Spisula Solidissima Oocytes Research Articles

Identification and isolation of maternal procentrosomes from oocytes of Spisula solidissima

Identification and isolation of maternal procentrosomes from oocytes of <i>Spisula solidissima</i>

Aurora B Kinase Maintains Chromatin Organization During the MI to MII Transition in Surf Clam Oocytes

Meiosis represents a specialized cell cycle whereby cells undergo two reductive divisions without an intervening S phase. In oocytes, the transition from meiosis I to II is brief, with paired sister chromatids remaining condensed throughout the interkinesis period. This stands in contrast to mitotic divisions where cytokinesis and the return to interphase is always accompanied by chromatin decondensation and nuclear envelope reformation. Because other aspects of M phase exit are normal, we probed the mechanisms that allow for polar body extrusion while retaining chromatin condensation in Spisula solidissima oocytes. If oocytes were activated in the presence of protein synthesis inhibitors, oocytes progressed normally through MI, but arrested in interkinesis with condensed chromatin, phosphorylated histone H3 and a disorganized MII spindle. Neither inhibition of CDK1- nor MAPK activity in arrested oocytes was sufficient to drive chromatin decondensation or nuclear envelope reformation, suggesting that these kinases were not responsible for the maintenance of chromatin condensation. However, inhibition of Aurora B kinase activity resulted in chromatin decondensation, loss of histone H3 phosphorylation and reformation of the nuclear envelope. Inhibition of Aurora B activity following MI also resulted in chromosome segregation defects during MII and blocked polar body formation, consistent with Aurora B’s well-established role in cytokinesis. Together, these results suggest that extended Aurora B activity between meiotic divisions maintains chromatin condensation, thus allowing for the rapid reassembly of the MII spindle and progression through meiosis.

Centriole duplication, centrosome maturation and spindle assembly in lysates of Spisula solidissima oocytes.

AbstractMarine oocytes and embryos have long been favored systems for the study of cell division and the cytoskeletal dynamics that accompany karyokinesis and cytokinesis. Several characteristics make marine egg systems ideal for the study of cell replication including: 1) the clarity of the eggs, which allows excellent real-time visualization of cytoskeletal components; 2) the natural synchrony of the cells, because the oocytes are typically arrested at a specific phase of the cell cycle until fertilized or artificially activated; 3) the rapid and synchronous cell divisions that follow fertilization or activation; and perhaps most importantly, 4) the large quantities of oocytes that can be obtained. Here we describe an in vitro system that utilizes cytoplasmic extracts prepared from surf clam, Spisula solidissima, oocytes for the analysis of the microtubule cytoskeleton during meiosis I spindle assembly.KeywordsGerminal VesicleHigh Voltage Electron MicroscopyGerminal Vesicle BreakdownSwing Bucket RotorSurf ClamThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Discovery of 5R-lipoxygenase activity in oocytes of the surf clam, Spisula solidissima

Arachidonic acid and 5-hydroxyeicosatetraenoic acid (5-HETE) are reported to induce reinitiation of meiosis in oocytes of the surf clam Spisula sachalinensis from the Sea of Japan (Varaksin et al., Comp. Biochem. Physiol. 101C, 627–630 (1992). As the Atlantic surf clam Spisula solidissima is a commonly used model for the study of meiosis reinitiation, we examined these cells for the possible occurrence of lipoxygenases and for the bioactivity of the products. Incubation of [ 14C]arachidonic acid with homogenates of S. solidissima oocytes led to the formation of two major metabolites: 5R-HETE, a novel lipoxygenase product, and 8R-HETE. The products were identified by HPLC, uv spectroscopy, and GC-MS. The corresponding hydroperoxy fatty acids, the primary lipoxygenase products, were isolated from incubations of ammonium sulfate fractionated oocyte cytosol. Arachidonic and eicosapentaenoic acids were identified as constituents of S. solidissima oocyte lipids and the free acids were equally good lipoxygenase substrates. We examined the activity of C18 and C20 polyunsaturated fatty acids and their lipoxygenase products on meiosis reinitiation in Spisula solidissima oocytes, using serotonin and ionophore A23187 as positive controls. The fatty acids and their derivatives were inactive. We conclude that in the surf clam, (as in starfish), there are responding and non-responding species in regard to the maturation-inducing activity of the oocyte lipoxygenase products, and that the lipoxygenase has another, as yet uncharacterized, function in oocyte physiology.

