Activation of Proteasome in Sea Urchin Sperm by Lysophosphatidylinositol and by Sperm Lipids

Effects of phospholipids, their metabolites and endogenous sperm lipids on the chymotrypsin-like activity of proteasome purified from sperm of the sea urchin, Strongylocentrotus intermedius were examined. Some lysophospholipids remarkably enhanced the activity. The most prominent activation was found in lysophosphatidylinositol (LPI) which enhanced about 12-fold at 2.5 μg/ml. On the other hand, higher concentrations (above 250 μg/ml) were required for the enhancement of the activity by some saturated fatty acids and phospholipids. Lipids extracted from sperm also were effective in the enhancement, and those from sperm which were treated for 15 sec in egg jelly were more effective than those from untreated sperm. These results suggest that certain metabolites belonging to lysophospholipids are produced during the acrosome reaction and activate sperm proteasome. Also, they are not inconsistent with our view that the chymotrypsin-like activity of sperm proteasome participates in the acrosome reaction (23, 24).     

The Acrosome Reaction Induced by Dimethylsulfoxide in Sea Urchin Sperm

The acrosome reaction in sea urchin sperm, as judged by disappearance of the acrosomal vesicles in Nomarski optics, was induced by dimethylsulfoxide (DMSO) at concentration above 0.1% in normal artificial sea water. The number of the acrosome-reacted spermatozoa increased in proportion to DMSO concentration. The DMSO-induced acrosome reaction, as well as the jelly water- or A23187-induced one, was inhibited by nifedipine and hardly occurred in Ca²⁺-free artificial sea water. However, the DMSO-induced acrosome reaction was found in a few number of spermatozoa in the presence of Ca²⁺at above 0.5 mM, though the jelly water- or A23187-induced acrosome reaction did not occur at external Ca²⁺levels lower than 1 mM. Dependency of the acrosome reaction by DMSO on external Ca²⁺is somewhat lower than that of the reaction by jelly water. In Ca²⁺-free artificial sea water, the acrosomal regions of DMSO-treated spermatozoa attached to their own tails. In some cases, spermatozoa thus treated with DMSO in Ca²⁺free artificial sea water caused formation of fertilization membrane in a few number of eggs kept in Ca²⁺-free artificial sea water. Even in the absence of extermal Ca²⁺, preliminary step of the acrosome reaction seems to be completed probably by DMSO-induced weak Ca²⁺-mobilization in spermatozoa.     

N-Linked Oligosaccharides of Sea Urchin Egg Jelly Induce the Sperm Acrosome Reaction

The sea urchin egg jelly coat (EJ) induces the acrosome reaction (AR) of sperm. We previously demonstrated that a fraction of EJ containing two glycoproteins of 82- and 138-kDa possess the AR inducing activity (8). Here we show that Peptide-N-Glycosidase-F treatment of EJ followed by precipitation and washing in 70% ethanol results in a substantial loss of AR inducing activity in the ethanol insoluble material. When a PNGase-F digest of EJ is chromatographed on a Sepacryl-200 gel filtration column, an AR inducing fraction elutes within the partitioning volume. Acrosome reaction inducing activity of undigested EJ does not elute within the partitioning volume. The chromatographed AR inducing fraction of the PNGase-F digest reacts strongly in the phenol-sulfuric assay demonstrating carbohydrate is present; silver stained gels do not detect the presence of protein. Harsh alkaline hydrolysis of EJ in an excess of NaBH₄, preserves a substantial amount of AR inducing activity. These data show that N-linked oligosaccharides released from EJ by PNGase-F digestion are capable of inducing the sperm acrosome reaction.     

Sperm Bind but Do not Unbind from the Fixed Sea Urchin Egg

In S. purpuratus sperm remained bound to aldehyde-fixed eggs and did not exhibit a detachment phase. Sperm bound in high numbers and the binding curve was similar to that for unfixed homologous gametes. Binding kinetics were analyzed using sequential still photographs of slightly flattened, fixed eggs under supported coverslips. In this way, a single egg could be observed. Alternatively, sperm remaining in the supernatant over settled eggs were counted at successive time points postmixing using spectrophotometry (340 nm) and hemocytometry. Additionally, aliquots of mixed gametes were fixed at successive time points and the numbers of bound sperm determined microscopically on egg perimeters. The maximum number of sperm bound by 2 min; this number remained at or near maximum for as long as 10 min when the experiments were terminated. When sperm were jelly-reacted before addition to eggs, fewer sperm bound, although binding curves were similar to the above experiments. It appears that the binding efficiency of sperm decreases with time after initiation of the acrosome reaction: (1) fewer prereacted sperm bound; and, (2) sperm did not continue to bind after 2 min even though the egg surface was not saturated. Whether these effects are related to motility or other factors is unclear. Furthermore, the above results indicate the importance of the cortical reaction to sperm unbinding. Such studies enable one to observe sperm behavior while precluding the effects of egg secretion during the initial stages of fertilization.     

