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.

Participation of PLA2 and PLC in DhL-induced activation of Rhinella arenarum oocytes

Zygote ◽  
2015 ◽  
Vol 24 (4) ◽  
pp. 495-501
Author(s):  
J. Zapata-Martínez ◽  
M.F. Medina ◽  
M.C. Gramajo-Bühler ◽  
G. Sánchez-Toranzo
Keyword(s):  
Aristolochic Acid ◽  
Specific Inhibitor ◽  
Competitive Inhibitor ◽  
Egg Activation ◽  
Inositol Triphosphate ◽  
Phosphoinositide Metabolism ◽  
Oocyte Activation ◽  
Rhinella Arenarum ◽  
Different Doses

SummaryRhinella arenarum oocytes can be artificially activated, a process known as parthenogenesis, by a sesquiterpenic lactone of the guaianolide group, dehydroleucodine (DhL). Transient increases in the concentration of cytosolic Ca2+ are essential to trigger egg activation events. In this sense, the 1-4-5 inositol triphosphate receptors (IP3R) seem to be involved in the Ca2+ transient release induced by DhL in this species. We analyzed the involvement of phosphoinositide metabolism, especially the participation of phospholipase A2 (PLA2) and phospholipase C (PLC) in DhL-induced activation. Different doses of quinacrine, aristolochic acid (ATA) (PLA2 inhibitors) or neomycin, an antibiotic that binds to PIP2, thus preventing its hydrolysis, were used in mature Rhinella arenarum oocytes. In order to assay the participation of PI-PLC and PC- PLC we used U73122, a competitive inhibitor of PI-PLC dependent events and D609, an inhibitor of PC-PLC. We found that PLA2 inhibits quinacrine more effectively than ATA. This difference could be explained by the fact that quinacrine is not a specific inhibitor for PLA2 while ATA is specific for this enzyme. With respect to the participation of PLC, a higher decrease in oocyte activation was detected when cells were exposed to neomycin. Inhibition of PC-PLC with D609 and IP-PLC with U73122 indicated that the last PLC has a significant participation in the effect of DhL-induced activation. Results would indicate that DhL induces activation of in vitro matured oocytes of Rhinella arenarum by activation of IP-PLC, which in turn may induce IP3 formation which produces Ca2+ release.

Tyrosine kinase receptor RON functions downstream of the erythropoietin receptor to induce expansion of erythroid progenitors

Blood ◽  
2004 ◽  
Vol 103 (12) ◽  
pp. 4457-4465 ◽  
Author(s):  
Emile van den Akker ◽  
Thamar van Dijk ◽  
Martine Parren-van Amelsvoort ◽  
Katja S. Grossmann ◽  
Ute Schaeper ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Erythropoietin Receptor ◽  
Mitogen Activated Protein Kinase ◽  
Tyrosine Kinase Receptor ◽  
Erythroid Progenitors ◽  
Downstream Target ◽  
Stress Erythropoiesis ◽  
Mitogen Activated Protein ◽  
Induced Signal

Abstract Erythropoietin (EPO) is required for cell survival during differentiation and for progenitor expansion during stress erythropoiesis. Although signaling pathways may couple directly to docking sites on the EPO receptor (EpoR), additional docking molecules expand the signaling platform of the receptor. We studied the roles of the docking molecules Grb2-associated binder-1 (Gab1) and Gab2 in EPO-induced signal transduction and erythropoiesis. Inhibitors of phosphatidylinositide 3-kinase and Src kinases suppressed EPO-dependent phosphorylation of Gab2. In contrast, Gab1 activation depends on recruitment and phosphorylation by the tyrosine kinase receptor RON, with which it is constitutively associated. RON activation induces the phosphorylation of Gab1, mitogen-activated protein kinase (MAPK), and protein kinase B (PKB) but not of signal transducer and activator of transcription 5 (Stat5). RON activation was sufficient to replace EPO in progenitor expansion but not in differentiation. In conclusion, we elucidated a novel mechanism specifically involved in the expansion of erythroblasts involving RON as a downstream target of the EpoR. (Blood. 2004;103:4457-4465)

scholarly journals Functional Analysis of H-Ryk, an Atypical Member of the Receptor Tyrosine Kinase Family

1999 ◽  
Vol 19 (9) ◽  
pp. 6427-6440 ◽  
Author(s):  
R. M. Katso ◽  
R. B. Russell ◽  
T. S. Ganesan
Keyword(s):  
Catalytic Activity ◽  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Catalytic Domain ◽  
Homology Modelling ◽  
Mitogen Activated Protein Kinase ◽  
In Vitro Mutagenesis ◽  
Chimeric Receptor ◽  
Activation Domain

ABSTRACT H-Ryk is an atypical receptor tyrosine kinase which differs from other members of this family at a number of conserved residues in the activation and nucleotide binding domains. Using a chimeric receptor approach, we demonstrate that H-Ryk has impaired catalytic activity. Despite the receptor’s inability to undergo autophosphorylation or phosphorylate substrates, we demonstrate that ligand stimulation of the chimeric receptor results in activation of the mitogen-activated protein kinase pathway. The ability to transduce signals is abolished by mutation of the invariant lysine (K334A) in subdomain II of H-Ryk. Further, by in vitro mutagenesis, we show that the amino acid substitutions in the activation domain of H-Ryk account for the loss of catalytic activity. In addition to the essential aspartate residue, either phenylalanine or glycine is required in the activation domain to maintain proper conformation of the catalytic domain and thus ensure receptor autophosphorylation. Homology modelling of the catalytic domain of H-Ryk provides a rationale for these findings. Thus, the signalling properties of H-Ryk are divergent from those of other classical receptor tyrosine kinases.

