RESPIRATION OF OOCYTES, UNFERTILIZED EGGS AND FERTILIZED EGGS FROM PSAMMECHINUS AND ASTERIAS

The lecture given by Dr Yasumasu should be considered from two points of view, namely its significance in the field of developmental biology and his own personal history as a developmental biologist.In 1908 Otto Warburg published a paper entitled ‘Beobachtungen üeber die Oxydationsprozesse im Seeigelei’ (Warburg, 1908). This is one of his earliest works, where he measured respiration of eggs with Winkler's method not with his manometer. This was the first paper describing the fact that the respiration in unfertilised eggs was considerably lower than that in fertilised ones. Many researchers confirmed his experiments and extended them. Borei (1948) measured oxygen consumption of oocytes and unfertilised and fertilised eggs and compared his results with those of other researchers. He observed that the respiration of eggs declined after they were removed from the ovary and placed into seawater and that it increased at fertilisation. He observed an exponential increase in respiration of fertilised eggs or embryos from the cleavage stage to the hatching blastula. He also observed an initial burst of respiration but failed to record it exactly. Ohnishi & Sugiyama (1963) measured the initial burst of respiration quantitatively with the oxygen electrode method. Thus, respiration of sea urchin eggs and early embryos was divided into three phases: respiration of unfertilised eggs, the initial burst of respiration at fertilisation and the respiration of fertilised eggs. The respiration of fertilised eggs increased exponentially with progression of development until hatching.

In eggs of the echiuroid Urechis unicinctus the respiration rate, which is not altered by fertilization, is inhibited by rotenone, antimycin A and cyanide. The respiration in echiuroid eggs is probably mediated by the mitochondrial respiratory chain. In fertilized eggs, the respiration was inhibited by oligomycin and stimulated by the uncouplers of oxidative phosphorylation 2,4-dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone, whereas respiration in unfertilized eggs was insensitive to these compounds. Insemination increased the respiratory rate in eggs in the presence of uncouplers and reduced it in the presence of oligomycin. These findings suggest that the capacity of electron transport in mitochondira is elevated by fertilization but becomes latent on fertilization-induced coupling of respiration with oxidative phosphorylation. Strong stimulation of the respiration in unfertilized eggs was induced by dichlorophenol indophenol, phenazine methosulfate and tetramethyl p-phenylenediamine, suggesting possible sites at which electron transport is regulated in unfertilized eggs. The resulting stimulation of respiration in unfertilized eggs was insensitive to uncouplers and oligomycin, but became sensitive to them after fertilization simultaneously with considerable decrease in its rate. Fertilization-induced coupling of the respiration seemed to reduce the respiratory rate enhanced artificially by these redox compounds.

Cytoplasmic tubulin purified from unfertilized sea urchin eggs self-assembles in the absence of microtubule-associated proteins (MAPs) [Suprenant and Rebhun, 1983; Detrich and Wilson, 1983] with a critical concentration for polymerization of 0.8 mg/ml at 15-18 degrees C, a value well below the 3 mg/ml tubulin present in these eggs [Pfeffer et al, 1976]. Studies of the calcium sensitivity of unfertilized S. purpuratus (sea urchin) egg tubulin were initiated to help understand how this tubulin is maintained unassembled in the unfertilized egg. Egg microtubules, assembled at physiological temperatures (15-18 degrees C) were depolymerized by a 100-fold lower free calcium concentration than egg microtubules assembled at the higher temperatures (25-37 degrees C) generally used to assemble mammalian brain microtubules. The initial rate of egg microtubule assembly was much more sensitive to calcium than was microtubule depolymerization at steady state at 37 degrees C. However, both processes were sensitive to near physiological free calcium concentrations at 18 degrees C. The co-assembly of bovine brain MAPs and sea urchin egg tubulin produced microtubules that required a 1,000-fold higher concentration of free calcium for depolymerization than microtubules assembled at 18 degrees C from egg tubulin alone. While calcium regulatory MAPs have not yet been found in sea urchin eggs, the fact that brain MAPs interact with egg tubulin and regulate both its critical concentration for polymerization [Suprenant and Rebhun, 1983] and its calcium sensitivity, suggests that such regulatory molecules exist. These results suggest that sea urchin egg tubulin assembly in vivo could be controlled by variations in intracellular calcium levels acting in concert with urchin egg proteins similar in function to brain MAPs.

