Free intracellular cations in echinoderm oocytes and eggs

The concentrations of Ca²⁺, Na⁺ and H⁺ in echinoderm oocytes and eggs were measured during maturation and activation using ion-selective microelectrodes. In both oocytes and eggs, from three species of starfish and two species of sea urchin, the resting level of cytosolic Ca²⁺ was about 10^-7 M. We did not detect any change in Ca²⁺ concentration either during hormone-induced oocyte maturation (starfish) or during egg activation (starfish and sea urchin) induced by spermatozoa or chemical agents. During 1-methyl-adenine induced maturation of starfish oocytes the intracellular level of Na⁺ increased from 12–35 mM to 40–90 mM, while the pH changed from 6.6–6.8 to 7.0–7.2 Aged oocytes, with intact germinal vesicles, also had elevated levels of Na⁺ and pH.     

Degradation of polyubiquitinated cyclin B is blocked by the MAPK pathway at the metaphase I arrest in starfish oocytes

In the starfish ovary, maturing oocytes stimulated by 1-methyladenine undergo synchronous germinal vesicle breakdown and then arrest in metaphase of the first meiotic division (metaphase I). Immediately after spawning, an increase of intracellular pH (pH(i)) from approximately 7.0 to approximately 7.3 is induced by Na(+)/H(+) antiporter in oocytes, and meiosis reinitiation occurs. Here we show that an endogenous substrate of the proteasome, polyubiquitinated cyclin B, was stable at pH 7.0, whereas it was degraded at pH 7.3. When the MAPK pathway was blocked by MEK inhibitor U0126, degradation of polyubiquitinated cyclin B occurred even at pH 7.0 without an increase of the peptidase activity of the proteasome. These results indicate that the proteasome activity at pH 7.0 is sufficient for degradation of polyubiquitinated cyclin B and that the MAPK pathway blocks the degradation of polyubiquitinated cyclin B in the maturing oocytes in the ovary. Immediately after spawning, the increase in pH(i) mediated by Na(+)/H(+) antiporter cancels the inhibitory effects of the MAPK pathway, resulting in the degradation of polyubiquitinated cyclin B and the release of the arrest. Thus, the key step of metaphase I arrest in starfish oocytes occurs after the polyubiqutination of cyclin B but before cyclin B proteolysis by the proteasome.     

Metaphase I arrest of starfish oocytes induced via the MAP kinase pathway is released by an increase of intracellular pH

Reinitiation of meiosis in oocytes usually occurs as a two-step process during which release from the prophase block is followed by an arrest in metaphase of the first or second meiotic division [metaphase I (MI) or metaphase II (MII)]. The mechanism of MI arrest in meiosis is poorly understood, although it is a widely observed phenomenon in invertebrates. The blockage of fully grown starfish oocytes in prophase of meiosis I is released by the hormone 1-methyladenine. It has been believed that meiosis of starfish oocytes proceeds completely without MI or MII arrest, even when fertilization does not occur. Here we show that MI arrest of starfish oocytes occurs in the ovary after germinal vesicle breakdown. This arrest is maintained both by the Mos/MEK/MAP kinase pathway and the blockage of an increase of intracellular pH in the ovary before spawning. Immediately after spawning into seawater, activation of Na+/H+ antiporters via a heterotrimeric G protein coupling to a 1-methyladenine receptor in the oocyte leads to an intracellular pH increase that can overcome the MI arrest even in the presence of active MAP kinase.     

Cryopreservation of starfish oocytes

Research from many laboratories over the past several decades indicates that invertebrate oocytes and eggs are extraordinarily difficult to freeze. Since starfish oocytes, eggs, and embryos are an important cell and developmental biology model system, there is great interest to cryopreserve these cells. Previous starfish oocyte cryopreservation studies using slow cooling protocols revealed that these cells are highly sensitive to osmotic stress and form intracellular ice at very high sub-zero temperatures, suggesting that common freezing methodologies may not prove useful. We report here that a short exposure to 1.5 M Me2SO/1 M trehalose in hypotonic salt solution followed by ultra-rapid cooling to cryogenic temperatures allows starfish oocytes to be cryopreserved with the average survival rate of 34% when normalized to control oocytes that were exposed to CPA, but not frozen. On average, 51% of the oocytes in 77% of the batches of frozen oocytes underwent meiotic maturation in response to the starfish maturation hormone, 1-methyladenine. In one experiment, eggs developing from thawed oocytes were capable of being fertilized and two developed into embryos. These data suggests that successful cryopreservation of starfish oocytes is possible, but will need further refinement to increase the numbers of fully competent embryos.     

