The onset and maintenance of hyperactivated motility of spermatozoa from the mouse

Estimates were made of the proportion of freely motile mouse spermatozoa displaying hyperactivated motility by an objective photographic method employing stroboscopic illumination under dark-field conditions and examining displacements of the sperm head and bend angles of the sperm tail. In media known to support in vitro fertilisation hyperactivation gradually appeared reaching about 40% by 6 hr incubation, and it was not promoted by 2 mM caffeine or 0.1 mM Bt₂ cAMP or washing the cells free of epididymal fluid. Raising the osmolarity of the medium to 400 mOSM with electrolytes, but not nonelectrolytes, did promote hyperactivation (60% by 2 hr) suggesting that the ionic strength of the medium was important. Hyperactivation in high ionic strength media could be prevented by removing or chelating Ca²⁺, or replacing Ca²⁺ with Ba²⁺ or Mg²⁺, when nonhyperactivated motility was maintained, but Sr²⁺, like Ca²⁺, permitted hyperactivated motility. Hyperactivation in low ionic strength medium could be promoted by the ionophore A23187, suggesting that Ca²⁺ movement into the cells is important. Of a range of glycolytic substrates tested supporting nonhyperactivated motility in the presence of lactate, only glucose supported hyperactivation. Addition to glucose— or Ca²⁺ — free, high ionic strength media after 2 hr increased hyperactivation immediately (glucose) or after a lag of 2 hr (Ca²⁺) suggesting that glucose acts on a Ca²⁺ — primed system. Removal from high ionic strength medium, chelation of Ca²⁺ or inhibition of glucose metabolism did not prevent hyperactivation continuing once it had been initiated, indicating different requirements for initiation and maintenance of this form of motility.     

Relationship between calcium,cyclic AMP,ATP, and intracellular pH and the capacity of hamster spermatozoa to express hyperactivated motility

Capacitation of hamster caudal spermatozoa at a density of 1 × 10⁶/ml is associated with a progressive rise in cAMP levels that precedes the onset of hyperactivated motility. This increase is not expressed by caput spermatozoa incubated under identical conditions. Both the incidence of hyperactivation and the rise in cAMP levels are severely attenuated in the absence of exogenous calcium. Neither factor is restored to control levels by the addition of the phosphodiesterase inhibitor IBMX, although in the presence of exogenous calcium, this reagent increased cAMP levels, stimulated percentage motility and advanced the appearance of hyperactivation. Treatment of spermatozoa at a density of 1 × 10⁶/ml with the calmodulin antagonist, calmidazolium (CZ), caused severe disruption of sperm motility and abolished hyperactivation, while causing only a slight reduction in cAMP content. Addition of IBMX in the presence of CZ elevated cAMP content to levels higher than normally observed during capacitation but did not restore either coordinated or hyperactivated motility. To determine both the mechanisms responsible for this elevation of cAMP content and the changes that occur during epididymal maturation to facilitate the expression of this increase, the free cytosolic calcium concentration, ATP levels, and intracellular pH of caput and caudal cells were compared. The calcium content of caudal spermatozoa rose significantly at a time when cAMP levels were increasing, while ATP content and intracellular pH fell. However, the inability of caput spermatozoa to express a rise in cAMP content was not due to deficiencies in any of these factors. These results indicate a positive role for the cAMP rise in the expression of hyperactivated motility and that the fundamental control mechanism governing both these events may be the influx of calcium that accompanies capacitation in this species.     

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.     

