Ability Of Nuclear Transfer Embryos Research Articles

Improvement of the Developmental Ability of Nuclear Transfer Embryos by Using Blastomeres from In Vitro Fertilized Embryos Selected According to the Early Developmental Stage and Cell Division Status as Donor Cells in Cattle

This study was conducted to improve the developmental ability of nuclear transfer (NT) embryos by using blastomeres from in vitro fertilized (IVF) embryos with high quality as donor cells. The IVF embryos selected at the 2-cell stage at 24-h postinsemination (hpi) and again at the ≥8-cell stage at 48 hpi (Selected-IVF-embryos) showed the highest blastocyst formation rate among embryos. When blastomeres from the Selected-IVF-embryos (Selected-NT group) or Nonselected-IVF-embryos (Non-selected-NT group) were used as donor cells for NT, the blastocyst formation rate in the Selected-NT group (25.6%) was significantly higher than that in the Non-selected-NT group (13.5%). When blastomeres from the Selected-IVF-embryos at 108 (contained many cells before cell division) and 126 hpi (contained many cells immediately after cell division) were used as donor cells for NT (108- and 126-NT groups, respectively), the 126-NT group showed a significantly higher blastocyst formation rate (32.1%) than the 108-NT group (16.8%). Embryo transfer of blastocysts in the 126-NT group showed that 11 of 23 recipients became pregnant; nine calves were obtained. For the NT embryos reconstructed using in vivo derived embryos, 9 of 20 recipients became pregnant; seven calves were obtained. These results indicate that the blastocyst formation rate of NT embryos can be improved by using blastomeres from IVF embryos selected at the early developmental stage, especially immediately after cell division, and that the resultant NT embryos have a high developmental ability to progress to term that is comparable to NT embryos reconstructed using in vivo derived embryos.

59 IN VITRO DEVELOPMENT OF FELINE CLONED EMBRYOS RECONSTRUCTED WITH PREADIPOCYTES

The technique of somatic cell nuclear transfer (NT) in domestic cats is expected to contribute to the conservation of wildcats, for which extinction is a concern. In this study, we examined in vitro developmental ability of cloned embryos produced using the preadipocytes of domestic cats as nuclear donors. Primary cultures of preadipocytes were established as reported previously (Yagi et al. 2004 Biochem. Biophys. Res. Commun. 321, 967–974). Briefly, fat tissue (2–3 g) was excised from an adult female cat and digested using 0.1% collagenase for 1 h at 37�C followed by centrifugation. Only mature adipocytes that were floating near the surface of the supernatant were collected and placed in a 12.5-cm2 culture flask filled with DMEM containing 20% FBS. The flask was filled with medium, tightly capped, and cultured upside down for 7–10 days, so that the floating adipocytes attached to the inner ceiling surface of the flask. When firm attachment of the cells to the ceiling surface of the flask was confirmed, the flask was then inverted and culture was continued using the routine cell culture technique for adherent cells. In vivo-matured oocytes were collected from the ovaries of domestic cats superovulated by eCG and hCG. IVM oocytes were obtained by culturing cumulus–oocyte complexes from the ovaries collected at local veterinary clinics in TCM199-based medium for 24 to 30 h. In vivo-matured and IVM oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm using a bevelled pipette in the presence of 7.5 µg mL–1 cytochalasin B. The nuclei of the donor cells were transferred to enucleated in vivo-matured and IVM oocytes by means of electric fusion (300 V mm–1, 30 µs, twice). The reconstructed embryos were activated electrically (125 V mm–1, 60 µs, twice), followed by treatment with 10 µg mL–1 cycloheximide and 5 µg mL–1 cytochalasin B. The cloned embryos were cultured in vitro for 7 days in MK-1 so that their developmental ability might be examined. The fusion rate of donor cells was similar between in vivo-matured and IVM oocytes (56.8%, 21/37 v. 54.5%, 42/77). The developmental ability of NT embryos reconstructed with in vivo-matured oocytes was similar to that of NT embryos reconstructed with IVM oocytes (cleavage: 52.4%, 11/21 v. 42.9%, 18/42; development to blastocysts: 9.5%, 2/21 v. 11.9%, 5/42). The results indicate that cloned feline embryos reconstructed using preadipocytes can develop in vitro into blastocysts.

