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From the * Institute of Zoology, Regent's Park,
London, United Kingdom; and the Laboratory of
Gamete Signalling, Babraham Institute, Babraham, Cambridge, United
Kingdom.
| Correspondence to: Dr R. A. P. Harrison, Laboratory of Gamete Signalling (540/2073), The Babraham Insitute, Cambridge CB2 4AT, United Kingdom (e-mail: robin.harrison{at}bbsrc.ac.uk ). |
| Received for publication December 10, 2001; accepted for publication February 25, 2002. |
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Abstract |
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Key words: Cyclic adenosine monophosphate, signal transduction, protein phosphatase, "cluster" analysis
Overall, motility expression appears to depend on the net level of phosphorylation of certain specific proteins, under the positive influence of protein kinases (especially protein kinase A [PKA]) and the negative influence of protein phosphatases (especially type PP1) (eg, Lindemann and Kanous, 1989; Tash, 1989; Smith et al, 1996; Si and Okuno, 1999). Current knowledge indicates that the system is critically controlled through modulation of PKA activity. This modulation is achieved by alterations in the intracellular concentration of the major sperm messenger molecule, cyclic adenosine monophosphate (cAMP), brought about principally by changes in adenylyl cyclase activity. Two important effectors of motility in the sperm's natural environment, bicarbonate and calcium, have been identified as direct activators of adenylyl cyclase (Morton et al, 1974; Okamura et al, 1985; Gross et al, 1987). Moreover, numerous studies have shown that pharmacological agents that bring about rises in intracellular levels of cAMP (eg, inhibitors of cyclic nucleotide phosphodiesterase such as caffeine or pentoxifylline) can stimulate poorly motile sperm samples. It is therefore generally supposed that motility shortcomings may often be due to the inability of the spermatozoa to produce and/or maintain sufficient levels of cAMP to stimulate PKA (eg, Magnus et al, 1993).
Studies on sperm motility activation (ie, change from slow nonlinear movement to linear forward progression) have nearly all concentrated on the overall population response, measuring global changes in motility parameters such as percentage motility, progressive velocity, and beat frequency. Much less attention has been paid to the response of the individual cells. In a recent study (Abaigar et al, 1999), however, we monitored bicarbonate-induced motility stimulation in boar sperm using the Hobson Sperm Tracker system in combination with videomicroscopy to provide data on motility parameters for individual cells. By applying "pattern" analysis (a form of "cluster" analysis) to the data, we were able to distinguish major subgroups of sperm whose motility characteristics differed substantially. We found that bicarbonate induced a much more linear and rapid movement in some of the spermatozoa, whereas it had little or no effect on others, even though these exhibited flagellar activity. We also observed clear differences in response between samples taken from different boars.
Boar spermatozoa are particularly good subjects for investigations of motility control mechanisms; poorly motile on release from the male reproductive tract, they activate rapidly on the addition of bicarbonate but are unresponsive to Ca2+ (Tajima et al, 1987). We have therefore used the bicarbonate-stimulated boar sperm model, in conjunction with our protocol for analyzing individual sperm motility characteristics, to investigate sperm response to effectors of the cAMP/PKA pathway. We posed the question "Is inadequacy of cAMP supply, with resultant failure of PKA activation, the main reason for differences between cells and between males?" A preliminary account of some of these experiments has been presented elsewhere (Harrison and Holt, 2000).
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Materials and Methods |
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Collection and Washing of Spermatozoa
Sperm-rich fractions of semen were collected from fertile boars, held
either at the Babraham Institute or kept by JSR Healthbred Limited at Thorpe
Willoughby (Selby, Yorkshire, United Kingdom) for commercial artificial
insemination; various breeds were represented. After filtration through gauze
to remove gel material, the semen from the Institute boars was diluted to a
concentration of approximately 3 x 107 cells/mL with
prewarmed Belts-ville Thawing Solution (BTS: 206 mM glucose, 20.4 mM
Na3-citrate, 14.9 mM NaHCO3, 10 mM KCl, 3.4 mM
Na2-EDTA, and 50 µg/mL kanamycin sulfate); it was then allowed
to cool slowly to ambient temperature (18°C-22°C) and was stored in
the dark for not more than 2 days. (BTS is a widely used ambient-temperature
extender for boar semen in which full fertility is preserved for at least 3
days—see Johnson et al,
1988.) The semen samples from JSR Healthbred were supplied already
diluted in BTS extender; they were received the day after collection and were
stored for not more than 1 further day.
