Spisula Solidissima Research Articles

Use of fresh or aged bovine oocytes for intracytoplasmic sperm injection (ICSI)

Following their incorporation into oocytes, sperm nuclei (SN) of the surf clam, Spisula solidissima, undergo an initial expansion, followed by condensation and then a dramatic enlargement during their development into male pronuclei. These changes are temporally correlated with alterations in the maternal chromatin: germinal vesicle breakdown (GVBD), meiotic maturation, and female pronuclear development, respectively. To analyze possible changes occurring in SN at fertilization, surf clam oocyte extracts; prepared before and after parthenogenetic activation, were examined for their ability to affect SN in vitro. Sperm heads were incubated in extracts for variable periods up to 5 hr. Extracts prepared from oocytes following GVBD (15 min postactivation) induced an expansion in ∼90% of SN by 60 min incubation. However, when SN were incubated in extracts from unactivated or 4-min-activated oocytes only ∼30% underwent expansion. Ultrastructural examination of specimens taken at increasing periods of incubation in oocyte extracts revealed that SN expansion in vitro resembled chromatin decondensation in vivo. SN incubated 1 to 5 hr in extracts prepared from oocytes following GVBD consisted of decondensed chromatin surrounded to varying degrees by membranous cisternae. Staining with anti-lamin antibody was variable: some specimens (60-70%) were positive while others (30-40%) were weak to negative. In contrast, all decondensed SN incubated in extracts from postmeiotic oocytes (65 min postactivation) were delimited by an intact nuclear envelope possessing nuclear pores and reactive to anti-lamin antibody. Decondensation of SN in 15- or 65-min extracts was blocked by EDTA, 2,6-dimethylami-nopurine, histone, and protamine. The presence (65-min extract) and absence (unactivated, 4- and 15-min extracts) of sperm nuclear envelope assembly in vitro is consistent with events in vivo, where such a structure forms after meiotic maturation in concert with the development of the female pronucleus.

ITQS as Collateral Rightly Understood: Preserving Commerce and Conserving Fisheries

INTRODUCTION number of vessels comprising the United States' fishing fleets has, in many fisheries, grown to such great numbers that their fishing capacity far exceeds fishery productivity. This excess capacity perpetuates a cycle of overinvestment and overfishing, contributing to dangerous depletion of fish stocks. In unsuccessful efforts to conserve threatened fish stocks, the United States has reduced fishing seasons and allowable harvest limits.(1) reduction of fishing seasons has itself contributed to overinvestment in fishing fleets. As fishing seasons were shortened, purchasing equipment capable of meeting limits in the shortest possible time became the business strategy of choice. A proposed alternative to these unsuccessful fishery management practices is the implementation of transferable fishing schemes, which may provide incentives to conserve fish stocks and simultaneously reduce the excessive numbers of active vessels within U.S. fisheries. Individual Transferable Quotas (ITQs), are tradable fishing rights created by the National Marine Fisheries Service (NMFS) and local fishery management councils in an attempt to alleviate the problem of overfishing, and achieve sustainable management in the United States.(2) Under ITQ management schemes, each qualifying fisher receives an individual quota, which is a specific percentage of the annual harvest limit--or total allowable catch (TAC)(3)--for a specific fishery. A fisher may use his percentage allocation to harvest fish himself, or he may transfer his allocation to someone else by lease or sale. The primary feature of ITQs is the assignment of . . . property rights to harvest common property resources such as fish and shellfish . . . Usually, ITQs are fully transferable (buy, sell, lease) to allow operators to optimize their business.(4) Critics of market-based environmental solutions point out the difficulty of applying economic models to environmental questions because [h]umans cannot . . . impose limited notions of order on a living world that, by its very nature, will not be pinned down.(5) Regardless of the validity of this criticism, the reality is that current policies value market mechanisms as superior to other alternatives.(6) implementation of transferable fishing schemes worldwide is evidence of this trend.(7) Transferable systems have been implemented in various fisheries around the world with distinct consequences for the economic structure and the conservation of fisheries. For example, in the Icelandic cod fishery, the implementation of such a management system has resulted in the concentration of fishing among a few large market participants.(8) In fact: This state of affairs has lead many to describe the system in feudal terms, with the quota kings or lords of the sea controlling most of the and profiting from renting it to tenant companies, who actually do much of the fishing. After paying the rental price, the tenant companies are left with only 60% of the value of the catch, while still bearing the normal expenses of fishing.(9) Currently, there are three ITQ management systems in the United States. These regulate the Atlantic Surf Clam and Ocean Quahog fisheries,(10) the Wreckfish fishery,(11) and the Alaskan Halibut and Sablefish fisheries.(12) While the character of each of these three fisheries is unique, the theory behind their ITQ management is the same: to provide for more efficient and sustainable management of the fish stock by limiting access through tradable fishing rights--ITQs--which are a percentage share of total allowable catch.(13) In theory, transferability of the will encourage needed fleet downsizing by giving marginal actors an asset they can sell to exit the market. Due to the nature of the initial allocation,(14) researchers point out that small actors, lacking the financial backing to acquire extra permanent quota-shares, must sell out. …

