Effects of wortmannin on the kinetics of gvbd and the activities of the maturation-promoting factor and mitogen-activated protein kinase during bovine oocyte maturation in vitro

2000 ◽  
Vol 53 (9) ◽  
pp. 1797-1806 ◽  
Author(s):  
M-K.I. Anas ◽  
A. Shojo ◽  
M. Shimada ◽  
T. Terada
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Mitogen Activated Protein Kinase ◽  
Bovine Oocyte ◽  
Maturation In Vitro ◽  
Mitogen Activated Protein ◽  
Kinetics Of ◽  
Maturation Promoting Factor

31 PROTOCOL OPTIMIZATION AND EVALUATION OF MATURATION PROMOTING FACTOR AND MITOGEN-ACTIVATED PROTEIN KINASE ACTIVITIES IN BOVINE CYTOPLASTS OBTAINED BY CHEMICAL ENUCLEATION TECHNIQUES

2014 ◽  
Vol 26 (1) ◽  
pp. 130
Author(s):  
N. Z. Saraiva ◽  
C. S. Oliveira ◽  
M. del Collado ◽  
M. R. de Lima ◽  
R. Vantini ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Activity ◽  
Significant Difference ◽  
Mitogen Activated Protein ◽  
Chi Squared ◽  
High Concentrations ◽  
Bovine Oocytes ◽  
Maturation Promoting Factor

Chemical enucleation using microtubule-depolymerizing drugs is an attractive procedure to simplify the enucleation process in nuclear transfer. The aim of this study was to optimize chemically assisted (CA) and chemically induced (CI) enucleation protocols using metaphase II (MII) and pre-activated bovine oocytes, respectively, and to evaluate the activity of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) in cytoplasts generated by these techniques. Initially, we determined the shortest effective treatment of MII and activated oocytes with 0.05 μg mL–1 demecolcine. Bovine oocytes in vitro matured (IVM) for 19 h (MII) or activated artificially with 5 μM ionomycin (5 min) and 10 μg mL–1 cycloheximide (5 h) after 26 h IVM were treated with demecolcine and samples were collected at 0, 0.25, 0.5, 1.0, 1.5, and 2.0 h of treatment. Oocytes were then stained with 10 μg mL–1 Hoechst 33342 and the protrusion or enucleation rates were determined. Next, we evaluated histone H1 and myelin basic protein (MBP) kinases, reflecting MPF and MAPK activities, respectively, in oocytes obtained from these treatments, and for that we used the method described by Kubelka et al. (2000 Biol. Reprod. 62, 292–302). Protrusion and enucleation rates were evaluated by the chi-squared (χ2) test, and MPF and MAPK activities were submitted to ANOVA and Tukey's test at 5% significance. For MII oocytes, effects of demecolcine were observed as early as 15 min, with a significant difference (P < 0.05) between control (12/112, 10.7%) and treated (33/114, 28.9%) groups in relation to protrusion rates. The largest number of protrusions was observed after 1.0 h of treatment (control: 15/113, 13.3%a; treated: 45/111, 40.5%b). In pre-activated oocytes, effects of demecolcine were also observed after 15 min, and in both techniques there were no significant differences between groups treated with demecolcine for 1.0, 1.5, or 2.0 h (CA: 40.5 to 52.5% of protrusion; CI: 35.2 to 46.7% of enucleation). In contrast to previous reports in which high concentrations of demecolcine for CA enucleation increased MPF activity, we observed no alterations in the activity of this factor at a demecolcine concentration of 0.05 μg mL–1. Activity of MAPK also did not differ significantly between the control and treated groups throughout evaluation. In the CI technique, a significant difference in MPF activity was observed after 0.5 h (70.3%) and 2.0 h of activation (39.1%), considering that the activity was 100% at the beginning of the evaluation. However, we observed no significant difference between the control and treated groups at any of the time points studied, as verified for MAPK activity. The exact effect of MPF on the nucleus in mammals is not well established. We believe that the use of low concentrations of demecolcine for short periods is less damaging to embryonic development and, until we have a better understanding of the effect of these kinases on the transferred nucleus, we recommend its use for chemical enucleation protocols in bovine. Financial support: FAPESP 2010/20744-6 and 2011/12983-3.

scholarly journals Mechanisms of FSH- and Amphiregulin-Induced MAP Kinase 3/1 Activation in Pig Cumulus-Oocyte Complexes During Maturation In Vitro

2019 ◽  
Vol 20 (5) ◽  
pp. 1179 ◽  
Author(s):  
Radek Prochazka ◽  
Lucie Nemcova
Keyword(s):  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Egf Receptor ◽  
De Novo ◽  
Cumulus Cells ◽  
Mitogen Activated Protein Kinase ◽  
Maturation In Vitro ◽  
Mitogen Activated Protein ◽  
Rapid Activation

