Phosphoprotein phosphatase inhibits flagellar movement of Triton models of sea urchin spermatozoa

Phosphoprotein phosphatase prepared from bovine cardiac muscle was used to study the roles of axonemal phosphoproteins in the flagellar motility of sea urchin spermatozoa. When isolated axonemes were incubated with cyclic AMP-dependent protein kinase, gamma-[32P]ATP and cyclic AMP, more than 15 polypeptides were phosphorylated. Most were dephosphorylated by treatment with phosphoprotein phosphatase. When Triton models of sea urchin spermatozoa were treated with phosphoprotein phosphatase followed by an addition of ATP, the flagellar motility of the models was drastically reduced in comparison with that of the untreated models. The motility of the phosphatase-treated Triton models was partially restored by an addition of cyclic AMP and cyclic AMP-dependent protein kinase. These data give strong support to the idea that the motility of eukaryotic flagella is controlled by a protein phosphorylation-dephosphorylation system.     

Evidence that cAMP-dependent protein kinase and a protein factor are involved in reactivation of triton X-100 models of sea urchin and starfish spermatozoa

A fraction obtained from detergent-extract of sea urchin or starfish spermatozoa using DEAE-cellulose chromatography reactivated Triton X-100 models of the spermatozoa in a cAMP-dependent manner. The DEAE fraction contained cAMP-dependent protein kinase with a high level of specific activity. Rabbit muscle inhibitor protein highly specific for cAMP-dependent protein kinases inhibited the ability of the deae fraction to induce reactivation of Triton X-100 models.l This inhibition paralleled inhibition of cAMP-dependent protein kinase activity of the DEAE fraction, suggesting participation of the enzyme in the cAMP-dependent reactivation of Triton X-100 models. However, cAMP-dependent protein kinase further purified from the DEAE fraction was incapable of reactivating these models by itself. A protein factor which was separated from the protein kinase in the course of purification of the enzyme was found to also be necessary for the reactivation. When cAMP-dependent protein kinase was pretreated with protein kinase inhibitor before addition of the protein factor, the reactivation of Triton X-100 models was no longer detected. However, after the protein factor had been incubated with cAMP and cAMP-dependent protein kinase, protein kinase inhibitor did not repress reactivation of Triton X-100 models. We propose that the reactivation needs phosphorylation of the protein factor by cAMP-dependent protein kinase.     

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.     

The cAMP-dependent protein kinase in sea urchin sperm tails: association of the enzyme with the flagellar axonemes

When sea urchin spermatozoa were treated with a Triton X-100 solution, cAMP-dependent protein kinase (cA-kinase) activity was extracted. Further extraction with Triton X-100 of axonemes isolated from the Triton-extracted sperm again released a considerable amount of the cA-kinase activity. The activity which remained after extraction three times with Triton X-100 was released by treatment with a low salt solution. These activities found in the various extracts were likely to be due to the same cA-kinase, which was a mammalian type II-like enzyme. The cA-kinase activity that remained in the axonemes after the first Triton X-100 extraction may be involved in the regulation of flagellar movement in the Triton-extracted sperm.     

Proteins associated with soluble adenylyl cyclase in sea urchin sperm flagella

Adenylyl cyclases (ACs) synthesize cAMP and are present in cells as transmembrane AC and soluble AC (sAC). In sperm, the cAMP produced regulates ion channels and it also activates protein kinase-A that in turn phosphorylates specific axonemal proteins to activate flagellar motility. In mammalian sperm, sAC localizes to the midpiece of flagella, whereas in sea urchin sperm sAC is along the entire flagellum. Here we show that in sea urchin sperm, sAC is complexed with proteins of the plasma membrane and axoneme. Immunoprecipitation shows that a minimum of 10 proteins is tightly associated with sAC. Mass spectrometry of peptides derived from these proteins shows them to be: axonemal dynein heavy chains 7 and 9, sperm specific Na+/H+ exchanger, cyclic nucleotide-gated ion channel, sperm specific creatine kinase, membrane bound guanylyl cyclase, cyclic GMP specific phosphodiesterase 5A, the receptor for the egg peptide speract, and alpha- and beta-tubulins. The sAC-associated proteins could be important in linking membrane signal transduction to energy utilisation in the regulation of flagellar motility.     

