Centrifugal bisection of starfish oocytes

Immature oocytes or mature eggs of starfish were centrifuged in a sucrose density gradient. They were then separated into two fractions of fragments, nucleate light fragments and anucleate heavy fragments. Vital-staining experiments showed that the oocytes were elongated along the animal-vegetal (AV) axis during the centrifugation in a contrast to centrifuged eggs whose centrifugal axis was not related to the AV axis. The light and heavy oocyte fragments were comprised of animal and vegetal halves of oocytes, respectively. When matured and fertilized, most of the light oocyte fragment-derived embryos failed gastrulation and developed into Dauerblastulae. Two-dimensional gel electrophoretic analysis of fragments revealed that three basic proteins were predominantly enriched in the heavy oocyte fragments but scarcely detected in the light oocyte fragments. One of these proteins, App20, was identified as a homologue of cyclophilin (peptidyl-prolyl cis-trans isomerase). The present study provides a simple means of separating a population of starfish oocytes into animal and vegetal halves, thereby enabling us to analyze any difference of components between animal and vegetal cytoplasm of the oocytes.     

Meiosis reinitiation of mussel oocytes involves L-type voltage-gated calcium channel

In the present work we assessed the involvement of L-type voltage opening Ca²⁺ channels in KCl-induced meiosis reinitiation of metaphase-arrested blue mussel (Mytilus galloprovincialis) oocytes by performing binding assays with a tritiated dihydropyridine analog (+)PN 200110. Our data reveal the existence of a single class of dihydropyridine receptors in plasma membrane-enriched rough microsome preparations of mussel oocytes. The apparent affinity (K_d) of characterized receptors equals 1.32 ± 0.21 μM while their maximal binding capacity (B_max) is 620 ± 150 pmol/mg protein. The comparison of the rank order of potency of analogs tested to: 1) inhibit [(+)-l³H]PN 200110 specific binding and 2) block KCl-induced meiosis reinitiation pointed to the pharmacological profile similar to but not identical with those previously described for mammalian dihydropyridine receptors. The efficiencies of all antagonists tested are linearly related (r = 0.995) in binding- (inhibition of [(+)-l³H]PN 200110 specific binding) and biological (inhibition of meiosis reinitiation) assays thus arguing for functional involvement of L-type Ca²⁺ channels in oocyte activation. Reversibility of antagonist actions on meiosis reinitiation and dependence of receptor binding characteristics on a membrane polarization state further suggested such a role. J. Cell. Biochem. 64:152–160. © 1997 Wiley-Liss, Inc.     

Initiation of cleavage in starfish eggs by the injection of triton-treated spermatozoa

Triton X-100-treated spermatozoa were injected into immature (fully grown, germinal vesicle stage) or mature (pronuclear stage) oocytes of the starfish, Asterina pectinifera, to study relation between initiation of cleavage and cortical reaction. Immature oocytes into which Triton X-100-treated spermatozoa were injected were treated with 1-methyladenine. Such immature oocytes initiated cleavage after completion of meiosis without formation of the fertilization membrane. The same results were obtained when Triton X-100-treated spermatozoa were injected into mature oocytes. Control oocytes into which only calcium-free sea water was injected did not cleave. These results indicate that the initiation of cleavage is independent of the cortical reaction but dependent on the existence of spermatozoa (spermatozoon) in the egg cytoplasm.     

Nuclear envelope breakdown in starfish oocytes proceeds by partial NPC disassembly followed by a rapidly spreading fenestration of nuclear membranes

Breakdown of the nuclear envelope (NE) was analyzed in live starfish oocytes using a size series of fluorescently labeled dextrans, membrane dyes, and GFP-tagged proteins of the nuclear pore complex (NPC) and the nuclear lamina. Permeabilization of the nucleus occurred in two sequential phases. In phase I the NE became increasingly permeable for molecules up to approximately 40 nm in diameter, concurrent with a loss of peripheral nuclear pore components over a time course of 10 min. The NE remained intact on the ultrastructural level during this time. In phase II the NE was completely permeabilized within 35 s. This rapid permeabilization spread as a wave from one epicenter on the animal half across the nuclear surface and allowed free diffusion of particles up to approximately 100 nm in diameter into the nucleus. While the lamina and nuclear membranes appeared intact at the light microscopic level, a fenestration of the NE was clearly visible by electron microscopy in phase II. We conclude that NE breakdown in starfish oocytes is triggered by slow sequential disassembly of the NPCs followed by a rapidly spreading fenestration of the NE caused by the removal of nuclear pores from nuclear membranes still attached to the lamina.     

