Search

Pluripotency relies on species-specific amino acid (AA) metabolism. In the mouse, inner cell mass (ICM) and ICM-derived pluripotent stem cells (PSCs) need threonine, which is catabolized by threonine dehydrogenase (TDH) into acetyl–CoA and glycine. Depleting (Δ) the culture medium of threonine (ΔT) or blocking TDH activity induces PSC death. By contrast, human PSCs do not survive without lysine (ΔK), leucine (ΔL), or methionine (ΔM). Since isolated bovine PSCs cannot be propagated in vitro, we screened for AAs that selectively support pluripotent ICM cells in intact bovine embryos. Five days (D5) post-IVF, embryos were transferred into glutamine-free synthetic oviduct fluid (gSOF) with Eagle’s nonessential (NE) and essential (E) AAs (gSOF_AA) plus BSA. Embryos were individually cultured until D8 under different conditions. Statistical significance was determined using Fisher’s exact test for blastocyst development (morphological grading to IETS standard) and t-tests for cell numbers (differential stain) and gene expression (quantitative or qPCR). Removal of BSA reduced grade 1–3 blastocyst (B1–3) development (37% v. 25%, n = 3; P 10-fold T-supplementation. Thus, 3-HNV protein incorporation, rather than acetyl-CoA reduction, may nonspecifically impair cellular function. In summary, we found that bovine ICM formation did not specifically depend on metabolizing threonine or any other single EAA. Research was supported by AgResearch Core Funding.

The inner cell mass (ICM) of mammalian blastocysts comprises 2 transient lineages, namely hypoblast and epiblast, which develop into extra-embryonic and embryonic tissues, respectively. In the mouse, epiblast cells autocrinally secrete fibroblast growth factor (FGF) to induce hypoblast differentiation, and pharmacological FGF/mitogen-activated protein kinase (MAPK) signal inhibition converts all ICM cells into epiblast. We conducted a chemical screen for additional signal enhancers of epiblast identity in bovine Day 8 blastocysts. From the morula stage onwards, in vitro-fertilised (IVF) embryos were cultured in the presence of 9 small molecule inhibitors, targeting 9 principal signal pathway components. Inhibitors included SB431542, LDN193189, BIBF1120, Forskolin, BI-D1870, A66/TGX 221/ZSTK474, and AZD1480, targeting TGFβ-RI, BMP-RI, VEGFR/PDGFR/FGFR, adenylate cyclase, ribosomal S6 kinase (RSK), PI3K, and JAK2 signalling, respectively. Using (1) blastocyst quality (by morphological grading), (2) cell numbers (by differential stain), and (3) lineage-specific candidate gene expression (by quantitative PCR) as readouts, we sought to identify positive and negative regulators of ICM development and lineage determination. Based on our previous digital mRNA profiling data (McLean et al. 2014 Biol. Reprod., in press), we selected discriminatory epiblast-specific (FGF4, NANOG) and hypoblast-specific (PDGFRα, SOX17) markers for qPCR analysis. Each inhibitor was compared, alone or in combination, to an appropriately diluted dimethylsulfoxide (DMSO) vehicle control in at least 3 biological replicates. Statistical significance was determined using a generalised linear mixed model with binomial distribution and logit link for developmental data and REML for log cell counts and log gene expression data, applying fixed treatment effects and random run and sample within run effects. Blocking TGFβ1-, BMP- or VEGF-/PDGF-/FGF-signalling did not affect blastocyst development, ICM v. trophectoderm (TE) cell numbers, or gene expression. Repression of PI3K signals via AG66 and TGX, but not ZSTK alone, modestly decreased grade 1–2 blastocyst development (P

