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a Livestock Improvement Corporation, Hamilton, New Zealand
b Department of Biological Sciences, University of Waikato, Hamilton, New Zealand
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Apart from tyrosine phosphorylation of soluble proteins involved in regulation of motility, a number of proteins from whole-cell extracts also display a time-dependent increase in tyrosine phosphorylation under capacitating conditions. It has also been demonstrated that the inhibition of phosphodiesterase induces an increase in tyrosine phosphorylation in ejaculated bovine sperm during capacitation [7].
There is growing evidence that reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radical, and superoxide, apart from being detrimental to sperm, also play a physiological role in sperm capacitation [8–12]. The increase in protein phosphorylation during capacitation and the role of ROS in capacitation are probably interrelated. Under some conditions, capacitation is mediated by ROS-regulated protein tyrosine phosphorylation [13, 14]. Generally, oxidizing conditions were found to promote tyrosine phosphorylation and stimulate sperm function, whereas an opposite effect was observed under reducing conditions when sperm were incubated in a medium promoting capacitation [13]. ROS also regulate protein tyrosine phosphorylation in other cells such as H-35 rat hepatoma cells [15].
We hypothesized that there would be changes in tyrosine phosphorylation of proteins from whole-cell sperm extracts and that the pattern of change in protein tyrosine phosphorylation would be significantly affected by both phosphodiesterase inhibition and oxygen tension in the incubation medium. To test this, sperm were incubated with a phosphodiesterase inhibitor and also under both aerobic and anaerobic conditions. Changes in the pattern of protein tyrosine phosphorylation in whole-cell extracts from sperm incubated under conditions described above were determined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Analytical grade chemicals were used in this study. Citric acid and D-glucose were obtained from Riedel-de Haën AG (Seelze, Germany). EGTA, theophylline, sodium orthovanadate, O-phospho-DL-tyrosine, n-[p-aminobenzenesulfonyl] acetamide (sulfacetamide) sodium salt, streptomycin sulfate, and penicillin were purchased from Sigma Chemical Co. (St. Louis, MO). BSA was acquired from Boehringer Ingelheim Bioproducts Partnership (Heidelberg, Germany). Catalase was extracted from livers of 18-mo-old, disease-free bulls owned by Livestock Improvement (Hamilton, New Zealand) by the method outlined by Summer and Myrback [16]. The gas mixture of 5% hydrogen and 95% nitrogen was purchased from British Oxygen Co. (Hamilton, New Zealand). Sodium chloride was purchased from Scientific Supplies Ltd. (Auckland, New Zealand). Tris was purchased from U.S. Biochemical (Cleveland, OH). Anti-phosphotyrosine monoclonal antibody 4G10 was purchased from Upstate Biotechnology (Lake Placid, NY). Complete protease inhibitor cocktail tablets were purchased from Boehringer Mannheim, GmbH (Mannheim, Germany). Pre-cast continuous gradient (10%-20%) gels for SDS-PAGE, supported nitrocellulose membrane, Laemmli sample buffer, 2-mercaptoethanol, Silver Stain Plus Kit, and polyoxyethylene sorbitan monolaurate (Tween 20) were purchased from Bio-Rad Laboratories (Hercules, CA). Horseradish peroxidase-conjugated anti-mouse sheep antibody was purchased from Amrad-Silenus (Melbourne, Australia). Enhanced chemiluminescence (ECL) Western blot detection reagents were purchased from Amersham International Plc (Buckinghamshire, England). X-OMAT AR Scientific Imaging Film was bought from Eastman Kodak (Rochester, NY). Propidium iodide and SYBR-14 were purchased as FertiLight Sperm Viability Kit from Molecular Probes (Eugene, OR). All other chemicals were purchased from BDH Chemicals Ltd. (Poole, England).