Hyperactivated motility of bull sperm is triggered at the axoneme by Ca2+ and not cAMP

Hyperactivated motility, a swimming pattern of mammalian sperm in the oviduct, is essential for fertilization in vivo. It is characterized by high-amplitude flagellar waves and, usually, highly asymmetrical flagellar beating. It had been suggested, but not tested, that Ca2+ and cAMP switch on hyperactivation by directly affecting the flagellar axoneme. In this study, the direct affects of these agents on the axoneme were tested by using detergent-demembranated bull sperm. As confirmed by TEM, treatment of sperm with 0.2% Triton X-100 disrupted the plasma, acrosomal, and inner mitochondrial membranes, leaving axonemes intact. In the presence of 2 mM ATP, the percentage of reactivated sperm that were hyperactivated increased to 80% when free Ca2+ was increased from 50 to 400 nM. The effect of the Ca2+ in this range was to increase beat asymmetry by increasing the curvature of the principal bend. No additional increases were observed above 400 nM free Ca2+, but motility was suppressed at 1 mM. The ability of Ca2+ to produce hyperactivation depended on ATP availability, such that more ATP was required to produce the high amplitude flagellar bends characteristic of hyperactivated motility than to produce activated motility. Cyclic AMP was not required for reactivation, nor for hyperactivation. Production of hyperactivated motility also required an alkaline environment (pH 7.9-8.5). These results suggest that, provided sufficient ATP is present and pH is sufficiently alkaline, Ca2+ switches on hyperactivation by enabling curvature of the principal bends to increase.     

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.     

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.     

Characterization of the intracellular calcium store at the base of the sperm flagellum that regulates hyperactivated motility

Hyperactivated sperm motility is usually characterized by high-amplitude flagellar bends and asymmetrical flagellar beating. There is evidence that an inositol 1,4,5-trisphosphate (IP3) receptor-gated Ca2+ store in the base of the flagellum provides Ca2+ to initiate hyperactivation; however, the identity of the store was not known. Ca2+ stores are membrane-bounded organelles, and the only two membrane-bounded organelles found in this region of sperm are the redundant nuclear envelope (RNE) and mitochondria. Transmission electron micrographs revealed two different compartments of RNE, one enriched with nuclear pores and the other containing few pores but extensive membranous structures with enlarged cisternae. Immunolabeling showed that IP3 receptors and calreticulin are located in the region containing enlarged cisternae. In other cell types, mitochondria adjacent to Ca2+ stores are actively involved in modulating Ca2+ signals by taking up Ca2+ released from stores and also may respond by increasing production of NADH and ATP to support increased energy demand. Nevertheless, bull sperm did not show an increase in NADH when Ca2+ was released from intracellular stores by thapsigargin to induce hyperactivation. Consistently, no net increase in ATP production was detected when sperm were hyperactivated, although ATP was hydrolyzed at a greater rate. Furthermore, blocking Ca2+ efflux from mitochondria by CGP-37157, a specific inhibitor of the mitochondrial Na+/Ca2+ exchanger, did not inhibit the development of hyperactivated motility. We concluded that the intracellular Ca2+ store is the part of RNE that contains enlarged cisternae and that Ca2+ is released directly to the axoneme to trigger hyperactivated motility without the active participation of mitochondria.     

Hyperactivation of monkey spermatozoa is triggered by Ca2+ and completed by cAMP

Digital image analysis of the flagellar movements of cynomolgus macaque spermatozoa hyperactivated by caffeine and cAMP was carried out to understand the change in flagellar movements during hyperactivation. The degree of flagellar bending increased remarkably after hyperactivation, especially at the base of the midpiece. Mainly two beating patterns were seen in the hyperactivated monkey sperm flagella: remarkably asymmetrical flagellar bends of large amplitude and relatively symmetrical flagellar bends of large amplitude. The asymmetrical bends were often seen in the early stage of hyperactivation, whereas the symmetrical bends executed nonprogressive, figure-of-eight movement. Beat frequency of the hyperactivated spermatozoa significantly decreased while wavelength of flagellar waves roughly doubled. To determine the conditions under which the axonemes of hyperactivated sperm flagella have asymmetrical or symmetrical bends, the plasma membranes of monkey spermatozoa were extracted with Triton X-100 and motility was reactivated with MgATP(2-) under various conditions. The asymmetrical flagellar bends were brought about by Ca(2+), whereas the symmetrical flagellar bends resulted from low levels of Ca(2+) and high levels of cAMP. Under these conditions, beat frequency and wavelength of flagellar waves of demembranated, reactivated spermatozoa were similar to those of the hyperactivated spermatozoa. These results suggest that during hyperactivation of monkey spermatozoa intracellular Ca(2+) concentrations first rise, and then decrease while cAMP concentrations increase simultaneously.     

