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Gary M. Wessel - One of the best experts on this subject based on the ideXlab platform.
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conservation of sequence and function in fertilization of the Cortical Granule serine protease in echinoderms
Biochemical and Biophysical Research Communications, 2014Co-Authors: Nathalie Oulhen, Gary M. WesselAbstract:Conservation of the Cortical Granule serine protease during fertilization in echinoderms was tested both functionally in sea stars, and computationally throughout the echinoderm phylum. We find that the inhibitor of serine protease (soybean trypsin inhibitor) effectively blocks proper transition of the sea star fertilization envelope into a protective sperm repellent, whereas inhibitors of the other main types of proteases had no effect. Scanning the transcriptomes of 15 different echinoderm ovaries revealed sequences of high conservation to the originally identified sea urchin Cortical serine protease, CGSP1. These conserved sequences contained the catalytic triad necessary for enzymatic activity, and the tandemly repeated LDLr-like repeats. We conclude that the protease involved in the slow block to polyspermy is an essential and conserved element of fertilization in echinoderms, and may provide an important reagent for identification and testing of the cell surface proteins in eggs necessary for sperm binding.
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synaptotagmin i is involved in the regulation of Cortical Granule exocytosis in the sea urchin
Molecular Reproduction and Development, 2006Co-Authors: Mariana Leguia, Sean D Conner, Linnea Berg, Gary M. WesselAbstract:Cortical Granules are stimulus-dependent secretory vesicles found in the egg cortex of most vertebrates and many invertebrates. Upon fertilization, an increase in intracellular calcium levels triggers Cortical Granules to exocytose enzymes and structural proteins that permanently modify the extracellular surface of the egg to prevent polyspermy. Synaptotagmin is postulated to be a calcium sensor important for stimulus-dependent secretion and to test this hypothesis for Cortical Granule exocytosis, we identified the ortholog in two sea urchin species that is present selectively on Cortical Granules. Characterization by RT-PCR, in-situ RNA hybridization, Western blot and immunolocalization shows that synaptotagmin I is expressed in a manner consistent with it having a role during Cortical Granule secretion. We specifically tested synaptotagmin function during Cortical Granule exocytosis using a microinjected antibody raised against the entire cytoplasmic domain of sea urchin synaptotagmin I. The results show that synaptotagmin I is essential for normal Cortical Granule dynamics at fertilization in the sea urchin egg. Identification of this same protein in other developmental stages also shown here will be important for interpreting stimulus-dependent secretory events for signaling throughout embryogenesis. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc.
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regulated proteolysis by Cortical Granule serine protease 1 at fertilization
Molecular Biology of the Cell, 2004Co-Authors: Sheila A Haley, Gary M. WesselAbstract:Cortical Granules are specialized organelles whose contents interact with the extracellular matrix of the fertilized egg to form the block to polyspermy. In sea urchins, the Granule contents form a fertilization envelope (FE), and this construction is critically dependent upon protease activity. An autocatalytic serine protease, Cortical Granule serine protease 1 (CGSP1), has been identified in the Cortical Granules of Strongylocentrotus purpuratus eggs, and here we examined the regulation of the protease activity and tested potential target substrates of CGSP1. We found that CGSP1 is stored in its full-length, enzymatically quiescent form in the Granule, and is inactive at pH 6.5 or below. We determined the pH of the Cortical Granule by fluorescent indicators and micro-pH probe measurements and found the Granules to be pH 5.5, a condition inhibitory to CGSP1 activity. Exposure of the protease to the pH of seawater (pH 8.0) at exocytosis immediately activates the protease. Activation of eggs at pH 6.5 or lower blocks activation of the protease and the resultant FE phenotypes are indistinguishable from a protease-null phenotype. We find that native Cortical Granule targets of the protease are beta-1,3 glucanase, ovoperoxidase, and the protease itself, but the structural proteins of the Granule are not proteolyzed by CGSP1. Whole mount immunolocalization experiments demonstrate that inhibition of CGSP1 activity affects the localization of ovoperoxidase but does not alter targeting of structural proteins to the FE. The mistargeting of ovoperoxidase may lead to spurious peroxidative cross-linking activity and contribute to the lethality observed in protease-null cells. Thus, CGSP1 is proteolytically active only when secreted, due to the low pH of the Cortical Granules, and it has a small population of targets for cleavage within the Cortical Granules.
