A trypsin-like proteinase localized in cortical granules isolated from unfertilized sea urchin eggs by zonal centrifugation. Role of the enzyme in fertilization

A trypsin-like proteinase was localized within a single subcellular compartment of unfertilized Strongylocentrotus purpuratus eggs, the cortical granules. Homogenates of eggs were fractionated by rate-zonal centrifugation. Enzymatic markers were used to determine the distribution of mitochondria (cytochrome oxidase), yolk platelets (acid nitrophenyl phosphatase), and cortical granules (β-1, 3-glucanase) in the sucrose density gradient. A bimodal distribution pattern was obtained for aryl esterase activity (substrate: β-naphthyl acetate), with one peak in the microsomal and the other in the cortical granule fractions. The cortical granule enzyme was characterized as a trypsin-like proteinase, since it also hydrolyzed another typical tryptic substrate α-N-benzoyl-l-arginine ethyl ester and was completely inactivated by soybean trypsin inhibitor (SBTI). The aryl esterase activity in the microsomal fractions was not inhibited by SBTI, while 50% of the total aryl esterase activity in the original egg homogenate was inactivated by SBTI. The identity of the enzyme(s) responsible for the aryl esterase activity associated with the microsomal particles is unknown at present.The cortical granule proteinase functions in the elevation of the fertilization membrane and establishment of the block to polyspermy at fertilization. Arbacia punctulata eggs inseminated in the presence of trypsin inhibitors, SBTI or tosyl lysine chloromethyl ketone (TLCK), failed to elevate normal fertilization membranes and became heavily polyspermic.On the basis of these results and observations made by other investigators with a wide variety of biological systems, it is proposed that trypsin-like proteinases function in the discharge of secretory granules from all types of cells.     

Respiration capacity of mitochondria isolated from unfertilized and fertilized sea urchin eggs

Mitochondria were isolated from unfertilized and fertilized eggs of the sea urchin, Strongylocentrotus purpuratus. Both preparations exhibited coupled adenosine 5'-diphosphate (ADP)-dependent) oxidation of flavin and pyridine-linked substrates and both yielded the expected P:O ratios with these substrates. Highest respiratory control indices (greater than 4.0) were observed when succinate or pyruvate + malate were used as substrates. Mitochondria from unfertilized and fertilized eggs exhibited sensitivity to respiratory and phosphorylation inhibitors and uncouplers and both preparations exhibited cross-over points at sites I, II and III of the respiratory chain. Low-temperature difference spectra revealed a normal complement of cytochromes c, b and aa3, although cytochrome c from unfertilized eggs appears to be more subject to extraction during the course of mitochondrial isolation than does cytochrome c from fertilized eggs. An unidentified pigment absorbing at approx. 570 nm was visible in low-temperature spectra of unfertilized eggs and unfertilized egg mitochondria.     

RNA synthesis in unfertilized sea urchin eggs

We have examined the synthesis of messenger-like RNA in unfertilized sea urchin eggs. Most of the RNA synthesized is restricted to the nucleus and sediments from 16 to 30S. A small fraction can be isolated from the postmitochondrial supernatant and displays a sedimentation profile typical of embryonic mRNA with peaks at 9 and 18S. This cytoplasmic RNA is largely present as free RNPs and we estimate that less than 20% of the RNA is in polysomes. The RNA made in the egg is unstable and reaches a steady state with a half-time of about 30 min. We have examined the accumulation of RNA in the egg and have calculated a rate of synthesis of 1.4 × 10^?14 g of RNA/min/egg which is similar, on a per-nucleus basis, to that found in the just-fertilized egg and very early embryo. It is approximately 10 times greater than the rate of RNA synthesis in the blastula nucleus. We estimate that the RNA synthesized by the unfertilized egg amounts to a maximum of 3 × 10^?13 g of potential mRNA at the time of fertilization, or 10–15% of its immediate needs. This RNA cannot account for the increase in protein synthesis that occurs after fertilization, which must be the result of the translation of another population of more stable egg or oogenic mRNA that is kinetically distinct from the RNA we have measured. The steady-state level of labeled RNA present in the egg does not change upon fertilization until after the first cleavage, at about 2.5 hr after fertilization. Thus the RNA synthesis that occurs in the just-fertilized zygote appears to be merely a continuation (at least quantitatively) of the RNA synthesis taking place in the egg.     

Nuclear histones of unfertilized sea urchin eggs

Histones have been isolated from the nuclei of unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. The electrophoresis of these histones exhibits a pattern different from that of the sperm or embryo of the same species.     

