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Interspecies somatic cell NT (SCNT) can be used in the conservation of endangered animals but only when there is an abundant source of compatible oocytes and recipients. The objective of this study was to compare the efficiency of intra- and interspecies SCNT in sheep using zona-free embryo reconstruction methods. Skin fibroblasts from either an argali (Ovis ammon) or control (Ovis aries) ram were used as donor cells for SCNT between passages 2 to 5 and following culture in medium containing 0.5% FCS for 4 to 6 days. Single cells were electrically fused to cytoplasts prepared following enucleation of in vitro-matured zona-free metaphase II-arrested oocytes obtained from domestic ewes. In an additional experiment with argali, a double cytoplast (DC-SCNT) procedure was used whereby a second cytoplast was fused to the first reconstruct within 1 h. Reconstructs were artificially activated ~25 h after the start of maturation using ionomycin and 6-DMAP. Zona-free parthenogenote (PG) control oocytes were activated around the same time. In each treatment, 10 to 12 zona-free embryos where cultured in microwells formed in 20-μL drops of modified synthetic oviduct fluid under oil. Half the medium was replaced on Day 3, and developing embryos were transferred to individual 5-μL drops on Day 6. Development on Day 7 was expressed as a percentage of cleaved embryos. Statistical significance was determined using Fisher’s exact test for embryo development and two-tailed t-test for embryo cell numbers. Total embryo development on Day 7 was significantly greater with intraspecies sheep SCNT compared with interspecies argali SCNT (34/157 = 21.7% v. 34/363 = 9.4%, respectively; P

Cloning by somatic cell nuclear transfer (SCNT) forces cells to lose their lineage-specific epigenetic marks and become totipotent again. This reprogramming process often results in epigenetic and transcriptional aberrations that compromise development. Development rates after SCNT can thus serve as a functional assay for genome-wide epigenetic reprogramming. Dolly the sheep, the first mammalian SCNT clone, was derived from a donor cell that was induced into quiescence by serum starvation. We hypothesized that quiescence alters the epigenetic status of donor cells and elevates their reprogrammability. To test this idea, we compared chromatin composition and cloning efficiency of serum-starved quiescent (G0) bovine adult male fibroblasts versus non-starved, diploid G1 controls. Mechanically synchronized G1 cells were generated by manual selection or mitotic shake-off and processed within 3 h post-mitosis. Based on morphological assessment and 5-ethyl-2′-deoxyuridine (EdU) incorporation during continuous labelling, >93% of cells were captured in G1. Using quantitative confocal immunofluorescence microscopy and fluorometric enzyme-linked immunosorbent assay (ELISA), we show that G0 fibroblasts were significantly hypomethylated at lysines (K) of histone 3 (H3), specifically H3K4me3, H3K9me2, H3K9me3, and H3K27me3, but not H3K9me1. They were also significantly hypoacetylated at H3K9 and H4K5, hyperacetylated at H4K12, and unchanged at H4K16 positions. Furthermore, G0 cells significantly down-regulated the nuclear abundance of RNA polymerase II, histone variant H2A.Z, as well as polycomb group proteins EED, SUZ12, PHC1, and RING2. Following NT into metaphase-arrested oocytes, G0 chromatin condensed slower than that of G1 cells, indicating a more relaxed configuration. After 7 days of in vitro culture, H3K9me3, but not H4K4me3, H3K27me3, SUZ12, and RING2, remained hypomethylated in G0- versus G1-derived NT blastocysts, both in the inner cell mass and trophectoderm (730 v. 550 nuclei from 55 v. 42 G0 v. G1 blastocysts, respectively; n = 7 NT runs). Reduced H3K9me3 levels correlated with significantly increased mRNA abundance of the H3K9me3-specific histone demethylase KDM4B (or JMJD2B) in NT blastocysts. Expression of other pluripotency-related factors (NANOG, SOX2, STELLA, and IIFITM3), imprinted genes (SNRPN), and histone demethylases (KDM4A) was not affected in G0-derived blastocysts (32 G0 v. 55 G1 blastocysts; n = 4). Following NT, G0 donors developed significantly better into cloned blastocysts (175/382 = 46% v. 122/332 = 37% for G0 v. G1, respectively; n = 7, P