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a rho signaling pathway mediates Cortical Granule translocation in the sea urchin oocyte
Mechanisms of Development, 2004Co-Authors: Fernando Coviannares, Gary M. Wessel, Guadalupe Martinezcadena, Juana Lopezgodinez, Ekaterina Voronina, Jesus GarciasotoAbstract:Cortical Granules are secretory vesicles of the egg that play a fundamental role in preventing polyspermy at fertilization. In the sea urchin egg, they localize directly beneath the plasma membrane forming a compact monolayer and, upon fertilization, undergo a Ca 2þ -dependent exocytosis. Cortical Granules form during early oogenesis and, during maturation, translocate from the cytosol to the oocyte cortex in a microfilament-mediated process. We tested the hypothesis that these Cortical Granule dynamics were regulated by Rho, a GTPase of the Ras superfamily. We observed that Rho is synthesized early in oogenesis, mainly in a soluble form. At the end of maturation, however, Rho associates with Cortical Granules. Inhibition of Rho with the C3 transferase from C. botulinum blocks Cortical Granule translocation and microfilaments undergo a significant disorganization. A similar effect is observed by GGTI-286, a geranylgeranyl transferase inhibitor, suggesting that the association of Rho with the Cortical Granules is indispensable for its function. In contrast, the anchorage of the Cortical Granules in the cortex, as well as their fusion at fertilization, are Rho-independent processes. We conclude that Rho association with the Cortical Granules is a critical regulatory step in their translocation to the egg cortex. q 2004 Elsevier Ireland Ltd. All rights reserved.
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Cortical Granule translocation is microfilament mediated and linked to meiotic maturation in the sea urchin oocyte
Development, 2002Co-Authors: Gary M. Wessel, Sean D Conner, Linnea BergAbstract:Cortical Granules exocytose after the fusion of egg and sperm in most animals, and their contents function in the block to polyspermy by creating an impenetrable extracellular matrix. Cortical Granules are synthesized throughout oogenesis and translocate en masse to the cell surface during meiosis where they remain until fertilization. As the mature oocyte is approximately 125 μm in diameter (Lytechinus variegatus), many of the Cortical Granules translocate upwards of 60 μm to reach the cortex within a 4 hour time window. We have investigated the mechanism of this coordinated vesicular translocation event. Although the stimulus to reinitiate meiosis in sea urchin oocytes is not known, we found many different ways to reversibly inhibit germinal vesicle breakdown, and used these findings to discover that meiotic maturation and Cortical Granule translocation are inseparable. We also learned that Cortical Granule translocation requires association with microfilaments but not microtubules. It is clear from endocytosis assays that microfilament motors are functional prior to meiosis, even though Cortical Granules do not use them. However, just after GVBD, Cortical Granules attach to microfilaments and translocate to the cell surface. This latter conclusion is based on organelle stratification within the oocyte followed by positional quantitation of the Cortical Granules. We conclude from these studies that maturation promoting factor (MPF) activation stimulates vesicle association with microfilaments, and is a key regulatory step in the coordinated translocation of Cortical Granules to the egg cortex.
Prue Talbot - One of the best experts on this subject based on the ideXlab platform.