Centrosomes isolated from Spisula solidissima oocytes contain rings and an unusual stoichiometric ratio of alpha/beta tubulin.

Centrosome-dependent microtubule nucleation involves the interaction of tubulin subunits with pericentriolar material. To study the biochemical and structural basis of centrosome-dependent microtubule nucleation, centrosomes capable of organizing microtubules into astral arrays were isolated from parthenogenetically activated Spisula solidissima oocytes. Intermediate voltage electron microscopy tomography revealed that each centrosome was composed of a single centriole surrounded by pericentriolar material that was studded with ring-shaped structures approximately 25 nm in diameter and <25 nm in length. A number of proteins copurified with centrosomes including: (a) proteins that contained M-phase-specific phosphoepitopes (MPM-2), (b) alpha-, beta-, and gamma-tubulins, (c) actin, and (d) three low molecular weight proteins of <20 kD. gamma-Tubulin was not an MPM-2 phosphoprotein and was the most abundant form of tubulin in centrosomes. Relatively little alpha- or beta-tubulin copurified with centrosomes, and the ratio of alpha- to beta-tubulin in centrosomes was not 1:1 as expected, but rather 1:4.6, suggesting that centrosomes contain beta-tubulin that is not dimerized with alpha-tubulin.

Modulation of serotonin binding sites in spisula solidissima oocytes by phorbol ester

In Spisula solidissima oocytes, serotonin (5-hydroxytryptamine, 5-HT)-dependent meiosis reinitiation is mediated via specific 5-HT membrane binding sites. This oocyte response is inhibited by the phorbol ester TPA. To assess whether the inhibitory effect of TPA was due to alteration of oocyte membrane binding sites, we studied their characteristics after TPA treatment. [ 3H]-5HT binding assays revealed that TPA decreased the affinity and, after prolonged treatment, increased the number of oocyte binding sites. Moreover, inhibitory actions of TPA on 5-HT-induced meiosis reinitiation paralleled its inhibitory effects on 5-HT binding site affinity. The inhibitory actions in biological assays were restricted to TPA (an inactive analog of TPA, TPA-met was inefficient) and were completely reversed by staurosporine. Our data thus suggest an inhibitory role for protein kinase C on oocyte 5-HT binding sites under physiological conditions.

Meiotic spindle assembly in cytoplasmic extracts of Spisula solidissima oocytes

Critical for karyokinesis in animal cells, is the assembly of a spindle-chromosome-aster complex which serves as the force-generation system for chromosome segregation. Spisula solidissima oocytes offer a unique system for the study of spindle assembly since they can be obtained in large quantities, are naturally synchronized in the cell cycle and can be induced to assemble meiotic spindles within 15 min after parthenogenetic activation. Oocytes contain a large nucleus [germinal vesicle (GV)] and are arrested at the G2/M border of meiosis I. No centrosomes or asters have been observed in oocytes at this stage by either electron microscopy or fluorescence microscopy using anti-tubulin antibodies. Observation of live oocytes with polarized light microscopy revealed that within 4-6 min after parthenogenetic activation, asters appeared and invaded the GV as it breaks down (GVBD). 15 min after activation a complete bipolar meiotic spindle was observed.As a first step towards the development of an in vitro biochemical reconstitution system for the study of spindle assembly, concentrated lysates were prepared at various times following parthenogenetic activation and analyzed by phase-contrast microscopy.

