Effects of transglutaminase substrates and inhibitors on the motility of demembranated reactivated spermatozoa

The effects of transglutaminase (TGase) substrates putrescine, dansylcadaverine, spermine, etc., and the TGase inhibitor cystamine were tested on the motility of demembranated mammalian spermatozoa. These products blocked within a few seconds the motility of demembranated reactivated spermatozoa at concentrations ranging from 0.25 to 5 mM. These minimal inhibitory concentrations could be decreased 5–150-fold when TGase substrates and inhibitor were incubated with demembranated spermatozoa for 15 min prior to the addition of Mg·ATP. The inhibition was reversed by higher concentrations of Mg·ATP but none of these TGase substrates or inhibitor could inhibit bull sperm dynein ATPase. TGase activities, as measured by the incorporation of ³H-putrescine into TCA-precipitable proteins, were present in both sperm Triton-soluble and -insoluble fractions. On the other hand, amine acceptor protein substrates for the TGase-catalyzed reaction were present only in the insoluble fraction. The Triton-soluble TGase was similar to the known “tissue” TGases; the Triton-insoluble TGase activity was calcium independent. The same TGase substrates and inhibitor that blocked the motility of reactivated spermatozoa also blocked TGase activities. Linear relationships were observed between the concentrations of these substances required to block sperm motility and those to block TGase activities. These data suggest the involvement of a TGase activity in sperm motility.     

Chymotrypsin-like protease activity associated with demembranated sperm of chum salmon.

Our previous study suggested that a chymotrypsin-like protease was involved in the motility of chum salmon sperm (Inaba K, Morisawa M, Biomed Res (1991) 12, 435-437). In this study, we examined the peptidase activity of demembranated sperm of chum salmon using ten synthetic peptides. When spermatozoa were treated with 0.04% Triton X-100 for extracting the plasma membrane and the suspension was separated into the Triton-soluble and insoluble fractions by centrifugation, only the hydrolytic activity towards succinyl (Suc)-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (MCA), a typical substrate for chymotrypsin-like protease, was mostly retained in the insoluble fraction. The bulk of the activities toward other substrates was detected in the soluble fraction. Flagellar axonemes isolated from demembranated sperm showed considerable hydrolytic activity toward Suc-Leu-Leu-Val-Tyr-MCA and the activity was still retained in the axoneme even after further washing. The hydrolysis was activated by a low concentration of SDS, suggesting that the protease associated with the axonemes is a multicatalytic ATP-dependent proteinase (proteasome). Motility of demembranated sperm was inhibited by Suc-Leu-Leu-Val-Tyr-MCA in an ATP-concentration-dependent manner. These results suggest that proteasomes associated with flagellar axoneme regulate flagellar 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.     

The effect of the trypsin inhibitor aprotinin (Trasylol) and TLCK on the gelatinolytic activity of acrosin and the motility of rabbit sperm in vitro

In a series of experiments the influence of the trypsin inhibitors aprotinin (Trasylol) and TLCK (N-p-tosyl-L-lysin chloromethyl ketone) on the gelatinolytic activity of acrosin and motility of rabbit spermatozoa was tested. Ejaculated, highly motile spermatozoa were washed in Brackett-Medium. 12.5 to 1000 microns Aprotinin and 50 to 1000 micrograms TLCK, respectively, were added to samples of 1 ml sperm suspension: the specimens were incubated at 37 degrees C. Increasing aprotinin concentrations reduced the gelatinolytic activity of acrosin and the sperm incubation at a concentration of 1000 micrograms Aprotinin/ml sperm. Spermatozoa in all TLCK specimens were entirely immotile 1.5 hours after incubation. The gelatinolytic activity of acrosin was obviously not inhibited at any TLCK concentration. These results suggest that, under these experimental conditions, aprotinin and TLCK may impair primarily the motility spermatozoa.     

