Sheldon S. Shen

Protein tyrosine kinase‐dependent release of intracellular calcium in the sea urchin egg

The aminoguanide, methylglyoxal bis(guanylhydrazone) (MGBG), was shown to stimulate phosphorylation of RR‐SRC, a synthetic protein tyrosine kinase (PTK) substrate, and different levels of tyrosyl phosphorylation of endogenous proteins in a sea urchin egg membrane‐cortex preparation. Stimulating protein tyrosine kinase activity in the sea urchin egg stimulated intracellular Ca2+ release, because microinjection of 1–5 mM of MGBG into unfertilized eggs triggered a transient rise in intracellular Ca2+ activity ([Ca2+]i) after a brief latent period. Pretreating eggs with PTK‐specific inhibitors, genistein or tyrphostin B42+, significantly inhibited the MGBG‐induced rise in [Ca2+]i. Methylglyoxal bis(guanylhydrazone) stimulation of PTK activities in the unfertilized sea urchin egg appeared to trigger Ca2+ release through phospholipase C (PLC)‐dependent inositol 1,4,5‐trisphosphate (InsP3) production. The MGBG‐induced Ca2+ response could be suppressed in eggs preloaded with the InsP3 receptor antagonist, heparin, and was reduced in eggs pretreated with U7312+, a PLC inhibitor. However, the response was unchanged in eggs treated with nicotinamide, an inhibitor of ADP‐ribosyl cyclase, or nifedipine, an inhibitor of nicotinic acid adenine dinucleotide phosphate activity. These results suggest that MGBG may be useful as a chemical agonist of PTK in sea urchin eggs and allow direct testing of the PTK requirement for the transient rise in [Ca2+]i in sea urchin eggs during fertilization. Although genistein was observed to significantly delay the onset, the sperm‐induced Ca2+ response in PTK inhibitor‐loaded eggs otherwise appeared normal. Therefore, it was concluded that sea urchin eggs contain a PTK‐dependent pathway that can mediate intracellular Ca2+ release, but PTK activity does not appear to be required for the fertilization response.

A synthetic peptide of the pseudosubstrate domain of protein kinase C blocks cytoplasmic alkalinization during activation of the sea urchin egg

Multiple second messenger pathways have been proposed for transduction of the sperm-egg fusion event during fertilization of sea urchin eggs. Cytoplasmic alkalinization due to increased Na(+)-H+ antiport has been causally linked to many of the metabolic events during fertilization. Two possible second messenger pathways coupling sperm-egg fusion and antiporter activity are activation of protein kinase C (PKC) and Ca2(+)-calmodulin kinase. A selective inhibitor of PKC is PKC(19-36), a synthetic peptide of the pseudosubstrate domain of the kinase. Injection of PKC(19-36) into unfertilized sea urchin eggs blocked cytoplasmic alkalinization during activation by phorbol 12-myristate 13-acetate, a PKC agonist. The rise in pH during fertilization was partially blocked by PKC(19-36), which suggested that multiple pathways regulate the antiporter during fertilization. The use of fluorescein chromophores to measure intracellular pH in sea urchin eggs is also discussed.

3 Mechanisms of Calcium Regulation in Sea Urchin Eggs and their Activities during Fertilization

Publisher Summary This chapter discusses the current evidence of Ca 2+ regulation in sea-urchin eggs during fertilization. Studies on calcium regulation during the fertilization of mammalian eggs and comparative changes in deuterostome eggs during fertilization are also discussed in the chapter, along with other events associated with egg membranes during fertilization. The regulation of intracellular Ca 2+ activity ([Ca 2+ ] i ) in a wide variety of cells is well established as an important event during signal transduction of numerous extrinsic stimuli. Different sources, including influx of external Ca 2+ and release from the intracellular stores, contribute to the rise in [Ca 2+ ] i . Irrespective of the cell type, resting [Ca 2+ ] is generally in the range of 50–200 n M and accounts for only a minute fraction of the total cellular Ca 2+ content, therefore the bulk of the cellular Ca 2+ is bound and may reside within the membrane-bound organelles.

