scholarly journals Involvement of Rabphilin-3A in cortical granule exocytosis in mouse eggs.

1996 ◽  
Vol 135 (6) ◽  
pp. 1741-1747 ◽  
Author(s):  
N Masumoto ◽  
T Sasaki ◽  
M Tahara ◽  
A Mammoto ◽  
Y Ikebuchi ◽  
...  
Keyword(s):  
Immunoblot Analysis ◽  
Cortical Granule ◽  
Cortical Region ◽  
Dependent Manner ◽  
Immunofluorescence Analysis ◽  
Putative Target ◽  
Terminal Fragment ◽  
Cortical Granule Exocytosis ◽  
Dose Dependent ◽  
Mouse Eggs

Rabphilin-3A is a putative target protein for Rab3A, a member of the small GTP-binding protein superfamily that has been suggested to play a role in regulated exocytosis in presynapses. In this study we determined the expression and the function of Rabphilin-3A in mouse eggs at fertilization. Rabphilin-3A mRNA and protein were detected by reverse transcriptase-PCR and immunoblot analysis, respectively, in metaphase II mouse eggs. Immunofluorescence analysis showed that Rabphilin-3A protein was distributed in the cortical region in eggs. Sperm induces cortical granule (CG) exocytosis via an increase in cytosolic Ca2+ at fertilization. We microinjected the NH2- or COOH-terminal fragment of recombinant Rabphilin-3A into metaphase II eggs. Neither treatments altered the sperm-induced cytosolic Ca2+ increase, but both inhibited CG exocytosis in a dose-dependent manner. The NH2-terminal fragment was more effective than the COOH-terminal fragment. Full-length Rabphilin-3A did not affect CG exocytosis, but it attenuated the inhibition of CG exocytosis by the NH2-terminal fragment. These results show that Rabphilin-3A is involved in Ca(2+)-dependent CG exocytosis at fertilization in mouse eggs.

SNAP-25 is essential for cortical granule exocytosis in mouse eggs

1998 ◽  
Vol 274 (6) ◽  
pp. C1496-C1500 ◽  
Author(s):  
Yoshihide Ikebuchi ◽  
Nobuyuki Masumoto ◽  
Tetsu Matsuoka ◽  
Takeshi Yokoi ◽  
Masahiro Tahara ◽  
...  
Keyword(s):  
Botulinum Neurotoxin ◽  
Endocrine Cells ◽  
Immunoblot Analysis ◽  
Cortical Granule ◽  
Rt Pcr ◽  
Botulinum Neurotoxin A ◽  
Granule Exocytosis ◽  
Cortical Granule Exocytosis ◽  
And Function ◽  
Mouse Eggs

Synaptosome-associated protein of 25 kDa (SNAP-25) has been shown to play an important role in Ca2+-dependent exocytosis in neurons and endocrine cells. During fertilization, sperm-egg fusion induces cytosolic Ca2+mobilization and subsequently Ca2+-dependent cortical granule (CG) exocytosis in eggs. However, it is not yet clear whether SNAP-25 is involved in this process. In this study, we determined the expression and function of SNAP-25 in mouse eggs. mRNA and SNAP-25 were detected in metaphase II (MII) mouse eggs by RT-PCR and immunoblot analysis, respectively. Next, to determine the function of SNAP-25, we evaluated the change in CG exocytosis with a membrane dye, tetramethylammonium-1,6-diphenyl-1,3,5-hexatriene, after microinjection of a botulinum neurotoxin A (BoNT/A), which selectively cleaves SNAP-25 in MII eggs. Sperm-induced CG exocytosis was significantly inhibited in the BoNT/A-treated eggs. The inhibition was attenuated by coinjection of SNAP-25. These results suggest that SNAP-25 may be involved in Ca2+-dependent CG exocytosis during fertilization in mouse eggs.

Initiation of the cortical reaction in hamster and sheep oocytes in response to inositol trisphosphate

1988 ◽  
Vol 91 (1) ◽  
pp. 139-144
Author(s):  
D.G. Cran ◽  
R.M. Moor ◽  
R.F. Irvine
Keyword(s):  
External Medium ◽  
Cortical Granule ◽  
Dependent Manner ◽  
Cortical Granules ◽  
Cortical Reaction ◽  
Ph Dependent ◽  
Cortical Granule Exocytosis ◽  
Gamma S ◽  
Dose Dependent ◽  
External Ph

Microinjection of inositol 1,4,5-triphosphate into sheep and hamster oocytes induces secretion of cortical granules in a dose-dependent manner. In the sheep, this effect is strongly pH-dependent with minimal exocytosis taking place at pH 6.8 but a full cortical reaction occurring at pH8.0. Exocytosis in the hamster is also affected by the pH of the external medium but to a lesser extent. Injection of GTP gamma S also induces exocytosis in both species but is more effective in the hamster. It is suggested that inositol metabolism stimulated by sperm-egg interaction with a GTP-binding protein may be part of the mechanism leading to cortical granule exocytosis and that this may be modulated by the external pH.

