DNA synthesis in unfertilized sea urchin eggs can be turned on and turned off by the addition and removal of procaine hydrochloride.

Unfertilized sea urchin eggs turn on thymidine transport, DNA synthesis and the chromosome cycle in response to procaine hydrochloride. The rates to which these processes activate depend on the extracellular concentration of procaine. Removing procaine turns off DNA synthesis and the chromosome cycle, re-adding procaine turns these processes on once more. Thymidine transport does not turn off after procaine removal. It remains on at the rate it had reached at the time of procaine removal. During a 12-hr period, unfertilized eggs in procaine complete four S-phases, while normal, fertilized embryos complete eight. Tritiated thymidine incorporation into DNA is very low in procaine-treated eggs. This is because in procaine the thymidine transport system is relatively inactive compared to that of fertilized eggs. The data suggest eggs may possess homeostatic mechanisms that actively suppress the DNA synthetic pathway and the chromosome cycle. This system for turning on and off DNA synthesis may prove useful in further analyses of the factors controlling DNA synthesis during early development.     

Increased uptake of thymidine in the activation of sea urchin eggs : Specificity of uptake and dependence on internal pH, the cortical reaction, and external sodium

The uptake of thymidine in sea urchin eggs is considered in terms of its specificity, the cortical reaction, and the increase of intracellular pH following fertilization. The rate of uptake increases greater than 50-fold after fertilization. All deoxyribonucleosides and ribonucleosides tested compete with thymidine for transport sites. Free pyrimidine and purine bases, deoxyribonucleotides, and amino acids do not compete, showing that the specificity of this uptake lies at the nucleoside level. Uptake may be turned on in unfertilized eggs by treatment with ammonia, a treatment known to by-pass the cortical reaction and raise intracellular pH. However, when compared with uptake in fertilized eggs, it proceeds later and at a lower rate. Both of these deficiencies are overcome by fertilizing the ammonia-treated eggs or treating them with butyric acid or ionophore A23187. These treatments induce the cortical reaction and stimulate an immediate and complete turn-on of thymidine uptake. Superseding these apparent involvements of the cortical reaction and mtracellular pH in thymidine uptake is an extremely strict requirement for extracellular Na⁺.     

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.     

eIF4E‐Binding proteins are differentially modified after ammonia versus intracellular calcium activation of sea urchin unfertilized eggs

Fertilization of sea urchin eggs triggers a rise of protein synthesis mainly dependent on the cap‐binding protein eIF4E, which is released from its repressor 4E‐BP and associates with eIF4G. Association of eIF4G with eIF4E is a crucial event for the onset of the first mitotic division following fertilization. Artificial activation of unfertilized eggs with the calcium ionophore A23187 results in the activation of protein synthesis comparable to the one triggered by fertilization, while increasing the intracellular pH by ammonia treatment results in partial activation of protein synthesis. Nevertheless, artificial activation does not induce the mitotic division. Here we investigate the effect of calcium ionophore and ammonia treatment of unfertilized eggs on eIF4E and its two antagonist partners, 4E‐BP and eIF4G. We show that the addition of calcium ionophore to unfertilized eggs induces permanent dissociation between eIF4E and 4E‐BP, whereas a reversible dissociation of the complex occurs after ammonia treatment. The regulation of the complex correlates with permanent or reversible 4E‐BP disappearance depending on the treatment used to trigger artificial activation. Furthermore, while calcium ionophore treatment of unfertilized eggs induces eIF4G modifications comparable to those observed following fertilization, ammonia treatment does not. These results suggest that ionophore and ammonia treatments of unfertilized eggs induce differential protein synthesis activation by targeting eIF4E availability and specific regulation through its two partners 4E‐BP and eIF4G. Mol. Reprod. Dev. 77: 83–91, 2010. © 2009 Wiley‐Liss, Inc.     