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.

Clam calamity; a recent supratidal storm-deposit as an analog for fossil shell beds

A winter storm struck the South Jersey shore near Atlantic City, New Jersey in 1992, excavating a population (108 individuals) of the burrowing infaunal surf-clam, Spisula solidissima, and depositing them above the high tide line. Nearly all individuals survived transport during the storm. Small clams at the base of the storm deposit reburrowed, larger individuals and small ones above the base of the deposit did not. The overburden of conspecifics most likely prevented them from attaining the requisite vertical burrowing orientation, or an underlying pavement of conspecifics blocked burrowing. Clams accumulated in piles or dunes standing as high as one meter on the beach and two meters in the lee of breakwaters and extending laterally <20 meters. Such accumulations might appear in the fossil record and as yet be unrecognized. Larger shells were often distinctively chipped at the anterior and posterior ends, probably as a result of collision with other shells during transport.

The Surf Clam ITQ Management: An Evaluation

Previous articleNext article No AccessThalassoramaThe Surf Clam ITQ Management: An EvaluationStanley WangStanley Wang Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by Marine Resource Economics Volume 10, Number 1Spring 1995 Article DOIhttps://doi.org/10.1086/mre.10.1.42629101 Views: 10Total views on this site Citations: 16Citations are reported from Crossref Copyright © 1995 Marine Resource FoundationPDF download Crossref reports the following articles citing this article:Jorge Holzer, Kenneth McConnell Extraction rights allocation with liquidity constraints, Resource and Energy Economics 71 (Feb 2023): 101345.https://doi.org/10.1016/j.reseneeco.2022.101345Andrew M Scheld, Jennifer Beckensteiner, Daphne M Munroe, Eric N Powell, Sarah Borsetti, Eileen E Hofmann, John M Klinck, David Secor The Atlantic surfclam fishery and offshore wind energy development: 2. Assessing economic impacts, ICES Journal of Marine Science 79, no.66 (Jun 2022): 1801–1814.https://doi.org/10.1093/icesjms/fsac109Devashish Kar Community-based fisheries management in different continents and countries across the world, (Jan 2021): 121–505.https://doi.org/10.1016/B978-0-12-821723-8.00005-1Eriko Hoshino, Ingrid van Putten, Sean Pascoe, Simon Vieira Individual transferable quotas in achieving multiple objectives of fisheries management, Marine Policy 113 (Mar 2020): 103744.https://doi.org/10.1016/j.marpol.2019.103744Jennifer J. Silver, Joshua S. Stoll How do commercial fishing licences relate to access?, Fish and Fisheries 20, no.55 (Jul 2019): 993–1004.https://doi.org/10.1111/faf.12393Katharina Jantzen, Ralf Döring, Leyre Goti, Lorena Fricke Individual Vessel Quotas in Germany and Denmark: A Fair Distribution Process?, (Jul 2017): 159–180.https://doi.org/10.1007/978-3-319-59169-8_8Kailin Kroetz, James N. Sanchirico, Julio Peña-Torres, and David Corderi Novoa Evaluation of the Chilean Jack Mackerel ITQ System, Marine Resource Economics 32, no.22 (Feb 2017): 217–241.https://doi.org/10.1086/690771Ayeisha A. Brinson, Eric M. Thunberg Performance of federally managed catch share fisheries in the United States, Fisheries Research 179 (Jul 2016): 213–223.https://doi.org/10.1016/j.fishres.2016.03.008Juan J. Agar, Jessica A. Stephen, Andy Strelcheck, and Assane Diagne The Gulf of Mexico Red Snapper IFQ Program: The First Five Years, Marine Resource Economics 29, no.22 (Jul 2015): 177–198.https://doi.org/10.1086/676825Dietmar Grimm, Ivan Barkhorn, David Festa, Kate Bonzon, Judd Boomhower, Valerie Hovland, Jason Blau Assessing catch shares' effects evidence from Federal United States and associated British Columbian fisheries, Marine Policy 36, no.33 (May 2012): 644–657.https://doi.org/10.1016/j.marpol.2011.10.014Lawrence J. White The Fishery as a Watery Commons: Lessons from the Experiences of Other Public Policy Areas for U.S. Fisheries Policy, SSRN Electronic Journal (Jan 2006).https://doi.org/10.2139/ssrn.945390Lawrence J. White Focusing More on Outputs and on Markets: What Financial Regulation Can Learn from Progress in Other Policy Areas, SSRN Electronic Journal (Jan 2006).https://doi.org/10.2139/ssrn.947895Richard McGarvey Demand-side fishery management: integrating two forms of input control, Marine Policy 27, no.33 (May 2003): 207–218.https://doi.org/10.1016/S0308-597X(02)00058-1Olivier Guyader, Olivier Thébaud Distributional issues in the operation of rights-based fisheries management systems, Marine Policy 25, no.22 (Mar 2001): 103–112.https://doi.org/10.1016/S0308-597X(00)00041-5Keith R Criddle, Seth Macinko A requiem for the IFQ in US fisheries?, Marine Policy 24, no.66 (Nov 2000): 461–469.https://doi.org/10.1016/S0308-597X(00)00025-7Bonnie J. McCay, Carolyn F. Creed, Alan Christopher Finlayson, Richard Apostle, Knut Mikalsen Individual transferable quotas (ITQs) in Canadian and US fisheries, Ocean & Coastal Management 28, no.1-31-3 (Jan 1995): 85–115.https://doi.org/10.1016/0964-5691(95)00068-2