scholarly journals Compensatory endocytosis occurs after cortical granule exocytosis in mouse eggs

2019 ◽  
Vol 235 (5) ◽  
pp. 4351-4360
Author(s):  
Matías D. Gómez‐Elías ◽  
Rafael A. Fissore ◽  
Patricia S. Cuasnicú ◽  
Débora J. Cohen
Keyword(s):  
Cortical Granule ◽  
Granule Exocytosis ◽  
Cortical Granule Exocytosis ◽  
Mouse Eggs

The dynamics of plasma membrane PtdIns(4,5)P2 at fertilization of mouse eggs

2002 ◽  
Vol 115 (10) ◽  
pp. 2139-2149 ◽  
Author(s):  
Guillaume Halet ◽  
Richard Tunwell ◽  
Tamas Balla ◽  
Karl Swann ◽  
John Carroll
Keyword(s):  
Plasma Membrane ◽  
De Novo ◽  
Confocal Imaging ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Vegetal Cortex ◽  
Living Mouse ◽  
Cortical Granule Exocytosis ◽  
Phosphatidylinositol 4 ◽  
Mouse Eggs

A series of intracellular Ca2+ oscillations are responsible for triggering egg activation and cortical granule exocytosis at fertilization in mammals. These Ca2+ oscillations are generated by an increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which results from the hydrolysis of phosphatidylinositol 4,5-bisphosphate[PtdIns(4,5)P2]. Using confocal imaging to simultaneously monitor Ca2+ and plasma membrane PtdIns(4,5)P2in single living mouse eggs we have sought to establish the relationship between the kinetics of PtdIns(4,5)P2 metabolism and the Ca2+ oscillations at fertilization. We report that there is no detectable net loss of plasma membrane PtdIns(4,5)P2either during the latent period or during the subsequent Ca2+oscillations. When phosphatidylinositol 4-kinase is inhibited with micromolar wortmannin a limited decrease in plasma membrane PtdIns(4,5)P2 is detected in half the eggs studied. Although we were unable to detect a widespread loss of PtdIns(4,5)P2, we found that fertilization triggers a net increase in plasma membrane PtdIns(4,5)P2 that is localized to the vegetal cortex. The fertilization-induced increase in PtdIns(4,5)P2 follows the increase in Ca2+, is blocked by Ca2+ buffers and can be mimicked, albeit with slower kinetics, by photoreleasing Ins(1,4,5)P3. Inhibition of Ca2+-dependent exocytosis of cortical granules, without interfering with Ca2+ transients, inhibits the PtdIns(4,5)P2 increase. The increase appears to be due to de novo synthesis since it is inhibited by micromolar wortmannin. Finally,there is no increase in PtdIns(4,5)P2 in immature oocytes that are not competent to extrude cortical granules. These studies suggest that fertilization does not deplete plasma membrane PtdIns(4,5)P2 and that one of the pathways for increasing PtdIns(4,5)P2 at fertilization is invoked by exocytosis of cortical granules.

Dynamics of cortical granule exocytosis at fertilization in living mouse eggs

1996 ◽  
Vol 270 (5) ◽  
pp. C1354-C1361 ◽  
Author(s):  
M. Tahara ◽  
K. Tasaka ◽  
N. Masumoto ◽  
A. Mammoto ◽  
Y. Ikebuchi ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cortical Granule ◽  
Free Calcium ◽  
Cortical Granules ◽  
Acetoxymethyl Ester ◽  
Living Mouse ◽  
Cortical Granule Exocytosis ◽  
Mouse Eggs

Sperm-egg fusion induces an intracellular free calcium concentration ([Ca2+]i) increase and exocytosis of cortical granules (CGs). Recently we used an impermeable fluorescent membrane probe, 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), to develop a method to evaluate the kinetics of exocytosis in single living cells. In this study we used digital imaging and confocal laser scanning microscopy to evaluate CG exocytosis in living mouse eggs with TMA-DPH. Time-related changes of CG exocytosis were estimated as the percent increase of TMA-DPH fluorescence. The increase of fluorescence in the egg started after sperm attachment, continued at an almost uniform rate, and ceased at 45-60 min. Whereas the [Ca2+]i increase at fertilization was transient or oscillatory, exocytosis was not always induced concomitantly with each [Ca2+]i peak. Next we used this method to determine some intracellular mediators of exocytosis in the egg. An intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, and a microfilament inhibitor, cytochalasin B, blocked sperm-induced exocytosis. A guanosine 5'-triphosphate-binding protein activator, AlF4-, induced exocytosis. These results suggest that [Ca2+]i, microfilament, and guanosine 5'-triphosphate-binding proteins may be involved in CG exocytosis. In conclusion, this method has significant advantages for studying exocytosis in living eggs.

scholarly journals Calcium uptake and release by isolated cortices and microsomes from the unfertilized egg of the sea urchin Strongylocentrotus droebachiensis.