Preparation of Diluents
The semen diluent used was 14G buffer [17], which consisted of trisodium citrate, 68 mM; glycine, 133.20 mM; D-glucose, 16.7 mM; dipotassium hydrogen orthophosphate 3-hydrate, 35 mM; glycerol, 162 mM; n-hexanoic acid, 2.15 mM; citric acid, 0.66 mM; magnesium chloride, 2.2 mM, and calcium chloride 2-hydrate, 2.5 mM; penicillin and streptomycin sulfate, each 1.25 megaunits per liter; sodium sulfacetamide, 0.1 µM; catalase, 800–1000 IU/ml. When standard incubation conditions were used, the diluent was purged with nitrogen for 30 min to reduce oxygen concentration. For experiments requiring anaerobic incubation conditions, the diluent was purged with nitrogen for 30 min and placed inside an anaerobic chamber (Coy Laboratory Products, Grass Lake, MI) to equilibrate overnight with a gas mixture of 5% hydrogen and 95% nitrogen. The chamber was equipped with an air lock to allow transfer of equipment and solutions without contaminating the internal atmosphere with air. A positive pressure was maintained inside the chamber, limiting the possibility of air access to the interior. Any residual oxygen inside the chamber was reduced to water by hydrogen in the gas mixture, aided by a palladium catalyst. Absence of oxygen in the atmosphere in the anaerobic chamber was confirmed by use of Anaerobic Indicator BR55 (Unipath Limited, Basingstoke, England). When sperm were incubated under aerobic conditions, the diluent was not gassed at all.
For motility analysis, BSA was added to the diluent to a final concentration of 1% to prevent sperm from adhering to glass slides.
Semen Collection and Preparation
Semen was collected from mature dairy bulls (Bos taurus) using an artificial vagina, and ejaculates were assessed visually for percentage motile sperm under an Olympus (Tokyo, Japan) BH2 microscope equipped with phase-contrast optics at x125 magnification. The concentration of sperm in the semen was measured using a Semen Concentration Photometer (Instruments Medecine Veterinaire, L'Aigle-Cedex, France). Only ejaculates containing more than 75% motile sperm were used in the experiments. For all treatments, semen of one ejaculate from each bull was initially diluted at 32°C in an anaerobic diluent, to a concentration of 240 x 106 sperm/ml, and allowed to cool to ambient temperature (18–20°C). It was then diluted again to a final concentration of 20 x 106 sperm/ml, in either aerobic, nitrogen-gassed, or anaerobic diluent at ambient temperature. Diluted sperm were incubated at ambient temperature in sealed 100-ml sterile Schott (Schott Glaswerke, Mainz, Germany) bottles wrapped in aluminum foil under aerobic, anaerobic, or nitrogen-gassed conditions, depending on the experiment.
For incubation under aerobic conditions, the bottles in which semen was incubated were opened daily and aerated by gentle shaking. Under nitrogen-gassed conditions, the headspace of bottles was filled with dry nitrogen. The anaerobic and nitrogen-gassed incubation bottles were shaken with the caps sealed. For incubation of sperm under anaerobic conditions, the sperm were suspended in an anaerobic medium and placed inside an anaerobic chamber as described above.
Assessing Percentage of Sperm with Intact Plasma Membrane
Aliquots of sperm were stained with FertiLight Sperm Viability Kit according to the manufacturer's instructions. The sperm with intact plasma membranes ("live") fluoresced green whereas sperm with damaged plasma membrane ("dead") fluoresced red. The proportion of sperm with intact plasma membranes was quantified by flow cytometry using a Becton Dickinson (San Jose, CA) fluorescence-activated cell sorter analytical flow cytometer with a built-in air-cooled argon ion laser. From each aliquot, a duplicate sample of 10 000 stained spermatozoa was analyzed for the log of its green and red fluorescence. Green fluorescence events were collected through a 515-nm band pass filter (FL1); red fluorescence events were collected through a 630-nm band pass filter (FL3). Both fluorochromes were excited with a 488-nm laser light. The results were analyzed on a Macintosh Quadra 650 computer with CellQuest Becton Dickinson software. Distributions of green- and red-stained sperm subpopulations were acquired as dot-plot cytograms for log FL1 and log FL3. Subpopulations on the cytograms were isolated and quantified using quadrant analysis. Percentage of live sperm was calculated by dividing number of events collected in the green fluorescence-only quadrant by the sum of numbers of cells collected in green-only, red-only, and double green and red fluorescence quadrants, and plotted against time of incubation.
