Energy metabolism of spermatozoa of the sea urchin Glyptocidaris crenularis

Energy metabolism in spermatozoa of the sea urchin Glyptocidaris crenularis was examined. The spermatozoa contained not only several kinds of phospholipids and cholesterol but also triacylglycerides (TG). Following dilution of the dry sperm in sea water, the TG content decreased rapidly. Other lipids, however, remained at constant levels, except for an increase in the level of free fatty acid. Oil red-O staining of spermatozoa showed that TG was principally located in part of the sperm midpiece. Also, high lipase activity was demonstrated in the spermatozoa. In both intact cells and a cell-free system, 14C-labeled fatty acids were oxidized to 14CO2. It is thus concluded that G. crenularis spermatozoa use TG as a substrate for energy metabolism.     

Synthetic study on the structure-activity relationship of sperm activating peptides from the jelly coat of sea urchin eggs

Various analogue peptides with substitution and deletion of amino acid residues have been synthesized by liquid phase method for Sperm Activating Peptides from the jelly coat of sea urchin eggs. The deletion of C-terminal Gly reduced the activity to about 1/3000, while removal of N-terminal Gly reduced the activity to 1/10. The residues Ser5 and Asp3 were replaced by Lys without significant loss of activity. Substitution of Phe2 by Gly, Ala or Pro markedly reduced the activity by the factor of 10(4)-10(6), in contrast to Tyr-substitution retaining almost full activity, indicating the essential role of the aromatic residue in exerting the activity. Substitutions, Asp3 to Glu and Gly10 to Pro, increased the activity 5-fold and 500-fold, respectively.     

Structure and action of sperm activating peptides from the egg jelly of a sea urchin, Anthocidaris crassispina

Three Sperm Activating Peptides (SAPs) have been isolated to homogeneity from the jelly coat of the eggs of a Japanese sea urchin, Anthocidaris crassispina, and two of them have been sequenced. At pH 6.8 they can stimulate the sperm respiration 20-30 fold, to the level in normal seawater (pH 8.2), and the half-maximal activation was achieved by SAPs as low as around 100 pM. The stimulative activity was both pH- and Na+-dependent. The chymotryptic fragments (res. 3-10) were 10(4)-10(5) times less active, and the thermolytic fragment (res. 4-10) was 10(6) times less active than the parent SAP. CD spectra of SAPs indicate that they have unordered structure in aqueous solution.     

极端温度对海刺猬(Glyptocidaris crenularis)存活、摄食、生长和性腺的影响

在实验室条件下研究了极端温度对海刺猬的存活率、摄食率、生长以及组织器官等方面的影响。实验设置两个处理组温度为30°C和-2℃,对照组为自然水温(19~23℃),每组设置3个重复,每个重复60枚海刺猬,实验周期为56 d。结果表明:高温组海刺猬实验温度升到30℃后2 d内全部死亡,其平均摄食量(5.19±1.31 g·ind-1)极显著小于对照组(15.15±1.58 g·ind-1)(P0.01);低温组和对照组之间海刺猬存活率差异不显著(P0.05);但低温组海刺猬平均摄食量(0.18±0.04 g·ind-1)极显著小于对照组(10.90±0.33 g·ind-1)(P0.01);56 d内低温组海刺猬个体湿重极显著小于对照组(P0.01);低温组海刺猬口器湿重、壳湿重、性腺湿重、壳干重、口器指数、壳指数极显著小于对照组(P0.01),性腺干重、口器干重显著小于对照组(P0.05);但性腺指数与对照组无显著差别(P0.05);低温组海刺猬最大承受压力极显著小于对照组(P0.01);在实验室条件下,海刺猬(2~3 g·ind-1)无法长时间在高温环境(30℃)下存活,而能在低温环境(-2℃)下存活但其摄食、生长和性腺性状影响极显著。     

Isolation and identification of novel sulfated and nonsulfated oligosialyl glycosphingolipids from sea urchin sperm

