Intracellular pH of bovine sperm increases during capacitation

The effect of heparin-induced capacitation on the intracellular pH (pH_i) of individual bovine sperm was determined with image analysis. Sperm were loaded with the acetoxymethyl ester of the pH sensitive fluorescent indicator, 2′,7′-bis(carboxyethyl)-5(6)-carboxy-fluorescein (BCECF). The pH_i of 5303 sperm was evaluated from a total of five bulls at .5, 2, 3, 4, and 5 h of incubation. The pH_i did not differ between the sperm head and mid-piece (P > 0.05). An increase in sperm head pH_i was seen in heparin-treated sperm at 3, 4, and 5 h of incubation relative to sperm incubated without heparin (control, P < 0.05). At 5 h of incubation, the pH_i in heparin-treated sperm was 6.92 ± 0.07, while control-treated sperm pH_i was 6.70 ± 0.03. Initially a normal frequency distribution was seen for sperm pH_i in both heparin- and control-treated sperm. As the incubation progressed, the frequency distribution began to skew towards higher pH_i in both samples but was more dispersed for the heparin-treated sperm. Following an NH₄Cl-induced alkaline load, the pH_i of both control- and heparin-treated sperm recovered toward the resting pH_i with a half-time of recovery of 1.5–1.7 min. The recovery of sperm pH_i was not due to leakage of NH⁴⁺ into sperm because recovery also occurred with trimethylamine. The instantaneous velocity of the pH_i recovery (v_i) was dependent on pH_i and decreased as pH_i decreased. Capacitation by heparin was associated with an 81% decrease in v_i at a pH_i of 7.00, but there was no effect of capacitation on the proton buffering power of the sperm, which was 87 ± 8 mM/pH unit. Results demonstrate that both the regulation of pH_i and resting pH_i were altered during capacitation of bovine sperm by heparin. © 1995 Wiley-Liss, Inc.     

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.     

Aquaporin inhibition changes protein phosphorylation pattern following sperm motility activation in fish

Our previous studies demonstrated that osmolality is the key signal in sperm motility activation in Sparus aurata spermatozoa. In particular, we have proposed that the hyper-osmotic shock triggers water efflux from spermatozoa via aquaporins. This water efflux determines the cell volume reduction and, in turn, the rise in the intracellular concentration of ions. This increase could lead to the activation of adenylyl cyclase and of the cAMP-signaling pathway, causing the phosphorylation of sperm proteins and then the initiation of sperm motility. This study confirms the important role of sea bream AQPs (Aqp1a and Aqp10b) in the beginning of sperm motility. In fact, when these proteins are inhibited by HgCl₂, the phosphorylation of some proteins (174 kDa protein of head; 147, 97 and 33 kDa proteins of flagella), following the hyper-osmotic shock, was inhibited (totally or partially). However, our results also suggest that more than one transduction pathways could be activated when sea bream spermatozoa were ejaculated in seawater, since numerous proteins showed an HgCl₂(AQPs)-independent phosphorylation state after motility activation. The role played by each different signal transduction pathways need to be clarified.     

Sorbitol can fuel mouse sperm motility and protein tyrosine phosphorylation via sorbitol dehydrogenase

Energy sources that can be metabolized to yield ATP are essential for normal sperm functions such as motility. Two major monosaccharides, sorbitol and fructose, are present in semen. Furthermore, sorbitol dehydrogenase (SORD) can convert sorbitol to fructose, which can then be metabolized via the glycolytic pathway in sperm to make ATP. Here we characterize Sord mRNA and SORD expression during mouse spermatogenesis and examine the ability of sorbitol to support epididymal sperm motility and tyrosine phosphorylation. Sord mRNA levels increased during the course of spermatogenic differentiation. SORD protein, however, was first detected at the condensing spermatid stage. By indirect immunofluorescence, SORD was present along the length of the flagella of caudal epididymal sperm. Furthermore, immunoelectron microscopy showed that SORD was associated with mitochondria and the plasma membranes of sperm. Sperm incubated with sorbitol maintained motility, indicating that sorbitol was utilized as an energy source. Sorbitol, as well as glucose and fructose, were not essential to induce hyperactive motility. Protein tyrosine phosphorylation increased in a similar manner when sorbitol was substituted for glucose in the incubation medium used for sperm capacitation. These results indicate that sorbitol can serve as an alternative energy source for sperm motility and protein tyrosine phosphorylation.     