Evidence for a New Subtype of Serotonin Receptor in Oocytes of the Surf Clam Spisula solidissima

The neurohormone serotonin 5-hydroxytryptamine (5-HT) triggers meiotic reinitiation in prophase-arrested oocytes of Spisula solidissima (Hirai et al., J. Exp. Zool . 245, 318-321, 1988). The original pharmacological profile of this oocyte response (Krantic et al., Dev. Biol. 146, 491-498, 1991) suggested a novel 5-HT receptor subtype. The present study employed [3H]5-HT binding assays to characterize these putative receptors. Kinetics of [3H]5-HT equilibrium binding saturation revealed that vitellin-deprived oocyte plasma membranes contained a homogeneous population of 5-HT-specific binding sites with an apparent affinity of 0.21 ± 0.03 μM and maximal binding capacity of 1.22 ± 0.62 pmol/mg protein. Analysis of [3H]5-HT-specific binding inhibition by various 5-HT-related compounds yielded a novel pharmacological profile for these binding sites. Moreover, the efficacies of most compounds in inhibiting the specific binding upon 5-HT membrane sites correlated with their efficacies in either triggering meiotic reinitiation (for agonists) or inhibiting the 5-HT-induced oocyte response (for antagonists). Such a correlation indicates that 5-HT binding sites located in the plasma membrane represent the physiological receptors involved in transduction of the biological effects of 5-HT in Spisula solidissima oocytes.

Meiosis reinitiation in surf clam oocytes is mediated via a 5-hydroxytryptamine5 serotonin membrane receptor and a vitelline envelope-associated high affinity binding site.

The neurohormone serotonin (5-hydroxytryptamine, 5HT) triggers meiosis reinitiation in prophase-arrested oocytes of Spisula solidissima. The original pharmacological profile of this response prompted us to examine whether it involved a novel type of 5HT receptor. In order to characterize these putative receptors, we performed [3H]5HT binding assays. Given the complexity of oocyte surface architecture, [3H]5HT-specific binding was measured in both plasma membrane and vitelline envelope fractions. Our data reveal the existence of a single class of 5HT-specific binding sites in each fraction. These binding sites appear distinct by their kinetic properties: 1) plasma membrane binding sites are of lower affinity but are more numerous than vitelline envelope sites; 2) [3H]5HT binding is more rapid in membrane fraction. The pharmacological profiles of both binding sites were defined in competition experiments by using sixteen 5HT-related and unrelated compounds. Both membrane and vitelline envelope sites displayed novel profiles. However, only the pharmacological profile determined for the plasma membrane sites corresponds to that observed for 5HT-induced meiosis reinitiation. These new 5HT5 binding sites are therefore the physiological receptors that mediate the effects of 5HT in Spisula oocytes. The putative role of vitelline envelope sites is discussed.

Centriole duplication in lysates of Spisula solidissima oocytes.

A cell-free system has been developed that executes centriole duplication. Surf clam (Spisula solidissima) oocytes, arrested at late prophase of meiosis I, do not contain centrioles, centrosomes, or asters. Serial section high-voltage electron microscopy (HVEM) of asters and spindles isolated from potassium chloride-activated oocytes indicates that within 4 minutes oocytes assemble a single centriole that is duplicated by 15 minutes when assembly of the first meiotic spindle is complete. A mixture of lysates from unactivated oocytes and potassium chloride-activated oocytes induces centriole formation and duplication. Astral microtubule content in these lysate mixtures increases with time.

The mechanism of serotonin-dependent control of meiosis reinitiation in spisula solidissima oocytes

The mechanism of serotonin-dependent control of meiosis reinitiation in spisula solidissima oocytes

Pharmacology of the serotonin-induced meiosis reinitiation in Spisula Solidissima oocytes

Germinal vesicle breakdown (GVBD) is the first visible response of the oocyte of Spisula solidissima to the neurohormone serotonin. Pharmacological characterization of this response was performed by using 24 serotonin-related compounds. Dose-response curves were assessed by quantification of GVBD. Rank orders of potency obtained were among agonists: serotonin > 8-hydroxy-2-(di- N-propylamino)tetralin hydrobromide > 2-methyl-serotonin > 1-(3-trifluoromethylphenyl)piperazine; among antagonists; ritanserin > ICS205930 > mianserin = ketanserin = propranolol > metoclopramide = yohimbine > spiperone. Various other monoaminergic compounds tested were inefficient, demonstrating the specificity of the oocyte response to serotonin. Transduction mechanisms underlying this response were then investigated. Ca 2+ appeared to be involved since serotonin induced an increase in the uptake of 45Ca 2+ and since it was inefficient in calcium-free sea water. The absence of synergy between serotonin and KCl suggested that both compounds use a common transduction pathway. Exposure of the oocyte to the protein kinase C activator TPA inhibited serotonin-dependent maturation. Our data thus point to an original, previously uncharacterized pharmacological profile and transduction mechanism by which serotonin induces oocyte meiosis reinitiation in Spisula solidissima

