Endogenous substrate for energy metabolism and ultrastructural correlates in spermatozoa of the sea urchin Diadema setosum

Energy metabolism in spermatozoa of the sea urchin Diadema setosum of the order Diadematoida was examined. The spermatozoa contained not only several kinds of phospholipids and cholesterol, but also triglyceride (TG). Glycogen and glucose were present at extremely low levels. Following dilution of dry sperm and incubation in seawater, the TG content decreased rapidly. Other lipids, however, remained at constant levels, except for a slight increase in the level of free fatty acid. High lipase activity was demonstrated in the spermatozoa. ¹⁴C-labeled fatty acid was oxidized to ¹⁴CO₂. Ultrastructural study also showed that lipid globules were present at the bottom of the midpiece. After incubation in seawater, morphological changes in the lipid globules were observed and some vacuoles appeared. Thus, the results obtained strongly suggest that D. setosum spermatozoa obtain energy through oxidation of fatty acid from TG stored in the lipid globules at the midpieces. © 1995 Wiley-Liss, Inc.     

Acetylcholinesterase in sea urchin spermatozoa

Flagella contain the bulk of spermatozoan acetylcholinesterase. Brief sonication of sea urchin sperm suspended in Tris-buffered (pH 8.0), Ca, Mg-free artificial sea water (F-ASW) containing 10 mM ethylene diaminetetracetic acid, (EDTA) doubled the specific activity over that of the intact spermatozoa. Lipids were removed from the solubilized supernatant of the tail membrane fraction by ether extraction. Hydrolysis of acetylthiocholine in the presence of dithiobisnitrobenzoic acid (DTNB) was monitored spectrophotometrically at 412 nm by the Ellman procedure. The enzyme was purified by affinity chromatography on a Sepharose cyanogen bromide gel to which the cholinesterase inhibitor trimethyl (para-aminophenyl) ammonium chloride was coupled. The enzyme was eluted from the column with a discontinous NaCl gradient (0.1–0.5 M). The active fraction recovered at 0.35 M NaCl contained 0.007% of the initial total sperm cell protein with a 500-fold increase in specific activity. Twenty-four hr centrifugation on a 5–20% sucrose density gradient at 50,000g in a Beckman L5-75 centrifuge yielded peaks at 14.7 S and 9.1 S. In the presence of 1% Triton X-100, three peaks appeared: 23.3 S, 13.7 S, and 9.1 S. These sedimentation coefficients resemble those of the electroplax acetylcholinesterase (AChE) forms A₈ and A₄. Eserine completely inhibited the activity of the purified enzyme, which exhibits a substrate optimum at 4 mM acetylcholine. The activity is depressed by 75% at 10 mM ACh and by 90% at 25 mM. The Km was 2.1 × 10^?4 M. In the sperm cell the enzyme that terminates the action of intracellularly synthesized ACh may be involved in controlling ionophoric channels that regulate transmembrane transport of calcium.     

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.     

Protein synthetic activity in swimming sea urchin spermatozoa

Summary

Free swimming Paracentrotus lividus spermatozoa show a significant rate of protein synthesis which remains nearly linear over a period of 90 min. This protein synthetic activity is scarcely affected by emetine but completely suppressed by chloramphenicol, suggesting its possible mitochondrial origin.     

Energy metabolism of sea urchin spermatozoa, with phosphatidylcholine as the preferred substrate

Phosphatidylcholine content in the spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, decreased rapidly during incubation with sea water. Sea urchin sperm contained approx. 85% phospholipid in total lipid. Phosphatidylcholine (PC) was the principal lipid. Other phospholipids, however, remained at constant levels during incubation. Although the free fatty acid content gradually increased following dilution of dry sperm in sea water, the amounts of triacylglycerol and cholesterol ester were extremely low. Analysis by gas-liquid chromatography indicated most of fatty acid moieties in PC to be polyenoic. PC composed in part of unsaturated fatty acids was consumed to a greater extent during incubation than that consisting of saturated fatty acids. Furthermore, 1-palmitoyl-2-[1-14C]linoleoylphosphatidylcholine was transformed to 14C-labelled free fatty acid in a subcellular system. Thus, possibly, phospholipase A2 is present in sea urchin sperm. Also, [1-14C]oleic acid was immediately oxidized to 14CO2 by sperm. It is thus concluded that sea urchin sperm use phosphatidylcholine as a substrate for energy metabolism.     