Inhibition of human sperm motility by contraceptive anti-eppin antibodies from infertile male monkeys: effect on cyclic adenosine monophosphate

Epididymal protease inhibitor (eppin [official symbol, SPINLW1]) is of interest as a male contraceptive target because of its specificity and location on the human sperm surface. We have examined the effect of anti-eppin antibodies from infertile male monkeys and the effect of recombinant human semenogelin on human sperm motility. Anti-eppin antibodies significantly decreased the progressive motility of human spermatozoa as measured by decreased total distance traveled, decreased straight-line distance, and decreased velocity. Anti-eppin treatment of spermatozoa significantly increased the amount of cAMP present in nonprogressive spermatozoa; however, approximately 25% of antibody-treated spermatozoa could be rescued by the addition of cAMP-acetoxymethyl ester, indicating that anti-eppin-treated spermatozoa have a compromised ability to utilize cAMP. Addition of recombinant human semenogelin has a concentration-dependent inhibitory effect on progressive motility (increased tortuosity and decreased velocity). We tested the hypothesis that anti-eppin antibodies bound to eppin would subsequently block semenogelin binding to eppin. Anti-eppin antibodies from infertile monkeys inhibited eppin from binding to semenogelin. Addition of affinity-purified antibodies made to the dominant C-terminal epitope of eppin had an inhibitory effect on progressive motility (increased tortuosity, decreased velocity, and straight distance). Our results suggest that the eppin-semenogelin binding site is critical for the removal of semenogelin in vivo during semen liquefaction and for the initiation of progressive motility. We conclude that the eppin-semenogelin binding site on the surface of human spermatozoa is an ideal target for a nonsteroidal male contraceptive.     

Motor apparatus in human spermatozoa that lack central pair microtubules

Electron microscopic examination of the spermatozoa from a man suffering from asthenozoospermia (poor or low sperm motility) showed that approximately 92% of the sperm flagella lacked central pair microtubules but possessed dynein arms and radial spokes while a small percentage of the spermatozoa had complete flagella. The characteristics of the motor apparatus of the spermatozoa and the effects of caffeine on the sperm motility were examined, as were the reactivation of demembranated spermatozoa and the sliding of doublet microtubules. Almost all spermatozoa were immotile in a Tyrode solution while only a small percentage of spermatozoa showed slow forward movement or feeble flagellar vibration, whereas addition of caffeine to the sperm suspension induced forward swimming of approximately half of the spermatozoa. The reactivation of demembranated spermatozoa with MgATP(2-) could not succeed because of disintegration of the demembranated flagella. However, when the demembranated spermatozoa were exposed to MgATP(2-) and then treated with elastase, the microtubular doublets of approximately half the number of the flagella slid from the end or middle of the flagella. These results suggest that the motor apparatus in the sperm flagella that lack the central pair microtubules is functionally assembled and intrinsically capable of undergoing flagellar movement but not strong enough to beat normally.     

Activation of sperm motility in striped bass via a cAMP-independent pathway

The objective of the present study was to identify the effect of osmolality, ions (K+, H+, Ca2+, Mg2+) and cAMP on the initiation of sperm motility in striped bass (Morone saxatilis). Striped bass spermatozoa remained motile in solutions isotonic to seminal plasma (350 mOsm/kg) until osmolality reached 600 mOsm/kg. K+ (0-100 mM) had no effect ( p>0.05 ) on sperm motility, and sperm displayed a high percentage of motility over a wide range of pH (6.0-8.5). Sperm motility could be initiated in Ca2+-free solutions. In contrast, sperm motility was inhibited (P<0.01) by solutions containing > or =10 mM Ca2+, and sperm could not be reactivated by a Ca2+-free solution. This Ca2+ inhibition was not affected by verapamil, a Ca2+ channel blocker. However, if sperm motility was first initiated in a Ca2+-free solution, the addition of Ca2+ solutions, up to 80 mM, failed to inhibit sperm motility, suggesting that Ca2+ inhibited the initiation of motility, but had no control of motile spermatozoa. Mg2+ solutions had similar inhibitory effects on sperm motility as Ca2+ solutions. Therefore, initiation of motility in striped bass sperm may be related to voltage-gated channels across the cell's plasma membrane. Membrane permeable cAMP did not initiate motility of quiescent, intact striped bass spermatozoa, and motility of demembranated sperm could be activated in the absence of cAMP.     

