Taking advantage of the use of supervised learning methods for characterization of sperm population structure related with freezability in the Iberian red deer

Using Iberian red deer as a model, this study presents a supervised learning method, the Support Vector Machines (SVM), to characterize sperm population structure related with freezability. Male freezability was assessed by evaluating motility, membrane status and mitochondrial membrane potential of sperm after a freezing-thawing procedure. The SVM model was generated using sperm motility information captured by computer-assisted sperm analysis (CASA) from thawed semen, belonging to six stags with marked differences on their freezability. A total of 1369 sperm tracks were recorded for seven kinematic parameters and assigned to four motility patterns based on them: weak motile, progressive, transitional and hyperactivated-like. Then, these data were split in two sets: the training set, used to train the SVM model, and the testing set, used to examine how the SVM method and three other unsupervised methods, a non-hierarchical, a hierarchical and a multistep clustering procedures, performed the sperm classification into subpopulations. The SVM was revealed as the most accurate method in the characterization of sperm subpopulations, showing all the sperm subpopulations obtained in this way high significant correlations with those sperm parameters used to characterize freezability of males. Given its superiority, the SVM method was used to characterize the sperm motile subpopulations in Iberian red deer. Sperm motile data from frozen-thawed semen belonging to 25 stags were recorded and loaded into the SVM model. The sperm population structure revealed that those males showing poor freezability were characterized by high percentages of sperm with a weak motility pattern. In opposite, males showing good freezability were characterized by higher percentages of sperm with a progressive and hyperactivated-like motility pattern and lower percentages of sperm with a weak motile pattern. We also identified a sperm subpopulation with a transitional motility pattern. This subpopulation increased as the freezability of males improved, and may be used as indicative of overall sperm motility.     

Action of metallic ions on the precocious development by rabbit sperm of motion patterns that are characteristic of hyperactivated motility

An earlier study demonstrated that rabbit sperm incubated for 16 hr under capacitation conditions acquire motility patterns identical to those seen in rabbit sperm capacitated in vivo. We now show that similar motion patterns develop after 0.5 hr incubation in a Trisbuffered medium, medium M. Development and decline of the motion patterns occurred in three phases each recognized by the character of the biphasic motion patterns. Hyperactivated sperm were objectively identified and quantified by a previously developed computer-directed model. The percentage of motile sperm that acquired hyperactivated motility and the period they remained in this state varied among sperm from different rabbits. The decline in hyperactivated motility was paralleled by a decrease in the average sperm curvilinear velocity (VCL) and average amplitude of lateral head displacement (AALH), but was not accompanied by a concomitant decrease in percentage of motile sperm. Pb²⁺ and Cd²⁺, at concentrations that did not inhibit motility, prevented development of hyperactivated motility. Inhibition of hyperactivated motility by Pb²⁺ was time- and concentration-dependent; the average percentage of hyperactivated sperm decreased from ～ 30% to<5% (n = 5) in 1 hr at a Pb²⁺ concentration of 25 μM. Cd²⁺ inhibition of hyperactivation was dependent only on concentration of the cation. At a concentration of 100 μM, the decrease in the percent of hyperactivated sperm was ～ 50% (n = 3). Hg²⁺, Zn²⁺, and Cr⁶⁺ at sublethal concentrations had no effect on hyperactivated motility development. These results suggest that Pb²⁺ and Cd²⁺, by virtue of their ability to prevent the wide curvature flagella beating that is characteristic of hyperactivation, can compromise fertilization at concentrations that do not inhibit sperm motility and act as a reproductive toxicant at a level other than spermatogenesis. © 1995 Wiley-Liss, Inc.     