During blastocyst development, the inner cell mass segregates into the epiblast and the hypoblast. These 2 tissues form morphologically and molecularly distinct cell populations that subsequently develop into the embryo proper and some extraembryonic components, respectively. In mouse, isolated epiblast cells can be directly converted into pluripotent embryonic stem cells, capable of differentiating into all cell types of an adult animal. Epiblast pluripotency is promoted by pharmacological inhibition of mitogen-activated protein kinase kinase (Mapkk). This shields epiblast cells from secreted fibroblast growth factor (Fgf), which would otherwise instruct them to exit pluripotency and differentiate into extraembryonic lineages. Indirect stimulation of the Wnt pathway by inhibiting glycogen synthase kinase 3 (GSK3) further antagonises inductive Fgf/Mapkk signalling. Thus the double inhibition (2i) of Mapkk and Gsk3 effectively promotes pluripotency (Q. L. Ying et al. 2008 Nature 453, 519–523; J. Nichols et al. 2009 Development 136, 3215–3222). We investigated the effect of 2i culture on bovine blastocysts. The IVF embryos were cultured in the presence of dimethyl sulfoxide or inhibitors of MAPKK (0.4 µM PD0325901) and GSK3 (3 µM CHIR99021) from the zygote (Day 1) stage onward. Compared to vehicle controls, 2i increased the abundance of cumulus cells in bovine IVF cultures, compromising blastocyst formation in cumulus-intact (248/823 = 30% v. 211/824 = 26%, respectively, n = 10; P 3-fold increased (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

We hypothesized that reprogramming a somatic cell following NT is a time-dependent process that can be improved by artificially prolonging the first cell cycle of the cloned embryo. Eleven candidate drugs were initially screened for their ability to reversibly delay the onset of the first cleavage in bovine parthenotes without affecting subsequent in vitro embryo development. After identifying the cyclin-dependent kinase inhibitor butyrolactone-1 (BLT1; BIOMOL International, Plymouth Meeting, PA, USA) as a suitable candidate, we determined its optimal concentration and exposure time. We then performed zona-free bovine NT with serum-starved male skin fibroblasts. Commencing 10 h after the start of IVC, reconstructed 1-cell embryos were treated with either 200 μM BLT1 or 0.4% DMSO in SOF culture medium for 8 to 11 h. After thorough washing, cleavage rates were recorded and culture continued until Day 7. Labeling with 5-bromo-2-deoxyuridine (BrdU) was used to determine DNA replication during the first cell cycle. Some embryos were also transferred singularly to recipient cows. Embryo development was analyzed by a generalized linear model with binomial variation and pregnancy rates by Fisher’s exact test. At 0, 2, 4, 6, and 10 h after the start of IVC, 0% (0/28), 8% (5/61), 67% (39/58), 90% (54/60), and 100% (16/16), respectively, of NT reconstructs had incorporated BrdU, indicating that all 1-cell NT embryos were in S-phase at the start of treatment. After 8 to 11 h of incubation in BLT1, only 28% (119/429) of NT embryos had cleaved, compared with 93% of DMSO-treated controls (297/319). After removing BLT1 in those embryos arrested at the 1-cell-stage, there was no BrdU incorporation over the subsequent 1 h (0/17), embryos entered mitosis and by 4 h, 90% had cleaved (86/96). Thus, BLT1-arrested embryos were at a post-replicative stage prior to M-phase. Rates of in vitro embryo development on Day 7, from late morula to expanded blastocyst stages, of either grade 1-3 or grade 1-2 quality, in the BLT1 treatment were not different compared with controls (129/275 = 47% v. 151/309 = 49% and 33% v. 33%, respectively). Nuclei counts in expanded blastocysts from the BLT1 treatment were not significantly different than controls (109 v. 121, n = 31). Embryo survival on Day 35 of pregnancy and for calves to 1 month ofage was also not different between BLT1 and control treatments (13/31 = 42% v. 12/29 = 41% and 6% v. 10%, respectively). In conclusion, treating 1-cell NT embryos in S-phase for 8 to 11 h with 200 μM BLT1 arrested embryos in G2 and delayed cleavage by approximately 6 h. Cell cycle arrest was fully reversible after drug withdrawal, with rates of cleavage and in vitro development comparable to that of controls. The prolongation of the first cell cycle in bovine NT embryos using this method did not, however, increase cloning efficiency. Arrest for longer periods, at other stages of the cell cycle, and using alternative reagents may be beneficial.