Activation of the proteasomes of sand dollar eggs at fertilization depends on the intracellular pH rise

The mechanism of the activation of intracellular proteasomes at fertilization was measured in living sand dollar eggs using the membrane-impermeant fluorogenic substrate, succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid. When the substrate was microinjected into unfertilized eggs, the initial velocity of hydrolysis of the substrate (V0) was low. V0 measured 5 to 10 min after fertilization was five to nine times the prefertilization level and remained high throughout the first cell cycle. Hydrolysis of the substrate was inhibited by clasto-lactacystin beta-lactone, a specific inhibitor of the proteasome. There has been in vitro evidence that calcium may be involved in regulation of proteasome activity to either inhibit the increase in peptidase activity associated with PA 28 binding to the 20S proteasome or stimulate activity of the PA 700-proteasome complex. Since both intracellular free Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) increase after fertilization, hydrolysis of the proteasome substrate was measured under conditions in which [Ca2+]i and pHi were varied independently during activation. When the pHi of unfertilized eggs was elevated by exposure to 15 mM ammonium chloride in pH 9 seawater, V0 increased to a level comparable to that measured after fertilization. In contrast, [Ca2+]i elevation without pHi change, induced by calcium ionophore in sodium-free seawater, had no effect on V0 in the unfertilized egg. Moreover, when unfertilized eggs were microinjected with buffers modulating pHi, V0 increased in a pH-dependent manner. These results indicate that the pHi rise at fertilization is the necessary prerequisite for activation of the proteasome, an essential component in the regulation of the cell cycle.     

Initiation of gynogenesis in Oreochromis niloticus following heterologous fertilization

Fertilization of tilapia ( Oreochromis niloticus ) eggs with heterologous sperm from carp ( Cyprinus carpio ), and from some other eyprinids resulted in higher levels of haploid gynogenetic development than in unfertilized eggs. Normal and irradiated carp sperm achieved similar rates of activation of tilapia eggs, but lower rates than those achieved by irradiated tilapia sperm. Diploidy in gynogenetic eggs fertilized with carp sperm was restored using pressure shocks and resulted in the development of healthy tilapia gynogens.     

Adaptation of cultured human lymphoblasts to growth in citrulline

DNA synthesis is initiated in unfertilized sea urchin eggs (Strongylocentrotus purpuratus and Lytechinus pictus) by exposing them to NH₄OH-sea water (ordinary sea water titrated to pH 9–9.1 with NH₄OH). The eggs are considered to be unfertilized eggs by visual and electro-biological criteria and because they can later be fertilized and then do give visible and electrobiological fertilization reactions. The incorporation of ³H-thymidine proceeds in rounds, the magnitude increasing in successive rounds. It is also reported that the treatment with NH₄OH activates the uptake of thymidine by the eggs, although the internal thymidine builds up more slowly in unfertilized eggs treated with NH₄OH than it does in fertilized eggs. The magnitude of the incorporation of exogenously supplied labelled thymidine into DNA is lower in the NH₄OH-treated unfertilized eggs than in normal fertilized eggs. This difference is not attributed to differences in the amount of DNA synthesized and the explanation is sought in thymidine uptake and nucleotide pathways.     

Intracellular pH shift leads to microtubule assembly and microtubule-mediated motility during sea urchin fertilization: correlations between elevated intracellular pH and microtubule activity and depressed intracellular pH and microtubule disassembly

The regulation of the microtubule-mediated motions within eggs during fertilization was investigated in relation to the shift in intracellular pH (pHi) that occurs during the ionic sequence of egg activation in the sea urchins Lytechinus variegatus and Arbacia punctulata. Microtubule assembly during formation of the sperm aster and mitotic apparatus was detected by anti-tubulin immunofluorescence microscopy, and the microtubule-mediated migrations of the sperm and egg nuclei were studied with time-lapse video differential interference contrast microscopy. Manipulations of intracellular pH were verified by fluorimetric analyses of cytoplasmic fluorescein incorporated as fluorescein diacetate. The ionic sequence of egg activation was manipulated i) to block the pHi shift at fertilization or reduce the pHi of fertilized eggs to unfertilized values, ii) to elevate artificially the pHi of unfertilized eggs to fertilized values, and iii) to elevate artificially or permit the normal pHi shift in fertilized eggs in which the pHi shift at fertilization was previously prevented. Fertilized eggs in which the pHi shift was suppressed did not assemble microtubules or undergo the normal microtubule-mediated motions. In fertilized eggs in which the pHi was reduced to unfertilized levels after the assembly of the sperm aster, no motions were detected. If the intracellular pH was later permitted to rise, normal motile events leading to division and development occurred, delayed by the time during which the pH elevation was blocked. Microtubule-mediated events occurred in eggs in which the intracellular pH was elevated, even in unfertilized eggs in which the pH was artificially increased. These results indicate that the formation and normal functioning of the egg microtubules is initiated, either directly or indirectly, by the shift in intracellular pH that occurs during fertilization.     

