The Experts below are selected from a list of 11913 Experts worldwide ranked by ideXlab platform
Marcela A. Michaut - One of the best experts on this subject based on the ideXlab platform.
-
Live imaging of Cortical granule exocytosis reveals that in vitro matured mouse oocytes are not fully competent to secrete their content
The Company of Biologists, 2018Co-Authors: Andrea I. Cappa, Matilde De Paola, Paula Wetten, Gerardo A. De Blas, Marcela A. MichautAbstract:Oocyte in vitro maturation does not entirely support all the nuclear and cytoplasmic changes that occur physiologically, and it is poorly understood whether in vitro maturation affects the competence of Cortical granules to secrete their content during Cortical Reaction. Here, we characterize Cortical granule exocytosis (CGE) in live mouse oocytes activated by strontium chloride using the fluorescent lectin FITC-LCA. We compared the kinetic of CGE between ovulated (in vivo matured, IVO) and in vitro matured (IVM) mouse oocytes. Results show that: (1) IVM oocytes have a severely reduced response to strontium chloride; (2) the low response was confirmed by quantification of remnant Cortical granules in permeabilized cells and by a novel method to quantify the exudate in non-permeabilized cells; (3) the kinetic of CGE in IVO oocytes was rapid and synchronous; (4) the kinetic of CGE in IVM oocytes was delayed and asynchronous; (5) Cortical granules in IVM oocytes show an irregular limit in regards to the Cortical granule free domain. We propose the analysis of CGE in live oocytes as a biological test to evaluate the competence of IVM mouse oocytes. This article has an associated First Person interview with the first author of the paper
-
Rab3A, a possible marker of Cortical granules, participates in Cortical granule exocytosis in mouse eggs.
Experimental cell research, 2016Co-Authors: Oscar D. Bello, Andrea Isabel Cappa, Matilde De Paola, Maria N Zanetti, Mitsunori Fukuda, Rafael A. Fissore, Luis S. Mayorga, Marcela A. MichautAbstract:Fusion of Cortical granules with the oocyte plasma membrane is the most significant event to prevent polyspermy. This particular exocytosis, also known as Cortical Reaction, is regulated by calcium and its molecular mechanism is still not known. Rab3A, a member of the small GTP-binding protein superfamily, has been implicated in calcium-dependent exocytosis and is not yet clear whether Rab3A participates in Cortical granules exocytosis. Here, we examine the involvement of Rab3A in the physiology of Cortical granules, particularly, in their distribution during oocyte maturation and activation, and their participation in membrane fusion during Cortical granule exocytosis. Immunofluorescence and Western blot analysis showed that Rab3A and Cortical granules have a similar migration pattern during oocyte maturation, and that Rab3A is no longer detected after Cortical granule exocytosis. These results suggested that Rab3A might be a marker of Cortical granules. Overexpression of EGFP-Rab3A colocalized with Cortical granules with a Pearson correlation coefficient of +0.967, indicating that Rab3A and Cortical granules have almost a perfect colocalization in the egg Cortical region. Using a functional assay, we demonstrated that microinjection of recombinant, prenylated and active GST-Rab3A triggered Cortical granule exocytosis, indicating that Rab3A has an active role in this secretory pathway. To confirm this active role, we inhibited the function of endogenous Rab3A by microinjecting a polyclonal antibody raised against Rab3A prior to parthenogenetic activation. Our results showed that Rab3A antibody microinjection abolished Cortical granule exocytosis in parthenogenetically activated oocytes. Altogether, our findings confirm that Rab3A might function as a marker of Cortical granules and participates in Cortical granule exocytosis in mouse eggs.
