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.     

Induction of apoptosis in starfish eggs requires spontaneous inactivation of MAPK (extracellular signal-regulated kinase) followed by activation of p38MAPK

Mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase) prevents DNA replication and parthenogenesis in maturing oocytes. After the meiotic cell cycle in starfish eggs, MAPK activity is maintained until fertilization. When eggs are fertilized, inactivation of MAPK occurs, allowing development to proceed. Without fertilization, highly synchronous apoptosis of starfish eggs starts 10 h after germinal vesicle breakdown, which varies according to season and individual animals. For induction of the apoptosis, MAPK should be activated for a definite period, called the MAPK-dependent period, during which eggs develop competence to die, although the exact duration of the period was unclear. In this study, we show that the duration of the MAPK-dependent period was approximately 8 h. Membrane blebbing occurred approximately 2 h after the MAPK-dependent period. Surprisingly, when MAPK was inhibited by U0126 after the MAPK-dependent period, activation of caspase-3 occurred earlier than in the control eggs. Thus, inactivation of MAPK is a prerequisite for apoptosis. Also, even in the absence of the inhibitor, MAPK was inactivated spontaneously when eggs began to bleb, indicating that inactivation of MAPK after the MAPK-dependent period acts upstream of caspase-3. Inactivation of MAPK also resulted in the activation of p38MAPK, which may contribute to apoptotic body formation.     

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.     

Involvement of caspase-9 in execution of the maternal program of apoptosis in Xenopus late blastulae overexpressed with S-adenosylmethionine decarboxylase

We previously demonstrated that overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus early embryos induces execution of maternal program of apoptosis shortly after midblastula transition, which likely serves as a fail-safe mechanism of early development to eliminate physiologically damaged cells before they entering the gastrula stage. To determine how caspases are involved in this process, we microinjected peptide inhibitors and "dominant-negative forms" of caspase-9 and -1 into Xenopus fertilized eggs, and found that inhibitors of caspase-9, but not caspase-1, completely suppress SAMDC-induced apoptosis. The lysate of SAMDC-overexpressing late blastulae contained activity to cleave in vitro-synthesized [(35)S]procaspase-9, but not [(35)S]procaspase-1, and mRNA for caspase-9, but not caspase-1, occurred abundantly in the unfertilized egg as maternal mRNA. We also found that overexpression of caspase-9 and -1 equally executes the apoptosis, but the apoptosis executed by these mRNAs was only partially rescued by Bcl-2 and rescued embryos did not develop beyond neurula stage. These results indicate that activation of caspase-9 is a key step for execution of the maternally preset program of apoptosis in Xenopus early embryos.     

Induction of starfish oocyte maturation by the beta gamma subunit of starfish G protein and possible existence of the subsequent effector in cytoplasm.

beta gamma subunits of G proteins were purified from starfish oocytes, and their role in the induction of oocyte maturation by 1-methyladenine was investigated. When injected into starfish oocytes, the purified beta gamma subunit of the starfish G protein induced germinal vesicle breakdown (GVBD) faster than that of bovine brain G protein. Injection of the starfish beta gamma into cytoplasm near the germinal vesicle (GV) induced GVBD earlier than when injected into the GV or the cytoplasm near the plasma membrane. Fluorescent-labeled beta gamma was retained in the injected area even after GVBD. Injected beta gamma also induced the formation of maturation-promoting factor as well as an increase of histone H1 kinase activity. These results suggest that beta gamma dissociates from alpha-subunit by the stimulation of 1-methyladenine and interacts with a cytoplasmic effector, which results in formation of active cdc2 kinase.     

Development of calcium release mechanisms during starfish oocyte maturation

In response to the maturation-inducing hormone 1-methyladenine, starfish oocytes acquire increased sensitivity to sperm and inositol trisphosphate (InsP3), stimuli that cause a release of calcium from intracellular stores and a rise in intracellular free calcium. In the immature oocyte, the calcium release in response to 10 sperm entries is less than that seen with a single sperm entry in the mature egg. Likewise, the sensitivity to injected InsP3 is less in the immature oocyte. Approximately 100 times as much InsP3 is required to obtain the same calcium release in an immature oocyte as in a mature egg. However, with saturating amounts of InsP3, immature oocytes and mature eggs release comparable amounts of calcium. These results indicate that although calcium stores are well-developed in the immature oocyte, mechanisms for releasing the calcium develop fully only during oocyte maturation.     