Compartmentation of thymidine kinase in unfertilized sea urchin eggs and possible release of thymidine kinase from particulates in activated eggs

The thymidine kinase activity of homogenates of unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, in 1 M NaCl was always lower than that of homogenates of the unfertilized eggs in hypotonic media or homogenates of the fertilized or ammonia-activated eggs in 1 M NaCl by 30–50%. Sonication of the unfertilized egg homogenates in 1 M NaCl resulted in the elevation of thymidine kinase activity up to a level in the fertilized or ammonia-activated egg homogenates which is not affected by sonication. Differential centrifugation of unfertilized egg homogenates in 1 M NaCl revealed that the latent thymidine kinase is associated with the 1500g pellet or even with the 200g pellet. Exposure of the 1500g pellet to sonication, hypotonic media, 0.3% Triton X-100 in 1 M NaCl, and 2 M propyleneglycol resulted in the elevation of thymidine kinase, which was eventually shown to be no longer bound to the pellet fraction. Latent thymidine kinase was not detected in the 1500g pellet prepared from the fertilized egg homogenate in 1 M NaCl. These findings seem to suggest that thymidine kinase in unfertilized eggs may be sequestered, at least partly, in some large intracellular structures but may be released from them upon fertilization or ammonia activation, in accordance with our earlier observation on the apparent activation of thymidine kinase afer fertilization.     

Taxol inhibits the nuclear movements during fertilization and induces asters in unfertilized sea urchin eggs

Taxol blocks the migrations of the sperm and egg nuclei in fertilized eggs and induces asters in unfertilized eggs of the sea urchins Lytechinus variegatus and Arbacia punctulata. Video recordings of eggs inseminated in 10 microM taxol demonstrate that sperm incorporation and sperm tail motility are unaffected, that the sperm aster formed is unusually pronounced, and that the migration of the egg nucleus and pronuclear centration are inhibited. The huge monopolar aster persists for at least 6 h; cleavage attempts and nuclear cycles are observed. Colcemid (10 microM) disassembles both the large taxol-stabilized sperm aster in fertilized eggs and the numerous asters induced in unfertilized eggs. Antitubulin immunofluorescence microscopy demonstrates that in fertilized eggs all microtubules are within the prominent sperm aster. Within 15 min of treatment with 10 microM taxol, unfertilized eggs develop numerous (greater than 25) asters de novo. Transmission electron microscopy of unfertilized eggs reveals the presence of microtubule bundles that do not emanate from centrioles but rather from osmiophilic foci or, at times, the nuclear envelope. Taxol-treated eggs are not activated as judged by the lack of DNA synthesis, nuclear or chromosome cycles, and the cortical reaction. These results indicate that: (a) taxol prevents the normal cycles of microtubule assembly and disassembly observed during development; (b) microtubule disassembly is required for the nuclear movements during fertilization; (c) taxol induces microtubules in unfertilized eggs; and (d) nucleation centers other than centrioles and kinetochores exist within unfertilized eggs; these presumptive microtubule organizing centers appear idle in the presence of the sperm centrioles.     

Relation of cytoplasmic alkalinization to microvillar elongation and microfilament formation in the sea urchin egg

Experiments have been carried out to test the proposal that the pH increase at fertilization in sea urchin eggs promotes microvillar elongation. Results presented herein show that microvillar elongation and microfilament formation occurred when sea urchin eggs were incubated in sodium-free seawater containing the calcium ionophore A23187, a treatment which initiates activation, i.e., induces a transient increase in intracellular free calcium, but prevents subsequent cytoplasmic alkalinization. Within elongated microvilli and cortices of these eggs, microfilaments were arranged in a loose meshwork. However, if the pH of the egg cytoplasm was increased experimentally, microfilament bundles appeared within individual microvilli. These findings suggest that: (1) microvillar elongation and microfilament formation in the sea urchin egg at fertilization may occur when cytoplasmic alkalinization is inhibited, and (2) formation of the microvillus bundle of microfilaments at egg activation is pH sensitive. Additionally, if the cytoplasmic pH of unfertilized eggs was experimentally elevated by NH₄Cl, microvilli failed to elongate. These data indicate that elevation of intracellular pH by this method is not sufficient to induce microvillar elongation.     