Prior to experimentation, sperm were isolated from the diluted semen by sedimentation through a 2-step gradient of iso-osmotic Percoll in a saline-based medium (Lynham and Harrison, 1998). After aspiration of the supernatant layers, the loose sperm pellets were resuspended in residual 70% Percoll-saline solution (final concentration approximately 4 x 108 sperm/mL). These preparations, whose viability as assessed by propidium iodide staining (Harrison and Vickers, 1990) was routinely greater than 90%, were used within 2 hours of washing.
Media and Reagents
The basal incubation medium ("Tyr"—see
Harrison and Miller, 2000) consisted of 116 mM NaCl, 3.1 mM KCl, 0.4 mM MgSO4, 0.3 mM
NaH2PO4, 5 mM glucose, 21.7 mM sodium lactate, 1 mM
sodium pyruvate, 1 mM ethyleneglycoltetraacetic acid (EGTA), 20 mM HEPES
(adjusted with NaOH to pH 7.6 at 20°C), 3 mg bovine serum albumin
(BSA)/mL, 100 µg kanamycin/mL, and 20 µg phenol red/mL; its final pH at
38°C was 7.4, and its osmolality was 300 mOsm/kg. The EGTA was included in
order to reduce head-to-head agglutination (which would have seriously
compromised motility measurements); preliminary experiments (data not shown)
confirmed the findings of Tajima et al
(1987) that external
Ca2+ was not required for bicarbonate to effect stimulation of
motility. Bicarbonate/CO2 was added in the form of suitable
aliquots of a 300-mM aqueous solution of NaHCO3 saturated with 100%
CO2 (a ratio of bicarbonate:CO2, yielding after dilution
pH 7.4 at 38°C—see Umbreit,
1957); thus, such additions did not disturb the pH of the medium.
Control incubations received similar aliquots of 300 mM NaCl. To prevent loss
of CO2 during subsequent incubation, the bicarbonate-containing
suspensions were maintained under 5% CO2 in air. Stock solutions of
effectors other than bicarbonate were prepared either in dimethylsulfoxide
(DMSO) or in Tyr.
Incubation Protocol
A volume (either 1 or 2 mL) of Tyr was prewarmed to 38°C in a capped
15-mL polystyrene tube (Sterilin, Stone, Staffs, United Kingdom), designated
the "experimental" tube. An aliquot (5-10 µL) of washed sperm
(final concentration approximately 2 x 106 cells/mL) was
added, and the suspension was incubated at 38°C for 10 minutes. A 60-µL
sample was then removed for motility analysis. Next, half the remaining
suspension was transferred to an empty prewarmed tube (designated the
"control" tube), and to it was added NaCl (as control for
bicarbonate addition) or Tyr medium or DMSO (as control for other effector
additions). Twelve minutes after the initiation of incubation, the
experimental treatment ("activator") was added to the rest of the
sperm suspension (in the "experimental" tube). Incubation of both
tubes was continued, and further 60-µL samples were taken for motility
analysis: from the "experimental" tube, 2, 7, 12, and 17 minutes
after the addition of activator, and from the "control" tube,
either 22 or 27 minutes after the "control" addition. In some
experiments, protein kinase or phosphatase inhibitors were added to the medium
in the "experimental" tube at the beginning of incubation in order
to observe their effects on subsequent activation treatments.
Motility Assessment
Sperm motility was recorded by videomicroscopy, essentially as described by
Holt et al (1996), except that
a 10x objective (negative high phase contrast) was used for microscopy
instead of a 20x objective, and 60-µL samples were taken from the
sperm suspensions. The recordings were analyzed quantitatively using a Hobson
Sperm Tracker (Hobson Tracking Systems, Sheffield, United Kingdom) operating
at a frame rate of 50 Hz. The "search radius" used was 5.9 µm,
and the "minimum track points" setting was 50 frames. The measured
parameters of sperm motion were curvilinear velocity (VCL), average path
velocity (VAP), straight-line velocity (VSL), amplitude of lateral head
displacement (ALH), beat cross frequency (BCF), linearity (LIN), straightness
(STR), and time (TIME). Details regarding the use of the Hobson Sperm Tracker
and a discussion of these parameters may be found elsewhere (see
Holt et al, 1996;
Abaigar et al, 1999, and
references therein).
