorescence in the entire tail (midpiece and entire tail; additional
staining in the principal piece and end piece;
Fig. 5). In all sperm, head
fluorescence had a granular-like character, differing, however in intensity
level from moderate with identifiable separate granules to a very high
intensity with an increased amount of granules, forming phosphorylated domains
within the membrane (Fig. 5).
These different patterns were combined with each other, resulting in 9 pattern
classes (Table 1). To analyze
the changes in tyrosine phosphorylation patterns and to order them in a
hierarchical sequence of capacitational events, different classification
models were used (Table 1).
Phosphorylation of tail and head were evaluated separately as well as in
combination with each other. Within two categories (nonphosphorylated
head/complete phosphorylated tail), the changes in single location and
intensity classes (from midpiece fluorescence, P10; to the entire tail
fluorescence, P20; and from the moderate granulated fluorescence, P21; to the
intensive cluster-like fluorescence, P22) were evaluated.
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Capacitation-Induced Changes in Tyrosine Phosphorylation— The percentage of nonphosphorylated spermatozoa decreased logarithmically as function of time (Fig. 6). The first significant changes occurred after 30 minutes (33.3% vs 59.9% at t = 0 minutes) and progressed toward 8.3% at 360 minutes. The percentage of cells with fluorescence localized on the head membrane without tail fluorescence decreased logarithmically during the incubation period (P < .05, Fig. 6), indicating that no additional head phosphorylation occurs before the tail became phosphorylated and that head-phosphorylated cells undergo rapid tail phosphorylation.
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An increase of the percentage of fluorescence pattern class E10 (phosphorylated proteins of the tail membrane without head membrane phosphorylation) was observed at 30 minutes (more than fourfold the initial value), which progressed up to 240 minutes of incubation (about sevenfold) and then decreased to 39.8% at 360 minutes. The temporal changes in this pattern class were fitted by a polynomial function (P < .05, Fig. 6). The sperm percentage in class E11 (both head and tail fluorescent) increased about 10-fold during incubation from 3 to 360 minutes. These changes also fitted a polynomial function (P < .05). The increase in E11 during the first 90 minutes ran parallel to the tail phosphorylation of nonphosphorylated sperm (E10), and the increase from 240 to 360 minutes in the E11 pattern class was complementary to the decrease in the class E10 during this incubation subperiod (Fig. 6).
There were significant influences of individual dogs on the portion values in classes E00, E01, and E10 (P < .001). The time course of tyrosine phosphorylation of the tail membrane proteins E10 for individual dogs is represented in Figure 7. In 2 dogs, the curve for time course of tail phosphorylation was biphasic, in 2 other dogs the increase was continuous and reached a plateau after 90 minutes, in the last dog, phosphorylation still progressed after 180 minutes.
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Over all time points, the major represented subclasses were P00, P01, P10, P11, P20, P21, and P22. The percentage of sperm in the classes P02 and P12 were consistently low (<5%) during incubation.
Correlation Between Tyrosine Phosphorylation and Motility— A correlation analysis between motility and tyrosine phosphorylation was performed for one experimental set in which motility and tyrosine phosphorylation were measured in parallel on the same ejaculates (see "Materials and Methods"). There was a significant positive correlation (P < .05) between the percentage of motile and forward motile cells and the percentage of cells without fluorescence (P00) or fluorescence localized in the midpiece (P10). Significant negative correlations (P < .05) were found between the percentage of motile and forward motile cells and the percentage of cells with fluorescence localized in the entire tail compartment (P20) (Table 2). Significant positive correlations were found between VCL, VSL, VAP, and the percentage of cells with fluorescence localized in the midpiece (P10). VCL, VAP, and the percentage of cells with fluorescence localized in the entire tail region (P20) correlated significantly negative. Lateral head displacement was negatively correlated with the percentage of cells with fluorescence localized in the entire tail region but nonfluorescent head (P20). There was also a negative correlation between beat cross-frequency (BCF) and the percentage of cells with entire tail/granulated head fluorescence (P21). Further correlations between fluorescence in the head region and motility parameters were not found.