Oocyte activation and passage through the metaphase/anaphase transition of the meiotic cell cycle is blocked in clams by inhibitors of HMG-CoA reductase activity.

Cell cycle progression for postembryonic cells requires the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-R), the enzyme which catalyzes the production of the isoprenoid precursor, mevalonate. In this study, we examine the requirements of HMG-R activity for cell cycle progression during the meiotic and early mitotic divisions using oocytes and dividing embryos from the surf clam, Spisula solidissima. Using two different inhibitors of HMG-R, we find that the activity of this enzyme appears to be required at three distinct points of the cell cycle during meiosis. Depending on the stage at which these inhibitors are added to synchronous clam cultures, a reversible cell cycle block is triggered at the time of activation or at metaphase of either meiosis I or II, whereas there is not block to the mitotic cell cycle. Inhibition of HMG-R activity in activated oocytes does not affect the transient activation of p42MAPK but results in a block at metaphase of meiosis I that is accompanied by the stabilization of cyclins A and B and p34cdc2 kinase activity. Our results suggest that metabolites from the mevalonate biosynthetic pathway can act to influence the process of activation, as well as the events later in the cell cycle that lead to cyclin proteolysis and the exit from M phase during clam meiosis.

Gametogenic Cycle in the Non-Native Atlantic Surf Clam, Spisula solidissima (Dillwyn, 1817), Cultured in the Coastal Waters of Georgia

This study describes the gametogenic cycle of the Atlantic surf clam. Spisula solidissima (Dillwyn, 1817), cultured from fall to spring in the coastal waters of Georgia, where it is non-native. Early active stages of gametogenic development began in November, with the majority (83%) of the animals in the early active stage by December. Gonadal indices increased to late active stages by March, with ripe individuals present in April. Spawning commenced in May and continued into June. Sex ratio (0.48 female to 1 .OO male) was significantly unequal. Results of this study indicate that clams achieved sexual maturity and spawned when cultured in the coastal waters of Georgia. An aquacultural enterprise in Georgia could obtain broodstock for the production of the following fall's seed crop from the prior year's growout field planted clams before their spring harvest.

