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1.
We studied the actin cytoskeleton state in Asterias amurensis oocytes within 30 min after the 1-methyladenine-induced maturation until the germinal vesicle breakdown. The total amount of actin remained unchanged during oocyte maturation. In immature oocytes, the major part of actin is not a part of filaments, but in the presence of 1-methyladenine massive actin polymerization began already within 20 min. Electron immunocytochemistry methods demonstrated joint localization of actin and alpha-protein in the cytoplasm. They were redistributed from the cortex to the cytoplasm in the presence of 1-methyladenine. A possible involvement of actin cytoskeleton in transmembrane transduction of the hormonal signal at the postreceptor stages is discussed. 相似文献
2.
Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes 总被引:2,自引:0,他引:2
Kyozuka K Chun JT Puppo A Gragnaniello G Garante E Santella L 《Developmental biology》2008,320(2):426-435
Before successful fertilization can occur, oocytes must undergo meiotic maturation. In starfish, this can be achieved in vitro by applying 1-methyladenine (1-MA). The immediate response to 1-MA is the fast Ca2+ release in the cell cortex. Here, we show that this Ca2+ wave always initiates in the vegetal hemisphere and propagates through the cortex, which is the space immediately under the plasma membrane. We have observed that alteration of the cortical actin cytoskeleton by latrunculin-A and jasplakinolide can potently affect the Ca2+ waves triggered by 1-MA. This indicates that the cortical actin cytoskeleton modulates Ca2+ release during meiotic maturation. The Ca2+ wave was inhibited by the classical antagonists of the InsP3-linked Ca2+ signaling pathway, U73122 and heparin. To our surprise, however, these two inhibitors induced remarkable actin hyper-polymerization in the cell cortex, suggesting that their inhibitory effect on Ca2+ release may be attributed to the perturbation of the cortical actin cytoskeleton. In post-meiotic eggs, U73122 and jasplakinolide blocked the elevation of the vitelline layer by uncaged InsP3, despite the massive release of Ca2+, implying that exocytosis of the cortical granules requires not only a Ca2+ rise, but also regulation of the cortical actin cytoskeleton. Our results suggest that the cortical actin cytoskeleton of starfish oocytes plays critical roles both in generating Ca2+ signals and in regulating cortical granule exocytosis. 相似文献
3.
In starfish, oocytes are released from prophase block by a hormone, which has been identified as 1-methyladenine. The action of 1-methyladenine is indirect in inducing oocyte maturation: it acts on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), the direct trigger of germinal vesicle breakdown (GVBD). Less than 5 min after hormone addition, thus about 10 min before appearance of the cytoplasmic maturation-promoting factor, a factor appears in the germinal vesicle, which triggers the production of cytoplasmic MPF, GVBD, and the subsequent events of meiotic maturation when transferred in the cytoplasm of any fully grown oocyte of the starfishes Marthasterias glacialis and Asterias rubens. Before hormone action, the germinal vesicle also contains a factor capable of inducing meiosis reinitiation in recipient oocytes, but in contrast with nuclear MPF, this factor acts exclusively when transferred in the cytoplasm of a special category of oocytes (the “competent” oocytes). In contrast to other oocytes (the “incompetent” oocytes) the competent oocytes are capable of producing MPF to some extent after enucleation, upon hormonal stimulation. Transfer of either nuclear or cytoplasmic MPF initially produced in hormone-treated maturing oocytes triggers the production of both cytoplasmic and nuclear MPF in non-hormone-treated recipient oocytes of both categories. 相似文献
4.
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. 相似文献
5.
Actin and spectrin localization in the oocytes of the starfish Asterias amurensis at hormonal induction of maturation until the destruction of the germinal vesicular membrane has been investigated by immunocytochemical and immunoblotting methods. In immature oocytes, spectrinlike protein and actin are detected to be colocalized in the undermembranous area of the cytoplasm and nuclear membrane. 1-Methyladenine causes redistribution of these proteins into intracellular structures. The actin-spectrin cytoskeleton rearrangement is shown to start at the animal pole of the oocyte and to spread then to its vegetative pole. 相似文献
6.