Activation of maternal centrosomes in unfertilized sea urchin eggs.

Centrosomes are undetectable in unfertilized sea urchin eggs, and normally the sperm introduces the cell's microtubule-organizing center (MTOC) at fertilization. However, artificial activation or parthenogenesis triggers microtubule assembly in the unfertilized egg, and this study explores the reappearance and behavior of the maternal centrosome. During activation with A23187 or ammonia, microtubules appear first at the cortex; centrosomal antigen is detected diffusely throughout the entire cytoplasm. Later, the centrosome becomes more distinct and organizes a radial microtubule shell, and eventually a compact centrosome at the egg center organizes a monaster. In these activated eggs, centrosomes undergo cycles of compaction and decompaction in synchrony with the chromatin, which also undergoes cycles of condensation and decondensation. Parthenogenetic activation with heavy water (50% D2O) or the microtubule-stabilizing drug taxol (10 microM) induces numerous centrosomal foci in the unfertilized sea urchin egg. Within 15 min after incubation in D2O, numerous fine centrosomal foci are detected, and they organize a connected network of numerous asters which fill the entire egg. Taxol induces over 100 centrosomal foci by 15 min after treatment, which organize a corresponding number of asters. The centrosomal material in either D2O- or taxol-treated eggs aggregates with time to form fewer but denser foci, resulting in fewer and larger asters. Fertilization of eggs pretreated with either D2O or taxol shows that the paternal centrosome is dominant over the maternal centrosome. The centrosomal material gradually becomes associated with the enlarged sperm aster. These experiments demonstrate that maternal centrosomal material is present in the unfertilized egg, likely as dispersed undetectable material, which can be activated without paternal contributions. At fertilization, paternal centrosomes become dominant over the maternal centrosomal material.     

Messenger ribonucleoprotein particles in unfertilized sea urchin eggs

The properties of poly(A)-containing messenger ribonucleoprotein particles (mRNPs) from unfertilized sea urchin eggs isolated under various ionic conditions were studied. Poly(A)-containing RNPs of eggs sediment with a modal value of 60–65 S under all conditions used. However, buoyant densities vary strikingly with conditions of particle preparation. Deproteinized poly(A)-containing mRNA has an average molecular weight of about 1 × 10⁶. RNPs prepared in 0.35 M Na⁺ in the absence of Mg²⁺ contain an average of 0.25 × 10⁶ daltons of protein, while particles prepared in 0.05 M Na⁺ in the absence of Mg²⁺ contain 0.35 to 11 × 10⁶ daltons of protein per RNA molecule. Particles prepared in 0.35 M Na⁺ plus 5 mM Mg²⁺ contain 1.4 × 10⁶ daltons of protein suggesting that Mg²⁺ may be necessary for maintenance of RNP intergrity if high Na⁺ concentrations are used to prevent nonspecific RNA-protein interactions. Particles prepared in 0.35 M K⁺ contain 0.9 × 10⁶ daltons of protein in both Mg²⁺ and EDTA. Mg²⁺ does not cause significant aggregation of particles, since the size of RNA extracted from RNPs is proportional to RNP sedimentation rate. Monovalent cation concentrations normally used in analysis of RNPs by sedimentation cause deproteinized poly(A)-containing RNA to sediment with abnormally high sedimentation coefficients, indicating that high sedimentation rates alone do not indicate that RNA is contained in an RNP.     

Comparison of Ca uptake characteristics of microsomal fractions isolated from unfertilized and fertilized sea urchin eggs

The microsomal fraction isolated from sea urchin H. pulcherrimus eggs has the ability to actively accumulate Ca²⁺ in the presence of ATP. The Ca²⁺ uptake was sustained by addition of oxalate and was apparently insensitive to sodium azide. The sequestered microsomal Ca was readily released by the divalent cation ionophore A23187. The microsomal fraction obtained from fertilized eggs accumulated Ca²⁺ about five times more quickly than did that from unfertilized eggs. The increased Ca²⁺ uptake by microsomal fraction obtained from fertilized eggs was due to an increase in the maximum velocity of Ca²⁺ uptake and there was no difference in K_m for calcium between the two fractions.     