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Perivitelline space: does it play a role in blocking polyspermy in mammals? Microsc Res Tech 2003;61:349–357
2015Co-Authors: Prue Talbot, Pramila DandekarAbstract:KEY WORDS fertilization; oocytes; Cortical Granules; extracellular matrix ABSTRACT The perivitelline space of mammalian oocytes changes in size and composition during preimplantation development. Often overlooked in the past, this space contains a hyaluro-nan-rich extracellular matrix prior to fertilization and a Cortical Granule envelope following release of the Cortical Granules at fertilization. The hyaluronan-containing matrix of unfertilized oocytes is well developed in some species such as opossums and humans but is scant in rodents including the hamster and mouse. The significance of the hyaluronan-rich matrix, which attaches to the plasma membrane of the oocytes, is not fully understood. However, hyaluronan, which can inhibit mem-brane fusion, is present in the perivitelline space (PVS) of unfertilized oocytes and must be negotiated by the fertilizing sperm. Following fertilization, the Cortical Granule envelope forms as the Cortical Granules disperse, thereby causing the PVS to increase significantly in size. Calcium is important in the dispersion of the Cortical Granules following exocytosis. Once formed, the Cortical Granule envelope in some species is about the same thickness as the zona pellucida, but it is not readily visualized unless it is stained with fluorescent probes or examined ultrastructurally after following stabilization with ruthenium red. The envelope contains proteins that remain in the PVS until the time of blastocyst hatching. Although little work has been done on the functions of the Cortical Granule envelope, several studies are consistent with the idea that it plays a role in blocking polyspermy. While nicotine increases polyspermy in sea urchins, its effects on polyspermy in human smokers have not been characterized, but could be addressed in human in vitro fertilizatio
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peptidylarginine deiminase pad is a mouse Cortical Granule protein that plays a role in preimplantation embryonic development
Reproductive Biology and Endocrinology, 2005Co-Authors: Min Liu, Patricia G Calarco, Michiyuki Yamada, Scott A Coonrod, Prue TalbotAbstract:Background While mammalian Cortical Granules are important in fertilization, their biochemical composition and functions are not fully understood. We previously showed that the ABL2 antibody, made against zona free mouse blastocysts, binds to a 75-kDa Cortical Granule protein (p75) present in a subpopulation of mouse Cortical Granules. The purpose of this study was to identify and characterize p75, examine its distribution in unfertilized oocytes and preimplantation embryos, and investigate its biological role in fertilization.
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perivitelline space does it play a role in blocking polyspermy in mammals
Microscopy Research and Technique, 2003Co-Authors: Prue Talbot, Pramila DandekarAbstract:The perivitelline space of mammalian oocytes changes in size and composition during preimplantation development. Often overlooked in the past, this space contains a hyaluro- nan-rich extracellular matrix prior to fertilization and a Cortical Granule envelope following release of the Cortical Granules at fertilization. The hyaluronan-containing matrix of unfertilized oocytes is well developed in some species such as opossums and humans but is scant in rodents including the hamster and mouse. The significance of the hyaluronan-rich matrix, which attaches to the plasma membrane of the oocytes, is not fully understood. However, hyaluronan, which can inhibit mem- brane fusion, is present in the perivitelline space (PVS) of unfertilized oocytes and must be negotiated by the fertilizing sperm. Following fertilization, the Cortical Granule envelope forms as the Cortical Granules disperse, thereby causing the PVS to increase significantly in size. Calcium is important in the dispersion of the Cortical Granules following exocytosis. Once formed, the Cortical Granule envelope in some species is about the same thickness as the zona pellucida, but it is not readily visualized unless it is stained with fluorescent probes or examined ultrastructurally after following stabilization with ruthenium red. The envelope contains proteins that remain in the PVS until the time of blastocyst hatching. Although little work has been done on the functions of the Cortical Granule envelope, several studies are consistent with the idea that it plays a role in blocking polyspermy. While nicotine increases polyspermy in sea urchins, its effects on polyspermy in human smokers have not been characterized, but could be addressed in human in vitro fertilization labs. Microsc. Res. Tech. 61:349 -357, 2003. © 2003 Wiley-Liss, Inc. WHAT IS THE PERIVITELLINE SPACE? The perivitelline space (PVS) is a "space" between the surface of the oocyte or more specifically the oo- lemma and the zona pellucida, an extracellular matrix synthesized by the oocyte. The term space is a misno- mer as it implies something that is empty and perhaps uninteresting. In fact, the PVS has contents that change during development and that appear to play various roles before, during, and after fertilization. The purpose of this review will be to discuss what is known about the PVS of mammals before and after fertiliza- tion and to examine the evidence that the contents of the PVS play a role in blocking polyspermy.
