Surf Clam Oocytes Research Articles

Discovery of Centrosomal RNA and Centrosomal Hypothesis of Cellular Ageing and Differentiation

In 2006, a group of scientists studying centrosomes of Spisula solidissima mollusc oocytes under the leadership of Alliegro (Alliegro, M.C.; Alliegro, M.A.; Palazzo, R.E. Centrosome-associated RNA in surf clam oocytes. Proc. Natl. Acad. Sci. USA 2006, 103(24), 9034–9038) reliably demonstrated the existence of specific RNA in centrosome, called centrosomal RNA (cnRNA). In their first article, five different RNAs (cnRNAs 11, 102, 113, 170, and 184) were described. During the process of full sequencing of the first transcript (cnRNA 11), it was discovered that the transcript contained a conserved structure—a reverse transcriptase domain located together with the most important centrosomal protein, γ-tubulin. In an article published in 2005, we made assumptions about several possible mechanisms for determining the most important functions of centrosomal structures and referred to one of them as a “RNA-dependent mechanism.” This idea about participation of hypothetic centrosomal small interference RNA and/or microRNA in the process was made one year prior to the discovery of cnRNA by Alliegro's group. The discovery of specific RNA in a centrosome is indirect evidence of a centrosomal hypothesis of cellular ageing and differentiation. The presence of a reverse transcriptase domain in this type of RNA, together with its uniqueness and specificity, makes the centrosome a place of information storage and reproduction.

Composition and Dynamics of the Nucleolinus, a Link between the Nucleolus and Cell Division Apparatus in Surf Clam (Spisula) Oocytes

The nucleolinus is a little-known cellular structure, discovered over 150 years ago (Agassiz, L. (1857) Contributions to the Natural History of the United States of America, First Monograph, Part IIL, Little, Brown and Co., Boston) and thought by some investigators in the late 19th to mid-20th century to function in the formation of the centrosomes or spindle. A role for the nucleolinus in formation of the cell division apparatus has recently been confirmed in oocytes of the surf clam, Spisula solidissima (Alliegro, M. A., Henry, J. J., and Alliegro, M. C. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 13718-13723). However, we know so little about the composition and dynamics of this compartment, it is difficult to construct mechanistic hypotheses or even to be sure that prior reports were describing analogous structures in the cells of mammals, amphibians, plants, and other organisms where it was observed. Surf clam oocytes are an attractive model to approach this problem because the nucleolinus is easily visible by light microscopy, making it accessible by laser microsurgery as well as isolation by common cell fractionation techniques. In this report, we analyze the macromolecular composition of isolated Spisula nucleolini and examine the relationship of this structure to the nucleolus and cell division apparatus. Analysis of nucleolinar RNA and protein revealed a set of molecules that overlaps with but is nevertheless distinct from the nucleolus. The proteins identified were primarily ones involved in nucleic acid metabolism and cell cycle regulation. Monoclonal antibodies generated against isolated nucleolini revealed centrosomal forerunners in the oocyte cytoplasm. Finally, induction of damage to the nucleolinus by laser microsurgery altered the trafficking of α- and γ-tubulin after fertilization. These observations strongly support a role for the nucleolinus in cell division and represent our first clues regarding mechanism.

High-throughput optofluidic system for the laser microsurgery of oocytes

This study combines microfluidics with optical microablation in a microscopy system that allows for high-throughput manipulation of oocytes, automated media exchange, and long-term oocyte observation. The microfluidic component of the system transports oocytes from an inlet port into multiple flow channels. Within each channel, oocytes are confined against a microfluidic barrier using a steady fluid flow provided by an external computer-controlled syringe pump. This allows for easy media replacement without disturbing the oocyte location. The microfluidic and optical-laser microbeam ablation capabilities of the system were validated using surf clam (Spisula solidissima) oocytes that were immobilized in order to permit ablation of the 5 μm diameter nucleolinus within the oocyte nucleolus. Oocytes were the followed and assayed for polar body ejection.