The maturation of mammalian oocytes in vitro can be stimulated by gonadotropins (follicle-stimulating hormone, FSH) or their intrafollicular mediator, epidermal growth factor (EGF)-like peptide—amphiregulin (AREG). We have shown previously that in pig cumulus-oocyte complexes (COCs), FSH induces expression and the synthesis of AREG that binds to EGF receptor (EGFR) and activates the mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathway. However, in this study we found that FSH also caused a rapid activation of MAPK3/1 in the cumulus cells, which cannot be explained by the de novo synthesis of AREG. The rapid MAPK3/1 activation required EGFR tyrosine kinase (TK) activity, was sensitive to SRC proto-oncogene non-receptor tyrosine kinase (SRC)-family and protein kinase C (PKC) inhibitors, and was resistant to inhibitors of protein kinase A (PKA) and metalloproteinases. AREG also induced the rapid activation of MAPK3/1 in cumulus cells, but this activation was only dependent on the EGFR TK activity. We conclude that in cumulus cells, FSH induces a rapid activation of MAPK3/1 by the ligand-independent transactivation of EGFR, requiring SRC and PKC activities. This rapid activation of MAPK3/1 precedes the second mechanism participating in the generation and maintenance of active MAPK3/1—the ligand-dependent activation of EGFR depending on the synthesis of EGF-like peptides.

scholarly journals 269 INACTIVATION OF MATURATION PROMOTING FACTOR AND MITOGEN-ACTIVATED PROTEIN KINASE IN PORCINE OOCYTES BY A SINGLE ELECTRICAL PULSE

2005 ◽  
Vol 17 (2) ◽  
pp. 284
Author(s):  
D.-B. Koo ◽  
J.-I. Chae ◽  
J.-S. Kim ◽  
G. Wee ◽  
B.-S. Song ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Electrical Pulse ◽  
Mitogen Activated Protein Kinase ◽  
Blastocyst Formation ◽  
Development Rates ◽  
Mitogen Activated Protein ◽  
Artificial Activation ◽  
Field Strengths ◽  
Maturation Promoting Factor

Activities of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) of mature oocytes should be decreased to begin subsequent development. In this study, activities of MPF and MAPK were investigated in porcine oocytes after artificial activation. To determine optimal electrical activation, porcine oocytes were exposed to 3 V AC pulse for 5 s followed by a single DC pulse of various electric field strengths (120, 150, 180, and 210 V/mm) and pulse durations (15, 30, 45, and 60 μs). For chemical activation, oocytes were exposed to 5 μM ionomycin for 5 min followed by 2 mM 6-dimethylaminopurine (6-DMAP) or 5 μg/mL cycloheximide for 4 h or 6 h. After activation, 40 to 50 oocytes were cultured in 50-μL drops of NCSU23 medium supplemented with 4 mg/mL BSA at 39°C, and 5% CO2 in air. After 6 days of culture, blastocyst formation was observed and then numbers of blastocyst nuclei were counted after staining with Hoechst 33342. in vitro development rates and numbers of blastocyst nuclei by the field strengths were not significantly different among experimental groups (P > 0.05). However, development rates to the blastocyst stage of porcine oocytes exposed to 15 and 30 μ­s were 27.4 and 24.4%, respectively, which were significantly higher than that (12.5%) of 60 μs (P < 0.05). Mean numbers of blastocyst nuclei in 15- and 30-μs groups (38.6 ± 13.4 and 37.9 ± 11.4, respectively) were significantly higher than that (25.8 ± 10.5) of the 60-μs group (P < 0.05). Blastocyst development after optimal electrical pulse exposure was compared with that after different chemical treatments. Electrical stimulation induced 22.9% of blastocyst formation, which was significantly higher (P < 0.01) than those induced by the chemical stimulators (3.4 and 2.7%). Based on these results, changes of constituent proteins (cdc2 and ERK) of MPF and MAPK after artificial activation were analyzed by immunoblotting using anti-PSTAIRE monoclonal antibody and anti-MAP kinase polyclonal antibody. Activities of both cdc2 and ERK in pig oocytes were reduced 4 h after electrical stimulus, but were maintained at optimal levels after treatment with ionomycin + 6-DMAP. Our results indicate that an optimal single electrical pulse is effective on the inactivation of MPF and MAPK in porcine oocytes, eventually resulting in activation of porcine oocytes produced in vitro.