Rabbit antisera against sea urchin unfertilized eggs (gelatinous coat removed), fertilized eggs and the gelatinous coat (fertilizin) of the unfertilized eggs have been found to inhibit the nuclear and cytoplasmic divisions of the fertilized egg. Antisera against whole sperm or sperm antifertilizin extracts were not effective in this regard, and absorption of the effective sera with sperm did not remove the blocking antibody of those sera. Extracts (calcium-free sea water) of whole fertilized eggs containing the ectoplasmic layer material were found to react with anti-egg sera after absorption with sperm, but not after absorption with eggs. This material in this extract has been tentatively described in this thesis as a carbohydrate. One such extract was found to agglutinate the sperm of the species, but not sand dollar sperm. Subsequent extracts have not shown this property. The respiration of fertilized eggs, blocked by the effective sera, was found to increase over the controls. The sodium content of fertilized eggs treated with immune blocking serum was not found to differ from the sodium content of eggs in normal serum. The tension at the surface of unfertilized eggs increases in the presence of anti-unfertilized egg serum, as judged by centrifugation experiments.

Unfertilized eggs of animals contain maternal messenger RNAs (mRNAs) and proteins, which are required for the maintenance of metabolism and regulation of development during the initial stages of embryogenesis. Unfertilized eggs are transcriptionally and translationally quiescent. After fertilization, activated translation of maternal mRNAs is one of the major forces that direct the early stages of embryogenesis before activation of the zygotic genome. However, a low rate and level of protein synthesis have been detected in unfertilized sea urchin eggs indicating that translation is not completely inhibited. Analysis of translatomes of unfertilized eggs and early embryos detected three sets of maternal mRNAs translated either before or after fertilization, or both before and after fertilization. Proteins encoded by maternal mRNAs, which are translated in unfertilized eggs, perform many different functions required for homeostasis, fertilization, egg activation, and early development. This suggests that translation in unfertilized sea urchin eggs may be required to renew the pool of proteins involved in these processes. Thus, translation may be necessary to maintain the fertility and developmental potential of sea urchin eggs during the long-term storage of eggs in ovaries until spawning begins.

It is well known that sea urchin eggs, which exhibit quite a low rate of respiration before fertilisation, undergo a sudden increase in the rate of respiration followed by its gradual decrease in about a 15 min period after fertilisation (Ohnishi & Sugiyama, 1963; Epel, 1969), in which the respiration is mediated mainly by Ca2+-activated non-mitochondrial respiratory systems (Foerder et al., 1978; Perry & Epel, 1985a,b). During this short period the rate of mitochondrial respiration gradually increases (Yasumasu et al., 1988) and stabilises at a higher rate than before fertilisation (Warburg, 1908, 1910; Whitaker, 1933; Yasumasu & Nakano, 1963), when the respiration due to non-mitochondrial respiratory systems is turned off. The rate of mitochondrial respiration, once enhanced upon fertilisation, increases further in the period between hatching and the gastrula stage, without any changes in the number of mitochondria or the capacity of electron transport in the mitochondrial respiratory chain (Fujiwara & Yasumasu, 1997; Fujiwara et al., 2000). It is likely that the respiratory rate is reduced by regulation of electron transport in the mitochondrial respiratory chain and increases due to the release of electron transport from the regulation upon fertilisation and after hatching.A marked increase in the respiratory rate after hatching is accompanied by an evident decrease in the ATP level without any change in the levels of ADP and AMP (Mita & Yasumasu, 1984). In isolated mitochondria, the rate of respiration, estimated in the presence of ADP at the same concentration as in embryos, is reduced by a high concentration of ATP as found in embryos before hatching but is not affected at a concentration as low as in gastrulae (Fujiwara & Yasumasu, 1997; Fujiwara et al., 2000) ATP at a high concentration probably blocks ATP release from mitochondria and consequently inhibits ADP uptake coupled to ATP release in the ATP/ADP translocation reaction in the mitochondrial membrane, causing a shortage of intra-mitochondrial ADP.

