Inositol-1,4,5-trisphosphate injection mimics fertilization potentials in sea urchin eggs

1986 ◽  
Vol 250 (2) ◽  
pp. C340-C344 ◽  
Author(s):  
B. E. Slack ◽  
J. E. Bell ◽  
D. J. Benos
Keyword(s):  
Membrane Potential ◽  
Partial Response ◽  
Sea Urchin ◽  
Time Course ◽  
Strongylocentrotus Purpuratus ◽  
Sea Urchin Eggs ◽  
Low Calcium ◽  
Inositol 1,4,5 Trisphosphate

The effects of inositol-1,4,5-trisphosphate (IP3) and of diacylglycerol (DAG) and its analogues on the membrane potential of eggs from the sea urchin Strongylocentrotus purpuratus were examined. Injection of IP3 into eggs resulted in a change in membrane potential that was similar in magnitude and time course to the fertilization potential elicited by sperm attachment. In low-calcium seawater, IP3 injection elicited a partial response. DAG and its analogues phorbol myristyl acetate and 1-oleoyl-2-acetylglycerol did not affect membrane potential either when applied by perfusion or when injected. The results indicate that IP3, but not DAG or its analogues, may be involved in the generation of the fertilization potential triggered by the interaction of sperm with sea urchin eggs.

The Respiratory and Glycolytic Enzymes of Sea-Urchin Eggs

1954 ◽  
Vol 31 (2) ◽  
pp. 208-217
Author(s):  
MARTYNAS YČAS
Keyword(s):  
Cytochrome C ◽  
Sea Urchin ◽  
Strongylocentrotus Purpuratus ◽  
Lactic Dehydrogenase ◽  
Glycolytic Pathway ◽  
Endogenous Respiration ◽  
Centrifugal Field ◽  
Lytechinus Pictus ◽  
Sea Urchin Eggs ◽  
Sea Urchin Egg

1. Activity corresponding to phosphoglucomutase, phosphohexoisomerase, aldolase, triosephosphate dehydrogenase, enolase and lactic dehydrogenase has been demonstrated in homogenates prepared from unfertilized sea-urchin eggs (Strongylocentrotus purpuratus and Lytechinus pictus). 2. The presence of cytochromes a and b1 has been confirmed. These cytochromes sediment in a relatively low centrifugal field. 3. No cytochrome c could be demonstrated, although cytochrome c is both reduced and oxidized by homogenates, and addition of cytochrome c increases the endogenous respiration and oxidation of succinate. 4. These results support the view that the usual glycolytic pathway operates in the sea-urchin egg and is the principal route of oxidation of carbohydrate.

scholarly journals Transition from mitosis to interphase in sea urchin first division: immunofluorescence studies of tubulin distribution in methacrylate sections.

1987 ◽  
Vol 35 (3) ◽  
pp. 343-349 ◽  
Author(s):  
P J Harris ◽  
B P Rubin
Keyword(s):  
Low Temperature ◽  
Methyl Methacrylate ◽  
Sea Urchin ◽  
Strongylocentrotus Purpuratus ◽  
Immunofluorescence Staining ◽  
Cleavage Cycle ◽  
Sea Urchin Eggs ◽  
Immunofluorescence Studies ◽  
Flat Embedding ◽  
Cell Thickness

Previous immunofluorescence studies of microtubule distribution in fertilized sea urchin eggs have suffered from poor resolution caused by cell thickness, unavoidable artifacts resulting from excessive flattening, or extraction by detergents of membranes and other lipid-containing structures that may be of interest in relation to the microtubules. To avoid these difficulties, we have developed a fixation and embedding protocol based on buffered paraformaldehyde fixation and butyl-methyl methacrylate embedment, which allows immunofluorescence staining of 0.5-1 micron sections. Polymerization artifacts are reduced by polymerizing the methacrylate at a relatively low temperature (40-45 degrees C) and by flat embedding for more uniform polymerization. Using this method, we have examined mitotic stages in the first cleavage cycle of the sea urchin Strongylocentrotus purpuratus. We provide evidence that the interphase microtubules that appear after first division are not derived from the mitotic asters but are new structures growing from organizing centers within the degenerating mitotic asters. During the transition from mitosis to interphase, there is a temporary overlap of old and new microtubules to form a very large composite aster at telophase before the old structure finally disappears.

