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Abstract: Poor success rates in somatic cell cloning are often attributed to abnormal early embryonic development as well as late abnormal fetal growth and placental development. Although promising results have been reported following chromatin transfer (CT), a novel cloning method that includes the remodeling of the donor nuclei in vitro prior to their transfer into enucleated oocytes, animals cloned by CT show placental abnormalities similar to those observed following conventional nuclear transfer. We hypothesized that the placental gene expression pattern from cloned fetuses was ontologically related to the frequently observed placental phenotype. The aim of the present study was to compare global gene expression by microarray analysis of Day 44–47 cattle placentas derived from CT cloned fetuses with those derived from in vitro fertilization (i.e. control), and confirm the altered mRNA and protein expression of selected molecules by qRT-PCR and immunohistochemistry, respectively. The differentially expressed genes identified in the present study are known to be involved in a range of activities associated with cell adhesion, cell cycle control, intracellular transport and proteolysis. Specifically, an imprinted gene, involved with cell proliferation and placentomegaly in humans (CDKN1C) and a peptidase that serves as a marker for non-invasive trophoblast cells in human placentas (DPP4), had mRNA and protein altered in CT placentas. It was concluded that the altered pattern of gene expression observed in CT samples may contribute to the abnormal placental development phenotypes commonly identified in cloned offspring, and that expression of imprinted as well as trophoblast invasiveness-related genes is altered in cattle cloned by CT. [Copyright &y& Elsevier]

Abstract: Oocyte cytoplasm plays a prominent role in cloned embryonic development. To investigate the influence of oocyte cytoplasmic amount on cloned embryo development, we generated bovine somatic cell nuclear transfer (SCNT) embryos containing high (30–40% of the cytoplasm was removed), medium (15–25% of the cytoplasm was removed) and low (<10% of the cytoplasm was removed) nucleocytoplasmic volume ratios (N/C) using enucleated metaphase II oocyte as recipient, and fibroblast as donor nucleus, and analyzed the expression levels of ND1, Cytb and ATPase6, as well as the embryonic quality. The results indicated: (1) the process of embryonic development was not influenced by <40% of cytoplasm removal; (2) the rate of blastocyst formation, the total number of blastomere and the ratio of ICM to TE were inversely proportional to the N/C; (3) SCNT embryos with reduced volume equal to 75–85% or >90% of an intact oocyte volume showed similar karyotype structure of the donor cells; (4) the number of mtDNA copy was larger in low N/C embryos than that in medium or high N/C embryos, and the expression levels of each gene hardly varied from the 2-cell to 8-cell stage, while the expression levels increased dramatically at the blastocyst stage; (5) from 16-cell to the blastocyst stage, the change of the expression level of each gene was not significant between low N/C embryos and IVF embryos, but it was more significant than those of high or medium N/C embryos. The results suggest that the decrease of mtDNA copy number and mitochondrial gene expression may be related to the impairment in early embryonic development, and removal of <10% adjacent cytoplasm volume may be optimal for bovine SCNT embryo development. [Copyright &y& Elsevier]

Abstract: In somatic cell-cloned animals, inefficient epigenetic reprogramming can result in an inappropriate gene expression and histone H4 acetylation is one of the key epigenetic modifications regulating gene expression. In this study, we investigated the levels of histone H4 acetylation of 11 development-related genes and expression levels of 19 genes in lungs of three normal control calves and nine aberrant somatic cell-cloned calves. The results showed that nine studied genes had decreased acetylation levels in aberrant clones (p <0.05) and two genes had no significant variations (p >0.05). Whereas 13 genes had significantly decreased expression (p <0.05) in aberrant clones, five genes showed no significant differences between controls and clones (p >0.05), and only one gene had higher expression level in clones (p <0.05). Furthermore, FGFR, GHR, HGFR and IGF1 genes showed lowered levels of both histone H4 acetylation and expression in aberrant clones than in controls, and the level of histone H4 acetylation was even more lowered in aberrant clones than those in controls. It was suggested that the lower levels of histone H4 acetylation in aberrant clones caused by the previous memory of cell differentiation might not support enough chromatin reprogramming, thus affecting appropriate gene expressions, and growth and development of the cloned calves. To our knowledge, this is the first study on how histone H4 acetylation affects gene expression in organs of somatic cell-cloned calves. [Copyright &y& Elsevier]

Abstract: Epigenetic reprogramming has a crucial role in establishing nuclear totipotency in normal development and in cloned animals. Insulin-like growth factor-2 receptor (Igf-2r) is a tissue-specifically and species-dependently imprinted gene, regulated by epigenetic modifications. The diversity of Igf-2r imprinting suggests that the success of animal cloning may be species-dependent. To determine the relation between epigenetic modifications and Igf-2r expression in cattle, and explore whether this gene was correctly imprinted and reprogrammed after nuclear transfer, we quantified Igf-2r mRNA in a cattle cell line after treated with an inhibitor of DNA methylation transferase or an inhibitor of histone deacetylase, and confirmed that DNA methylation and histone acetylation could regulate this gene expression. CpG island searching showed that there is a conservative imprinting control region (ICR) within the second intron of Igf-2r in cattle, analogous to mice and sheep, regulating this gene imprinting. DNA methylation analysis in sperm and blood cells showed that DNA methylation at Igf-2r ICR2 was reprogrammed in normal cattle. The methylation at Igf-2r ICR2 showed significant variation in tissues, such as blood, liver, brain, heart and heart. It suggested that Igf-2r imprinting was tissue-specifically regulated. In cloned cattle, DNA methylation at Igf-2r ICR2 was markedly altered in comparison with normal fetus, while patterns of DNA methylation at Igf-2r 3′-UTR (3-terminal untranslated region) were similar to normal fetus, it indicated that 3′-UTR was not significantly altered by cloning procedures, but DNA methylation at the locus of gene imprinting was disrupted and not completely reprogrammed after nuclear transfer. [Copyright &y& Elsevier]