Multivariate Analyses of Motility Parameters
For each experiment, the motility data from all sampling times and all
replicates were merged and subjected to multivariate analysis using the
computer program PATN (Belbin,
1993) running on a personal computer. The program uses a series of
procedures to analyze and compare the motility parameter values associated
with each spermatozoon so as to identify subgroups within the sperm population
("patterns"). The identification of the subgroups and their
hierarchical classification is carried out by the program entirely
independently of the investigator, who is simply required to judge to what
degree subgroups may be combined to yield a sufficiently small number to allow
practical comparison.
A fuller description and illustration of the use of PATN analysis (a special form of "cluster" analysis) to identify subpopulations within boar sperm samples is given by Abaigar et al (1999). However, key points may be emphasized. PATN analysis is performed using data from all individual spermatozoa within a single experiment, prepared by merging raw data files from every measured sperm sample within that experiment. As each of these files may contain multivariate data from 400-500 spermatozoa, the combined data sets contain many thousands of cases (sets of values for each cell). Unlike many conventional cluster analysis software packages, PATN is not limited by the computer memory, and such large amounts of data can be analyzed on a conventional personal computer. Upon completion of the PATN analysis, each individual spermatozoon is categorized as belonging to one of the small number of groups or subpopulations described above. In the present study, the groups were distinguished on the basis of multivariate combinations of motion parameters. Qualitative consideration of the parameter values pertaining to the group centroids enabled a descriptive interpretation of the sperm motion behavior, which each group represented. (Note that the precise parameter values inevitably differed slightly between experiments. The key issue, however, was the identification of subpopulations and their essential characteristics.)
Once the subpopulations had been identified, the relative percentages of spermatozoa belonging to each group were calculated for each (boar x treatment x incubation time) combination. Percentages were then combined across replicate experiments, and statistical analysis was carried out by analysis of variance using Statistica for Windows (Statsoft UK, Letchworth, United Kingdom) after transformation of the percentages to logs or angles (both are recognized approaches).
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Results |
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Analysis of the various motility parameters (Table 1) revealed that the enhancement of motility was characterized as an increase in VAP, VSL, and LIN of trajectory and a decrease in ALH. When this increase was explored by plotting LIN against VAP, it became clear that the changes were not globally expressed in every cell, but only in a subpopulation (compare Figure 2a and b). After stimulation, there was a very significant increase in the number of cells showing progressive motility (increased LIN), but at the same time, there was clearly a subpopulation of cells whose motility had been only very little altered (if at all) by bicarbonate.
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PATN analysis enabled us to identify subpopulations with different motility characteristics within our samples (see Table 2), of which one increased in proportion greatly in response to bicarbonate (see Figure 3a). Given the much higher VSL and LIN of this subpopulation, we refer to its members henceforth as "fast linear" sperm. The repeatability of the bicarbonate stimulation as detected by PATN analysis was tested on sperm from 2 boars, samples being tested shortly after being washed and at 40, 80, and 120 minutes thereafter. Although the 2 sets of samples differed from each other, within-boar differences between the 4 replicate samples were insignificant (P =.785, F3,16 = 0.355 for boar X; P =.424, F3,16 = 0.988 for boar Y). As well as confirming the validity of the analytical approach, these results showed that the ability of the spermatozoa to respond was maintained for up to 2 hours after washing and that the decline in bicarbonate-stimulated motility noted above was not due to an inherent decline in response due to "in vitro" aging.
