Characterization of hatching-associated changes in the sea urchin fertilization envelope

The embryo of the sea urchin Strongylocentrotus purpuratus hatches from the fertilization envelope (FE) via synthesis and secretion of a hatching enzyme and by ciliary activity. Although the basic characteristics of the hatching enzyme are known, little is understood about changes in the FE during hatching. We have studied the biochemical changes in FEs during hatching. Polyacrylamide gel analysis revealed an increasingly complex polypeptide spectrum of the extractable fraction of FEs isolated during development. Immunoblotting of these polypeptides (using antiserum against the soluble polypeptides extracted from FEs isolated at 30 minutes postinsemination) revealed a decrease in the soluble FE components during hatching. Immunochemical analysis of hatching medium showed a strong correlation between the soluble FE components released and the hatching interval. Immunoblotting of hatching media indicated the presence of soluble FE polypeptides of similar and lower molecular weights than those obtained for extracts of FEs. These results imply that the hatching-associated changes in the FE of S purpuratus occur via proteolysis of FE components, which are derived from the paracrystalline protein fraction, a subset of cortical granule proteins.     

Rho-kinase in sea urchin eggs and embryos

The activation of sea urchin eggs at fertilization provides an ideal system for studying the molecular events involved in cellular activation. Rho GTPases, which are key signaling enzymes in eukaryotes, are involved in sustaining the activation of sea urchin eggs; however, their downstream effectors have not yet been characterized. In somatic cells, RhoA regulates a serine/threonine kinase known as Rho-kinase (ROCK). The activity of ROCK in early sea urchin development has been inferred, but not tested directly. A ROCK gene was identified in the sea urchin (Strongylocentrotus purpuratus) genome and the sequence of its cDNA determined. The sea urchin ROCK (SpROCK) sequence predicts a protein of 158 kDa with >72% and 45% identities with different protein orthologues of the kinase catalytic domain and the complete protein sequence, respectively. SpROCK mRNA levels are high in unfertilized eggs and decrease to 35% after 15 min postfertilization and remain low up to the 4 cell stage. Antibodies to the human ROCK-I kinase domain revealed SpROCK to be concentrated in the cortex of eggs and early embryos. Co-immunoprecipitation assays indicate that RhoA and SpROCK are physically associated. This association is destroyed by treatment with the C3 exoenzyme and with the ROCK antagonist H-1152. H-1152 also inhibited DNA synthesis in embryos. We conclude that the Rho-dependent signaling pathway, via SpROCK, is essential for early embryonic development.     

Ionophore-induced efflux of sodium and potassium ions from sea urchin eggs

The efflux of K⁺ and Na⁺ from sea urchin eggs during Ca²⁺ ionophore A23187-induced parthenogenesis was studied in a K⁺ and Na⁺-free artificial seawater using extracellular ion-specific electrodes. We have probed this model system with monovalent cation-specific ionophores to determine if they affect K⁺ efflux in the unfertilized egg and whether any changes in ionophore sensitivity are observed during egg activation. In 500 mM choline chloride, 10 mM CaCl₂, 50 mM MgCl₂, 10 mM Tris-Cl pH 8.0, A23187 induced a rapid efflux of K⁺ and Na⁺ from the eggs after a short lag time (10–15 seconds). After the burst, the rate of K⁺ efflux remained higher than the pre-activation rate, but was lower than during the burst phase, while the rate of Na⁺ efflux became nearly zero. Monovalent cation-specific ionophores (valinomycin, gramicidin and nigericin) had no effect on K⁺ efflux from the unfertilized eggs in our model system. However, once the egg was activated by A23187, each of the above ionophores caused a prolongation of the burst phase for many minutes. These results show that the unfertilized egg plasma membrane (using our artificial conditions) is not susceptible to the monovalent cation-specific antibiotics and suggest that either the inserted cortical granule membrane or the developing fertilization envelope interacts with these ionophores to cause the change in rate-limiting step for K⁺ efflux observed egg activation.     

