Donor cell type, cell-cycle stage, and passage number of cultured cells all affect the developmental potential of cloned embryos. Because acetylation of the histones on nuclear chromatin is an important aspect of gene activation, the present study investigated the differences in histone acetylation of bovine fibroblast and cumulus cells at various passages and cell-cycle stages. The acetylation was qualitatively analyzed by epifluorescent confocal microscopy and quantitatively by immunofluorescent flow cytometry. Specifically, we studied levels of histone H4 acetylated at lysine 8 and histone H3 acetylated at lysine 18; acetylation at these lysine residues is among the most common for these histone molecules. We also studied levels of linker histone H1 in donor cells. Our results show that stage of cell cycle, cell type, and number of cell passages all had an effect on histone content. Histone H1 and acetyl histone H3 increased with cell passage (passages 5-15) in G0/G1- and G2/M-stage cumulus and fibroblast cells. We also found that acetyl histone H4 was lower in early versus late cell passages (passage 5 vs. 15) for G0/G1-stage cumulus cells. In both cell types examined, acetyl histones increased with cell-cycle progression from G0/G1 into the S and G2/M phases. These results indicate that histone acetylation status is remodeled by in vitro cell culture, and this may have implications for nuclear transfer.

We have previously shown that bovine oocytes parthenogenetically activated after 40 hours (hr) of in vitro maturation proceed through the cell cycle faster than those after 20 hr of maturation. In the present study, we used this model of different speed of nuclear progression to investigate the correlation of two hallmarks of nuclear events, exit of metaphase arrest and pronuclear formation, with dynamics of MPF and MAPK. Bovine oocytes were matured in vitro for 20 hr (young) or 40 hr (aged) and activated in 7% ethanol followed by incubation in cycloheximide for 0, 0.5, 1, 3, 5, or 7 hr. Activity of MPF and MAPK was lower in aged than young oocytes. The responses to oocyte activation by both the two kinases and nuclear progression were faster in aged than in young oocytes. The activity of MPF declined to undetectable levels (P < 0.05) as early as 0.5 hr after activation in aged oocytes, while this did not happen in young oocytes until 3 hr after activation. The inactivation of MAPK occurred approximately 2 hr earlier in aged oocytes (5 hr post-activation) than in young oocytes (7 hr post-activation). Furthermore, the decline in MPF activity preceded that of MAPK in both young and aged oocytes by about 2 hr. The decrease in activity of MPF and MAPK corresponded with the exit from meiosis and pronuclei formation regardless of the speed of nuclear progression. Despite dramatic changes in activity of MPF and MAPK, the levels of Cdc2 and Erk2 proteins were unchanged (P > 0.05) during the first 7 hr of activation. These observations suggest that inactivation of MPF and MAPK are pre-requisite for the release from metaphase arrest and formation of pronuclei in bovine oocytes.

Premature chromosome condensation (PCC) was believed to promote nuclear reprogramming and to facilitate cloning by somatic cell nuclear transfer (NT) in mammalian species. However, it is still uncertain whether PCC is necessary for the successful reprogramming of an introduced donor nucleus in cattle. In the present study, fused NT embryos were subjected to immediate activation (IA, simultaneous fusion and activation), delayed activation (DA, activation applied 4 h postfusion), and IA with aged oocytes (IAA, activation at the same oocyte age as group DA). The morphologic changes, such as nuclear swelling, the occurrence of PCC, and microtubule/aster formation, were analyzed in detail by laser-scanning confocal microscopy. When embryos were subjected to IA in both IA and IAA groups, the introduced nucleus gradually became swollen, and a pronuclear-like structure formed within the oocyte, but PCC was not observed. In contrast, delaying embryo activation resulted in 46.5%-91.2% of NT embryos exhibiting PCC. This PCC was observed beginning at 4 h postcell fusion and was shown as one, two, or multiple chromosomal complexes. Subsequently, a diversity of pronuclear-like structures existed in NT embryos, characterized as single, double, and multiple nuclei. In the oocytes exhibiting PCC, the assembled spindle structure was observed to be an interactive mass, closely associated with condensed chromosomes, but no aster had formed. Regardless of whether they were subjected to IA, IAA, or DA treatments, if the oocytes contained pronuclear-like structures, either one or two asters were observed in proximity to the nuclei. A significantly higher rate of development to blastocysts was achieved in embryos that were immediately activated (IA, 59.1%; IAA, 40.7%) than in those for which activation was delayed (14.2%). The development rate was higher in group IA than in group IAA, but it was not significant (P = 0.089). Following embryo transfer, there was no statistically significant difference in the pregnancy rates (Day 70) between two of the groups (group IA, 11.7%, n = 94 vs. group DA, 12.3%, n = 130; P > 0.05) or live term development (group IA, 4.3% vs. group DA, 4.6%; P > 0.05). Our study has demonstrated that the IA of bovine NT embryos results in embryos with increased competence for preimplantational development. Moreover, PCC was shown to be unnecessary for the reprogramming of a transplanted somatic genome in a cattle oocyte.