Measurements of Sperm Velocities and Percentage of Motile Sperm
The percentage of motile sperm and subjective velocity score (SVS) were assessed visually in six observation fields under x10 negative phase-contrast objective. The SVS graded sperm motility from 0 (immotile sperm) to 10 (rapid forward motility).
For computer-aided sperm analysis (CASA), aliquots were drawn from incubations on Days 0, 1, 3, 5, 7; six video recordings of 1.5-min duration were made with an Olympus BX50 microscope attached to a JVC TKS-350 video camera (Victor Company of Japan, Ltd., Yokohama, Japan) using a x10 negative phase objective. Disposable semen analysis chambers of 20-µm depth (Fertility Technologies, Natick, MA) were used for CASA; volume of the analyzed drop was 7 µl. All the recordings were conducted on a warm stage set at 37°C ± 1°C. Curvilinear velocities (VCL) and straight-line velocities (VSL) were analyzed from the video recordings using the Hobson sperm tracker (Hobson Tracking Systems Ltd., Sheffield, UK). The analyzer was calibrated for linear distances using a haemocytometer grid (Neubauer; Fortuna, Wertheim, Germany). Velocities of all the sperm tracks recorded in each 1-min time window were measured using the following analysis settings: minimum tracking time 0.3 sec, maximum tracking time 40 sec, search radius 18.50 µm, framing rate 50 Hz. Median values of VCL and VSL were obtained.
Washing of Sperm Cells to Remove Seminal Plasma Proteins Prior to Protein Extraction
To 35 ml of suspension containing 20 x 106 sperm cells/ml, 10 ml of Tris-buffered saline (TBS) consisting of 150 mM NaCl, 25 mM Tris (pH 7.4), supplemented with 1 mM sodium orthovanadate, and 5 ml of MilliQ water (Millipore, Bedford, MA) was added. This suspension was centrifuged at 3000 x g for 15 min. The supernatant was carefully aspirated, avoiding removal of any sperm cells and leaving 5 ml of the buffer above the pellet. Sperm were subsequently resuspended in 50 ml of TBS supplemented with 1 mM sodium orthovanadate and centrifuged as before. The pellet, after aspiration of the supernatant, was resuspended with 1 ml TBS, carefully transferred to a 1.5 ml Eppendorf (Hamburg, Germany) tube, and centrifuged again at 16 000 x g for 5 min. The supernatant was carefully aspirated, and the pellet was frozen and stored in liquid nitrogen until extraction of the proteins.
Extraction of Soluble Proteins from Sperm Pellets
Soluble protein extracts were obtained using a method described by Vijayaraghavan et al. [3] with minor modifications. The frozen sperm pellets were thawed and resuspended in 600 µl of ice-cold homogenization buffer (10 mM Tris, pH 6.7, 1 mM EGTA, 1 mM sodium orthovanadate, 3.2 mg/ml of Complete protease inhibitors) and sonicated using a microprobe (Sonicator W380 Heat Systems-Ultrasonic, Farmingdale, NY), with four 5-sec bursts, with power output set at 4 and percentage duty cycle at 90%. The sperm sonicate was centrifuged at 16 000 x g for 15 min at 4°C, and the collected supernatant was centrifuged again at 100 000 x g for 1 h at 4°C. A volume of 400 µl of the 100 000 x g supernatant, called the soluble fraction in the rest of this report, was mixed and boiled for 5 min with 800 µl of Laemmli sample buffer containing 5% 2-mercaptoethanol immediately before SDS-PAGE.