The small organic compound HMN-176 delays satisfaction of the spindle assembly checkpoint by inhibiting centrosome-dependent microtubule nucleation

HMN-176 is a potential new cancer therapeutic known to retard the proliferation of tumor cell lines. Here, we show that this compound inhibits meiotic spindle assembly in surf clam oocytes and delays satisfaction of the spindle assembly checkpoint in human somatic cells by inducing the formation of short and/or multipolar spindles. HMN-176 does not affect centrosome assembly, nuclear envelope breakdown, or other aspects of meiotic or mitotic progression, nor does it affect the kinetics of Spisula or mammalian microtubule (MT) assembly in vitro. Notably, HMN-176 inhibits the formation of centrosome-nucleated MTs (i.e., asters) in Spisula oocytes and oocyte extracts, as well as from isolated Spisula or mammalian centrosomes in vitro. Together, these results reveal that HMN-176 is a first-in-class anticentrosome drug that inhibits proliferation, at least in part, by disrupting centrosome-mediated MT assembly during mitosis.

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.

Centrosome-associated RNA in surf clam oocytes.

Centrosomes are the major microtubule-organizing center in animal cells. They are composed of a pair of [9(3) + 0] centrioles surrounded by a relatively ill-defined pericentriolar matrix, provide the ciliary centriole-kinetosome (basal body) progenitor, and organize the assembly of microtubules into the mitotic spindle during cell division. Despite >100 years of microscopic observation and their obvious significance, our understanding of centrosome composition, dynamic organization, and mechanism of action is limited when compared with that of other cellular organelles. Centrosomes duplicate only once per cell cycle to ensure development of a normal bipolar spindle. The initial event in centrosome duplication is centriole replication, which is generative, semiconservative, and independent of the nucleus. Such observations led to the proposal that centrosomes contain their own complement of nucleic acids, possibly representative of an organellar genome comparable with those described for mitochondria and chloroplasts. The consensus in the field is that centrosomes lack DNA but may contain RNA. We isolated centrosomes from oocytes of the surf clam, Spisula solidissima, and purified from them a unique set of RNAs. We show here by biochemical means and subcellular in situ hybridization that the first transcript we analyzed is intimately associated with centrosomes. Sequence analysis reveals that this centrosome-associated RNA encodes a conserved RNA-directed polymerase domain. The hypothesis that centrosomes contain an intrinsic complement of specific RNAs suggests new opportunities to address the century-old problem of centrosome function, heredity, and evolution.

Increase of cAMP upon release from prophase arrest in surf clam oocytes.

Surf clam (Spisula solidissima) oocytes are spawned at the prophase I stage of meiosis, and they remain arrested at this stage until fertilization. Full oocyte meiosis reinitiation, first evidenced by germinal vesicle breakdown (GVBD), may be induced by artificial activators mimicking sperm, such as high K(+) or serotonin. Previous reports indicated that treatments thought to increase the level of oocyte cAMP inhibited sperm- or serotonin-induced, but not KCl-induced, GVBD in clam oocytes. These observations extend the well known requirement for a drop in oocyte cAMP levels in mammalian, amphibian or starfish oocytes and support the view that such a drop is universally important throughout the animal kingdom. We have re-examined the cAMP dependency of GVBD in clam oocytes and found that various treatments that raise oocyte cAMP levels did not, surprisingly, affect either KCl- or serotonin-induced GVBD. Such treatments, however, inhibited GVBD upon insemination of the oocytes, but this was due to the failure of sperm to fuse/penetrate the oocytes; thus, it was not an inhibition of oocyte activation as such. Direct measurements of oocyte cAMP levels after activation by serotonin, KCl or sperm showed that, contrary to expectations, there is a rise in cAMP levels before GVBD. Using SQ22536, an adenylyl cyclase inhibitor, the increase in oocyte cAMP level was partly prevented and GVBD proceeded, but with a significant retardation, indicating that the normal cAMP rise facilitates GVBD. Our work sheds light on the diversity of upstream pathways leading to activation of MPF and provides a unique model whereby the onset of meiosis reinitiation is associated with an increase, not a decrease, in oocyte cAMP levels.