Transmembrane adenylyl cyclase regulates amphibian sperm motility through protein kinase A activation

Sperm motility is essential for achieving fertilization. In animals with external fertilization as amphibians, spermatozoa are stored in a quiescent state in the testis. Spermiation to hypotonic fertilization media triggers activation of sperm motility. Bufo arenarum sperm are immotile in artificial seminal plasma (ASP) but acquire in situ flagellar beating upon dilution. In addition to the effect of low osmolarity on sperm motility activation, we report that diffusible factors of the egg jelly coat (EW) regulate motility patterns, switching from in situ to progressive movement. The signal transduction pathway involved in amphibian sperm motility activation is mostly unknown. In the present study, we show a correlation between motility activation triggered by low osmotic pressure and activation of protein kinase A (PKA). Moreover, this is the first study to present strong evidences that point toward a role of a transmembrane adenyl-cyclase (tmAC) in the regulation of amphibian sperm motility through PKA activation.     

Regulation of protein phosphorylation and motility of sperm by cyclic adenosine monophosphate and calcium

Motility and protein phosphorylation have been measured under identical experimental conditions in ejaculated dog sperm lysed with low concentrations of Triton X-100 and reactivated with [gamma-32P]ATP. Cyclic AMP stimulates motility and protein phosphorylation while calcium inhibits motility and the overall incorporation of phosphate into endogenous proteins. Analysis of 32P-labeled sperm proteins on 1- and 2-dimensional polyacrylamide gels demonstrates that an enhanced phosphorylation of a defined number of specific proteins is associated with cAMP-stimulated motility. A major axonemal proteins, namely tubulin, has been tentatively identified as a phosphoprotein subject to regulation by cAMP. The phosphorylation of tubulin is almost completely dependent upon cAMP and is not affected by microM calcium. On the other hand, the cAMP-dependent stimulated phosphorylation of the other sperm proteins still occurs, but in most instances at a reduced rate in the presence of calcium. Two high molecular weight (Mr) phosphoproteins (350,000 and 260,000 daltons) whose phosphorylation states are modified by cAMP and calcium also were identified. It is suggested that 1 or both these proteins may be high Mr subunits of dynein. The phosphorylation of 1 of these proteins is stimulated by cAMP, but not affected by calcium; the other is stimulated by cAMP and inhibited by calcium. Three major cAMP-independent phosphoproteins of Mr 98,000, 43,000 and 26,000 have been identified. The phosphorylation of the 98,000 Mr protein is markedly reduced by micromolar calcium and not restored by cAMP. Using anticalmodulin drugs to inhibit motility, we suggest that the inhibitory effects of calcium on flagellar motility may be mediated in part by calmodulin. We conclude that the regulation of flagellar motility in cAMP and calcium includes mechanisms involving the control of the phosphorylation state of sperm proteins, some of which may be axonemal components.     

A role for phosphorylation of glycogen synthase kinase-3alpha in bovine sperm motility regulation

The long-term goal of our work is to understand biochemical mechanisms underlying sperm motility and fertility. In a recent study we showed that tyrosine phosphorylation of a 55-kDa protein varied in direct proportion to motility. Tyrosine phosphorylation of the protein was low in immotile compared to motile epididymal sperm. Inhibition or stimulation of motility by high calcium levels or cAMP, respectively, results in a corresponding decrease or increase in tyrosine phosphorylation of the 55-kDa protein. Here we report purification and identification of this motility-associated protein. Soluble extracts from bovine caudal epididymal sperm were subjected to DEAE-cellulose, Affi-Gel blue, and cellulose phosphate chromatography. Tyrosine phosphate immunoreactive fractions contained glycogen synthase kinase-3 (GSK-3) activity, suggesting a possible correspondence between these proteins. This suggestion was verified by Western blot analyses following one-dimensional and two-dimensional gel electrophoresis of the purified protein using monoclonal and affinity-purified polyclonal antibodies against the catalytic amino-terminus and carboxy-terminus regions of GSK-3. Further confirmation of the identity of these proteins came from Western blot analysis using antibodies specific to the tyrosine phosphorylated GSK-3. Using this antibody, we also showed that GSK-3 tyrosine phosphorylation was high in motile compared to immotile sperm. Immunocytochemistry revealed that GSK-3 is present in the flagellum and the anterior portion of the sperm head. These data suggest that GSK-3, regulated by phosphorylation, could be a key element underlying motility initiation in the epididymis and regulation of mature sperm function.     