Whole-Cell Protein Extracts
Whole-cell protein extracts were obtained as described by Galantino-Homer et al. [7]. The frozen sperm pellet obtained from the washing procedure to remove seminal plasma (described above) was thawed and resuspended in 800 µl of Laemmli sample buffer without 2-mercaptoethanol. It was then boiled for 5 min and centrifuged at 16 000 x g for 10 min. The supernatant was recovered, and 2-mercaptoethanol was added to reach a final concentration of 5%. The mixture was then boiled for 5 min immediately before SDS-PAGE.
SDS-PAGE and Western Blotting
Proteins of whole-cell sperm extracts (5 µl) or of the soluble fraction (35 µl) were separated by SDS-PAGE, using Modular Mini-PROTEAN Electrophoresis System (Bio-Rad) under constant voltage conditions (150 V) until the tracer front ran to the edge of the gel. The proteins were then electrophoretically transferred to supported nitrocellulose (0.2 µm) membrane using a wet transfer system (100 V, 1 h). The nonspecific protein binding sites on the membrane were then blocked with 5% BSA in TBS for 1 h at room temperature. The membrane was then incubated overnight at 4°C with anti-phosphotyrosine monoclonal antibody 4G10 at 1:3000 dilution (0.3 µg/ml) in TBS containing 0.1% Tween 20 (TTBS) and 1% BSA.
After three 15-min washes with TTBS containing 1% BSA, the blots were incubated at room temperature for 2 h with sheep anti-mouse immunoglobulin antibody conjugated with horseradish peroxidase at 1:3000 dilution (0.3 µg/ml) in TTBS. The blots were then subjected to three 15-min-long washes in 40 ml of TTBS, and the protein bands containing phosphotyrosine residues were detected with an ECL detection system. Parallel gels were run to determine relative protein loading in each well [18]. These gels were silver-stained to determine total protein, according to manufacturer's instructions.
Quantification of Tyrosine Phosphorylation and Total Protein Loading
The silver-stained gels and the ECL contact photographs of tyrosine-phosphorylated protein bands on Western blot membranes were scanned with a Model GS-700 Imaging Densitometer (Bio-Rad) using reflectance mode at 600 dpi resolution. Scanned images were analyzed using a Power Macintosh computer equipped with Molecular Analyst software (Bio-Rad).
To estimate variations in total protein concentrations between samples loaded on different lanes, rectangular boxes were drawn around four major protein bands in each loading well lane on the digital image of the silver-stained gel. The optical density of all the pixels within the rectangle was integrated and adjusted for background (optical density of the region on the gel image without protein loaded). The highest value of integrated optical density of the same molecular weight proteins from different loading lanes was assigned a value of one. Relative protein concentration of the same molecular weight proteins from other loading lanes was calculated as a fraction of this value. For each loading lane, representing a different day of sperm incubation, a mean value of the relative protein loading of all four measured protein bands was calculated and was termed the relative protein loading. Relative protein loading did not vary by more than 25% between samples, and variations were random without a time-dependent trend.
To quantify changes in protein tyrosine phosphorylation of different protein bands, rectangular boxes were drawn around bands on scanned digital images of ECL contact photographs of Western blots, and adjusted integrated optical density values were obtained for each band. These values are proportional to the degree of tyrosine phosphorylation of the protein bands. Integrated optical density values of bands from Western blots were divided by the relative protein loading value of the same sperm sample. Resulting values of normalized phosphotyrosine signal (PY signal) in arbitrary units are displayed in figures under blot images.
