Analysis of actinomycin D treated cattle oocytes and their use for somatic cell nuclear transfer

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• 作者：Marcelo Tigre Moura ; Regivaldo Vieira de Sousa ; Ligiane de Oliveira Leme ; Rodolfo Rumpf
• 关键词：Oocyte ; Enucleation ; Actinomycin D ; Nuclear Transfer
• 刊名：Animal Reproduction Science
• 出版年：2008
• 出版时间：December 2008
• 年：2008
• 卷：109
• 期：1-4
• 页码：40-49
• 全文大小：280 K

文摘

The present work aimed to evaluate the transcription and replication inhibitor, actinomycin D, for oocyte chemical enucleation. Cattle oocytes matured in vitro were treated with actinomycin D according to the following treatments: T1, control; T2 = 1.0 μg/ml for 16 h; T3 = 1.0 μg/ml for 14 h; T4 = 2.5 μg/ml for 14 h; T5 = 5.0 μg/ml for 14 h. The oocytes were denuded and activated during 24–26 h of maturation. Oocytes were fixed to determine the maturation status and for chromosome morphology evaluation. Furthermore, oocytes treated with actinomycin D were used for somatic cell nuclear transfer (SCNT). Parthenogenetic and SCNT embryos were fixed to evaluate the percentage of apoptotic nuclei by the TUNEL assay. 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 = 1.8 % ; T3 = 1.3 % ; T4 = 0.9 % ; T5 = 0.0 % ) after actinomycin D treatment were significantly different. There was a significant chromosome uncoiling when treated with greater concentrations (2.5 and 5.0 μg/ml). After SCNT, the cleavage rate (61.3 % ) was similar to the actinomycin D-treated control group (61.3 % ) and less than the non-treated control (70.2 % ), although the blastocyst rate was greater in the SCNT group (11.8 % ) comparing with the treated control (3.6 % ) and less than the untreated control (38.0 % ). Treated parthenogenetic embryos had more apoptotic cells than the parthenogenetic controls (24.2 % compared with 4.8 % ). However, the SCNT group using treated cytoplasts was similar from the SCNT control (9.3 compared with 13.0 % ). Actinomycin D treatment was efficient in blocking embryonic development. Moreover, it was possible to obtain reconstructed embryos that possess an apoptotic cell index indistinguishable from controls.