Protein phosphorylation during activation of surf clam oocytes.

We have investigated the increase of phosphorylated proteins upon activation of surf clam (Spisula solidissima) oocytes, by measuring the cumulative incorporation of 32P in proteins and by performing an SDS-PAGE and autoradiographic analysis of 32P-labeled proteins, from oocytes initially radiolabeled with 32P-orthophosphate. The phosphorylation inhibitor 6-dimethylaminopurine (6-DMAP) inhibits both germinal vesicle breakdown (GVBD) and the normal increase in phosphorylated proteins observed upon activation by KCl, in a reversible and dose-dependent manner. Using different artificial seawaters (normal, Ca(2+)-free, Na(+)-free), we observed that the increase of phosphorylated proteins, upon K+ stimulation, occurs only when GVBD is allowed to proceed along with an increased Ca2+ influx, in normal or Na(+)-free seawater. Stimulation of oocytes by ammonia, which directly raises intracellular pH (pHi) but does not trigger GVBD, is without effect on the level or pattern of phosphorylated proteins. The link between the Ca2+ influx and the level of phosphorylated proteins was further investigated using conditions altering the duration or the level of Ca2+ influx upon K+ stimulation. In all conditions tested, both GVBD and the level of phosphorylated proteins were similarly affected by alterations of the Ca2+ influx, indicating that these processes are tightly coupled one with another. Upon activation of oocytes, six major proteins of estimated molecular weights of 31, 41, 48, 56, 80 and 86 kDa undergo an increased phosphorylation that is reversibly sensitive to 6-DMAP. Our results suggest that increased protein phosphorylation, sensitive to 6-DMAP, is necessary for GVBD and that it is indirectly linked to the increased Ca2+ influx that stands as an upstream trigger for activation, while an elevated pHi alone has no effect on these processes.     

Nuclear envelope disassembly and nuclear lamina depolymerization during germinal vesicle breakdown in starfish

During germinal vesicle breakdown (GVBD) in starfish, the nuclear envelope disassembles before the nuclear lamina completely depolymerizes, judging from correlative ultrastructural, immunolabeling, and light microscopic analyses. At 13 degrees C, prophase-arrested oocytes of Pisaster ochraceus begin GVBD and rapidly undergo nuclear envelope disassembly about 50 min after addition of the maturation-inducing hormone 1-methyladenine (1-MA). The nuclear lamina of these oocytes, however, remains present for 10-20 min following the vesiculation of the nuclear envelope. Completion of GVBD, as evidenced by a blending of the nuclear contents with the surrounding cytoplasm, occurs within about 15 min after the nuclear lamina has fully depolymerized. Immunofluorescence studies also indicate that a marked increase in the phosphorylations of nuclear proteins precedes the structural reorganizations of the nuclear envelope and nuclear lamina during GVBD.     

Meiotic breakdown of nuclear envelope in oocytes of Spisula solidissima involves phosphorylation and release of nuclear lamin

During meiotic nuclear envelope breakdown (NEBD) in maturing oocytes of the surf clam, Spisula solidissima, the 67-kDa lamin is extensively phosphorylated, concurrently with its solubilization. This is accompanied by a reduction of the nuclear diameter. Quercetin, a protein kinase inhibitor, does not affect lamin phosphorylation and release, nor NEBD per se, but specifically inhibits the early phosphorylation of a set of proteins, on which NEBD seems to depend. Our results suggest that meiotic NEBD in Spisula oocytes may be controlled by a mechanism which involves lamin phosphorylation, similar to that which is thought to operate in mitosis.     

Thapsigargin induces meiotic maturation in surf clam oocytes.