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p62 p56 are Cortical Granule proteins that contribute to formation of the Cortical Granule envelope and play a role in mammalian preimplantation development
Molecular Reproduction and Development, 2001Co-Authors: Tanya Hoodbhoy, Pramila V. Dandekar, P Calarco, Prue TalbotAbstract:The purpose of this study was to identify specific Cortical Granule protein(s) that form the Cortical Granule envelope and examine their role(s) in fertilization and preimplantation development. The polyclonal antibody A-BL2 was used to show that the Cortical Granules of mice, rats, hamsters, cows, and pigs contain a pair of proteins designated p62/p56. These proteins are released from hamster Cortical Granules at fertilization and contribute to formation of the Cortical Granule envelope, an extracellular matrix present in the perivitelline space of fertilized mammalian oocytes. P62/p56 were present in the Cortical Granule envelope throughout preimplantation development and were found in blastomere cortices of 4-cell to blastocyst stage embryos. Hamster oocytes fertilized in vivo in the presence of A-BL2 were all monospermic, suggesting that p62/p56 do not function in blocking polyspermy. Likewise treatment of morula to blastocyst stage hamster embryos with A-BL2 had no effect on the implantation of blastocysts. However, cleavage divisions were inhibited in vivo in a dose-dependent manner when fertilized oocytes or 2-cell embryos were treated with A-BL2. Inhibition of cell division was more pronounced in 2-cell embryos than in fertilized oocytes. This study identifies p62/p56 as Cortical Granule proteins that contribute to the formation of the Cortical Granule envelope and further supports the idea that after their release at fertilization, p62/p56 function in regulating preimplantation development at the level of oocyte and blastomere cleavage.
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characterization fate and function of hamster Cortical Granule components
Molecular Reproduction and Development, 2001Co-Authors: Tanya Hoodbhoy, Prue TalbotAbstract:Little is known about the compo- sition and function of mammalian Cortical Granules. In this study, lectins were used as tools to: (1) estimate the number and molecular weight of glycoconjugates in hamster Cortical Granules and show what sugars are associated with each glycoconjugate; (2) identify Cortical Granule components that remain associated with the oolemma, Cortical Granule envelope, and/or zona pellucida of fertilized oocytes and preimplantation embryos; and (3) examine the role of Cortical Granule glycoconjugates in preimplantation embryogenesis. Microscopic examination of unfertilized oocytes re- vealed that the lectins PNA, DBA, WGA, RCA120, Con A, and LCA bound to hamster Cortical Granules. Moreover, LCA and Con A labeled the zona pellucida, Cortical Granule envelope, and plasma membrane of fertilized and artificially activated oocytes and two and eight cell embryos. Lectin blots of unfertilized oocytes had at least 12 glycoconjugates that were recognized by one or more lectins. Nine of these glycoconjugates are found in the Cortical Granule envelope and/or are associated with the zona pellucida and plasma membrane following fertilization. In vivo functional studies showed that the binding of Con A to one or more mannosylated Cortical Granule components in- hibited blastomere cleavage in two-cell embryos. Our data show that hamster Cortical Granules contain ap- proximately 12 glycoconjugates of which nine remain associated extracellularly with the fertilized oocyte after the Cortical reaction and that one or more play a role in regulating cleavage divisions. Mol. Reprod. Dev. 58:223-235, 2001. fl 2001 Wiley-Liss, Inc.
Ronald W Holz - One of the best experts on this subject based on the ideXlab platform.