The effects of 1-methyladenine on oxygen consumption and adenine nucleotide levels were examined in oocytes of Pisaster ochraceus and Patiria miniata. Oocytes of both genera to which 1-methyladenine was added consumed more oxygen than control oocytes beginning 1 to after 1-methyladenine addition. The increase in oxygen consumption was correlated with maturation changes in the oocytes and particularly with germinal vesicle breakdown. Pre-fertilization oxygen consumption of eggs did not differ significantly from post-fertilization oxygen consumption of eggs in either genus for after fertilization. ATP and AMP concentrations within the oocytes decreased during 1-methyladenine induced maturation, while the ADP concentration increased. It was suggested that increases in ADP concentration and decreases in ATP concentration within maturing starfish oocytes occurred in response to greater energy demands. The simultaneous increase in oocyte oxygen consumption was interpreted as an indicator of increased oxidative phosphorylation acting to restore initial nucleotide concentrations. 相似文献
7.
Keiichiro Kyozuka Jong T. Chun Agostina Puppo Gianni Gragnaniello Ezio Garante Luigia Santella 《PloS one》2009,4(7)
Background
Starfish oocytes are arrested at the first prophase of meiosis until they are stimulated by 1-methyladenine (1-MA). The two most immediate responses to the maturation-inducing hormone are the quick release of intracellular Ca2+ and the accelerated changes of the actin cytoskeleton in the cortex. Compared with the later events of oocyte maturation such as germinal vesicle breakdown, the molecular mechanisms underlying the early events involving Ca2+ signaling and actin changes are poorly understood. Herein, we have studied the roles of G-proteins in the early stage of meiotic maturation.Methodology/Principal Findings
By microinjecting starfish oocytes with nonhydrolyzable nucleotides that stabilize either active (GTPγS) or inactive (GDPβS) forms of G-proteins, we have demonstrated that: i) GTPγS induces Ca2+ release that mimics the effect of 1-MA; ii) GDPβS completely blocks 1-MA-induced Ca2+; iii) GDPβS has little effect on the amplitude of the Ca2+ peak, but significantly expedites the initial Ca2+ waves induced by InsP3 photoactivation, iv) GDPβS induces unexpectedly striking modification of the cortical actin networks, suggesting a link between the cytoskeletal change and the modulation of the Ca2+ release kinetics; v) alteration of cortical actin networks with jasplakinolide, GDPβS, or actinase E, all led to significant changes of 1-MA-induced Ca2+ signaling.Conclusions/Significance
Taken together, these results indicate that G-proteins are implicated in the early events of meiotic maturation and support our previous proposal that the dynamic change of the actin cytoskeleton may play a regulatory role in modulating intracellular Ca2+ release. 相似文献8.
Luigia Santella Laura De Riso Giovanni Gragnaniello Keiichiro Kyozuka 《Experimental cell research》1999,248(2):567
Cortical granules (secretory vesicles located under the cortex of mature oocytes) release their contents to the medium at fertilization. Their exocytosis modifies the extracellular environment, blocking the penetration of additional sperm. The granules translocate to the surface during the maturation process, and it has been suggested that they move to the cortex via cytoskeletal elements. In this paper we show that the increase in intracellular Ca2+, which the maturing hormone 1-methyladenine (1-MA) induces in starfish through the activation of inositol 1,4,5-trisphosphate (InsP3) receptors, triggers changes in filamentous actin, which then direct the correct movement and reorientation of the cortical granules and the elevation of the fertilization envelope. 相似文献
9.
Cellular mechanisms generating the polarized redistribution of maternal Type I postplasmic/PEM mRNAs in ascidian oocytes remain unknown. We have previously shown that PEM-1 mRNA is associated with a network of rough cortical Endoplasmic Reticulum (cER) polarized along the animal-vegetal (a-v) axis forming a cER-mRNA domain in mature oocytes. We now investigate the a-v polarization of this cER-mRNA domain during meiotic maturation using H. roretzi and C. intestinalis. We show that the cER and Hr-PEM-1 aggregate as interconnected cortical patches at the cell periphery before maturation, which uniformly spread out during maturation and form a reticulated organization enriched in the vegetal hemisphere at the end of maturation. Time-lapse video recordings coupled with micromanipulations reveal that stereotyped surface, cortical and cytoplasmic flows accompany the vegetal shift of the cER-mRNA domain and mitochondria-rich myoplasm. Treatments with cytochalasin B and nocodazole indicate that both polarization of the cER-mRNA domain and mitochondria-rich myoplasm and cortical and cytoplasmic flows depend on actin cytoskeleton, but not microtubules. Using cortical fragments prepared from maturing oocytes coupled with high resolution immuno/in situ localization, we have further analyzed the effects of these inhibitors on the reorganizations the cER network and Hr-PEM-1 mRNA. We show that before maturation starts, Hr-PEM-1 mRNAs are already associated with the cER, and actin cytoskeleton inhibitors disturb their association. Finally, we hypothesize that Germinal Vesicle Break Down (GVBD) triggers an actomyosin-dependant cortical flow which directs the a-v polarization of ascidian oocytes. 相似文献
10.