Teniposide, a topoisomerase II inhibitor, prevents chromosome condensation and separation but not decondensation in fertilized surf clam ( Spisula solidissima) oocytes

DNA topoisomerase II has been implicated in regulating chromosome interactions. We investigated the effects of the specific DNA topoisomerase II inhibitor, teniposide on nuclear events during oocyte maturation, fertilization, and early embryonic development of fertilized Spisula solidissima oocytes using DNA fluorescence. Teniposide treatment before fertilization not only inhibited chromosome separation during meiosis, but also blocked chromosome condensation during mitosis; however, sperm nuclear decondensation was unaffected. Chromosome separation was selectively blocked in oocytes treated with teniposide during either meiotic metaphase I or II indicating that topoisomerase II activity may be required during oocyte maturation. Teniposide treatment during meiosis also disrupted mitotic chromosome condensation. Chromosome separation during anaphase was unaffected in embryos treated with teniposide when the chromosomes were already condensed in metaphase of either first or second mitosis; however, chromosome condensation during the next mitosis was blocked. When interphase two- and four-cell embryos were exposed to topoisomerase II inhibitor, the subsequent mitosis proceeded normally in that the chromosomes condensed, separated, and decondensed; in contrast, chromosome condensation of the next mitosis was blocked. These observations suggest that in Spisula oocytes, topoisomerase II activity is required for chromosome separation during meiosis and condensation during mitosis, but is not involved in decondensation of the sperm nucleus, maternal chromosomes, and somatic chromatin.

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.

Sperm nuclear transformations consist of enlargement and condensation coordinate with stages of meiotic maturation in fertilized Spisula solidissima oocytes

Rates of sperm nuclear expansion were measured and correlated with processing of the maternal chromatin in synchronous populations of fertilized surf clam ( Spisula solidissima) oocytes fixed at regular intervals following insemination and stained with the DNA fluorochrome Hoechst 33342. Sperm nuclei expanded in four distinct phases each temporally coordinate with events of meiotic maturation: germinal vesicle stage (phase A), germinal vesicle breakdown (phase B), polar body formation (phase C), and female pronuclear development (phase D). Sperm nuclei were essentially unchanged during phase A (rate = 0.1 μm 2/min, enlarged during phases B (rate = 8.2 μm 2/min) and D (rate = 6.2 μm 2/min), and condensed during phase C (rate = −1.9 μm 2/min). Sperm nuclear enlargement during phase D was significantly less in polyspermic and polygynic zygotes. The effects of various treatments (temperature, microtubule disruption, pH alterations, and metabolic and protein synthesis inhibitions) which perturbed sperm nuclear enlargement and meiotic processing of the maternal chromatin indicated that the two processes are coupled and may be linked by common regulatory agents.

Nuclear envelope proteins from Spisula solidissima germinal vesicles

Biochemical characterization of the nuclear pore complex requires quantities of highly enriched nuclear pore complex material which could not be obtained with available procedures. We have developed a technique for the mass isolation of nuclear envelopes from germinal vesicles of Spisula solidissima oocytes. The nuclear pore complex is intact after isolation as judged ultrastructurally. The nuclear envelope and the pore complex fibrous lamina fraction are highly purified with respect to nuclear and cytoplasmic protein contaminants. The fibrous lamina pore complex (FLPC) as presently isolated consists of about eight major proteins, three of which are phosphorylated. Comparison of the FLPC of clams with that of rat reveals three proteins of similar molecular weights, which may be pore complex-specific proteins. The clam nuclear envelope has only one protein (67000) in the molecular weight range which is comparable to the three lamina of rat nuclei. The solubility, intermolecular cross-linking and in vitro phosphorylation of this protein resemble that of one of the lamina of rat nuclei. The other lamina of the rat nuclear envelope are not essential proteins of the pore complex because they are not present in the clam FLPC preparation. They also seem non-essential for the maintenance of the fibrous lamina.