Axokinin phosphorylation by cAMP-dependent protein kinase is sufficient for activation of sperm flagellar motility

Using a selective inhibitor of cAMP-dependent protein kinase, N-[2(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), the requirement for cAMP-dependent phosphoproteins in the initiation of dog sperm flagellar motility was examined. H-8 inhibited motility of live as well as reactivated sperm in a dose-dependent manner. The half-maximal inhibition of reactivated motility (32 microM) paralleled the inhibition of pure catalytic subunit of cAMP-dependent protein kinase (50 microM) measured under the same conditions. H-8 inhibited protein phosphorylation both in whole models and in isolated Nonidet P-40 (NP-40) extracts of sperm. Axokinin, the heat-stable NP-40-soluble protein whose phosphorylation is required for flagellar reactivation, represented 97% of the de novo phosphate incorporation in the NP-40 extract after stimulation by cAMP. 500 microM H-8 inhibited axokinin phosphorylation by 87%. When sperm were reactivated in the presence of up to 5 mM H-8 with NP-40 extract that had been prephosphorylated with cAMP-dependent protein kinase, then neither cAMP nor cAMP-dependent protein kinase activity was required for full flagellar reactivation. If sperm were rendered completely immotile by pretreatment with H-8, then the resulting model remained immotile in the continued presence of H-8 unless prephosphorylated axokinin was added. These results suggest that phosphorylated axokinin is not only required for flagellar reactivation but is sufficient as well.     

Regulation of sperm flagellar motility by calcium and cAMP-dependent phosphorylation

There is substantial evidence that cAMP-dependent phosphorylation is involved in the activation of motility of spermatozoa as they are released from storage in the male reproductive tract. This evidence includes observations that in vivo activation of motility can be inhibited by protein kinase inhibitors, can be reversed by protein phosphatase treatment of demembranated spermatozoa, and is associated with phosphorylation of sperm proteins, and observations that spermatozoa that have not been activated in vivo can be activated in vitro by cAMP-dependent phosphorylation. Activation in vivo can often be triggered by conditions that increase intracellular pH, but the relevance of this to in vivo activation under natural conditions and the steps between pH increase and cAMP increase have not been fully established. The relationships between changes in the protein substrates for cAMP-dependent phosphorylation and changes in axonemal function are still unknown. Sperm chemotaxis to egg secretions is widespread; in the sea urchin Arbacia, the egg jelly peptide resact has been identified as a chemoattractant. Response to chemoattractants involves changes in asymmetry of flagellar bending waves, and similar changes in asymmetry can be produced in vitro by increases in [Ca++]. Temporal changes in resact receptor occupancy might lead to transient changes in intracellular [Ca++] and the asymmetry of flagellar bending, but many links in this hypothetical sequence remain to be established. Both of these signalling systems offer immediate opportunities for investigations of biochemical pathways leading to easily assayable biological responses. However, complications resulting from interactions between these two systems need to be considered.     