Following nuclear transfer (NT), the most stringent measure of extensive donor cell reprogramming is development into viable offspring. This is referred to as cloning efficiency and quantified as the proportion of cloned embryos transferred into surrogate mothers that survive into adulthood. Cloning efficiency depends on the ability of the enucleated recipient cell to carry out the reprogramming reactions (‘reprogramming ability’) and the ability of the nuclear donor cell to be reprogrammed (‘reprogrammability’). It has been postulated that reprogrammability of the somatic donor cell epigenome is inversely proportional to its differentiation status. In order to test this hypothesis, reprogrammability was compared between undifferentiated stem cells and their differentiated isogenic progeny. In the mouse, cells of divergent differentiation status from the neuronal, haematopoietic and skin epithelial lineage were tested. In cattle and deer, skeletal muscle and antler cells, respectively, were used as donors. No conclusive correlation between differentiation status and cloning efficiency was found, indicating that somatic donor cell type may not be the limiting factor for cloning success. This may reflect technical limitations of the NT-induced reprogramming assay. Alternatively, differentiation status and reprogrammability may be unrelated, making all cells equally difficult to reprogramme once they have left the ground state of pluripotency.

Previous studies in the mouse have shown treatment of somatic cell nuclear transfer (SCNT) embryos with histone deacetylase inhibitors (HDACi) to significantly increase cloning efficiency (Kishigami S et al. 2006 BBRC 340, 183–189; van Thuan N 2007 Asian Reproductive Biology Society 4, 9 abst). Increasing histone acetylation may open donor chromatin allowing better access for oocyte cytoplasmic factors to facilitate reprogramming. Here, we determined the effect of two HDACi, Trichostatin A (TSA), and scriptaid (Sigma-Aldrich, Castle Hill, NSW, Australia), on bovine cloning efficiency. Zona-free SCNT was performed with serum starved fibroblasts fused to enucleated MII-arrested IVM oocytes. After 4 h, reconstructs were activated with 5 μm ionomycin and 2 mm 6-dimethylaminopurine (DMAP) and cultured individually in 5 μL drops of AgResearch synthetic oviduct fluid (SOF) medium. Treatment with HDACi commenced concomitant with the 4 h DMAP incubation and continued in SOF for the remainder of the treatment period; totalling either 18 or 48 h post activation (hpa). TSA concentrations examined were: 0, 5, 50, and 500 nm, with all treatments containing 0.5% DMSO (n = 1121). Following TSA treatment, increased histone (H) acetylation at lysine (K) of H4K5 was confirmed by semi-quantitative immunofluorescence at the eight-cell stage. Scriptaid concentrations examined were: 0, 5, 50, 250, and 1000 nm, with all treatments containing 0.5% DMSO during DMAP and 0.1% DMSO during IVC (n = 1059). In vitro development on Day 7 was expressed in terms of transferable quality embryos as a percentage of reconstructs cultured. Data were analyzed using a generalized linear model with binomial variation and logit link. Embryos from selected treatments were transferred singularly to recipient cows on Day 7 with pregnancy data analyzed using Fisher’s exact test. Day 7 in vitro development was significantly greater with 5 nm TSA treatment for 18 hpa compared to controls (47.1% v. 34.5%; P < 0.02). Treatment of embryos with TSA for 48 hpa had no effect at any concentration tested. In contrast, scriptaid treatment for 18 hpa had no effect in vitro, while exposure for 48 hpa at 1000 nm significantly increased the development of transferable quality embryos compared to 0 nm (44.0% v. 32.4%; P < 0.005). There was no significant difference in embryo survival rates at D150 of gestation between embryos treated with 0 or 5 nm TSA for 18 hpa (8/48 v. 10/48; 16.7% v. 20.8%). However, in vivo development at Day 150 of gestation following treatment of embryos with 1000 nm scriptaid for 48 hpa was significantly lower compared to controls (1/37 v. 6/31; 2.7% v. 19.4%; P < 0.05). Contrary to the mouse, TSA or scriptaid treatment as used in this study did not increase cloning efficiency in cattle. The use of various HDACi either alone or in combination with DNA demethylating agents may still prove beneficial for reprogramming following nuclear transfer. Supported by FRST C10X0303.