Incubation of Sperm for Extended Periods at Ambient Temperature
Three in vitro incubation experiments were carried out. In experiment 1, semen was collected and diluted as described above and incubated under standard conditions (nitrogen-gassed diluent) for 8 days. The percentage of sperm with intact plasma membrane was determined by flow cytometry on Days 0, 2, 5, and 8 after ejaculation. On the same days, aliquots of sperm were incubated in the medium containing 0 or 5.5 mM theophylline at 37°C for 30 min, and SVS and percentage of motile sperm were assessed. The cells were then harvested by centrifugation from both treatments, washed, rapidly frozen and stored in liquid nitrogen until protein extraction. The degree of tyrosine phosphorylation of soluble and whole-cell proteins extracted from the frozen spermatozoa was determined by Western blotting. Velocities of theophylline-stimulated and nonstimulated spermatozoa were measured using CASA on Days 0, 1, 3, 5, and 7 of incubation.
In the second experiment, semen was divided after collection and dilution; one half was incubated under aerobic and the other under anaerobic conditions. SVS, percentage motile sperm, and percentage of sperm with intact plasma membrane were assessed on Days 0, 4, 6, 8, 10, and 12 after ejaculation for sperm incubated under aerobic and anaerobic conditions. On the same days, sperm were washed and frozen as before for assessment of the state of protein tyrosine phosphorylation by Western blotting.
In the third experiment, semen was divided after collection and dilution and incubated under anaerobic and aerobic conditions. On Day 5 of in vitro incubation, one half of the semen incubated under anaerobic conditions was transferred from an anaerobic chamber to an aerobic environment. Similarly, one half of the semen incubated under aerobic conditions was transferred to the anaerobic chamber. On Days 6, 9, and 12, sperm cells were harvested, washed, and frozen as before, from the four treatments (i.e., semen incubated throughout under anaerobic conditions, semen transferred on Day 5 from an anaerobic to an aerobic environment, semen incubated throughout under aerobic conditions, semen transferred on Day 5 from an aerobic to an anaerobic environment). These samples were then used for assessment of the state of protein tyrosine phosphorylation by Western blotting.
Statistical Analysis of Results
The results from the Hobson sperm tracker were analyzed by a least squares ANOVA (SAS Institute, Cary, NC) fitting the effects of treatment (anaerobic and aerobic, theophylline-stimulated and nonstimulated) using median VSL and VCL as the dependent variables. The bull effects were treated as a random variable (n = 3), and time was treated as a covariate. Trend lines (linear or third-order polynomial) were fitted to obtain the best fit for the data shown in Figures 3, 8, and 9. The differences in means for all other results were subjected to a t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The percentage of motile sperm declined from around 85% on day of collection (Day 0) to 15% on Day 8 after collection (Fig. 1). In the samples stimulated with theophylline, the percentage of motile sperm increased significantly (P < 0.05) compared with that for nonstimulated samples, the difference ranging from 10% on Day 2 to approximately 35% on Day 8. The SVS declined during incubation in both stimulated and nonstimulated sperm, but scores were significantly higher in samples stimulated with theophylline on all sampling days (P < 0.05). The results of visual assessment of sperm motility were confirmed by sperm velocity measurements using CASA (Fig. 2). The VSL and VCL values of sperm incubated for more than 3 days were significantly higher (P < 0.05) after stimulation with theophylline than those for the nonstimulated sperm. The percentage of sperm with intact plasma membranes (live) decreased only slightly during incubation. Theophylline seemed to accelerate disintegration of the plasma membrane in sperm previously incubated for extended time at ambient temperature, as the percentage of live sperm was lower in sperm stimulated with theophylline than in nonstimulated sperm on later days of incubation (Fig. 3).
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Effects of Incubation of Sperm Under Standard Conditions (Nitrogen-Gassed Diluent) on Protein Tyrosine Phosphorylation of the Soluble Protein Extracts
In all the samples, two major bands of Mr 67 000 and Mr 36 000 were immunodetected with the anti-phosphotyrosine 4G10 antibody. Densitometry showed that there was no increase in the intensity of either of the two bands in any sample over the period during which sperm were incubated in the nitrogen-gassed diluent. Because the results between bulls differed substantially, Western blots of all three bulls are presented. Although changes in tyrosine phosphorylation of the proteins from soluble fraction were not consistent between bulls, a general downward trend corresponding to declining sperm motility was observed in the sum of signals from Mr 67 000 and Mr 36 000 proteins. This was most noticeable in bull 3.