Ca(2+) response to cADPr during maturation and fertilization of starfish oocytes.

During the reinitiation of the meiotic cycle (maturation) induced by the hormone 1-methyladenine (1-MA), starfish oocytes undergo structural and biochemical changes in preparation for successful fertilization. Previous work has shown that the sensitivity of internal Ca(2+) stores to InsP(3) increases during maturation of the oocytes. Since Astropecten auranciacus oocytes also respond to cADPr, we have studied whether the response to cADPr also changes during maturation. We have found that the photoactivation of injected cADPr in immature oocytes immediately induces multiple patches of Ca(2+) release in the cortical region. The Ca(2+) signal then spreads from these initial points of increase to the entire cell. In mature oocytes, the uncaging of cADPr induces instead a single (or at most a dual) initial point of Ca(2+) release, which is immediately followed by the formation of a cortical Ca(2+) flash and then by the globalization of the wave and by the elevation of the fertilization envelope. External Ca(2+) plays a role in the Ca(2+) responses. Inhibition of L-type Ca(2+) channels does not affect the initial Ca(2+) release, but abolishes the cortical flash and impairs the elevation of the fertilization envelope. External Ca(2+) has other effects, as shown by the irregular appearance of the surface of oocytes incubated in Ca(2+)-free sea water. The sequence of Ca(2+) responses induced by cADPr in mature oocytes mimics those seen at fertilization, i.e., a first localized Ca(2+) increase followed by a cortical flash and by the globalization of the Ca(2+) signal. As in the case of maturation, L-type Ca(2+) channel blockers abolish the sperm induced cortical flash.     

Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes

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.     

Cyclin B synthesis and rapamycin-sensitive regulation of protein synthesis during starfish oocyte meiotic divisions

Translation of cyclin mRNAs represents an important event for proper meiotic maturation and post-fertilization mitoses in many species. Translational control of cyclin B mRNA has been described to be achieved through two separate but related mechanisms: translational repression and polyadenylation. In this paper, we evaluated the contribution of global translational regulation by the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-binding protein) on the cyclin B protein synthesis during meiotic maturation of the starfish oocytes. We used the immunosupressant drug rapamycin, a strong inhibitor of cap-dependent translation, to check for the involvement of this protein synthesis during this physiological process. Rapamycin was found to prevent dissociation of 4E-BP from the initiation factor eIF4E and to suppress correlatively a burst of global protein synthesis occurring at the G2/M transition. The drug had no effect on first meiotic division but defects in meiotic spindle formation prevented second polar body emission, demonstrating that a rapamycin-sensitive pathway is involved in this mechanism. While rapamycin affected the global protein synthesis, the drug altered neither the specific translation of cyclin B mRNA nor the expression of the Mos protein. The expression of these two proteins was correlated with the phosphorylation and the dissociation of the cytoplasmic polyadenylation element-binding protein from eIF4E.     

Nuclear envelope breakdown may deliver an inhibitor of protein phosphatase 1 which triggers cyclin B translation in starfish oocytes

In vertebrates, enhanced translation of mRNAs in oocytes and early embryos entering M-phase is thought to occur through polyadenylation, involving binding, hyperphosphorylation and proteolytic degradation of Aurora-activated CPEB. In starfish, an unknown component of the oocyte nucleus is required for cyclin B synthesis following the release of G2/prophase block by hormonal stimulation. We have found that CPEB cannot be hyperphosphorylated following hormonal stimulation in starfish oocytes from which the nucleus has been removed. Activation of Aurora kinase, known to interact with protein phosphatase 1 and its specific inhibitor Inh-2, is also prevented. The microinjection of Inh-2 restores Aurora activation, CPEB hyperphosphorylation and cyclin B translation in enucleated oocytes. Nevertheless, we provide evidence that CPEB is in fact hyperphosphorylated by cdc2, without apparent involvement of Aurora or MAP kinase, and that cyclin B synthesis can be stimulated without previous degradation of phosphorylated CPEB. Thus, the regulation of cyclin B synthesis necessary for progression through meiosis can be explained by an equilibrium between CPEB phosphorylation and dephosphorylation, and both aspects of this control may rely on the sole activation of Cdc2 and subsequent nuclear breakdown.     