Acid Release at Activation and Fertilization of Starfish Oocytes

Fertilization or activation by ionophore A 23187 induces a transient acid release in prophase-blocked and in maturing oocytes of Asterias rubens and Marthasterias glacialis. 1-Methyladenine-induced maturation is not accompanied by acid release. There is no significant difference in the kinetic and amount of acid release related to the nature of activation or the stage of oocytes in each species. The amount of acid released per oocyte volume is smaller than total "fertilization acid" of sea urchin eggs but comparable to its Na-insensitive component. Cortical reaction can be initiated without significant acid release in ammonia treated oocytes. A burst of sodium influx occurs at activation or fertilization of oocytes. Kinetic and amount of Na influx are comparable to acid release. Vitelline membrane elevation is impaired upon activation of oocytes in the absence of extracellular sodium but a significant although smaller release of acid occurs. This suggests that starfish oocytes release acid by a mechanism differing from the Na⁺-H⁺ exchange of sea urchin eggs.     

Revised sequence and expression of cyclin B cDNA from the starfish Asterina pectinifera.

Cyclin B cDNA was cloned from the ovary of the starfish Asterina pectinifera and analyzed by RT-PCR and 3'- and 5'-RACE techniques. The cDNA consists of a 0.13-kb upstream untranslated region, a 1.22-kb coding region, and a 0.86-kb downstream untranslated region. The open reading frame encoded a polypeptide of 404 amino acid residues with a calculated molecular weight of 45,692. All the characteristic sequences, such as destruction and cyclin boxes, cyclin B motif, and cytoplasmic retention and nuclear export signals, were found in the newly cloned cyclin B cDNA. The deduced amino acid sequence of the cyclin B cDNA was highly homologous in the middle and carboxy terminal regions to that from mature eggs of the same organism, but quite different in the amino terminal region. Evidence was obtained which suggested that this cyclin B is expressed in immature and maturing oocytes and is the same as that cloned from mature eggs.     

The role of the actin cytoskeleton in calcium signaling in starfish oocytes

Ca(2+) is the most universal second messenger in cells from the very first moment of fertilization. In all animal species, fertilized eggs exhibit massive mobilization of intracellular Ca(2+) to orchestrate the initial events of development. Echinoderm eggs have been an excellent model system for studying fertilization and the cell cycle due to their large size and abundance. In preparation for fertilization, the cell cycle-arrested oocytes must undergo meiotic maturation. Studies of starfish oocytes have shown that Ca(2+) signaling is intimately involved in this process. Our knowledge of the molecular mechanism of meiotic maturation and fertilization has expanded greatly in the past two decades due to the discovery of cell cycle-related kinases and Ca(2+)-mobilizing second messengers. However, the molecular details of their actions await elucidation of other cellular elements that assist in the creation and transduction of Ca(2+) signals. In this regard, the actin cytoskeleton, the receptors for second messengers and the Ca(2+)-binding proteins also require more attention. This article reviews the physiological significance and the mechanism of intracellular Ca2+ mobilization in starfish oocytes during maturation and fertilization.     

Involvement of mitogen-activating protein kinase and intracellular pH in the duration of the metaphase I (MI) pause of starfish oocytes after spawning

The metaphase I (MI) arrest of starfish oocytes is released after spawning. In this study using starfish Asterina pectinifera , the duration of MI after spawning was ~20 min and ~30 min in fertilized and unfertilized oocytes, respectively. This prolongation of MI in unfertilized oocytes, referred to as the MI pause, was maintained by mitogen-activating protein kinase (MAPK) as well as low intracellular pH (~7.0). Contrary to previous reports, MI arrest was not maintained by MAPK, since it was inactive in the oocytes arrested at MI in the ovary and activated immediately after spawning. Also, cyclin B was not degraded at pH 6.7 in the cell-free preparation without MAPK activity, whereas it was degraded at pH 7.0, suggesting that MI arrest was solely maintained by lower pH (< 7.0). Normal development occurred when the spawned oocytes were fertilized before the first polar body formation, whereas fertilization after the first polar body formation increased the rate of abnormal development. Thus, due to MI pause and MI arrest, the probability for fertilization before the polar body formation might be increased, leading to normal development.     