Purification of a Sperm Lectin Extracted from Spermatozoa of the Sea Urchin Hemicentrotus pulcherrimus

Hemagglutinating activity for human type A erythrocytes was detected in a sperm extract obtained by treatment with Triton X-100 of spermatozoa from the sea urchin Hemicentrotus pulcherrimus. Among tested sugars only N-acetyl-D-galactosamine had any inhibitory effect on the hemagglutinating activity of the sperm extract. The lectin was purified by a combination of affinity chromatography and ion-exchange chromatography. A single band was obtained after SDS-polyacrylamide gel electrophoresis of the purified lectin, corresponding to an apparent molecular weight of 15,000 daltons. Trypsin-generated fragments of the surface of eggs significantly inhibited hemagglutination of erythrocytes by the purified lectin. The biological role of the sperm lectin is discussed.     

THE MECHANISM OF ACTION OF COLCHICINE : Colchicine Binding Properties of Sea Urchin Sperm Tail Outer Doublet Tubulin

The thermal depolymerization procedure of Stephens (1970. J. Mol. Biol. 47:353) has been employed for solubilization of Strongylocentrotus purpuratus sperm tail outer doublet microtubules with the use of a buffer during solubilization which is of optimal pH and ionic strength for the preservation of colchicine binding activity of chick embryo brain tubulin. Colchicine binding values were corrected for first-order decay during heat solubilization at 50°C (t_½ = 5.4 min) and incubation with colchicine at 37°C in the presence of vinblastine sulfate (t_½ = 485 min). The colchicine binding properties of heat-solubilized outer doublet tubulin were qualitatively identical with those of other soluble forms of tubulin. The solubilized tubulin (mol wt, 115,000) bound 0.9 ± 0.2 mol of colchicine per mol of tubulin, with a binding constant of 6.3 x 10⁵ liters/mol at 37°C. The colchicine binding reaction was both time and temperature dependent, and the binding of colchicine was prevented in a competitive manner by podophyllotoxin (K_i = 1.3 x 10^-6 M). The first-order decay of colchicine binding activity was substantially decreased by the addition of the vinca alkaloids, vinblastine sulfate or vincristine sulfate, thus demonstrating the presence of a vinca alkaloid binding site(s) on the outer doublet tubulin. Tubulin contained within the assembled microtubules did not decay. Intact outer doublet microtubules bound less than 0.001 mol of colchicine per mol of tubulin contained in the microtubules, under conditions where soluble tubulin would have bound 1 mol of colchicine per mol of tubulin (saturating concentration of colchicine, no decay of colchicine binding activity). The presence of colchicine had no effect on the rate of solubilization of outer doublet microtubules during incubation at 37°C. Therefore, the colchicine binding site on tubulin is blocked (not available to bind colchicine) when the tubulin is in the assembled outer doublet microtubules.     

Aster Formation in Sea Urchin Eggs Induced by the injection of Enzyme-Treated Sperm Centrioles

To determine the responsible components of isolated sperm centrioles for the aster induction in sea urchin eggs, the sperm centriolar fraction was treated with various enzymes and was injected into the unfertilized eggs, then the aster formation in first division was observed after fertilization.
Treatment with 1 μg/ml or higher concentration of trypsin inhibited the centriolar activity for aster induction, whereas the treatment with 50 μg/ml of DNase 1, 80 μg/ml of RNase A, 40 μg/ml of RNase T1, or 0.1 μg/ml of trypsin had no inhibitory effect to induce asters. Injection of 0.5 μg/ml of RNase A or 1 mUg/ml of RNase T1 into the egg caused the detention of mitosis at the streak stage. To examine the temperature effect for aster induction, the centriolar fraction was pre-treated with boiling temperature, and it was found that the fraction became incapable to induce any aster.
Results obtained suggest that the effective components of the sperm centriolar fraction to induce asters in the fertilized sea urchin eggs are the proteins but not the nucleic acids. The aster inducing activity is destroyed by heat treatment.     

