Utilização da actinomicina D como método de enucleação química de ovócitos bovinos destinados à transferência nuclear

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

Cloning by Nuclear Transfer (NT) is a low efficiency and labor intense technique. The process, in technical terms, is one of the main factors responsible for both embryo quality and quantity. Enucleation is the most aggressive step. Although alternative methods have been proposed, it remains mainly as it has been first described in the early fifties. The objective of the present work was to evaluate the effect of transcription and replication inhibitor actinomycin D as a chemical enucleation method for oocytes. Oocytes were matured in vitro (MIV) for 4 or 6 hours and exposed to actinomycin D (T1, control; T2 = 1,0 μg ml- / 16hs; T3 = 1,0 μg ml- / 14hs; T4 = 2,5 μg ml- / 14hs; T5 = 5,0 μg ml- / 14hs). The oocytes were denuded 20 hours after MIV and activated 24-26 hours after MIV. The cleavage rate was recorded 48 hours post activation and the blastocyst at 168 (D7) and 192 (D8) hours of culture. Some oocytes were fixed and stained with lacmoyd or orcein to determine the maturation status or for chromosome morphology evaluation, respectively. Furthermore, oocytes treated with actinomycin D were used as recipient cytoplasts for NT, while some embryos (D3) were fixed to evaluate the percentage of apoptotic nuclei by the TUNEL assay. The data relative to the maturation, cleavage and blastocyst rate were analyzed by the chi-square test and apoptosis percentages by the Mann-Whitney test. The maturation (T1 = 90,4%; T2 = 82,3%; T3 = 79,1%; T4 = 83,4%; T5 = 74,7%), cleavage (T1 = 68,9%; T2 = 46,0%; T3 = 49,7%; T4 = 33,4%; T5 = 29,3%) and blastocyst rate at D8 (T1 = 41,1%; T2 = 1,4%; T3 = 1,3%; T4 = 0,9%; T5 = 0,0%) after actinomycin D treatment was significantly different. Analyzing the fixed oocytes, 63,2% were in MII. While evaluating the chromosomes, it was observed a significant uncoiling when exposed to higher concentrations (2,5 and 5,0 μg ml-). The remaining groups had no apparent perceptible modifications. Furthermore, oocytes treated with 1 μg ml- for 14 hours were used as recipient cytoplasts. The cleavage rate (61,3%) was similar to the actinomycin D treated control group (61,3%) and lower than the non-treated control (70,2%), although the blastocyst rate was higher in the NT group (11,8%) comparing to the treated control (3,6%) and lower to the untreated control (38,0%). After analyzing the embryos for apoptotic cells, the treated parthenogenetic embryos had a higher index than the parthenogenetic control (24,2% vs. 4,8%). However, the NT embryos generated by treated cytoplasts wasnt statistically different from the NT control (9,3 vs. 13,0%). The actinomycin D treatment is efficient to block embryonic transcription and replication. Moreover, it was possible to obtain reconstructed embryos that possess an apoptotic cell index indistinguishable from the control.

ASSUNTO(S)

nuclear transfer. bovino actinomycin d reproducao animal actinomicina d transferência nuclear enucleação oocyte enucleation bovine ovócito

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