scholarly journals Cell cycle-dependent phosphorylation of the 77 kDa echinoderm microtubule-associated protein (EMAP) in vivo and association with the p34cdc2 kinase

1996 ◽  
Vol 109 (12) ◽  
pp. 2885-2893 ◽  
Author(s):  
E. Brisch ◽  
M.A. Daggett ◽  
K.A. Suprenant
Keyword(s):  
Cell Cycle ◽  
Sea Urchin ◽  
Time Course ◽  
Mitotic Apparatus ◽  
Sea Urchin Eggs ◽  
Spindle Poles ◽  
A Cell ◽  
Cycle Dependent ◽  
Phosphopeptide Mapping

The most abundant microtubule-associated protein in sea urchin eggs and embryos is the 77 kDa echinoderm microtubule-associated protein (EMAP). EMAP localizes to the mitotic spindle as well as the interphase microtubule array and is a likely target for a cell cycle-activated kinase. To determine if EMAP is phosphorylated in vivo, sea urchin eggs and embryos were metabolically labeled with 32PO4 and a monospecific antiserum was used to immunoprecipitate EMAP from 32P-labeled eggs and embryos. In this study, we demonstrate that the 77 kDa EMAP is phosphorylated in vivo by two distinct mechanisms. In the unfertilized egg, EMAP is constitutively phosphorylated on at least five serine residues. During the first cleavage division following fertilization, EMAP is phosphorylated with a cell cycle-dependent time course. As the embryo enters mitosis, EMAP phosphorylation increases, and as the embryo exits mitosis, phosphorylation decreases. During mitosis, EMAP is phosphorylated on 10 serine residues and two-dimensional phosphopeptide mapping reveals a mitosis-specific site of phosphorylation. At all stages of the cell cycle, a 33 kDa polypeptide copurifies with the 77 kDa EMAP, regardless of phosphorylation state. Antibodies against the cdc2 kinase were used to demonstrate that the 33 kDa polypeptide is the p34cdc2 kinase. The p34cdc2 kinase copurifies with the mitotic apparatus and immunostaining indicates that the p34cdc2 kinase is concentrated at the spindle poles. Models for the interaction of the p34cdc2 kinase and the 77 kDa EMAP are presented.

scholarly journals Ca2+ release triggered by nicotinate adenine dinucleotide phosphate in intact sea urchin eggs

1995 ◽  
Vol 312 (3) ◽  
pp. 955-959 ◽  
Author(s):  
C M Perez-Terzic ◽  
E N Chini ◽  
S S Shen ◽  
T P Dousa ◽  
D E Clapham
Keyword(s):  
Sea Urchin ◽  
Specific Inhibitor ◽  
Intact Cells ◽  
Sea Urchin Eggs ◽  
Sea Urchin Egg ◽  
Cyclic Adp Ribose ◽  
Inositol 1,4,5 Trisphosphate ◽  
Dose Dependent Increase ◽  
Dose Dependent

Nicotinate adenine dinucleotide phosphate (NAADP) was recently identified [Lee and Aarhus (1995) J. Biol. Chem. 270, 2152-2157; Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] as a potent Ca(2+)-releasing agent in sea urchin egg homogenates. NAADP triggered Ca2+ release by a mechanism that was distinct from inositol 1,4,5-trisphosphate (InsP3)- and cyclic ADP-ribose (cADPR)-induced Ca2+ release. When NAADP was microinjected into intact sea urchin eggs it induced a dose-dependent increase in cytoplasmic free Ca2+ which was independent of the extracellular [Ca2+]. The Ca2+ waves elicited by microinjections of NAADP originated at the site of injection and swept across the cytosol. As previously found in sea urchin egg homogenates, NAADP-induced Ca2+ release in intact eggs was not blocked by heparin or by prior desensitization to InsP3 or cADPR. Thio-NADP, a specific inhibitor of the NAADP-induced Ca2+ release in sea urchin homogenates [Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] blocked NAADP (but not InsP3 or cADPR) injection-induced Ca2+ release in intact sea urchin eggs. Finally, fertilization of sea urchin eggs abrogated subsequent NAADP-induced Ca2+ release, suggesting that the NAADP-sensitive Ca2+ pool may participate in the fertilization response. This study demonstrates that NAADP acts as a selective Ca(2+)-releasing agonist in intact cells.