Nuclear transfer in sheep embryos: The effect of cell-cycle coordination between nucleus and cytoplasm and the use of in vitro matured oocytes

The developmental ability of nuclear transplant sheep embryos derived from in vitro matured oocytes was studied by controlling cell-cycle coordination of donor embryonic nuclei and recipient cytoplasts. Oocytes were recovered from nonatretic antral follicles of adult sheep ovaries and cocultured with follicle shells in M199-based medium supplemented with gonadotrophins in a nonstatic system. Effective activation of IVM oocytes was obtained by applying two pulses of 1.0 kv/cm 22 min apart in inositol-based electroporation medium to oocytes matured in vitro for 27 hr. Synthesis of DNA (S-phase) was assessed by BrdU incorporation and was found to initiate around 5 hpa (hours postactivation) and to persist until 18 hpa. Mitotic blastomeres were induced by treating embryos with 6.6 μM nocodazole for 14–17 hr. Three types of transfers were compared directly: “S → S,” early embryonic nuclei (mostly in S-phase) were transferred to presumptive S-phase cytoplasts; “M → MII,” nocodazole-treated embryonic nuclei (most in M-phase) were transferred to MII-phase cytoplasts; and control (S → MII), conventional nuclear transfer of fusion and activation simultaneously. The results showed that fusion and recovery rates did not differ among the three groups. However, after 6 days of in vivo culture, the morula and blastocyst formation rate was significantly higher for the M → MII combination than for the control (28.3% vs. 8.1%, P < 0.05), while no significant differences in developmental rate were observed between S → S and M → MII, and between S → S and control, though developmental rate was also increased for S → S compared to control (20.9% vs. 8.1%, P > 0.05). Transfer of blastocysts derived from M → MII or S → S nuclear cytoplasmic reconstitution to synchronized recipient ewes resulted in the birth of lambs. These data suggest that in vitro matured oocytes can support full-term development of nuclear transplant sheep embryos when the cell cycle of nucleus and cytoplasm is coordinated, and that M → MII nuclear transfer might be an efficient and simple way to improve the developmental competence of the reconstituted embryos. Mol. Reprod. Dev. 47:255–264, 1997. © 1997 Wiley-Liss, Inc.     

A study of quantitative dynamics of F-actin during oocyte maturation in the starfish Asterias amurensis

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 α_i-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.     

Pharmacological characterization of NAADP-induced Ca2+ signals in starfish oocytes

The recently discovered second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) is central to the onset of intracellular Ca2+ signals induced by several stimuli, including fertilization. The nature of the Ca2+ pool mobilized by NAADP is still controversial. Depending on the cell type, NAADP may target either an acidic compartment with lysosomal properties or ryanodine receptors (RyRs) on endoplasmic reticulum. In addition, NAADP elicits a robust Ca2+ influx into starfish oocytes by activating a Ca2+-mediated current across the plasma membrane. In the present study, we employed the single-electrode intracellular recording technique to assess the involvement of either acidic organelles or RyRs in NAADP-elicited Ca2+ entry. We found that neither drugs which interfere with acidic compartments nor inhibitors of RyRs affected NAADP-induced depolarization. These data further support the hypothesis that a yet unidentified plasma membrane Ca2+ channel is the target of NAADP in starfish oocytes.     

Calcium and calcium-binding proteins in the nucleus

Calcium has long been known to play a role as a key cytoplasmic second messenger, but until relatively recently its possible involvement in nuclear signal transduction and the regulation of nuclear events has not been extensively studied. Evidence revealing the presence of transmembrane nuclear Ca²⁺ gradients and a variety of intranuclear Ca²⁺ binding proteins has fueled renewed interest in this key ion and its involvement in cell-cycle timing and division, gene expression, and protein activation. This review will offer an overview of the current state of knowledge and theory regarding calcium orchestration of nuclear functions and events and discuss possible future directions in this field of study.     