Activation of Xenopus eggs by proteases: possible involvement of a sperm protease in fertilization

Egg activation in cross-fertilization between Xenopus eggs and Cynops sperm may be caused by a protease activity against Boc-Gly-Arg-Arg-MCA in the sperm acrosome. To determine the role of the sperm protease in fertilization, the protease was purified from Cynops sperm using several chromatographic techniques. We found that purified sperm protease readily hydrolyzes Boc-Gly-Arg-Arg-MCA and Z-Arg-Arg-MCA, that protease activity was inhibited by the trypsin inhibitors aprotinin and leupeptin, and that not only the purified protease, but also cathepsin B, induces activation in Xenopus eggs. We inseminated unfertilized Xenopus eggs with homologous sperm in the presence of various peptidyl MCA substrates or protease inhibitors and demonstrated that trypsin inhibitors or MCA substrates containing Arg-Arg-MCA reversibly inhibited fertilization of both fully jellied and denuded eggs. Sperm motility was not affected by the reagents. An extract obtained from Xenopus sperm showed hydrolytic activity against Boc-Gly-Arg-Arg-MCA, Z-Arg-Arg-MCA, and Arg-MCA. These results suggest that the tryptic protease in Xenopus sperm is involved in fertilization, most likely by participating in egg activation.     

Roles of pH and cyclic adenosine monophosphate in the acquisition of potential for sperm motility during migration from the sea to the river in chum salmon

In the natural process of the migration of chum salmon from the sea to the river, spermatozoa moved from the testis to the sperm duct, and the pH value of seminal plasma, concentration of cyclic adenosine monophosphate (AMP) in the sperm cells, and potential for sperm motility increased. Cyclic AMP levels and the potential for motility gradually increased when testis spermatozoa with no capacity for movement were incubated in the artificial seminal plasma of which the pH was much the same as, or higher than, the pH of natural seminal plasma from the sperm duct. Such correlation in motility, pH, and cyclic AMP suggests that the increases in seminal pH and intracellular cyclic AMP level during passage of spermatozoa from the testis to the sperm duct cause the acquisition of potential for motility. Motility of testicular spermatozoa demembranated with Triton X-100 was very low in fish caught in the sea, while motility of spermatozoa from the posterior portion of the sperm duct was much higher in fish caught in the river. Furthermore, nondemembranated, intact spermatozoa showed a lag in the timing of the acquisition of potential for motility vs. demembranated spermatozoa: The demembranated sperm exhibited the potential earlier than the nondemembranated sperm. These data suggest that increase in activity of the motile apparatus, the axoneme, is a prerequisite, in part, for the acquisition of sperm motility, whereas the development of some function of the plasma membrane also contributes to this phenomenon. © 1993 Wiley-Liss, Inc.     

The possible role of protein-carboxyl methylation in the regulation of flagellar movement of fowl spermatozoa

Both intact and demembranated fowl spermatozoa were incubated at 30 degrees C and 40 degrees C with adenosine, 3-deazaadenosine and homocysteine thiolactone. This combination of products is known to block intracellular protein-carboxyl methylation reaction. The motility of intact spermatozoa incubated at 30 degrees C was vigorous but decreased markedly after the addition of 100 microM adenosine+100 microM 3-deazaadenosine+100 microM homocysteine thiolactone. During this incubation period, the intracellular ATP concentrations of spermatozoa were maintained at approximately 40 nmol ATP/10(9) cells, in spite of the inhibition of motility. The motility of demembranated spermatozoa at 30 degrees C was not inhibited by the same concentrations of blocker. At 40 degrees C, the motility of intact spermatozoa without any effectors was almost negligible. The addition of blocker did not appreciably affect the motility of spermatozoa, which remained almost negligible. In contrast, motility became vigorous even at 40 degrees C when intact spermatozoa were suspended in fluid to which had been added 1 mM CaCl(2) or 100 nM calyculin A, a specific inhibitor of protein phosphatase-type 1 and -type 2. Stimulation of motility by Ca(2+) or calyculin A was inhibited by the presence of a blocker. Contrary to that of intact spermatozoa, the motility of demembranated spermatozoa stimulated by protein phosphatase inhibitor at 40 degrees C was not inhibited by the presence of a blocker. These results suggest that protein-carboxyl methylation may be involved in the regulation of fowl sperm motility. Furthermore, it appears that the methylating enzyme may be present in the cytoplasmic matrix and/or the plasma membrane but not retained in the axoneme and/or accessory cytoskeletal components.     