I report here that thapsigargin, an inhibitor of Ca(2+)-ATPase activities in internal Ca2+ stores, induces meiotic maturation in prophase I-arrested surf clam (Spisula solidissima) oocytes. The half-maximal dose for triggering germinal vesicle breakdown (GVBD) is 120 nM. Thapsigargin-induced GVBD is followed by all normal subsequent steps of meiotic maturation including extrusions of first and second polar bodies, with almost normal timing as compared with K(+)-induced activation. Thapsigargin-induced GVBD requires the presence of external Ca2+ at a half-maximal concentration of 0.6 mM. In normal sea water, thapsigargin-induced activation is accompanied by a slightly increased 45Ca2+ uptake by the oocytes and by an intracellular pH rise of 0.3 U. These results show that thapsigargin-sensitive Ca2+ pools regulating Ca2+ fluxes exist in surf clam oocytes, and they also further establish that Ca2+ ions are the major initial trigger for meiosis resumption in this species.     

Inositol trisphosphate, inositol phospholipid metabolism, and germinal vesicle breakdown in surf clam oocytes

Others have reported that microinjection of inositol 1,4,5-trisphosphate (InsP3) releases stored intracellular Ca2+ and causes fertilization envelope elevation, part of the activation process normally initiated by fertilization in deuterostome eggs. In the protostome, Spisula solidissima, germinal vesicle breakdown (GVBD) is the first visible response of the egg to fertilization. To test the effects of InsP3 on egg activation in this organism, we microinjected the compound into oocytes. Microinjection of 0.4-7.0 x 10(-21) moles of InsP3 (equivalent to 5-80 pM if distributed throughout the cell) elicited GVBD in a dose-dependent manner, demonstrating that increased oocyte InsP3 can mimic part of the activation process in this protostome. Synthesis of InsP3 occurs in vivo when phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is hydrolyzed by phospholipase C. To determine whether stimulus-induced synthesis of InsP3 occurs after fertilization of Spisula oocytes, we labeled oocyte lipids with [32P]orthophosphate and measured the radioactivity in phospholipids after insemination. Fertilization resulted in a rapid, transient loss of radioactivity from PtdInsP2. Because the radioactivity in phosphatidylinositol 4-phosphate and other phospholipids did not change, the loss of radioactivity from PtdInsP2 is most likely due to its hydrolysis, yielding InsP3 and diacylglycerol. The latter compound activates protein kinase C which has also been shown to be involved in regulating Spisula oocyte GVBD. Since both of these compounds appear to be early products of fertilization, they could coordinately activate Ca2+- and protein kinase C-dependent processes involved in Spisula oocyte GVBD. These data indicate that egg activation in this protostome includes pathways similar to those found in deuterostome eggs and in other eukaryotic cells.     

Nuclear envelope breakdown requires overcoming the mechanical integrity of the nuclear lamina

In prophase cells, lamin B1 is the major component of the nuclear lamina, a filamentous network underlying the nucleoplasmic side of the nuclear membrane, whereas lamin A/C is dissociated from the scaffold. In vivo fluorescence microscopy studies have shown that, during the G2/M transition, the first gap in the nuclear envelope (NE) appears before lamin B1 disassembly and is caused by early spindle microtubules impinging on the NE. This result suggests that the mechanical tearing of the NE by microtubules plays a central role to the progression of mitosis. To investigate whether this microtubule-induced NE deformation is sufficient for NE breakdown, we assess the mechanical resilience of a reconstituted lamin B1 network. Quantitative rheological methods demonstrate that human lamin B1 filaments form stiff networks that can resist much greater deformations than those caused by microtubules impinging on the NE. Moreover, lamin B1 networks possess an elastic stiffness, which increases under tension, and an exceptional resilience against shear deformations. These results demonstrate that both mechanical tearing of the lamina and biochemical modification of lamin B1 filaments are required for NE breakdown.     