A soluble adenylyl cyclase from sea urchin spermatozoa

A previously identified, calmodulin-binding, sea urchin sperm flagellar adenylyl cyclase (AC) was cloned and sequenced and found to be a homologue of mammalian sperm soluble adenylyl cyclase (sAC). Compared to the mammalian sAC, the sea urchin sAC (susAC) has several long amino acid insertions, some of which contain protein kinase A phosphorylation sites. The enzymatic activity of susAC shows a steep pH dependency curve, the specific activity doubling when the pH is increased from 7.0 to 7.5. This suggests that like sperm dynein ATPase, the susAC is probably activated by increases in intracellular pH occurring upon spawning into seawater and also when sperm respond to contact with the egg jelly layer. The susAC is strongly activated by manganese, but has low activity in magnesium. Gene database searches identified sAC homologues in species known to have cyclic AMP-dependent sperm motility. This implies (as shown in mouse) that susAC has a role in sperm motility, most probably through axonemal protein phosphorylation or ion channel regulation.     

Molecular Cloning and Characterization of a Radial Spoke Head Protein of Sea Urchin Sperm Axonemes: Involvement of the Protein in the Regulation of Sperm Motility

Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used to study regulatory mechanisms involved in flagellar motility. Here, we report that one of these antibodies, monoclonal antibody D-316, has an unusual perturbating effect on the motility of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pattern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE, D-316 recognized a single polypeptide of 90 kDa. This protein was purified following its extraction by exposure of axonemes to a brief heat treatment at 40°C. The protein copurified and coimmunoprecipitated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI = 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protein sequences in databases indicated that the protein is related to radial spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii, which share 37% and 25% similarity, respectively, with p63. However, the sea urchin protein possesses structural features distinct from RSP4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form α-helical coiled-coil secondary structures. These results suggest a major role for p63 in the maintenance of a planar form of sperm flagellar beating and provide new tools to study the function of radial spoke heads in more evolved species.     

Endogenous activity of cyclic nucleotide-dependent protein kinase in plasma membranes isolated from Strongylocentrotus purpuratus sea urchin sperm.

Activity of cyclic nucleotide-dependent protein kinase was investigated in flagellar plasma membranes of sea urchin sperm (S. purpuratus). Membranes incubated with [gamma-32P]ATP showed in the presence of 1 microM cAMP an increased phosphorylation in multiple polypeptides. Half maximal response was seen at 0.6 microM of cAMP. In contrast, higher concentrations (100 microM) of cGMP were required to cause the same amount of protein phosphorylation. 80% of the protein kinase activity stimulatable by cAMP was resistant to extraction by 10 mM EGTA and sonication but it was entirely recovered in a detergent-solubilized fraction. Membranes pretreated with 200 microM cAMP, ultracentrifuged and resuspended in buffer solution did not undergo cAMP-stimulated phosphorylation in their polypeptides. This study demonstrates that flagellar plasma membranes isolated from S. purpuratus sea urchin sperm have an endogenous cAMP-dependent protein kinase, which may be bound to the membrane via its regulatory subunit.     