Intracellular pH Increase Driven by an Na+/H+Exchanger upon Activation of Surf Clam Oocytes

Intracellular pH (pHi) measurements were performed in surf clam (Spisula solidissima) oocytes before and after artificial activation or fertilization [evidenced by germinal vesicle breakdown (GVBD)] by the dimethyloxazolidinedione (DMO) and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) methods. Results using both methods showed increases of pHiof 0.3 pH unit after activation by excess K+. Using BCECF, we found an increase of similar magnitude after fertilization or after the addition of serotonin. By contrast, GVBD did not occur when the pHiwas increased to similar or even higher levels by exposing the oocytes to ammonia. In sodium-free seawater, excess K+induced GVBD but the pHiof K+-activated oocytes decreased significantly below the resting level of unactivated oocytes. The pHiincreases in K+-activated oocytes were otherwise proportional to the external Na+concentration. The amiloride derivatives dimethylamiloride and hexamethylene amiloride (at 10–50 μM) efficiently inhibited the K+-induced increase of pHibut did not block GVBD. These two derivatives were able, however, to retard K+-induced GVBD, hexamethylene amiloride being the more efficient. This retardation of K+-induced GVBD could be abolished by the simultaneous addition of ammonia. Taken altogether, these results show that a pHiincrease, driven by a typical Na+/H+exchanger, follows activation of surf clam oocytes but that this pHiincrease is neither sufficient nor required for GVBD, though it does allow its progression at an optimal rate.

Chapter 19 Methods for Studying in Vitro Assembly of Male Pronuclei Using Oocyte Extracts from Marine Invertebrates Sea Urchins and Surf Clams

Cell-free systems derived from oocytes, eggs, or embryos constitute powerful tools to investigate the morphological and biochemical transformations of the sperm nucleus that follow fertilization. The ability of cell-free extracts to support pronuclear formation depends on the state of maturation and activation of the egg cytoplasm from which they are prepared. Extracts from unfertilized and unactivated oocytes or eggs support only early stages of sperm chromatin decondensation. In contrast, extracts from artificially activated or fertilized eggs support complete male pronuclear formation, including nuclear envelope and lamina assembly and DNA replication. This chapter describes the procedures used for assembling male pronuclei in cell-free extracts from sea urchin eggs and, to a lesser degree, from surf clam oocytes. Shortly after fertilization, the sperm nuclear envelope is rapidly broken down inside the egg cytoplasm. In vivo removal of the sperm nuclear envelope is documented by electron microscopy in both the sea urchin and the surf clam. An advantage of cell-free systems is the potential to evaluate independently cytosolic and membrane vesicle (MV) contributions to nuclear development. Membrane-free cytosol is obtained after the ultracentrifugation of crude lysates and MVs can be recovered from the pellets.

Parthenogenetic activation of mouse oocytes by 6-dimethylaminopurine

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

Mechanisms of activation of surf clam (Spisula solidissima) oocytes

Summary Surf clam (Spisula solidissima) oocytes, when shed, are at prophase I of meiotic maturation. Fertilization is the normal activation process leading to germinal vesicle breakdown (GVBD) and further steps of meiosis, followed by successive embryonic mitoses. We review here work suggesting that early rises in intracellular Ca2+ and pH (pHi) are normally associated with oocyte activation. Experimental evidence suggesting a pivotal role for the Ca2+ response and a more modest contribution of pHi are presented. The mechanisms of activation of Spisula oocytes by various artificial agents mimicking sperm are discussed in relation to their potential relevance to the physiological process of fertilization. We emphasize that serotonin-induced activation of Spisula oocytes is probably the best available model system to gain knowledge on the signal transducing systems stimulated by both serotonin or sperm. The potential physiological role of serotonin receptors present on oocytes is critically considered. Futu...

Intracellular pH Measurements During Fertilization of Surf Clam (Spisula solidissima) Oocytes.

Previous articleNext article No AccessFERTILIZATION AND DEVELOPMENTAL BIOLOGYIntracellular pH Measurements During Fertilization of Surf Clam (Spisula solidissima) OocytesF. Dube and W. R. EckbergF. Dube Search for more articles by this author and W. R. Eckberg Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Biological Bulletin Volume 191, Number 2October 1996 Published in association with the Marine Biological Laboratory Article DOIhttps://doi.org/10.1086/BBLv191n2p279 Views: 19Total views on this site Copyright © 1996 by Marine Biological LaboratoryPDF download Crossref reports no articles citing this article.