The oocyte lamin persists as a single major component of the nuclear lamina during embryonic development of the surf clam

Nuclei and nuclear lamina-enriched fractions, isolated from 1 to 5-day-old embryos of the surf clam, Spisula solidissima, contain only one major lamin protein, which appears to be identical to the oocyte lamin (L67), as judged by 2D IEF/SDS PAGE, reactivity with a polyclonal antibody directed against L67 and 125I tryptic peptide mapping. The same protein is also present in liver, muscle, nerve and testis from adult animals. No proteins--recognized by several poly- and monoclonal antibodies, specific for somatic lamins from different vertebrate species or the oocyte lamin LIII of Xenopus- have been detected in nuclei or NL-enriched preparations, isolated from embryos or adult tissues. Synthesis of L67 is detectable in embryos 2h after fertilization; it reaches a maximum in 6h-old embryos and gradually declines thereafter. These results argue that the composition of the NL bears no obvious relationship to the structural and functional changes that take place during the embryonic development of this invertebrate.     

Partial activation of surf clam oocytes by hypertonic treatment.

In prophase I-arrested surf clam oocytes, fertilization is the normal trigger for resumption of meiosis, first evidenced by germinal vesicle breakdown (GVBD). Various artificial agents are able to induce GVBD and most of them require the presence of external Ca²⁺ to be efficient. One exception to this rule is the reported possibility of inducing GVBD by an hypertonic treatment, using high concentrations of glycerol, in the complete absence of external Ca²⁺. We have investigated the processes underling glycerol-induced activation and found that, under this condition, GVBD shows very slow kinetics and is not followed by any normal subsequent steps of meiosis, such as formation of metaphase chromosomes or polar body extrusion. Glycerol-activated oocytes do not show the normal response in protein synthesis but they undergo increased protein phosphorylation which is inhibited by 6-dimethylaminopurine (6-DMAP), which also inhibits GVBD. We conclude that the hypertonic treatment by glycerol, in the complete absence of external Ca²⁺, induces a partial program of activation through increased protein phosphorylation but that the normal full response requires an increased Ca²⁺ influx as triggered by other well-known artificial activating agents.     

Membrane potential,pH and the activation of surf clam oocytes

Unfertilized oocytes of the surf clam, Spisula solidissima, have resting membrane potentials of ?18 ± 7 mV (n = 20). Within five seconds of sperm addition, an electrophysiologically detectable response was apparent, which was characterized by a rapid and prolonged depolarrization depolarization followed four to five minutes post-insemination by the beginning of the beginning of a steady hyperpolarization to approximatelv ?70 mV. This final hyperpolarization was completed within ten minutes of sperm addition. The initial rapid depolarization following insemination may result from a transient increase in sodium conductance, and it may be crucial in preventing polyspermy, since the degree of polyspermy in Spisula oocytes was sensitive to external sodium ion concentrations. Evidence was obtained that changes in intracellular pH are essential for oocvte activation. Using germinal vesical breakdown (GVB) as a marker for activation, it was shown that agents that raise intracellular pH (ammonia and procaine) induced GVB, whereas agents that lower intracellular pH pH (Na-acetate or Na-propionate seawater) inhibited GVB.     

Protein kinase C and meiotic maturation of surf clam oocytes

We report here that phorbol ester, a potent activator of protein kinase C, induces germinal vesicle breakdown in surf clam oocytes. However, phorbol ester-induced activation is slow and is not accompanied by an increased Ca2+ influx. Simultaneous additions of phorbol ester and various amounts of K+ ions, which induce Ca2+ influx of different amplitudes, result in successful activation within the normal time schedule at K+ concentrations inefficient alone in activating the oocytes. In vivo, increased protein phosphorylation triggered by phorbol ester amounts to about one third that seen after fertilization. These results suggest that increased Ca2+ influx and protein kinase C activation act in synergy to cause resumption of meiotic maturation in these oocytes.     