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evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory Granule membrane comparison of exocytosis of injected bovine chromaffin Granule membranes and endogenous Cortical Granules in xenopus laevis oocytes
Cellular and Molecular Neurobiology, 1994Co-Authors: Donalyn Scheuner, Ronald W HolzAbstract:1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin Granule membranes and of endogenous Cortical Granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca2+. Exocytosis of chromaffin Granule membranes was detected by the appearance of dopamine-β-hydroxylase of the chromaffin Granule membrane in the oocyte or egg plasma membrane. Cortical Granule exocytosis was detected by release of Cortical Granule lectin, a soluble constituent of Cortical Granules, from individual cells. 2. Injected chromaffin Granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca2+ induced by incubation with ionomycin. 3. Elevated Ca2+ triggered Cortical Granule exocytosis in eggs but not in oocytes. 4. Injected chromaffin Granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous Cortical Granules. 5. Protein kinase C activation by phorbol esters stimulates Cortical Granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin Granule membrane exocytosis in oocytes, indicating that although Cortical Granules and chromaffin Granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both. 6. These results suggest that structural or biochemical characteristics of the chromaffin Granule membrane result in its ability to respond to a Ca2+ stimulus. In the oocytes, Cortical Granule components necessary for Ca2+-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca2+ stimulus and downstream events.
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evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory Granule membrane comparison of exocytosis of injected bovine chromaffin Granule membranes and endogenous Cortical Granules in xenopus laevis oocyt
Cellular and Molecular Neurobiology, 1994Co-Authors: Donalyn Scheuner, Ronald W HolzAbstract:SUMMARY 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaflin Granule membranes and of endogenous Cortical Granules to undergo exocytosis in Xenopus laevis oocytes and eggs in response to cytosolic Ca ~÷. Exocytosis of chromatfin Granule membranes was detected by the appearance of dopamine-/3-hydroxylase of the chromaffin Granule membrane in the oocyte or egg plasma membrane. Cortical Granule exocytosis was detected by release of Cortical Granule lectin, a soluble constituent of Cortical Granules, from individual cells.
Tom Ducibella - One of the best experts on this subject based on the ideXlab platform.
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identification of a translocation deficiency in Cortical Granule secretion in preovulatory mouse oocytes
Biology of Reproduction, 2001Co-Authors: Allison L Abbott, Rafael A. Fissore, Tom DucibellaAbstract:Preovulatory, germinal vesicle (GV)-stage mouse oocytes are unable to undergo normal Cortical Granule (CG) secretion. Full secretory competence is observed by metaphase II (MII) of meiosis and involves the development of calcium response mechanisms. To identify the deficient or inhibited step in CG secretion, preovulatory GV-stage oocytes were stimulated and tested for their ability to undergo translocation, docking, and/or fusion. The mean CG distance to the plasma membrane was not reduced in fertilized or sperm fraction-injected, GV-stage oocytes relative to that in control GV-stage oocytes. In addition, analysis of individual CG distances to the plasma membrane indicated no subpopulation of CGs competent to translocate. Further analysis demonstrated that secretory incompetence likely is not due to a lack of proximity of CGs to the egg's primary calcium store, the endoplasmic reticulum. Calcium/calmodulin-dependent protein kinase II (CaMKII), which is reportedly involved in secretory Granule translocation and secretion in many cells, including eggs, was investigated. A 60-kDa CaMKII isoform detected by Western blot analysis increased 150% during oocyte maturation. The CaMKII activity assays indicated that MII-stage eggs correspondingly have 110% more maximal activity than GV-stage oocytes. These data demonstrate that the primary secretory deficiency is due to a failure of CG translocation, and that a maturation-associated increase in CaMKII correlates with the acquisition of secretory competence and the ability of the egg to undergo normal activation.
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calcium and the control of mammalian Cortical Granule exocytosis
Frontiers in Bioscience, 2001Co-Authors: Allison L Abbott, Tom DucibellaAbstract:Abstract At fertilization, the release of intracellular calcium is necessary and sufficient for most, if not virtually all, of the major events of egg activation that are responsible for the onset of embryonic development. In mammalian eggs, repetitive calcium oscillations stimulate egg activation events through calcium-dependent effectors, such as calmodulin, protein kinases, and specific proteins involved in exocytosis. One of the earliest calcium-dependent events is the exocytosis of Cortical Granules (CGs), a secretory event resulting in the block to polyspermy and the prevention of triploidy. Emerging studies suggest that CG release in mature eggs is dependent upon calcium-dependent proteins similar to those in somatic cells employed to undergo calcium-regulated exocytosis. In contrast, pre-ovulatory oocytes are incompetent to undergo CG exocytosis due to deficiencies in the ability to release and respond to increases in intracellular calcium. The development of competence to release and respond to calcium is relevant to both animal and human in vitro fertilization programs that largely utilize ovarian oocytes not all of which are fully activation competent.
