Cyclin B synthesis is required for sea urchin oocyte maturation

Sea urchins are members of a limited group of animals in which meiotic maturation of oocytes is completed prior to fertilization. This is different from oocytes of most animals such as mammals and amphibians in which fertilization reactivates an arrested meiotic cycle. Using a recently developed technique for in vitro maturation of sea urchin oocytes, we analyzed the role of cyclin B, the regulatory component of maturation-promoting factor, in the control of sea urchin oocyte meiotic induction and progression. Oocytes of the sea urchin Lytechinus variegatus accumulate significant amounts of cyclin B mRNA and protein during oogenesis. We analyzed cyclin B synthetic requirements in oocytes and early embryos by inhibiting cyclin B synthesis with DNA and morpholino antisense oligonucleotides. Cyclin B synthesis is not necessary for the entry of G2-arrested oocytes into meiosis; however, it is required for the proper progression through meiotic divisions. Surprisingly, mature sea urchin eggs contain significant cyclin B protein following meiosis that serves as a maternal store for early cleavage divisions. We also find that cyclin A can functionally substitute for cyclin B in early embryos but not in oocytes. These studies provide a foundation for understanding the mechanism of meiotic maturation independent of the zygotic cell cycle.     

Sea urchin fertilization stimulates CaM kinase-II (multifunctional [type II] Ca2+/CaM kinase) activity and association with p34cdc2

Upon fertilization, the sea urchin egg synthesizes proteins which impart a Ca²⁺ dependence to M-phase onset. A potential target of this Ca²⁺ dependence may be CaM kinase-II (the multifunctional [type II] Ca²⁺/calmodulin [CaM]-dependent protein kinase) which is necessary for nuclear envelope breakdown in fertilized sea urchin eggs. This study was intended to determine whether sea urchin CaMK-II is activated after fertilization and whether it interacts with other known M-phase regulators, such as p34^cdc2. We report that total CaMK-II activity, measured by solution assays, increases after fertilization, peaking just prior to cleavage. Interestingly, total CaMK-II activity continues to fluctuate, peaking again prior to second and third cleavage. Gel assays also reveal enhanced levels of the 56 and 62 kDa potential CaMK-II phosphoproteins after fertilization. Finally, CaMK-II activity and only the 62 kDa phosphoprotein physically associate with p34^cdc2, but again only after fertilization. These changes in CaMK-II activity and p34^cdc2-association after fertilization may ensure that Ca²⁺ signals are targeted to the M-phase machinery at the appropriate developmental times.     

Subcellular distribution of aster-nucleated microtubule length: A more or less mitotic status of cytoplasmic areas during meiosis I of starfish oocytes

Summary— Shortening of aster-nucleated microtubules is a mitotic feature, which is proportional to the M-phase promoting factor kinase activity. We have taken advantage of this characteristic to appraise the mitotic status of cytoplasmic territories in polyspermic starfish oocytes. In early prometaphase, asters are small in the nuclear area, where cyclin B accumulated before GVBD, while in the remaining of the oocytes asters are large. In metaphase all asters are small. This coincides with a change in behaviour of cyclin B, whose association with microtubules increases in late prometaphase. We discuss the possibility that uneven distribution of cyclin B-dependent kinase is a general feature of mitosis and that it allows the cell to build the convenient spindle at the right place.     

Inhibition of mitosis in fertilized sea urchin eggs by inhibition of the cyclic AMP-dependent protein kinase

Inhibition of cAMP-dependent protein kinase activity by microinjection of a specific physiologic protein inhibitor into sea urchin eggs inhibits the first cleavage after fertilization. Inhibition apparently occurs at some time prior to or during formation of the mitotic spindle. Measurement of the total protein kinase activity of sea urchin egg homogenates after fertilization showed that cAMP-dependent phosphorylation increases after fertilization and then declines prior to or at the time of the first cleavage. It is concluded that a cAMP-dependent phosphorylation plays a significant role in events leading to regulation of mitotic spindle assembly.     

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.     