Lamin B methylation and assembly into the nuclear envelope

Lamin B is reversibly methyl-esterified and phosphorylated during the mammalian cell cycle. In order to study the role of methylation in lamin B function, we isolated mitotic cells in the presence of the microtubule inhibitor, nocodazole. Following removal of nocodazole, methylation of mitotic lamin B was found to precede its assembly into the nuclear envelope as cells exited mitosis. Very little additional methylation took place on assembled lamins. We were able to slow the rate of lamin B methylation with methylthioadenosine (MTA). A delay in lamin B methylation was accompanied by a corresponding delay in assembly of lamin B into the envelope. The delay was approximately 20-30 min beyond the typical 60-70 min usually required. Assembly of lamins A and C, which are not methylated, was also delayed by MTA, although to a lesser degree, suggesting that an interaction between the lamins is necessary for formation of the nuclear envelope. Chromatin decondensation was also slowed in the presence of MTA. Other inhibitors of methylation which had no inhibitory effect on the methyl esterification of lamin B were tested and found to have no effect on envelope assembly or chromatin decondensation. These results were obtained with Chinese hamster ovary cells as well as with the stem cell line, PC 13. Dephosphorylation of lamin B normally follows a time course similar to that of nuclear envelope assembly. In the presence of MTA, however, lamin B assembly was slowed with little effect on dephosphorylation. This resulted in a large population of dephosphorylated, but unassembled, lamin B protein, demonstrating that dephosphorylation is not sufficient for envelope assembly. The lack of effect on the time course of dephosphorylation also suggests that MTA is not acting upstream of the methylation event.     

Identification of microtubule-associated proteins in the meiotic spindle of surf clam oocytes

Meiotic spindles isolated from surf clam oocytes to morphological purity are biochemically complex, consisting of many polypeptides. These proteins fall into two classes: (a) polypeptides that are apparently cytoplasmic proteins and are not specifically associated with the spindle; and (b) polypeptides that are specifically associated with the spindle. A subset of the spindle-associated proteins, including a 250,000 mol wt component, remain with spindle tubulin through cycles of cold depolymerization and warm polymerization, showing that they are microtubule-associated proteins.     

Nuclear envelope breakdown proceeds by microtubule-induced tearing of the lamina.

The mechanism of nuclear envelope breakdown (NEBD) was investigated in live cells. Early spindle microtubules caused folds and invaginations in the NE up to one hour prior to NEBD, creating mechanical tension in the nuclear lamina. The first gap in the NE appeared before lamin B depolymerization, at the site of maximal tension, by a tearing mechanism. Gap formation relaxed this tension and dramatically accelerated the rate of chromosome condensation. The hole produced in the NE then rapidly expanded over the nuclear surface. NE fragments remaining on chromosomes were removed toward the centrosomes in a microtubule-dependent manner, suggesting a mechanism mediated by a minus-end-directed motor.     

Comparative patterns of protein phosphorylation during activation of surf clam oocytes by different artificial agents

Oocytes from the surf clam Spisula solidissima are arrested at prophase I of meiotic maturation, until fertilization, We analyzed the patterns of phosphorylated proteins under procedures mimicking, to various degrees, the normal sperm-induced activation process. High K⁺-seawater, the phorbol ester TPA, serotonin, or a combination of these were used to analyze their effects on both germinal vesicle breakdown (GVBD) and protein phosphorylation. Oocytes were preloaded with ³⁶S-methionine or ³²P-phosphate, and the pattern of labeled proteins was analyzed by polyacrylamide gel electrophoresis followed by autoradiography. When comparing, in high K⁺-activated oocytes, the pattern of phosphorylated proteins with that of synthesized proteins, it appeared that these two processes were largely unrelated to one another. Activation induced by TPA was slower (60 min for GVBD) than that induced by high K⁺ or serotonin (12–15 min for GVBD), but was similarly sensitive to the protein phosphorylation inhibitor, 6-dimethylaminopurine, and resulted in a qualitatively similar pattern of phosphorylated proteins appearing with slower kinetics, reflecting slower GVBD. When both serotonin and TPA were added to oocytes, the kinetics of GVBD was intermediate (30 min), and so was the appearance of phosphorylated proteins. Finally, the kinetics of development of H1 kinase activities was evaluated in oocytes activated by serotonin, TPA, or both. Similar to the general pattern of phosphorylated proteins, increased histone H1 kinase activities developed to similar degrees but with kinetics reflecting those of GVBD in each case. In conclusion, activations by different artificial agents, utilizing different pathways, resulted in GVBD with different kinetics but similar overall patterns of phosphorylated proteins after a lag typical of the agent used. This suggests that diverse pathways may initially be used to activate oocytes, but that these different pathways eventually merge into a common one, resulting in a highly conserved and regulated sequence of phosphorylation processes. © 1996 Wiley-Liss, Inc.     