Changes in the motility patterns of spermatozoa from the rabbit epididymis as assessed by computer-aided sperm motion analysis

Sperm maturation in the epididymis includes changes in their potential for motility that enables spermatozoa to reach the egg and penetrate its investments. The motility characteristics of spermatozoa from the testis, the epididymis, and vas deferens of the rabbit were investigated by computer-assisted sperm analysis (CASA). Various forms of motility were displayed by sperm from different regions of the epididymis released into incubation medium Testicular sperm were motile, although nonprogressive. The maximum percentage motility was expressed by sperm in the proximal cauda epididymidis, and forward progression was developed by spermatozoa from the distal caput. Once forward progression was established, the curvilinear velocity was about the same for sperm from all regions of the tract, whereas straight-line velocity increased between the mid-corpus and cauda and paralleled the decline in lateral displacement of the head. The maintenance of motility in vitro was best maintained by sperm from the distal regions of the tract although sperm from the distal caput maintained motility better than sperm from the proximal and midcorpus regions. Analysis of the motile sperm cells revealed several types of trajectories (“irregular,” “small circular,” “large circular and arcs,” “jagged” and “straight-line”) that were analyzed by discriminant analysis using the variables generated by CASA. Accuracy of classification varied from 70% to 96%, depending on the type of track. The classification function was then applied to the changes that occurred during incubation and showed that irregular trajectories gave way to small and then large circular tracks and progressive forms as sperm matured. © 1996 Wiley-Liss, Inc.     

Identification of sperm subpopulations with defined motility characteristics in ejaculates from Ile de France rams

The aims of this study were to identify different motile sperm subpopulations in fresh ejaculates from six Ile de France rams, by using a computer-assisted sperm motility analysis (CASA) system, and to evaluate the effects of individual ram and season on population distribution. Overall sperm motility and individual kinematic parameters of motile spermatozoa were evaluated for 125,312 spermatozoa, defined by curvilinear velocity (VCL), linear velocity (VSL), average path velocity (VAP), linearity coefficient (LIN), straightness coefficient (STR), wobble coefficient (WOB), mean amplitude of lateral head displacement (ALH) and frequency of head displacement (BCF). A multivariate cluster analysis was carried out to classify these spermatozoa into a reduced number of subpopulations according to their movement patterns. The statistical analysis clustered the whole motile sperm population into five separate groups: subpopulation 1, constituted by rapid, progressive and non sinuous spermatozoa (VCL=126.41 μm/s, STR=92.87% and LIN=86.47%); subpopulation 2, characterized by progressive spermatozoa with moderate velocity (VCL=74.74 μm/s and STR=84.03%); subpopulation 3, represented by rapid, progressive and sinuous spermatozoa (VCL=130.45 μm/s, STR=76.02% and LIN=47.68%); subpopulation 4 represents rapid nonprogressive spermatozoa (VCL=128.69 μm/s and STR=44.09%); subpopulation 5 includes poorly motile, nonprogressive spermatozoa with a very irregular trajectory (VCL=36.81 μm/s and STR=47.04%). Our results show the existence of five subpopulations of motile spermatozoa in ram ejaculates. The frequency distribution of spermatozoa within subpopulations was quite similar for the six rams, and the five subpopulations turned out to be very stable along seasons.     

Tyrphostin-A47 inhibitable tyrosine phosphorylation of flagellar proteins is associated with distinct alteration of motility pattern in hamster spermatozoa

To acquire fertilizing potential, mammalian spermatozoa must undergo capacitation and acrosome reaction. Our earlier work showed that pentoxifylline (0.45 mM), a sperm motility stimulant, induced an early onset of hamster sperm capacitation associated with tyrosine phosphorylation of 45-80 kDa proteins, localized to the mid-piece of the sperm tail. To assess the role of protein tyrosine phosphorylation in sperm capacitation, we used tyrphostin-A47 (TP-47), a specific protein tyrosine kinase inhibitor. The dose-dependent (0.1-0.5 mM) inhibition of tyrosine phosphorylation by TP-47 was associated with inhibition of hyperactivated motility and 0.5 mM TP-47-treated spermatozoa exhibited a distinct circular motility pattern. This was accompanied by hypo-tyrosine phosphorylation of 45-60 kDa proteins, localized to the principal piece of the intact-sperm and the outer dense fiber-like structures in detergent treated-sperm. Sperm kinematic analysis (by CASA) of spermatozoa, exhibiting circular motility (at 1st hr), showed lower values of straight line velocity, curvilinear velocity and average path velocity, compared to untreated controls. Other TP-47 analogues, tyrphostin-AG1478 and -AG1296, had no effect either on kinematic parameters or sperm protein tyrosine phosphorylation. These studies indicate that TP-47-induced circular motility of spermatozoa is compound-specific and that the tyrosine phosphorylation status of 45-60 kDa flagellum-localized proteins could be key regulators of sperm flagellar bending pattern, associated with the hyperactivation of hamster spermatozoa.     

