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Nuclear transfer of bovine embryonic disc cells
Theriogenology
322
NUCLEAR TRANSFER OF BOVINE EMBRYONIC DISC CELLS T. Itoh, Y. Aoyagi, M. Konishi, H. Itakura, T. Takedomi, S. Yazawa and K. Akane Embryo Transfer Laboratory Central Research Institute for Feed and Livestock, ZEN-NOH Tsukuba, Ibaraki 300-42, Japan Embryonic stem cells are valuable in that they can provide a large population of identical cells for use by nuclear transfer to produce clonal offspring. The present study was undertaken to assess the totipotency of bovine embryonic disc (ED) cells by nuclear transfer. Therefore, the development in vitro and survival rate after transfer to recipient cows of bovine embryos reconstructed from two cell types (ED cells and blastomere cells of the morula) were examined. Bovine embryos (Japanese Black cattle) were obtained by IVM/IVF/IVC. Five days after fertilization, a portion of the morula stage embryos were used as donors for conventional nuclear transfer. The others were cultured for one week. Then, inner cell mass cells isolated from blastocysts using calcium ionophore (A23 187) were plated on feeder layers of mitotically inactivated primary mouse embryonic fibroblasts in Dulbecco’s Modified Eagle’s medium containing 20% fetal calf serum. The trophectoderm was manually removed from the ED cells which had attached to the monolayers. Seven days after isolation, ED cells were used as donors for nuclear transfer. In this experiment, the procedures for nuclear transfer and in vitro culture were conducted as described previously (Aoyagi et al., 1994, Theriogenology, 41: 157). The developmental rates to the compacted morula and blastocyst stages were determined at day 6 after nuclear transfer. Thereafter, a portion of the compacted morula and blastocyst stage embryos were transferred to synchronized holstein heifers. Table 1. Develooment of embryos reconstructed from ED cells and blastomere cells of the morula No. (%) Donor No. No. (%) of No. of No. (%) pregnant pulsed cleaved morulae / blastocysts recipients cell type Day30 Day60 9 33 (30) 14 89 (82) 3 21 (1l)b 10 131 (66)b 4bDifferent superscripts denote significant differences (P < 0.05, Chi-square test), morula
109
7w
ED cell
199
3 (30,
As shown in Table 1, the incidence of embryos reconstructed from ED cells developing to the morulalblastocyst stages was significantly lower than the incidence of embryos reconstructed from blastomeres of the morula. Therefore, further studies am needed to improve the developmental ability of reconstructed embryos derived from ED cells. Among the three ED cell-derived pregnancies, one cow aborted before day 100 of pregnancy, one gave birth to a normal calf. In addition, the third maintain a normal condition at the moment, beyond 100 days. These results demonstrate that bovine ED cells are totipotent by nuclear transfer. In the near future, it appears that the nuclear transfer protocol using ED cells will be useful for obtaining a large number of cloned calves.
322
NUCLEAR TRANSFER OF BOVINE EMBRYONIC DISC CELLS T. Itoh, Y. Aoyagi, M. Konishi, H. Itakura, T. Takedomi, S. Yazawa and K. Akane Embryo Transfer Laboratory Central Research Institute for Feed and Livestock, ZEN-NOH Tsukuba, Ibaraki 300-42, Japan Embryonic stem cells are valuable in that they can provide a large population of identical cells for use by nuclear transfer to produce clonal offspring. The present study was undertaken to assess the totipotency of bovine embryonic disc (ED) cells by nuclear transfer. Therefore, the development in vitro and survival rate after transfer to recipient cows of bovine embryos reconstructed from two cell types (ED cells and blastomere cells of the morula) were examined. Bovine embryos (Japanese Black cattle) were obtained by IVM/IVF/IVC. Five days after fertilization, a portion of the morula stage embryos were used as donors for conventional nuclear transfer. The others were cultured for one week. Then, inner cell mass cells isolated from blastocysts using calcium ionophore (A23 187) were plated on feeder layers of mitotically inactivated primary mouse embryonic fibroblasts in Dulbecco’s Modified Eagle’s medium containing 20% fetal calf serum. The trophectoderm was manually removed from the ED cells which had attached to the monolayers. Seven days after isolation, ED cells were used as donors for nuclear transfer. In this experiment, the procedures for nuclear transfer and in vitro culture were conducted as described previously (Aoyagi et al., 1994, Theriogenology, 41: 157). The developmental rates to the compacted morula and blastocyst stages were determined at day 6 after nuclear transfer. Thereafter, a portion of the compacted morula and blastocyst stage embryos were transferred to synchronized holstein heifers. Table 1. Develooment of embryos reconstructed from ED cells and blastomere cells of the morula No. (%) Donor No. No. (%) of No. of No. (%) pregnant pulsed cleaved morulae / blastocysts recipients cell type Day30 Day60 9 33 (30) 14 89 (82) 3 21 (1l)b 10 131 (66)b 4bDifferent superscripts denote significant differences (P < 0.05, Chi-square test), morula
109
7w
ED cell
199
3 (30,
As shown in Table 1, the incidence of embryos reconstructed from ED cells developing to the morulalblastocyst stages was significantly lower than the incidence of embryos reconstructed from blastomeres of the morula. Therefore, further studies am needed to improve the developmental ability of reconstructed embryos derived from ED cells. Among the three ED cell-derived pregnancies, one cow aborted before day 100 of pregnancy, one gave birth to a normal calf. In addition, the third maintain a normal condition at the moment, beyond 100 days. These results demonstrate that bovine ED cells are totipotent by nuclear transfer. In the near future, it appears that the nuclear transfer protocol using ED cells will be useful for obtaining a large number of cloned calves.