Translational initiation factors from sea urchin eggs and embryos: Functional properties are highly conserved

The cortical actin cytoskeleton undergoes dramatic rearrangements during fertilization of sea urchin eggs. To characterize these changes further, we quantified the relative changes in filamentous actin (F-actin) during fertilization and the first cell cycle in both intact eggs and in isolated cortices by quantitative fluorescence microscopy. The level of F-actin in the intact egg decreased after fertilization and continued to decrease throughout the first cell cycle. By 60 min after fertilization, the level of F-actin had decreased to 50% of the unfertilized sea urchin egg. By cytokinesis, the level of F-actin had decreased to 30% of the unfertilized egg. After completion of cell division, individual blastomeres had 10% of the F-actin in the unfertilized egg. In contrast, there was an increase in cortical F-actin to 370% of the level in the unfertilized egg after fertilization. This increase corresponded to the formation of microvilli. There was little change in the level of cortical F-actin during the first cell cycle. We draw parallels to other systems that increase the amount of F-actin in the Triton-insoluble cytoskeleton by recruiting actin from a Triton-soluble pool of F-actin. © 1996 Wiley-Liss, Inc.

The effect of D2O on the crystallization of polymerizable tubulin in sea urchin egg cytoplasm was investigated by estimating the yield of "vinblastine (VB)-crystals" by directly measuring the dimensions of the crystals produced and by protein assays of the crystal isolates. The yield of VB-crystals in mature unfertilized eggs was fairly constant; it neither increased nor decreased in the presence of D2O. On fertilization, the yield of crystals decreased markedly as compared with yields from unfertilized eggs; but, the yield was restored to the value for unfertilized eggs when an adequate concentration of D2O was present during incubation. These results are evidence that tubulin molecules in unfertilized sea urchin eggs are in the polymerizable state but become masked and partly unpolymerizable after fertilization and the D2O releases the masked state and converts unpolymerizable tubulin molecules into active and polymerizable state.

The in Vivo Rate of Glucose-6-Phosphate Dehydrogenase Activity in Sea Urchin Eggs Determined with a Photolabile Caged Substrate

The cortical actin cytoskeleton undergoes dramatic rearrangements during fertilization of sea urchin eggs. To characterize these changes further, we quantified the relative changes in filamentous actin (F-actin) during fertilization and the first cell cycle in both intact eggs and in isolated cortices by quantitative fluorescence microscopy. The level of F-actin in the intact egg decreased after fertilization and continued to decrease throughout the first cell cycle. By 60 min after fertilization, the level of F-actin had decreased to 50% of the unfertilized sea urchin egg. By cytokinesis, the level of F-actin had decreased to 30% of the unfertilized egg. After completion of cell division, individual blastomeres had 10% of the F-actin in the unfertilized egg. In contrast, there was an increase in cortical F-actin to 370% of the level in the unfertilized egg after fertilization. This increase corresponded to the formation of microvilli. There was little change in the level of cortical F-actin during the first cell cycle. We draw parallels to other systems that increase the amount of F-actin in the Triton-insoluble cytoskeleton by recruiting actin from a Triton-soluble pool of F-actin.

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

An investigation has been made into the effect of light and carbon monoxide on the respiration of sea-urchin eggs (Psammechinus miliaris), with special reference to unfertilized eggs. The O2 uptake of unfertilized eggs in air is 38% higher in the dark than in light. This inhibitory action of light persists when gas mixtures containing CO and O2 are substituted for air. Fertilized and unfertilized eggs differ in that the former’s respiration is only about 10% inhibited by light. In 80% CO in O2 in the dark, the O2 uptake of unfertilized eggs is 55% higher than in 80% N2 in O2 in the dark. In 95% CO in O2 in the dark, the O2 uptake of unfertilized eggs is only 7% higher than in 95 % N2 in O2 in the dark, showing that at this partial pressure, CO exerts an inhibitory action on respiration. There is no difference between O2 uptake in 80% CO in O2 in light and dark. O2 uptake in 95 % CO in O2 in light is 45 % higher than in this gas mixture in the dark, showing that at this partial pressure of CO, photo-reversal of respiratory inhibition occurs. Results similar to those in 80% CO in O2 were obtained with gas mixtures containing lower partial pressures of CO. Cytochromes a and were identified spectroscopically in unfertilized eggs. It is concluded that respiration is mediated through the cytochrome system before and after fertilization. In theories which postulate that cytochrome is ‘thrown into circulation’ at fertilization, the simultaneous light-inhibition of respiration, stimulation of O2 uptake by CO, and photo-reversible inhibition of O2 uptake by CO have not been taken into consideration.