Dynein inner arm heavy chain identification in cAMP‐activated flagella using class‐specific polyclonal antibodies

While studying cAMP-dependent dynein alpha-heavy chain phosphorylation, we found previously [Stephens and Prior, 1992: J. Cell Sci. 103:999-1012] that high salt extraction of sperm flagella from the mussel Mytilus edulis or the clam Spisula solidissima removed most visible dynein arms, accompanied by an amount of Mg+2-ATPase that correlated with the mass of dynein alpha- and beta-heavy chains removed. However, although almost devoid of ATPase activity, such extracted axonemes retained one third of the heavy chain mass as two sets of electrophoretically-distinct, vanadate-cleavable, non-phosphorylated proteins. To explore the nature of these dynein-like proteins, antibodies to the alpha- and beta-heavy chains were blot affinity-purified from a rabbit antiserum raised against gradient-purified Spisula 18-20S flagellar outer arm dynein. Although able to recognize common epitopes of the opposite chain type, neither the alpha- nor the beta-heavy chain antibody recognized the tightly-bound proteins in either species, proving that they are immunologically distinct. While the beta-antibody recognized its heavy chain homolog in gill cilia, the alpha-antibody did not, demonstrating immunological distinction between flagellar and ciliary dynein alpha-heavy chains. Immunization of a mouse with nitrocellulose strips containing one of the two tightly-bound Spisula flagellar proteins produced an antiserum that cross-reacted with each tightly-bound protein in both species and also recognized alpha- and beta-heavy chains. The anti-molluscan serum cross-reacted strongly with sea urchin sperm flagellar dynein B-, C-, and D-bands, considered to be inner arm components, but not with sea urchin outer arm alpha- or beta-heavy chains. These data indicate that the electrophoretically and immunologically distinct, tightly-bound proteins of molluscan flagella are inner arm dynein heavy chains.

Centrosomal components immunologically related to tektins from ciliary and flagellar microtubules.

Centrosomes are critical for the nucleation and organization of the microtubule cytoskeleton during both interphase and cell division. Using antibodies raised against sea urchin sperm flagellar microtubule proteins, we characterize here the presence and behavior of certain components associated with centrosomes of the surf clam Spisula solidissima and cultured mammalian cells. A Sarkosyl detergent-resistant fraction of axonemal microtubules was isolated from sea urchin sperm flagella and used to produce monoclonal antibodies, 16 of which were specific- or cross-specific for the major polypeptides associated with this microtubule fraction: tektins A, B and C, acetylated alpha-tubulin, and 77 and 83 kDa polypeptides. By 2-D isoelectric focussing/SDS polyacrylamide gel electrophoresis the tektins separate into several polypeptide spots. Identical spots were recognized by monoclonal and polyclonal antibodies against a given tektin, indicating that the different polypeptide spots are isoforms or modified versions of the same protein. Four independently derived monoclonal anti-tektins were found to stain centrosomes of S. solidissima oocytes and CHO and HeLa cells, by immunofluorescence microscopy. In particular, the centrosome staining of one monoclonal antibody specific for tektin B (tekB3) was cell-cycle-dependent for CHO cells, i.e. staining was observed only from early prometaphase until late anaphase. By immuno-electron microscopy tekB3 specifically labeled material surrounding the centrosome, whereas a polyclonal anti-tektin B recognized centrioles as well as the centrosomal material throughout the cell cycle. Finally, by immunoblot analysis tekB3 stained polypeptides of 48-50 kDa in isolated spindles and centrosomes from CHO cells.

Accumulation of paralytic shellfish toxins by surfclams, Spisula solidissima (Dillwyn, 1897) in the Gulf of Maine: seasonal changes, distribution between tissues, and notes on feeding habits.

Accumulation of paralytic shellfish poisoning (PSP) toxins by surfclams, Spisula solidissima, was studied over a period of two years at two inshore locations in southern Maine and at six stations on Georges Bank in the Gulf of Maine. Whole animals as well as individual tissues (siphon, mantle, digestive gland, foot, adductor muscle, gill) were analyzed for PSP-toxicity levels using the standard AOAC mouse bioassay. Analyses of gut contents were carried out on surfclams from both inshore and offshore locations to identify the type of particles ingested. Surfclams feed primarily on phytoplankton and detrital material characteristic of the overlying seawater and surface sediment. No evidence was found for any selection based on particle size or type. Elevated levels of PSP toxins were noted in surfclams from Georges Bank more than two years after initial toxification. Toxins were not evenly distributed among the various tissues of surfclams. Initially, maximum toxicity among surfclam tissues was found in digestive glands; however, subsequent analyses of samples collected later in the year indicated that toxicity in gill and mantle tissues had increased relative to initial values. No toxicity was detected in adductor muscles. Surfclams are characterized by a high variation in total toxin load among individual animals, with a tendency for decreasing variation as toxin levels increase. Archived data from the Main Department of Marine Resources revealed annual and seasonal patterns of toxin accumulation by surfclams, i.e., toxin accumulation is an annual event, with initial increases in toxicity usually occurring in early spring.

