Contrasting effects of G1.2/G2.2 and SOF1/SOF2 embryo culture media on pre- and post-implantation development of non-transgenic and transgenic cloned goat embryos.

This study compared the efficiency of two embryo culture media (SOF1/SOF2 and G1.2/G2.2) for pre- and post-implantation development of somatic cell nuclear transfer goat embryos derived from non-transgenic and transgenic (for htPA and hrcfIX genes) fibroblasts. Despite similar cleavage rates, G1.2/G2.2 supported significantly higher blastocyst development than SOF1/SOF2 (30-35% versus 21%; P < 0.05), irrespective of cell transgenesis. However, following embryo transfer, pregnancy outcomes (establishment, full-term development and live birth) were all significantly higher (P < 0.05) for embryos developed in SOF1/SOF2 versus G1.2/G2.2. Gene expression profiling of 17 developmentally important genes revealed that: (i) SOX2, FOXD3, IFNT, FZD, FGFR4, ERK1, GCN5, PCAF, BMPR1, SMAD5, ALK4, CDC25 and LIFR were significantly induced in blastocysts developed in SOF1/SOF2 but not G1.2/G2.2; (ii) OCT4, CTNNB and CDX2 were similarly expressed in both groups; and (iii) AKT was significantly higher in G1.2/G2.2 than SOF1/SOF2 (P < 0.05). Following IVF, although blastocyst development in G1.2/G2.2 was significantly higher than SOF1/SOF2 counterparts, the majority of assessed genes were similarly expressed in blastocysts developed in both groups. It was concluded that the long-term programming effects of embryo culture medium and/or embryo production method may irreversibly affect post-implantation development of cloned embryos through defined molecular pathways.

Improved in vitro development of cloned bovine embryos using S-adenosylhomocysteine, a non-toxic epigenetic modifying reagent.

In this study, fibroblast cells were stably transfected with mouse POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) to investigate the effect of S-adenosylhomocysteine (SAH), the reversible non-toxic inhibitor of DNA-methyltransferases (DNMTs), at different intervals post-fusion on in vitro development of cloned bovine embryos. Treatment with SAH for 12 hr resulted in 54.6 ± 7.7% blastocyst production, which was significantly greater than in vitro fertilized embryos (IVF: 37.2 ± 2.7%), cloned embryos treated with SAH for 72 hr (31.0 ± 7.6%), and control cloned embryos (34.6 ± 3.6%). The fluorescence intensities of the EGFP-POU5F1 reporter gene at all intervals of SAH treatment, except of 72 hr, were significantly higher than control somatic cell nuclear transfers (SCNT) embryos. The intensity of DNA-methylation in cloned embryos treated with SAH for 48 hr was similar to that of IVF embryos, and was significantly lower than the other SCNT groups. The levels of H3K9 acetylation in all SCNT groups were significantly lower than IVF embryos. Real-time PCR analysis of gene expression revealed significantly higher expression of POU5F1 in cloned versus IVF blastocysts. Neither embryo production method (SCNT vs. IVF) nor the SAH treatment interval affected expression of the BCL2 gene. Cloned embryos at all intervals of SAH treatment, except for 24 hr, had significantly increased VEGF transcript compared to IVF and control SCNT embryos. It was suggested that the time interval of DNMT inhibition may have important consequences on different in vitro features of bovine SCNT, and the improving effects of DNMT inhibition on developmental competency of cloned embryos are restricted to a specific period of time preceding de novo methylation.

Epigenetic modification does not determine the time of POU5F1 transcription activation in cloned bovine embryos.

