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Effects of puromycin and actinomycin D on fertilized rabbit eggs cultured in vitro
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 puromycin- or 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.
Insights
Puromycin and actinomycin D disrupt early rabbit embryo development by inhibiting cell division and DNA synthesis. These findings suggest that newly synthesized proteins are essential for fertilization events.
Area of Science:
- Reproductive biology
- Developmental biology
- Molecular biology
Background:
- Fertilization and early embryonic development involve complex molecular processes.
- Understanding the role of protein synthesis is crucial for comprehending developmental events.
Purpose of the Study:
- To investigate the effects of puromycin and actinomycin D on rabbit zygote fertilization and early development.
- To determine the necessity of nascent protein synthesis for fertilization.
Main Methods:
- Rabbit zygotes were cultured with varying concentrations of puromycin and actinomycin D.
- Inhibition of mitosis, cytokinesis, leucine incorporation, and DNA synthesis was assessed.
- Autoradiography and scintillation counting were used to measure DNA synthesis.
Main Results:
- Puromycin and actinomycin D inhibited mitosis and cytokinesis in fertilized rabbit eggs.
- Nuclear envelope breakdown and chromatin association were impaired by both agents.
- DNA synthesis was reduced in zygotes treated with puromycin and actinomycin D.
Conclusions:
- Nascent protein synthesis appears to be required for key events during fertilization.
- Puromycin and actinomycin D serve as valuable tools for studying protein synthesis requirements in early development.