Sea urchin sperm peroxidase is competitively inhibited by benzohydroxamic acid and phenylhydrazine

1986 ◽  
Vol 64 (12) ◽  
pp. 1333-1338 ◽  
Author(s):  
Herbert Schuel ◽  
Regina Schuel
Keyword(s):  
Sea Urchin ◽  
Strongylocentrotus Purpuratus ◽  
Kinetic Studies ◽  
Biochemical Properties ◽  
Benzohydroxamic Acid ◽  
Somatic Tissues ◽  
Competitive Inhibitors ◽  
Triton X ◽  
Tetramethyl Benzidine ◽  
Sea Urchin Sperm

Sea urchin sperm contain a phenylhydrazine-sensitive peroxidase that is believed to use hydrogen peroxide produced by the fertilized egg to reduce sperm fertility and thereby assist in the prevention of polyspermy. Strongylocentrotus purpuratus sperm were treated initially with hypotonic phosphate buffer (pH 7.0) to remove catalase and then extracted with 0.5% Triton X-100 in 0.5 M acetate buffer (pH 5.0). Peroxidase activity in this detergent extract was assayed using 3,3′,5,5′-tetramethyl benzidine (TMB) as oxidizable substrate. Kinetic studies showed that the Km for TMB is 250 μM. Benzohydroxamic acid and phenylhydrazine are known to be competitive inhibitors of a variety of plant and animal peroxidases. These substances were found to competitively inhibit the sea urchin sperm peroxidase: for benzohydroxamic acid, Ki = 51.2 μM, mean inhibitory dose (ID50) = 146.7 μM; for phenylhydrazine, Ki = 201 nM, ID50 = 303 nM. These findings (i) indicate that the biochemical properties of the sea urchin sperm peroxidase resembles those of peroxidases found in somatic tissues where oxygen radicals are produced by phagocytes to kill bacteria and (ii) support our hypothesis that the sperm peroxidase has a functional role in the prevention of polyspermy during fertilization.

scholarly journals Properties of an antiserum against native dynein 1 from sea urchin sperm flagella.

1977 ◽  
Vol 73 (1) ◽  
pp. 182-192 ◽  
Author(s):  
K Ogawa ◽  
D J Asai ◽  
C J Brokaw
Keyword(s):  
Atpase Activity ◽  
Sea Urchin ◽  
Strongylocentrotus Purpuratus ◽  
Beat Frequency ◽  
Bend Angle ◽  
Tryptic Fragment ◽  
Effective Inhibition ◽  
Dynein Arms ◽  
Sea Urchin Sperm ◽  
Anthocidaris Crassispina

Effects of an antiserum against native dynein 1 from sperm flagella of the sea urchin Strongylocentrotus purpuratus were compared with effects of an antiserum previously obtained against an ATPase-active tryptic fragment (fragment 1A) of dynein 1 from sperm flagella of the sea urchin, Anthocidaris crassispina. Both antisera precipitate dynein 1 and do not precipitate dynein 2. Only the fragment 1A antiserum precipitates fragment 1A and produces a measurable inhibition of dynein 1 ATPase activity. Both antisera inhibit the movement and the movement-coupled ATP dephosphorylation of reactivated spermatozoa. The inhibition of movement by the antiserum against dynein 1 is much less than by the antiserum against fragment 1A, suggesting that a specific interference with the active ATPase site may be required for effective inhibition of movement. Both antisera reduce the bend angle as well as the beat frequency of reactivated S. purpuratus spermatozoa, suggesting that the bend angle may depend on the activity of the dynein arms which generate active sliding.

Crosstalk between protein kinases A and C regulates sea urchin sperm motility

Zygote ◽  
2021 ◽  
pp. 1-12
Author(s):  
Arlet Loza-Huerta ◽  
Hiram Pacheco-Castillo ◽  
Alberto Darszon ◽  
Carmen Beltrán
Keyword(s):  
Protein Kinase ◽  
Sperm Motility ◽  
Sea Urchin ◽  
Sea Urchins ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Competitive Inhibitors ◽  
Motility Regulation ◽  
Dependent Protein ◽  
Sea Urchin Sperm