Activation of follicle cell surface phospholipase by tyrosine kinase dependent pathway is an essential event in ascidian fertilization

Eggs of Ascidia ceratodes and Phallusia mammillata block polyspermy by releasing a phosphatidylinositol‐linked glycosidase from the follicle cell and egg surface that binds to and blocks all unoccupied sperm binding sites on the vitelline coat. Release of this glycosidase is thought to be under the control of a membrane‐bound phospholipase. To elucidate the mechanism of phospholipase activation, intact eggs and isolated follicle cells are activated by either sperm or the tyrosine kinase activator 9,10‐dimethyl‐1,2‐benzanthracene (DMBA). Both treatments caused release of comparable quantities of glycosidase activity, the earliest event following fertilization. A corresponding increase in phospholipase activity accompanied this glycosidase release. The tyrosine kinase inhibitor genistein blocked release by DMBA at concentrations as low as 1 μM, but had no effect on sperm‐induced release even when used up to 100 μM. Tyrphostin A23, another tyrosine kinase inhibitor, when used at 200 μM blocked glycosidase release and decreased phospholipase activity following both DMBA activation and fertilization. Western blot analysis probing for phosphotyrosine content of disrupted intact eggs with their follicle cells revealed the absence of a band in tyrphostin‐treated eggs corresponding to a 40 kDa protein that was present in both unfertilized and fertilized egg samples. Based on these results, we propose that phosphorylation of specific tyrosine residues is necessary for phospholipase activation and is sufficient to trigger subsequent glycosidase release. Mol. Reprod. Dev. 54:69–75, 1999. © 1999 Wiley‐Liss, Inc.     

Calcium influx following fertilization of Urechis caupo eggs.

Measurements of ⁴⁵Ca flux into and out of Urechis eggs indicate that, during the first 10 min after insemination, the eggs take up 0.24 pmole of Ca/egg. Total egg Ca measured by atomic absorption (AA) spectroscopy increased by 0.23 pmole of Ca/egg (0.56, 0.79, and 0.76 pmole of Ca/egg for unfertilized, 10-min fertilized, and 60-min fertilized eggs, respectively). Thus, the total change in egg Ca is accounted for by the influx even though the rate of efflux, measured as a release of ⁴⁵Ca from preloaded eggs, increases to twice the unfertilized rate by 15 min. The fertilization influx follows saturation kinetics (K_a = 1.3 mM). It is competitively inhibited by procaine, but is not inhibited by dinitrophenol, mersalyl acid, or ruthenium red. Ten percent of the total Ca influx has occurred by 10 sec, and it is, therefore, the most rapid response to fertilization yet known in these eggs. The influx is also observed in eggs partially activated by insemination in pH 7 seawater (SW); the other fertilization responses, except sperm penetration, do not occur in pH 7 SW. Although Ca influx alone is insufficient to activate the eggs, it may be a prerequisite for cytoplasmic activation and development, inducing other secondary responses which are prevented by low external pH.     

Mammalian sperm-egg interaction: fertilization of mouse eggs triggers modification of the major zona pellucida glycoprotein, ZP2