Cyclic assembly-disassembly of cortical microtubules during maturation and early development of starfish oocytes

An extensive array of cortical microtubules in oocytes of the starfish Pisaster ochraceus undergoes multiple cycles of disappearance and reappearance during maturation and early development. These events were studied in isolated fragments of the oocyte cortex stained with antitubulin antibodies for indirect immunofluorescence. The meshwork of long microtubules is present in the cortex (a) of immature oocytes, i.e., before treatment with the maturation-inducing hormone 1-methyladenine, (b) for 10-20 min after treatment with 1-methyladenine, (c) after formation of the second polar body (in reduced numbers in unfertilized oocytes), and (d) in the intermitotic period between first and second cleavage divisions. The array of cortical microtubules is absent in oocytes (a) undergoing germinal vesicle breakdown, (b) during the two meiotic divisions (polar body divisions), and (c) during mitosis of the first and, perhaps, subsequent cleavage divisions. The cycle of assembly-disassembly of cortical microtubules is synchronized to the cycle of nuclear envelope breakdown and reformation and to the mitotic cycle; specifically, cortical microtubules are present when a nucleus is intact (germinal vesicle, female pronucleus, zygote nucleus, blastomere nucleus) and are absent whenever a meiotic or mitotic spindle is present. These findings are discussed in terms of microtubule organizing centers in eggs, possible triggers for microtubule assembly and disassembly, the eccentric location of the germinal vesicle, and the regulation of oocyte maturation and cell division.     

6-Dimethylaminopurine blocks starfish oocyte maturation by inhibiting a relevant protein kinase activity

The puromycin analog N6,N6-dimethyladenine (6-dimethylaminopurine or 6-DMAP) was found to inhibit meiosis reinitiation in starfish oocytes stimulated by the natural hormone 1-methyladenine. Increasing concentrations of this agent delayed and eventually blocked germinal vesicle breakdown. They were found to be effective even when applied during the hormone-independent period, after the oocytes had been already committed to reinitiate meiosis. 6-DMAP mimics most of the effects of emetine since it induces protein dephosphorylation, inhibits polar body formation, and promotes the precocious appearance of resting nuclei. However, unlike emetine, 6-DMAP does not affect protein synthesis. The effect of this agent cannot be accounted for by a stimulation of the protease or phosphoprotein phosphatase activities since the rate and extent of protein dephosphorylation do not increase in its presence. Data from in vivo and in vitro endogenous protein phosphorylation experiments suggest rather that 6-DMAP may directly or indirectly affect the activity of a relevant c-AMP and Ca2+-independent protein kinase which is stimulated after hormone addition and seems to support starfish oocyte maturation.     

Fertilization of the starfish Astropecten aurantiacus

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

Phosphorylated ERK5/BMK1 transiently accumulates within division spindles in mouse oocytes and preimplantation embryos

MAP kinases of the ERK family play important roles in oocyte maturation, fertilization, and early embryo development. The role of the signaling pathway involving ERK5 MAP kinase during meiotic and mitotic M-phase of the cell cycle is not well known. Here, we studied the localization of the phosphorylated, and thus potentially activated, form of ERK5 in mouse maturing oocytes and mitotically dividing early embryos. We show that phosphorylation/dephosphorylation, i.e. likely activation/inactivation of ERK5, correlates with M-phase progression. Phosphorylated form of ERK5 accumulates in division spindle of both meiotic and mitotic cells, and precisely co-localizes with spindle microtubules at metaphase. This localization changes drastically in the anaphase, when phospho-ERK5 completely disappears from microtubules and transits to the cytoplasmic granular, vesicle-like structures. In telophase oocytes it becomes incorporated into the midbody. Dynamic changes in the localization of phospho-ERK5 suggests that it may play an important role both in meiotic and mitotic division.     

Alkaline pH favors microtubule self-assembly in surf clam, Spisula solidissima, oocyte extracts

Microtubule assembly in surf clam oocytes is dependent upon events that occur during fertilization. Prior to fertilization there are few, if any microtubules, but within minutes after fertilization microtubules assemble to form the meiotic apparatus. This study demonstrates that the assembly of microtubules after fertilization may be dependent on the fertilization-induced pH change of the cytoplasm. Since the magnitude of the intracellular pH (pHi) change in Spisula oocytes has not been determined, surf clam microtubule assembly was examined at pH values that reflect the pHi change that occurs during sea urchin fertilization. The results indicate that microtubule assembly in crude oocyte extracts is favored at alkaline pH. In contrast, purified surf clam tubulin assembles to a greater extent at pH 6.6 than at pH 7.2. These results reveal that the tubulin in unfertilized oocytes can assemble into microtubules at pH 6.6 but that they are prevented from doing so by pH-dependent cytoplasmic regulatory factors in the oocyte.     

Organization and regulation of cortical microtubules during the first cell cycle of Xenopus eggs.