Aprotinin and a seminal plasma factor inhibit the motility of demembranated reactivated rabbit spermatozoa

The demembranation and reactivation of ejaculated rabbit spermatozoa have been studied. ATP, Mg, glutamate, dithiothreitol (DTT), and Tris-HCl were found to be essential for a good reactivation. With this experimental model, we investigated the effects of protease inhibitors on the reinitiation of movement by ATP and on the movement of already motile spermatozoa. Soybean trypsin inhibitor (STI) prolonged the length of reactivation by 7- to 8-fold, whereas pepstatin, antithrombin III, phenylmethylsulfonyl fluoride (PMSF), and alpha-1-antitrypsin had no significant effect. Aprotinin (1.5 micrograms/ml) and leupeptin (50 micrograms/ml) completely prevented the reinitiation of movement by ATP; aprotinin at the same concentration even blocked the movement of motile spermatozoa. A tissue-specific seminal plasma factor could also prevent both the reinitiation of movement and the movement of motile spermatozoa. However, it took 2-3 times the amount of seminal plasma to stop the movement than to prevent the reinitiation of movement. The inhibitor in the seminal plasma is most probably not a protease or an aprotinin-like protease inhibitor since a partially purified preparation of the seminal plasma inhibitor does not hydrolyze a trypsin substrate, is not inhibited by protease inhibitors and has no significant capacity to inhibit trypsin. The data suggest that aprotinin and the seminal plasma inhibitor block movement through different mechanisms. Aprotinin and the seminal plasma inhibitor represent two new tools to study the regulation of sperm movement.     

Regulation of microtubule sliding by a 36-kDa phosphoprotein in hamster sperm flagella

Cyclic AMP has been shown essential for activation of sperm motility. When immotile hamster caudal epididymal spermatozoa were suspended in a Ca2+-deficient solution, they showed a sluggish motility. Spermatozoa were demembranated and transferred to an ATP-containing reactivation solution. Demembranated spermatozoa did not exhibit reactivated flagellar movement unless cAMP was added. Conversely, when the immotile epididymal spermatozoa were suspended in a Ca2+-containing solution, they were immediately activated to display a vigorous motility; demembranated spermatozoa also exhibited reactivated flagellar movement in the reactivation solution without cAMP. Further investigation of microtubule sliding properties revealed that the effects of Ca2+ on live spermatozoa were identical with the effects of cAMP on demembranated spermatozoa both in microtubule sliding velocity and sliding disintegration pattern. Moreover, a 36-kDa flagellar protein was found to be phosphorylated in a cAMP-dependent manner and coupled to the motility activation. A polyclonal antibody against this protein was developed and showed specific immunolocalization and significant inhibitory effects on microtubule sliding disintegration. These results indicate that extracellular Ca2+ owes its effect to triggering intracellular cAMP production, and cAMP-dependent phosphorylation of a 36-kDa phosphoprotein activates hamster sperm motility through regulation of microtubule sliding properties.     