In bull 1, the tyrosine phosphorylation of the soluble protein of Mr 67 000 declined almost 2-fold in nonstimulated samples. In samples stimulated with theophylline, tyrosine phosphorylation of the protein of Mr 67 000 was similar on Days 0–2 and then increased on Days 5–8 (Fig. 4). Tyrosine phosphorylation of the soluble protein of Mr 36 000 initially declined between Days 0 and 2 and then increased slightly from Day 5 to 8 in a nonstimulated sample. In the presence of theophylline, tyrosine phosphorylation of the soluble protein of Mr 36 000 declined more than 7-fold between Days 0 and 8 of incubation.
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In bull 2, the tyrosine phosphorylation state of the larger protein remained relatively unchanged in the absence of theophylline; but, under stimulation with theophylline, it declined almost 3-fold over 8 days of incubation. In contrast, tyrosine phosphorylation of the smaller protein in samples not stimulated with theophylline declined approximately 5-fold. Phosphotyrosine signal of the smaller protein from samples stimulated with theophylline, normalized for protein loading, declined slightly between Days 0 and 5 and then more markedly on Day 8 (Fig. 4).
In bull 3, the larger protein band on Western blot was too faint to quantify by densitometry in both theophylline-treated and untreated samples. Tyrosine phosphorylation of the smaller protein declined approximately 3-fold over 8 days in both theophylline-treated and untreated samples (Fig. 4).
Effect of Incubation of Sperm Under Standard Conditions (in Nitrogen-Gassed Diluent) on Protein Tyrosine Phosphorylation of the Whole-Cell Protein Extract
There was a time-dependent increase in tyrosine phosphorylation of a number of proteins from whole-cell extracts during long-term incubation of sperm in nitrogen-gassed diluent (Fig. 5). Two major bands of Mr 220 000 and Mr 80 000 showed the most obvious increase in tyrosine phosphorylation. There was no substantial difference in the pattern of phosphorylation between extracts from sperm stimulated with theophylline and those from nonstimulated sperm cells. The increase in tyrosine phosphorylation with incubation ranged from approximately 60-fold for the protein with Mr 220 000 to 8-fold for the protein with Mr 80 000.
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Effect of Incubation of Sperm Under Anaerobic and Aerobic Conditions on Tyrosine Phosphorylation of the Proteins from Whole-Cell Extracts
Tyrosine phosphorylation of a number of proteins extracted from sperm diluted in the anaerobic medium increased in a time-dependent manner between Day 0 and Day 12 of incubation (Fig. 6). In the case of the protein with Mr 220 000 the increase was more than 25-fold; for the protein of Mr 110 000 the increase was 6-fold, for the protein of Mr 80 000 the increase was 17-fold, and for the protein of Mr 25 000 it was more than 4-fold. No increase in protein tyrosine phosphorylation was observed in sperm incubated under aerobic conditions (Fig. 6). The same trends were observed in sperm incubated under aerobic and anaerobic conditions for 12 days in a subsequent experiment (Fig. 7). When sperm were transferred from anaerobic to aerobic conditions after 5 days of incubation, the same proteins that were phosphorylated during anaerobic incubation were dephosphorylated between Days 6 and 12 of aerobic incubation (Fig. 7). Conversely, when sperm initially incubated under aerobic conditions were transferred to an anaerobic environment, proteins that did not display increased protein tyrosine phosphorylation until this point became progressively phosphorylated on tyrosine residues between Days 6 and 12 (Fig. 7).