Hamster sperm motility transformation during development of hyperactivation in vitro and epididymal maturation

The transformation of hamster sperm motility during capacitation in vitro and during maturation in the caudal epididymis was analyzed and compared using videomicrography. Sperm recovered from the distal portion of the caudal epididymis, as well as ejaculated sperm recovered from the uterus exhibited low amplitude, planar flagellar beating. By 3 hr of incubation under capacitating conditions, the caudal epididymal sperm were swimming in helical patterns apparently produced by significantly increased acuteness of flagellar bending and by torsion seen as abrupt, periodic turning of the head. By 4 hr, most sperm were hyperactivated, swimming in circles resulting from asymmetrical, planar flagellar bending that was significantly more acute than the preceding patterns. When motility parameters of fresh sperm were compared with those of sperm swimming in the transitional helical pattern and with hyperactivated sperm, transitional sperm had significantly higher net and average path velocities than the others, indicating that they covered space at the greatest rate. This suggests that the transitional phase plays an important role in sperm transport. Sperm recovered from the proximal region of the caudal epididymis, near the corpus, swam in either the helical or hyperactivated patterns, or a mixture of the two. The means of their flagellar curvature ratios and linear indices were intermediate between helical and hyperactivated mean values. Thus, sperm undergoing final maturation in the caudal epididymis reverse the pattern of development of hyperactivation. Also, the development of hyperactivated motility must therefore entail induction of a preexisting potential for flagellar movement, rather than a maturational process.     

Stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators

Capacitation of rhesus monkey spermatozoa was assessed by monitoring sperm flagellar beat and trajectory changes during incubation in vitro and by determining sperm penetration into rhesus oocytes and hamster zona-free ova. Rhesus sperm capacitation in vitro depended on the addition to the culture medium of the cyclic nucleotide mediators, caffeine and dibutyryl cyclic AMP. Capacitation was correlated with the development of hyperactivated motility. Spermatozoa treated with the cyclic nucleotide mediators, and showing hyperactivated motility, penetrated 57.4% of all rhesus oocytes and fertilized 88.9% of mature rhesus oocytes that were morphologically normal. Control spermatozoa did not penetrate any of the eggs. Some sperm penetration into hamster ova occurred but was not statistically significant. These data provide a basis for achieving in-vitro fertilization in the rhesus monkey and information on specific sperm motility characteristics associated with fertilizing ability.     

Minimum and maximum extracellular Ca2+ requirements during mouse sperm capacitation and fertilization in vitro

