Mitogen activated protein kinase plays a significant role in metaphase II arrest, spindle morphology, and maintenance of maturation promoting factor activity in bovine oocytes

2001 ◽  
Vol 59 (1) ◽  
pp. 106-114 ◽  
Author(s):  
Ana Carla Gordo ◽  
Chang Li He ◽  
Sallie Smith ◽  
Rafael A. Fissore
Keyword(s):  
Protein Kinase ◽  
Significant Role ◽  
Mitogen Activated Protein Kinase ◽  
Factor Activity ◽  
Mitogen Activated Protein ◽  
Bovine Oocytes ◽  
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 Activities of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are not required for the global histone deacetylation observed after germinal vesicle breakdown (GVBD) in porcine oocytes

Reproduction ◽  
2006 ◽  
Vol 131 (3) ◽  
pp. 439-447 ◽  
Author(s):  
Tsutomu Endo ◽  
Kunihiko Naito ◽  
Sachi Kume ◽  
Yukio Nishimura ◽  
Koji Kashima ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Protein Kinase ◽  
Germinal Vesicle ◽  
Histone Deacetylation ◽  
Mitogen Activated Protein Kinase ◽  
Germinal Vesicle Breakdown ◽  
Immature Oocytes ◽  
Mitogen Activated Protein ◽  
Maturation Promoting Factor

The acetylation of nuclear core histone has been suggested to work as an epigenetic mark for transmitting gene expression patterns to daughter cells. Global histone deacetylations, presumably involved in the reprogramming of the gene expression, have been observed after germinal vesicle breakdown (GVBD) in a cell cycle-dependent manner during meiotic maturation of mouse and porcine oocytes, although the regulation mechanism of histone deacetylation has not been studied well. In the present study, we examined the involvement of a crucial cell-cycle-regulator, maturation-promoting factor (MPF), and a meiosis-related kinase, mitogen-activated protein kinase (MAPK), in the global histone deacetylation during porcine oocyte maturation. In order to know whether the activities of MPF and MAPK were required, or the breakdown of GV membrane was sufficient, for the global histone deacetylation observed after GVBD, we artificially destroyed the GV membrane of the porcine immature oocytes. The artificial GV destruction (AGVD) induced histone deacetylation without the activation of MPF and MAPK. This deacetylation after AGVD was not affected by an MPF inhibitor, roscovitine, or an inhibitor of protein synthesis, cycloheximide, but was completely prevented by an inhibitor of histone deactylases (HDACs), trichostatine A. HDAC1 was present in the GV of the immature oocytes and localized on chromosomes after GVBD and AGVD. These results suggest that the MPF and MAPK activities were dispensable and the breakdown of the GV membrane was sufficient for the global histone deacetylation, which was catalyzed by HDAC activity

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