Quantitation of the dynein pool in unfertilized sea urchin eggs

A dynein-like ATPase activity has been isolated previously from soluble extracts of unfertilized sea urchin eggs. However, the use of non-quantitative isolation techniques, in particular affinity for microtubules or Ca2+/calmodulin, has precluded accurate estimates of dynein pool size. We have taken the unique approach of using dynein-like ATPase activity to quantitate the egg dynein pool. This approach is based on the isolation by anion-exchange chromatography on DEAE-Sephacel of a peak of dynein-like ATPase activity comprising 65% of soluble ATPase activity in the cytosolic extract. Identification of cytoplasmic dynein was based on dose-dependent inhibition by erythro-9-[3-(2-hydroxynonyl)]adenine and orthovanadate, low GTPase activity and a sedimentation coefficient of 12 S. Two high molecular weight polypeptides corresponding to the A- and D-bands of axonemal dynein were shown to copurify with dynein-like ATPase activity and to undergo specific photocrosslinking with [alpha-32P]ATP, suggesting that they were egg dynein catalytic polypeptides. The specific ATPase activity of these putative catalytic polypeptides was determined to be 1.2 mumol.min-1.mg-1. The specific dynein-like ATPase activity of the crude soluble extract of unfertilized sea urchin eggs was determined to be 0.004 mumol.min-1.mg-1. The concentration of putative dynein catalytic polypeptides was therefore determined from the ratio of the specific activities of crude to pure cytoplasmic dynein catalytic polypeptide to be 0.33% of soluble protein, or 99 pg per egg. This is approximately 3-fold greater than the mass of dynein catalytic polypeptides estimated to be present in cilia at the blastula stage of sea urchin embryonic development. The large amount of cytoplasmic dynein in unfertilized eggs suggests that it could act as a precursor of embryonic ciliary dynein. Three minor peaks of ATPase activity were also resolved from cytosolic extracts and shown to be dynein-like. However, their GTPase activities were 2-4-fold higher than that of cytoplasmic dynein, raising the possibility that egg cytoplasm may contain several isoforms of dynein.     

Characterization of water in unfertilized and fertilized sea urchin eggs

The water in unfertilized and fertilized sea urchin eggs was characterized with a proton nuclear magnetic resonance (NMR) titration method assuming fast proton diffusion (FPD) between water compartments. This method involves stepwise dehydration with sequential T1 relaxation time and water content determinations. The results analyzed by the FPD model give evidence of intracellular water compartments with three different correlation times: 6 X 10(-12) sec (bulk water), 1 X 10(-10) sec (structured water) and about 2 X 10(-9) sec (bound water). Fertilization is accompanied by a substantial increase in bulk water (from 111 to 414 g H2O per 100 g dry mass) and by a decrease in the water of hydration (from 128 g to 56 g per 100 g dry mass). This study shows that 54% of the water in the unfertilized sea urchin egg has motional properties different from bulk water and that this percentage decreases dramatically shortly after fertilization. Most of the change in T1 relaxation rate observed at fertilization can be accounted for by uptake of bulk water associated with elevation of the fertilization membrane.     

The "lecithotrophic" sea urchin Heliocidaris erythrogramma lacks typical yolk platelets and yolk glycoproteins

The sea urchin Heliocidaris tuberculata undergoes typical development, forming an echinoid pluteus larva, whereas H. erythrogramma undergoes direct development via a highly modified, nonfeeding larva. Using a polyclonal antibody prepared against yolk glycoproteins from the typical developer Stronglyocentrotus purpuratus, we found that H. tuberculata contains cross-reactive proteins in abundance, but H. erythrogramma does not. In addition, we used immunoelectron microscopy to demonstrate that unfertilized eggs of H. tuberculata contain yolk platelets, but those of H. erythrogramma do not.     

Thymidine kinase activation in unfertilized sea urchin eggs by homogenization and fertilization

Kinetics of in vivo phosphorylation of ³H-thymidine taken up by sea urchin eggs was compared between unfertilized and fertilized eggs. The percentage of phosphorylated ³H-thymidine in the total acid-soluble radioactivity in the cell increased with increasing incubation time within the first several minutes of incubation in the unfertilized eggs, while nearly 100% of phosphorylation of thymidine was observed without regards to the incubation time and in spite of a tremendous increase in the net uptake of thymidine in the fertilized eggs, suggesting possible activation of thymidine kinase occurring soon after fertilization.In contrast to the in vivo finding, the thymidine kinase activity in unfertilized egg homogenates was found in general to be almost as large as that in fertilized egg homogenates. However, when the enzyme activity was assayed within a short period (30 min) after homogenization of unfertilized eggs, the activity was found to increase more or less with time after homogenization, reaching a level equal to that in fertilized egg homogenates. This enzyme activation after homogenization was especially marked in case of Pseudocentrotus eggs and sometimes amounted to a several fold increase.Preliminary investigations revealed possible involvement of some redox reaction(s) in the thymidine kinase activation during and/or after homogenization of unfertilized sea urchin eggs.