Effects of puromycin and actinomycin D on fertilized rabbit eggs cultured in vitro

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Abstract
Rabbit zygotes were cultured in media containing varying concentrations of puromycin and actinomycin D in order to determine what effect these agents have on events of fertilization and early development. Concentrations of puromycin greater than 10−5 M inhibited mitosis, cytokinesis of fertilized eggs and leucine incorporation. In approximately 50% of the zygotes treated with puromycin (10−5 M or greater) the female pronucleus remained at its site of formation, the cortex, and did not migrate centrad. In these instances the male pronucleus moved from its site of development to the female pronucleus. Concentrations of actinomycin D greater than 10−7 M also inhibited mitosis and cytokinesis of fertilized eggs. Breakdown of the nuclear envelopes and association of the paternally‐ and maternally‐derived chromatin did not occur in either puromycinor actinomycin D‐treated embryos. Actinomycin D at all of the concentrations employed (10−9 to 10−4 M) had little effect upon the structure of nucleolus‐like bodies within male and female pronuclei. Male and female pronuclei of puromycin (10−4 M) and actinomycin D (10−5 M) treated zygotes, incubated with tritiated thymidine and prepared for scintillation counting and autoradiography, synthesized DNA at a reduced level when compared to control specimens. Although “side effects” of puromycin and actinomycin D treatment are not excluded, these results suggest that nascent proteins may be required for events of fertilization.