Endogenous protein phosphorylation in oocytes of Marthasterias glacialis was examined by incubating living oocytes with [32P]phosphate and cortical and endoplasmic fractions with [γ-32P]ATP. Individual phosphorylated proteins were detected by autoradiography after bidimensional and monodimensional electrophoresis, using SDS-polyacrylamide gradient gel slabs. An increased phosphorylation of several protein species was observed as early as 5 min following in vivo hormonal stimulation by 1-methyladenine. No dephosphorylation nor any change in protein staining of the gels was observed. In vitro phosphorylation patterns were consistent with those observed in vivo. They did not change upon in vitro 1-methyladenine addition and remained unaffected when the incubations were carried out in the presence of cAMP or beef heart protein kinase inhibitor. Cortical phosphorylation was inhibited by calcium ions. The results suggest that the hormone promotes alterations in the availability of phosphorylation sites in some proteins already present in the control oocytes which, as well as the corresponding activated cAMP-independent protein kinases, may play a significant role during formation of the maturation promoting factor. 相似文献
11.
Reproduction may be affected by stressful events changing the female endocrine or metabolic profile. An altered environment during oocyte development could influence the delicate process of oocyte maturation. Here, the effect of simulated stress by media supplementation with blood plasma from sows after adrenocorticotropic hormone (ACTH) administration during the preovulatory period was assessed. Oocytes were matured for 46 hours in the presence of plasma from ACTH-treated sows, or plasma from NaCl-treated control sows, or medium without plasma (BSA group). The plasma used had been collected at 36 and 12 hours (±2 hours) before ovulation (for the first 24 hours + last 22 hours of maturation, respectively). Subsequent fertilization and embryo development were evaluated. Actin cytoskeleton and mitochondrial patterns were studied by confocal microscopy both in the oocytes and the resulting blastocysts. Nuclear maturation did not differ between treatments. Subtle differences were observed in the actin microfilaments in oocytes; however, mitochondrial patterns were associated with the treatment (P < 0.001). These differences in mitochondrial patterns were not reflected by in vitro outcomes, which were similar in all groups. In conclusion, an altered hormonal environment provided by a brief exposure to plasma from ACTH-treated sows during in vitro oocyte maturation could induce alterations in actin cytoskeleton and mitochondrial patterns in oocytes. However, these changes might not hamper the subsequent in vitro embryo development. 相似文献
12.
Background
When preparing for fertilization, oocytes undergo meiotic maturation during which structural changes occur in the endoplasmic reticulum (ER) that lead to a more efficient calcium response. During meiotic maturation and subsequent fertilization, the actin cytoskeleton also undergoes dramatic restructuring. We have recently observed that rearrangements of the actin cytoskeleton induced by actin-depolymerizing agents, or by actin-binding proteins, strongly modulate intracellular calcium (Ca2+) signals during the maturation process. However, the significance of the dynamic changes in F-actin within the fertilized egg has been largely unclear.Methodology/Principal Findings
We have measured changes in intracellular Ca2+ signals and F-actin structures during fertilization. We also report the unexpected observation that the conventional antagonist of the InsP3 receptor, heparin, hyperpolymerizes the cortical actin cytoskeleton in postmeiotic eggs. Using heparin and other pharmacological agents that either hypo- or hyperpolymerize the cortical actin, we demonstrate that nearly all aspects of the fertilization process are profoundly affected by the dynamic restructuring of the egg cortical actin cytoskeleton.Conclusions/Significance
Our findings identify important roles for subplasmalemmal actin fibers in the process of sperm-egg interaction and in the subsequent events related to fertilization: the generation of Ca2+ signals, sperm penetration, cortical granule exocytosis, and the block to polyspermy. 相似文献13.