A specific inhibitor of p34cdc2/cyclin B suppresses fertilization-induced calcium oscillations in mouse eggs

Abstract Fertilization-induced Ca2+ oscillations in mouse eggs cease at the time of pronuclear formation when maturation-promoting factor (MPF) is inactivated, but the Ca2+ oscillations are ceaseless if eggs are arrested at metaphase by colcemid, which maintains the activity of MPF. To determine the possible role of MPF in regulation of cytoplasmic Ca2+ excitability, roscovitine, a specific inhibitor of p34cdc2/cyclin B kinase, was used to inactivate MPF, and its effect on fertilization-induced Ca2+ oscillations was investigated. Our results showed that roscovitine at ≥ 50 μM suppressed fertilization-induced Ca2+ oscillations in normal and colcemid-treated metaphase II (MII) eggs after the first 1–2 Ca2+ spikes. Roscovitine inhibition of fertilization-induced Ca2+ oscillations could be reversed by extensive washing of the eggs. Histone H1 kinase activity in colcemid-treated MII eggs was similarly inhibited by roscovitine, which suggested that the cessation of fertilization-induced Ca2+ oscillations is due to the inactivation of MPF. Thimerosal-induced Ca2+ oscillations in Ca2+-, Mg2+-free medium was also suppressed by roscovitine, suggesting a general inhibitory effect of roscovitine on Ca2+ oscillations. The inhibition may be achieved by disruption of Ca2+ release and refilling of the calcium store. Thapsigargin, an inhibitor of the endoplasmic reticulum Ca-ATPase, induced significantly less Ca2+ release in roscovitine-treated eggs than in the non-drug-treated eggs. Taken together, our results suggest that MPF plays an important role in regulation of the cytoplasmic Ca2+ excitability in mouse eggs.

Na+H+ antiport during fertilization of the sea urchin egg is blocked by W-7 but is insensitive to K252a and H-7

Fertilization of the sea urchin egg initiates or accelerates a number of metabolic activities, which have been causally linked to a rise in cytoplasmic pH due to increased Na+-H+ antiport. Two possible regulatory pathways linking sperm-egg fusion to the activity of the antiporter are activation of protein kinase C (PKC) and Ca2+, calmodulin (CaM)-dependent kinase. This report presents the effects of protein kinase inhibitors on antiporter activation during fertilization and treatment with PKC agonists, dioctanoylglycerol or phorbol diester. Protein kinase inhibitors, K252a and H-7 blocked the action of PKC agonists, without inhibiting cytoplasmic alkalinization during fertilization. In contrast, W-7 blocked fertilization-induced rise in cytoplasmic pH, without altering the actions of PKC agonists. These results suggest that the Na+-H+ antiporter may be regulated by PKC or Ca2+, CaM-dependent kinase activities, but activation of the antiporter during fertilization is Ca2+, CaM-dependent, despite production of diacylglycerols by hydrolysis of phosphatidylinositols.

Synergistic release of calcium in sea urchin eggs by caffeine and ryanodine

A transient rise in intracellular Ca2+ during fertilization is necessary for activation of the quiescent sea urchin egg. Several mechanisms contribute to the rise in Ca2+ including influx across the egg plasma membrane and release from intracellular stores. The egg contains both IP3-sensitive and -insensitive Ca2+ release mechanisms and in this study we have used single-cell spectrofluorimetry to examine the effects of caffeine and ryanodine on Ca2+ release in eggs preloaded with fura 2. Caffeine induced a small Ca2+ release that was insensitive to heparin or ruthenium red. Ca2+ liberation by caffeine could be augmented by prior treatment with thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPase. Variable Ca2+ releases were observed in response to microinjection of ryanodine. The action of ryanodine appeared to be enhanced by prior injection of heparin and partially inhibited by ruthenium red. The release of Ca2+ by caffeine or ryanodine was generally insufficient to trigger cortical granule exocytosis, thus these eggs could be fertilized and a second Ca2+ release during fertilization was measured. Unlike the caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release mechanism in somatic cells, the graded responses in eggs suggested this caffeine- and ryanodine-sensitive release mechanism is not sensitive to sudden changes in Ca2+. Thus we could examine the combined actions of caffeine and ryanodine on Ca2+ release, which were synergistic. Caffeine treatment of ryanodine-injected eggs or ryanodine injection of caffeine-treated eggs stimulated a Ca2+ release significantly larger than the release by either drug independently. The experiments presented here suggest that sea urchin eggs liberate Ca2+ in response to caffeine and ryanodine; however, the regulation of this release differs from that described for caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release of somatic cells.