Calcium/calmodulin and calmodulin kinase II stimulate hyperactivation in demembranated bovine sperm

Hyperactivated motility is observed among sperm in the mammalian oviduct near the time of ovulation. It is characterized by high-amplitude, asymmetrical flagellar beating and assists sperm in penetrating the cumulus oophorus and zona pellucida. Elevated intracellular Ca2+ is required for the initiation of hyperactivated motility, suggesting that calmodulin (CALM) and Ca2+/CALM-stimulated pathways are involved. A demembranated sperm model was used to investigate the role of CALM in promoting hyperactivation. Ejaculated bovine sperm were demembranated and immobilized by brief exposure to Triton X-100. Motility was restored by addition of reactivation medium containing MgATP and Ca2+, and hyperactivation was observed as free Ca2+ was increased from 50 nM to 1 microM. However, when 2.5 mM Ca2+ was added to the demembranation medium to extract flagellar CALM, motility was not reactivated unless exogenous CALM was readded. The inclusion of anti-CALM IgG in the reactivation medium reduced the proportion hyperactivated in 1 microM Ca2+ to 5%. Neither control IgG, the CALM antagonist W-7, nor a peptide directed against the CALM-binding domain of myosin light chain kinase (MYLK2) inhibited hyperactivation. However, when sperm were reactivated in the presence of CALM kinase II (CAMK2) inhibiting peptides, hyperactivation was reduced by 75%. Furthermore, an inhibitor of CAMK2, KN-93, inhibited hyperactivation without impairing normal motility of intact sperm. CALM and CAMK2 were immunolocalized to the acrosomal region and flagellum. These results indicate that hyperactivation is stimulated by a Ca2+/CALM pathway involving CAMK2.     

Hepatitis B Virus S Protein Enhances Sperm Apoptosis and Reduces Sperm Fertilizing Capacity In Vitro

Objective

Studying the impact of Hepatitis B virus S protein (HBs) on early apoptotic events in human spermatozoa and sperm fertilizing capacity.

Methodology/Principal Findings

Spermatozoa were exposed to HBs (0, 25, 50, 100 µg/ml) for 3 h, and then fluo-4 AM calcium assay, Calcein/Co²⁺ assay, protein extraction and ELISA, ADP/ATP ratio assay, sperm motility and hyperactivation and sperm-zona pellucida (ZP) binding and ZP-induced acrosome reaction (ZPIAR) tests were performed. The results showed that in the spermatozoa, with increasing concentration of HBs, (1) average cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) rose; (2) fluorescence intensity of Cal-AM declined; (3) average levels of cytochrome c decreased in mitochondrial fraction and increased in cytosolic fraction; (4) ADP/ATP ratios rose; (5) average rates of total motility and mean hyperactivation declined; (6) average rate of ZPIAR declined. In the above groups the effects of HBs exhibited dose dependency. However, there was no significant difference in the number of sperms bound to ZP between the control and all test groups.

Conclusion

HBs could induce early events in the apoptotic cascade in human spermatozoa, such as elevation of [Ca²⁺]_i, opening of mitochondrial permeability transition pore (MPTP), release of cytochrome c (cyt c) and increase of ADP/ATP ratio, but exerted a negative impact on sperm fertilizing capacity.     