Studies on the appearance and nature of a maturation-inducing factor in the cytoplasm of amphibian oocytes exposed to progesterone 总被引:20,自引:0,他引:20
Full-grown oocytes of amphibians respond in vitro to exogenous progesterone by undergoing physiological maturation (breakdown of the germinal vesicle (GVBD), meiosis, and acquisition of the capacity for activation). Both cytoplasm and “cytosol” from maturing oocytes have been shown to produce similar events when injected into unstimulated oocytes. This activity appeared within 4 hr after hormone treatment in Rana pipiens and Xenopus laevis and represents the earliest detectable, specific response of the oocyte yet observed, i.e., 6–8 hr before GVBD in Rana. Maturing oocytes retained activity as long as 100 hr after exposure to progesterone, and activity was also obtained from ovulated eggs and cleaving embryos. In addition, cytoplasm from Rana pipiens, Xenopus laevis, or Ambystoma mexicanum was effective in inducing maturation in oocytes of each other, indicating a lack of specificity.Recipient oocytes of Xenopus laevis consistently began to mature within 1.5–3 hr after injection of maturing cytoplasm, well before progesterone-treated controls. The timing of the response was closely related to the quantity of cytoplasm transferred, suggesting the presence of both a minimum and threshold level of cytoplasmic factor. Serial cytoplasmic transfer in Xenopus oocytes showed no significant loss of activity through 10 injections. 相似文献
14.
Protein phosphorylation with specific protein kinases plays the key role in the regulation of meiotic maturation of oocytes. However, little is known about the contribution of kinases to the temporal and positional regulation of the cytoskeleton rearrangement in maturing oocytes, including the actin cytoskeleton. In order to study a relationship between the kinase activities and actin cytoskeleton rearrangement, we analyzed protein phosphorylation in the isolated actin cytoskeleton of Xenopus laevis oocytes. Analysis of the full grown oocytes and eggs injected with [-32P]ATP has revealed phosphorylation of many proteins associated with the actin cytoskeleton and shown the appearance of three additional major phosphoproteins, 20, 43, and 69 kDa, during oocyte maturation. A significant number of these phosphoproteins were also found after incubation of the isolated cytoskeleton with [-32P]ATP in vitro, thus confirming that the kinases modifying these substrates are also specifically associated with actin. The in vivo and in vitro kinase activities were also stimulated during maturation. Analysis of kinase self-phosphorylation in situ and protein phosphorylation in solutions and substrate containing gels revealed a set of actin-associated kinases, including cAMP- and Ca2+-dependent kinases, as well as MAP, p34cdc2, and tyrosine kinase activities. Their level was the highest in the eggs. The involvement of kinases in the actin cytoskeleton rearrangement during oocyte maturation is discussed. 相似文献
15.
Lim D Ercolano E Kyozuka K Nusco GA Moccia F Lange K Santella L 《The Journal of biological chemistry》2003,278(43):42505-42514
The resumption of the meiotic cycle (maturation) induced by 1-methyladenine in prophase-arrested starfish oocytes is indicated by the breakdown of the germinal vesicle and is characterized by the increased sensitivity of the Ca2+ stores to inositol 1,4,5-trisphosphate (InsP3) to InsP3 starting at the animal hemisphere (where the germinal vesicle was originally located) and propagating along the animal/vegetal axis of the oocyte. This initiates Ca2+ signals around the germinal vesicle before nuclear envelope breakdown. Previous studies have suggested that the final activation of the maturation-promoting factor (MPF), a cyclin-dependent kinase, which is the major element controlling the entry of eukaryotic cells into the M phase, occurs in the nucleus. MPF is then exported to the cytoplasm where its activity is autocatalytically amplified following a similar animal/vegetal spatial pattern. We have investigated whether activated MPF was involved in the increased sensitivity of the Ca2+ response to InsP3. We have found that the development of increased sensitivity of the Ca2+ stores to InsP3 receptors together with the Ca2+ signals in the perinuclear region was blocked in oocytes treated with the specific MPF inhibitor roscovitine. That the nuclear MPF activation is indeed required for changes of the InsP3 receptors sensitivity was shown by enucleating or by dissecting oocytes into vegetal and animal hemispheres prior to the addition of 1-MA. MPF activity 50 min after 1-methyladenine addition was much lower in the enucleated oocytes and in the vegetal hemisphere, which did not contain the germinal vesicle, as compared with the animal hemisphere, which did contain it. The Ca2+ increase induced by InsP3 under these experimental conditions correlated with the changes in actin cytoskeleton induced by MPF. 相似文献
16.