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A comparison of the bicarbonate response of the 7 independent sperm samples showed clear differences with respect to proportions of fast linear sperm (compare Figure 3b through h). In some samples prior to stimulation, there were few such cells, whereas in others, there were many. The degree and time course of stimulation (ie, increase in the proportion of fast linear sperm) engendered by bicarbonate were also variable. Although stimulation by bicarbonate was less obvious in samples in which there were already many fast linear sperm (eg, Figure 3f), there were marked differences in the degree of stimulation induced in samples initially containing relatively few such sperm (eg, compare Figure 3b and c). It should be noted that low stimulation (as in Figure 3b, d, and g) was not due to the presence of many dead sperm, for viability in all of these samples was greater than 80%; moreover, the proportion of fast linear spermatozoa was that with respect to the motile population, since in our assessments, the Hobson Sperm Tracker was detecting only those cells that showed tail movement.
Involvement of cAMP in Motility Stimulation
Using semen samples selected for low basal motility but good response to
bicarbonate, the effect of adding the cAMP analog cBIMPS (10, 30, or 100
µM) was compared to that of adding 5 mM bicarbonate. The analog induced an
increase in the proportion of spermatozoa showing fast linear progression in a
similar fashion to bicarbonate (compare
Figure 2b and c). As might be
expected, the time course of the effect of cBIMPS
(Figure 4) was slower than that
of bicarbonate (the analog would permeate the plasma membrane much more slowly
than bicarbonate/CO2). Also predictably, 100 µM cBIMPS
stimulated most rapidly, reaching a peak at 7 minutes of incubation; however,
its stimulatory effect waned with time compared with the lower concentrations,
declining significantly by 17 minutes (P =.028). (A corresponding
decline was not observed with the 5-mM bicarbonate treatment: P
>.8). Notably, none of the concentrations tested was able to induce a
significantly higher proportion of "fast linear" sperm than
bicarbonate.
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Involvement of Protein Kinases in Motility Stimulation
These studies involved sperm samples preselected as described in the
previous section. Preincubation with H89, a specific inhibitor of PKA
(Chijiwa et al, 1990), lowered
the proportion of fast linear sperm in unstimulated samples and also
completely abolished the ability of 3 mM bicarbonate to stimulate (compare
Figure 5b with a and
Figure 2d with b).
Preincubation with GF109203X, a specific inhibitor of protein kinase C (PKC)
isoforms 
, ß, and 
(Toullec et al, 1991), had no
effect on the proportion of fast linear sperm prior to bicarbonate
stimulation, nor did it inhibit the effect of bicarbonate
(Figure 5c). On the other hand,
lavendustin A, a specific inhibitor of protein tyrosine kinases (PTKs)
(Onoda et al, 1989;
O'Dell et al, 1991), though it
did not affect the stimulatory process itself, caused a significant decline in
the stimulated motility with time (Figure
5d). (Lower levels of bicarbonate were used in these experiments
in order to maximize the sensitivity of the kinase inhibitor tests.)
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Processes involving protein phosphorylation are generally controlled via a balance between kinase activity, which adds a phosphate residue to the target protein, and phosphatase activity, which removes it. To test the effect of blocking phosphoprotein breakdown, sperm were pre-incubated with calyculin, a potent inhibitor of protein phosphatase types PP1 and PP2A (Ishihara et al, 1989), before being challenged with bicarbonate. The results are shown in Figure 6. Preincubation with calyculin alone stimulated motility markedly in comparison to control samples (compare bicarbonate-free values in Figure 6a with those in 6b). The addition of bicarbonate to calyculin-treated suspensions stimulated motility further. However, the maximum level of stimulation achieved did not exceed that obtained by treatment with bicarbonate alone (compare maximum values in Figure 6a with those in b). Notably, in the presence of calyculin, this level of stimulation was maintained during further incubation, whereas in the presence of bicarbonate alone, the stimulation declined. Such an effect of calyculin could have been due to its preventing the breakdown of phosphorylated motility-control proteins, as PKa activity declined concomitantly with falling cAMP levels (see postulated explanation above for decline in bicarbonate stimulation).
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Discussion |
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Because treatment with phosphodiesterase inhibitors such as caffeine and pentoxifylline can enhance the motility of some poorly motile samples (eg, Garbers et al, 1971; Lewis et al, 1994; see also Abaigar et al, 1999), it has been hypothesized that such samples are unable for some reason to maintain sufficient levels of intracellular cAMP to activate the motility mechanism (see Magnus et al, 1993). At the same time, studies on t-locus mouse strains (Olds-Clarke, 1996; Herrmann et al, 1999) have indicated that lesions in motility expression can be genetic in origin.
