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.

Enhanced developmental potential of heat-shocked porcine parthenogenetic embryos is related to accelerated mitogen-activated protein kinase dephosphorylation

2009 ◽  
Vol 21 (7) ◽  
pp. 892 ◽  
Author(s):  
S. Clay Isom ◽  
Randall S. Prather ◽  
Edmund B. Rucker III
Keyword(s):  
Heat Shock ◽  
Protein Kinase ◽  
Elevated Temperatures ◽  
Mitogen Activated Protein Kinase ◽  
Oocyte Activation ◽  
Developmental Potential ◽  
Mapk Activity ◽  
Mitogen Activated Protein ◽  
Parthenogenetic Embryos

Recently, we demonstrated that a 9-h heat shock of 42°C can have marked stimulatory effects on porcine parthenogenetic embryo development if applied immediately after oocyte activation. Developmental discrepancies between heat-shocked (HS) and non-HS embryos were manifest as early as 3 h after activation, suggesting involvement of maturation promoting factor (MPF) and/or mitogen-activated protein kinase (MAPK). Analysis of cdc2 kinase activity showed that MPF inactivation occurred at similar rates in HS and control embryos upon oocyte activation. However, MAPK dephosphorylation was accelerated in HS embryos compared with controls. Okadaic acid, a protein phosphatase inhibitor, maintained MAPK activity at high levels in both non-HS and HS embryos and sensitised HS embryos to the effects of elevated temperatures. No increase in heat shock proteins was observed in pronuclear-stage HS embryos. These data suggest that the acceleration of development observed in HS porcine parthenogenetic embryos is associated with a precocious inactivation of the MAPK signalling cascade. The faster cleavage divisions observed in HS embryos may be linked physiologically to their enhanced developmental potential in vitro.

Mitogen-activated protein kinase regulates normal transition from metaphase to interphase following parthenogenetic activation in porcine oocytes

Zygote ◽  
2001 ◽  
Vol 9 (1) ◽  
pp. 15-23 ◽  
Author(s):  
Hideki Tatemoto ◽  
Norio Muto
Keyword(s):  
Protein Kinase ◽  
Time Course ◽  
Polar Body ◽  
Specific Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Cellular Functions ◽  
Mapk Activity ◽  
Mitogen Activated Protein ◽  
Second Polar Body

The decrease in maturation-promoting factor (MPF) activity precedes that in mitogen-activated protein kinase (MAPK) activity after egg activation, but the cellular functions of this delayed inactivation of MAPK are still unclear. The present study was conducted to examine the essential role of MAPK activity for supporting the transition from metaphase to interphase in porcine oocytes matured in vitro. The increases in the phosphorylated forms of MAPK and the activities of MAPK and histone H1 kinase (H1K) were shown in oocytes arrested at the metaphase II (MII) stage. After additional incubation of MII-arrested oocytes in medium with added U0126, a specific inhibitor of MAPK kinase, 24% of oocytes completed the second meiotic division and underwent entry into interphase with pronucleus (PN) formation, but not second polar body (PB-2) emission. The intensities of the phosphorylated forms of MAPK and the activities of MAPK and H1K in matured oocytes treated with U0126 were significantly decreased by the treatment with U0126. Electrostimulation to induce artificial activation caused both H1K and MAPK inactivation; the inactivation of H1K preceded the inactivation of MAPK and sustained high levels of MAPK activity were detected during the period of PB-2 emission. However, the time sequence required for MAPK inactivation was significantly reduced by the addition of U0126 to the culture medium following electrostimulation, resulting in the dramatic inactivation of MAPK distinct from that of H1K. In these oocytes, PB-2 emission was markedly inhibited but little difference was found in the time course of PN formation compared with oocytes not treated with U0126. These findings suggest that the decrease in MAPK activity is partly involved in driving matured oocytes out of metaphase to induce PN development, and that the delayed MAPK inactivation after the onset of MPF inactivation in activated oocytes has a crucial role for PB-2 emission to accomplish the transition from meiosis to mitosis.

