scholarly journals Strontium-induced rat egg activation

Reproduction ◽  
2005 ◽  
Vol 130 (4) ◽  
pp. 467-474 ◽  
Author(s):  
R Tomashov-Matar ◽  
D Tchetchik ◽  
A Eldar ◽  
R Kaplan-Kraicer ◽  
Y Oron ◽  
...  
Keyword(s):  
Calcium Concentration ◽  
Cytosolic Calcium ◽  
Egg Activation ◽  
Cortical Granule ◽  
Strontium Chloride ◽  
Quantitative Immunohistochemistry ◽  
Cortical Granule Exocytosis ◽  
Rat Eggs ◽  
Trisphosphate Receptor ◽  
Mouse Eggs

Parthenogenetic agents that evoke cytosolic calcium concentration ([Ca2+]i) oscillations similar to those evoked by sperm, mimic fertilization more faithfully than agents that trigger a single [Ca2+]itransient. Strontium chloride (SrCl2) binds to and activates the Ca2+-binding site on the inositol 1,4,5-trisphosphate receptor and evokes [Ca2+]ioscillations. Although SrCl2has been reported to activate mouse eggs, little is known regarding the pattern of the [Ca2+]ioscillations it evokes in rat eggs and their effect on the early events of egg activation: cortical granule exocytosis (CGE) and completion of meiosis (CM). In the current study we investigated the effect of various concentrations of SrCl2(2, 4 or 6 mM) on [Ca2+]i, by monitoring [Ca2+]ioscillations in fura-2-loaded rat eggs. Treatment with 2 mM SrCl2was optimal for inducing the first [Ca2+]itransient, which was similar in duration to that triggered by sperm. However, the frequency and duration of the subsequent [Ca2+]ioscillations were lower and longer in SrCl2-activated than in sperm-activated eggs. The degree of CGE was identical in eggs activated by either sperm or SrCl2, as assessed by semi-quantitative immunohistochemistry combined with confocal microscopy. Evoking 1, 2 or 10 [Ca2+]ioscillations (8, 15 or 60 min in SrCl2respectively) had no effect on the intensity of fluorescent CGE reporter dyes, while 60-min exposure to SrCl2caused a delay in CM. Our results demonstrate that SrCl2is an effective parthenogenetic agent that mimics rat egg activation by sperm, as judged by the generation of [Ca2+]ioscillations, CGE and CM.

scholarly journals Association between myristoylated alanin-rich C kinase substrate (MARCKS) translocation and cortical granule exocytosis in rat eggs

Reproduction ◽  
2006 ◽  
Vol 131 (2) ◽  
pp. 221-231 ◽  
Author(s):  
Efrat Eliyahu ◽  
Nataly Shtraizent ◽  
Alina Tsaadon ◽  
Ruth Shalgi
Keyword(s):  
Secretory Process ◽  
Egg Activation ◽  
Cortical Granule ◽  
Granule Exocytosis ◽  
Kinase Substrate ◽  
Kinase C ◽  
Cortical Granule Exocytosis ◽  
Rat Eggs ◽  
Mammalian Eggs ◽  
Pkc Activation

Cortical granule exocytosis (CGE), following egg activation, is a secretory process that blocks polyspermy and enables successful embryonic development. CGE can be triggered independently by either a rise in intracellular calcium concentration ([Ca2+]i) or activation of protein kinase C (PKC). The present study investigates the signal transduction pathways leading to CGE through activation of PKC or stimulation of a rise in [Ca2+]i. Using Western blot analysis, co-immunoprecipitation and immunohistochemistry, combined with various inhibitors or activators, we investigated the link between myristoylated alanin-rich C kinase substrate (MARCKS) translocation and CGE. We were able to demonstrate translocation of MARCKS from the plasma membrane to the cortex, in fertilized as well as in parthenogenetically activated eggs. MARCKS phosphorylation was demonstrated upon PKC activation, whereas a PKC inhibitor (myrPKCψ) prevented both MARCKS translocation and CGE in 12-O-tetradecanoyl phorbol-13-acetate (TPA)-activated eggs. We have further shown that upon egg activation the amount of phosphorylated MARCKS (p-MARCKS) and the amount of calmodulin bound to MARCKS were increased. MARCKS translocation in ionomycin activated eggs was also inhibited by the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide hydrochloride (W7). These results complement other studies showing MARCKS requirement for exocytosis and imply that upon fertilization, MARCKS translocation is followed by CGE. These findings present a significant contribution to our understanding of CGE in mammalian eggs in particular, as well as cellular exocytosis in general.