The principal theme of these investigations concerns the inhibited state of mature unfertilized sea urchin eggs with respect to uridine uptake and protein synthesis. Part I demonstrates that unfertilized-eggs are relatively impermeable to uridine. Fertilized eggs, however, develop during the first hour an energy-dependent, uptake mechanism for uridine accumulation. Labeled uridine assimilated by fertilized eggs is recovered as phosphorylated nucleosides, primarily triphosphates. Experiments support the idea that uridine penetration into sea urchin eggs depends upon the phosphorylation of the 5' carbon atom at the cell surface. Tests with puromycin show that protein synthesis is unnecessary for the generation of uridine uptake after fertilization. The evidence favors the view that uridine kinase is sequestered within the unfertilized egg and thus incapable of activity at the cell surface until after fertilization. Parts II and III use biochemical and autoradiographic methods to show that growing oocytes of sea urchins, in contrast to many other organisms, undergo considerable RNA and protein synthesis. Protein synthesis in isolated oocytes occurs throughout the germinal vesicle and cytoplasm and takes place on polyribosomes. RNA synthesis is localized in the nucleolus. Mature eggs, however, synthesize only little protein even in mixed suspensions with oocytes. Long-term maintenance of spawned female sea urchins, after but one injection of labeled uridine, produces ripe unfertilized eggs possessing highly radioactive RNA. The distribution of label in the extracted RNAs is 70-80% ribosomal, 10-20% heterogeneous, and 5-10% soluble. Part IV is an electron microscopic and biochemical examination of RNA-labeled mature unfertilized and fertilized eggs. The findings are correlated with the difference in protein synthesizing activity before and after fertilization. The results show that unfertilized eggs synthesize protein upon RNase-sensitive polyribosomes. The large increase in protein synthesis after fertilization occurs in association with the assembly of additional polyribosomes. Homogenates of unfertilized eggs also possess synthetically inactive, RNase-resistant, ribosomal aggregates. Evidence suggests that trypsin followed by RNase disperses the aggregates. Homogenates of fertilized eggs, however, contain very few RNase-resistant ribosomal aggregates. By forty minutes after fertilization, about 70% of the new protein synthesis can be attributed to the new polyribosomes. The weight of the evidence indicates that the remaining 30% of the stimulation of protein synthesis is due to the activation of masked polyribosomes. Appendix 1 shows that, for unfertilized and fertilized eggs, competition for uptake of amino acids occurs primarily among those belonging to the same charge group. Appendix 2 demonstrates that one amino acid can displace another of the same category from intact eggs both before and after fertilization. By combination of these facts, then, it is possible to achieve greater labeling of egg-proteins than has been previously realized.

A maternal store of histones in unfertilized sea urchin eggs is demonstrated by two independent criteria. Stored histones are identified by their ability to assemble into chromatin of male pronuclei of fertilized sea urchin eggs in the absence of protein synthesis, suggesting a minimum of at least 25 haploid equivalents for each histone present and functional in the unfertilized egg. In addition, electrophoretic analysis of proteins from acid extracts of unfertilized whole eggs and enucleated merogons reveals protein spots comigrating with cleavage stage histone standards, though not with other histone variants found in later sea urchin development or in sperm. Quantification of the amount of protein per histone spot yields an estimate of several hundred haploid DNA equivalents per egg of stored histone. The identity of some of the putative histones was verified by a highly sensitive immunological technique, involving electrophoretic transfer of proteins from the two-dimensional polyacrylamide gels to nitrocellulose filters. Proteins in amounts less than 2 x 10(-4) micrograms can be detected by this method.