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Figure 8a and b shows the temporal behavior of VCL vs T2 (entire tail phosphorylation) and VSL vs T1 (midpiece phosphorylation) for 2 individual ejaculates of 2 dogs. The course of curves for VCL and T2 is nearly complementary (decrease in velocity is accompanied by am increase in the percentage of spermatozoa with entire tail phosphorylation, illustrating negative correlation during incubation); the course of curves for VSL and T1 is almost parallel (increase/decrease in velocity is accompanied by an increase or decrease in midpiece phosphorylation, illustrating positive correlations).
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The time course of motility parameters differed from the time course in the first experimental series. There were slight increases in BCF, VAP, and VCL at 90 minutes, and increases in LHD at 30 and 180 minutes. However, no regular oscillations were observed. Analysis of motility changes for each individual dog and pair-wise comparison showed that the changes in velocities, BCF, and LHD occurred with a time phase shift.
Changes in Subclasses of Phosphorylation— In the next step, the sequential changes in subclasses of tyrosine phosphorylation (phosphorylation of midpiece and endpiece with nonphosphorylated head) were approximated. Figure 9 shows the time course of these phosphorylation processes. Phosphorylation of the midpiece (P10) increases rapidly and is nearly completed within 30 minutes, followed by an exponential decrease during the entire incubation period. This decrease is complementary to the increase in principal piece tyrosine phosphorylation (P20) during 30–90 minutes of incubation. After 90 minutes, phosphorylation of the end piece dominates and is maintained more or less at the same level. The few fluctuations are related to individual differences (individual data omitted). Between 240 and 360 minutes, a significant decrease was observed for all dogs tested in this set.
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For the spermatozoa with entire tail phosphorylation, the changes in the head membrane (P21 and P22) were analyzed separately. These changes count for essential changes in E11 pattern during second half of incubation (Fig. 6), whereas in the first half of incubation, the changes in E11 were noticed as a tail phosphorylation of the already head-phosphorylated spermatozoa (P01 and P02). The percentages of spermatozoa with granulated moderate fluorescence of the head (P21) and with cluster-like intensive fluorescence (P22) both increased linearly, reaching 27.5% and 12.8%, respectively, at the end of the incubation period (n = 3 dogs, n = 6 ejaculates). When plotted together with the graph of capacitation time course, recorded by CTC staining, the time courses of capacitation and AR seem to develop approximately at the same rate as head phosphorylation of sperm with phosphorylated tail membranes (Fig. 10).
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Discussion |
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Sperm path velocity, VCL, linearity, and LHD are used in most algorithms to characterize hyperactivated movement: higher values of VCL and LHD reflect both the proportion of hyperactivated spermatozoa and the vigor of their movement (Rota et al, 1999). Cyclical increases in VCL, VSL, and VAP observed during the first 3 hours appear to reflect the changes induced by bicarbonate. The cyclical-like character of velocity changes may be caused by a number of reasons. Stimulation of motility may be a consequence of bicarbonate-stimulated adenylate cyclase. In porcine spermatozoa, the initial rise in cAMP levels occurs very rapidly within 1 minute, and, following a decrease at 10 minutes, rises again, reaching a peak at 25 minutes (Harrison and Miller, 2000). This may explain the first periodic-like changes in velocities. Alternatively, hyperactivated motility may develop at different rates and with different phases for individual males, leading to artifactual increases and decreases. The latter possibility appears to be unlikely for the first experimental set, because the oscillate character of velocity changes was observed for all tested beagles. It can not be excluded that successively capacitating sperm subpopulations within one ejaculate develop hyperactivated motility at different rates resulting in cyclical-like behavior. However, the strong regularity of cycles during first 180 minutes indicates that the variation in hyperactivation rates cannot solely account for the observed changes, as continuously increasing proportions of cells with higher velocities would rather develop stochastically. The most likely hypothesis seems to be the combination of bicarbonate effects on intracellular cAMP levels and of heterogeneity of individual cells/cohorts response within one ejaculate to capacitating conditions. Differences between males can count for the absence of strong oscillations in the second motility experiment, because the phase differences for velocities and LHD changes were observed for different beagles.