Summary Fertilization, a crucial event for species preservation, in sea urchins, as in many other organisms, requires sperm motility regulation. In Strongylocentrotus purpuratus sea urchins, speract, a sperm chemoattractant component released to seawater from the outer egg layer, attracts sperm after binding to its receptor in the sperm flagellum. Previous experiments performed in demembranated sperm indicated that motility regulation in these cells involved protein phosphorylation mainly due to the cAMP-dependent protein kinase (PKA). However, little information is known about the involvement of protein kinase C (PKC) in this process. In this work, using intact S. purpuratus sea urchin sperm, we show that: (i) the levels of both phosphorylated PKA (PKA substrates) and PKC (PKC substrates) substrates change between immotile, motile and speract-stimulated sperm, and (ii) the non-competitive PKA (H89) and PKC (chelerythrine) inhibitors diminish the circular velocity of sperm and alter the phosphorylation levels of PKA substrates and PKC substrates, while the competitive inhibitors Rp-cAMP and bisindolylmaleimide (BIM) do not. Altogether, our results show that both PKA and PKC participate in sperm motility regulation through a crosstalk in the signalling pathway. These results contribute to a better understanding of the mechanisms that govern motility in sea urchin sperm.

scholarly journals PROPERTIES OF FLAGELLAR "RIGOR WAVES" FORMED BY ABRUPT REMOVAL OF ADENOSINE TRIPHOSPHATE FROM ACTIVELY SWIMMING SEA URCHIN SPERM

1974 ◽  
Vol 63 (3) ◽  
pp. 970-985 ◽  
Author(s):  
Barbara H. Gibbons ◽  
I. R. Gibbons
Keyword(s):  
Sea Urchin ◽  
Sperm Suspension ◽  
Complete Relaxation ◽  
Viscous Shear ◽  
Radial Spokes ◽  
Wave Forms ◽  
Cross Bridges ◽  
Triton X ◽  
The Waves ◽  
Sea Urchin Sperm

Sea urchin sperm were demembranated and reactivated with a solution containing 0.04% Triton X-100 and 0.03 mM ATP. The ATP concentration was then lowered abruptly by diluting the sperm suspension 50-fold into reactivating solution containing no ATP. The flagella of the sperm in the diluted suspension were not motile, but they were bent into a variety of stationary rigor wave forms closely resembling the wave forms occurring at different stages of the flagellar bending cycle during normal movement. The form of these rigor waves was unchanged upon storage for several hours in the presence of dithiothreitol and EDTA. Addition of 1 µM ATP induced slow relaxation of the waves, with most of the sperm becoming partially straightened over a period of about 30 min; somewhat higher concentrations gave a more rapid and complete relaxation. Concentrations of ATP above 10 µM induced resumption of normal beating movements. Addition of ITP, GTP, or GDP (up to 1 mM) produced no relaxation of the rigor waves. Digestion with trypsin to an extent sufficient to disrupt the radial spokes and the nexin links caused no change in the rigor wave forms, suggesting that these wave forms could be maintained by the dynein cross-bridges between the outer doublet tubules of the flagellar axoneme. Study of the effects of viscous shear on the rigor wave axonemes has shown that they are resistant to distortion by bending, although they can be twisted relatively easily.

Calcium-dependent bidirectional power stroke of the dynein arms in sea urchin sperm axonemes

1996 ◽  
Vol 109 (12) ◽  
pp. 2833-2842 ◽  
Author(s):  
S. Ishijima ◽  
M. Kubo-Irie ◽  
H. Mohri ◽  
Y. Hamaguchi
Keyword(s):  
Light Microscopy ◽  
Sea Urchin ◽  
Electron Microscopic Examination ◽  
Dark Field ◽  
Power Stroke ◽  
Electron Microscopic ◽  
Calcium Dependent ◽  
Dynein Arms ◽  
Triton X ◽  
Sea Urchin Sperm