PURPOSE: To investigate the effect of epigenetic modification on pattern, time and capacity of transcription activation of POU5F1, the key marker of pluripotency, in cloned bovine embryos. METHODS: Bovine fibroblasts were stably transfected with POU5F1 promoter-driven enhanced green fluorescent protein (EGFP). This provided a visible marker to investigate the effect of post-activation treatment of cloned bovine embryos with trichostatin A (TSA) on time and capacity of POU5F1 expression and its subsequent effect on in vitro development of cloned bovine embryos. RESULTS: Irrespective of TSA treatment, POU5F1 expression was not detected until 8-16 cell stage, but was detected in both inner cell mass and trophectoderm at the blastocyst stage. TSA treatment significantly increased POU5F1 expression, and the yield and quality of cloned embryo development compared to control. CONCLUSION: The POU5F1 expression of cloned embryos is strictly controlled by the stage of embryo development and may not be altered by TSA-mediated changes occur in DNA-methylation and histone-acetylation of the genome.

Sperm status and DNA dose play key roles in sperm/ICSI-mediated gene transfer in caprine.

In relation to the growing recent interest in the establishment of sperm-mediated gene transfer (SMGT) technology as a convenient and effective method for the simple production of transgenic animals, in this study the possibility of using SMGT to produce transgenic caprine embryos was investigated for the first time. Buck sperm were directly incubated with different concentrations (0-500 ng) of pcDNA/his/Lac-Z plasmid and used for IVF or ICSI. Sperm used for ICSI were categorized into motile or live-immotile group before being injected into oocytes. In a separate experiment, dead sperm prepared by repeated freezing/thawing were used for DNA-incubation before ICSI. Sham injection was carried out by intracytoplasmic injection of approximately the same volume of media containing different doses of DNA using an ICSI needle. Transgene expression and transmission were detected by X-Gal staining and PCR analysis of developed embryos, respectively. A reasonable blastocyst rate was observed in all the groups. Only embryos in the sham group were negative for transgene transmission. Transgene expression was completely dependent on the delivery technique and status of sperm, and was only observed in the live-immotile and dead ICSI groups. The results of this study showed that the technique (IVF vs. ICSI vs. sham injection), sperm status (motile vs. live-immotile vs. dead) and to some extent DNA concentration affect embryo development, transgene transmission and expression.

Enucleated ovine oocyte supports human somatic cells reprogramming back to the embryonic stage.

Increased possibility of universality of ooplasmic reprogramming factors resulted in a parallel increased interest to use interspecies somatic cell nuclear transfer (iSCNT) to address basic questions of developmental biology and to improve the feasibility of cell therapy. In this study, the interactions between human somatic cells and ovine oocytes were investigated. Nuclear remodeling events were first observed 3 h post-iSCNT as nuclear swelling, chromosome condensation, and spindle formation. A time-dependent decrease in maturation promoting activity of inactivated reconstructs coincided with increased aberrations in chromosome and spindle organization of the newly developed embryos. The sequence and duration of nuclear remodeling events were irrespective of donor cell type used. Although the majority of the reconstituted embryos arrested before embryonic genome activation (8-16-cell) stage, less than 5% of them could progress beyond transcription-requiring developmental stage and formed blastocyst-like structures with distinct inner cell mass and trophectoderm at days 7 and 8 post-SCNT. Importantly, real-time assessment of three developmentally important genes (Oct4, Sox2, and Nanog) indicated their upregulation in iSCNT blastocysts. Blastocyst-derived outgrowths had alkaline phosphatase activity that was lost upon passage. Collectively, this study introduced ovine oocyte as a credible cytoplast for remodeling and reprogramming of human somatic cells back to the embryonic stage and provided a platform for further studies to unravel possible differences exist between reprogramming ability of oocytes of different mammalian species.

Developmental Competence and Pluripotency Gene Expression of Cattle Cloned Embryos Derived from Donor Cells Treated with 5-aza-2'-deoxycytidine.