High cytosolic free calcium level signals apoptosis through mitochondria-caspase mediated pathway in rat eggs cultured in vitro

The present study was aimed to find out whether an increase of cytosolic free calcium level induces egg apoptosis through mitochondria-caspase mediated pathway. To increase cytosolic free calcium level and morphological apoptotic changes, ovulated eggs were cultured in Ca²⁺/Mg²⁺ free media-199 with or without various concentrations of calcium ionophore (0.5, 1, 2, 3, 4 μM) for 3 h in vitro. The morphological apoptotic changes, cytosolic free calcium level, hydrogen peroxide (H₂O₂) concentration, catalase activity, cytochrome c concentration, caspase-9 and caspase-3 activities and DNA fragmentation were analyzed. Calcium ionophore induced morphological apoptotic features in a concentration-dependent manner followed by degeneration at higher concentrations (3 and 4 μM). Calcium ionophore increased cytosolic free calcium level, induced generation of hydrogen peroxide (H₂O₂) and inhibited catalase activity in treated eggs. The increased H₂O₂ concentration was associated with increased cytochrome c concentration, caspase-9 and caspase-3 activities that resulted in the induction of morphological features characteristic of egg apoptosis. The increased caspase-3 activity finally induced DNA fragmentation as evidenced by TUNEL positive staining in calcium ionophore-treated eggs. These findings suggest that high cytosolic free calcium level induces generation of H₂O₂ that leads to egg apoptosis through mitochondria-caspase mediated pathway.     

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.     

Okadaic acid stimulates caspase-like activities and induces apoptosis of cultured rat mesangial cells

Glomerular mesangial cells play an important role in the development of glomerulosclerosis. Mesangial cell apoptosis has been shown to be involved in different stages of development of glomerulonephritis. The aim of the present study was to evaluate the effect of inhibition of serine/threonine phosphatases by okadaic acid, a shell fish toxin, on rat mesangial cell apoptosis and to examine the molecular mechanisms particularly the role of caspases. Okadaic acid significantly induced mesangial cell apoptosis, as measured by an increase in cytoplasmic nucleosome-associated DNA fragmentation. The induction of apoptosis was dependent on protein synthesis, because cyclohexamide, a protein synthesis inhibitor, blocked okadaic acid-induced apoptosis. In addition, okadaic acid stimulated caspase activities (as measured by caspase substrate peptide hydrolysis) in cultured rat mesangial cells at different time points. After 12 h treatment, okadaic acid caused a modest increase in caspase-8 (IETD-pNAse)(159.3 ± 6.7%) activity, while after 18 h treatment, okadaic acid caused a significant increase in caspase-3 (DEVD-pNAse)(906 ± 245%) activity. Okadaic acid-stimulated caspase-3 activity was inhibited by Z-IETD-FMK (caspase-8 inhibitor) suggesting that the caspase-3 activity is downstream of caspase-8 activity. Both caspase-3 and caspase-8 inhibitors blocked okadaic acid-stimulated apoptosis. These data suggest that inhibition of protein phosphatases by okadaic acid induces apoptosis in rat mesangial cells by activating caspase-3- and -8-like activities and that caspase-3-like activity is downstream of caspase-8-like activity.     

Okadaic acid stimulates caspase-like activities and induces apoptosis of cultured rat mesangial cells

Glomerular mesangial cells play an important role in the development of glomerulosclerosis. Mesangial cell apoptosis has been shown to be involved in different stages of development of glomerulonephritis. The aim of the present study was to evaluate the effect of inhibition of serine/threonine phosphatases by okadaic acid, a shell fish toxin, on rat mesangial cell apoptosis and to examine the molecular mechanisms particularly the role of caspases. Okadaic acid significantly induced mesangial cell apoptosis, as measured by an increase in cytoplasmic nucleosome-associated DNA fragmentation. The induction of apoptosis was dependent on protein synthesis, because cyclohexamide, a protein synthesis inhibitor, blocked okadaic acid-induced apoptosis. In addition, okadaic acid stimulated caspase activities (as measured by caspase substrate peptide hydrolysis) in cultured rat mesangial cells at different time points. After 12 h treatment, okadaic acid caused a modest increase in caspase-8 (IETD-pNAse) (159.3 +/- 6.7%) activity, while after 18 h treatment, okadaic acid caused a significant increase in caspase-3 (DEVD-pNAse) (906 +/- 245%) activity. Okadaic acid-stimulated caspase-3 activity was inhibited by Z-IETD-FMK (caspase-8 inhibitor) suggesting that the caspase-3 activity is downstream of caspase-8 activity. Both caspase-3 and caspase-8 inhibitors blocked okadaic acid-stimulated apoptosis. These data suggest that inhibition of protein phosphatases by okadaic acid induces apoptosis in rat mesangial cells by activating caspase-3- and -8-like activities and that caspase-3-like activity is downstream of caspase-8-like activity.     