In this study, the tyrosine phosphorylation of canine sperm during capacitation treatment was recorded. In mice, efflux of cholesterol from the sperm membrane during capacitation has been shown to enhance membrane fluidity and, consequently, to initiate further alterations such as reorganization of the sperm surface and tyrosine phosphorylation of a subset of sperm proteins (Visconti et al, 1995a,b; Visconti and Kopf, 1998). This tyrosine phosphorylation has been shown to be a capacitation-induced event in boar spermatozoa (Flesch et al, 1999). Little is known about tyrosine phosphorylation of dog sperm membrane proteins. Recent studies by Rigau et al (2002) demonstrated the dependence of tyrosine phosphorylation on glucose and fructose, both inducing a fast and intense increase in overall tyrosine phosphorylation of dog spermatozoa. In earlier studies by Tash and Means (1982), a relationship between enhanced phosphorylation of a number of specific proteins and cAMP-stimulated motility was found. However, neither the time course of changes in the phosphorylated proteins nor their relationship to hyperactivated motility assessed under fertilizing conditions was estimated until now for dog sperm, nor has appropriate kinetic evidence been given to reveal specific order in their appearance in other species.
During capacitation treatment, sperm proteins of tail and head compartments became phosphorylated as capacitation progressed. By using mathematical model, it is possible to approximate the proportion of changes in different phosphorylation classes to characterize their kinetics precisely and to compare different kinetics (Petrunkina et al, 2001a). Analysis of the model functions confirmed the observation that head phosphorylation occurs later than tail phosphorylation. Indeed, the percentage of cells showing fluorescence of the head region but not the tail decreased in a strong logarithmic fashion from the beginning to the end of the incubation period, giving evidence that no additional head phosphorylation occurs before the tail phosphorylation has occurred. In contrast, the proportion of cells showing fluorescence in the midpiece and entire tail increased continuously as a polynomial function of time up to 240 minutes. The increase in cells phosphorylated in both head and tail compartments during the first 90 minutes ran parallel to the tail phosphorylation of nonphosphorylated sperm (cf. Fig. 6), pointing to a conclusion that tail phosphorylation of nonphosphorylated cells and cells that had head-phosphorylated progressed at similar rates. After 240 minutes, tail phosphorylation decreased concomitantly with the increase of cells phosphorylated in both compartments. This indicates a continuous transformation from tail-phosphorylated cells to both head-and tail-phosphorylated cells at about 240 minutes of incubation, and demonstrates that the phosphorylation of head proteins is coordinated with tail phosphorylation. The tail phosphorylation process could be divided into 2 additives: by proportions of cells phosphorylated in the midpiece and by proportions of cells with the entire tail fluorescence. The former exhibit a rapid increase within 30 minutes of incubation and a following decrease, complementary to the increase in sperm with fluorescence of the entire tail (this transition of cells from the first category in the second category can be interpreted as successive phosphorylation process). These two phases correspond to different stages of earlier capacitational changes. The first phase, associated with phosphorylation of midpiece proteins, correlated with motility changes. The increase in midpiece fluorescence was accompanied by an increase in VCL, VAP, and VSL. Principal piece and end piece fluorescence was accompanied by a loss in velocities, LHD, and BCF. A missing correlation of motility parameters with the phosphorylation of the head proteins indicates that these two processes are not directly related. These results are similar to those reported for monkey epididymal sperm or hamster sperm, showing that the protein tyrosine phosphorylation status is related to the flagellar motion (Si, 1999; Si and Okuno, 1999; Yeung et al, 1999). Furthermore, head phosphorylation seems to complete the series of destabilizing events corresponding to the active "capacitation window." Two phases of head phosphorylation of the already tail-phosphorylated cells are developing in parallel. During incubation, a redistribution of fluorescence patterns occurred. Cells shifted from class H1 to class H2 (sperm with intensive cluster-like fluorescence). A similar shift of cell proportions within two classes of head fluorescence during incubation under capacitating conditions was observed for boar sperm in our previous study (Petrunkina et al, 2001a); thus it is likely to be a general phenomenon. The comparison of kinetics for these changes with the kinetics of changes in CTC patterns B and AR suggest that sperm with entire tail and granulated head phosphorylation can be classified as capacitated, cells with cluster fluorescence as just undergoing the AR (time course of the changes in tyrosine phosphorylation and CTC patterns kinetics are nearly equivalent). Unlike the responses in tyrosine phosphorylated proteins in monkey sperm, which were in great contrast to the responses in the AR (Yeung et al, 1999), this last stage of protein tyrosine phosphorylation response in dog sperm appears to be immediately involved in the precipitation of the AR. The upper limit of a window of destabilization (Harrison, 1996) is reached after an intense cluster-like phosphorylation of head membrane proteins has happened. At this threshold, a spontaneous AR is possible. Beyond this limit, the continuing degeneration leads to cell death. That the percentage of cells in P11 was maintained at a rather low level and that almost no cells were observed in classes P02 and P12 gives also additional indication that the final phosphorylation steps (corresponding to completion of the sequence of capacitational changes leading to spontaneous acrosome reaction) do not occur before the entire tail became phosphorylated.