Requirement of extracellular calcium ions for various stages of fertilization and fertilization-related phenomena in the hamster

Using a semi-chemically defined medium, the requirement of extracellular Ca²⁺ for survival, capacitation, and acrosome reaction of spermatozoa as well as various stages of fertilization in the hamster was studied. A Ca²⁺-deficient environment is unfavorable for long-term survival of spermatozoa. Sperm capacitation may occur in Ca²⁺-deficient media, but not as efficiently as in normal media. The acrosome reaction definitely requires extracellular Ca²⁺. Other processes or phenomena that require extracellular Ca²⁺ are initiation and maintenance of hyperactivated motility of spermatozoa, penetration of acrosome-reacted spermatozoa into the zona pellucida, fusion of the spermatozoa with eggs, and the development of pronuclear eggs into two-cell embryos. Extracellular Ca²⁺ is apparently unnecessary for the attachment of spermatozoa to the zona and egg surfaces, decondensation of the sperm nucleus, and the development of sperm and egg pronuclei within the egg. These results were compared with data obtained in other species such as the sea urchin, mouse, rat and guinea pig.     

Regulation of activation state and flagellar wave form in epididymal rat sperm: evidence for the involvement of both Ca2+ and cAMP

Rat sperm from the cauda epididymis exhibit increased motility, longevity, and a distinct circular pattern of flagellar curvature in response to 5 mM procaine-HCl or 0.1 mM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), reagents that are thought to play a role in the immobilization of free cellular calcium. Triton X-100-extracted sperm models will exhibit the same pattern of motility and curvature as procaine- or TMB-8-activated cells, but only when calcium is removed by a strong chelating agent, and in the presence of cAMP (3 microM). Demembranated sperm models produced from epididymal rat sperm are quiescent unless cAMP is added. In these sperm models, the presence or absence of free calcium mediates a transition in flagellar curvature. The increased activity of the procaine-treated intact cells was not accompanied by a change in cellular ATP content, nor was ATP availability the limiting factor in the quiescent sperm. Therefore, the increased motility produced by procaine is probably mediated by a fall in free intracellular Ca2+ accompanied by a rise in cAMP. Our finding that calcium controls the curvature of sperm flagella may explain altered patterns of flagellar beating, such as the hyperactivated motility that sperm exhibit in the female reproductive tract.     

Glucose regulation of guinea-pig sperm motility.

Washed guinea-pig spermatozoa from the vas deferens re-acquired progressive motility within 1-2 min of incubation in minimal culture medium containing pyruvate and lactate. When glucose was added, either at the beginning or after 15 min of incubation, the cells showed stimulated motility (increased straight-line velocity, linearity and beat-cross frequency, P less than 0.01). Re-acquisition of progressive motility was preceded by a significant (P less than 0.005) transient increase in sperm concentration of cyclic adenosine 5'-phosphate (cAMP) with or without glucose in the medium. Papaverine caused another large significant (P less than 0.001) increase in cAMP concentration; and 5.56mM glucose with papaverine caused a further stimulation in cAMP beyond that with papaverine alone (P less than 0.005). Although 0.05 or 5.56mM glucose plus alpha-chlorohydrin stimulated sperm motility, they did not further stimulate the increase in cAMP after 30 s of incubation. Thus, there was no apparent correlation between the glucose-stimulating effect on sperm motility and the enhancement of cAMP at 30 s. However, there was a close correlation between glucose-stimulated motility and enhancement of ATP (P less than 0.05) by glucose even under incubation conditions in which glucose caused the Crabtree effect (decrease in respiration rate).     

An inositol 1,4,5-trisphosphate receptor-gated intracellular Ca(2+) store is involved in regulating sperm hyperactivated motility.

Hyperactivated motility, a swimming pattern displayed by mammalian sperm in the oviduct around the time of ovulation, is essential to fertilization. Ca(2+) has been shown to be crucial for the initiation and maintenance of hyperactivated motility. Nevertheless, how Ca(2+) reaches the axoneme in the core of the flagellum to switch on hyperactivation is unknown. Ca(2+)-releasing agents were used to determine whether an intracellular store provides Ca(2+) to the axoneme. Hyperactivation was induced immediately in bull sperm by thapsigargin, caffeine, and thimerosal. The responses were dose-dependent and were induced in both capacitated and uncapacitated sperm. When external Ca(2+) was buffered below 50 nM with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, the response to caffeine was significantly reduced; however, the responses to thapsigargin and thimerosal were not affected. This indicates caffeine-induced hyperactivation depends on external Ca(2+) influx, whereas hyperactivation by thapsigargin and thimerosal do not. Acrosome reactions were not induced by these treatments; therefore, an acrosomal store was probably not involved. Indirect immunofluorescence labeling showed type I inositol 1,4,5-trisphosphate receptors (IP(3)R) in the acrosome and neck region, but no ryanodine receptors (RyR) were found using anti-RyR antibodies or BODIPY FL-X ryanodine. These data indicate that there is an IP(3)R-gated Ca(2+) store in the neck region of sperm that regulates hyperactivated motility.     