Purification and Characterization of Trypsin like Enzyme of Sperm of the Sea Urchin, Hemicentrotus pulcherrimus

Trypsin like enzyme has been isolated from sperm of the sea urchin, Hemicentrotus pulcherrimus , using tryptophane methyl ester-Sepharose 4B and soybean trypsin inhibitor-Sepharose 4B affinity chromatographies.
The isolated enzyme preparation is homogenous in polyacrylamide gel electrohoresis at pH 2.3. The molecular weight of the enzyme estimated by gel filtration is about 33,000, and the enzyme separates into two subunits of 10,900 and 20,500 on sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence of β-mercaptoethanol.
This enzyme is active to N-α-benzoyl arginine ethyl ester (BAEE), N-α-toluenesulfonyl-L-arginine ethyl ester (TAME), and N-α-benzoyl-DL-arginine-p-nitroanilide, but not N-acetyl-L-tyrosine ethyl ester, N-benzoyl-L-tyrosine ethyl ester, Hippuryl-L-arginine, and Hippuryl-L-phenylalanine. The optimal pH of this enzyme is about 8.0. The Michaelis constants for BAEE and TAME are 3.3 × 10⁻⁶M, and 8.2 × 10⁻⁵M, respectively.
Soybean trypsin inhibitor and lima bean trypsin inhibitor completely inhibit the activity of this enzyme, while N-α-tosyl-L-lysine chloromethyl ketone, ovomucoid trypsin inhibitor, and α-1-antitrypsin partially inhibit. L-1-tosylamide-2-phenyl chloromethyl ketone, chyrnostatin, and aporotinine are without effect.
This enzyme is stable at pH 2.0–3.0 and labile at pH 8.0. Ca²⁺ and Mg²⁺ activate this enzyme, but do not stabilize at pH 8.0. Seawater, NaCl, and KCl inhibit this enzyme activity.
Release of this enzyme from the acrosomal vesicle is suggested.     

Iqcg Is Essential for Sperm Flagellum Formation in Mice

Mammalian spermatogenesis comprises three successive phases: mitosis phase, meiosis phase, and spermiogenesis. During spermiogenesis, round spermatid undergoes dramatic morphogenesis to give rise to mature spermatozoon, including the condensation and elongation of nucleus, development of acrosome, formation of flagellum, and removal of excessive cytoplasm. Although these transformations are well defined at the morphological level, the mechanisms underlying these intricate processes are largely unknown. Here, we report that Iqcg, which was previously characterized to be involved in a chromosome translocation of human leukemia, is highly expressed in the spermatogenesis of mice and localized to the manchette in developing spermatids. Iqcg knockout causes male infertility, due to severe defects of spermiogenesis and resultant total immobility of spermatozoa. The axoneme in the Iqcg knockout sperm flagellum is disorganized and hardly any typical (“9+2”) pattern of microtubule arrangement could be found in Iqcg knockout spermatids. Iqcg interacts with calmodulin in a calcium dependent manner in the testis, suggesting that Iqcg may play a role through calcium signaling. Furthermore, cilia structures in the trachea and oviduct, as well as histological appearances of other major tissues, remain unchanged in the Iqcg knockout mice, suggesting that Iqcg is specifically required for spermiogenesis in mammals. These results might also provide new insights into the genetic causes of human infertility.     

Purification and Characterization of Sperm Creatine Kinase and Guanylate Cyclase of the Sea Urchin Hemicentrotus pulcherrimus

Creatine kinase and guanylate cyclase were purified from Hemicentrotus pulcherrimus spermatozoa. The molecular weight of the purified sperm tail creatine kinase was estimated to be 137,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sperm tail guanylate cyclase was purified by chromatography on a WGA-Sepharose column connected to a Concanavalin A-Sepharose column, and a Superose 12 HR column. The molecular weight of the tail guanylate cyclase was estimated to be 128,000 by SDS-PAGE. The specific activity of the purified enzyme was 8.25 μmol of cGMP formed/min/mg protein. Sperm-activating peptide I (SAP-I) causes an electrophoretic mobility shift of H. pulcherrimus sperm guanylate cyclase from 131 kDa to 128 kDa. The 131 kDa form of guanylate cyclase was co-purified with a 76 kDa protein, whose molecular mass is similar to that of a SAP-I receptor. The purified 131 kDa form of guanylate cyclase had higher activity than the 128 kDa form. The 131 kDa and 128 kDa forms of guanylate cyclase contained 23.83 ± 0.65 and 4.16 ± 0.45 moles of phosphate per mol protein (mean ± S.D.; n = 3), respectively. The activities of guanylate cyclase and creatine kinase increased during the testis development. During spermatogenesis, sperm tail creatine kinase was detected immunohistochemically only in mature spermatozoa.     