The correlations between motility and tyrosine phosphorylation indicate that the control of the phosphorylation state of sperm proteins can be involved in the mechanisms that regulate motility. Tash and Means (1982) found that motility of dog spermatozoa was regulated by cAMP and calcium. Particularly, cAMP stimulated motility and tyrosine phosphorylation, whereas calcium inhibited motility and did not affect phosphorylation of the major axonemal protein, tubulin. On the other hand, the cAMP-stimulated phosphorylation of other sperm proteins still occurred, but at a reduced rate in the presence of calcium ions. This indicates the importance of bicarbonate for initiation of capacitation-related protein tyrosine phosphorylation as an adenylate cyclase-stimulating agent. The dependence of tyrosine phosphorylation on bicarbonate and calcium has already been demonstrated in other species. For hamster sperm it could be shown that an absence of either Ca2+ or NaHCO3 in the capacitation medium delayed the protein phosphorylation, but without both there was a significant decrease in the phosphorylation of the proteins throughout the period of capacitation (Kulanand and Shivaji, 2001). In earlier studies, Tash and coworkers (1984, 1986) have shown that activation of flagellar motility is dependent on a cAMP-dependent phosphorylation of a soluble sperm protein. The molecular mechanisms responsible for activation or deactivation of motility observed in the present work deserve a further study. A major role of a calmodulin-dependent protein phosphatase in the calcium-dependent regulation of flagellar motility can be ruled out from the results of Tash et al (1988). They found that inhibition of motility and phosphorylation by calcium was catalyzed by a calmodulin-dependent protein phosphatase. Dog sperm contained the subunits of this enzyme, localized both in the flagellum and head regions. Preincubation of purified phosphatase stimulated VCL and LHD (Tash et al, 1988). In this context, stimulation of motility during the first 30–90 minutes of capacitation treatment may be related to phosphatase activity, preventing end piece phosphorylation. The following decrease of motility parameters may be caused by changes in the activity of this enzyme.
Although there were individual differences in protein tyrosine phosphorylation, the same characteristic course of phosphorylation was observed in all dogs: primary phosphorylation of midpiece proteins, followed by principal piece and end piece phosphorylation, which was maintained for 1–2 hours before the succeeding phosphorylation of the head proteins occurred.
Concluding Remarks
The protein phosphorylation changes in canine sperm are coincident with
series of destabilizing events during incubation under fertilizing conditions:
hyperactivation, increase in cytosolic calcium levels, capacitation, and AR,
followed by a loss of motility and cell death. There is a specific order in
the signal appearance; head tyrosine phosphorylation is kept at a low level
before the two stages of tail phosphorylation (related to hyperactivation) are
completed. This mechanism may be important in vivo during sperm transit in the
female genital tract to ensure appropriate timing of full capacitation
coordinated with that of ovulation. As far as we know, this study is the first
to provide evidence that capacitation-associated protein tyrosine
phosphorylation is linked to hyperactivation in canine spermatozoa. As a
parameter reflecting the individuality of response of single males to
fertilizing conditions, the kinetics of phosphorylation is a good marker of
the capacitation process and may be useful for diagnostic purposes. The
identification and characterization of phosphorylated sperm proteins as well
as the estimation of mechanisms of signal propagation by tyrosine
phosphorylation still need to be studied.
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