-
Cortical granule exocytosis is mediated by alpha snap and n ethilmaleimide sensitive factor in mouse oocytes
PLOS ONE, 2015Co-Authors: Maria Matilde De Paola, Oscar D. Bello, Marcela A. MichautAbstract:Cortical granule exocytosis (CGE), also known as Cortical Reaction, is a calcium- regulated secretion that represents a membrane fusion process during meiotic cell division of oocytes. The molecular mechanism of membrane fusion during CGE is still poorly understood and is thought to be mediated by the SNARE pathway; nevertheless, it is unkown if SNAP (acronym for soluble NSF attachment protein) and NSF (acronym for N-ethilmaleimide sensitive factor), two key proteins in the SNARE pathway, mediate CGE in any oocyte model. In this paper, we documented the gene expression of α-SNAP, γ-SNAP and NSF in mouse oocytes. Western blot analysis showed that the expression of these proteins maintains a similar level during oocyte maturation and early activation. Their localization was mainly observed at the Cortical region of metaphase II oocytes, which is enriched in Cortical granules. To evaluate the function of these proteins in CGE we set up a functional assay based on the quantification of Cortical granules metaphase II oocytes activated parthenogenetically with strontium. Endogenous α-SNAP and NSF proteins were perturbed by microinjection of recombinant proteins or antibodies prior to CGE activation. The microinjection of wild type α-SNAP and the negative mutant of α-SNAP L294A in metaphase II oocytes inhibited CGE stimulated by strontium. NEM, an irreversibly inhibitor of NSF, and the microinjection of the negative mutant NSF D1EQ inhibited Cortical Reaction. The microinjection of anti-α-SNAP and anti-NSF antibodies was able to abolish CGE in activated metaphase II oocytes. The microinjection of anti-γ SNAP antibody had no effect on CGE. Our findings indicate, for the first time in any oocyte model, that α-SNAP, γ-SNAP, and NSF are expressed in mouse oocytes. We demonstrate that α-SNAP and NSF have an active role in CGE and propose a working model.
Robert Aarhus - One of the best experts on this subject based on the ideXlab platform.
-
a derivative of nadp mobilizes calcium stores insensitive to inositol trisphosphate and cyclic adp ribose
Journal of Biological Chemistry, 1995Co-Authors: Robert AarhusAbstract:We have previously shown that alkaline treatment of NADP generates a derivative which can mobilize Ca2+ from sea urchin egg homogenates (Clapper, D. L., Walseth, T. F., Dargie, P. J., and Lee, H. C. (1987) J. Biol. Chem. 262, 9561-9568). In this study, the active derivative was purified and shown by high pressure liquid chromatography to be distinct from NADP and NADPH. However, its proton NMR spectrum was virtually identical to that of NADP. The mass of its molecular ion was measured by high resolution mass spectrometry to be 743.0510, one mass unit larger than the corresponding ion of NADP. These results are consistent with the active derivative being nicotinic acid adenine dinucleotide phosphate (NAADP). Ca2+ release induced by NAADP was saturable with a half-maximal concentration of about 30 nM. The release was specific since NADP and nicotinic acid adenine dinucleotide were ineffective even at 10-40-fold higher concentrations. The NAADP-dependent Ca2+ release showed desensitization and was insensitive to heparin and a specific antagonist of cyclic ADP-ribose (cADPR), 8-amino-cADPR. The release mechanism did not require calmodulin. This is similar to the inositol trisphosphate-sensitive release but distinct from that of cADPR. That the NAADP-sensitive Ca2+ stores were different from those sensitive to inositol trisphosphate- or cADPR was further indicated by their differences in distribution on Percoll density gradients. Microinjection of NAADP into live sea urchin eggs induced transient elevation of intracellular Ca2+ and triggered the Cortical Reaction, indicating the NAADP-dependent mechanism is operative in intact cells.
Prue Talbot - One of the best experts on this subject based on the ideXlab platform.
-
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.
-
Perivitelline space of marsupial oocytes: Extracellular matrix of the unfertilized oocyte and formation of a Cortical granule envelope following the Cortical Reaction
Molecular reproduction and development, 1995Co-Authors: Pramila V. Dandekar, Karen E. Mate, Prue TalbotAbstract:The purpose of this study was to characterize the structure of the vestments surrounding unfertilized and Cortical granule-reacted oocytes from a marsupial, the grey short-tailed opossum Monodelphis domestica and to determine if a Cortical granule envelope (CGE) forms in the perivitelline space (PVS) following the Cortical Reaction. Unfertilized oocytes collected from mature ovarian follicles and oviducal oocytes that had undergone a Cortical Reaction were fixed for electron microscopy in the presence of ruthenium red which stabilizes extracellular matrices (ECM) and facilitates demonstration of a CGE. Unfertilized oocytes were surrounded by a zona pellucida and had a PVS which contained a thick ECM comprised of granules and filaments. This matrix appeared to attach to the oolemma and was structurally similar to matrices reported previously in the PVS of unfertilized oocytes from eutherian mammals and two other marsupials, the Virginia opossum and the fat-tailed dunnart. The cortex of unfertilized oocytes contained Cortical granules which were absent in oocytes recovered from the oviducts of mated females. Oviducal oocytes which lacked Cortical granules exhibited a new coat within the PVS between the zona pellucida and the tips of the oocyte microvilli. This coat, the CGE, appeared structurally similar to CGEs described previously around fertilized eutherian oocytes. The CGE of the grey short-tailed opossum is approximately 1 microns thick and is made up of numerous small dense granules. The coats of the opossum oocyte are compared to those present around other marsupial and eutherian oocytes.