Effects of antibodies against tubulin on the movement of reactivated sea urchin sperm flagella

Antibodies binding to sea urchin flagellar outer-doublet tubulin have been isolated from rabbit sera by tubulin-affinity chromatography employing electrophoretically purified tubulin as the immobilized substrate. This procedure provides "induced" antitubulin antibody from immune sera and "spontaneous" antitubulin antibody from preimmune sera. These antitubulins were characterized in terms of their specificity, ability to bind to sea urchin axonemes, and effects on the motility of reactivated spermatozoa. Induced antitubulin antibody specifically reduced the bend angle and symmetry of the movement of demembranated reactivated spermatozoa without affecting the beat frequency. At identical concentrations, spontaneous antitubulin had no effect on motility. Affinity-purified induced antitubulins from three other rabbits all gave specific bend-angle inhibition, whereas their corresponding spontaneous antitubulins had no effect on the flagellar movement. The effects of antitubulin on microtubule sliding were examined by observing the sliding disintegration of elastase-digested axonemes induced by MgATP2+-. Affinity-purified induced antitubulin antibody, in quantities sufficient to completely paralyze reactivated flagella, did not inhibit microtubule sliding. The amplitude-inhibiting effect of induced antitubulin on reactivated spermatozoa may be caused by action on a mechanism responsible for controlling flagellar bending rather than by interference with the active sliding process. This is the first report of an antitubulin antibody having an inhibitory activity on microtubule-associated movement.     

Adrenergic stimulation of sea urchin sperm cells

The noradrenergic agonists norepinephrine and isoproterenol elicit greater stimulatory swim ming responses in sea urchin spermatozoa than epinephrine. The β-blocker atenolol induces an even greater motile rate, while the α-blocker phentolamine has only a moderate effect, it also causes a minimal reduction in the sperm cells' response to atenolpol. Caffeine increases the motility but to a lesser degree than 8-Br-cAMP. In drug interaction assays, both caffeine and 8-Br-cAMP depress the adrenergic effects. Agents that affect access of calcium to the flagellar apparatus (verapamil and trifluoperazine) depress the motility below the level of the controls when incubated separately with the sperm suspensions and counteract the stimulation due to atenolol. Adrenergic modulation of sperm motility thus appears to be both a calcium-dependent and a cyclic nucleotide-dependent process.     

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.     

Digital image analysis of the flagellar beat of activated and hyperactivated suncus spermatozoa

The flagellar beat of hyperactivated Suncus spermatozoa was analyzed by digital imaging and was compared to that of the nonhyperactivated (activated) spermatozoa in order to examine the function of the accessory fibers during the flagellar beat and the sliding filament mechanism inducing the motility of the hyperactivated spermatozoa. Unusual large and long characteristics of the accessory fibers were involved in generating the gently curved bends and a low beat frequency. Examination of the motility parameters of the flagellar beat of the activated and hyperactivated spermatozoa attached to a slide glass by their heads revealed that there were two beating modes: a frequency-curvature dependent mode in the activated flagellar beat and a nearly constant frequency mode in the hyperactivated flagellar beat. The hyperactivated flagellar beat was characterized by sharp bends in the proximal midpiece and a low beat frequency. The sharp bends in the proximal midpiece were induced by the increase in the total length of the microtubule sliding at the flagellar base. The rate of microtubule sliding (sliding velocity) in the axoneme remained almost constant in the flagellar beat of both the activated and hyperactivated spermatozoa. Comparison of the sliding velocity in Suncus, golden hamster, monkey, and sea urchin sperm flagella with their stiffness suggests that the sliding velocity is determined by the stiffness at the flagellar base and that the same sliding microtubule system functions in both mammalian and echinoderm spermatozoa.     