1One of the several factors that contribute to the low efficiency of mammalian somatic 2 cloning is the poor fusion between the small somatic donor cell and the large recipient 3 oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity 4 on fusion rate, and subsequent developmental potential of cloned bovine embryos. The 5 toxicity of PHA was established by examining its effects on the development of 6 parthenogenetic bovine oocytes treated with different dosages (Expt 1), and for different 7 durations (Expt 2). The effective dosage and duration of PHA treatment (150 µg/mL, 20 8 min incubation) was selected and used to compare membrane fusion efficiency and 9 embryo development following somatic cell nuclear transfer (Expt 3). Cloning with 10 somatic donor fibroblasts vs. cumulus cells was also compared, both with and without 11 PHA treatment (150 µg/mL, 20 min). Our results showed that the fusion rate of nuclear 12 donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61 % 13 (P<0.05), and from 59 to 88% (P<0.05) with cumulus cell nuclear donors. The nuclear 14 transfer (NT) efficiency per oocyte used was improved following PHA treatment, for 15 both fibroblast (13 vs. 22%), as well as cumulus cell (17 vs. 34%) (P<0.05). The cloned 16 embryos, both with and without PHA treatment, were subjected to vitrification and 17 embryo transfer testing, and resulted in similar survival (approximately 90% hatching) 18 and pregnancy rates (17 to 25%). Three calves were born following vitrification and 19 embryo transfer of these embryos; two from the PHA-treated group, and one from non-20 PHA control group. We conclude that PHA treatment can significantly improve the 21 fusion efficiency of somatic NT in cattle, and therefore, increase the development of 22 cloned blastocysts. Furthermore, within a determined range of dosage and duration, PHA 23 3 has no detrimental effect on embryo survival post vitrification, nor on pregnancy or 1 calving rates following embryo transfer. 2 3 4

The objective of this study was to determine the effect of donor nuclear exposure in MII oocyte cytoplasm on nuclear reprogramming events and subsequent development of cloned embryos in cattle. Somatic nuclear transfer (NT) was performed by electrofusion of the enucleated MII oocytes with cultured cumulus cells by ovum pickup. Donor cell-cytoplast pairs were fused by applying two direct current pulses at 2 kV/cm for 10 μs. Fused NT embryos were randomly divided in to Treatment A (immediate activation) and Treatment B (delayed activation, 4-h exposure in MII cytoplast before activation). In both treatment groups, the activation protocol was identical and consisted of incubation in cycloheximide (10 μg/mL) plus cytochalasin D (2.5 μg/mL) in M199 + 7.5% FBS for 1 h, followed by culture in cycloheximide (10 μg/mL) for an additional 4 h. Reconstructed embryos in both groups were subsequently cultured in CR1aa in 5% CO2, 5% O2, and 90% N2 at 39°C. Samples from both treatments were fixed at 0, 1, 2, 4, 6, 12, 18, 24, 30, 36, and 44 h after fusion. All fixed samples were double stained for tubulin and DNA, and observed with a laser-scanning confocal microscope for changes in nuclei and microtubules. The experiment was replicated three times. Cleavage rate and blastocyst rate were recorded and analyzed by Studen's t-test (SPSS 11.0, Chicago, IL). The staining revealed an absence of premature chromosome condensation (PCC) in all embryos in Treatment A. However, delayed activation (Treatment B) resulted in a high incidence of PCC, probably due to high levels of MPF in the MII cytoplasts. Chromosome condensation was observed in Treatment B at 4 h (82%, n = 17), 6 h (80%, n = 10), 12 h (36%, n = 25), 18 h (71%, n = 24), 24 h (50%, n = 16) and 30 h (6%, n = 18) after fusion. Subsequent culture results of these cloned embryos (Table 1) indicated that there were significantly higher cleavage rates and blastocyst development in Treatment A than in Treatment B. This study clearly demonstrated that PCC is not essential to support bovine cloned blastocyst development. Direct exposure of donor nuclei in a MII cytoplast enviroment for a very short time was sufficient for nuclear reprogramming. Table 1. Effect of donor nuclear exposure duration in MII cytoplasm on the development potential of cloned bovine embryos