Absence of Species Specificity of Germinal Vesicle Factor Required for the Cytoplamic Cycle During Meiotic Division

Germinal vesicles (GV) of starfish oocytes are known to contain some factor(s) indispensable for inducing cyclic cytoplasmic activity, which may be involved in driving meiotic cell cycle. We have examined species specificity of the factor by injecting enucleated oocytes of the starfish, Asterina pectinifera , with GV contents of either different starfish ( Asterias amurensis, Astropecten scoparius ) or sea cucumber ( Holothuria moebi ). The injected oocytes showed cyclic changes in cortical tension after treatment with 1-methyladenine. Thus the nature of the factor appears similar among echinoderms. Growing oocytes of A. pectinifera much smaller (75% in diameter) than fully-grown oocytes did contain the factor to rescue the enucleated oocytes, even though such small oocytes themselves did not respond to 1-methyladenine. This result suggests that the factor begins to accumulate at the earlier stage of oogenesis.     

Long-term effects of methoxyfenozide on the adult reproductive processes and longevity of Spodoptera exigua (Lepidoptera: Noctuidae)

The long-term effects of methoxyfenozide on the longevity and reproductive processes of beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), adults were assessed after exposure by ingestion. Methoxyfenozide significantly reduced adult male longevity compared with females by 1.1 and 1.5 d at 75 and 150 mg (AI)/liter, respectively. Fecundity decreased by >60% with both concentrations at 72 and 96 h after treatment, but at 48 h, no significant effect was observed. The carbohydrate, protein, and lipid content in the eggs were determined as representatives of the biochemical effects of methoxyfenozide associated with the disruption of reproductive processes. The content of carbohydrates in the eggs laid 48 h at treatment was similar to that of controls, but it increased by approximately 1.5 and 2-fold in eggs laid after 72 and 96 h, respectively, compared with controls (15 microg per egg). Protein content was reduced approximately 2.5 and approximately 3-fold for each treatment concentration, respectively, compared with the controls (25 and 23 microg per egg for 75 and 150 mg [AI]/liter, respectively) in eggs collected 72 and 96 h after treatment. Lipid content significantly decreased by approximately 1.6-fold in both treatment concentrations in eggs collected at 48 and 96 h after treatment compared with the controls (24 and 21 microg per egg for 48 and 96, respectively), but it was similar to controls (approximately 19 microg per egg) at 72 h (approximately 15 microg per egg) for both concentrations. The biochemical effects of methoxyfenozide on S. exigua egg formation detected in this work are consistent with the reduction in fertility observed, as reported previously.     

家蚕卵显微注射操作对蚕卵孵化及畸形蚕发生的影响

早期胚胎显微注射是目前获得转基因家蚕Bombyx mori的主要途径。显微注射操作对蚕卵的损伤导致注射后的蚕卵孵化率降低, 是家蚕转基因工作的主要障碍之一。本研究对不同卵龄的蚕卵进行了开孔或注射实验, 并对产后5 h的蚕卵上背侧、 腹侧、 前极、 后极和中央等5个不同的位置进行了开孔实验, 调查了卵孵化率和体形异常蚕的产生情况。结果表明： 较早卵龄期的注射或从蚕卵背侧的注射可以获得高的孵化率。腹侧注射产生大量的体形异常蚕而背侧注射的蚕完全正常。通过调整注射时期和注射位置避开上述影响可以减少死卵和畸形蚕, 提高孵化率。本研究为改进家蚕转基因操作技术提供了有效的参考。     

Extraction and preliminary characterization of maturation-promoting factor from starfish oocytes

Cytoplasm of maturing starfish oocytes possesses a factor which induces maturation upon injection into immature oocytes. Such maturation-promoting factor (MPF) was extracted from maturing oocytes of Asterina pectinifera and characterized preliminarily. After 1-methyladenine (1-MeAde) treatment, maturing oocytes were packed in a centrifuge tube to remove jelly and excess medium, and then crushed by centrifugation. The turbid supernatant was homogenized with a buffer containing NaF, Na-beta-glycerophosphate, ATP, EGTA and leupeptin, followed by centrifugation. MPF extracted in the supernatant was purified partially by ammonium sulfate precipitation, hydrophobic chromatography on pentyl-agarose and gel filtration on Sephacryl S-300. The final material induced maturation in the recipient starfish oocytes when 0.5 ng of protein was injected in a volume of 400 pl. The maturation response included germinal vesicle breakdown, and formation of polar bodies and egg pronucleus. Such MPF preparation induced maturation in oocytes of Xenopus laevis as well. Further, starfish MPF was found to be a heat-labile protein; its molecular weight (MW) was estimated as 300 X 10(3) D by gel filtration and its sedimentation coefficient value as 5S by centrifugation on sucrose density gradients.     