While differences in semen quality between males have been the focus of many investigations over many years, the degree of functional heterogeneity to be found within any given sperm sample has only recently been openly discussed. It has been postulated that the heterogeneity represents varying degrees of maturation caused by peristaltic and ciliary movement and consequent mixing of sperm within the epididymal duct (see Amann et al, 1993). On the other hand, Herrmann et al (1999) have speculated that the genetic variability resulting from crossing over during spermatogenic meiosis might result in each spermatozoon producing and retaining its own set of protein products, whence functional heterogeneity could be expected. Since suboptimal motility expression has profound implications with respect to fertility, identification of the molecular basis of this inadequacy would be of great practical importance, given the modern powers of gene mapping and focused breeding programs.
In an earlier study (Abaigar et al, 1999), by applying PATN analysis to data on motility characteristics from individual sperm, we could show clearly that bicarbonate treatment of boar sperm stimulated some cells but not others. Bicarbonate initiates the protein phosphorylation pathway controlling motility expression by activating adenylyl cyclase to produce increased levels of cAMP; thus, our model and analytical method seemed especially suited for investigating the molecular cause of this heterogeneity of response. The goal of the present study was to find out if between-cell and between-ejaculate differences in motility expression were due to shortcomings in cAMP supply such that PKA was insufficiently active to maintain optimum phosphorylation states in key motility control proteins.
As a first stage, we confirmed our earlier preliminary observations, namely that bicarbonate stimulates motility by inducing a faster, more linear type of progression but that only a proportion of the sperm population responds. In addition, we showed that the changes took full effect within 2 minutes of the addition of 15 mM bicarbonate, thus essentially confirming the observations of Tajima et al (1987). Moreover, we saw considerable differences among ejaculates, both in the level of motility expression in unstimulated samples and in the degree of stimulation achieved by bicarbonate addition.
The compound cBIMPS is a very useful cAMP analog with which to activate PKA. It is relatively permeable, is highly resistant to hydrolysis by cyclic nucleotide phosphodiesterases, and has a much higher affinity for PKA than for protein kinase G (Sandberg et al, 1991). Treatment with cBIMPS therefore effectively bypasses the adenylyl cyclase/phosphodiesterase section of the PKA signaling pathway. Incubation of sperm with cBIMPS stimulated motility in a similar fashion to bicarbonate, causing an increase in the proportion of cells exhibiting fast linear motility. The increase was dose-dependent in that higher concentrations of cBIMPS stimulated motility more rapidly. However, the proportions of cells that were stimulated by the different concentrations of cBIMPS were essentially similar to those stimulated by bicarbonate, and many cells remained unaffected. Since it can be expected that all cells are essentially equally permeable to cBIMPS, there should be little difference between cells with respect to the rate of entry of the analog; moreover, once in, its resistance to hydrolysis by phosphodiesterases would imply that intracellular levels would remain closely similar between cells. Because cBIMPS was unable to stimulate cells to a greater extent than bicarbonate, we conclude that intercell differences in response were not due to differences in the ability of bicarbonate to stimulate adenylyl cyclase or to differences in phosphodiesterase activity. The same argument can be applied to differences between males: cBIMPS did not eliminate such differences (see SEM bars in Figure 4); therefore, they were not due to differences in efficiency of cAMP provision.
H89 is a well-characterized and specific inhibitor of PKA (Chijiwa et al, 1990). In our experiments, H89 was not only able to block any motility-stimulating effect of bicarbonate, but it also reduced basal motility to very low levels (Figures 2d and 5b). Ain et al (1999) have also reported strong inhibitory effects of H89 on hamster sperm motility. Although Leclerc et al (1996) found H89 to be only slightly inhibitory toward human sperm motility, the concentration they used (10 µM) may have been insufficient (see Harrison and Miller, 2000). Taken together with the motility-stimulating action of cBIMPS, the ability of H89 to inhibit both basal motility and stimulation by bicarbonate further confirms a central and direct role of PKA in the control of boar sperm motility (Lindemann and Kanous, 1989; Tash, 1989). Its effect runs counter to suggestions that the role of cAMP in motility stimulation might be mediated by actions other than the stimulation of PKA (eg, activation of ion channels or stimulation of exchange factors in Ras signaling pathways—see Aitken, 2000).