Cell confluency is as efficient as serum starvation for inducing arrest in the G0/G1 phase of the cell cycle in granulosa and fibroblast cells of cattle

The cell cycle stage of donor cells is an important factor influencing developmental ability of nuclear transfer embryos. In the present experiment, cumulus and fibroblast cells of cattle were subjected to flow cytometric cell cycle analysis before being used in somatic cloning experiments. The following experimental groups were analyzed for each cell type: (1) actively dividing cells, (2) cells confluent for 4 days, (3) cells starved for 1, 2, 3 or 5 days. Using the propidium iodide flow cytometric assay, there were no significant differences ( P ≥ 0.05) in the percentage of cells in G0/G1 regardless of origin and type of cell, after confluency or serum starvation. Differences with the growing cells were found ( P ≤ 0.01). To determine what subset of cells in G0/G1 were in the G0 subphase of the cell cycle, an immunofluorescence analysis was conducted using monoclonal anti-PCNA antibodies in a FACS assay. There were not statistically significant differences in the percentage of cells that enter G0, between confluent and any starved group for either type of cells. Bovine fibroblast cells, confluent or serum starved for 3 days, were used in nuclear transfer experiments. A slight trend for a more desirable fusion rate in starved cells was detected, and embryo cleavage was greater in starved cells, however, in vitro development to blastocysts was similar between groups. Data indicate that prolonged culture of cells in the absence of serum does not imply a shift in the percentage of cells that enter G0/G1 or G0 alone, and that confluency is sufficient to induce quiescence. This finding can be beneficial in nuclear transfer programs, because there are negative effects such as apoptosis, associated with serum starvation.

Nuclear Transfer in Rats Using an Established Embryonic Cell Line and Cumulus Cells.

The present study was performed to establish a rat cell line from blastocysts and to examine the developmental ability of nuclear transfer embryos reconstituted from the established cells and cumulus cells with enucleated mature oocytes. No colonies of embryo-derived cells were observed when morulae were cultured. In contrast, 89% and 40% of the attached embryos formed colonies when blastocysts and hatched blastocysts were cultured, respectively. When the colonies were subcultured in the absence of feeder cells, a cell line with round cell morphology was obtained. This cell morphology was stable up to at least passage 23. The percentage of these embryo-derived cells at G0/G1 was increased when cells were cultured in medium with 0.5% FCS for 48 and 72 h. Ninety-three percent of fresh, non-cultured cumulus cells were at G0/G1. The percentages of reconstructed embryos survival (44-48%), and developed to the 2-cell stage (35%), were significantly higher when the nuclei of cumulus cells were used than when the nuclei of blastocyst-derived cells were used (29% and 3%). The results of the present study indicate that a rat cell line can be established from blastocysts and maintained in vitro for a long period, and that reconstructed embryos obtained by transferring the blastocyst-derived cell nuclei into enucleated oocytes have poorer development potential than those obtained using cumulus cell nuclei. No reconstructed embryos developed beyond the 2-cell stage in vitro.

Establishment of a porcine cell line from in vitro-produced blastocysts and transfer of the cells into enucleated oocytes.

The present study was conducted to establish a porcine cell line from blastocysts produced in vitro and to examine the developmental ability of nuclear transfer embryos reconstituted with the cells and enucleated mature oocytes. When hatched blastocysts were cultured in Dulbecco's modified Eagle's medium with supplements, no colonies of embryo-derived cells were observed. In contrast, 56% of embryos that were attached to feeder layers of STO cells formed colonies in NCSU-23 with supplements. When the colonies were subcultured in the absence of feeder cells, a cell line with an epithelial-like cell morphology was obtained. This cell morphology was stable up to at least passage 30. Although no fused embryos were observed when a pulse of 100 V/mm was applied, the fusion rate increased significantly at 150 V/mm (28%) and 200 V/mm (64%). At 200 V/mm, 39% of fused embryos cleaved, but no embryos developed beyond the 3-cell stage. When cocultured with electro-activated oocytes, percentages of reconstructed embryos cleaved (65%) and developed to the 4-cell stage (23%) were significantly higher than percentages for those (cleavage: 38%; 4-cell stage: 3%) in the absence of activated oocytes. At 7 days after culture, one reconstructed embryo successfully developed to the blastocyst stage in the presence of activated oocytes. When green fluorescent protein-expressing cells and enucleated oocytes were fused and the fused embryos were cultured with electro-activated oocytes, 3 of 102 reconstructed embryos developed to the blastocyst stage. All of the blastocysts were positive for fluorescent green under ultraviolet light. The results of the present study indicate that a porcine cell line can be established from the hatched blastocyst and maintained in vitro for a long period, and that reconstructed embryos obtained by transferring the blastocyst-derived cells into enucleated oocytes have the ability to develop to the blastocyst stage in vitro.