Fertilization of the starfish Astropecten aurantiacus

In the starfish Astropecten aurantiacus the acrosome reaction occurs when the spermatozoon contacts the outer surface of the jelly layer. A long thin acrosomal filament is extruded from the anterior region of the spermatozoon and establishes contact with the oocyte surface. This latter interaction initiates the movement of the spermatozoon to the oocyte surface, formation of the fertilization cone and the cortical reaction. The first detectable electrical change across the oocyte plasma membrane during interaction with the spermatozoon is the fertilization potential (FP) which occurs simultaneously with the cortical reaction. The FP is probably the electrical result of the modification of the oocyte plasma membrane during cortical exocytosis. There are no primary step-like depolarizations during fertilization of starfish oocytes, which contrasts with the situation in sea urchin eggs [see 13]. We suggest that the difference in electrical response to fertilization of starfish oocytes and sea urchin eggs may be attributed to the location of the acrosome reaction in these animals and not to their different meiotic states.     

A metaphase pause: Hormone-induced maturation progresses through a long pause at the first meiotic metaphase in oocytes of the starfish, Pisaster ochraceus

In several species of starfish, it has been reported that the meiotic divisions in fertilized oocytes occur precociously compared to those in unfertilized oocytes. The nature of the 'acceleration' of meiosis was studied using Pisaster ochraceus oocytes. The extent of the acceleration of first polar body formation was found to be completely dependent on the time of fertilization (or artificial activation); fertilization at about 100 min after 1–methyladenine application accelerated meiosis I the most, while earlier or later fertilization resulted in a smaller extent of accelerations of meiosis I. Observation of isolated meiotic spindles and fluorescent visualization of meiotic spindles in whole oocytes showed that progression of meiosis I in Pisaster oocytes pauses transiently at metaphase I for more than 40min unless they are activated. The activation shortened the duration of metaphase I, which resulted in the acceleration of first polar body formation. A new term 'metaphase pause' is proposed to define this long duration of metaphase I in starfish oocytes.     

Site of production of meiosis-inducing substance in ovary of starfish

The site of production of meiosis-inducing substance (MIS), produced in the ovary under the influence of gonad-stimulating substance (GSS) taken from radial nerves, was studied with the starfish, Asterina pectinifera. The rate of oocyte maturation observed in isolated oocytes with follicles kept in sea water containing GSS (100 μg/ml) was generally low when a small number of eggs per unit quantity of sea water was used. However, with increased number of oocytes per milliliter of GSS-sea water, the maturation rate increased up to 100% (10⁴ eggs/ml). The supernatant of such an incubation mixture of oocytes and GSS contained an appreciable amount of MIS. When a large number of oocytes (10⁴/ml) was used, the rate of oocyte maturation increased with the rise in concentration of GSS. Isolated follicles were found to produce MIS when incubated in sea water containing GSS, suggesting that the site of production of MIS is the follicle cells. The physiological role of the follicle cells surrounding the full grown oocytes seems to be to produce MIS just before spawning under the influence of GSS.     

Intracellular Localization of DNA Polymerases in the Oocyte of Starfish, Asterina pectinifera

Oocytes of the starfish, Asterina pectinifera , were separated into karyoplasts and cytoplasts by centrifugation in a sucrose density gradient in the presence of cytochalasin B. More than 90% of the DNA polymerase activity of whole oocytes was recovered in the karyoplasts, and less than 10% in the cytoplasts. DNA polymerase α was specifically localized in karyoplasts. Of the DNA polymerase β activity of whole oocytes, 70–80% was found in the karyoplasts, and about 15% in the cytoplasts. The problem of whether DNA polymerases are localized within germinal vesicles in starfish oocytes is discussed.     