299 LEPTIN ACCELERATES PRONUCLEAR FORMATION FOLLOWING INTRACYTOPLASMIC SPERM INJECTION OF PORCINE OOCYTES: POSSIBLE ROLE OF MITOGEN-ACTIVATED PROTEIN KINASE INACTIVATION

2007 ◽  
Vol 19 (1) ◽  
pp. 265
Author(s):  
Y. X. Jin ◽  
X. S. Cui ◽  
W. K. Chang ◽  
T. Kim ◽  
N. H. Kim
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Intracytoplasmic Sperm Injection ◽  
Treated Group ◽  
Mitogen Activated Protein Kinase ◽  
Parthenogenetic Activation ◽  
Mitogen Activated Protein ◽  
Cytoplasmic Maturation ◽  
Pronuclear Formation

Although evidence suggests that leptin modulates oocyte maturation in vitro, few studies have examined the direct effect of leptin on cytoplasmic maturation and pronuclear formation following fertilization of porcine oocytes.The aim of this studywas to determine microtubule and microfilament assembly following oocyte maturation, and to assess blastocyst formation, mitogen-activated protein kinase (MAPK) activity, and pronuclear formation following parthenogenetic stimuli or intracytoplasmic sperm injection (ICSI) of leptin-treated oocytes.A symmetrical shape of the meiotic spindle was considered to be the normal microtubule assembly, and a clear, thick microfilament area containing a second metaphase chromatin was considered to be morphologically normal. Significant differences were determined using Tukey's multiple range test with P &lt; 0.05 being considered significant. Addition of 10 ng mL-1 of leptin to the in vitro maturation (IVM) medium significantly increased the percentage of zygotes that developed to the blastocyst stage (26.1 � 2.1%; P &lt; 0.05) compared with that of the control (0 ng mL-1: 16.3 � 2.8%) or 1 ng mL-1 (18.6 � 2.6%) leptin treatment groups following parthenogenetic activation. After IVM for 44 h, the percentage of oocytes with normal microtubules was higher in the leptin (10 ng mL-1) group than in the control group (89.1 � 4.3% vs. 78.5 � 4.0%, respectively; P &lt; 0.05). However, the percentage of normal microfilament assembly was similar in the leptin-treated and control groups (85.6 � 4.3% and 82.9 � 5.0%, respectively). In oocytes matured in vitro in the presence of 10 ng mL-1 of leptin and subsequently induced by parthenogenetic activation via chemical stimulation, there was a significant increase in the formation of 2 pronuclei (2PN: 52.1 � 5.2%; P &lt; 0.05) at 6 h, compared with the control non-leptin-treated oocytes (40.7 � 4.0%). Similarly, addition of 10 ng mL-1 of leptin to the IVM medium also increased 2PN formation at the same time point following ICSI (leptin treatment: 47.9 � 4.0; control: 36.2 � 3.8%; P &lt; 0.05). The addition of 10 ng mL-1 of leptin to the IVM medium decreased the level of phospho-ERK1/2 at 6 and 9 h and was lower in the leptin-treated group compared with the control non-leptin-treated group (P &lt; 0.01). These results suggest that exogenous leptin enhances cytoplasmic maturation and pronuclear formation following fertilization, possibly via the MAPK pathway.

scholarly journals Mechanistic Studies of the Mitotic Activation of Mos

2006 ◽  
Vol 26 (14) ◽  
pp. 5300-5309 ◽  
Author(s):  
Jianbo Yue ◽  
James E. Ferrell
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Tyrosine Kinases ◽  
Spindle Assembly Checkpoint ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Egg Extracts ◽  
Mitogen Activated Protein

ABSTRACT The protein kinase Mos is responsible for the activation of MEK1 and p42 mitogen-activated protein kinase during Xenopus oocyte maturation and during mitosis in Xenopus egg extracts. Here we show that the activation of Mos depends upon the phosphorylation of Ser 3, a residue previously implicated in the regulation of Mos stability; the dephosphorylation of Ser 105, a previously unidentified phosphorylation site conserved in Mos proteins; and the regulated dissociation of Mos from CK2β. Mutation of Ser 3 to alanine and/or mutation of Ser 105 to glutamate produces a Mos protein that is defective for M-phase activation, as assessed by in vitro kinase assays, and defective for induction of oocyte maturation and maintenance of the spindle assembly checkpoint in extracts. Interestingly, Ser 105 is situated at the beginning of helix αC in the N-terminal lobe of the Mos kinase domain. Changes in the orientation of this helix have been previously implicated in the activation of Cdk2 and Src family tyrosine kinases. Our work suggests that Ser 105 dephosphorylation represents a novel mechanism for reorienting helix αC.

scholarly journals Cross-talk between IRAK-4 and the NADPH oxidase1

2007 ◽  
Vol 403 (3) ◽  
pp. 451-461 ◽  
Author(s):  
Sandrine Pacquelet ◽  
Jennifer L. Johnson ◽  
Beverly A. Ellis ◽  
Agnieszka A. Brzezinska ◽  
William S. Lane ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nadph Oxidase ◽  
P38 Mapk ◽  
Interleukin 1 ◽  
Mitogen Activated Protein Kinase ◽  
Free System ◽  
Mitogen Activated Protein ◽  
A Cell ◽  
Tlr4 Activation

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

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