Tyrosine phosphorylation signaling regulates Ca2+ entry by affecting intracellular pH during human sperm capacitation

Capacitation is a mandatory process for the acquisition of mammalian sperm fertilization competence and involves the activation of a complex and still not fully understood system of signaling pathways. Under in vitro conditions, there is an increase in both protein tyrosine phosphorylation (pTyr) and intracellular Ca²⁺ levels in several species. In human sperm, results from our group revealed that pTyr signaling can be blocked by inhibiting proline-rich tyrosine kinase 2 (PYK2). Based on the role of PYK2 in other cell types, we investigated whether the PYK2-dependent pTyr cascade serves as a sensor for Ca ²⁺ signaling during human sperm capacitation. Flow cytometry studies showed that exposure of sperm to the PYK2 inhibitor N-[2-[[[2-[(2,3-dihydro-2-oxo-1 H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]- N-methyl-methanesulfonamide hydrate (PF431396) produced a significant and concentration-dependent reduction in intracellular Ca ²⁺ levels during capacitation. Further studies revealed that PF431396-treated sperm exhibited a decrease in the activity of CatSper, a key sperm Ca ²⁺ channel. In addition, time course studies during capacitation in the presence of PF431396 showed a significant and sustained decrease in both intracellular Ca ²⁺ and pH levels after 2 hr of incubation, temporarily coincident with the activation of PYK2 during capacitation. Interestingly, decreases in Ca ²⁺ levels and progressive motility caused by PF431396 were reverted by inducing intracellular alkalinization with NH ₄Cl, without affecting the pTyr blockage. Altogether, these observations support pTyr as an intracellular sensor for Ca ²⁺ entry in human sperm through regulation of cytoplasmic pH. These results contribute to a better understanding of the modulation of the polymodal CatSper and signaling pathways involved in human sperm capacitation.     

Hyperactivation of hamster sperm motility by temperature-dependent tyrosine phosphorylation of an 80-kDa protein.

Protein phosphorylation and dephosphorylation are believed to play key roles in regulation of sperm motility. Here we examine the effect of temperature on hamster sperm motility and protein tyrosine phosphorylation status. As in previous work, a decrease from 37 degrees C to 22 degrees C caused loss of hyperactivated motility. We now find that cooling also produces a dephosphorylation of several 48-80-kDa flagellar peptides. A return to 37 degrees C restored hyperactivation but resulted in rephosphorylation of only an 80-kDa protein. Conversely, hyperactivation and phosphorylation of the 80-kDa component were insensitive to incubation temperature for sperm incubated with the protein phosphatase inhibitor, calyculin A, or for sperm demembranated by detergent extraction. These results strongly indicate that the temperature-sensitive tyrosine phosphorylation status of an 80-kDa sperm flagellar peptide explains the sensitivity of hyperactivation to temperature.     

Study of bovine sperm motility in shear-thinning viscoelastic fluids

To elucidate the process whereby sperm arrive at an egg in the female reproductive organs, it is essential to investigate how rheological properties of the fluid around mammalian spermatozoa affect their motility. We examined the motility and flagellar waveform of bovine sperm swimming in a fluid with similar rheological properties as mammalian cervical mucus. The results indicated that the surrounding rheological properties largely affected the flagellar waveform of mammalian spermatozoa; in particular, shear-thinning viscoelastic fluid increased the progressive motility of the sperm. To investigate the influence of flagellar waveform on sperm motility in more detail, the waveform was expressed as a function and the progressive thrust of the sperm was calculated based on the empirical resistive force theory. The results of this study showed that the progressive thrust increased with the curvature of the flagellar tip. Moreover, we calculated the thrust efficiency of motile sperm. Results showed that the thrust efficiency in shear-thinning viscoelastic fluids was larger than that in Newtonian fluids, regardless of viscosity. This suggests that motile sperm in cervical mucus move efficiently by means of a motion mechanism that is suited to their surrounding environment.     

Inhibition of sperm motility by bovine serum components.

Various sources and components of mammalian sera were evaluated for their ability to maintain or inhibit sperm motility. Human, rabbit, hamster, and porcine sera were equal in ability to maintain motility of human sperm. Four sources of fetal calf serum and one source of neonatal calf serum were unable to maintain motility of human sperm or sperm-fertilizing potential. In the presence of human serum, fetal calf serum actually inhibited human sperm motility. Fetuin, a component of fetal calf serum, contained the inhibitory activity. An inhibitory effect of fetuin on porcine and caprine sperm motility was also observed. The inhibitory activity resided in the second peak when fetuin was separated by isoelectric focusing. The sperm head membranes remained impermeable to dye, and mitochondrial membrane potential was maintained after motility had been reduced to almost zero by incubation with fetuin and fetuin fractions. Fetuin or the active portion of the molecule may be a useful component of a vaginal contraceptive and in research where inhibition of motility is desirable.     