Relation between nuclear envelope and nuclear lamina in nuclear assemblyin vitro

Xenopus laevis egg extracts cell-free nuclear assembly system was used as an experimental model to study the process of nuclear lamina assembly in nuclear reconstitutionin vitro. The experimental results showed that lamin was involved in the nuclear assemblyin vitro. The assembly of nuclear lamina was preceded by the assembly of nuclear matrix, and probably, inner nuclear matrix assembly provided the basis for nuclear lamina assembly. Inhibition of normal assembly of nuclear Iknina, by preincubating egg extracts cell-free system with anti-lamin antibodies, resulted in abnormal assembly of nuclear envelope, suggesting that nuclear envelope assembly is closely associated with nuclear lamina assembly.     

Relation between nuclear envelope and nuclear lamina in nuclear assembly in vitro

Xenopus laevis egg extracts cell-free nuclear assembly system was used as an experimental model to study the process of nuclear lamina assembly in nuclear reconstitutionin vitro. The experimental results showed that lamin was involved in the nuclear assemblyin vitro. The assembly of nuclear lamina was preceded by the assembly of nuclear matrix, and probably, inner nuclear matrix assembly provided the basis for nuclear lamina assembly. Inhibition of normal assembly of nuclear Iknina, by preincubating egg extracts cell-free system with anti-lamin antibodies, resulted in abnormal assembly of nuclear envelope, suggesting that nuclear envelope assembly is closely associated with nuclear lamina assembly. Project supported by the National Natural Science Foundation of China.     

Relation between nuclear envelope and nuclear lamina in nuclear assembly in vitro

Xenopus laevis egg extracts cell-free nuclear assembly system was used as an experimental model to study the process of nuclear lamina assembly in nuclear reconstitution in vitro. The experimental results showed that lamin was involved in the nuclear assembly in vitro. The assembly of nuclear lamina was preceded by the assembly of nuclear matrix, and probably, inner nuclear matrix assembly provided the basis for nuclear lamina assembly. Inhibition of normal assembly of nuclear lamina, by preincubating egg extracts cell-free system with anti-lamin antibodies, resulted in abnormal assembly of nuclear envelope, suggesting that nuclear envelope assembly is closely associated with nuclear lamina assembly.     

Acid release following activation of surf clam (Spisula solidissima) eggs.

Acid release was observed after activation of Spisula eggs with excess KCI. This acid release begins within 20 sec after the activation and continues for 9–15 min. The amount of acid released was 6.8 μmole per milliliter of packed eggs. In Ca-free or Na-free sea water, the acid release is completely inhibited; subsequent addition of the deficient ion leads to acid release and breakdown of germinal vesicles. These results suggest that Spisula eggs release protons after activation in a manner similar to that of sea urchin eggs, and that acid release with concomitant increase in cytoplasmic pH is probably a general event on activation of marine eggs.     

Disappearance and reformation of the nuclear lamina structure during specific stages of meiosis in oocytes

The nuclear lamina is a rigid, proteinaceous layer underlying the inner nuclear membrane of eucaryotic cells. It is present in somatic cell nuclei, disappears during mitosis, and is absent from male meiotic cells. We have investigated the disappearance and reformation of the nuclear lamina during meiosis in oocytes, using immunofluorescence and electron microscopy. We find that the status of the nuclear lamina during meiosis of oocytes differs from the reversible depolymerization seen in mitosis in two respects. First, the lamina disappears during meiotic prophase without affecting the structure of the nuclear membranes or the nuclear pores. Second, the proteins of the dissociated lamina are undetectable by immunological methods in pachytene oocytes, whereas they persist in the cytoplasm during mitosis.