The minimum and maximum extracellular Ca2+ concentrations required to promote capacitation, the acrosome reaction, hyperactivated motility, zona penetration and gamete fusion in the mouse have been established. The traces of free calcium in Ca2+-deficient medium were shown not to enhance capacitation since the inclusion of EGTA to chelate free ions during a 120 min preincubation failed to alter the kinetics of capacitation from those observed in the absence of EGTA; 1 h after addition of 1.80 mM-Ca2+, both suspensions were highly fertile. Complete capacitation, when suspensions were immediately functional upon the addition of 1.80 mM-Ca2+, required the presence of greater than or equal to 90 microM-Ca2. Considerably higher concentrations were required to initiate optimal sperm responses: acrosome reaction, 900 microM; gamete fusion, 900 microM; hyperactivated motility, 1.80 mM; zona penetration, 1.80 mM. None of these changes was effected when Ca2+ was less than 450 microM. The responses to elevated Ca2+ were dependent on the length of incubation, being initially positive and then negative. A short (30 min) exposure to 3.40 mM-Ca2+ (x 2 the standard) accelerated capacitation, as evidenced by significantly increased acrosome loss, precocious expression of hyperactivated motility and enhanced fertilizing ability when Ca2+ was reduced to 1.80 mM. However, extended (120 min) preincubation irreversibly damaged sperm function. In the presence of 7.20 mM-Ca2+ (x 4), fertilizing ability was inhibited at both 30 and 120 min, despite a high incidence of acrosome loss. The primary deleterious effect appeared to be on motility which was judged to be more erratic than in 1.80 mM-Ca2+, possibly due to elevated intracellular Ca2+. Because of the considerable difference in threshold Ca2+ concentrations, it is now possible to dissociate the Ca2+-dependent events of capacitation from those of the acrosome reaction and motility changes.     

Computer assisted sperm analysis of motility patterns of postthawed epididymal spermatozoa of springbok (Antidorcas marsupialis), impala (Aepyceros melampus), and blesbok (Damaliscus dorcus phillipsi) incubated under conditions supporting domestic cattle in vitro fertilization

The need for information on the reproductive physiology of different wildlife species is important for ex situ conservation using such methods as in vitro fertilization (IVF). Information on species reproductive physiology and evaluation of sperm quality using accurate, objective, repeatable methods, such as computer-assisted sperm analysis (CASA) for ex situ conservation has become a priority. The aim of this study was to evaluate motility patterns of antelope epididymal spermatozoa incubated for 4 h under conditions that support bovine IVF using CASA. Cauda epididymal spermatozoa were collected postmortem from testicles of springbok (N = 38), impala (N = 26), and blesbok (N = 42), and cryopreserved in biladyl containing 7% glycerol. Spermatozoa were thawed and incubated in Capacitation media and modified Tyrode lactate (m-TL) IVF media using a protocol developed for domestic cattle IVF. The study evaluates 14 motility characteristics of the antelope epididymal sperm at six time points using CASA. Species differences in CASA parameters evaluated under similar conditions were observed. Several differences in individual motility parameters at the time points were reported for each species. Epididymal sperm of the different antelope species responded differently to capacitation agents exhibiting variations in hyperactivity. Motility parameters that describe the vigor of sperm decreased over time. Spermatozoa from the different antelope species have different physiological and optimal capacitation and in vitro culture requirements. The interspecies comparison of kinematic parameters of spermatozoa between the antelopes over several end points contributes to comparative sperm physiology which forms an important step in the development of species specific assisted reproductive techniques (ARTs) for ex situ conservation of these species.     

The effect of heparin on motility parameters and protein phosphorylation during bovine sperm capacitation

It is generally accepted that incubation with heparin is required to induce capacitation of ejaculated bovine spermatozoa in vitro. The capacitation process implicates many biochemical events, and is correlated with modified sperm motility and the phosphorylation of specific proteins on tyrosine residues. To better understand the molecular basis of heparin-induced capacitation, bovine spermatozoa were incorporated with a radioactive substrate of protein kinases [gamma32P]-ATP, to observe protein phosphorylation dynamics over time. Sperm motion parameters including the percentage of motile spermatozoa, amplitude of lateral head displacement (ALH) and flagellar beat cross frequency (BCF) were assessed to determine whether the protein phosphorylation patterns induced by heparin also promote changes in motility. Capacitation was confirmed using the chlortetracycline fluorescence assay and the appearance of 'pattern B' stained spermatozoa. Evaluation of the different motility parameters during capacitation reveal that heparin has a marked negative effect, over time, on the percentage of motile spermatozoa, consistent with hyperactivation. Indeed, the presence of heparin greatly increases the BCF of bull spermatozoa and induces a significant increase in the ALH compared to spermatozoa incubated without heparin. We detected several sperm proteins that are phosphorylated over time. A 45 kDa protein is the most intensely phosphorylated of the sperm proteins. However, it is visible regardless of the presence of heparin. Interestingly, a second phosphorylated protein of approximately 50 kDa undergoes more intense phosphorylation with heparin than without. In summary, the present study demonstrated that heparin induces physiological changes in several sperm motility parameters including ALH, BCF and the percentage of motile spermatozoa. Heparin also increases the intensity of phosphorylation of a 50 kDa sperm protein. These results suggest that capacitation of bovine spermatozoa and capacitation-associated motility changes may be regulated by a mechanism that includes protein phosphorylation, and that a presently unknown protein kinase is involved.     

