, Volume 22, Issue 1, pp 61-66

In vivo treatment with cycloheximide or actinomycin D inhibits early embryonic development in rainbow trout (Oncorhynchus mykiss)

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Abstract

Early embryonic development is dependent on proteins translated from messenger ribonucleic acid (mRNA) molecules of maternal origin and the embryonic genome. The present study examined the importance of maternal mRNA during early embryogenesis in the rainbow trout (Oncorhynchus mykiss), and developmentally, when the embryonic genome becomes transcriptionally active. To address the significance of maternal mRNA, eggs were exposed to the translational inhibitor cycloheximide (CHX) for 12 h post-fertilization. Concentrations of CHX beginning at 1 μM significantly delayed the initial stages of cleavage. Exposed embryos had only reached the 4-cell stage, while a significant proportion of the eggs treated with 0.1 μM CHX or no CHX (i.e., control) had attained the 8-cell stage. Treatment of fertilized eggs with the transcriptional inhibitor actinomycin D (AMD) was utilized to demonstrate when synthesis of embryonic mRNA begins. Rainbow trout eggs exposed to 16 μM AMD were unaffected after 12 h, but by 9 days post-fertilization had not developed the embryonic keel characteristic of the controls. To determine when transcriptional inhibition could first be demonstrated AMD exposed embryos were dissected free of the egg, between days 2 and 6 of incubation, and individual blastomeres prepared for diameter measurements. After 3 days of exposure to 16 μM AMD embryonic blastomeres were significantly larger than the control group (P<0.05), which normally decrease significantly in size due to repeated cellular division. This showed that AMD interfered with nuclear transcription preventing the progression of cellular events necessary for normal cleavage, and indicates that transcriptional activation of the embryonic genome in rainbow trout occurs about 3 days post-fertilization.