Characterization of the mouse sperm plasma membrane zona-binding site sensitive to trypsin inhibitors

The first contact of mammalian gametes is the binding of the spermatozoon to the zona pellucida of the egg. Previous work has shown that binding of the spermatozoon to the zona in the mouse occurs prior to the acrosome reaction and that trypsin inhibitors block this initial binding. This suggests that the sperm surface contains a trypsinlike binding site that functions by an active site mechanism to effect initial zona binding. When suspensions of twice-washed spermatozoa were incubated with the serine protease active site titrant, 4-methylumbelliferyl p-guanidinobenzoate (MUGB), the titrant was hydrolyzed at a rate of 8 pmoles/min-10(6) cells. MUGB was found to inhibit the binding of spermatozoa to the zona pellucida. The degree of inhibition and the rate of hydrolysis of MUGB by washed spermatozoa depend on the concentration of titrant, with half maximal effects at 13 microM and a linear correlation with r = 0.99. The analogous lysyl and arginyl trypsin substrates containing 7-amino-4-methylcoumarin as the fluorogenic leaving group were not hydrolyzed under the same conditions and did not inhibit zona binding. Both binding of sperm to zona-intact eggs and the hydrolysis of MUGB by sperm are inhibited by p-nitrophenyl guanidinobenzoate, soybean trypsin inhibitor, and acid-solubilized zonae. The linear correlation coefficients of the inhibition of sperm binding and MUGB hydrolysis by these three substances are greater than 0.92. This "trypsinlike" sperm site is essential for sperm binding to the zona: its stereospecificity is unique in that it reacts with trypsin inhibitors but not with trypsin substrates.     

Regulation of flagellar motility by temperature-dependent phosphorylation of a 43 kDa axonemal protein in fowl spermatozoa.

Phosphorylation of fowl sperm proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after incubating the demembranated spermatozoa with [gamma-32P]ATP at 30 degrees C or 40 degrees C. A marked difference of phosphorylation between 30 degrees C and 40 degrees C was observed in a 43 kDa protein. This protein was slightly phosphorylated at 40 degrees C, but strongly phosphorylated at 30 degrees C in a cAMP-independent manner. The motility of demembranated spermatozoa was negligible at 40 degrees C, but vigorous movement was observed at 30 degrees C. These results showed that phosphorylation of a 43 kDa protein is likely to be a regulatory step in the maintenance of fowl sperm motility.     

A monoclonal antibody against the dynein IC1 peptide of sea urchin spermatozoa inhibits the motility of sea urchin, dinoflagellate, and human flagellar axonemes.

To investigate the role of axonemal components in the mechanics and regulation of flagellar movement, we have generated a series of monoclonal antibodies (mAb) against sea urchin (Lytechinus pictus) sperm axonemal proteins, selected for their ability to inhibit the motility of demembranated sperm models. One of these antibodies, mAb D1, recognizes an antigen of 142 kDa on blots of sea urchin axonemal proteins and of purified outer arm dynein, suggesting that it acts by binding to the heaviest intermediate chain (IC1) of the dynein arm. mAb D1 blocks the motility of demembranated sea urchin spermatozoa by modifying the beating amplitude and shear angle without affecting the ATPase activity of purified dynein or of demembranated immotile spermatozoa. Furthermore, mAb D1 had only a marginal effect on the velocity of sliding microtubules in trypsin-treated axonemes. This antibody was also capable of inhibiting the motility of flagella of Oxyrrhis marina, a primitive dinoflagellate, and those of demembranated human spermatozoa. Localization of the antigen recognized by mAb D1 by immunofluorescence reveals its presence on the axonemes of flagella from sea urchin spermatozoa and O. marina but not on the cortical microtubule network of the dinoflagellate. These results are consistent with a dynamic role for the dynein intermediate chain IC1 in the bending and/or wave propagation of flagellar axonemes.     

Characterization of sperm motility in sea bass: the effect of heavy metals and physicochemical variables on sperm motility