Differences in Tyrosine Phosphorylation of Oocyte Key Proteins during 5HT-Induced Meiosis Reinitiation in Two Bivalve Species

The neurohormone serotonin (5HT) triggers meiosis reinitiation in oocytes of the pelecypod molluses Spisula solidissima and Ruditapes philippinarum. However, while Spisula oocytes complete maturation, Ruditapes oocytes arrest in metaphase I. Anti-phosphotyrosine antibody revealed that serotonin triggered an early tyrosine phosphorylation of p42 mapk which was transient in Spisula, but persisted in Ruditapes . Moreover, dephosphorylation of tyrosine residues of p34 cdc2 was only observed in Spisula oocytes, simultaneously with germinal vesicle breakdown. The possibility is discussed that such differences may account for maintenance of the metaphase I block.

Sperm Nuclear Transformations in Cytoplasmic Extracts from Surf Clam (Spisula solidissima) Oocytes

Following their incorporation into oocytes, sperm nuclei (SN) of the surf clam, Spisula solidissima, undergo an initial expansion, followed by condensation and then a dramatic enlargement during their development into male pronuclei. These changes are temporally correlated with alterations in the maternal chromatin: germinal vesicle breakdown (GVBD), meiotic maturation, and female pronuclear development, respectively. To analyze possible changes occurring in SN at fertilization, surf clam oocyte extracts; prepared before and after parthenogenetic activation, were examined for their ability to affect SN in vitro. Sperm heads were incubated in extracts for variable periods up to 5 hr. Extracts prepared from oocytes following GVBD (15 min postactivation) induced an expansion in ∼90% of SN by 60 min incubation. However, when SN were incubated in extracts from unactivated or 4-min-activated oocytes only ∼30% underwent expansion. Ultrastructural examination of specimens taken at increasing periods of incubation in oocyte extracts revealed that SN expansion in vitro resembled chromatin decondensation in vivo. SN incubated 1 to 5 hr in extracts prepared from oocytes following GVBD consisted of decondensed chromatin surrounded to varying degrees by membranous cisternae. Staining with anti-lamin antibody was variable: some specimens (60-70%) were positive while others (30-40%) were weak to negative. In contrast, all decondensed SN incubated in extracts from postmeiotic oocytes (65 min postactivation) were delimited by an intact nuclear envelope possessing nuclear pores and reactive to anti-lamin antibody. Decondensation of SN in 15- or 65-min extracts was blocked by EDTA, 2,6-dimethylami-nopurine, histone, and protamine. The presence (65-min extract) and absence (unactivated, 4- and 15-min extracts) of sperm nuclear envelope assembly in vitro is consistent with events in vivo, where such a structure forms after meiotic maturation in concert with the development of the female pronucleus.

Composition, preservation, and use of sea clam viscera as a protein supplement for growing pigs2

Use of surf clam viscera as an animal feed was assessed by measuring nutrient content, susceptibility to spoilage, preservation with organic acid, and replacement value for soybean meal when fed to growing pigs. Viscera exiting the processing plant averaged 88% moisture and 71% CP in DM. Amino acid contents (51% in DM) and profiles were similar to those of soybean meal. Storage and feeding quality of wet viscera could be maintained up to 3 wk by adding formic or propionic acid (3.5%, wt/wt). Pigs readily ate corn-based diets containing wet viscera preserved with propionic acid. Growth rate was similar for pigs from 18 kg to market weight (92 kg) when soybean meal and(or) clam viscera were fed as protein supplements. However, cooked loin roasts from pigs fed one-half or all supplemental protein from viscera (5 or 10% viscera in diet DM) had distinctive fishy aromas. To produce acceptable pork, future studies must identify lower dietary levels or withdrawal periods for viscera.

Characterization of a Major Nucleoplasmin-like Germinal Vesicle Protein Which Is Rapidly Phosphorylated before Germinal Vesicle Breakdown in Spisula solidissima

Oocytes of the surf clam, Spisula solidissima, are arrested at the G2/M boundary of meiotic prophase I. At this stage, they possess a prominent germinal vesicle (GV), comprising about 25% of the total oocyte volume, in which pools of mRNAs and proteins that facilitate the rapid rounds of cell division which occur early in development are stored. We have isolated and characterized an abundant 49-kDa phosphoprotein, localized exclusively to the GV, which shares properties with nucleoplasmin. Like nucleoplasmin, this 49-kDa protein is a heat-stable, highly acidic phosphoprotein [containing over 32% (Glx + Asx) with an isoelectric point of about 4.0] and is soluble in 80% ammonium sulfate. In contrast to the pentameric nucleoplasmin, much of this protein is isolated as a disulfide-linked multimer which migrates at 120 kDa. In addition, a fraction of the 49-kDa protein is associated via disulfide bonds to a doublet of 42-kDa proteins. In vivo, this 49-kDa protein is phosphorylated prior to germinal vesicle breakdown (GVBD) and at 5 min after oocyte activation rapidly incorporates 32P to 60% of maximal level, suggesting that this protein plays a major role in the cascade of events which lead to oocyte activation and GVBD.