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.

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.

scholarly journals Knockin’ on Egg’s Door: Maternal Control of Egg Activation That Influences Cortical Granule Exocytosis in Animal Species

2021 ◽  
Vol 9 ◽  
Author(s):  
Japhet Rojas ◽  
Fernando Hinostroza ◽  
Sebastián Vergara ◽  
Ingrid Pinto-Borguero ◽  
Felipe Aguilera ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Animal Species ◽  
Egg Activation ◽  
Cortical Granule ◽  
Secretory Vesicles ◽  
Cortical Granules ◽  
Maternal Control ◽  
Molecular Machinery ◽  
Cortical Granule Exocytosis ◽  
Critical Aspects

Fertilization by multiple sperm leads to lethal chromosomal number abnormalities, failed embryo development, and miscarriage. In some vertebrate and invertebrate eggs, the so-called cortical reaction contributes to their activation and prevents polyspermy during fertilization. This process involves biogenesis, redistribution, and subsequent accumulation of cortical granules (CGs) at the female gamete cortex during oogenesis. CGs are oocyte- and egg-specific secretory vesicles whose content is discharged during fertilization to block polyspermy. Here, we summarize the molecular mechanisms controlling critical aspects of CG biology prior to and after the gametes interaction. This allows to block polyspermy and provide protection to the developing embryo. We also examine how CGs form and are spatially redistributed during oogenesis. During egg activation, CG exocytosis (CGE) and content release are triggered by increases in intracellular calcium and relies on the function of maternally-loaded proteins. We also discuss how mutations in these factors impact CG dynamics, providing unprecedented models to investigate the genetic program executing fertilization. We further explore the phylogenetic distribution of maternal proteins and signaling pathways contributing to CGE and egg activation. We conclude that many important biological questions and genotype–phenotype relationships during fertilization remain unresolved, and therefore, novel molecular players of CG biology need to be discovered. Future functional and image-based studies are expected to elucidate the identity of genetic candidates and components of the molecular machinery involved in the egg activation. This, will open new therapeutic avenues for treating infertility in humans.

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.

Parthenogenetic activation of mouse eggs by microinjection of a truncated c-kit tyrosine kinase present in spermatozoa

Development ◽  
1997 ◽  
Vol 124 (11) ◽  
pp. 2267-2274 ◽  
Author(s):  
C. Sette ◽  
A. Bevilacqua ◽  
A. Bianchini ◽  
F. Mangia ◽  
R. Geremia ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Catalytic Domain ◽  
Specific Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Egg Activation ◽  
Tyrosine Kinase Receptor ◽  
Cortical Granule ◽  
Oocyte Activation ◽  
Cos Cells ◽  
Mouse Eggs

A truncated form of the c-kit tyrosine kinase receptor, corresponding to the phosphotransferase portion of the cytoplasmic catalytic domain and the carboxyterminus (tr-kit), is accumulated during late mouse spermiogenesis. Here we report that tr-kit is specifically localized in the residual sperm cytoplasm, with maximal accumulation in the midpiece of the flagellum, suggesting that it can enter the egg during fertilization. Microinjection of extracts from COS cells expressing a recombinant tr-kit protein into metaphase II-arrested mouse oocytes caused complete oocyte activation, including cortical granule exocytosis, completion of the 2nd meiotic division, formation of a parthenogenetic pronucleus and progression through cleavage stages. No activation above background levels was obtained with extracts from mock-transfected COS cells. Similar results were obtained by microinjection of in vitro synthesized tr-kit mRNA into metaphase II-arrested oocytes. Tr-kit-induced parthenogenetic egg activation was completely inhibited by oocyte preincubation with the Ca2(+)-chelating agent BAPTA-AM or with a specific inhibitor of phospholipase C activity. Tr-kit-induced egg activation was associated with a decrease in activity of mitogen-activated protein kinase, an essential component of the cytostatic factor. These results candidate tr-kit as a putative sperm factor required for triggering activation of mouse eggs at fertilization.

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