Anti-tubulin antibodies and confocal immunofluorescence microscopy were used to examine the organization and regulation of cytoplasmic and cortical microtubules during the first cell cycle of fertilized Xenopus eggs. Appearance of microtubules in the egg cortex temporally coincided with the outgrowth of the sperm aster. Microtubules of the sperm aster first reached the animal cortex at 0.25, (times normalized to first cleavage), forming a radially organized array of cortical microtubules. A disordered network of microtubules was apparent in the vegetal cortex as early as 0.35. Cortical microtubule networks of both animal and vegetal hemispheres were reorganized at times corresponding to the cortical rotation responsible for specification of the dorsal-ventral (D-V) axis. Optical sections suggest that the cortical microtubules are continuous with the microtubules of the sperm aster in fertilized eggs, or an extensive activation aster in activated eggs. Neither assembly and organization, nor disassembly of the cortical microtubules coincided with MPF activation during mitosis. However, cycloheximide or 6-dimethylaminopurine, which arrest fertilized eggs at interphase, blocked cortical microtubule disassembly. Injection of p13, a protein that specifically inhibits MPF activation, delayed or inhibited cortical microtubule breakdown. In contrast, eggs injected with cyc delta 90, a truncated cyclin that arrest eggs in M-phase, showed normal microtubule disassembly. Finally, injection of partially purified MPF into cycloheximide-arrested eggs induced cortical microtubule breakdown. These results suggest that, despite a lack of temporal coincidence, breakdown of the cortical microtubules is dependent on the activation of MPF.     

Protein synthesis requirements during resumption of meiosis in the hamster oocyte: early nuclear and microtubule configurations.

The organization of chromatin and cytoplasmic microtubules changes abruptly at M-phase entry in both mitotic and meiotic cell cycles. To determine whether the early nuclear and cytoplasmic events associated with meiotic resumption are dependent on protein synthesis, cumulus-enclosed hamster oocytes were cultured in the presence of 100 micrograms/ml puromycin or cycloheximide for 5 hr. Both control (untreated) and treated oocytes were analyzed by fluorescence microscopy after staining with Hoechst 33258 and tubulin antibodies. Freshly isolated oocytes exhibit prominent nucleoli and diffuse chromatin within the germinal vesicle as well as an interphase network of cytoplasmic microtubules. After 4-4.5 hr in culture, most oocytes were in prometaphase I of meiosis as characterized by a prominent spindle with fully condensed chromosomes and numerous cytoplasmic asters. After 5-5.5 hr in culture, microtubule asters are no longer detected in most cells, and the spindle is the only tubulin-positive structure. Incubation for 5 hr in the presence of inhibitors does not impair germinal vesicle breakdown, chromatin condensation, kinetochore microtubule assembly, or cytoplasmic aster formation in the majority of oocytes examined; however, under these conditions, a population of oocytes retains a germinal vesicle, exhibiting variable degrees of chromatin condensation and cytoplasmic aster formation. Meiotic spindle formation is inhibited in all oocytes. These effects are fully reversible upon culture of treated oocytes in drug-free medium for 5 hr. The data indicate that meiotic spindle assembly is dependent on ongoing protein synthesis in the cumulus-enclosed hamster oocyte; in contrast, chromatin condensation and aster formation are not as sensitive to protein synthesis inhibitors during meiotic resumption.     

Cyclin E/Cdk2 is required for sperm maturation, but not DNA replication, in early sea urchin embryos

The cell cycle is driven by the activity of cyclin/cdk complexes. In somatic cells, cyclin E/cdk2 oscillates throughout the cell cycle and has been shown to promote S-phase entry and initiation of DNA replication. In contrast, cyclin E/cdk2 activity remains constant throughout the early embryonic development of the sea urchin and localizes to the sperm nucleus following fertilization. We now show that cyclin E localization to the sperm nucleus following fertilization is not unique to the sea urchin, but also occurs in the surf clam, and inhibition of cyclin E/cdk2 activity by roscovitine inhibits the morphological changes indicative of male pronuclear maturation in sea urchin zygotes. Finally, we show that inhibition of cyclin E/cdk2 activity does not block DNA replication in the early cleavage cycles of the sea urchin. We conclude that cyclin E/cdk2 activity is required for male pronuclear maturation, but not for initiation of DNA replication in early sea urchin development.     

Regulation of mitogen-activated protein kinase phosphorylation, microtubule organization, chromatin behavior, and cell cycle progression by protein phosphatases during pig oocyte maturation and fertilization in vitro