Tuning sperm chemotaxis by calcium burst timing

Marine invertebrate oocytes establish chemoattractant gradients that guide spermatozoa towards their source. In sea urchin spermatozoa, this relocation requires coordinated motility changes initiated by Ca²⁺-driven alterations in sperm flagellar curvature. We discovered that Lytechinus pictus spermatozoa undergo chemotaxis in response to speract, an egg-derived decapeptide previously noted to stimulate non-chemotactic motility alterations in Strongylocentrotus purpuratus spermatozoa. Sperm of both species responded to speract gradients with a sequence of turning episodes that correlate with transient flagellar Ca²⁺ increases, yet only L. pictus spermatozoa accumulated at the gradient source. Detailed analysis of sperm behavior revealed that L. pictus spermatozoa selectively undergo Ca²⁺ fluctuations while swimming along negative speract gradients while S. purpuratus sperm generate Ca²⁺ fluctuations in a spatially non-selective manner. This difference is attributed to the selective suppression of Ca²⁺ fluctuations of L. pictus spermatozoa as they swim towards the source of the chemoattractant gradient. This is the first study to compare and characterize the motility components that differ in chemotactic and non-chemotactic spermatozoa. Tuning of Ca²⁺ fluctuations and associated turning episodes to the chemoattractant gradient polarity is a central feature of sea urchin sperm chemotaxis and may be a feature of sperm chemotaxis in general.     

The velocity of microtubule sliding: its stability and load dependency

It is now well understood that ATP-driven active sliding between the doublet microtubules in the sperm axoneme generates flagellar movement. However, much remains to be learned about how this movement is controlled. Detailed analyses of the flagellar beating of the mammalian spermatozoa revealed that there were two beating modes at a constant rate of microtubule sliding: that is, a nearly constant-curvature beating in nonhyperactivated spermatozoa and a nearly constant-frequency beating in hyperactivated spermatozoa. The constant rate of microtubule sliding suggests that the beat frequency and waveform of the flagellar beating are dependently regulated. Comparison of the sliding velocity of several mammalian and sea urchin sperm flagella with their mechanical property clarified that the sliding velocity of the microtubule was determined by the stiffness of the flagellum at its base, and that its relationship was expressed by a logarithmic equation that is similar to the classical force-velocity equation of the muscle contraction. Data from sea urchin spermatozoa also satisfied the equation, suggesting that the same microtubule sliding system functions in both the mammalian and echinoderm spermatozoa.     

Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm

Preliminary data demonstrated that the inhibition of reactivated sperm motility by calcium was correlated with inhibited protein phosphorylation. The inhibition of phosphorylation by Ca2+ was found to be catalyzed by the calmodulin-dependent protein phosphatase (calcineurin). Sperm from dog, pig, and sea urchin contain both the Ca2+-binding B subunit of the enzyme (Mr 15,000) and the calmodulin-binding A subunit with an Mr of 63,000. The sperm A subunit is slightly higher in Mr than reported for other tissues. Inhibition of endogenous calmodulin-dependent protein phosphatase activity with a monospecific antibody revealed the presence of 14 phosphoprotein substrates in sperm for this enzyme. The enzyme was localized to both the flagellum and the postacrosomal region of the sperm head. The flagellar phosphatase activity was quantitatively extracted with 0.6 M KCl from isolated flagella from dog, pig, and sea urchin sperm. All salt-extractable phosphatase activity was inhibited with antibodies against the authentic enzyme. Preincubation of sperm models with the purified phosphatase stimulated curvolinear velocity and lateral head amplitude (important components of hyperactivated swimming patterns) and inhibited beat cross frequency suggesting a role for this enzyme in axonemal function. Our results suggest that calmodulin-dependent protein phosphatase plays a major role in the calcium-dependent regulation of flagellar motility.     

Identification of sea urchin sperm adenylate cyclase

Calmodulin (CaM) affinity chromatography of a detergent extract of sea urchin sperm yielded approximately 20 major proteins. One of these proteins, of Mr 190,000, was purified and used to immunize rabbits. After absorption with living sperm, the serum reacted monospecifically on one- and two-dimensional Western immunoblots with the Mr 190,000 protein. The anti-190-kD serum inhibited 94% of the adenylate cyclase (AC) activity of the CaM eluate. An immunoaffinity column removed 95% of the AC activity, and the purified (but inactive) Mr 190,000 protein was eluted from the column. The antiserum also inhibited 23% of the activity of bovine brain CaM-sensitive AC and 90% of the activity of horse sperm CaM-sensitive AC. These data support the hypothesis that the Mr 190,000 protein is sea urchin sperm AC. Although this AC bound to CaM, it was not possible to demonstrate directly a Ca2+ or CaM sensitivity. However, two CaM antagonists, calmidazolium and chlorpromazine, both inhibited AC activity, and the inhibition was released by added CaM, suggesting the possibility of regulation of this AC by CaM. Indirect immunofluorescence showed the Mr 190,000 protein to be highly concentrated on only the proximal half of the sea urchin sperm flagellum. This asymmetric localization of AC may be important to its function in flagellar motility. This is the first report of the identification of an AC from animal spermatozoa.     