Initiation of Sperm Motility Induced by Cyclic AMP in Hamster and Boar

When the plasma membrane of hamster and boar spermatozoa was extraced by treatment with Triton X-100 and the demembranated spermatozoa were transferred to a reactivating medium containing only ATP, axonemes were initially immotile, and then gradually became motile. Under these experimental conditions, the cAMP content in the reactivating medium increased soon. This suggests that cAMP is synthesized from ATP by adenylate cyclase involved in incompletely removed or solubilized residual sperm membrane and that the autosynthesized cAMP causes the delay in motility initiation. This delayed initiation of motility did not occur when phosphodiesterase was added to the reactivating medium and the phosphodiesterase-dependent quiescent sperm became motile instantaneously at any time when excess cAMP was supplemented. Furthermore, demembranated sperm which were diluted in the reactivating medium containing ATP and cAMP, immediately became motile. cAMP levels in the cell increased during the initiation of sperm motility in both species. These results suggest that cAMP is the real factor indispensable for the initiation of sperm motility at ejaculation in mammals.     

Relationship of protein tyrosine phosphorylation state with tolerance to frozen storage and the potential to undergo cyclic AMP‐dependent hyperactivation in the spermatozoa of Japanese Black bulls

The aim of this study was to elucidate the relationship between protein tyrosine phosphorylation state and sperm characteristics in frozen‐stored spermatozoa of Japanese Black bulls. The spermatozoa were washed with PBS containing polyvinyl alcohol and then incubated with cell‐permeable cAMP analog cBiMPS to induce flagellar hyperactivation. Before and after incubation, the spermatozoa were used for immunodetection of tyrosine‐phosphorylated proteins, assessment of morphological acrosome condition and evaluation of motility. In bulls whose frozen‐stored spermatozoa were classified as having a high‐grade acrosome condition before incubation, sperm tyrosine‐phosphorylated proteins, including the 33‐kDa tyrosine‐phosphorylated SPACA1 protein, were localized in the anterior region of the acrosome and equatorial subsegment. The immunodetection level of the 41‐ and 33‐kDa sperm tyrosine‐phosphorylated proteins in the Western blots and the immunofluorescence of tyrosine‐phosphorylated proteins and SPACA1 proteins in the anterior region of the sperm acrosome were lower in bulls whose frozen‐stored sperm were classified as having a low‐grade acrosome condition. On the other hand, after incubation with cBiMPS, immunodetection levels of at least 10 tyrosine‐phosphorylated proteins increased in the connecting and principal pieces of spermatozoa, coincident with the induction of flagellar hyperactivation. Many of the spermatozoa also exhibited detection patterns similar to those of boar hyperactivated spermatozoa. These results are consistent with the suggestion that immunodetection levels of tyrosine‐phosphorylated proteins are valid markers that can predict the level of tolerance to frozen storage and the potential to undergo cAMP‐dependent hyperactivation for the spermatozoa of individual Japanese Black bulls. Mol. Reprod. Dev. 77:910–921, 2010. © 2010 Wiley‐Liss, Inc.     