Ultrastructural Study of Asters Induced by Microinjection with Sperm Centriolar Fraction in Sea Urchin Eggs

Multiple asters can be artificially induced in sea urchin fertilized eggs by the microinjection of the centriolar fraction of sperm homogenate. Investigation was continued by the electron microscopy to determine whether the multi-aster formation was due to the centrioles or the contaminants in the injected sperm fraction. Thirty three asters in 3 operated eggs were thoroughly examined, and we confirmed that the presence of centrioles in the central region of 26 asters. We considered that the rest of them might contained the centrioles in the sections lost during the preparation procedures. Fragmented axoneme, the plug of electron dense material, and the centriolar fossa, which were usually accompanied with the isolated centrioles, disappeared from the centrioles in these multiple asters. However, electron dense, amorphous materials were formed associating with the triplet blades and distributed around the centrioles. Many astral microtubules were terminated in these pericentriolar materials. Results obtained suggest that, although the pericentriolar material is acting as the microtubule organizing center, all multiple asters, except those derived from fertilization (2 asters per egg), are most likely induced by the injected centrioles and not by the contaminants.     

Different Migration Patterns of Sea Urchin and Mouse Sperm Revealed by a Microfluidic Chemotaxis Device

Chemotaxis refers to a process whereby cells move up or down a chemical gradient. Sperm chemotaxis is known to be a strategy exploited by marine invertebrates such as sea urchins to reach eggs efficiently in moving water. Less is understood about how or whether chemotaxis is used by mammalian sperm to reach eggs, where fertilization takes place within the confinement of a reproductive tract. In this report, we quantitatively assessed sea urchin and mouse sperm chemotaxis using a recently developed microfluidic model and high-speed imaging. Results demonstrated that sea urchin Arbacia punctulata sperm were chemotactic toward the peptide resact with high chemotactic sensitivity, with an average velocity V_x up the chemical gradient as high as 20% of its average speed (238 μm/s), while mouse sperm displayed no statistically significant chemotactic behavior in progesterone gradients, which had been proposed to guide mammalian sperm toward eggs. This work demonstrates the validity of a microfluidic model for quantitative sperm chemotaxis studies, and reveals a biological insight that chemotaxis up a progesterone gradient may not be a universal strategy for mammalian sperm to reach eggs.     

Sperm Flagellum Volume Determines Freezability in Red Deer Spermatozoa

The factors affecting the inter-individual differences in sperm freezability is a major line of research in spermatology. Poor sperm freezability is mainly characterised by a low sperm velocity, which in turn is associated with low fertility rates in most animal species. Studies concerning the implications of sperm morphometry on freezability are quite limited, and most of them are based on sperm head size regardless of the structural parts of the flagellum, which provides sperm motility. Here, for the first time, we determined the volumes of the flagellum structures in fresh epididymal red deer spermatozoa using a stereological method under phase contrast microscopy. Sperm samples from thirty-three stags were frozen and classified as good freezers (GF) or bad freezers (BF) at two hours post-thawing using three sperm kinetic parameters which are strongly correlated with fertility in this species. Fourteen stags were clearly identified as GF, whereas nineteen were BF. No significant difference in sperm head size between the two groups was found. On the contrary, the GF exhibited a lower principal piece volume than the BF (6.13 µm³ vs 6.61 µm³, respectively, p = 0.006). The volume of the flagellum structures showed a strong negative relationship with post-thawing sperm velocity. For instance, the volume of the sperm principal piece was negatively correlated with sperm velocity at two hours post-thawing (r = −0.60; p<0.001). Our results clearly show that a higher volume of the sperm principal piece results in poor freezability, and highlights the key role of flagellum size in sperm cryopreservation success.     

Matrix Metalloproteinases in a Sea Urchin Ligament with Adaptable Mechanical Properties

