Developmental capacity of Bos indicus oocytes after inhibition of meiotic resuption by 6-dimethylaminopurine.
Several reports have suggested that a treatment before in vitro maturation might improve oocyte competence and increase its developmental potential. Therefore, the objectives of the present study were to establish the kinetics of IVM in Zebu oocytes, to assess the effect of 6-dimethylaminopurine (6-DMAP), a phosphorylation inhibitor, on meiotic resumption, and to verify the developmental potential of the blocked oocytes after removal of the inhibitory conditions. To establish the kinetics of in vitro maturation 1422 oocytes were obtained from Nellore cows ovaries and matured in presence and absence of gonadotropins. Samples of oocytes were taken from culture at 0, 6, 9, 12, 15, 18, 21 and 24h, and the oocytes were fixed, stained and evaluated for nuclear morphology. Germinal vesicle break down (GVBD) occurred between 6 and 12h of culture in both groups. By 21h the majority of the oocytes had reached metaphase II in presence (71%) and absence (62%) of gonadotropins. In order to examine the inhibitory effect of 6-DMAP, 585 oocytes were cultured for 12, 18 and 24h in the presence or absence of 2mM of 6-DMAP. At each time point the oocytes were evaluated for nuclear morphology. To test the reversibility of meiotic inhibition 366 oocytes were incubated for 0, 12, 18 and 24h in the presence of 6-DMAP and then were transferred to the maturation medium and cultured for further 24h. A total of 429 oocytes were used to evaluate the developmental potential after meiotic inhibition. The oocytes were cultured in the presence of 6-DMAP for 0, 12, 18 and 24h, and then were matured, fertilized and cultured in vitro. Culture of bovine oocytes in the presence of 6-DMAP up to 24h completely blocked GVBD with more than 90% of the oocytes at GV stage. The inhibitory effect of 6-DMAP was fully reversible since maturation rates were similar (P>0.05) among all treatment groups. The evaluation of embryo development after various periods of meiotic blockage showed that inhibition, regardless the time period, had no effect (P>0.05) on penetration and cleavage rates. However, the proportion of embryos at blastocyst stage was reduced after inhibition for 12 (20.2%), 18 (20.1%) and 24h (19.0%) compared with the control group (35.6%). 6-DMAP has a reversible effect on maintenance of meiotic arrest, but reduced further embryo development.
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Embrapa Gado de Corte, Rodovia BR 262 km 4, Caixa Postal 154, CEP, Campo Grande, MS, Brazil. margot@cnpgc.embrapa.br
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D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D008540:Meiosis / Q000187:drug effects*; D009865:Oocytes / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D006080:Ovarian Follicle / Q000166:cytology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors
D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; C001020:N(6),N(6)-dimethyladenine; D000225:Adenine