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.

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.

Partial activation of surf clam oocytes by hypertonic treatment.

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

Thapsigargin induces meiotic maturation in surf clam oocytes

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

Particulate Tubulin in Interphase and Metaphase Extracts of Oocytes of Spisula.

In 1972 Weisenberg reported that surf clam oocytes contained a particulate and sedimentable pool of tubulin that could be isolated in buffers containing hexylene glycol. This "interphase particulate tubulin" (IPT) copurified with 10-20 {mu}m granular spheres, which were identified as the "tubulin-containing structures" (TCS). Approximately one TCS per oocyte was isolated and the TCS disappeared after nuclear envelope breakdown. Weisenberg postulated that the TCS was comprised of a stored form of tubulin or a microtubule-assembly intermediate. To characterize this intriguing form of stored tubulin, IPT was isolated in hexylene glycol-containing buffers as described by Weisenberg (1972, J. Cell Biol. 54, 266-278) and the amount of sedimentable tubulin was quantitated by immunoblotting during the first meiotic cell cycle. Approximately 10% of the total tubulin in Spisula oocytes sediments at g forces that are too small to pellet tubulin dimers or even single microtubules. Granular spheres, approximately 15 {mu}m in diameter, are present in the sedimentable tubulin fractions. During the first cell cycle, the granular spheres disappear while the sedimentable tubulin levels gradually decrease. Although the disappearance of the spheres corresponds with the loss of sedimentable tubulin, the spheres do not contain tubulin. An initial centrifugation of the oocyte homogenates at 650g leaves most of the tubulin in the supernatant and the granular spheres in the pellet. The tubulin-containing fractions are composed of membranes and an amorphous unidentified material associated with short microtubules. Sedimentable tubulin is not detected in homogenates prepared at 0{deg}C or in the absence of hexylene glycol, conditions that favor microtubule disassembly. It is likely that sedimentable tubulin is composed of hexylene glycol-induced polymers and not unique particulate structures that sequester tubulin. Finally, the granular spheres that contaminate the tubulin preparations are identified as nucleoli. They are morphologically identical to the nucleoli of the intact oocyte and they fluoresce brightly when stained with the Hoechst DNA dye 22358.

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.

Oocyte Membrane Components Preventing Trypsin-Induced Germinal Vesicle Breakdown in the Surf Clam Oocytes.

Maturation of surf clam oocytes can be induced with sperm, KCl, serotonin or calcium ionophore. We now report that the oocytes treated with trypsin can also induce germinal vesicle breakdown (GVBD). Oocytes incubated in 0.1% trypsin solution for 0.5 to 1 h, subsequently washed and incubated in control medium for 1 to 2 h underwent maturation. Treatment with glycosidase, DNase and RNase did not induce GVBD. The optimal condition for the induction of maturation was to incubate oocytes in 0.1 % trypsin solution for 1 h and to transfer the oocytes to a medium composed of 0.4 M Tris-HCl, pH 7.4, 20 mM CaCl2. The occurrence of spontaneous GVBD was dependent upon the presence of Ca2+ in the suspending medium. The optimal concentration of Ca2+ was 20 mM. To show that a membrane component is involved in sustaining maturation arrest, oocytes were incubated in a medium containing 1 M urea, 5 mM EDTA, 10 mM Tris-HCl, pH 7.4, for 30 min. The extracted membrane protein at final concentrations of 0.4 to 0.8 mg/ml blocked GVBD of trypsin treated oocytes. To identify the active component, the extracted membrane protein preparations were fractionated by gel filtration through a Sephadex G-100 column. The active principle had an estimated Mr of 18 kD based on elution position of standard proteins on gel filtration. The partially purified product was active at a concentration of 0.1-0.2 mg/ml. The present findings suggest that maturation arrest is sustained by an oocyte membrane protein and the resumption of oocyte maturation results from the hydrolysis of this substance(s) by a protease in the presence of Ca2+.

Protein phosphorylation during activation of surf clam oocytes

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