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Abstract Pronucleus microinjection is a commonly used method for the preparation of transgenic mice. The influence of its injection process may be one of the main reasons for low developmental capacity of the pronucleus microinjected embryos. And that the cytoskeleton system is closely related with the pronucleus migration and early cleavage, etc. To observe the dynamic changes of α-tubulin and F-actin, during the embryonic development in KM mouse(Mus musculus) embryos after pronucleus microinjection, and lay the foundation for improving the developmental capacity of the pronucleus microinjected embryos, immunofluorescence and laser confocal microscopy were used to detect the location of α-tubulin and F-actin in the first meiotic division of the microinjected zygotes. The results indicated that: (1)The survival rate was (75.11±2.10)%, the cleavage rate was (79.70±1.75)%, the blastocyst rate was (44.85±3.21)%, and those were highly significant lower (P<0.01) than that of the control group(the cleavage rate: (79.70±1.75)%, the blastocyst rate: (44.85±3.21)% of the microinjected zygotes. (2)At the pronucleus stage of the microinjected zygotes, α-tubulin got a diffuse distribution. Meanwhile, there was no obviously cytaster in cytoplasmic, and fibrous α-tubulin didn't concentrate around the two pronuclei. During the stage of pronucleus migration and fusion, fibrous α-tubulin gradually concentrated around the two pronuclei, and the midbody dissolved gradually. At the beginning of the first mitosis, α-tubulin participated in the formation of spindle. At the 2-cell stage, α-tubulin concentrated in the midbody at the junction of the two blastomeres. At the pronucleus stage of the control mouse zygotes, fibrous α-tubulin composed midbody and cytaster remodeling around male and female pronucleus to pull the pronuclei to get close. With further development, the distribution of α-tubulin had no significant differences from the stage of pronucleus migration and fusion until the 2-cell stage. (3)At the pronucleus stage of the microinjected zygotes, F-actin distributed around the two pronuclei and the cortex regions. Following the pronucleus migration and fusion, F-actin distributed in cytoplasmic gradually decreased, obviously tended to concentrated around the two pronuclei and the cortex regions. At the beginning of the first mitosis, F-actin distributed in cytoplasmic almost disappeared, while the distribution of F-actin was concentrated in the cortex regions. At the 2-cell stage, F-actin mainly distributed in the cleavage furrow. As for the normal mouse zygotes, F-actin concentrated in the cortex regions before the first mitosis. Following the development, the distribution of F-actin had no significant differences from the first mitosis until the 2-cell stage. Our study indicated that the effects of pronucleus microinjection on the microtubule network mainly concentrated at the pronucleus stage. However, it could recover before the stage of pronucleus fusion, while the effects of pronucleus microinjection on the microfilament network mainly concentrate at the beginning of the first mitosis, moreover, it has no significant effect on the distribution of the cortex regions.
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Received: 29 November 2012
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