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.     

Fatty chain composition of phospholipids in sea urchin spermatozoa

1. An analysis was made of lipids extracted from the spermatozoa of the sea urchins, Hemicentrotus pulcherrimus and Anthocidaris crassispina. 2. Nearly all the lipids from both species consisted of phospholipids (about 80%) and cholesterol (about 14%). Triglyceride and cholesterol ester were present in trace amounts. 3. The fatty acid composition of each phospholipid was determined by gas-liquid chromatography. In both species, the fatty acid consisting of phosphatidylethanolamine was of the unsaturated type for the most part, while cardiolipin was comprised to a considerable degree of saturated fatty acids. In phosphatidylcholine and phosphatidylserine from H. pulcherrimus sperm, unsaturated fatty acid content was somewhat higher than that in phospholipids from A. crassispina sperm.     

Metabolism of lipid and carbohydrate in sea urchin spermatozoa

When the dry sperm of the sea urchin, Hemicentrotus pulcherrimus, were diluted 100 times in artificial sea water at 0°C and at 20°C, they became motile and the levels of ATP and creatine phosphate decreased rapidly. The level of ADP hardly changed, and the AMP level increased after the dilution. After the dilution, the respiratory rate at 2°C was almost one fifth that of 20°C. Both phospholipid and glycogen were used for the energy sources in sea urchin sperm. The level of phospholipid was 10-fold higher than that of glycogen in the dry sperm. The phospholipid level decreased after dilution at 20°C, though the level hardly changed at 0°C, suggesting that phospholipid was hardly metabolized the lower temperature. The level of α -glycerophosphate increased at 20°C after the dilution but did not change at 0°C. The level of glycogen decreased after the dilution, regardless of the temperature. The glycolysis was also activated after the dilution. Of the intermediates of the tricarboxylic acid cycle, the citrate concentration increased at 0°C and the malate concentration also increased at 0°C and especially strongly at 20°C.     

Effect of Na+-free seawater on energy metabolism in sea urchin spermatozoa with special reference to coenzyme A and carnitine derivatives

When the dry sperm of sea urchin, Hemicentrotus pulcherrimus, were diluted 100 times in artificial seawater, they became motile and the level of ATP decreased. However, after dilution in Na⁺-free seawater, the sperm were still immotile and the ATP level remained constant. CoA, carnitine, and their derivatives as intermediates of lipid metabolism were also measured in sperm. The levels of CoA derivatives were much higher than those of carnitine derivatives. The free and acid-soluble CoA level decreased and the long-chain acyl-CoA level increased after dilution in artificial seawater, However, they did not change after dilution in Na⁺-free seawater. The levels of carnitine derivatives hardly changed after the dilution in both sorts of artificial seawater, The respiratory rate was extremely low in Na⁺-free seawater, Furthermore, concentrations of pyruvate, lactate, citrate, and malate remained essentially constant after dilution in the Na⁺-free seawater, whereas they changed markedly after dilution in the artificial seawater.     

Poly A metabolism in sea urchin embryos

Three different methods for the detection and isolation of RNA that contains poly A sequences are compared. All three methods selectively bind poly A containing sequences. However only the poly dT cellulose technique gave reproducible results and did not modify the binding characteristics of the RNA initially bound to the poly dT cellulose. The proportion of the total cellular RNA that contains poly A is about the same in the blastula and gastrula stages and is lower at cleavage. The kinetics of accumulation of RNA containing poly A for the blastula stage indicates extensive turnover of poly A containing material. RNA containing poly A is characterized by its heterogeneous sedimentation. Some of the RNA containing poly A is as large as 0.5 × 10⁶ Daltons.     