Formation of polar-body-like structures induced in polyspermic starfish oocytes that had been inseminated at the germinal vesicle stage

Immature starfish oocytes, which are arrested at the first meiotic prophase and contain a large nucleus called the germinal vesicle (GV), are known to accept multiple sperm on insemination. We found that if these polyspermic starfish oocytes are induced to mature, they often form small protrusion(s) adjacent to the first polar body emitted shortly earlier. We refer to these protrusion(s) as 'polar-body-like structures (PLS).' Fluorescent staining of PLS indicated that they were not merely cytoplasmic protrusions, but contained some chromatin. Maturing process of these polyspermic oocytes was examined by immnofluorescent staining, which showed that: (i) numerous sperm asters were observed after the onset of GV breakdown; (ii) before the first polar body (PB1) emission, a complex microtubular structure resembling a multipolar spindle was formed; and (iii) several isolated asters were observed after PB1 emission. These results indicate that PLS formation may be induced by interaction of meiosis-I spindle with paternal centrosomes incorporated at GV stage.     

Nanosecond electric pulses penetrate the nucleus and enhance speckle formation

Nanosecond electric pulses generate nanopores in the interior membranes of cells and modulate cellular functions. Here, we used confocal microscopy and flow cytometry to observe Smith antigen antibody (Y12) binding to nuclear speckles, known as small nuclear ribonucleoprotein particles (snRNPs) or intrachromatin granule clusters (IGCs), in Jurkat cells following one or five 10 ns, 150 kV/cm pulses. Using confocal microscopy and flow cytometry, we observed changes in nuclear speckle labeling that suggested a disruption of pre-messenger RNA splicing mechanisms. Pulse exposure increased the nuclear speckled substructures by ∼2.5-fold above basal levels while the propidium iodide (PI) uptake in pulsed cells was unchanged. The resulting nuclear speckle changes were also cell cycle dependent. These findings suggest that 10 ns pulses directly influenced nuclear processes, such as the changes in the nuclear RNA-protein complexes.     

Nucleolar targeting of 5S RNA in Xenopus laevis oocytes: Somatic-type nucleotide substitutions enhance nucleolar localization

In Xenopus laevis oocytes, 5S RNA is stored in the cytoplasm until vitellogenesis, at which time it is imported into the nucleus and targeted to nucleoli for ribosome assembly. This article shows that throughout oogenesis there is a pool of nuclear 5S RNA which is not nucleolar-associated. This distribution reflects that of oocyte-type 5S RNA, which is the major 5S RNA species in oocytes; only small amounts of somatic-type, which differs by six nucleotides, are synthesized. Indeed, ³²P-labeled oocyte-type 5S RNA showed a degree of nucleolar localization similar to endogenous 5S RNA (33%) after microinjection. In contrast, ³²P-labeled somatic-type 5S RNA showed significantly enhanced localization, whereby 70% of nuclear RNA was associated with nucleoli. A chimeric RNA molecule containing only one somatic-specific nucleotide substitution also showed enhanced localization, in addition to other somatic-specific phenotypes, including enhanced nuclear import and ribosome incorporation. The distribution of ³⁵S-labeled ribosomal protein L5 was similar to that of oocyte-type 5S RNA, even when preassembled with somatic-type 5S RNA. The distribution of a series of 5S RNA mutants was also analyzed. These mutants showed various degrees of localization, suggesting that the efficiency of nucleolar targeting can be influenced by many discrete regions of the 5S RNA molecule. J. Cell. Biochem. 69:490–505, 1998. © 1998 Wiley-Liss, Inc.     

核不均一性核糖核蛋白在RNA加工过程中的作用

在真核细胞中,初始转录产物（前体mRNA）经过一系列复杂的转录后加工过程形成成熟的mRNA.在这一过程中,大量蛋白质和加工因子有序汇集在核糖核蛋白复合体中并参与对前体RNA的加工过程. 该复合体中的蛋白质部分主要由一类约20种称为核不均一性核糖核蛋白的多肽分子构成.除了早期了解的一些结构性功能外,近来已有许多证据显示这些蛋白质在细胞中RNA的代谢及其他活动方面具有更加广泛和积极的作用.