Hyaluronic acid delays boar sperm capacitation after 3 days of storage at 15 degrees C

The present study was undertaken to determine the effects of the addition of hyaluronic acid (HA), ranged from 12.5 to 200 microg/ml, on boar sperm capacitation status during a storage time (up to 3 days) at 15 degrees C in Beltsville thawing solution (BTS). The raw extender was the negative control whereas different concentrations of caffeine (CAF), ranged from 0.25 to 8mM, served as positive controls. Sperm viability, motility, morphology, and osmotic resistance were also determined before and after assessing the treatments. Samples were obtained from 28 healthy and post-pubertal Piétrain boars and sperm parameters were tested immediately after the addition of treatments and after 1, 2 and 3 days of refrigeration at 15 degrees C. Sperm capacitation status was determined by chlortetracycline (CTC) staining and sperm viability by means of a multiple fluorochrome-staining test. Sperm motility and morphology were assessed using phase-contrast microscopy accompanied by a computer assisted sperm analysis system (CASA). Whereas HA delayed sperm capacitation, CAF increased the frequency of capacitated spermatozoa after 2 days of cooling. Moreover, HA did not modify other sperm parameters, such as sperm velocity, whereas CAF increased progressive motility during the first 2 days of cooling and then decreased. It can be concluded that the addition of HA at 50 and 100 microg/ml to the BTS extender may delay sperm capacitation after 3 days of cooling.     

Identification of capacitation-associated phosphoproteins in porcine sperm electroporated with ATP-gamma-(32)P.

Our objectives were to incorporate ATP-gamma-(32)P into boar sperm to radiolabel endogenous phosphoproteins and compare phosphorylation patterns from sperm incubated in capacitating (CM) and non-capacitating conditions (NCM). Sperm were electroporated (1000 V/cm, 125 microF/cm, 65 Omega/cm, 0.3 msec) with ATP-gamma-(32)P which moderately decreased sperm viability (P < 0.01), but did not affect motility (P = 0.34) or the appearance of spontaneous acrosome reactions (P = 0.49). Sperm incubated in CM for 3 hr underwent capacitation, determined by the ability to undergo ionophore-induced acrosome reactions (P 0.05) and the 57 kDa phosphoprotein increased after capacitation (P /= 0.02). ATP-gamma-(32)P can, therefore, be incorporated into porcine sperm to radiolabel endogenous phosphoproteins, and the different profiles from sperm incubated in NCM versus CM suggest that capacitation is mediated by signaling events involving protein phosphorylation.     