Response of rainbow smelt, Osmerus mordax (Mitchill), eggs to low pH

The eggs of rainbow smelt were incubated at pH c . 4.5, c . 5.5 and c ontrol ( c . 6.8) for durations of 2–14 days to assess their vulnerability to acid precipitation. These eggs are likely to be exposed to marked pH depressions during their incubation in small streams owing to run-off from snow melt and spring rains. Survival after 48 h exposure was not related to pH, although longer exposure to low pH significantly decreased survival. The developmental stage at initial exposure was also important in determining survival. Overall egg survival varied from 0–87% at pH c . 4.5,0–89% at pH c . 5.5, and 44–100% at control pH levels, (c. 6.8).     

Molecular characterization of the starfish inositol 1,4,5-trisphosphate receptor and its role during oocyte maturation and fertilization.

The release of calcium ions (Ca(2+)) from their intracellular stores is essential for the fertilization of oocytes of various species. The calcium pools can be induced to release Ca(2+) via two main types of calcium channel receptor: the inositol 1,4,5-trisphosphate receptor (IP(3)R) and the ryanodine receptor. Starfish oocytes have often been used to study intracellular calcium mobilization during oocyte maturation and fertilization, but how the intracellular calcium channels contribute to intracellular calcium mobilization has never been understood fully, because these molecules have not been identified and no specific inhibitors of these channels have ever been found. In this study, we utilized a novel IP(3)R antagonist, the "IP(3) sponge," to investigate the role of IP(3) during fertilization of the starfish oocyte. The IP(3) sponge strongly and specifically competed with endogenous IP(3)R for binding to IP(3). By injecting IP(3) sponge into starfish oocyte, the increase in intracellular calcium and formation of the fertilization envelope were both dramatically blocked, although oocyte maturation was not blocked. To investigate the role of IP(3)R in the starfish oocyte more precisely, we cloned IP(3)R from the ovary of starfish, and the predicted amino acid sequence indicated that the starfish IP(3)R has 58-68% identity to mammalian IP(3)R types 1, 2, and 3. We then raised antibodies that recognize starfish IP(3)R, and use of the antibodies to perform immunoblot analysis revealed that the level of expression of IP(3)R remained unchanged throughout oocyte maturation. An immunocytochemical study, however, revealed that the distribution of starfish IP(3)R changes during oocyte maturation.     

Different routes lead to apoptosis in unfertilized sea urchin eggs

Results obtained in various species, from mammals to invertebrates, show that arrest in the cell cycle of mature oocytes is due to a high ERK activity. Apoptosis is stimulated in these oocytes if fertilization does not occur. Our previous data suggest that apoptosis of unfertilized sea urchin eggs is the consequence of an aberrant short attempt of development that occurs if ERK is inactivated. They contradict those obtained in starfish, another echinoderm, where inactivation of ERK delays apoptosis of aging mature oocytes that are nevertheless arrested at G1 of the cell cycle as in the sea urchin. This suggests that the cell death pathway that can be activated in unfertilized eggs is not the same in sea urchin and in starfish. In the present study, we find that protein synthesis is necessary for the survival of unfertilized sea urchin eggs, contrary to starfish. We also compare the effects induced by Emetine, an inhibitor of protein synthesis, with those triggered by Staurosporine, a non specific inhibitor of protein kinase that is widely used to induce apoptosis in many types of cells. Our results indicate that the unfertilized sea urchin egg contain different mechanisms capable of leading to apoptosis and that rely or not on changes in ERK activity, acidity of intracellular organelles or intracellular Ca and pH. We discuss the validity of some methods to investigate cell death such as measurements of caspase activation with the fluorescent caspase indicator FITC-VAD-fmk or acidification of intracellular organelles, methods that may lead to erroneous conclusions at least in the sea urchin model.     

CHANGES IN THE LEVEL OF ARGININE PHOSPHATE FOLLOWING FERTILIZATION OF ECHINODERM EGGS*

The content of arginine phosphate was measured following fertilization of sea-urchin eggs and starfish oocytes. In sea-urchin eggs, a rise in the level of arginine phosphate occurred within 2 min after insemination: this was not accompanied by any detectable alteration in the level of ATP. On the other hand, the level of arginine phosphate in starfish oocytes did not change on fertilization.     