Mouse lipocalin as an enhancer of spermatozoa motility

The 24p3 protein is a 25 kDa glycoprotein that is secreted into the uterine fluid during the proestrous phase of mice. We assessed the effects on spermatozoa motility and on the functions of mouse spermatozoa using the computer-assisted sperm analysis method, cytochemical staining and detection of the protein tyrosine phosphorylation pattern. Compared with the control cells, sperm motility was stimulated by the addition of 24p3 protein into the medium. Introducing 24p3 protein enhanced progressive motility but did not promote the appearance of hyperactivated movement. The presence of 24p3 protein in the medium did not allow the cells to undergo the capacitated protein tyrosine phosphorylation pattern and acrosome reaction. The tyrosine phosphorylation pattern shows phosphoproteins in the range of Mr 50000–106000 correlated with the sperm progressive motility after the addition of 24p3 protein into the medium. Using flow cytometry, we assessed the changes in the intracellular pH and measured the intracellular cAMP concentration with an immunodetection kit. The results indicated that the elevation in intracellular pH from 6.67 to 6.89, increase of intracellular cAMP accumulation, and protein tyrosine phosphorylation might be the factors in enhancement of sperm motility as the 24p3 protein bound to the spermatozoa. The 24p3 protein may have a role in regulating flagellar motility.     

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.     

Phosphorylation of triton X-100 soluble and insoluble protein substrates in a demembranated/reactivated human sperm model

Sperm motility is a process which involves a cascade of events mediated by cAMP and Ca²⁺, cAMP in the initiation of flagellar movement, and Ca²⁺ in the regulation of beat asymmetry, and it has been suggested that these two messengers act through phosphorylation/dephosphorylation of axonemal proteins. Only a few studies on human sperm protein phosphorylation have been reported and no relation of this process with motility or other function has been established. In the present study, phosphorylation of human sperm proteins was performed using detergent-demembranated spermatozoa, in which motility is reactivated by the addition of ATP. This system allows direct accessibility of intracellular kinases to [³²P]-γATP and allows some relation between protein phosphorylation and flagellar movements. After electrophoresis and autoradiography, numerous phosphoproteins were detected. Phosphorylation of 2 proteins (36 and 51 kDa) was stimulated by cAMP in a concentration-dependent manner, and this increase was prevented by inhibitors of cAMP-dependent protein kinase. In order to characterize phosphoproteins originating from the cytoskeleton or axoneme, detergent extracted spermatozoa were also subjected to phosphorylation. Three major phosphorylated proteins (14.8, 15.3, and 16.2 kDa) were detected, the first two expressing cAMP-dependency according to their cAMP concentration-dependent increase in phosphorylation and the reversal of this effect by inhibitors of cAMP-dependent protein kinase. Proteins phosphorylation during the reactivation of demembranated spermatozoa previously immobilized H₂O₂, xanthine + xanthine oxidase-generated reactive oxygen species, or the oxidative phosphorylation uncoupler rotenone, revealed increases in cAMP-independent phosphorylation of proteins of 16.2, 46, and 93 kDa. These results documenting human sperm phosphoproteins form a base for further studies on the role of protein phosphorylation in sperm functions. © 1996 Wiley-Liss, Inc.     

CRISP1 as a novel CatSper regulator that modulates sperm motility and orientation during fertilization

Ca²⁺-dependent mechanisms are critical for successful completion of fertilization. Here, we demonstrate that CRISP1, a sperm protein involved in mammalian fertilization, is also present in the female gamete and capable of modulating key sperm Ca²⁺ channels. Specifically, we show that CRISP1 is expressed by the cumulus cells that surround the egg and that fertilization of cumulus–oocyte complexes from CRISP1 knockout females is impaired because of a failure of sperm to penetrate the cumulus. We provide evidence that CRISP1 stimulates sperm orientation by modulating sperm hyperactivation, a vigorous motility required for penetration of the egg vestments. Moreover, patch clamping of sperm revealed that CRISP1 has the ability to regulate CatSper, the principal sperm Ca²⁺ channel involved in hyperactivation and essential for fertility. Given the critical role of Ca²⁺ for sperm motility, we propose a novel CRISP1-mediated fine-tuning mechanism to regulate sperm hyperactivation and orientation for successful penetration of the cumulus during fertilization.