Mutually exclusive expression of the Strongylocentrotus purpuratus Spec1 gene and its Lytechinus pictus homologue in cells of hybrid embryos

Development ◽  
1992 ◽  
Vol 114 (1) ◽  
pp. 193-201 ◽  
Author(s):  
P.E. Nisson ◽  
M.F. Gaudette ◽  
B.P. Brandhorst ◽  
W.R. Crain
Keyword(s):  
In Situ Hybridization ◽  
Sea Urchin ◽  
Time Course ◽  
Strongylocentrotus Purpuratus ◽  
Hybridization Analysis ◽  
Reciprocal Hybrid ◽  
Serial Sections ◽  
Lytechinus Pictus ◽  
Hybrid Embryo

The expression of the Spec1 gene of Strongylocentrotus purpuratus and its Lytechinus pictus homologue LpS1 was analyzed in reciprocal hybrid embryos of these two species of sea urchin. While the time course of accumulation of Spec1 mRNA was nearly normal in hybrid embryo populations, the accumulation of LpS1 mRNA was not. This was particularly evident in plutei, where the level of LpS1 mRNA was less than 5% that in normal L. pictus plutei. In situ hybridization analysis of serial sections indicated that LpS1 mRNA was detectable in only about 2% of hybrid plutei in either cross, whereas Spec1 mRNA was present in nearly all hybrid plutei; expression of either homologue was appropriately restricted to the aboral ectoderm. In crosses of L. pictus eggs with S. purpuratus sperm (LpSp), about 1% of hybrid plutei expressed LpS1 RNA in most or all aboral ectoderm cells at normal levels, and did not express Spec1 RNA; in another 1% of the LpSp hybrid plutei the Spec1 and LpS1 transcripts were present at normal levels in complementary, non-overlapping patches of contiguous aboral ectoderm cells. In the reciprocal SpLp cross, each hybrid pluteus expressed either only the LpS1 gene (about 2%) or only the Spec1 gene throughout the aboral ectoderm. In SpLp hybrid gastrulae the level of LpS1 mRNA was less restricted; about 2% of the embryos contained only LpS1 RNA, and about half expressed only Spec1 transcripts, but in the remaining embryos Spec1 and LpS1 transcripts were coexpressed in the same aboral ectoderm cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Increase of cGMP, cADP-ribose and inositol 1,4,5-trisphosphate preceding Ca2+ transients in fertilization of sea urchin eggs

Development ◽  
2001 ◽  
Vol 128 (22) ◽  
pp. 4405-4414 ◽  
Author(s):  
Ritsu Kuroda ◽  
Kenji Kontani ◽  
Yasunari Kanda ◽  
Toshiaki Katada ◽  
Takashi Nakano ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Sea Urchin ◽  
Guanylate Cyclase ◽  
Soluble Guanylate Cyclase ◽  
Sea Urchin Eggs ◽  
Direct Measurements ◽  
Cyclic Adp Ribose ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor ◽  
Receptor Channel

Transient increases, or oscillations, of cytoplasmic free Ca2+ concentration, [Ca2+]i, occur during fertilization of animal egg cells. In sea urchin eggs, the increased Ca2+ is derived from intracellular stores, but the principal signaling and release system involved has not yet been agreed upon. Possible candidates are the inositol 1,4,5-trisphosphate receptor/channel (IP3R) and the ryanodine receptor/channel (RyR) which is activated by cGMP or cyclic ADP-ribose (cADPR). Thus, it seemed that direct measurements of the likely second messenger candidates during sea urchin fertilization would be essential to an understanding of the Ca2+ signaling pathway. We therefore measured the cGMP, cADPR and inositol 1,4,5-trisphosphate (IP3) contents of sea urchin eggs during the early stages of fertilization and compared these with the [Ca2+]i rise in the presence or absence of an inhibitor against soluble guanylate cyclase. We obtained three major experimental results: (1) cytosolic cGMP levels began to rise first, followed by cADPR and IP3 levels, all almost doubling before the explosive increase of [Ca2+]i; (2) most of the rise in IP3 occurred after the Ca2+ peak; IP3 production could also be induced by the artificial elevation of [Ca2+]i, suggesting the large increase in IP3 is a consequence, rather than a cause, of the Ca2+ transient; (3) the measured increase in cGMP was produced by the soluble guanylate cyclase of eggs, and inhibition of soluble guanylate cyclase of eggs diminished the production of both cADPR and IP3 and the [Ca2+]i increase without the delay of Ca2+ transients. Taken together, these results suggest that the RyR pathway involving cGMP and cADPR is not solely responsible for the initiating event, but contributes to the Ca2+ transients by stimulating IP3 production during fertilization of sea urchin eggs.

scholarly journals Regulation of cortical vesicle exocytosis in sea urchin eggs by inositol 1,4,5-trisphosphate and GTP-binding protein.