Novel sulfated and nonsulfated oligosialyglycosphingolipids were isolated from sperm of the sea urchin,Hemicentrotus pulcherrimus, and their structures were established as follows: ±HSO₃Neu5Ac2(8Neu5Ac2)_n6G1c11´Cer, wheren=0, 1, 2, 3. This provides the first evidence for the natural occurrence of a tetrasialic acid structure in glycosphingolipids. The finding of sulfated oligosialyl chains is especially noteworthy in that the sulfate group exclusively resides on the C-8 of the nonreducing terminal residues of oligo/polysialyl chains and that sulfation appears to be a termination signal for elongation of oligosialyl chains. Sulfation at the nonreducing terminal Neu5Ac residues of oligosialyl chains was also found to facilitate the formation of an inter-residue lactone between the carboxyl group at the nonreducing terminal sulfated Neu5Ac and the hydroxyl group at C-9 of the penultimate Neu5Ac residue. The long chain base was 4-hydroxysphinganine (t18:0) and the major fatty acid species were identified as C20:1, C21:1, and C22:1.Abbreviations C:M chloroform:methanol - FAB-MS fast atom bombardment mass spectrometry - GLC gas-liquid chromatography - GSL glycosphingolipid - HPTLC high performance thin-layer chromatography - Neu5Ac8HSO₃ or HSO₃ Neu5Ac 5-N-acetyl-8-O-sulforylneuraminic acid - Neu5Gc9HSO₃ 5-N-glycolyl-9-O-sulforylneuraminic acid - NMR nuclear magnetic resonance - PB sodium phosphate buffer, pH 7.2 - Sia sialic acid - HSO₃ Sia sulfated sialic acid - polySia polysialic acid - TFA trifluoroacetic acid Since the dividing line between oligo- and polysialic acids is not rigidly defined, we propose here that those containing more than penta-sialic acid chains can be referred to as the polysialic acid group. The rationale for this is that 2 8-linked pentasialic acid is the minimum chain length to exhibit the conformational property of polysialic acid [of Michon, F, Brisson, J-R, Jennings, H (1987)Biochemistry 26: 8399–405] and to act as a substrate for the endosialidase, Endo-N [Hallenbeck, PC, Vimr, ER, Yu, F, Bassler, B, Troy, FA (1987)J Biol Chem 262: 3553–61].     

Movement of sea urchin sperm flagella

The motion of the sea urchin sperm flagellum was analyzed from high-speed cinemicrographs. At all locations on the flagellum the transversal motion and the curvature were found to vary sinusoidally in time. The curvatures of the flagella increase strongly near the proximal junction. Two sperm are described in transient from rest to normal motion. The full wave motion developed in both sperm within 40 ms.     

The effects of prolonged food deprivation on the covering behavior of the sea urchins Glyptocidaris crenularis and Strongylocentrotus intermedius

In this study, we investigated effects of 24 weeks of food deprivation on covering behavior of Glyptocidaris crenularis and Strongylocentrotus intermedius. Neither G. crenularis nor S. intermedius significantly reduced covering behavior during both short- and long-term food deprivation. However, G. crenularis and S. intermedius had significantly different behavioral patterns over the 24 weeks. Covering behavior of G. crenularis greatly increased from the first to the second week, with decreasing reaction time and increasing covering ability, while the covering behavior of S. intermedius generally remained unchanged during the experiment of 24 weeks. As prolonged food deprivation does not significantly affect covering behavior of G. crenularis and S. intermedius, the present study highlights the innate nature and biological significance of covering behavior in sea urchins, providing new insights into its behavioral mechanism.     

A new hyperpolarization-activated, cyclic nucleotide-gated channel from sea urchin sperm flagella

A sea urchin sperm flagellar hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel is known (SpHCN1) that is modulated by cAMP. Here, we describe a second flagellar HCN channel (SpHCN2) cloned from the same sea urchin species. SpHCN2 is 638 amino acids compared to 767 for SpHCN1. SpHCN2 has all the domains of an HCN channel, including six transmembrane segments (S1-S6), the ion pore, and the cyclic nucleotide-binding domain. The two full-length proteins are 33% identical and 51% similar. The six transmembrane segments vary from 46-79% identity. S4, which is the voltage sensor, is 79% identical between the two proteins. The ion selectivity filter sequence is GYG in the ion pore of SpHCN1 and GFG in SpHCN2. By sequence, SpHCN2 is 73.5kDa, but it migrates on SDS-PAGE at 64kDa. Western immunoblots show localization to flagella, which is confirmed by immunofluorescence. A neighbor-joining tree shows that SpHCN2 is basal to all known HCN channels. SpHCN2 might be the simplest pacemaker channel yet discovered.     

