The availability of cow ovaries from the slaughterhouse has been very limited in Taiwan. To maximize the use of cow ovaries for research purposes, whole ovary dissection was performed and the developmental competence of the oocytes derived from different sizes of follicles was assessed by the rates of in vitro maturation (IVM) and parthenogenetic activation of the oocytes in Experiment 1 (Exp 1). Cumulus-oocyte complexes (COCs) derived from small (1-2 mm) and large (3-8 mm) follicles were subjected to standard IVM culture for 24 h. Mature oocytes were selected and then parthenogenetically activated using A23187 (5 microm, 5 min) or thimerosal (200 microm, 10 min) alone or combined with 6-dimethylaminopurine (2.5 mm and 3.5 h, respectively). Activation rates of the oocytes, neither from the large nor small follicles, were affected by different activation treatments (single or combined stimuli). Whereas maturation rates for the oocytes from large follicles were superior to those from small follicles in both the single (59% vs 45%) and combined treatments (76% vs 40%; p < 0.05). To understand how prolonged heat shock (HS) influences cytoskeletal configurations of mature bovine oocytes, in Experiment 2 (Exp 2), matured oocytes derived from large follicles were randomly allocated to different durations of HS treatments at 41.5 degrees C for 0 (C0h, control, n = 12), 1 (HS1h, n = 28), 2 (HS2h, n = 31), and 4 h (HS4h, n = 30). An additional control group was cultured for 4 h without HS (38.5 degrees C, 4 h, n = 35). Alterations in nuclear structures, microtubules (MTs), and microfilaments (MFs) of the oocytes were examined. Abnormalities in the chromosomes, spindle MTs and the percentages of oocytes with cytoplasmic MTs increased with time of HS treatment. The intensity of the MF distribution in the HS oocytes was also altered. Significant changes in the cytoskeleton after HS may be associated with the reduced development under hyperthermia and, perhaps, with the low pregnancy rates of the animals during hot seasons.

Synchronization of the cell cycle stages in G0/G1 phase is one of the key factors determining the success of nuclear transplantation. Serum deprivation, contact inhibition and chemical inhibitors are widely used methods for this purpose. In this study, cell cycle stages of foetal fibroblasts and cumulus cells were determined using flow cytometry [fluorescence-activated cell scan (FACS)]. Foetal fibroblasts (in vitro cultured for 72-120 h) and fresh cumulus cells were analysed in Experiment 1. Fifty to 55% proliferating fibroblasts remained in G0/G1 phase compared with 78% in confluent culture (p <0.05). In contrast to foetal fibroblasts, fresh cumulus cells maintained 90% of the population in the G0/G1 stage. When serum was retrieved from the proliferating fibroblasts from day 1 to day 5 (Experiment 2), proportions of G0/G1 cells increased from the initial ratio of 53 to 87% at day 4 of starvation, which was significantly higher than the non-starved proliferating cells (p <0.05). In Experiment 3, fibroblasts were treated with aphidicolin (0.1 microg/ml, 6 h), demicolcine (0.5 microg/ml, 10 h), or a combination of these two chemicals to synchronize the cell cycle stages. Surprisingly, no differences or significantly lower in the proportions of G0/G1)phase cells were detected (25-50%) compared with the uncontrolled growing cells (53%). These results suggested that fresh cumulus cells rest their cell cycle in G0/G1 stage. Serum deprivation became effective in the first 24 h and reached the highest proportions during days 4-5 after deprivation. Chemical synchronization of the cell cycle stage of rabbit foetal fibroblasts to G0/G1 phase appeared less effective compared to serum deprivation.

In Expt 1, goat antisera against rabbit blastocysts were induced using spleen cell injection and skin-graft for immunosurgical isolation of ICM cells. Goats received rabbit spleen cell suspension (4 x 10(8) cells/ml) intravenously once a week for three consecutive weeks, plus an additional dose (boost injection) 10 days after the third injection, or a piece of rabbit skin (3 x 3 cm) transplantation. Blood samples were collected starting from the day after the last cell injection for 21 days. Serum was separated, heat inactivated and stored in frozen condition before titre analysis. Results showed that the antisera/antibodies derived by spleen cell injection reached their peak titre 7 days after the last cell injection, compared with 5 days by the skin-grafted group. In Expt 2, morphologically normal blastocysts were collected for isolating ICMs immunosurgically or for direct culture of zona-free whole blastocysts. In both methods, ICM cells started attaching to the feeder layer and outgrowing from the centre portion of the cells on day 3 after the onset of culture. ICM outgrowths increased in size during days 4-5, and most cells differentiated morphologically after day 6. One colony derived from isolated ICM developed into morphologically ES-like cells expressing alkaline phosphatase activity. Our results indicated that both skin-grafting and spleen cell injection were effective inducing antisera against rabbit embryonic cells. More studies are required to optimize the culture system for rabbit ES cells.

No abstract

Most studies of mouse cloning successfully achieved activation of the reconstructed oocytes by strontium (Sr) combined with cytochalasin B (CB) treatment. A protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), was used to inhibit the activity of maturation promoting factor for activation of oocytes, but it has never been successfully applied in mouse cloning. This study investigates the activation efficiency of 6-DMAP in mouse somatic cell nuclear transfer (SCNT). Higher parthenogenetic blastocyst rates (71-72%, p < 0.05) were achieved in the oocytes treated with Sr6D (10 mM Sr combined with 2 mM 6-DMAP for 4 h) and Sr6D + SrCB (Sr6D for 2 h then Sr combined with 5 mug/ml CB for another 2 h), and a higher rate of hatching and hatched blastocyst was observed in the Sr6D + SrCB group (31%, p < 0.01) compared with other treatment groups (1-8%). For mouse cloning, cumulus cells of enhanced green fluorescent protein (EGFP)-expressed ESC chimera F1 were used as donor nuclei. Following activation, better development of the cloned embryos was observed in Sr6D + SrCB treatment. Moreover, different media, i.e. KSOM-AA, MEM-alpha and MK, for culturing cloned embryos were also compared in this study. Better morula/blastocyst (40%) and blastocyst (29%) rates were achieved in the embryos cultured in MEM-alpha medium (p < 0.05). Consequently, four EGFP cloned mice were generated in the activation treatment containing 6-DMAP following embryo transfer. In conclusion, treatment with 6-DMAP in combination with other activation stimuli successfully activates mouse reconstructed oocytes and support full-term development of the transgenic SCNT cloned embryos.