PARENT SESSION
Preimplantation Embryo/Fetal Development
173
USE OF METAPHASE I PORCINE CYTOPLAST FOR RECONSTRUCTION OF EMBRYOS BY NUCLEAR TRANSFER.
Rzucidlo, S. Jacek1, Miyoshi, Kazuchika1,2, Arat, Sezen1,3, Gibbons, John1, Stice, Steven1, 1 2 3
ABSTRACT- The potential of metaphase (M) I porcine cytoplast to reprogram transferred somatic cell nucleus and its ability to support embryo development to the blastocyst stage were examined. MI and MII oocytes were enucleated and confluent (CON) or nocodazole-treated (NOC) fibroblasts were introduced into the perivitelline space. After fusion, reconstructed MI embryos were cultured in the in vitro maturation medium with or without cytochalasin B until the activation. MI and MII reconstructed embryos were activated simultaneously and then cultured in G1.2 medium. Embryos reconstructed with CON cells were cultured for 2 h after activation in G1.2 medium supplemented with cytochalasin B. The chromatin configuration was examined before (MI only) and 6 h after activation (MI and MII) in a subset of reconstructed embryos. Remaining embryos continued to be cultured and transferred into G2.2 medium at 72 h after culture. Before activation, condensed chromatin was observed in 85.4 ± 3.5% and 54.9 ± 6.8 %of MI oocytes reconstructed with CON fibroblasts (MI+CON) and MI oocytes reconstructed with NOC fibroblasts (MI+NOC), respectively. In 9.0 ± 5.9% of MI+NOC condensed chromatin with one polar body was observed. Swollen nucleus was observed in only 5.4 ± 2.9% and 8.3 ± 8.3% of MI+CON and MI+NOC, respectively. After activation, swollen nucleus was present in 53.7 ± 8.1%, 25.1 ± 9.0%, 90.6 ± 4.8%, and 30.1 ± 5.8% of MI+CON, MI+NOC, MII+CON, and MII+NOC reconstructed oocytes, respectively. Swollen nucleus with polar body was present in 6.1 ± 6.1% of MI+NOC and 23.3 ± 18.6% of MII+NOC. Regardless of the type of donor cell used, the cleavage rate of embryos reconstructed using MII cytoplast was significantly higher than those reconstructed from MI cytoplast (26.7 ± 3.0% vs. 8.0 ± 2.6%, p<0.05). Proportion of blastocysts in MI+CON (0.4 ± 0.4%) was lower than in MII+CON (7.6 ± 0.5%), (p<0.05). Regardless of type of cytoplast used, no blastocysts were produced using NOC fibroblasts in contrast to 4.0 ± 0.3% (p<0.05) of blastocysts reconstructed from CON cells. Our data indicate that the porcine MI cytoplast has the potential to reprogram the donor nucleus and to support the development of reconstructed embryos to the blastocyst stage; however the development was significantly compromised in comparison to the MII reconstructed embryos.
KEY WORDS: pig oocytes, nuclear transfer, reprograming, metaphase I