In vitro phosphorylation of sea urchin sperm adenylate cyclase by cyclic adenosine monophosphate-dependent protein kinase

Addition of [gamma -32P]ATP to a 2% Brij-78 40,000g supernatant of sea urchin sperm results in the cAMP-dependent phosphorylation of eight to ten proteins. One phosphoprotein of Mr 190 kD is sperm adenylate cyclase (AC). An antiserum to the AC immunoprecipitates the Mr 190 kD protein. Peptide maps of immunoprecipitates show that the AC is the only phosphoprotein present in the Mr 200 kD range. With respect to the in vitro phosphorylation of AC, the endogenous kinase has a Km for ATP of 5.2 microM and is maximally stimulated by 4-8 microM cAMP. The protein kinase inhibitors H8 (9 microM) and PKI (30 U/ml) inhibit the phosphorylation of the AC. The catalytic subunit of bovine cAMP-dependent protein kinase phosphorylates the AC on the same peptides as the endogenous protein kinase. Cyanogen bromide generated peptide maps of the phosphorylated AC show a minimum of five sites of phosphorylation. No change in the Km or Vmax of the sperm AC resulted from the additional phosphorylation by bovine kinase. Calcium ions at submicromolar concentrations completely block the in vitro phosphorylation of the AC, suggesting the presence in the preparation of a Ca2(+) -activated protein phosphatase. To our knowledge, this is the first report of the phosphorylation of an AC by cAMP-dependent protein kinase.     

Alteration of lipid organization following fertilization of sea urchin eggs

The fluorescent probe merocyanine 540 was used to examine the organization of the lipids in the external leaflet of the plasma membrane after fertilization of sea urchin eggs. These lipids in unfertilized eggs are closely packed, as evidenced by their inability to bind the dye, whereas in fertilized eggs and cells of embryos up to at least the gastrula stage, the membrane becomes more loosely organized, and stains with bright ring fluorescence. Induction of late fertilization events with ammonia failed to induce this change in staining behavior. Sperm components are not required to induce this alteration since parthenogenetically activated eggs stained. However, treatment of eggs with procaine, which specifically inhibits the early event of cortical granule fusion, was effective in suppressing staining. These results indicate that cortical granule fusion after fertilization results in a change in the organization of the lipids of the plasma membrane of sea urchin eggs.     

Activity of some glycolytic and pentose phosphate pathway enzymes during the development of adventitious roots

Activities of phosphofructokinase (PFK, EC 2.7.1.11), glyceraldehyde 3-phosphate (NAD) dehydrogenase [G-3-PD(NAD), EC 1.2.1.12], glucose 6-phosphate dehydrogenase (G-6-PD, EC 1.1.1.49), and 6-phosphogluconate dehydrogenase (6-PGD, EC 1.1.1.44) were determined in bean cuttings (Phaseolus vulgaris L. cv. Top Crop) over 4 days, encompassing adventitious root primordium initiation and development. Effects of applied auxin and “endogenous root-forming stimulus”(ERS) on enzyme activities, concentrations of reducing sugars, and primordium development were also determined during the first 4 days of propagation. Effects of auxin were determined through use of applied indole-3-acetic acid (IAA) or 2,3,5-triiodobenzoic acid. Effects of ERS were evaluated by means of decapitation of cuttings. Increased basipetal transport and increased metabolism of reducing sugars occurred in leafy cuttings in response to applied IAA and to ERS. Primordium development and activities of the four enzymes increased in leafy cuttings under conditions that simultaneously increased basipetal transport and metabolism of reducing sugars. Three types of enzyme activity response were found: (i) activity increased over time by ERS and by applied IAA [G-3-PD(NAD)], (ii) activity increased over time by ERS but not by applied IAA (PFK, G-6-PD), (iii) activity increased over time but not by ERS or applied IAA (6-PGD). Increases in G-3-PD(NAD), G-6-PD, and PFK activity in leafy cuttings were positively related to primordium development. 6-PGD activity increased in leafy cuttings during primordium development and may have supported it. However, equal increases occurred in decapitated cuttings, in which the long-term development of primordia was supressed. Results for G-3-PD(NAD) that were obtained in an experiment with jack pine (Pinus banksiana Lamb.) seedling cuttings were similar to results for the same enzyme in bean cuttings. G-3-PD(NAD) activity in naphthaleneacetic acid-treated jack pine cuttings increased with time, in comparison with untreated cuttings, before root emergence.     