Freezing tolerance in embryos and spermatozoa of the sea urchin

Late embryos of the sea urchin survive freezing, at least for a short period of time, at ?196 °C in the presence of a cryoprotectant. The freezing tolerance in glycolated embryos is greater in advanced developmental stages. High rates of both cooling and warming during a freezethaw sequence were more dangerous in one-cell embryos than in late ones. Both ethylene glycol and DMSO exerted a significant protection against freezing injury on embryos in all the stages after fertilization, but not on unfertilized egg cells. Cryopreservation of sea urchin sperm at ?196 °C in the presence of 1.5 m ethylene glycol has been achieved for 3 days. The fertilizability of eggs inseminated by frozen-thawed sperm was more than two-thirds. Nearly all the eggs thus fertilized developed to normal gastrulae.     

Purification and characterization of calmodulin from sea urchin spermatozoa

Calmodulin was purified to apparent homogeneity from sea urchin spermatozoa by heat-treatment at 85 degrees C, ammonium sulphate precipitation at pH 4.2, DEAE-Sephacel chromatography and gel filtration on Sephadex G-100. Approximately 8.3 micrograms calmodulin were recovered per 10(10) sperm cells. The sperm calmodulin had an apparent molecular weight of 17 800. The purified calmodulin activated calmodulin-deficient phosphodiesterase from pig coronary arteries, with half-maximal activation occurring at approximately 40 ng calmodulin/ml. Trifluoperazine also inhibited the sperm calmodulin activity. These results demonstrate that calmodulin is present in high amounts in sea urchin spermatozoa, and that it is essentially the same as the calmodulin isolated from various other tissues.     

Cross fertilization between sea urchin eggs and oyster spermatozoa

Interphylum crossing was examined between sea urchin eggs (Temnopleurus hardwicki) and oyster sperm (Crassostrea gigas). The eggs could receive the spermatozoa with or without cortical change. The fertilized eggs that elevated the fertilization envelope began their embryogenesis. Electron microscopy revealed that oyster spermatozoa underwent acrosome reaction on the sea urchin vitelline coat, and their acrosomal membrane fused with the egg plasma membrane after the appearance of an intricate membranous structure in the boundary between the acrosomal process and the egg cytoplasm. Oyster spermatozoa penetrated sometimes into sea urchin eggs without stimulating cortical granule discharge and consequently without fertilization envelope formation. The organelles derived from oyster spermatozoa seemed to be functionally inactive in the eggs whose cortex remained unchanged.     

Localization and characterization of phosphatidylcholine in sea urchin spermatozoa.

Sea urchin spermatozoa obtain energy for movement through oxidation of endogenous phospholipids, particularly phosphatidylcholine (PC). This study was undertaken to determine the localization of PC available for utilization in energy metabolism in spermatozoa of the sea urchin, Hemicentrotus pulcherrimus. Following incubation with sea water, the PC content in sperm heads decreased significantly, while that in sperm tails did not change. PC was abundant in sperm heads, particularly the midpieces. PC composed of unsaturated fatty acids was consumed to a greater extent during incubation than that consisting of saturated fatty acids. Analysis by gas-liquid chromatography indicated most of fatty acid moieties in the midpieces PC to be unsaturated. Phospholipase A2 activity was also distributed in sperm heads, particularly the midpieces. It thus appears that PC as a substrate for energy metabolism is located in the midpieces of sea urchin spermatozoa.     

Reactions of the sea urchin oocyte to foreign spermatozoa

Spermatozoa of the mussel Cyprina islandica and the nemertine Malacobdella grossa have been adddd to oocytes and mature eggs of the sea urchin Psammechinus miliaris. No spermatozoa were found to attach to the surface of the mature eggs which also remained unactivated throughout the experiments. Spermatozoa of both species were found to reach the oocyte surface and to become attached there. The interaction between egg and sperm was different in the two species and different from the situation of a sea urchin sperm on the sea urchin oocyte. The nemertine sperm was found to penetrate the cortex of the oocyte in a fashion resembling phagocytosis. The mussel sperm was partly surrounded by thin protrusions from the sea urchin oocyte which extended along a major portion of the sperm head.