To produce live cloned mammals from adult somatic cells the nuclei of these cells must be first reprogrammed from a very restricted, cell lineage-specific gene expression profile to an embryo-like expression pattern, compatible with embryonic development. Although this has been achieved in a number of species the efficiency of cloning remains very low. Inadequate reprogramming of epigenetic marks in the donor cells correlated with aberrant embryonic gene expression profiles has been identified as a key cause of this inefficiency. Some of the most common epigenetic marks are chemical modifications of histones, the main structural proteins of chromatin. A range of different histone modifications, including acetylation and methylation, exists and can be attributed to either repression or activation of genes. One epigenetic mark which is known to be very stable and difficult to remove during reprogramming is the trimethylation of lysine 9 in histone H3 (H3K9Me3). To test the hypothesis that H3K9Me3 marks are a major stumbling block for successful cloning we are attempting to remove these marks by overexpression of the H3K9Me3 specific histone demethylase, jmjd2b, in donor cells, prior to their use for nuclear transfer. We have engineered mouse embryonic stem (ES) cells for the tet inducible expression of a fusion protein with a functional jmjd2b or non-functional mutant jmjd2b histone demethylase. Approximately 94% and 88% of the cells can be induced for the expression of functional and mutant jmjd2b-EGFP in the respective ES cell lines. Immunofluorescence analyses have shown that induction of functional jmjd2b-EGFP results in an approximately 50% reduction of H3K9Me3 levels compared to non-induced cells and induced mutant jmjd2b-EGFP cells. The comparison of the in-vitro embryo development following nuclear transfer with induced and non-induced donor cells show significantly better overall development to blastocysts and morulae from induced donor cells with reduced H3K9Me3 levels.

The cloning of cattle by somatic cell nuclear transfer (NT) is associated with a high incidence of abnormal placentation, excessive fluid accumulation in the fetal sacs (hydrops syndrome) and fetal overgrowth (Lee RSF et al. 2004 Biol. Reprod. 70, 1–11). Early embryonic loss in bovine NT pregnancies may also be due to immunological rejection (Hill JR et al. 2002 Biol. Reprod. 67, 55–63). As a means of overcoming placental abnormalities and improving pregnancy outcome in bovine NT, reconstructed blastocysts were produced by combining immunosurgically isolated inner cell masses (ICM) from Day 7 NT embryos with the trophectoderm (TE) of Day 7 IVF embryos. Oocytes for the production of NT and IVF embryos were obtained from abattoir-collected ovaries of dairy cows. The semen used for IVF was from the bull from which the cell line for NT was derived. The NT blastocysts were produced as described previously (Oback B et al. 2003 Cloning Stem Cells 5, 3–12) except that two one-cell embryos were aggregated together after NT (2NT). Blastocyst reconstruction was achieved using a modified procedure (Rorie RW et al. 1994 Vet. Record 135, 186–187). Embryos from four experimental groups were transferred individually to synchronized recipient heifers on Day 8 of culture: (1) ICM from 2NT embryos reconstructed with IVF TE (R-2NT, n = 15); (2) ICM from IVF embryos reconstructed with IVF TE (R-IVF, n = 15); (3) control 2NT (n = 10); and (4) control IVF (n = 10). Pregnancy rates were recorded and treatments compared using Fisher's exact test. After slaughter between Days 149 and 161 of gestation, morphometric measurements were determined for the fetuses, fetal organ weights, fluid volumes, and placentomes. Data were rank transformed; treatments were compared using Student's t-test with standard errors calculated from the pooled variation. Pregnancy rates on Day 35 were R-2NT (60%), R-IVF (47%), 2NT (90%), and IVF (10%). Pregnancy rates on Day 150 were R-2NT (40%), R-IVF (40%), 2NT (70%), and IVF (10%). The reason for the low IVF pregnancy rate was unknown. Previously, pregnancy rates using the same sire and cell line (but using Day 7 embryo transfer) on Day 35 were 63% (n = 40) and 69% (n = 42) for IVF and single, non-aggregated NT, respectively, and 50% and 33% for IVF and NT on Day 150. The single NT pregnancy rate was not significantly different from that for the 2NT embryos. There was no significant difference in pregnancy rates on Day 35 and Day 150 between R-2NT v. 2NT, R-2NT v. R-IVF, or 2NT v. R-IVF. The blastocyst reconstruction procedure did not have any impact on fetal development or influence pregnancy rates. All fetuses recovered were male. No significant differences were found between R-2NT and 2NT fetuses in terms of fetal weight, fluid volume, total placentome weight, and placentome numbers or in the relative and absolute weights of the brain, heart, liver, and kidneys. Thus, replacement of the TE in NT embryos with TE from IVF embryos did not overcome placental abnormalities or decrease fetal overgrowth prevalence.