Inhibition of sperm motility by the volatile anesthetic halothane

The inhalational anesthetic halothane reversibly inhibits the motility of sea urchin sperm dose-dependently at concentrations up to 5 mM. Experiments with Triton X-100 extracted, trypsinized axonemes showed that halothane has no effect on the rate of axonemal disintegration in the presence of ATP. These results suggest that halothane inhibits flagellar activity by acting at a site other than the dynein ATPase component of the flagellum.     

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.     

Evidence for functional differences between two flagellar dynein ATPases

Energy coupling in flagellar motility was investigated using demembranated, reactivated sea urchin spermatozoa (Arbacia punctulata). The ATP-dependence of ATPase activity was investigated for ATP concentrations ranging from 4 microM to 600 microM ATP. Using Eadie-Scatchard plot analysis, we identified two axonemal dynein ATPase activities. Their apparent Michaelis constants were calculated to be equal to 4 microM and 161 microM ATP, and they were referred to, respectively, as the high-affinity dynein ATPase (HADA) and the low-affinity dynein ATPase (LADA). Investigation of movement-coupled ATPase activity (difference between the ATPase activities of reactivated and broken, immotile spermatozoa) indicated that HADA and LADA were both 65% movement-coupled. The apparent Michaelis constants of movement-coupled HADA and LADA, 12 microM and 271 microM ATP, respectively, were two- to four-fold greater than the apparent Michaelis constants of movement-uncoupled HADA and LADA. The apparent Michaelis constants for force generation and beat frequency of reactivated spermatozoa were determined to be 24 microM and 290 microM ATP, respectively. These results raise the possibility that flagellar force generation is controlled primarily by movement-coupled HADA, and that flagellar beat frequency is controlled primarily by movement-coupled LADA. Thus, mechanochemical activity in flagellar motility may be divided between two enzymatically and functionally distinct classes of flagellar dyneins.     

Cytoplasm calcium-binding proteins of germ cells and embryos of the sea urchin

Synchronous, demonstrative, easily reproducible fertilization with the following embryonic development makes the process in the sea urchin extremely attractive for studying many biological enigmas. In particular, germ and embryonic cells of the sea urchin present a wide opportunity for investigating different associated phenomena launched by an increase in concentration of Ca²⁺ in cells ([Ca²⁺]_i).Ca²⁺ ions participate in the activation of diverse processes of respiration and sperm motility (Shapiro et al., 1990; Brokaw, 1991), chemotaxis of spermatozoa to components of the egg jelly (Ward et al., 1985), acrosomal reaction (Trimmer et al., 1986; Shapiro et al., 1990), cortical reaction, formation of the fertilization membrane (Sasaki, 1984; Sardet and Chang, 1987), cellular division in the embryo (Poenie et al., 1985; Silver, 1986; Whitaker and Patel, 1990), their adhesion (McClay and Matranga, 1986), differentiation and formation of spicules (Mitsunaga et al., 1988) and metamorphosis (Carpenter et al., 1984).The present review combines information on the function of calcium-binding proteins and their targets, calmodulin regulation of NAD-kinase, exocytosis of cortical granules, Ca²⁺- and calmodulin-dependent protein phosphatase, Ca²⁺-dependent protein phosphorylation, regulation of ion-exchanger in the germ and embryonic cells as well as Ca²⁺- and calmodulin control of sperm motility in sea urchins.