Anionic and cationic components from protein aggregates in bovine seminal plasma and their effects of sperm motility

Bovine seminal plasma proteins are in an aggregated form of high molecular weight in their native state. By immobilisation on a cation exchanger with exposure to disaggregating conditions (i.e., acetonitrile and low pH), the high-molecular-weight aggregates could be dissociated to slowly release the low-molecular-weight components. The anionic component released from the cation exchanger during disaggregation was collected by adsorption on a hydrophobic interaction column. The cationic component remaining on the cation exchanger was eluted with NaOH. Both components were found on gel permeation chromatography to be <5 kDa. SDS—PAGE of the various fractions showed that component of low molecular weight were still in an aggregated form. These components resulting from the disaggregation process have detrimental effects on sperm motility and the effects were more substantial compared with that of whole seminal plasma. All the cationic components were significantly detrimental to sperm motility, especially the fractions of low molecular weight. The anionic fractions reduced sperm motility when in an aggregated state. The isolated anionic peptide was not detrimental in its free form. In all fractions the peptides tended to re-aggregate to a higher molecular weight under neutral conditions, however, the isolated anionic peptide (molecular weight <1,500) failed to do so. © Wiley-Liss, Inc.     

Effect of kininase II inhibitors on bradykinin-stimulated bovine sperm motility

It has been found that the enzyme kininase II present in the seminal plasma inactivates the kinins produced by the kallikreins, thus blocking their beneficial effects on the motility of post-ejaculated mammalian spermatozoa. In this study we examined the influence of 2 kininase II inhibitors (captopril and enalapril maleate) on bradykinin-induced bovine sperm motility. Ejaculates were collected from Holstein-Friesian bulls, and semen samples exhibiting reduced sperm motility (30 to 60%; mean 53 +/- 3.8%) were used. Each semen sample was divided into 6 portions: 1) control; 2) treated with bradykinin (M-8); 3) treated with captopril (M-2); 4) treated with enalapril maleate (M-3); 5) treated with bradykinin + captopril; and 6) treated with bradykinin + enalapril maleate. Total sperm motility was recorded over 4 h at 1-h intervals. It was found that in the second hour after treatment both the combinations of bradykinin + captopril and bradykinin + enalapril maleate considerably enhanced sperm motility compared with that of the controls (P < 0.01 and P < 0.05, respectively). Total sperm motility rates of semen samples treated with these combinations were also higher than in the samples treated either with inhibitors alone or with bradykinin alone. We concluded that the stimulatory effect of bradykinin on bovine sperm motility was considerably potentiated and prolonged by the addition of the kininase II inhibitors.     

Caffeine-stimulated ATP-reactivated motility in a detergent-treated bovine sperm model.

Mammalian sperm cells contain most of the components of a cyclic AMP-mediation system. To determine if the cyclic AMP-dependent protein kinase has a role in the control of bovine sperm motility, a sperm model was developed that was permeable to exogenously added ATP. Treatment of bovine epididymal spermatozoa with dithiothreitol and Brij-35 (polyoxyethylene alcohol), a nonionic surfactant, resulted in a sperm model with caffeine-stimulated, ATP-reactivatable motility. The results of the data obtained using this sperm model can be summarized as follows. (1) Brij partially solubilized the cyclic AMP-dependent protein kinase activity and released nearly half of the total acid-extractable nucleotides of the cells. (2) Brij treatment severely damaged the sperm mitochondria as judged by their lack of respiration. (3) Brij-treated spermatozoa lose their motility but were reactivated with ATP; the reactivated motility was stimulated by caffeine. (4) Despite the caffeine stimulation of motility in Brij-treated spermatozoa, increased protein phosphorylation did not accompany reactivation of motility, nor could a cyclic AMP effect be demonstrated on reactivated motility or on kinase activity in the sperm model.