Active sliding between doublet microtubules of sea urchin sperm axonemes that were demembranated with Triton X-100 in the presence or absence of calcium was induced with ATP and elastase at various concentrations of Ca2+ to examine the effects of Ca2+ on the direction of the power stroke of the dynein arms. Dark-field light microscopy of microtubule sliding revealed that the sliding from the axonemes demembranated with Triton and millimolar calcium and disintegrated with ATP and elastase showed various patterns of sliding disintegration, including loops of doublet microtubules formed near the head or the basal body. These loops were often thicker than the remaining axonemal bundle. In contrast, only thinner loops were found from the axonemes demembranated with Triton in the absence of calcium and disintegrated with ATP and elastase at high Ca2+ concentrations. Electron microscopic examination of the direction of microtubule sliding showed that the doublet microtubules in the axonemes demembranated in the presence of millimolar calcium moved toward the base of the axonemes by the dynein arms on the adjacent doublet microtubule as well as by their own dynein arms. Doublet microtubules in the axonemes demembranated in the absence of calcium moved toward the base of the axonemes only by their own dynein arms. Similar observations have been obtained from the axonemes from which the outer dynein arms were selectively extracted. From these observations, we can conclude that the dynein arms generate force in both directions and this feature of the dynein arms arises from at least the inner dynein arms.

The Effect of Partial Extraction of Dynein Arms on the Movement of Reactivated Sea-Urchin Sperm

1973 ◽  
Vol 13 (2) ◽  
pp. 337-357 ◽  
Author(s):  
BARBARA H. GIBBONS ◽  
I. R. GIBBONS
Keyword(s):  
Sea Urchin ◽  
Room Temperature ◽  
Beat Frequency ◽  
Wave Form ◽  
Bending Waves ◽  
Flagellar Beat ◽  
Elastic Forces ◽  
Dynein Arms ◽  
Triton X ◽  
Sea Urchin Sperm

Sea-urchin sperm were extracted with o.5 M KCl for 45 s at room temperature in the presence of Triton X-100, and then transferred to reactivating solution containing 1 mM ATP. The flagellar beat frequency of these KCl-extracted sperm (16 beats/s) was only about half that of control Triton-extracted sperm that had not been exposed to 0.5 M KCl (31 beats/s), although the form of their bending waves was not significantly altered. Examination by electron microscopy showed that the extraction with 0.5 M KCl removed the majority of the outer arms from the doublet tubules, leaving the inner arms apparently intact. By varying the duration of the KCl-extraction, it was shown that the rate of decrease in beat frequency paralleled the rate of disappearance of the arms. Prolonging the extraction time beyond 45 s at room temperature, or 4 min at o °C, had little further effect on beat frequency. ATPase measurements suggested that 6o-65% of the dynein in the original axonemes had been solubilized when the extraction with KCl was permitted to go to completion. These results indicate that the generation and propagation of flagellar bending waves of essentially typical form are not prevented by the removal of the outer row of dynein arms from the doublet tubules. In terms of the sliding filament model of flagellar bending, the results suggest that the rate of sliding between tubules under these conditions is proportional to the number of dynein arms present. The lack of significant change in wave form implies that the total amount of sliding that occurs during each bending cycle is not affected by the reduced number of dynein arms, but is regulated independently in some manner by the elastic forces generated by other structures in the bent axoneme.

Speract-receptor interaction and the modulation of ion transport in Strongylocentrotus purpuratus sea urchin sperm

Zygote ◽  
1999 ◽  
Vol 8 (S1) ◽  
pp. S20-S21 ◽  
Author(s):  
Blanca-Estela Galindo ◽  
Takuya Nishigaki ◽  
Esmeralda Rodríguez ◽  
Daniel Sánchez ◽  
Camen Beltrán ◽  
...  
Keyword(s):  
Ion Transport ◽  
Sea Urchin ◽  
Conformational Changes ◽  
Sea Urchins ◽  
Strongylocentrotus Purpuratus ◽  
Dissociation Rate ◽  
Receptor Interaction ◽  
Jelly Coat ◽  
Dissociation Rate Constants ◽  
Sea Urchin Sperm

We are studying the regulation of ion transport in sperm physiology. Sperm ion permeability is modulated by components from the outer layer of the egg which, depending on the species, regulate sperm motility, Chemotaxis and the acrosome reaction (AR). This reaction is required for sperm to fertilise the egg in many species from sea urchins to man (Darszon et al., 1999).Speract, a decapeptide from the external layer of Strongylocentrotus purpuratus sea urchin eggs, influences sperm respiration, motility and possibly the AR. Signal transduction starts when speract binds to a protein of 77 kDa closely coupled to sperm guanylyl cyclase (Garbers, 1989). Our recent receptor binding experiments using fluorescent-labelled speract (fluorescein and rhodamine) have allowed estimates of the association (kon 2.4 × 107 M−1s−1) and dissociation rate constants (koff 1.3 × 10−4 s−1). Furthermore, studies with fluorescent speract analogues indicate that the receptor undergoes conformational changes that depend on intracellular pH (pHi). The overall results are consistent with the possibility that speract may induce in sea urchin sperm a hyperactivated-like flagellar movement inside the jelly coat to accelerate sperm penetration through this layer.

scholarly journals Modulation of the asymmetry of sea urchin sperm flagellar bending by calmodulin.