Ricardo D Moreno - One of the best experts on this subject based on the ideXlab platform.
-
control of membrane fusion during spermiogenesis and the acrosome Reaction
Biology of Reproduction, 2002Co-Authors: Joao Ramalhosantos, Gerald Schatten, Ricardo D MorenoAbstract:Membrane fusion is important to reproduction because it occurs in several steps during the process of fertilization. Many events of intracellular trafficking occur during both spermiogenesis and oogenesis. The acrosome Reaction, a key feature during mammalian fertilization, is a secretory event involving the specific fusion of the outer acrosomal membrane and the sperm plasma membrane overlaying the principal piece of the acrosome. Once the sperm has crossed the zona pellucida, the gametes fuse, but in the case of the sperm this process takes place through a specific membrane domain in the head, the equatorial segment. The Cortical Reaction, a process that prevents polyspermy, involves the exocytosis of the Cortical granules to the extracellular milieu. In lower vertebrates, the formation of the zygotic nucleus involves the fusion (syngamia) of the male pronucleus with the female pronucleus. Other undiscovered membrane trafficking processes may also be relevant for the formation of the zygotic centrosome or other zygotic structures. In this review, we focus on the recent discovery of molecular machinery components involved in intracellular trafficking during mammalian spermiogenesis, notably related to acrosome biogenesis. We also extend our discussion to the molecular mechanism of membrane fusion during the acrosome Reaction. The data available so far suggest that proteins participating in the intracellular trafficking events leading to the formation of the acrosome during mammalian spermiogenesis are also involved in controlling the acrosome Reaction during fertilization.
Gary M Wessel - One of the best experts on this subject based on the ideXlab platform.
-
sea urchin ovoperoxidase oocyte specific member of a heme dependent peroxidase superfamily that functions in the block to polyspermy
Mechanisms of Development, 1998Co-Authors: Gary J Lafleur, Yuka Horiuchi, Gary M WesselAbstract:Abstract Ovoperoxidase is one of several oocyte-specific proteins that are stored within sea urchin Cortical granules, released during the Cortical Reaction, and incorporated into the newly formed fertilization envelope. Ovoperoxidase plays a particularly important role in this process, crosslinking the envelope into a hardened matrix that is insensitive to biochemical and mechanical challenges and thus providing a permanent block to polyspermy. Here we present the primary structures of two ovoperoxidases as predicted from cDNAs cloned from the sea urchins Strongylocentrotus purpuratus (AF035380) and Lytechinus variegatus (AF035381). We also present a proposed scheme for the post-translational processing of ovoperoxidase based upon comparisons between the cDNA and protein structures and taking into account previously published reports. The sea urchin ovoperoxidase sequences conform to a profile shared by members of a heme-dependent animal peroxidase family, including the mammalian myelo-, lacto-, eosinophil, and thyroid peroxidases. Using in situ RNA hybridizations, we showed that the mRNA of S . purpuratus ovoperoxidase (4 kb) is present exclusively in oocytes, and is turned over rapidly following germinal vesicle breakdown. Taking into account our immunoblot and N-terminal sequencing data along with reports from similar peroxidases, we propose that ovoperoxidases are synthesized in a pre-pro form and proteolytically processed to result in the 70 and 50 kDa forms that are found in the fertilization envelope. The sequence and structural data presented here will facilitate our continuing studies of the biogenesis of Cortical granules and the fertilization envelope. Additionally, since ovoperoxidase activities have been reported in a wide range of animals, these cDNAs will be useful in uncovering similar peroxidases used in the fertilization Reactions of other metazoan eggs.