Protein kinase C from sea urchin eggs.

1. Protein kinase C is considered to be ubiquitous in tissues and organs; however, its isolation and characterization have been principally with adult mammalian tissues. 2. There is increasing evidence for the importance of this enzyme during early development. 3. In this study, protein kinase C has been identified and partially characterized in cytosolic fraction from sea urchin eggs. 4. The enzyme was resolved from other protein kinase activities by ion exchange chromatography. 5. Phosphatidylserine and Ca2+ were required for protein kinase C to be active. 6. Diacylglycerol and phorbol ester enhanced the activation of the enzyme.

The mechanisms of cell membrane resealing in rabbit corneal epithelial cells.

Purpose: To examine membrane repair mechanisms in rabbit corneal epithelial (RCE) cells. Methods: Microneedle puncture and fluorescent dye loss were used to wound membranes and assay resealing, respectively. Different repair mechanisms were detected pharmacologically and with antisense oligonucleotides. Results: The RCE cells rapidly reseal plasma membranes by calcium-dependent exocytotic mechanisms that exhibit both facilitated and potentiated responses to multiple wounding. The facilitated response was inhibited by specific inhibitors of protein kinase C (PKC) and brefeldin A, and the potentiated response was blocked by inhibitors of cAMP-dependent protein kinase (PKA). Reduction of myosin IIA inhibited the facilitated response, and reduction of IIB inhibited the initial resealing. Conclusions: RCE cells rapidly repair plasma membrane disruptions. At a second wound at the same site, PKC stimulated vesicle formation from the Golgi apparatus, resulting in more rapid membrane resealing for a facilitated response. The RCE cell also contains a PKA-dependent global potentiation of membrane resealing.

Calcium signaling at fertilization

Calcium is well established as a second messenger in a diverse array of cell activities. Changes in intracellular Ca2+ activities range from localized releases to complex oscillations, which may encode specific cellular signals. The full variety of calcium responses is observed during the fertilization of different animal oocytes and eggs. Current research has focused on the cellular mechanisms that generate these Ca(2+)-activity changes.

Molecular Cloning and Characterization of Protein Kinase C from the Sea Urchin Lytechinus pictus

Protein kinase C (PKC) has been shown to play a role in events involved in fertilization such as activation of the Na+/H+antiporter and an NADPH dependent oxidase. In addition, it is involved in cell fate programming later in development of the sea urchin embryo. In order to further address the role of PKC in sea urchin development, we have screened a Lytechinus pictus ovary tissue cDNA library and identified one clone for sea urchin protein kinase C (suPKC1). This clone encodes a deduced protein with a molecular mass of 72.4 kDa, which shows strong homology to invertebrate and mammalian protein kinase C (PKC) sequences. PKC has been partially purified from eggs of L. pictus. This kinase activity has been shown to be dependent upon phosphatidylserine, diacylglycerol and Ca2+. In agreement with this biochemical data, suPKC1 has a C2 or Ca2+‐binding domain suggesting its activity would be Ca2+‐dependent. Polyclonal antibodies raised against peptides of the suPKC1 sequence recognize an antigen of approximately 71 kDa in DE52 fractions that contain PKC activity; this reactivity is not observed in fractions that lack PKC activity. Using a ribonuclease protection assay, we have demonstrated the presence of suPKC1 message throughout developmental stages of the sea urchin embryo.