BACKGROUND: Reconstructed embryos from terminally differentiated somatic cells have revealed high levels of genomic methylation which results in inappropriate expression patterns of imprinted and non-imprinted genes. These aberrant expressions are probably responsible for different abnormalities during the development of clones. Improvement in cloning competency may be achieved through modification of epigenetic markers in donor cells. MATERIALS AND METHODS: Our objective was to determine if treatment of donor cells for 72 hours with 5-aza-2'-deoxycytidine (5-aza-dc; 0-0.3 µM), a DNA methyl transferase inhibitor, improved development and expression of Oct-4. RESULTS: In comparison with untreated cells, 0.01 and 0.08 µM 5-aza-dc treated cells insignificantly decreased the blastocyst rate (32.1% vs. 28.6% and 27.2%, respectively) while it was significant for 0.3 µM treated cells (6.5%). Embryo quality as measured by the total cell number (TCN) decreased in a dose-related fashion, which was significant at 0.08 and 0.3 µM 5-aza-dc treated cells when compared with 0 and 0.01 µM 5-aza-dc treated cells. Although reconstructed embryos from 0.08 and 0.3 µM 5-aza-dc treated cells showed lower levels of DNA methylation and histone H3 acetylation, development to blastocyst stage was decreased. The epigenetic markers of embryos cloned from 0.01 µM 5-aza-dc remained unchanged. CONCLUSION: These results show that 5-aza-dc is not a suitable choice for modifying nuclear reprogramming. Finally, it was concluded that the wide genomic hypomethylation induced by 5-aza-dc deleteriously impacts the developmental competency of cloned embryos.

Effect of ovarian cyclic status on in vitro embryo production in cattle.

BACKGROUND: The relationship between cyclic status of cattle ovaries on in vitro embryo development up to the blastocyst stage was investigated. MATERIALS AND METHODS: Cattle ovaries were collected immediately after slaughter and divided into three categories based on their cyclic status, which included: 1. the presence of a large follicle (LF), 2. the presence of a corpus luteum (CL) and 3. ovaries without LF or CL (WLCF). Oocytes of these ovaries were obtained and used for in vitro maturation and fertilization. Presumptive zygotes were then cultured up to the blastocyst stage in synthetic oviductal fluid culture medium. RESULTS: There were no significant differences between cleavage rates of the three groups. The rate of embryos in the compact morula stage for the CL group was 48.2% which was significantly higher than the related rate of the LF group (36.6%), but non-significantly higher than that of the ST group (45.7%). The highest blastocyst rate belonged to the CL group (54.6%) which was significantly greater than the WLCF group (32.9%) and non-significantly higher than the LF group (52.4%). There was no significant difference in blastocyst rates in the CL and LF groups. CONCLUSION: Preselection of oocyte donor ovaries containing a CL or LF can be used as a feasible and non- invasive criterion to obtain the most competent oocytes capable of development to the blastocyst stage.

Somatic cell-induced hyperacetylation, but not hypomethylation, positively and reversibly affects the efficiency of in vitro cloned blastocyst production in cattle.

5-Aza-2'-deoxycytidine (AzC), trichostatin A (TSA), and its natural mimetic, sodium butyrate (NaB), are antineoplastic drugs that can modify the epigenetic status of donor cells prior to somatic cell nuclear transfer (SCNT). In this study, we used fibroblast cells treated with these drugs to investigate the direct and indirect effects of induced changes in DNA methylation and acetylation of the lysine 9 residue of histone H3 (H3K9). Additionally, we assayed cellular characteristics (cell growth, cell proliferation, cell cycle progression, and apoptosis) and SCNT efficiency in response to these drugs as well as monitoring these effects 24 h after removing the drugs. We observed the following: (1) AzC, TSA, and NaB all showed dose-dependent effects on different cellular characteristics; (2) TSA and NaB induced H3K9 hyperacetylation accompanied by DNA hypermethylation, whereas AzC induced DNA hypomethylation with no effect on H3K9 hyperacetylation; (3) TSA and NaB improved cloning efficiency, whereas AzC reduced it; and (4) unlike AzC, the effects of TSA and NaB on cellular characteristics and SCNT efficiency were reversed following drug removal. Our results indicate that somatic cells treated with TSA and NaB show better survival and recovery rates following the removal of these drugs. Moreover, H3K9 hyperacetylation (induced with TSA and NaB), but not DNA hypomethylation (induced with AzC), favors cloning efficiency.