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.     

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.     

Maternal messenger RNA and histone synthesis in embryos of the surf clam Spisula solidissima.

Messenger RNA has been isolated from the postribosomal supernatant of Spisula solidissima eggs. This mRNA directs the synthesis of several proteins when added to the ascites or wheat germ cell free system. No histone except F1 is coded for by Spisula egg mRNA, in contrast to what has been reported previously for sea urchin egg mRNA. In sea urchin eggs histone mRNA is among the abundant species of maternal mRNA.Histones have been prepared from Spisula embryos at different development stages and histone synthesis followed by incubation with (¹⁴C)lysine. The analysis by electrophoresis on acrylamide gels indicates that the pattern of synthesis of histones changes during development and that a new histone F1 fraction is actively synthesized from the 32–64 cells stage. In earlier embryos a different F1 histone is synthesized and the mRNA for this protein may be the only histone mRNA present in eggs.     

Localization and quantification of gonad serotonin during gametogenesis of the surf clam, Spisula solidissima

In the surf clam, Spisula solidissima, serotonin was reported to induce spawning when injected into the gonads. At nanomolar concentrations, it facilitates the fertilizability of oocyte by sperm, at micromolar concentration, it triggers the meiotic maturation of prophase 1-arrested oocytes, thus mimicking the effect of sperm. To further understand the role of serotonin in the gametogenic and spawning processes, we used both immunohistochemistry and high-pressure liquid chromatography linked with electrochemical detection to detect serotonin in the gonads of the surf clam. We found serotonin-containing varicose fibers covering the surface of the germinal epithelium in both sexes. The area occupied by the serotonergic innervation field encircling gonad acini varied according to the gonadal stages (active phase, ripe phase, partially spawned phase, spent phase). We also found large variations in the serotonin concentration between specimens during the gametogenic cycle. The serotonin concentration was correlated with gonad growth: it decreased in the ripe phase in comparison with the previous phase, the active phase. We attribute the decrease to the increase of total gonad mass in this stage. In contrast, as spawning begins, the total gonad mass declines while the gonad serotonin concentration increases to a level similar to that found in active phase. The finding that prior to spawning, serotonin is present in the gonads within fibers exhibiting distinct varicosities suggests that it is implicated in spawning.     

Surface alterations of fertilized surf clam (Spisula solidissima) eggs induced by concanavalin A

Fertilized Spisula eggs, incubated in ConA, were examined at periodic intervals to determine the effects of lectin binding on events of fertilization and cleavage. ConA was localized to specific regions of the vitelline layer and plasma membrane by reacting lectin-treated eggs with horseradish peroxidase and diaminobenzidine. In contrast to eggs, little reaction product was associated with the plasma membrane of spermatozoa. Sperm that fused with ConA-treated eggs failed to move into the cortex of the ovum and were observed as bulbous appendages at the surface of the zygote. Reorganization of sperm nuclei was inhibited, and male pronuclei failed to develop. ConA also inhibited polar body formation and cleavage. The maternally derived chromatin underwent meiosis, and the chromosomes normally taken into the first and second polar bodies were retained within the zygote. All of the maternally derived chromatin was organized within four or more female pronuclei which subsequently entered mitosis. The effects of ConA binding on events at the surface of fertilized Spisula eggs were abrogated by α-methyl-d-mannoside; succinyl-ConA only partially inhibited fertilization-related processes. The effects of ConA are discussed in terms of possible cross-linking of surface components of fertilized Spisula eggs which may inhibit deformation of the zygote cortex.     

Microtubule cycles in oocytes of the surf clam, Spisula solidissima: an immunofluorescence study

Oocytes of the surf clam, Spisula solidissima, underwent germinal vesicle breakdown and two meiotic divisions to give off polar bodies when they were fertilized or parthenogenetically activated with KCl. Fertilized eggs further proceeded to mitosis and cleaved, while parthenogenetically activated eggs remained uncleaved. We examined changes in microtubule-containing structures during meiotic divisions and subsequent mitotic processes by immunofluorescence. A monoclonal anti-tubulin antibody was applied to alcohol-fixed eggs from which the vitelline membrane had been removed by protease digestion. Up to the stage of second polar body formation, the pattern of microtubule organization in the first and second meiotic spindles was identical in both fertilized and parthenogenetically activated eggs. However, while fertilized eggs formed a sperm aster and mitotic spindles later, activated eggs formed only monaster- or ring-shaped microtubule-containing structures which underwent cycles of alternating formation and breakdown. Lactoorecin staining of parthenogenetically activated eggs revealed that the chromosome cycle could occur in these eggs, in phase with this microtubule cycle.     

Temporal and spatial expression of embryonic alkaline phosphatase activity in the surf clam,Spisula solidissima

The temporal appearance and spatial distribution of alkaline phosphatase (EC 3.1.3.1) have been examined during the development of the surf clam, Spisula solidissima. Enzyme activity was detectable at low levels from fertilization onward with a sharp linear increase in activity occurring at the trochophore stage, 14 h post-fertilization. Histochemical localization of enzyme activity at the light microscope level showed the increased activity associates initially with a limited number of large macromeres and is progressively restricted to the cells forming the tip of the growing archenteron. Electron microscopy shows the activity is restricted to the luminal and lateral plasma membrane of these cells.     