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Effect of Incubation of Sperm Under Anaerobic and Aerobic Conditions on Motility and Viability
The SVS, the percentage of motile sperm (Fig. 8), and the percentage of sperm with intact plasma membrane (Fig. 9) declined under both anaerobic and aerobic conditions. For the first 5 days of in vitro incubation, the rate of decline of those parameters was similar under anaerobic and aerobic conditions, after which sperm incubated under aerobic conditions deteriorated more rapidly (P < 0.005).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Apart from improved motility, exposure of spermatozoa to theophylline, after a period of incubation, appears to promote plasma membrane disintegration (Fig. 3). This finding is similar to earlier results in reports describing ultrastructural damage to sperm membranes caused by incubation with another phosphodiesterase inhibitor, caffeine [20, 21]. It has been previously demonstrated that stimulation of sperm motility via inhibition of cyclic nucleotide phosphodiesterase leads to increased respiration rate in intact bull spermatozoa [22]. It is therefore highly probable that during incubation with theophylline in our experiments the respiration rate was elevated, leading to increased ROS production. This could result in damage to plasma membranes that are already compromised by the extended in vitro incubation.
The pattern of changes in tyrosine phosphorylation of soluble proteins upon incubation was not repeatable between ejaculates from different bulls in our study and is therefore difficult to interpret. One clear trend, however, is observed. The sum of the phosphotyrosine signal from larger and smaller soluble proteins in the same loading lane declined with time of incubation in all bulls and both treatments. This is the pattern that would be observed if the smaller detected protein was a product of proteolytic cleavage of the larger one and if tyrosine phosphorylation of this protein declined with incubation. Variations in the degree of proteolysis of different samples would explain variations in the band pattern observed. It is probable that samples from bull 3 had undergone complete cleavage at a specific site, resulting in detection of only the smaller tyrosine-phosphorylated fragment, while in other samples incomplete proteolysis resulted in detection of two proteins. Efforts to clarify this by isolating and identifying both tyrosine-phosphorylated proteins will be undertaken in our laboratory. The between-bull variations in tyrosine phosphorylation of soluble sperm proteins could be attributable to differing rates of sperm cell degradation during incubation of diluted ejaculates of different bulls.
Our Western blots of tyrosine-phosphorylated proteins from soluble fractions of ejaculated sperm were quite different from those presented by Vijayaraghavan et al. [3]. The single soluble tyrosine-phosphorylated protein of Mr 55 000 described by Vijayaraghavan et al. [3] was not detected in any of the samples from three different bulls. Two tyrosine-phosphorylated proteins were detected on most of the blots in our study. The differences may be due to the fact that we used ejaculated sperm while the previous researchers used cauda and epididymal sperm. Upon ejaculation, sperm cells are mixed with seminal plasma, which contains several motility activators [23]. It is conceivable that these activators initiate tyrosine phosphorylation on a different set of proteins from those phosphorylated in the epididymis. Also, some components of our diluent are different from those in the diluent used by Vijayaraghavan et al., and this may have altered the pattern of tyrosine phosphorylation of soluble sperm proteins. However, solely on the basis of the molecular weight estimation we cannot exclude the possibility that the larger protein detected on our blots is indeed the same as that described by Vijayaraghavan et al. Purification and identification of this protein should answer the question whether the phosphorylated proteins from soluble extracts of ejaculated sperm are related to the soluble tyrosine-phosphorylated protein of Mr 55 000 from epididymal sperm described by Vijayaraghavan et al. [3].
Incubation with theophylline does not seem to increase tyrosine phosphorylation of soluble proteins (Fig. 4). This is in contrast to the observation of Vijayaraghavan et al. [3] that the increase in cAMP concentration led to increased tyrosine phosphorylation of a cytosolic protein of Mr 55 000. We do not know whether the decline we have detected in the degree of tyrosine phosphorylation of soluble proteins has a causal link to the time-dependent decline of sperm motility or whether the two processes are simply occurring at the same time without any cause-effect relationship.