1986 ◽  
Vol 102 (6) ◽  
pp. 2205-2210 ◽  
Author(s):  
J A Oberdorf ◽  
J F Head ◽  
B Kaminer
Keyword(s):  
Sea Urchin ◽  
Calcium Uptake ◽  
Inositol Trisphosphate ◽  
Calcium Ionophore ◽  
Strongylocentrotus Droebachiensis ◽  
Cortical Granule ◽  
Free Calcium ◽  
Cortical Region ◽  
Ionophore A23187 ◽  
Dependent Manner

Isolated cortices from unfertilized sea urchin eggs sequester calcium in an ATP-dependent manner when incubated in a medium containing free calcium levels characteristic of the resting cell (approximately 0.1 microM). This ATP-dependent calcium uptake activity was measured in the presence of 5 mM Na azide to prevent mitochondrial accumulation, was increased by oxalate, and was blocked by 150 microM quercetin and 50 microM vanadate (known inhibitors of calcium uptake into the sarcoplasmic reticulum). Cortical regions preloaded with 45Ca in the presence of ATP were shown to dramatically increase their rate of calcium efflux upon the addition of (a) the calcium ionophore A23187 (10 microM), (b) trifluoperazine (200 microM), (c) concentrations of free calcium that activated cortical granule exocytosis, and (d) the calcium mobilizing agent inositol trisphosphate. This pool of calcium is most likely sequestered in the portion of the egg's endoplasmic reticulum that remains associated with the cortical region during its isolation. We have developed a method for obtaining a high yield of purified microsomal vesicles from whole eggs. This preparation also demonstrates ATP-dependent calcium sequestering activity which increases in the presence of oxalate and has similar sensitivities to calcium transport inhibitors; however, the isolated microsomal vesicles did not show any detectable release of calcium when exposed to inositol trisphosphate.

Leishmanolysin (gp63 metallopeptidase)-like activity extracellularly released byHerpetomonas samuelpessoai

Parasitology ◽  
2005 ◽  
Vol 132 (1) ◽  
pp. 37-47 ◽  
Author(s):  
C. G. R. ELIAS ◽  
F. M. PEREIRA ◽  
B. A. SILVA ◽  
C. S. ALVIANO ◽  
R. M. A. SOARES ◽  
...  
Keyword(s):  
Proteolytic Activity ◽  
Protein Profile ◽  
Immunoblot Analysis ◽  
Secretory Protein ◽  
Biochemical Properties ◽  
Maximum Activity ◽  
Dependent Manner ◽  
Sds Page ◽  
Dose Dependent ◽  
Anti Gp63

In previous studies, we showed thatHerpetomonas samuelpessoaiproduced a large amount of a surface-located metallopeptidase that presented similar biochemical properties to that of gp63 fromLeishmaniaspp., which is a well-known virulence factor expressed by these digenetic parasites. The present study aims to identify the proteolytic activity released by livingH. samuelpessoaicells. In this context, the parasites were incubated in phosphate buffer up to 4 h, and the supernatants were obtained by centrifugation and filtration steps and were then applied on SDS–PAGE to determine the secretory protein profile and on gelatin-SDS–PAGE to identify the proteolytic activity. The results demonstrated thatH. samuelpessoaisecreted at least 12 polypeptides and an extracellular peptidase of 66 kDa. This enzyme had its activity diminished by 1,10-phenanthroline, EDTA and EGTA. This metallopeptidase was active in a broad spectrum of pH, showing maximum activity at pH 6·0 at 37 °C. Casein was also cleaved by this secretory proteolytic enzyme, while bovine serum albumin and haemoglobin were not degraded under these conditions. Fluorescence microscopy and flow cytometry using anti-gp63 antibody against leishmanolysin ofL. amazonensisdemonstrated the presence of similar molecules on the cell-surface ofH. samuelpessoai. Moreover, immunoblot analysis showed the presence of a reactive polypeptide in the cellular extract and in the supernatant fluid ofH. samuelpessoai, which suggests immunological similarities between these two distinct trypanosomatids. The zinc-metallopeptidase inhibitor 1,10-phenanthroline was able to inhibit the secretion of the 66 kDa metallopeptidase in a dose-dependent manner, while the phospholipase C inhibitor (p-CMPS) did not alter the secretion pattern. Additionally, anti-cross-reacting determinant (CRD) antibody failed to recognize any secreted polypeptide fromH. samuelpessoai. Collectively, these results suggest that the gp63-like molecule was released from theH. samuelpessoaisurface by proteolysis instead of phospholipolysis, in a similar mechanism to that observed inLeishmania.

scholarly journals Rab3A, a possible marker of cortical granules, participates in cortical granule exocytosis in mouse eggs

2016 ◽  
Vol 347 (1) ◽  
pp. 42-51 ◽  
Author(s):  
Oscar Daniel Bello ◽  
Andrea Isabel Cappa ◽  
Matilde de Paola ◽  
María Natalia Zanetti ◽  
Mitsunori Fukuda ◽  
...  
Keyword(s):  
Cortical Granule ◽  
Cortical Granules ◽  
Granule Exocytosis ◽  
Cortical Granule Exocytosis ◽  
Mouse Eggs
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