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incompetence of preovulatory mouse oocytes to undergo Cortical Granule exocytosis following induced calcium oscillations
Developmental Biology, 1999Co-Authors: Allison L Abbott, Rafael A. Fissore, Tom DucibellaAbstract:Immature oocytes of many species are incompetent to undergo Cortical Granule (CG) exocytosis upon fertilization. In mouse eggs, CG exocytosis is dependent primarily on an inositol 1,4,5-trisphosphate (IP3)-mediated elevation of intracellular calcium ([Ca2+]i). While deficiencies upstream of [Ca2+]i release are known, this study examined whether downstream deficiencies also contribute to the incompetence of preovulatory mouse oocytes to release CGs. The experimental strategy was to bypass upstream deficiencies by inducing normal, fertilization-like [Ca2+]i oscillations in fully grown, germinal vesicle (GV) stage oocytes and determine if the extent of CG exocytosis was restored to levels observed in mature, metaphase II (MII)-stage eggs. Because IP3 does not stimulate a normal Ca2+ response in GV-stage oocytes, three alternate methods were used to induce oscillations: thimerosal treatment, electroporation, and sperm factor injection. Long-lasting oscillations from thimerosal treatment resulted in 64 and 10% mean CG release at the MII and GV stages, respectively (P < 0.001). Three electrical pulses induced mean [Ca2+]i elevations of approximately 730 and 650 nM in MII- and GV-stage oocytes, respectively, and 31% CG release in MII-stage eggs and 9% in GV-stage oocytes (P < 0.001). Sperm factor microinjection resulted in 86% CG release in MII-stage eggs, while similarly treated GV-stage oocytes exhibited < 1% CG release (P < 0.001). Taken together, these results demonstrate a deficiency downstream of [Ca2+]i release which is developmentally regulated in the 12 h prior to ovulation.
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study of protein kinase c antagonists on Cortical Granule exocytosis and cell cycle resumption in fertilized mouse eggs
Molecular Reproduction and Development, 1997Co-Authors: Tom Ducibella, Leah LefevreAbstract:Although pharmacological agonists of protein kinase C (PKC) stimulate some events of mammalian egg activation, including Cortical Granule (CG) exocytosis, it is not known if these events are dependent on PKC activation during the normal process of fertilization. In order to examine the potential role of PKC in CG exocytosis, this study investigated whether PKC agonists faithfully mimic CG release and whether PKC antagonists block fertilization-induced CG release in mature mouse eggs. Phorbol ester (TPA, 2.5 ng/ml) treatment resulted in an atypical pattern of CG release in which there was a greater net loss of CGs in the equatorial region of the egg than in the region opposite the spindle. This pattern also was in contrast to that during fertilization, in which CG release occurred randomly throughout the cortex. Fertilization experiments utilized two different PKC inhibitors, bisindolyl-maleimide (5 μM) and chelerytherine (0.8 μM), targeted to both the “conserved” substrate and ATP binding domains of PKC. Simultaneous use of both inhibitors at maximal concentrations (compatible with fertilization and above their IC50S) resulted in no detectable inhibition of CG release in treated fertilized eggs compared to controls. In addition, no inhibition of anaphase onset was observed in treated fertilized eggs. Activity of the inhibitors was verified by demonstrating that they blocked the induction of CG loss by TPA. Moreover, 1 μM staurosporine, a potent but less specific antagonist of PKC, also did not block CG loss, whereas the metaphase-anaphase transition was temporarily inhibited. The results indicate that TPA does not faithfully mimic CG release in fertilized eggs, that a role for PKC in CG release at fertilization remains to be established, and that other calcium-dependent effectors may be involved in CG exocytosis. Mol Reprod Dev 46:216–226, 1997. © 1997 Wiley-Liss, Inc.