Naor and colleagues (see Kalina et al, 1995, and references therein) have reported that motility expression in human spermatozoa involves PKC action. We were unable to detect alterations in bicarbonate-induced motility stimulation in our boar sperm that had been pretreated with GF109203X (bis-indolyl-maleimide I). This latter is a specific inhibitor (Toullec et al, 1991) of the "conventional" Ca2+-dependent PKC forms (Newton, 1995) reported to be present in human (Kalina et al, 1995) and bull spermatozoa (Chaudhry and Casillas, 1992). However, it may be that PKC involvement in sperm motility expression is species related, reflecting the ability of Ca2+ to stimulate motility: the ion has no effect in boar sperm (Tajima et al, 1987; see also "Materials and Methods"). There have also been reports that tyrosine phosphorylation plays a role in the control of motility (Vijayaraghavan et al, 1997; Ashizawa et al, 1998; Si and Okuno, 1999), although Ain et al (1999) found no effect of the PTK inhibitor tyrphostin A-47 on hamster sperm motility. In our experiments, sperm preincubated with the general PTK inhibitor lavendustin A (Onoda et al, 1989; O'Dell et al, 1991) showed no changes either in basal motility or in the initial degree to which bicarbonate was able to stimulate motility. However, lavendustin treatment caused a more rapid decline in the bicarbonate-induced stimulation thereafter (Figure 5d), indicating that tyrosine phosphorylation may be involved in processes underlying the maintenance of motility expression.
Cellular functions controlled via protein phosphorylation involve protein phosphatases as well as protein kinases. The control is exerted through the net balance of the 2 opposing activities. Sperm motility is no exception, for it has been demonstrated that protein phosphatase inhibitors such as calyculin A and okadaic acid stimulate motility (Ashizawa et al, 1995a; Smith et al, 1996), presumably by reducing the rate at which dephosphorylation of key proteins takes place and thereby increasing their net phosphorylation state. We found that calyculin stimulated the motility of boar spermatozoa, increasing considerably the percentage of "fast" cells. The addition of bicarbonate to the calyculin-treated sperm increased this percentage further. However (and importantly), the overall induction of "fast" cells by a combination of calyculin and bicarbonate was no greater than that brought about by bicarbonate alone. Thus, we deduce that the failure of sperm to respond fully to bicarbonate stimulation does not stem from an imbalance in the PKA/protein phosphatase axis.
In conclusion, the experiments described in this paper indicate that between-cell and between-animal differences in motility expression in boar spermatozoa are not due either to inadequacies of cAMP supply or to inadequacies in PKA-catalyzed phosphorylation. Although the permeable cAMP analog cBIMPS, which is highly resistant to phosphodiesterase attack, stimulated sperm motility in a manner closely similar to bicarbonate, it was unable to increase the number of sperm showing rapid linear motion in excess of that induced by bicarbonate. Tests of the effect of protein kinase inhibitors showed that motility stimulation was directly dependent on PKA and did not appear to involve phosphorylation events catalyzed by PKC or PTK (though the latter may have a role in maintaining stimulation). The protein phosphatase inhibitor calyculin was able to stimulate motility in the same way as bicarbonate, indicating that it was acting to increase the net phosphorylation state of PKA target proteins. However, the inclusion of calyculin did not increase the overall level of stimulation induced by bicarbonate. Thus, the failure to respond to stimulation was not due to insufficient PKA capacity or to overactive dephosphorylation of its products. The molecular basis for variability in sperm motility expression is therefore deduced to lie principally either downstream of PKA (possibly involving kinases other than PKC or PTK: cf Ashizawa et al, 1995b, 1997) or in a parallel pathway, which affects the efficiency of the main PKA-dependent signaling cascade.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Ain R, Uma Devi K, Shivaji S, Sechagiri PB.
Pentoxifylline-stimulated capacitation and acrosome reaction in hamster
spermatozoa: involvement of intracellular signalling molecules. Mol
Hum Reprod. 1999;5:618
-626.
