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.     

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.     

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.     

Phosphatidylcholine metabolism for energy production in sea urchin spermatozoa

Sea urchin spermatozoa use endogenous phosphatidylcholine (PC) to produce energy for swimming. The catabolism of PC was studied in Hemicentrotus pulcherrimus spermatozoa. Following incubation in sea water, the content of PC decreased and that of choline increased gradually, whereas phosphocholine maintained a constant level. Measurement of the radioactivity in metabolites converted from 1-palmitoyl-2-[1-14C]linoleoyl-PC, [choline-methyl-14C]dipalmitoyl-PC and 1-[1-14C]palmitoyl-lysophosphatidylcholine (LysoPC) showed that the major degradative pathway is PC----LysoPC----glycerophosphocholine----choline. 1-Palmitoyl-2-[1-14C]linoleoyl-PC and [1-14C]oleic acid were oxidized to 14CO2 in a cell-free system of spermatozoa. Sea urchin spermatozoa thus appear to quite likely obtain energy through the oxidation of fatty acid(s) from PC.     

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.     

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.     

Developmental studies of sea urchin chromatin. Chromatin isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus

Chromatin was isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus. The isolated chromatin shows less absorptivity ratio of 230 nm : 260 nm and possesses less protein than does embryonic chromatin. The ratio of histone : DNA is 1.02; nonhistone : DNA 0.13; RNA : DNA 0.04. Sperm chromatin melts in two steps with T_ms 70°C and 84°C in 2.5 × 10⁻⁴, M EDTA in contrast to embryonic chromatin with a single T_m = 72°C. Disc electrophoresis of basic proteins of sperm revealed one minor component with extremely fast mobility and three major components. The one with the slowest mobility is characteristic of sperm. The embryo has in turn its characteristic histone which also migrated slowly in disc electrophoresis. Both of these unique histone fractions are selectively extracted from chromatin by 5% perchloric acid. Amino acid analyses of these chromatographically purified unique fractions show that both contain a large amount of lysine, while that from sperm, in addition, contains also a large amount of arginine. Minimal molecular weights of 33,000 for sperm and 16,200 for embryo unique histone were estimated from these analyses. Sperm chromatin supports a level of RNA synthesis in vitro with exogeneously supplied RNA polymerase about 2% that of the corresponding free DNA.     

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.     

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.     

2-Chloro adenosine triphosphate as substrate for sea urchin axonemal movement

The 2-substituted ATP analog 2-Chloro ATP was tested for its capacity to support axonemal movement. The movement of sea urchin axonemes reactivated with 2-Cl ATP appeared very similar to that with ATP. Detailed waveform analysis indicated that bend angle and shear amplitude were not significantly different for ATP and 2-Cl ATP. Although wavelength differs at particular nucleotide concentrations, if normalized to the beat frequency, it is similar for ATP and 2-Cl ATP. The main difference in the movement with the two analogs was seen in beat frequency and sliding velocity. The Vmax for beat frequency and mean sliding velocity was lower for 2-Cl ATP. The apparent Km for beat frequency and sliding velocity was much lower for 2-Cl ATP. The ratio of these two effects, that is, (Vmax/Km) is higher for 2-Cl ATP. Thus 2-Cl ATP is a good substrate for axonemal movement. The significantly lower Km of 2-Cl ATP was also demonstrated by its ability to support oscillatory motion at concentrations below that for ATP. The observations identify the structures and conformation of substrate necessary to support axonemal movement.     

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.     

Phosphorylation of membrane-bound guanylate cyclase of sea urchin spermatozoa

When Arbacia punctulata spermatozoa are incubated in seawater containing ammonium hydroxide (pH 8.8), the sperm plasma membrane-bound guanylate cyclase is dephosphorylated, its electrophoretic mobility increases (from an apparent molecular mass of 160 to 150 kD), and its enzymatic activity decreases 3.5-fold. Transfer of these cells into ammonium-free seawater (pH 7.4) results in the rephosphorylation of the cyclase, its reconversion to 160 kD, and recovery of the enzymatic activity lost upon dephosphorylation. This is the first direct demonstration that the activity of membrane-bound guanylate cyclase can be regulated by phosphorylation. A plasma membrane preparation is described that specifically supports the in vitro phosphorylation of the guanylate cyclase. This preparation will be useful in more detailed studies on the relationship between phosphorylation state and enzymatic activity of membrane-bound guanylate cyclase.