We used okadaic acid (OA), a potent inhibitor of protein phosphatases 1 and 2A, to study the regulatory effects of protein phosphatases on mitogen-activated protein (MAP) kinase phosphorylation, morphological changes in the nucleus, and microtubule assembly during pig oocyte maturation and fertilization in vitro. When germinal vesicle (GV) stage oocytes were exposed to OA, MAP kinase phosphorylation was greatly accelerated, being fully activated at 10 min. However, MAP kinase was dephosphorylated by long-term (>20 h) exposure to OA. Correspondingly, premature chromosome condensation and GV breakdown were accelerated, whereas meiotic spindle assembly and meiotic progression beyond metaphase I stage were inhibited. OA also quickly reversed the inhibitory effects of butyrolactone I, a specific inhibitor of maturation-promoting factor (MPF), on MAP kinase phosphorylation and meiosis resumption. Treatment of metaphase II oocytes triggered metaphase II spindle elongation and disassembly as well as chromosome alignment disruption. OA treatment of fertilized eggs resulted in prompt phosphorylation of MAP kinase, disassembly of microtubules around the pronuclear area, chromatin condensation, and pronuclear membrane breakdown, but inhibited further cleavage. Our results suggest that inhibition of protein phosphatases promptly phosphorylates MAP kinase, induces premature chromosome condensation and meiosis resumption as well as pronucleus breakdown, but inhibits spindle organization and suppresses microtubule assembly by sperm centrosomes in pig oocytes and fertilized eggs.     

Protein phosphatases are involved in the in vivo activation of histone H1 kinase in mouse oocyte

Histone H1 kinase and protein phosphorylation have been studied in mouse oocyte. Histone H1 kinase activity increases when the oocyte enters M-phase at the time of GVBD and is paralleled with a burst of protein phosphorylation. This activity dramatically drops after parthenogenetic activation induced by puromycin. Okadic acid (OA), a potent inhibitor of protein phosphatases, induces GVBD when oocytes are arrested in the first meiotic prophase by dbc-AMP; the continuous presence of the phosphatase inhibitor, however, inhibits the polymerization of metaphase microtubules. Following activation of metaphase II-arrested mouse eggs by puromycin, OA can induce the breakdown of the nuclear envelope and the activation of histone H1 kinase. This indicates that in the absence of protein synthesis, and therefore of cyclin synthesis, inhibition of protein phosphatases may be sufficient to induce the entry into M-phase during the first cell cycle of the mouse parthenogenetic activated oocyte.     

6－Dimethylaminopurine（6－DMAP） Spontaneously Induces Interphase Transition Of Metaphase Mouse Oocytes

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Different routes lead to apoptosis in unfertilized sea urchin eggs

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

Change in the heterogeneous distribution of tubulin isotypes in mitotic microtubules of the sea urchin egg by treatment with microtubule depolymerizing or stabilizing drugs.

In the mitotic sea urchin egg, the spindle microtubules were composed of different tubulin isotypes from those of astral microtubules using monoclonal antibodies [Oka et al. (1990) Cell Motil. Cytoskeleton, 16, 239-250]. Three of the antibodies, D2D6, DM1B, and YL1/2, were specific for spindle microtubules, astral microtubules and reactive with both microtubules, respectively. The mitotic sea urchin egg was treated with microtubule depolymerizing (colcemid and nocodazole) and stabilizing (hexylene glycol) drugs and change in the heterogeneous distribution of the tubulin isotypes was investigated by the immunofluorescence procedure using these three monoclonal anti-tubulin antibodies. We observed that: (1) the microtubule depolymerizing drugs caused quick depolymerization of most mitotic microtubules, and a small number of spindle microtubules remaining were stained with all three antibodies; (2) hexylene glycol induced many microtubules in the mitotic apparatus, which was stained with D2D6 but was not stained with DM1B; (3) hexylene glycol also induced a great number of miniasters in the cytoplasm, and they were stained with three antibodies. These results suggest that these drugs altered the distribution of tubulin isotypes in the mitotic microtubules during depolymerization or polymerization within a short time.     

6-Dimethylaminopurine (6-DMAP), a reversible inhibitor of the transition to metaphase during the first meiotic cell division of the mouse oocyte

The first meiotic cell division (meiotic maturation) of dictyate stage mouse oocytes removed from the follicle resumes spontaneously in vitro. We used the puromycin analog 6-dimethylaminopurine (6-DMAP) to test the respective roles of protein synthesis and protein phosphorylation in driving this process. While protein synthesis inhibitors do not block meiosis resumption, 6-DMAP was found to inhibit germinal vesicle breakdown (GVBD), by inhibiting the burst of protein phosphorylation without changing the rate of incorporation of [35S]methionine into proteins. This effect is reversible; it depends both upon drug concentration and the particular female. When added after GVBD and before the emission of the first polar body, 6-DMAP decreases the level of protein phosphorylation and induces decondensation of the chromosomes and reformation of the nuclear envelope. In contrast, 6-DMAP did not trigger these processes in metaphase II oocytes which only produce resting nuclei when treated by protein synthesis inhibitors. From these data, we conclude that (1) the early appearance and stability of mouse MPF in Metaphase I oocytes depend on protein phosphorylation rather than on protein synthesis, and (2) protein synthesis is necessary to maintain the condensation of the chromosomes in metaphase II oocytes.