A role for MAP kinase in regulating ectodomain shedding of APLP2 in corneal epithelial cells

2001 ◽  
Vol 281 (2) ◽  
pp. C603-C614 ◽  
Author(s):  
Ke-Ping Xu ◽  
Driss Zoukhri ◽  
James D. Zieske ◽  
Darlene A. Dartt ◽  
Christian Sergheraert ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Culture Medium ◽  
Kinase Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial ◽  
Mapk Activity ◽  
Mitogen Activated Protein

We previously reported an increased secretion of amyloid precursor-like protein 2 (APLP2) in the healing corneal epithelium. The present study sought to investigate signal transduction pathways involved in APLP2 shedding in vitro. APLP2 was constitutively shed and released into culture medium in SV40-immortalized human corneal epithelial cells as assessed by Western blotting, flow cytometry, and indirect immunofluorescence. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) caused significant increases in APLP2 shedding. This was inhibited by staurosporine and a PKC-ε-specific, N-myristoylated peptide inhibitor. Epidermal growth factor (EGF) also induced APLP2 accumulation in culture medium. Basal APLP2 shedding as well as that induced by PMA and EGF was blocked by a mitogen-activated protein kinase (MAPK) kinase inhibitor, U-0126. Our results suggest that MAPK activity accounts for basal as well as PKC- and EGF-induced APLP2 shedding. In addition, PKC-ε may be involved in the induction of APLP2 shedding in corneal epithelial cells.

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.

scholarly journals Identification of Functional MKK3/6 and MEK1/2 Homologs from Echinococcus granulosus and Investigation of Protoscolecidal Activity of Mitogen-Activated Protein Kinase Signaling Pathway Inhibitors In Vitro and In Vivo

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Chuanshan Zhang ◽  
Jing Li ◽  
Tuerganaili Aji ◽  
Liang Li ◽  
Xiaojuan Bi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Echinococcus Granulosus ◽  
Sensu Stricto ◽  
Mitogen Activated Protein Kinase ◽  
Content Type ◽  
Mapk Cascades ◽  
Significant Difference ◽  
Mitogen Activated Protein

ABSTRACT Cystic echinococcosis is a zoonosis caused by the larval stage of Echinococcus granulosus sensu lato. There is an urgent need to develop new drugs for the treatment of this disease. In this study, we identified two new members of mitogen-activated protein kinase (MAPK) cascades, MKK3/6 and MEK1/2 homologs (termed EgMKK1 and EgMKK2, respectively), from E. granulosus sensu stricto. Both EgMKK1 and EgMKK2 were expressed at the larval stages. As shown by yeast two-hybrid and coimmunoprecipitation analyses, EgMKK1 interacted with the previously identified Egp38 protein but not with EgERK. EgMKK2, on the other hand, interacted with EgERK. In addition, EgMKK1 and EgMKK2 displayed kinase activity toward the substrate myelin basic protein. When sorafenib tosylate, PD184352, or U0126-ethanol (EtOH) was added to the medium for in vitro culture of E. granulosus protoscoleces (PSCs) or cysts, an inhibitory and cytolytic effect was observed via suppressed phosphorylation of EgMKKs and EgERK. Nonviability of PSCs treated with sorafenib tosylate or U0126-EtOH, and not with PD184352, was confirmed through bioassays, i.e., inoculation of treated and untreated protoscoleces into mice. In vivo treatment of E. granulosus sensu stricto-infected mice with sorafenib tosylate or U0126-EtOH for 4 weeks demonstrated a reduction in parasite weight, but the results did not show a significant difference. In conclusion, the MAPK cascades were identified as new targets for drug development, and E. granulosus was efficiently inhibited by their inhibitors in vitro. The translation of these findings into in vivo efficacy requires further adjustment of treatment regimens using sorafenib tosylate or, possibly, other kinase inhibitors.

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