The changes in tyrosine phosphorylation of proteins from whole-cell extracts are very slow when compared to typical receptor-mediated tyrosine phosphorylation, or to those observed in ejaculated bovine [7] and human [24] sperm incubated under capacitating conditions. For example, insulin receptor kinase requires only 15 sec to reach maximum tyrosine autophosphorylation after insulin binding or 10 min when stimulated with hydrogen peroxide [15]. Capacitation-associated protein tyrosine phosphorylation reaches a maximum after 4 h of incubation [7, 25]. During prolonged in vitro incubation we have observed tyrosine phosphorylation still occurring even after 10 days of incubation (Figs. 6 and 7).
We did not observe major differences in the pattern of protein tyrosine phosphorylation of whole sperm cell extracts between theophylline-stimulated and nonstimulated sperm (Fig. 5). In contrast, inhibition of phosphodiesterase in bovine sperm incubated under capacitating conditions strongly promotes protein tyrosine phosphorylation and is even able to overcome glucose inhibition of tyrosine phosphorylation [7].
In order to determine whether the exclusion of oxygen, and thus respiration, during incubation would affect protein tyrosine phosphorylation, sperm were incubated in vitro under aerobic and anaerobic conditions. We found that contrary to our expectation, a time-dependent increase in tyrosine phosphorylation was observed under anaerobic but not under aerobic conditions (Fig. 6). One possible reason for the lack of protein tyrosine phosphorylation under aerobic conditions may be the composition of the incubation medium, which is a commercial diluent empirically optimized over the years to maintain sperm viability at ambient temperature [1]. This diluent does not contain heparin, BSA, or sodium bicarbonate, which are known to be required for capacitation and tyrosine phosphorylation associated with capacitation [25, 26]. On the contrary, it contains glucose, which has been shown to inhibit bovine sperm capacitation [27] and tyrosine phosphorylation [7], and is also a mild reducing agent. Other strong, cell permeable-reducing agents such as 2-mercaptoethanol suppress tyrosine phosphorylation under capacitating conditions [13]. Catalase, which was present in the medium, also inhibits capacitation-related tyrosine phosphorylation in bovine sperm via the rapid removal of hydrogen peroxide from the diluent [14]. The medium also contains Ca2+, which has been shown to promote capacitation in murine sperm [26] but has been reported to negatively modulate capacitation-associated tyrosine kinase activity in human sperm [18]. We can speculate that the capacitation-related increase in protein tyrosine phosphorylation, which is promoted by oxidative conditions, would be inhibited in this medium under both aerobic and anaerobic conditions.
When sperm incubated under anaerobic conditions are transferred to aerobic conditions, tyrosine phosphorylation of proteins that were phosphorylated during incubation is reversed. When sperm incubated for 5 days under aerobic conditions are transferred to an anaerobic environment, the same set of proteins that are phosphorylated during incubation under anaerobic conditions get phosphorylated in a time-dependent manner (Fig. 7). This indicates that both protein tyrosine kinase and phosphatase activities are regulated by the redox status of the cell, probably through the generation of ROS [14].
An increase in tyrosine phosphorylation of proteins from whole-cell extracts observed during extended incubation of spermatozoa at ambient temperature is not modified by the phosphodiesterase inhibition, nor is it enhanced by oxidative conditions. On the contrary, it is inhibited in the presence of oxygen and is reversed upon transfer of sperm cells from an anaerobic to aerobic environment. It takes place in a medium devoid of known capacitating agents across a time span of 10 days. The role of protein tyrosine phosphorylation during long-term sperm incubation at ambient temperature, and its relation to other tyrosine phosphorylation-mediated processes, can be understood only when the proteins that are phosphorylated during incubation are isolated and identified.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This research was financially supported by Livestock Improvement Corporation and by Technology New Zealand through the Graduate Research in Industry Fellowship. 
2 Correspondence: R. Vishwanath, Livestock Improvement Corporation Ltd., Private Bag 3016, Hamilton, New Zealand. FAX: 64 7 858 2741; rvish{at}lic.co.nz
Accepted: February 3, 2000.
Received: July 2, 1999.
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