Evidence for the Involvement of Internal Calcium Stores during Serotonin-Induced Meiosis Reinitiation in Oocytes of the Bivalve Mollusc Ruditapes philippinarum

In contrast to the situation found in the bivalves Barnea candida and Spisula solidissima , prophase-arrested oocytes of Ruditapes philippinarum cannot be fertilized when removed from the ovary. They must first undergo germinal vesicle breakdown under the influence of the neurohormone serotonin (5-HT), which drives them to a second block occurring in metaphase of the first maturation division. In the studies described in this paper, we investigate the possibility that calcium is involved as a second messenger in controlling this first step in the reinitiation of meiosis.Our data show that, in addition to 5-HT, ionophore, thapsigargin, and the weak bases ammonia and procaine can also induce prophase-arrested oocytes of Ruditapes to resume meiosis. 5-HT, thapsigargin, and ammonia all trigger a surge of intracellular Ca2+ and are effective even in the absence of external Ca2+. That such Ca2+ transients, which are enhanced in the presence of external Ca2+, actually play a key role in the process of meiosis reinitiation is shown by the fact that loading the oocytes with BAPTA/AM or treating them with D-600 blocks maturation.In contrast, excess KCl, which has been shown to trigger meiosis reinitiation of prophase-arrested oocytes of Barnea and Spisula and to activate metaphase I-arrested oocytes of Ruditapes, does not produce any significant intracellular Ca2+ transient nor does it reinitiate meiosis, when added to Ruditapes prophase-arrested oocytes. These data suggest that such voltage-operated Ca2+ channels may only appear during the course of maturation and that both intracellular and extracellular Ca2+ are involved in triggering 5-HT-dependent release from the prophase block in this species.

Optimization of fertilization parameters for rearing surf clams ( Spisula solidissima)

Laboratory experiments were conducted to determine optimal conditions for “in vitro” fertilization of surf clam, Spisula solidissima, oocytes. Critical variables such as concentration of gametes, number of spermatozoa per oocyte, temperature, pH and salinity were investigated and assessed in relation to the proportion of monospermic and polyspermic fertilizations. The optimal concentrations of sperm and oocytes for monospermic fertilizations were 0.8–4×10 6 spermatozoa/ml and 5–30×10 3 oocytes/ml. Good percentages of monospermic fertilizations (80%) were thus obtained at sperm: oocyte ratios of 30:1 to 500:1 with optimal success (95% monospermic fertilizations) at a sperm: oocyte ratio of 50:1 to 100:1. Higher ratios induced increased polyspermic fertilizations while, at lower values, the oocytes remained unfertilized. Temperature, pH and salinity of natural sea water were varied separately from the standard values of 18°C, pH 8.0 and 24‰ and the success of fertilization was assessed. Temperatures ranging from 6 to 24°C had no detrimental effects upon fertilization and temperatures of 8 to 20°C appeared adequate for optimal fertilization. Fertilization rates, not affected at pH values ranging from 7.5 to 10.5, decreased rapidly at other values. Fertilization was not affected at salinities ranging from 20 to 35‰ and a salinity range of 23 to 30‰ was ideal for fertilization.

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.

Developmental Biology of Marine Organisms

In contrast to the situation found in the bivalves Barnea Candida and Spisula solidissima prophase arrested oocytes of Ruditapes philippinarum cannot be fertilized when extracted from the ovary. They must first undergo germinal vesicle breakdown (GVBD) under the influence of the neurohormone serotonin (5-HT) which drives them to a second block occurring in metaphase of the first maturation division. We used Fluo-3 to investigate the possibility that calcium was involved as a second messenger in controlling this first step in meiosis reinitiation. Our data showed that, in addition to 5-HT, ionophore, thapsigargin and the weak bases ammonia and procaine could induce prophase-arrested oocytes of Ruditapes to resume meiosis.

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.