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effects of calcium bapta buffers and the calmodulin antagonist w 7 on mouse egg activation
Developmental Biology, 1996Co-Authors: Leah Lefevre, Tom Ducibella, Richard M Schultz, Gregory S KopfAbstract:Results of numerous experiments indicate that the transient rise in intracellular Ca2+ following sperm-egg fusion is essential for the subsequent events that constitute egg activation. Some events of egg activation, e.g., Cortical Granule exocytosis, however, appear more sensitive to intracellular Ca2+ than other events, e.g., cell cycle resumption. To examine if specific events of egg activation have different thresholds for Ca2+, we manipulated buffered intracellular Ca2+ concentrations by microinjecting Ca2+-BAPTA buffers and then examined the effect on the Cortical Granule exocytosis, recruitment of maternal mRNAs, and cell cycle resumption. We find that whereas Cortical Granule exocytosis occurs over a narrow threshold range of injected free Ca2+ concentrations between 0.5 and 1.0 microM, recruitment of maternal mRNAs is only partially stimulated at injected free Ca2+ concentrations of 2.5 microM, and no evidence for cell cycle resumption was observed (up to 2.5 microM Ca2+). Although the Ca2+- and phospholipid-dependent protein kinase, protein kinase C, is implicated in aspects of egg activation, calmodulin is also a potential target for the transient increase in Ca2+ that occurs following fertilization. Whereas incubation of eggs in the presence of the calmodulin antagonist W-7 followed by insemination does not block Cortical Granule exocytosis, cell cycle resumption, as assessed by the metaphase-to-anaphase transition, a decrease in histone H1 kinase activity and the time course for the emission of the second polar body are significantly delayed/inhibited.
Donalyn Scheuner - One of the best experts on this subject based on the ideXlab platform.
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evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory Granule membrane comparison of exocytosis of injected bovine chromaffin Granule membranes and endogenous Cortical Granules in xenopus laevis oocytes
Cellular and Molecular Neurobiology, 1994Co-Authors: Donalyn Scheuner, Ronald W HolzAbstract:1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin Granule membranes and of endogenous Cortical Granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca2+. Exocytosis of chromaffin Granule membranes was detected by the appearance of dopamine-β-hydroxylase of the chromaffin Granule membrane in the oocyte or egg plasma membrane. Cortical Granule exocytosis was detected by release of Cortical Granule lectin, a soluble constituent of Cortical Granules, from individual cells. 2. Injected chromaffin Granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca2+ induced by incubation with ionomycin. 3. Elevated Ca2+ triggered Cortical Granule exocytosis in eggs but not in oocytes. 4. Injected chromaffin Granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous Cortical Granules. 5. Protein kinase C activation by phorbol esters stimulates Cortical Granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin Granule membrane exocytosis in oocytes, indicating that although Cortical Granules and chromaffin Granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both. 6. These results suggest that structural or biochemical characteristics of the chromaffin Granule membrane result in its ability to respond to a Ca2+ stimulus. In the oocytes, Cortical Granule components necessary for Ca2+-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca2+ stimulus and downstream events.
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evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory Granule membrane comparison of exocytosis of injected bovine chromaffin Granule membranes and endogenous Cortical Granules in xenopus laevis oocyt
Cellular and Molecular Neurobiology, 1994Co-Authors: Donalyn Scheuner, Ronald W HolzAbstract:SUMMARY 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaflin Granule membranes and of endogenous Cortical Granules to undergo exocytosis in Xenopus laevis oocytes and eggs in response to cytosolic Ca ~÷. Exocytosis of chromatfin Granule membranes was detected by the appearance of dopamine-/3-hydroxylase of the chromaffin Granule membrane in the oocyte or egg plasma membrane. Cortical Granule exocytosis was detected by release of Cortical Granule lectin, a soluble constituent of Cortical Granules, from individual cells.