Tomoya Kotani Kyota Yasuda Ryoma Ota Masakane Yamashita 《The Journal of cell biology》2013,202(7):1041-1055
Temporal control of messenger RNA (mRNA) translation is an important mechanism for regulating cellular, neuronal, and developmental processes. However, mechanisms that coordinate timing of translational activation remain largely unresolved. Full-grown oocytes arrest meiosis at prophase I and deposit dormant mRNAs. Of these, translational control of cyclin B1 mRNA in response to maturation-inducing hormone is important for normal progression of oocyte maturation, through which oocytes acquire fertility. In this study, we found that dormant cyclin B1 mRNA forms granules in the cytoplasm of zebrafish and mouse oocytes. Real-time imaging of translation revealed that the granules disassemble at the time of translational activation during maturation. Formation of cyclin B1 RNA granules requires binding of the mRNA to Pumilio1 protein and depends on actin filaments. Disruption of cyclin B1 RNA granules accelerated the timing of their translational activation after induction of maturation, whereas stabilization hindered translational activation. Thus, our results suggest that RNA granule formation is critical for the regulation of timing of translational activation. 相似文献
17.
Oocyte maturation (meiosis re-initiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Following hormonal stimulation of the oocyte, an intracellular Maturation Promoting Factor (MPF) appears in the cytoplasm which triggers nuclear envelope breakdown and maturation divisions. α-Naphthylphosphate (α-NP), a widely used phosphatase inhibitor/substrate, was found to induce oocyte maturation when microinjected intracellularly (50% maturation at 3.5 mM; 100% above 6 mM, final intracellular concentration) into oocytes of Marthasterias and Asterias but not of Astropecten. As 1-MeAde, α-NP triggers a complete maturation, i.e. germinal vesicle breakdown, extrusion of the two polar bodies and formation of the female pronucleus. The kinetics of α-NP-induced maturation (35–45 min) is, however, longer than the kinetics of 1-MeAde-induced maturation (18–20 min). The addition of α-NP externally to oocytes does not trigger maturation. Among several reported phosphatase inhibitors, including two natural protein phosphatase inhibitors and several products structurally related to α-NP, only α-NP was found capable of inducing maturation when microinjected into oocytes. α-NP triggers the appearance of MPF activity in the cytoplasm of oocytes into which it has been injected. Although α-NP-induced maturation is insensitive to inhibitors whose action is known to be restricted to the hormone-dependent period (such as the protease inhibitor leupeptin), it is blocked by inhibitors of MPF action (such as nicotinamide and lithium). Finally it was found that α-NP-induced maturation is inhibited by simultaneous microinjection of protein phosphatase-2A; also, α-NP, classically used as an inhibitor of acid and alkaline phosphatases, is able to inhibit protein phosphatases 1 and 2 A. The addition of α-NP to oocytes increases the level of phosphorylated proteins. These results constitute direct evidence that an elevated level of phosphorylated proteins is sufficient to trigger MPF activity and to induce maturation. 相似文献
18.
Barbara Wabik-Sliz 《Molecular reproduction and development》1986,15(1):87-94
The time taken to dissolve the zona pellucida was compared with fertilizability as well as the meiotic maturation rate of the oocytes from the same (KE × CBA) F2 females. The presence of granular material in oocyte cytoplasm was also examined. It was found that for F2 females in which the zona pellucida digestion was fast, the number of fertilized oocytes was high; for F2 females with zonae pellucidae more resistant to enzyme, the number of fertilized oocytes was low. The correlation between the two characters was significant, indicating their common genetic and/or physiological control. The low or high solubility of zona pellucida did not correlate with the rate of meiotic maturation of the oocyte. This suggests separate factors controlling these two characters. A separate factor seems to control the appearance of granules in cytoplasm since their presence interfered neither with zona pellucida solubility nor with maturation rate of the oocyte. 相似文献
19.
20.
KIYOSHI SANO TAKEO KISHIMOTO SUMI L. KOIDE HARUO KANATANI SAMUEL S. KOIDE 《Development, growth & differentiation》1979,21(5):457-464
Nicotinamide inhibited both germinal vesicle breakdown (GVBD) and polar body formation (PBF) in surf clam and starfish oocytes. In the surf clam nicotinamide at 0.3 mM completely blocked PBF in the fertilized oocytes. For blockage of GVBD higher concentration was required. In the starfish, nicotinamide (30 mM) prevented PBF but not GVBD, when added 7 min after the commencement of 1-methyladenine (1-MeAde) administration. These results suggest that PBF is blocked by nicotinamide independent of its effect on GVBD. In the case of starfish, NAD+was more effective than nicotinamide in inhibiting oocyte maturation. Nicotinamide also blocked GVBD induced by microinjection of the cytoplasm containing maturation-promoting factor (MPF) obtained from 1-MeAde-treatcd oocytes. These results suggest that nicotinamide prevents the action of MPF rather than inhibiting the interaction of 1-McAde with cell membrane or the induction of MPF. 相似文献