Aitken RJ. Possible redox regulation of sperm motility activation. J Androl. 2000;21:491 -496.[Medline]
Amann RP, Hammerstedt RH, Veeramachaneni DNR. The epididymis and sperm maturation: a perspective. Reprod Fertil Dev.1993; 5:361 -381.[Medline]
Ashizawa K, Hashimoto K, Higashio M, Tsuzuki Y. The addition of mitogen-activated protein kinase and p34cdc2 kinase substrate peptides inhibits the flagellar motility of demembranated fowl spermatozoa. Biochem Biophys Res Commun.1997; 240:116 -121.[Medline]
Ashizawa K, Higashio M, Tsuzuki Y. Effects of tyrosine kinase inhibitor on the motility and ATP concentrations of fowl spermatozoa. Mol Reprod Dev.1998; 49:196 -202.[Medline]
Ashizawa K, Magome A, Tsuzuki Y. Stimulation of motility and respiration of intact fowl spermatozoa by calyculin A, a specific inhibitor of protein phosphatase-1 and -2A, via a Ca2+-dependent mechanism. J Reprod Fertil.1995a; 105:109 -114.
Ashizawa K, Wishart GJ, Nishinakama K, Sakamoto T, Tsuzuki Y. Regulatory mechanisms of fowl sperm motility: possible role of endogenous myosin light chain kinase-like protein. J Reprod Fertil. 1995b;104:141 -148.
Belbin L. PATN, Pattern Analysis Package. Canberra, Australia: CSIRO; 1993.
Chaudhry PS, Casillas ER. Isotypes of protein kinase C in bovine sperm. Arch Biochem Biophys.1992; 295:268 -272.[Medline]
Chijiwa T, Mishima A, Hagiwara M, et al. Inhibition of
forskolin-induced neurite outgrowth and protein phosphorylation by a newly
synthesized selective inhibitor of cyclic AMP-dependent protein kinase,
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide
(H-89), of PC12D pheochromocytoma cells. J Biol Chem.1990; 265:5267
-5272.
Garbers DL, First NL, Sullivan JJ, Lardy HA. Stimulation and maintenance of ejaculated bovine spermatozoan respiration and motility by caffeine. Biol Reprod.1971; 5:336 -339.[Abstract]
Gross MK, Toscano DG, Toscano WA. Calmodulin-mediated adenylate
cyclase from mammalian sperm. J Biol Chem.1987; 262:8672
-8676.
Harrison RAP, Holt WV. Modelling boar sperm signalling systems. In: Johnson LA, Guthrie HD, eds. Boar Semen Preservation IV. Lawrence, KS: Allen Press; 2000:13 -19.
Harrison RAP, Miller NGA. cAMP-Dependent protein kinase control of plasma membrane lipid architecture in boar sperm. Mol Reprod Dev. 2000;55:220 -228.[Medline]
Harrison RAP, Vickers SE. Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. J Reprod Fertil. 1990;88:343 -352.
Herrmann BG, Koschorz B, Wertz K, McLaughlin KJ, Kispert A. A protein kinase encoded by the t complex responder gene causes non-mendelian inheritance. Nature.1999; 402:141 -146.[Medline]
Holt C, Holt WV, Moore HDM. Choice of operating conditions to
minimize sperm subpopulation sampling bias in the assessment of boar semen by
computer-assisted semen analysis. J Androl.1996; 17:587
-596.
Ishihara H, Martin BL, Brautigan DL, et al. Calyculin A and okadaic acid: inhibitors of protein phosphatase activity. Biochem Biophys Res Commun. 1989;159:871 -877.[Medline]
Johnson LA, Aalbers JG, Grooten HJG. Artificial insemination of swine: fecundity of boar semen stored in Beltsville TS (BTS), Modified Modena (MM), or MR-A and inseminated on one, three and four days after collection. Zuchthygiene.1988; 23:49 -55.
Kalina M, Socher R, Rotem R, Naor Z. Ultrastructural localization
of protein kinase C in human sperm. J Histochem
Cytochem. 1995;43:439
-445.