Mice have been cloned from somatic and embryonic cells; however, only 0–3% of the reconstructed embryos develop into viable offspring. In addition, the piezo microinjection method widely used for mouse nuclear transfer (NT) is difficult to master. Our objective was to compare cumulus and ES cells as nuclear donors using a simplified method of zona-free NT. In cattle, zona-free NT is simpler, faster, easier to learn and more reproducible than zona-intact NT (Oback et al. 2003 Cloning Stem Cells 5, 3–12). Oocytes were recovered at metaphase II stage (13 h after hCG injection) from the oviducts of C57BL/6J × DBA/2 F1 females (8–10 weeks of age). Cumulus cells were removed with hyaluronidase (300 units/mL) and the zona pellucida digested with pronase (0.5%) at 37°C for 3 min. Oocytes were then enucleated under UV light in cytochalasin B (5 μg/mL) after a 5-min staining with Hoechst (5 μL/mL). The metaphase DNA was removed in an enucleation pipette (16–20 μm, perpendicular break) by separating karyoplast and cytoplast with a simple separation pipette (60–80 μm, perpendicular break, closed round tip). Embryonic stem (ES) cells were cultured for 3 days and serum-starved for 16 h before use. Cells from this line had yielded offspring by the piezo procedure. Cumulus cells were used freshly. Donor cells were attached to the cytoplasts with phytohemagglutinin (10 μg/mL) and couplets were electrically fused in 0.2 mM mannitol buffer. Reconstructed embryos were activated 1–2 h after fusion for 5–6 h in CZB medium containing 10 mM strontium chloride and 5 μg/mL of cytochalasin B. Embryos were cultured individually in 5-μL droplets in CZB. Morulae and blastocysts were transferred into the uteri (Day 2.5) of pseudopregnant surrogate mothers (C57BL/6J × CBA/2J). Recipient mothers were sacrificed at 19.5 days postcoitum and pups removed. Airways were cleaned to remove fluid and the pups were held in a warm box before being fostered by a lactating mother. During development of the technique, we assessed the frequency of fusion, cleavage of reconstructed embryos, and development to morula/blastocyst stage. Fusion (58.1 ± 6.7% vs. 24.2 ± 1.7%, P < 0.001) and cleavage (66.4 ± 4.2% vs. 50.5 ± 5.4%, P < 0.05), all respectively, were higher when cumulus cells were used as donors, as compared with ES cells. However, the percentage of embryos developing to morula/blastocyst stage was greater when ES cells were used (22.2 ± 4.2% vs. 5.3 ± 2.7%, P < 0.01). Using ES cells as donors, 19/94 (20.2%) reconstructed embryos reached compacted morula/blastocyst stage. After transfer to five recipients, one pup was born (5.2%). It was larger and heavier than uncloned pups of the same age. The pup is healthy and now 12 weeks old. Genotype was confirmed by microsatellite analysis. The birth of a healthy cloned mouse pup from zona-free NT provides “proof of principle” of a technology that promises to increase throughput, ease of operation, and reproducibility of mouse cloning.