RNA 3' cleavage and polyadenylation in oocytes and unfertilized eggs of Xenopus laevis

Xenopus laevis histone H4 and H1 genes were transcribed in vitro to generate artificial precursor mRNAs (pre-mRNAs). These pre-mRNAs were microinjected into oocytes, matured oocytes, and unfertilized eggs of Xenopus laevis and their 3' cleavage and polyadenylation were investigated. In the oocyte nucleus both H4 and H1 pre-mRNAs were 3' cleaved but were not detectably polyadenylated. In the oocyte cytoplasm there was neither 3' cleavage nor polyadenylation of these histone pre-mRNAs. When injected into either matured oocytes or unfertilized eggs, the pre-mRNAs underwent 3' cleavage but this was inefficient when compared to the oocyte nucleus. In addition approximately 50% of the remaining uncleaved pre-mRNA was subject to a polyadenylation activity which added A tails of approximately 70 A residues. In contrast, artificial mouse beta-globin pre-mRNAs were not detectably 3' cleaved or polyadenylated in either microinjected oocytes or unfertilized eggs.     

Microinjection of the monoclonal anti-tubulin antibody YL1/2 inhibits cleavage of sand dollar eggs

Two monoclonal antibodies against alpha-tubulin (YL1/2 and D2D6) were microinjected into the egg of the sand dollar Clypeaster japonicus, and their effects on cleavage of the egg were investigated. They had already been shown by immunoblotting to react specifically with egg tubulin and by immunofluorescence to stain the mitotic apparatus [OKA et al., (1990). Cell Motil. Cytoskel. 16:239-250]. Injection of YL1/2 prevented chromosome movement and cleavage, although the cleavage furrow developed in some cases. In all eggs injected at prometaphase, metaphase, or anaphase, the birefringence of the mitotic apparatus disappeared immediately after injection. Injection of D2D6 had no significant effect on mitosis or cleavage of whole eggs injected after nuclear disappearance, although it prevented the disappearance of the nuclear envelope in 54% of the eggs injected before the disappearance. FITC-conjugated D2D6 did not accumulate in the spindle when injected into the dividing sand dollar egg. These results indicate that YL1/2 disassembled microtubules, whereas D2D6 did not bind to microtubules in the living cell.     

Identification of a starfish egg PLC-gamma that regulates Ca2+ release at fertilization

At fertilization, eggs undergo a cytoplasmic free Ca2+ rise, which is necessary for stimulating embryogenesis. In starfish eggs, studies using inhibitors designed against vertebrate proteins have shown that this Ca2+ rise requires an egg Src family kinase (SFK) that directly or indirectly activates phospholipase C-gamma (PLC-gamma) to produce IP3, which triggers Ca2+ release from the egg's endoplasmic reticulum (ER) [reviewed in Semin. Cell Dev. Biol. 12 (2001) 45]. To examine in more detail the endogenous factors in starfish eggs that are required for Ca2+ release at fertilization, an oocyte cDNA encoding PLC-gamma was isolated from the starfish Asterina miniata. This cDNA, designated AmPLC-gamma, encodes a protein with 49% identity to mammalian PLC-gamma1. A 58-kDa Src family kinase interacted with recombinant AmPLC-gamma Src homology 2 (SH2) domains in a specific, fertilization-responsive manner. Immunoprecipitations of sea urchin egg PLC-gamma using an affinity-purified antibody directed against AmPLC-gamma revealed fertilization-dependent phosphorylation of PLC-gamma. Injecting starfish eggs with the tandem SH2 domains of AmPLC-gamma (which inhibits PLC-gamma activation) specifically inhibited Ca2+ release at fertilization. These results indicate that an endogenous starfish egg PLC-gamma interacts with an egg SFK and mediates Ca2+ release at fertilization via a PLC-gamma SH2 domain-mediated mechanism.