Expression of multiple Src family kinases in sea urchin eggs and their function in Ca release at fertilization

Egg activation at fertilization in deuterostomes requires a rise in intracellular Ca²⁺, which is released from the egg's endoplasmic reticulum. In sea urchins, a Src Family Kinase (SpSFK1) is necessary for the PLCγ-mediated signaling event that initiates this Ca²⁺ release (Giusti, A.F., O'Neill, F.J., Yamasu, K., Foltz, K.R. and Jaffe, L.A., 2003. Function of a sea urchin egg Src family kinase in initiating Ca2+ release at fertilization. Dev. Biol. 256, 367-378.). Annotation of the Strongylocentrotus purpuratus genome sequence led to the identification of additional, predicted SFKs (Bradham, C.A., Foltz, D.R., Beane, W.S., Amone, M.I., Rizzo, F., Coffman, J.A., Mushegian, A., Goel, M., Morales, J., Geneviere, A.M., Lapraz, F., Robertson, A.J., Kelkar, H., Loza-Coll, M., Townley, I.K., Raisch, M., Roux, M.M., Lepage, T., Gache, C., McClay, D.R., Manning, G., 2006. The sea urchin kinome: a first look. Dev. Biol. 300, 180-193.; Roux, M.M., Townley, I.K., Raisch, M., Reade, A., Bradham, C., Humphreys, G., Gunaratne, H.J., Killian, C.E., Moy, G., Su, Y.H., Ettensohn, C.A., Wilt, F., Vacquier, V.D., Burke, R.D., Wessel, G. and Foltz, K.R., 2006. A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation. Dev. Biol. 300, 416-433.). Here, we describe the cloning and characterization of these 4 additional SFKs and test their function during the initial Ca²⁺ release at fertilization using the dominant-interfering microinjection method coupled with Ca²⁺ recording. While two of the new SFKs (SpFrk and SpSFK3) are necessary for Ca²⁺ release, SpSFK5 appears dispensable for early egg to embryo transition events. Interestingly, SpSFK7 may be involved in preventing precocious release of Ca²⁺. Binding studies indicate that only SpSFK1 is capable of direct interaction with PLCγ. Immunolocalization studies suggest that one or more SpSFK and PLCγ are localized to the egg cortex and at the site of sperm-egg interaction. Collectively, these data indicate that more than one SFK is involved in the Ca²⁺ release pathway at fertilization.     

Characterization of valine transport in sea urchin eggs

In unfertilized eggs, the mechanism of valine uptake can be summarized as follows. It is saturable over the external concentration of valine and insensitive to the presence of external sodium, depletion of cellular energy supplies and intracellular acidosis. The activation energy for the transport reaction (16.3 kcal/mol) is within the range of values reported for active transport of small molecules. In fertilized eggs, the total rate of valine uptake can be divided into two components: (i) a Na⁺-insensitive uptake which accounts for about 7% of total absorption as shown by studies in Na⁺-free medium seems to possess the same characteristics as in unfertilized eggs, (ii) a Na⁺-dependent transport of valine which constitutes the main entry is formed about 5 min after fertilization. It follows Michaelis-Menten kinetics characterized by 15-fold increase in $\text{V}{}_{\max }$ with no change in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. These two mechanisms have characteristics in common, such as their insensitivity to metabolic energy supply, their energy of activation and their ability to concentrate valine. The relationship between the establishment of the Na⁺-dependent valine uptake and the ionic events triggered by fertilization is discussed.     