Starfish oocytes form intracellular ice at unusually high temperatures

Starfish oocytes, eggs, and embryos are popular models for studying meiotic maturation, fertilization, and embryonic development. Their large (170- to 200-microm) oocytes are obtainable in copious amounts and are amenable to manipulations that mammalian oocytes are not. The most formidable obstacle to working with marine oocytes is their seasonal availability, yet a successful means of preserving them for use during the nonreproductive season has not been reported. The aim of this study was to investigate the response of starfish oocytes to freezing with rapid and slow cooling rates under a variety of conditions to develop a cryopreservation protocol for these cells. Cryomicroscopic observation revealed that starfish oocytes in isotonic medium undergo intracellular ice formation (IIF) at very high subzero temperatures, such that the mean difference between the temperature of extracellular ice formation (T(EIF)) and IIF (TI(IF)) was less than 3 degrees C and the average T(IIF) was approximately between -4 and -6 degrees C. Neither partial cellular dehydration nor addition of the cryopreservative dimethyl sulfoxide significantly depressed the T(IIF). Under some conditions, we observed ice nucleation at multiple locations within the cytoplasm, suggesting that several factors contribute to the unusually high T(IIF) during controlled-rate freezing and thus vitrification may be a more suitable method for cryopreserving these cells.     

Worker laying in leafcutter ant Acromyrmex subterraneus brunneus （Formicidae, Attini）

We studied the process of offspring production in queenless colonies of Acromyrmex subterraneus brunneus, and particularly evaluated the ovary development of workers as a function of their age. For this, subcolonies were set up and evaluated at different periods of isolation from the queen （2, 4 and 6 months）, besides individually labeled age groups. The subcolonies were assessed according to offspring production and ovaries containing oocytes or not. The evaluations showed worker oviposition and development of males originating from worker-laid eggs. At 2 months＇ absence of the queen, eggs and larvae were found, with eggs in a higher proportion than larvae. After 4 months, the proportion of eggs had reduced while larvae had increased, and a pupa was found in one subcolony. At 6 months, besides a higher share of larvae, one pupa and one adult male were found. Dissection of workers revealed ovaries containing oocytes during the periods of evaluation. Only a group of medium-sized and large workers, 23.3%, 20.9% and 37.5% of the population from each period assessed in queenless subcolonies respectively, presented developed oocytes in the ovary. The same was observed in colonies with a queen, with 17.6%, 19.6% and 7.8% of the group of dissected workers from each time period, respectively. With respect to worker age, we observed by dissection of the ovary, that the greatest percentage of individuals with ovarioles containing oocytes occurred at 45 days （6 weeks） up to 90 days （12 weeks）. These results probably are associated with the workers reproduction and the laying of trophic and reproductive eggs in colonies with and without a queen; these eggs have distinct functions in each situation.     

Antibody against the actin-binding protein depactin attenuates Ca signaling in starfish eggs

Being present in starfish oocytes, the cofilin/ADF (actin-depolymerizing factor) family protein depactin severs actin filaments. Previously, we reported that exogenous cofilin microinjected into starfish eggs significantly augmented the Ca²⁺ release in response to inositol 1,4,5-trisphosphate (InsP₃) or fertilizing sperm, raising the possibility that intracellular Ca²⁺ signaling could be modulated by the actin cytoskeleton. In this communication, we have targeted the endogenous depactin by use of the specific antibody that was raised against its actin-binding domain. The anti-depactin antibody microinjected into the starfish oocytes and eggs effectively altered the structure of the actin cytoskeleton, and significantly delayed the meiotic progression induced by 1-methyladenine. When microinjected into the mature eggs, the anti-depactin antibody markedly reduced the amplitude of the Ca²⁺ response in a dose-dependent manner, corroborating the results of our previous study with cofilin. In addition, the eggs microinjected with the anti-depactin antibody displayed reduced rate of successful elevation of the fertilization envelope and an elevated tendency of polyspermic interaction. Taken together, our data suggest that the actin cytoskeleton is implicated not only in meiotic maturation and intracellular Ca²⁺ signaling, but also in the fine regulation of gametes interaction and cortical granules exocytosis.