1985 ◽  
Vol 100 (6) ◽  
pp. 1875-1883 ◽  
Author(s):  
C J Brokaw ◽  
S M Nagayama
Keyword(s):  
Sea Urchin ◽  
Gel Electrophoresis ◽  
Binding Activity ◽  
Calmodulin Binding ◽  
Bending Waves ◽  
Affinity Resin ◽  
The Asymmetry ◽  
Triton X ◽  
Asymmetric Bending ◽  
Sea Urchin Sperm

Sea urchin spermatozoa demembranated with Triton X-100 in the presence of EGTA, termed potentially asymmetric, generate asymmetric bending waves in reactivation solutions containing EGTA. After they are converted to the potentially symmetric condition by extraction with Triton and millimolar Ca++, they generate symmetric bending waves in reactivation solutions containing EGTA. In the presence of EGTA, their asymmetry can be restored by addition of brain calmodulin or the concentrated supernatant obtained from extraction with Triton and millimolar Ca++. These extracts contain calmodulin, as assayed by gel electrophoresis, radioimmunoassay, activation of brain phosphodiesterase, and Ca++-dependent binding of asymmetry-restoring activity to a trifluorophenothiazine-affinity resin. Conversion to the potentially symmetric condition can also be achieved with trifluoperazine substituted for Triton during the exposure to millimolar Ca++, which suggests that the calmodulin-binding activity of Triton is important for this conversion. These observations suggest that the conversion to the potentially symmetric condition is the result of removal of some of the axonemal calmodulin and provide additional evidence for axonemal calmodulin as a mediator of the effect of Ca++ on the asymmetry of flagellar bending.

scholarly journals The axonemal axis and Ca2+-induced asymmetry of active microtubule sliding in sea urchin sperm tails.

1986 ◽  
Vol 102 (6) ◽  
pp. 2042-2052 ◽  
Author(s):  
W S Sale
Keyword(s):  
Sea Urchin ◽  
Pair Interaction ◽  
Central Pair ◽  
Sperm Tail ◽  
Lytechinus Pictus ◽  
Motile Cells ◽  
Definition Of ◽  
Triton X ◽  
Explicit Discussion ◽  
Sea Urchin Sperm

Structural studies of stationary principal bends and of definitive patterns of spontaneous microtubule sliding disruption permitted description of the bending axis in sea urchin sperm tail axonemes. Lytechinus pictus sperm were demembranated in a buffer containing Triton X-100 and EGTA. Subsequent resuspension in a reactivation buffer containing 0.4 mM CaCl2 and 1.0 mM MgATP2- resulted in quiescent, rather than motile, cells and each sperm tail axoneme took on an extreme, basal principal bend of 5.2 rad. Thereafter, such flagellar axonemes began to disrupt spontaneously into two subsets of microtubules by active sliding requiring ATP. Darkfield light microscopy demonstrated that subset "1" is composed of microtubules from the inside edge of the principal bend. Subset "2" is composed of microtubules from the outside edge of the principal bend and always scatters less light in darkfield than subset 1. Subset 2, which always slides in the proximal direction, relative to subset 1, results in a basal loop of microtubules, and the subset 2 loop is restricted to the bend plane during sliding disruption. Electron microscopy revealed that doublets 8, 9, 1, 2, 3 and the central pair comprise subset 1, and doublets 4, 5, the bridge, 6, and 7 comprise subset 2. The microtubules of isolated subset 2 are maintained in a circular arc in the absence of spoke-central pair interaction. Longitudinal sections show that the bending plane bisects the central pair. We therefore conclude that the bend plane passes through doublet 1 and the 5-6 bridge and that doublet 1 is at the inside edge of the principal bend. Experimental definition of the axis permits explicit discussion of the location of active axonemal components which result in Ca2+-induced stationary basal bends and explicit description of components responsible for alternating basal principal and reverse bends.

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