The relationships between early ionic events, the pattern of protein synthesis, and oocyte activation in the surf clam, Spisula solidissima

The ionic events linked to activation of surf clam (Spisula solidissima) oocytes include a transient increased Ca2+ influx and an acid release. The aim of the present work was to further elucidate the respective roles of these two ionic events and to clarify the possible role of protein kinase C in the sequence of events leading to oocyte activation. K+-enriched seawater, ammonium chloride, and the phorbol ester 12-O-tetradecanoyl-13-phorbol acetate (TPA), a protein kinase C activator, were tested for their ability to promote germinal vesicle breakdown (GVBD), an acid release, increased 45Ca2+ uptake, and a shift in the pattern of protein synthesis. Oocytes activated by addition of K+ ions release an amount of H+ similar to that induced by fertilization, with the same time course, show an increased, verapamil-sensitive, 45Ca2+ uptake that is proportional to the amount of added K+, and undergo a shift in their pattern of protein synthesis, which requires the presence of external Ca2+. Ammonium chloride, at concentrations causing a higher production of acid than that induced by K+ ions or fertilization, does not trigger GVBD nor any increased 45Ca2+ uptake or any detectable shift in the pattern of protein synthesis. Combined additions of ammonium chloride with subthreshold concentrations of K+ ions allow GVBD to occur, thus revealing a synergistic effect of ammonia and K+ ions. TPA slowly induces GVBD, an Na+-dependent acid release, and a shift in the pattern of protein synthesis, in the absence of increased 45Ca2+ uptake. Our results lead us to propose the following sequence of events for the activation of Spisula oocytes: an increased Ca2+ influx contributes to activate protein kinase C which causes a Na+-dependent acid release leading to a rise of pHi. This rise of pHi, although insufficient by itself, may set the pHi in a permissive range for activation to occur through the action of other protein kinase C-sensitive events leading to the production of meiosis-inducing proteins.     

Early development of the serotonergic and dopaminergic nervous system in Spisula solidissima (surf clam) larvae.

We have defined the development of the serotonergic and dopaminergic components of the central nervous system in the early Spisula solidissima (surf clam) embryo using HPLC and immunocytochemistry. HPLC analysis reveals norepinephrine, dopamine, and serotonin are present at 24 h post-fertilization. Immunocytochemistry shows that the serotonergic nervous system emerges during the late trochophore stage with the development of a single serotonergic cell, C/A1, in the cerebral/apical ganglion. After 48 h, a second serotonergic cell forms, C/A2, which is connected to C/A1 by two serotonergic processes, and a single serotonergic cell emerges in the visceral ganglion, V1. At 72 h, a new serotonergic cell body develops in the cerebral/apical ganglion, C/A3. After 96 h, the cerebral/apical ganglion and visceral ganglion are connected by a serotonergic process. Expression of the dopamine receptor, D2, begins by 24 h with a generalized expression in the region of the developing gut. D2 expression in the gut ceases by 48 h. At 48 h, a network of fibers forms dorsolateral to the mouth. By 72 h, D2 expressing projections emerge from this network.     

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.     

Nascent protein requirement for completion of meiotic maturation and pronuclear development: examination of fertilized and A-23187-activated surf clam (Spisula solidissima) eggs

The involvement of newly synthesized proteins and calcium in meiotic processes, sperm nuclear transformations, and pronuclear development was examined in emetine-treated, fertilized, and A-23187-activated Spisula eggs by observing changes in the morphogenesis of the maternal and paternal chromatin. Emetine treatment (50 micrograms/ml) initiated 30 min before fertilization or A-23187 activation inhibited incorporation of [3H]leucine into TCA-precipitable material and blocked second polar body formation. Sperm incorporation and the initial enlargement of the sperm nucleus were unaffected; however, the dramatic enlargement and transformation of the sperm nucleus into a male pronucleus, which normally follow polar body formation, were delayed 10 to 20 min. Unlike the situation in untreated, control eggs, male pronuclear development took place while the maternally derived chromosomes remained condensed. It was not until approximately 20 min after the normal period of pronuclear development that the maternal chromosomes dispersed and formed a female pronucleus in emetine-treated, fertilized eggs. Formation of pronuclei, however, was unaffected in both emetine-treated, A-23187-activated eggs and fertilized eggs incubated with A-23187. These observations indicate that germinal vesicle breakdown, first polar body formation, and initial transformations of the sperm nucleus are independent of newly synthesized proteins. Inhibition of second polar body formation and the delay in pronuclear development brought about by emetine, as well as the appearance of silver grains over pronuclei in autoradiographs of control eggs incubated with [3H]leucine demonstrate that nascent proteins are involved with the completion of meiotic maturation and the development of male and female pronuclei. The ability of A-23187 to override the inhibitory effects of emetine on pronuclear development suggests that both nascent protein and calcium signals are involved in regulating the status of the maternal and paternal chromatin during pronuclear development.