Novel procedures for collection of sea urchin egg cortical granule exudate: Partial characterization and evidence for postsecretion processing

We have developed two procedures to collect total cortical granule exudate in a soluble form from eggs of the sea urchin Strongylocentrotus purpuratus. Egg suspensions were either treated with dithiothreitol to disrupt the vitelline envelope or divalent cations were removed postinsemination to prevent the normal vitelline-to-fertilization envelope transition. Rapid acidification of the insemination mixture (dithiothreitol-treated eggs) to pH 6.0 prevented precipitation of the paracrystalline protein fraction described by Bryan [1970a]. Exudate was partitioned into three fractions. The pH 8.0-insoluble fraction appeared to be identical to the paracrystalline protein fraction. The pH 8.0-soluble fraction was separated into pH 4.0-soluble and-insoluble fractions. Analysis for peroxidase and protease activities showed that peroxidase activity was localized in all three fractions whereas protease activity was restricted to the pH 4.0 insoluble fraction as reported [Carroll and Epel, 1975]. A minimum of six major proteins were detected on native polyacrylamide gels of total exudate. Under reducing and denaturing conditions, 12 polypeptides ranging from 19,000 to 165,000 in molecular weight were detected in total exudate; six polypeptides were recovered in the pH 8.0-insoluble fraction. To test the hypothesis that protease and peroxidase activities process cortical granule proteins after secretion, we inseminated eggs in solutions containing peroxidase and protease inhibitors. The paracrystalline protein fraction crystallized slowly from insemination mixtures containing both inhibitors compared to controls and there were dramatic differences in exudate electrophoretic patterns. We suggest that cortical granule protease and peroxidase activities process the exudate so that the paracrystalline protein fraction rapidly crystallizes during normal fertilization.     

Transient recovery from trypsin-caused reduction in fertilizability of sea urchin eggs

Summary The effect of trypsin on the fertilizability of sea urchin eggs was studied withParacentrotus lividus andPseudocentrotus depressus. The main effects were two reductions of fertilizability, with a transient increase intervening. The first decrease was probably caused by degradation of sperm-binding sites at the vitelline sheet and the second by degradation of binding sites on the plasma membrane.     

Cross fertilization between sea urchin eggs and oyster spermatozoa

Interphylum crossing was examined between sea urchin eggs (Temnopleurus hardwicki) and oyster sperm (Crassostrea gigas). The eggs could receive the spermatozoa with or without cortical change. The fertilized eggs that elevated the fertilization envelope began their embryogenesis. Electron microscopy revealed that oyster spermatozoa underwent acrosome reaction on the sea urchin vitelline coat, and their acrosomal membrane fused with the egg plasma membrane after the appearance of an intricate membranous structure in the boundary between the acrosomal process and the egg cytoplasm. Oyster spermatozoa penetrated sometimes into sea urchin eggs without stimulating cortical granule discharge and consequently without fertilization envelope formation. The organelles derived from oyster spermatozoa seemed to be functionally inactive in the eggs whose cortex remained unchanged.     

A MAPK pathway is involved in the control of mitosis after fertilization of the sea urchin egg

Activation and role of mitogen-activated protein (MAP) kinase (MAPK) during mitosis are still matters of controversy in early embryos. We report here that an ERK-like protein is present and highly phosphorylated in unfertilized sea urchin eggs. This MAPK becomes dephosphorylated after fertilization and a small pool of it is transiently reactivated during mitosis. The phosphorylated ERK-like protein is localized to the nuclear region and then to the mitotic poles and the mitotic spindle. Treatment of eggs after fertilization with two different MEK inhibitors, PD 98059 and U0126, at low concentrations capable to selectively induce dephosphorylation of this ERK-like protein, or expression of a dominant-negative MEK1/2, perturbed mitotic progression. Our results suggest that an ERK-like cascade is part of a control mechanism that regulates mitotic spindle formation and the attachment of chromosomes to the spindle during the first mitosis of the sea urchin embryo.     

Distal cis-acting elements restrict expression of the CyIIIb actin gene in the aboral ectoderm of the sea urchin embryo