Leclerc P, de Lamirande E, Gagnon C. Cyclic adenosine 3',5' monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. Biol Reprod.1996; 55:684 -692.[Abstract]
Lewis SEM, McKinney KA, Thompson W. Influence of pentoxifylline on human sperm motility in asthenozoospermic individuals using computer-assisted analysis. Arch Androl.1994; 32:175 -183.[Medline]
Lindemann CB, Kanous KS. Regulation of mammalian sperm motility. Arch Androl.1989; 23:1 -22.[Medline]
Lynham JA, Harrison RAP. Use of stored pig eggs to assess boar
sperm fertilizing functions in vitro. Biol Reprod.1998; 58:539
-550.
Magnus Ø, Åbyholm T, Brekke I, Purvis K. Provocation testing of human sperm motility using energy substrates and activators of the cyclic nucleotide system. II. Studies on sperm from asthenozoospermic subjects. Int J Fertil.1993; 38:123 -128.[Medline]
Morton B, Harrigan-Lum J, Albagli L, Jooss T. The activation of motility in quiescent hamster sperm from the epididymis by calcium and cyclic nucleotides. Biochem Biophys Res Commun.1974; 56:372 -379.[Medline]
Newton AC. Protein kinase C: structure, function, and regulation.
J Biol Chem.1995; 270:28495
-28498.
O'Dell TJ, Kandel ER, Grant SGN. Long-term potentiation in the hippocampus is blocked by tyrosine kinase inhibitors. Nature. 1991;353:558 -560.[Medline]
Okamura N, Tajima Y, Soejima A, Masuda H, Sugita Y. Sodium
bicarbonate in seminal plasma stimulates the motility of mammalian spermatozoa
through direct activation of adenylate cyclase. J Biol
Chem. 1985;260:9699
-9705.
Olds-Clarke P. How does poor motility alter sperm fertilizing
ability? J Androl.1996; 17:183
-186.
Onoda T, linuma H, Sasaki Y, et al. Isolation of a novel tyrosine kinase inhibitor, lavendustin A, from Streptomyces griseolavendus.J Nat Prod. 1989;52:1252 -1257.[Medline]
Sandberg M, Butt E, Nolte C, et al. Characterization of Sp-5,6-dichloro-1-ß-D-ribofuranosyl-benzimidazole-3',5'-monophosphorothioate (Sp-5,6-DCl-cBIMPS) as a potent and specific activator of cyclic AMP-dependent protein kinase in cell extracts and intact cells. Biochem J. 1991;279:521 -527.
Si Y, Okuno M. Role of tyrosine phosphorylation of flagellar
proteins in hamster sperm hyperactivation. Biol
Reprod. 1999;61:240
-246.
Smith GD, Wolf DP, Trautman KC, Silva E, Greengard P, Vijayaraghavan S. Primate sperm contain protein phosphatase 1, a biochemical mediator of motility. Biol Reprod.1996; 54:719 -727.[Abstract]
Tajima Y, Okamura N, Sugita Y. The activating effects of bicarbonate on sperm motility and respiration at ejaculation. Biochim Biophys Acta.1987; 924:519 -529.[Medline]
Tash JS. Protein phosphorylation; the second messenger signal transducer of flagellar motility. Cell Motil Cytoskel.1989; 14:332 -339.[Medline]
Toullec D, Pianetti P, Coste H, et al. The bisindolylmaleimide
GF109203X is a potent and selective inhibitor of protein kinase C.
J Biol Chem.1991; 266:15771
-15781.
Umbreit WW. Carbon dioxide and bicarbonate. In: Umbreit WW, Burris RH, Stauffer JF, eds. Manometric Techniques. 3rd ed. Minneapolis, Minn: Burgess Publishing Co; 1957:18 -27.
Vijayaraghavan S, Trautman KD, Goueli SA, Carr DW. A tyrosine-phosphorylated 55-kilodalton motility-associated bovine sperm protein is regulated by cyclic adenosine 3',5'-monophosphates and calcium. Biol Reprod.1997; 56:1450 -1457.[Abstract]
Yeung CH, Wagenfeld A, Nieschlag E, Cooper TG. The cause of
infertility of male c-ros tyrosine kinase receptor knockout mice.
Biol Reprod.2000; 63:612
-618.
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, bicarbonate absent. **: different from
"zero-time" value, P <.001.
,