1986 ◽  
Vol 102 (1) ◽  
pp. 70-76 ◽  
Author(s):  
P R Turner ◽  
L A Jaffe ◽  
A Fein
Keyword(s):  
Sea Urchin ◽  
Intracellular Free Calcium ◽  
Free Calcium ◽  
Lytechinus Variegatus ◽  
Nucleotide Analogs ◽  
Sea Urchin Eggs ◽  
Sequence Of Events ◽  
Inositol 1,4,5 Trisphosphate ◽  
Vesicle Exocytosis ◽  
Nucleotide Binding Proteins

To investigate the roles of inositol 1,4,5-trisphosphate (InsP3) and guanyl nucleotide binding proteins (G-proteins) in the transduction mechanism coupling fertilization and exocytosis of cortical vesicles in sea urchin eggs, we microinjected InsP3 and guanyl nucleotide analogs into eggs of Lytechinus variegatus. Injection of 28 nM InsP3 caused exocytosis. However, if the egg was first injected with EGTA ([Cai] less than or equal to 0.1 microM; EGTA = 1.6 mM), InsP3 injection did not cause exocytosis, supporting the hypothesis that InsP3 acts by causing a rise in intracellular free calcium. Injection of 28 microM guanosine-5'-0-(3-thiotriphosphate) (GTP-gamma-S), a hydrolysis-resistant analog of GTP, caused exocytosis, but exocytosis did not occur if the egg was pre-injected with EGTA. Injection of 3 mM guanosine-5'-0-(2-thiodiphosphate) (GDP-beta-S), a metabolically stable analog of GDP, prevented sperm from stimulating exocytosis. However, injection of GDP-beta-S did not prevent the stimulation of exocytosis by InsP3. These results suggested the following sequence of events. The sperm activates a G-protein, which stimulates production of InsP3. InsP3 elevates intracellular free calcium, which causes exocytosis.

Inositol 1,4,5-trisphosphate microinjection activates sea urchin eggs

Nature ◽  
1984 ◽  
Vol 312 (5995) ◽  
pp. 636-639 ◽  
Author(s):  
Michael Whitaker ◽  
R. F. Irvine
Keyword(s):  
Sea Urchin ◽  
Sea Urchin Eggs ◽  
Inositol 1,4,5 Trisphosphate

Cyclic ADP-ribose activates caffeine-sensitive calcium channels from sea urchin egg microsomes

1998 ◽  
Vol 274 (2) ◽  
pp. C430-C439 ◽  
Author(s):  
Claudio F. Pérez ◽  
Juan José Marengo ◽  
Ricardo Bull ◽  
Cecilia Hidalgo
Keyword(s):  
Sea Urchin ◽  
Microsomal Fraction ◽  
Ruthenium Red ◽  
Single Channels ◽  
Channel Activity ◽  
Sea Urchin Eggs ◽  
Sea Urchin Egg ◽  
Cyclic Adp Ribose ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca

Adenosine 5′-cyclic diphosphoribose [cyclic ADP-ribose (cADPR)], a metabolite of NAD+ that promotes Ca2+ release from sea urchin egg homogenates and microsomal fractions, has been proposed to act as an endogenous agonist of Ca2+ release in sea urchin eggs. We describe experiments showing that a microsomal fraction isolated from Tetrapigus nyger sea urchin eggs displayed Ca2+-selective single channels with conductances of 155.0 ± 8.0 pS in asymmetric Cs+ solutions and 47.5 ± 1.1 pS in asymmetric Ca2+ solutions. These channels were sensitive to stimulation by Ca2+, ATP, and caffeine, but not inositol 1,4,5-trisphosphate, and were inhibited by ruthenium red. The channels were also activated by cADP-ribose in a Ca2+-dependent fashion. Calmodulin and Mg2+, but not heparin, modulated channel activity in the presence of cADP-ribose. We propose that these Ca2+ channels constitute the intracellular Ca2+-induced Ca2+ release pathway that is activated by cADP-ribose in sea urchin eggs.

Sign in / Sign up

Export Citation Format

Share Document