The distal region of the S. purpuratus actin CyIIIb gene, between −400 and −1400 nucleotides, contains at least three distinct cis-acting elements (C1R, C1L and E1) which are necessary for correct expression of fusion reporter genes in transgenic sea urchin embryos. The contribution of these elements in the temporal and spatial regulation of the gene was analyzed by single and double site-directed mutagenesis in fusion constructs which carry the bacterial chloramphenicol acetyl transferase (CAT) gene as a reporter. Following microinjection of the transgenes in sea urchin embryos, the activity of the mutants was compared to the wild type in time and space by measuring CAT activity at the blastula and pluteus embryonic stages and by in situ hybridization to the CAT mRNA at pluteus stage. Our results indicate that E1 involved in the temporal regulation of CyIIIb and that all three elements are necessary and sufficient to confer aboral (dorsal) ectoderm specificity to the proximal promoter. This is achieved by suppressing the promoter's activity in all other tissues by the cooperative interaction of the cis-acting elements. The C1R element, binding site of the nuclear receptors SpCOUP-TF and SpSHR2, is by itself sufficient to restrict expression in the ectoderm, whereas the aboral ectoderm restricted expression requires in addition the presence of both C1L adn E1. It is therefore evident, that the actin CyIIIb gene is exclusively expressed in the aboral ectoderm by a combinatorial repression in all other cell lineages of the developing embryo.     

Acid release,cytoplasmic alkalinization,and chromosome condensation during sea urchin fertilization and amine-lnduced parthenogenesis

We have studied the relationship between acid release, cytoplasmic alkalinization, and the extent of chromosome condensation during parthenogenetic activation of sea urchin eggs. The relative rate of acid release in Strongylocentrotus purpuratus eggs was determined from pH measurements of egg suspensions. Acid release in inseminated eggs began after a lag of 0.4 min and the relative rate increased 108-fold, declined, and release was essentially complete by 8-min postinsemination. An average of 3.8 ± 0.23 × 10^?12moles H⁺ cell^? was released as determined by backtitration with NaOH. Acid release characteristics of eggs parthenogenetically activated with either NH₄C1, methylamine ethylamine, n-propylamine, n-butylamine, or benzylamine were qualitatively similar. There was no detectable lag peroid and the increase in relative rate of acid release was directly proportional to the carbon number of the amine used, eg, from 8.3-fold methylamine to 470-fold with benzylamine. The total equivalents of acid released ranged from 0.50–8.2 × 10^?12 moles H⁺·cell^? in direct proportion to the concentration of amine used. The degree fo cytoplasmic alkalinization induced as a function of methylamine and benzylamine concentration was determined by pH measurements fo egg homogenates; egg cultures were also prepared for microscopic examination of chromosome condensation. None of the eggs had condensed chromosomes at 0.5-mM methylamine whereas a cytoplasmic alkalinization of 0.6 pH units was observed. Increased methylamine levels up to 10mM resulted in chromiosome condensation in only 20% of the eggs. A similar result was found with benzylamine. We conclude that acid release and cytoplasmic alkalinization during chemical parthenogenesis are insufficient to mimic sperm induction of chromiosome condensation and suggest that an additional factor(s) is required for chromosome condensation by low concentration of amines.     

Ultrastructural organization of heterochromatin within sea urchin sperm nuclei

Chromatin within swollen or lysed isolated sperm nuclei of the sea urchin, Strongylocentrotus purpuratus, was examined by electron microscopy. Spread preparations of lysed sperm nuclei demonstrated dense aggregates of nondispersed material and beaded filaments radiating from these aggregates. These beaded fibers are similar in size and appearance to the “beads-on-a-string” seen as characteristic of chromatin spreads from numerous interphase nuclei. The beads are nucleosomes that have an average diameter of 130 Å. The interconnecting string is 40 Å indiameter and corresponds to the spacer DNA. In thin sections of swollen nuclei the sperm chromatin appears to be composed of 400 Å superbeads that are closely apposed to form 400 Å fibers. As the chromatin disperses, the superbeads are seen to be attached to one another by chromatin fibers of 110 Å diameter. In thin sections, the 400 Å superbeads appear to disperse directly into the 110 Å fibers with no intervening structures. This work demonstrates that the heterochromatin in Strongylocentrotus purpuratus sperm nuclei is composed of nucleosomes that form 100 Å filaments that are compacted into 400 Å superbeads. The superbeads coalesce to give the morphological appearance of 400 Å fibers.     