Computer assisted sperm analysis (CASA) was used to characterize the motility of sea bass Dicentrarchus labrax spermatozoa and to study the effect of several physicochemical variables and heavy metals on sperm swimming performance. Duration of sperm motility in sea bass was very short (<50 s). During the first 20 s all the motility variables measured remained approximately constant, the velocity and linearity of the movement being maximum during this period, while both variables decreased sharply later. While slight variations in pH did not significantly modify sperm swimming performance, changes in osmolality affected all the measured motility variables. Two of the heavy metals tested, Cu²⁺ and Pb²⁺, did not affect sperm motility when the activating media contained up to 100 ppm of the metal salts. In contrast, Hg²⁺ modified the morphology of post-swimming spermatozoa at 0·4–1 ppm (sperm dilution rate 1:39) and completely arrested sperm motility at concentrations as low as 0·1 ppm (sperm dilution rate 1:2500). Assuming a covalent binding to sperm cells, this revealed a finite number of c. 10 million Hg²⁺ binding sites per spermatozoon. Complementary results using demembranated spermatozoa suggested that the main target of HgCl₂ would be located in the plasma membrane and that HgCl₂ would inhibit water channels, hence preventing sperm motility.     

Inhibition of in vitro capacitation of hamster spermatozoa by nitric oxide synthase inhibitors.

In an attempt to understand the role of nitric oxide(NO) in sperm capacitation, in the present study, hamster spermatozoa were used to evaluate the effects of NO on motility, viability, hyperactivation, capacitation and protein tyrosine and serine phosphorylation using specific inhibitors of nitric oxide synthase (NOS); namely L-NAME (N-nito-L-aginine methyl ester) and 7-Ni (7-nitroindazole). The results indicated that L-NAME inhibits sperm motility, hyperactivation and acrosome reaction where as 7-Ni inhibits only hyperactivation and acrosome reaction thus implying that NOS inhibitors exhibit subtle differences with respect to their effects on sperm functions. This study also provides evidence that NOS inhibitors inhibit sperm capacitation by their ability to modulate protein tyrosine phosphorylation. However, the inhibitors had no effect on the protein serine phosphorylation of hamster spermatozoa during capacitation. Thus, these results indicate that NO is required     

Purification and characterization of a sperm motility-dynein ATPase inhibitor from boar seminal plasma.

A sperm motility inhibitor from boar seminal plasma was purified. The purification procedure included dialysis against 0.1 M Tris-HCl containing 0.1 mM DTT and chromatographies on SP-Sephadex C-25 and Phenyl-Sepharose CL-4B. With this procedure, the seminal plasma motility inhibitor (SPMI) preparation was highly purified with a 18% recovery of inhibitory activity. The molecular weight of SPMI in native conditions has been estimated at 50,000 by molecular sieving, but 3 polypeptides with molecular weights of 14,000, 16,000 and 18,000 were observed following polyacrylamide gel electrophoresis in denaturing conditions. SPMI is a thermolabile basic protein that is stable between pH 6 and pH 11. The observations that SPMI effects on motility of demembranated spermatozoa are reversed by Mg.ATP and that SPMI inhibited bull dynein ATPase in a concentration-dependent manner suggest that this protein blocks the motility of demembranated spermatozoa by interfering with dynein arm function.     

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.     

Initiation,prolongation, and reactivation of the motility of salmonid spermatozoa

The motility of salmonid spermatozoa initiated by dilution of the milt with ovarian fluid or isotonic saline is brief duration; it was believed that it can be activated only once in the life of the spermatozoon. Dilution of the milt with an equal volume of isotonic saline (0.12 M-NaCl) containing 5 mM-3-isobutyl-1-methylxanthine (MIX) prolonged and intensified sperm motiliy. When motility had stopped after initial mobilization with saline or ovarian fluid, it could be reactivated by addition of MIX; reactivated spermatozoa fertilized eggs. Dilution with saline containing K⁺ (24 mEq/liter) did not initiate sperm motility even in the presence of MIX. The spermatozoa were mobilized by subsequent with 0.12 M-NaCl. The concentration of adenosine triphosphate (ATP) in sperm suspensions dropped on dilution with saline and rose as motility ceased, but declined without subsequent recovery following dilution with MIX-saline. The concentration of cyclic adenosine monophosphate (cAMP) rose and fell sharply on initiation of motility and rose again after motility had declined. While salmonid spermatozoa can be mobilized by dilition with saline alone, the effectiveness of MIX in reactivating “spent” spermatozoa supports the assumption that cAMP plays a role in the initiation of sperm motility.