Activation of amphibian oocytes by sperm extracts

Zygote ◽  
2008 ◽  
Vol 16 (4) ◽  
pp. 303-308 ◽  
Author(s):  
F. Bonilla ◽  
M. T. Ajmat ◽  
G. Sánchez Toranzo ◽  
L. Zelarayán ◽  
J. Oterino ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Dna Replication ◽  
Egg Activation ◽  
Cortical Granule ◽  
Oocyte Activation ◽  
Granule Exocytosis ◽  
Bufo Arenarum ◽  
Sperm Extract ◽  
Cortical Granule Exocytosis

SummaryIn the fertilization of most animals, egg activation is accompanied by an increase in cytoplasmatic Ca2+; however, the mechanism through which the fertilizing sperm induce this phenomenon is still controversial. An increase in intracellular free Ca2+ is required to trigger egg activation events, a process that includes cortical granule exocytosis, resumption and completion of meiosis and DNA replication, and culminates in the first mitotic cleavage. In this work, we investigated the effect of microinjection and incubation of different fractions of homologous sperm extract on the activation of Bufo arenarum oocytes matured in vitro. Two heat treatment-sensitive fractions obtained by chromatography were able to induce oocyte activation. The sperm fraction, which contained a 24 kDa protein, induced 90% activation when it was microinjected into the oocytes. Whilst the sperm fraction, which contained a 36 kDa protein, was able to induce about 70% activation only when it was applied on the oocyte surface.

Calcium- and meiotic-spindle-independent activation of pig oocytes by the inhibition of staurosporine-sensitive protein kinases

Zygote ◽  
1997 ◽  
Vol 5 (1) ◽  
pp. 75-82 ◽  
Author(s):  
Weihua Wang ◽  
Qingyuan Sun ◽  
Misa Hosoe ◽  
Yasuo Shioya
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Meiotic Spindle ◽  
Cortical Granule ◽  
Oocyte Activation ◽  
Cell Stage ◽  
Nuclear Activation ◽  
Intracellular Ca

SummaryThe dependence of pig oocyte activation (both nuclear activation and cortical granule exocytosis) induced by staurosporine on intracellular Ca2+ rise and spindle assembly was studied. Nuclear activation was evaluated by pronuclear (PN) formation, cleavage and their developmental ability, and cortical granule (CG) exocytosis was assessed by electron microscopy and laser confocal microscopy of oocytes labelled with fluorescein isothiocyanate-peanut agglutinin. Exposure of pig oocytes of 0.3 and 3μM protein kinase inhibitor staurosporine for 30 min resulted in the nuclear activation in 71.8% and 85.7% of the oocytes, respectively. The pronuclei in activated oocytes contained several compact nucleoli. When the cleaved 2-cell oocytes were further cultured in vitro, 93.5% developed beyond the 4-cell stage, and 12.9% developed to the morula stage after 4 days of culture. Of the oocytes treated with 3μM staurosporine, 62.5% and 9.4% released their CGs partially and completely, respectively. The nuclear activation induced by staurosporine was overcome by the prior treatment of oocytes with okadaic acid, resulting in only 33.3% of the oocytes undergoing nuclear activation. However, when oocytes were exposed first to 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethanal ester), a cell permeate calcium chelator, or Colcemid, a meiotic spindle disrupter, and then to staurosporine, nuclear activation was observed in 74.2% and 82.3% of the oocytes, espectively. These data were the same as those in oocytes treated only with staurosporine (85.7%). The present study indicates that pig oocytes can be activated by the inhibition of staurosporine-sensitive protein kinase(s), and that this activation is dependent upon mitogen-activated protein kinase but independent of the intracellular Ca2+ rise and spindle integrity.

FGFR1 is fused to the centrosome-associated proteinCEP110 in the 8p12 stem cell myeloproliferative disorder with t(8;9)(p12;q33)

Blood ◽  
2000 ◽  
Vol 95 (5) ◽  
pp. 1788-1796 ◽  
Author(s):  
Géraldine Guasch ◽  
Gary J. Mack ◽  
Cornel Popovici ◽  
Nicole Dastugue ◽  
Daniel Birnbaum ◽  
...  
Keyword(s):  
Stem Cell ◽  
Tyrosine Kinase ◽  
Leucine Zipper ◽  
Catalytic Domain ◽  
Coiled Coil ◽  
Myeloproliferative Disorder ◽  
Tyrosine Kinase Receptor ◽  
Cell Cycle Distribution ◽  
Leucine Zippers ◽  
Unique Cell

The hallmark of the 8p12 stem cell myeloproliferative disorder (MPD) is the disruption of the FGFR1 gene, which encodes a tyrosine kinase receptor for members of the fibroblast growth factor family.FGFR1 can be fused to at least 3 partner genes at chromosomal regions 6q27, 9q33, or 13q12. We report here the cloning of the t(8;9)(p12;q33) and the detection of a novel fusion betweenFGFR1 and the CEP110 gene, which codes for a novel centrosome-associated protein with a unique cell-cycle distribution. CEP110 is widely expressed at various levels in different tissues and is predicted to encode a 994-amino acid coiled-coil protein with 4 consensus leucine zippers [L-X(6)-L-X(6)-L-X(6)-L]. Both reciprocal fusion transcripts are expressed in the patient's cells. The CEP110-FGFR1 fusion protein encodes an aberrant tyrosine kinase of circa 150-kd, which retains most of CEP110 with the leucine zipper motifs and the catalytic domain of FGFR1. Transient expression studies show that the CEP110-FGFR1 protein has a constitutive kinase activity and is located within the cell cytoplasm.

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.

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