Selective translation of mRNA controls the pattern of protein synthesis during early development of the surf clam, Spisula solidissima

Striking changes in the pattern of protein synthesis occur shortly after the fertilization of Spisula solidissima oocytes. These changes include a strong reduction in the synthesis of prominent oocyte-specific proteins and a large increase in the synthesis of at least three proteins whose labeling dominates the pattern of protein synthesis in early embryos. Several independent lines of evidence suggest that these changes are modulated at the translational level: 1. —Oocytes and embryos contain identical sets of mRNA, as measured by cell-free translation of phenol-extracted RNA from the two developmental stages. 2. —Different sets of these messages are associated with ribosomes at the two stages. 3. —Cell-free translation of mixtures of the unextracted 12,000 × g supernatants of oocyte or embryo homogenates with reticulocyte lysate gives patterns of protein synthesis that accurately resemble the patterns of synthesis from intact cells of the cognate stage, despite the fact that these homogenates contain identical sets of mRNA. These results provide strong evidence that the alterations in the pattern of protein synthesis at fertilization are due to stage-specific utilization of different subsets of messenger RNA from a common maternal mRNA population at the two stages of development. The fact that phenol extraction of these mRNAs renders them all equally available for translation in the reticulocyte lysate implies that discrimination is achieved by selective repression of availability by some phenol-soluble component of the cells.

Induction of nuclear breakdown and meiosis in Spisula solidissima oocytes by calcium ionophore.

Induction of germinal vesicle breakdown and reinitiation of the meiotic process resulted following exposure of immature Spisula solidissima oocytes to divalent ionophore A23187. Meiosis continued to completion in the presence of the ionophore; however, parthenogenetic development of the gametes did not occur. Incubation of oocytes in calcium deficient media (Ca++ free or Ca++Mg++ free seawater) but not Mg++ free seawater inhibited ionophore induced maturation. Effects of calcium deficiency were reversible. The results suggest that nuclear disintegration and the meiotic process induced by sperm are mediated by ionic stimuli and that the establishment of meiotic arrest is linked to the availability of calcium within the oocyte.

Regulation of Tubulin Organization During Meiosis

Meiotic spindle formation in Spisula solidissima oocytes has been studied in vivo and in vitro. Measurements were made of polymerized tubulin content during the first meiotic division. The amount of polymer was high prior to activation of the eggs, fell to a minimum of about 5 min after activation and at 15 min (metaphase) returned to approximately its initial value. The polymerized tubulin in the unactivated eggs appears to be organized into granular spheres about 10–20 microns in diameter attached to the egg cortex. This particle contains few microtubules but is composed primarily of a granular matrix and fibrous material. The granular matrix may be a polymorphic aggregate of tubulin and could be a storage form of tubulin or an intermediate in microtubule assembly. The polymerization and organization of microtubules has been studied in vitro, using crude homogenates of Spisula oocytes. Microtubules can be formed in homogenates of both activated and unactivated eggs; however, in homogenates of eggs in which nuclear membrane breakdown has occurred, microtubules form around a central phase dense particle resulting in a structure which resembles a spindle aster. The central particle appears to be a microtubule organizing center (MTOC). The MTOC can be pelleled by centrifugation and will induce aster formation when remixed with the supernatant. Aster formation can be obtained using supernatants prepared from either activated or unactivated eggs, while the pellet must be obtained from activated eggs. Thus, tubulin subunits appear to be capable of spindle formation at all stages, while MTOC formation or activation does not occur until about the time of nuclear membrane breakdown.

STUDIES OF EARLY CLEAVAGE IN THE SURF CLAM, SPISULA SOLIDISSIMA, USING METHYLENE BLUE AND TOLUIDINE BLUE AS VITAL STAINS

1. Techniques for vitally staining oocytes of Spisula solidissima with certain basic dyes are described. 2. Such staining reveals small particles in the cytoplasm of the egg. 3. After fertilization the particles show very specific movements and localization changes which can be described by saying that the particles follow the centrioles through cleavage. 4. Spindle movements and position changes can easily be followed and are described in detail. 5. The results are discussed in the light of related work in the literature.