Sperm-egg interaction in mammals is initiated by binding of sperm to the zona pellucida, an acellular coat completely surrounding the plasma membrane of unfertilized eggs and preimplantation embryos. Fertilization results in transformation of the zona pellucida (“zona reaction”), such that additional sperm are unable to bind to the zona pellucida of fertilized eggs and embryos, and sperm that had partially penetrated the zona pellucida of eggs prior to fertilization are prevented from further penetration after fertilization. The failure of sperm to bind to fertilized mouse eggs and embryos is attributable to modification of the sperm receptor, ZP3, an 83,000-molecular weight glycoprotein present in zonae pellucidae isolated from both eggs and embryos [Bleil, J. D., and Wassarman, P. M. (1980). Cell, 20, 873–882]. In this investigation, ZP2, the major glycoprotein found in mouse zonae pellucidae [Bleil, J. D., and Wassarman, P. M. (1980). Develop. Biol., 76, 185–202] was analyzed by gel electrophoresis under a variety of conditions in order to determine whether or not it undergoes modification as a result of fertilization. Under nonreducing conditions, ZP2 present in solubilized zonae pellucidae that were isolated individually from mouse oocytes, eggs, and embryos migrates on SDS-polyacrylamide gels with an apparent molecular weight of 120,000. However, under reducing conditions, ZP2 from embryos, but not from oocytes or unfertilized eggs, migrates with an apparent molecular weight of 90,000 and has been designated ZP2_f. The evidence presented suggests that modification of ZP2 following fertilization involves proteolysis of the glycoprotein, but that intramolecular disulfide bonds prevent the release of peptide fragments. It is shown that the same change in ZP2 can be generated in vitro by artificial activation of unfertilized mouse eggs with the calcium ionophore A23187, thus eliminating the possibility that a sperm component is responsible for the modification of ZP2 following fertilization. These results suggest that some of the changes in the biochemical and biological properties of zonae pellucidae, observed following fertilization or activation of mouse eggs, result from modification of the major zona pellucida glycoprotein, ZP2.     

Dinitrocresol and phosphate stimulation of the oxygen consumption of a cell-free oxidative system obtained from sea urchin eggs

1. A cell-free system capable of using oxygen with oxalacetate as substrate has been prepared from both unfertilized and fertilized sea urchin eggs. The oxygen uptake by this system is about twice that of an equivalent quantity of intact unfertilized eggs and half that of an equivalent quantity of intact fertilized eggs. 2. The oxygen consumption of this cell-free oxidative system can be stimulated by addition of suitable concentrations of 4,6-dinitro-o-cresol or by inorganic phosphate. This confirms, with a cell-free system obtained from sea urchin eggs, the observations of Loomis and Lipmann regarding stimulation of oxygen consumption by a system obtained from rabbit kidney. 3. A preliminary but unsuccessful attempt has been made to determine the conditions under which cell-free, aerobic, phosphorylating systems may be obtained from either unfertilized or fertilized sea urchin eggs.     

Kinetics of actin assembly attending fertilization or artificial activation of sea urchin eggs

Changes in the state of actin assembly triggered by fertilization or by artificial activation of sea urchin eggs were quantified using the DNase I inhibition assay. Insemination of Lytechinus pictus or Strongylocentrotus purpuratus eggs induces a cyclic variation in the level of G-actin as follows: between 0 and 30 s after insemination, the G-actin content decreases. This is followed by an increase in the amount of monomeric actin between 30 and 60 s, and then from 60 s to 5 min postinsemination there is a progressive decrease in the egg's level of G-actin. This latter decrease is more pronounced in S. purpuratus eggs than in L. pictus eggs. Using sperm mimetics that trigger an increase in intracellular calcium concentration (A23187 in sodium-free seawater), a cytoplasmic alkalinization (NH4Cl), a plasma membrane depolarization (seawater enriched with potassium ions), or all three of these phenomena (A23187 in normal seawater), each phase depicted at fertilization correlates with the following metabolic events accompanying egg awakening: phase 1, of uncertain origin (possibly related to plasma membrane depolarization); phase 2, elevation of intracellular calcium concentration; phase 3, alkalinization of the intracellular milieu but only if the transient intracellular calcium rise has taken place.     

A novel procedure for obtaining denuded sea urchin eggs and observations on the role of the vitelline layer in sperm reception and egg activation

A procedure is described for the complete removal of the vitelline layer of the eggs of the sea urchin, Strongylocentrotus purpuratus. The method involves treatment of unfertilized eggs with an S. purpuratus cortical granule protease preparation followed by incubation in an alkaline dithiothreitol seawater solution. Eggs denuded of their vitelline layers react metabolically to parthenogenetic agents and sperm like unfertilized eggs, whereas the fertilizability of denuded eggs and receptivity to sperm is much less than controls. The present method is superior to previous methods using mercaptans in that all of the vitelline layer is removed and to procedures using other proteolytic enzymes in that no ¹²⁵I-labelled plasma membrane proteins are extensively modified. Thus the cortical granule protease dithiothreitol procedure is ideal for studies of the plasma membrane of the unfertilized egg and for studies on the role of the vitelline layer in normal fertilization and development.     