Quantification and identification of sperm subpopulations using computer-aided sperm analysis and species-specific cut-off values for swimming speed

Abstract

Motility is an essential characteristic of all flagellated spermatozoa and assessment of this parameter is one criterion for most semen or sperm evaluations. Computer-aided sperm analysis (CASA) can be used to measure sperm motility more objectively and accurately than manual methods, provided that analysis techniques are standardized. Previous studies have shown that evaluation of sperm subpopulations is more important than analyzing the total motile sperm population alone. We developed a quantitative method to determine cut-off values for swimming speed to identify three sperm subpopulations. We used the Sperm Class Analyzer^® (SCA) CASA system to assess the total percentage of motile spermatozoa in a sperm preparation as well as the percentages of rapid, medium and slow swimming spermatozoa for six mammalian species. Curvilinear velocity (VCL) cut-off values were adjusted manually for each species to include 80% rapid, 15% medium and 5% slow swimming spermatozoa. Our results indicate that the same VCL intervals cannot be used for all species to classify spermatozoa according to swimming speed. After VCL intervals were adjusted for each species, three unique sperm subpopulations could be identified. The effects of medical treatments on sperm motility become apparent in changes in the distribution of spermatozoa among the three swimming speed classes.     

Soluble adenylyl cyclase (sAC) is indispensable for sperm function and fertilization

We previously demonstrated that male mice deficient in the soluble adenylyl cyclase (sAC) are sterile and produce spermatozoa with deficits in progressive motility and are unable to fertilize zona-intact eggs. Here, analyses of sAC(-/-) spermatozoa provide additional insights into the functions linked to cAMP signaling. Adenylyl cyclase activity and cAMP content are greatly diminished in crude preparations of sAC(-/-) spermatozoa and are undetectable after sperm purification. HCO(3)(-) is unable to rapidly accelerate the flagellar beat or facilitate evoked Ca(2+) entry into sAC(-/-) spermatozoa. Moreover, the delayed HCO(3)(-)-dependent increases in protein tyrosine phosphorylation and hyperactivated motility, which occur late in capacitation of wild-type spermatozoa, do not develop in sAC(-/-) spermatozoa. However, sAC(-/-) sperm fertilize zona-free oocytes, indicating that gamete fusion does not require sAC. Although ATP levels are significantly reduced in sAC(-/-) sperm, cAMP-AM ester increases flagellar beat frequency, progressive motility, and alters the pattern of tyrosine phosphorylated proteins. These results indicate that sAC and cAMP coordinate cellular energy balance in wild-type sperm and that the ATP generating machinery is not operating normally in sAC(-/-) spermatozoa. These findings demonstrate that sAC plays a critical role in cAMP signaling in spermatozoa and that defective cAMP production prevents engagement of multiple components of capacitation resulting in male infertility.     

Coupling biochemistry and hydrodynamics captures hyperactivated sperm motility in a simple flagellar model

Hyperactivation in mammalian sperm is characterized by highly asymmetrical waveforms and an increase in the amplitude of flagellar bends. It is important for the sperm to be able to achieve hyperactivated motility in order to reach and fertilize the egg. Calcium (Ca²⁺) dynamics are known to play a large role in the initiation and maintenance of hyperactivated motility. Here we present an integrative model that couples the CatSper channel mediated Ca²⁺ dynamics of hyperactivation to a mechanical model of an idealized sperm flagellum in a 3-d viscous, incompressible fluid. The mechanical forces are due to passive stiffness properties and active bending moments that are a function of the local Ca²⁺ concentration along the length of the flagellum. By including an asymmetry in bending moments to reflect an asymmetry in the axoneme's response to Ca²⁺, we capture the transition from activated motility to hyperactivated motility. We examine the effects of elastic properties of the flagellum and the Ca²⁺ dynamics on the overall swimming patterns. The swimming velocities of the model flagellum compare well with data for hyperactivated mouse sperm.     

Hyperactivated motility induced in mouse sperm by calcium ionophore A23187 is reversible

The reversibility of hyperactivated motility was tested in caudal epididymal mouse sperm by treating them with 1 microM calcium ionophore A23187 in dimethyl sulfoxide (DMSO), followed 2 min later by the addition of medium containing high levels of bovine serum albumin (BSA) (final concentrations: 0.5 microM A23187, 22 mg/ml BSA). Controls received DMSO alone, followed by BSA. Immediately following treatment with A23187, motility was weak and vibratory. Two minutes after the addition of high levels of BSA, motility was hyperactivated, as determined by videotape analysis of linearity of trajectory and acuteness of flagellar bending. Ten minutes after the addition, the movement pattern returned to that of fresh, uncapacitated epididymal sperm. Control sperm retained the linear swimming pattern of fresh caudal epididymal sperm during the 10 min of observation. Ninety minutes later, however, both control and treated sperm became hyperactivated. The percentage of motile sperm was not affected by treatment or time. Thus, ionophore-induced hyperactivation is reversible and does not interfere with the normal development of hyperactivation during incubation under capacitating conditions in vitro.     