Genome-wide analysis of glucose-6-phosphate dehydrogenases in Arabidopsis

In green tissues of plants under illumination, photosynthesis is the primary source of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which is utilized in reductive reactions such as carbon fixation and nitrogen assimilation. In non-photosynthetic tissues or under non-photosynthetic conditions, the oxidative pentose phosphate pathway contributes to basic metabolism as one of the major sources of NADPH. The first and committed reaction is catalyzed by glucose-6-phosphate dehydrogenase (G6PDH). We characterized the six members of the G6PDH gene family in Arabidopsis. Transit peptide analysis predicted two cytosolic and four plastidic isoforms. Five of the six genes encode active G6PDHs. The recombinant isoforms showed differences in substrate requirements and sensitivities to feedback inhibition. Plastidic isoforms were redox sensitive. One cytosolic isoform was insensitive to redox changes, while the other was inactivated by oxidation. The respective genes had distinct expression patterns that did not correlate with the activity of the proteins, implying a regulatory mechanism beyond the control of mRNA abundance. Two cytosolic and one plastidic isoform were detected in vivo using zymograms, and the respective genes were identified using T-DNA insertion lines. The activity of a plastidic isoform was detected in all tissues including photosynthetic tissues despite its sensitivity to reduction observed in vitro. Genomic data, gene expression, and in vivo enzyme activity data were integrated with in vitro biochemical data to propose in vivo roles for individual G6PDH isoforms in Arabidopsis.     

Calcium-mediated inactivation of the MAP kinase pathway in sea urchin eggs at fertilization.

We have evaluated the regulation of a 43-kDa MAP kinase in sea urchin eggs. Both MAP kinase and MEK (MAP kinase kinase) are phosphorylated and active in unfertilized eggs while both are dephosphorylated and inactivated after fertilization, although with distinct kinetics. Reactivation of MEK or the 43-kDa MAP kinase prior to or during the first cell division was not detected. Confocal immunolocalization microscopy revealed that phosphorylated (active) MAP kinase is present primarily in the nucleus of the unfertilized egg, with some of the phosphorylated form in the cytoplasm as well. Incubation of unfertilized eggs in the MEK inhibitor U0126 (0.5 microM) resulted in the inactivation of MEK and MAP kinase within 30 min. Incubation in low concentrations of U0126 (sufficient to inactivate MEK and MAP kinase) after fertilization had no effect on progression through the embryonic cell cycle. Microinjection of active mammalian MAP kinase phosphatase (MKP-3) resulted in inactivation of MAP kinase in unfertilized eggs, as did addition of MKP-3 to lysates of unfertilized eggs. Incubation of unfertilized eggs in the Ca(2+) ionophore A23187 led to inactivation of MEK and MAP kinase with the same kinetics as observed with sperm-induced egg activation. This suggests that calcium may be deactivating MEK and/or activating a MAP kinase-directed phosphatase. A cell-free system was used to evaluate the activation of phosphatase separately from MEK inactivation. Unfertilized egg lysates were treated with U0126 to inactivate MEK and then Ca(2+) was added. This resulted in increased MAP kinase phosphatase activity. Therefore, MAP kinase inactivation at fertilization in sea urchin eggs likely is the result of a combination of MEK inactivation and phosphatase activation that are directly or indirectly responsive to Ca(2+).     

Clonal response to cold tolerance in creeping bentgrass and role of proline-associated pentose phosphate pathway

Single seed origin creeping bentgrass (‘Penncross’) clonal lines were screened to find genetic heterogeneity, which reflected diversity of phenolic production linked to cold stress within a cross-pollinated cultivar. In this study, total soluble phenolic and antioxidant activity varied among 20 creeping bentgrass clonal lines, confirming wide heterogeneity in this cross-pollinated species. Correlations between phenolic content and proline-associated pentose phosphate pathway were also found among the clonal lines. The active metabolic role of proline in cellular metabolic adjustment to cold stress and its support for likely energy synthesis via mitochondrial oxidative phosphorylation was inferred in creeping bentgrass clonal lines based on the activity of proline dehydrogenase. Results of photochemical efficiency of these clonal lines after cold temperature treatment (4 °C) also indicated a close association between stress tolerance and proline-associated pentose phosphate pathway regulation for phenolic biosynthesis and antioxidant response. This study provides a sound metabolic based rationale to screen bentgrass clonal lines for enhanced cold stress tolerance.