Mutable collagenous tissues (MCTs) of echinoderms show reversible changes in tensile properties (mutability) that are initiated and modulated by the nervous system via the activities of cells known as juxtaligamental cells. The molecular mechanism underpinning this mechanical adaptability has still to be elucidated. Adaptable connective tissues are also present in mammals, most notably in the uterine cervix, in which changes in stiffness result partly from changes in the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). There have been no attempts to assess the potential involvement of MMPs in the echinoderm mutability phenomenon, apart from studies dealing with a process whose relationship to the latter is uncertain. In this investigation we used the compass depressor ligaments (CDLs) of the sea-urchin Paracentrotus lividus. The effect of a synthetic MMP inhibitor - galardin - on the biomechanical properties of CDLs in different mechanical states (“standard”, “compliant” and “stiff”) was evaluated by dynamic mechanical analysis, and the presence of MMPs in normal and galardin-treated CDLs was determined semi-quantitatively by gelatin zymography. Galardin reversibly increased the stiffness and storage modulus of CDLs in all three states, although its effect was significantly lower in stiff than in standard or compliant CDLs. Gelatin zymography revealed a progressive increase in total gelatinolytic activity between the compliant, standard and stiff states, which was possibly due primarily to higher molecular weight components resulting from the inhibition and degradation of MMPs. Galardin caused no change in the gelatinolytic activity of stiff CDLs, a pronounced and statistically significant reduction in that of standard CDLs, and a pronounced, but not statistically significant, reduction in that of compliant CDLs. Our results provide evidence that MMPs may contribute to the variable tensility of the CDLs, in the light of which we provide an updated hypothesis for the regulatory mechanism controlling MCT mutability.     

Does Phospholipase A2 Participate in the Acrosome Reaction of Sea Urchin Sperm?: A Pharmacological Study

We examined whether phospholipase A₂ (PLA₂) is involved in the initiation of the acrosome reaction of sperm of the sea urchin, Strongylocentrotus intermedius , using inhibitors and an activator of this enzyme. Quinacrine and p-bromophenacyl bromide (PBPB) inhibited the egg jelly-induced acrosome reaction at 100 μM, but not the ionomycin-induced one. Depression of egg jelly-induced increase of intracellular free Ca²⁺concentration ([Ca²⁺]_i) by these reagents was expected and examined using fura 2. Quinacrine interfered with the flourescence of fura 2, but PBPB was found to depress at concentrations which could inhibit the acrosome reaction. Furthermore, melittin, which is known to stimulate PLA², caused a [Ca²⁺]_i increase and a formation of acrosomal process-like structure on sperm head. These results suggest that PLA₂ participates in the early step(s) of the acrosome reaction of sea urchin sperm.     

Micromechanics of Sea Urchin Spines

The endoskeletal structure of the Sea Urchin, Centrostephanus rodgersii, has numerous long spines whose known functions include locomotion, sensing, and protection against predators. These spines have a remarkable internal microstructure and are made of single-crystal calcite. A finite-element model of the spine’s unique porous structure, based on micro-computed tomography (microCT) and incorporating anisotropic material properties, was developed to study its response to mechanical loading. Simulations show that high stress concentrations occur at certain points in the spine’s architecture; brittle cracking would likely initiate in these regions. These analyses demonstrate that the organization of single-crystal calcite in the unique, intricate morphology of the sea urchin spine results in a strong, stiff and lightweight structure that enhances its strength despite the brittleness of its constituent material.     

Properties of Intracellular Hatching enzyme in the Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

The intracellular hatching enzyme was confirmed to be particulate-bound in the sea urchin, Hemicentrotus pulcherrimus. The enzyme was solubilized most effectively by sonication in buffer containing 12.5 mM CaCl₂, and 0.5 M KCl. The intracellular hatching enzyme is suggested to be activated by an antipain- or elastatinal-susceptible protease(s) on its solubilization. Since the intracellular hatching enzyme solubilized in the absence of protease inhibitors was inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin, the active hatching enzyme is concluded to be a chymostatin-sensitive serine protease. The enzyme required CaCl₂, and KCl or NaCl for both stability and activity. The preference of the enzyme of anions as sodium salts was as follows: Cl⁻ > NO₃⁻ > I⁻ > SCN⁻. The apparent molecular weights of the intracellular hatching enzyme (IHE) and the hatching enzyme secreted from the blastula with or without the fertilization envelope (SHE or dSHE) were estimated as 89,000, 135,000, 80,000, respectively. On incubations with isolated fertilization envelopes as an enzyme substrate, the apparent molecular weights of dSHE and IHE increased to 128,000 and 105,000, respectively.     

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.     

Eutacticity in Sea Urchin Evolution

An eutactic star, in a n-dimensional space, is a set of N vectors which can be viewed as the projection of N orthogonal vectors in a N-dimensional space. By adequately associating a star of vectors to a particular sea urchin, we propose that a measure of the eutacticity of the star constitutes a measure of the regularity of the sea urchin. Then, we study changes of regularity (eutacticity) in a macroevolutive and taxonomic level of sea urchins belonging to the Echinoidea class. An analysis considering changes through geological time suggests a high degree of regularity in the shape of these organisms through their evolution. Rare deviations from regularity measured in Holasteroida order are discussed.