The comparison of assessment of pigeon semen motility and sperm concentration by conventional methods and the CASA system (HTM IVOS)

The aim of these experiments was to compare conventional, microscopic methods of evaluating pigeon sperm motility and concentration to those measured by computer-assisted sperm analysis (CASA system). Semen was collected twice a week from two groups of pigeons, each of 40 males (group I: meat-type breed; group II: fancy pigeon) using the lumbo-sacral and cloacal region massage method. Ejaculates collected in each group were diluted 1:100 in BPSE solution and divided into two equal samples. One sample was examined subjectively by microscope and the second one was analysed using CASA system. The sperm concentration was measured by CASA using the anti-collision (AC) system and fluorescent staining (IDENT). There were not any significant differences between the methods of evaluation of sperm concentration. High positive correlations in both groups were observed between the sperm concentration estimated by Thom counting chamber and AC (r=0.87 and r=0.91, respectively), and between the sperm concentration evaluated by Thom counting chamber and IDENT (r=0.85 and r=0.90, respectively). The mean values for CASA measurement of proportion of motile spermatozoa (MOT) and progressive movement (PMOT) were significantly lower than the values estimated subjectively in both groups of pigeons (p< or =0.05 and p< or =0.01, respectively). Positive correlations in MOT and PMOT were noted between both methods of evaluation. The CASA system is very rapid, objective and sensitive method in detecting subtle motility characteristics as well as sperm concentration and is recommended for future research into pigeon semen.     

Different signaling pathways in bovine sperm regulate capacitation and hyperactivation

Hyperactivated sperm motility is characterized by high-amplitude and asymmetrical flagellar beating that assists sperm in penetrating the oocyte zona pellucida. Other functional changes in sperm, such as activation of motility and capacitation, involve cross talk between the cAMP/PKA and tyrosine kinase/phosphatase signaling pathways. Our objective was to determine the role of the cAMP/protein kinase A (PKA) signaling pathway in hyperactivation. Western blot analyses of detergent extracts of whole sperm and flagella were performed using antiphosphotyrosine antibody. Bull sperm capacitated by 10 microg/ml heparin and/or 1 mM dibutyryl-cAMP plus 100 microM 3-isobutyl-1-methylxanthine exhibited increased protein tyrosine phosphorylation without becoming hyperactivated. Procaine (5 mM) or caffeine (10 mM) immediately induced hyperactivation in nearly 100% of motile sperm but did not increase protein tyrosine phosphorylation. After 4 h of incubation with caffeine, sperm expressed capacitation-associated protein tyrosine phosphorylation but hyperactivation was significantly reduced. Sperm initially hyperactivated by procaine or caffeine remained hyperactivated for at least 4 h in the presence of Rp-cAMPS (cAMP antagonist) or PKA inhibitors H-89 or H-8. Pretreatment with inhibitors also failed to block induction of hyperactivation; however, the inhibitors did block protein tyrosine phosphorylation when sperm were incubated with capacitating agents, thereby verifying inhibition of the cAMP/PKA pathway. While induction of hyperactivation did not depend on cAMP/PKA, it did require extracellular Ca(2+). These findings indicate that hyperactivation is mediated by a Ca(2+) signaling pathway that is separate or divergent from the pathway associated with acquisition of acrosomal responsiveness and does not involve protein tyrosine phosphorylation downstream of the actions of procaine or caffeine.