Sperm nuclear dispersion coordinate with meiotic maturation in fertilized Spisula solidissima eggs

Investigations were carried out with fertilized Spisula solidissima eggs, in which changes in incorporated sperm nuclei were determined by measurement of the diameter of dispersing paternal chromatin. Results of such an analysis demonstrated that sperm nuclear dispersion does not proceed at a constant rate and consists of four phases (1–4), coordinate with major changes in the status of the maternal chromatin. (1) The first phase was a short lag period prior to germinal vesicle breakdown in which the size of the sperm nucleus increased only slightly. (2) This was followed by a rapid dispersion of the sperm nucleus coordinate with germinal vesicle breakdown. With the development of the first meiotic spindle, sperm chromatin dispersion slowed dramatically; this phase (3) lasted until the completion of the meiotic divisions at which time the sperm chromatin underwent a second rapid increase in size (4) that was correlated with development of the female pronucleus. When zygotes were treated with agents that inhibited germinal vesicle breakdown (verapamil, sodium-free seawater, and chloroquine), sperm nuclear dispersion did not occur. Evidence is presented indicating that nucleocytoplasmic interactions coincident with germinal vesicle breakdown induce sperm nuclear dispersion in Spisula zygotes.     

Actin in triton-treated cortical preparations of unfertilized and fertilized sea urchin eggs

Triton-treated cortical fragments of unfertilized and fertilized sea urchin eggs prepared in the presence of greater than or equal to 5 mM EGTA contain 15-30% of the total egg actin. However, actin filaments are not readily apparent by electron microscopy on the cortical fragments of unfertilized eggs but are numerous on those of fertilized eggs. The majority of the actin associated with cortical fragments of unfertilized eggs is solubilized by dialysis against a low ionic strength buffer at pH 7.5. This soluble actin preparation (less than 50% pure actin) does not form proper filaments in 0.1 M KCl and 3 mM MgCl2, whereas actin purified from this preparation does, as judged by electron microscopy. Optical diffraction analysis reveals that these purified actin filaments have helical parameters very similar to those of muscle actin. Furthermore, the properties of the purified actin with regard to activation of myosin ATPase are similar to those of actin from other cell types. The possibility that actin is maintained in a nonfilamentous form on the inner surface of the unfertilized egg plasma membrane and is induced to assemble upon fertilization is discussed.     

Does the low respiratory rate in unfertilized eggs result mainly from depression of the redox reaction catalyzed by flavoproteins? Analysis of the respiratory system by light-induced release of CO-mediated inhibition

In unfertilized eggs of the sea urchin, the quite low respiratory rate is enhanced by tetramethyl- p -phenylenediamine (TMPD), phenazine methosulfate (PMS) and sperm and this augmentation is completely inhibited by carbon monoxide (CO). Exposure to light releases eggs from this CO-mediated inhibition. The action spectra for photoreactivation of CO-inhibited cytochrome c oxidase in isolated mitochondria and CO-blocked respiration in TMPD-treated eggs were found to be similar to the absorption spectrum of CO-bound cytochrome aa ₃. In PMS-treated eggs and fertilized eggs, the maximum photoreactivation of CO-inhibited respiration occurred at a light fluence rate higher than that for maximum photoreactivation of CO-inhibited respiration in TMPD-treated eggs, with peaks at the same wavelengths as those in the absorption spectrum of reduced cytochrome b. A similar phenomenon was seen for NADH cytochrome c reductase in mitochondria. Thus, cytochrome c oxidase and NADH cytochrome c reductase, whose activities are not altered by fertilization, seem to be functional, even in unfertilized eggs. In unfertilized eggs, difference spectra indicated that PMS and sperm augmented cytochrome b reduction and that TMPD accelerated cytochrome c reduction without cytochrome b reduction. Therefore, it is likely that depression of electron transport to cytochrome b , which is augmented by PMS and sperm, is responsible for the low respiratory rate in unfertilized eggs.