Polypeptide subunits of dynein 1 from sea urchin sperm flagella

A high-resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis system has been used to show the presence, in both whole sperm and isolated flagellar axonemes, of eight polypeptides migrating in the 300,000–350,000 molecular weight range characteristic of the heavy chains of dynein ATPase. Previously, only five such chains have been discernible. Extraction of isolated axonemes for 10 min at 4°C with a solution containing 0.6 M NaCl, pH 7, releases a mixture of particles that separate, in sucrose density gradient centrifugation, into a major peak, dynein 1 ATPase, sedimenting at 21 S and a minor peak at 12–14S. The polypeptide compositions of these two peaks are different. The dynein 1 peak, which contains most of the protein on the gradient, contains approximately equal quantities of two closely migrating heavy chains, with a small amount of a third, more slowly migrating chain; no other heavy chains appear in this peak. Two groups of smaller polypeptides (three intermediate chains, within the apparent molecular weight range 76,000–122,000 and four newly discovered light chains, within the apparent molecular weight range 14,000–24,000) cosediment with the 21 S peak. The heavy chain composition of the 12–14S peak is more complex, all eight heavy chains occurring in approximately the same ratios as occur in intact axonemes.     

Isolation of intact sperm asters from fertilized sea urchin eggs

We have developed a procedure for isolating intact sperm asters in quantity from fertilized sea urchin eggs. This procedure is based on detergent-extraction methods developed previously for the bulk isolation of mitotic apparatuses. Using this protocol it is possible to isolate sperm asters as soon as they appear in the fertilized egg or at any subsequent point in their brief existence.     

Structural components of sea urchin sperm nuclei

The sea urchin sperm nucleus rapidly loses its conoid morphology and becomes more voluminous and spherical upon its entry into the egg cytoplasm during fertilization. This investigation has attempted to determine what are the structural constraints placed upon the sperm nucleus, so that further investigations might determine the egg cytoplasmic factors that are responsible for modifying nuclear morphology. Isolated sperm nuclei were subjected to various extraction procedures in order to remove the majority of the proteins (histones) and also the DNA; subsequently, the residual structures were processed for and examined by electron microscopy. The data presented in this investigation demonstrate the removal of the sperm nuclear histones plus other nonhistone proteins has no effect on the conoid morphology of the sperm nucleus, yet this protein removal has a profound effect on the structure of the nuclear chromatin. It is also shown that removal of the majority of the nuclear DNA has no effect on the shape of the sperm nucleus. These results indicate that there are other components (possibly a nuclear matrix) associated with the sperm nucleus that are responsible for maintaining its conoid morphology.     

Ca2+ channels from the sea urchin sperm plasma membrane

Ca2+ influx across the sea urchin sperm plasma membrane is a necessary step during the egg jelly-induced acrosome reaction. There is pharmacological evidence for the involvement of Ca2+ channels in this influx, but their presence has not been directly demonstrated because of the small size of this cell. Sea urchin sperm Ca2+ channels are being studied by fusing isolated plasma membranes into planar lipid bilayers. With this strategy, a Ca2+ channel has been detected with the following characteristics: (a) the channel exhibits a high mainstate conductance (gamma MS) of 172 pS in 50 mM CaCl2 solutions with voltage-dependent decaying to smaller conductance states at negative Em; (b) the channel is blocked by millimolar concentrations of Cd2+, Co2+, and La3+, which also inhibit the egg jelly-induced acrosome reaction; (c) the gamma MS conductance sequence for the tested divalent cations is the following: Ba2+ greater than Sr2+ greater than Ca2+; and (d) the channel discriminates poorly for divalent over monovalent cations (PCa/PNa = 5.9). The sperm Ca2+ channel gamma MS rectifies in symmetrical 10 mM CaCl2, having a maximal slope conductance value of 94 pS at +100 mV applied to the cis side of the bilayer. Under these conditions, a different single-channel activity of lesser conductance became apparent above the gamma MS current at positive membrane potentials. Also in 10 mM Ca2+ solutions, Mg2+ permeates through the main channel when added to the cis side with a PCa/PMg = 2.9, while it blocks when added to the trans side. In 50 mM Ca2+ solutions, the gamma MS open probability has values of 1.0 at voltages more positive than -40 mV and decreases at more negatives potentials, following a Boltzmann function with an E0.5 = -72 mV and an apparent gating charge value of 3.9. These results describe a novel Ca2(+)-selective channel, and suggest that the main channel works as a single multipore assembly.     

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.     

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.