Contents The present study compared the efficiency of transgenic (TG) cloned embryo production by somatic cell nuclear transfer (SCNT) with fetal-derived fibroblast cells (FFCs) which were transfected with pEGFP-N1 to in vitro-fertilized (IVF), parthenogenetic and SCNT counterparts by evaluating the rates of cleavage and blastocyst formation, apoptosis rate at different developmental stages, cell number, ploidy and gene expression in blastocysts. In SCNT and TG embryos, the rates of cleavage and blastocyst formation were significantly lower (p < 0.05) than those of IVF controls, but it did not differ between SCNT and TG embryos. In IVF control, 86.7% embryos displayed diploid chromosomal complements and the rates were significantly (p < 0.05) higher than those of SCNT and TG embryos. Most TG embryos (79%) with FFCs expressed the gene by both PCR and under fluorescence microscopy. The expression of apoptosis by TUNEL was first detected at six to eight cell stages in all embryos of IVF, SCNT and TG groups, but the expression rate at each developmental stages was significantly higher (p < 0.05) in SCNT and TG embryos than in IVF counterparts. The expression rate in inner cell mass (ICM) of TG embryos was significantly higher (p < 0.05) than in SCNT and IVF embryos. These results indicate that the high occurrence of apoptosis observed in SCNT and TG embryos compared with IVF counterparts might influence the developmental competence. Moreover, the SCNT embryos derived using non-transfected donor cells exhibited a lower apoptosis expression in ICM cells than in TG embryos derived using pEGP-N1-transfected donor cells suggesting a possible role of negative gene effect in TG embryos. [ABSTRACT FROM AUTHOR]

Cloning by somatic nuclear transfer is an inefficient process in which many of the cloned animals died shortly after birth and displayed organ abnormalities. In an effort to determine the possible genetic causes of neonatal death and organ abnormalities, we have examined expression patterns of four genes that modified chromatin (DNMT1, PCAF, MeCP2 and EED) in six organs (heart, liver, spleen, lung, kidney and brain) of both neonatal death cloned bovines (n=9) and normal control calves produced by artificial insemination (Al) using real-time quantitative RT-PCR. The effect of the age of the fibroblast donor cell on the gene expression profiles was also investigated. Aberrant expressions of DNMT1 and PCAF were found in some studied tissues, but the expression of MeCP2 and EED had similar levels to those of the normal controls. The expression of DNMT1 showed a higher level in heart, liver and brain of both cloned bovines. A higher expression level of PCAF was seen in heart and liver of both cloned bovines, but a lower level was seen only in spleen of adult fibroblast (AF) cell-derived clones. Our results suggest that aberrant expression in gene that modified chromatins were found in cloned bovine tissues of neonatal death. Because DNMT1 and PCAF play an important role in DNA methylation and histone acetylation on nuclear chromatin respectively, and normal expression of DNMT1 and PCAF is needed for precious reprogramming of donor nuclear, the aberrant transcription patterns of DNMT1 and PCAF in these clones may contribute to the defects of organs reported in neonatal death of clones. [ABSTRACT FROM AUTHOR]

Newborn ovary homeobox (NOBOX) is a homeobox gene that is preferentially expressed in the oocytes and is essential for folliculogenesis in mice. NOBOX knockout mice are infertile, and lack of NOBOX perturbs the expression of many germ-cell-specific genes and microRNAs. Furthermore, mutations in the NOBOX gene associated with premature ovarian failure have been described in humans. However, the temporal and cell-specific expression of NOBOX in bovine oocytes and the potential function of NOBOX in early embryogenesis have not been described previously. The objectives of this study were to clone the complementary (c)DNA encoding for bovine NOBOX, analyze the expression of NOBOX mRNA in bovine tissues including fetal ovaries of different developmental stages, and characterize the temporal expression patterns of NOBOX mRNA during oocyte maturation and early embryogenesis. Based on the sequence of a predicted cDNA for bovine NOBOX, we successfully amplified, using RT-PCR, a cDNA fragment representing the coding region of bovine NOBOX from bovine fetal ovary cDNA. Additional 5′ and 3′ sequences were obtained using rapid amplification of cDNA ends (RACE) procedures. The assembled full-length NOBOX cDNA is 2275 bp with an open reading frame encoding a protein of 500 amino acids with a conserved homeodomain and typical nuclear localization signal. The predicted NOBOX protein shares 61% and 49% amino acid sequence identity with its human and mouse counterparts, respectively. A BLAST search of the bovine genome database at the National Center for Biotechnology Information (NCBI) revealed that the bovine NOBOX gene is located on chromosome 4, spans approximately 5.5 kb, and is encoded by 7 exons. Northern blot analysis revealed an approximately 2.3-kb bovine NOBOX RNA transcript. RT-PCR analysis of RNA samples from a panel of 14 different bovine tissues revealed that expression of NOBOX mRNA is restricted to ovarian samples and can be detected in fetal ovaries harvested as early as 105 days of gestation, when primary follicles start to form. Further RT-PCR analysis using RNA isolated from oocytes and granulosa and cumulus cells of antral follicles indicates that bovine NOBOX is expressed in oocytes but not in other follicular cells. Real-time PCR analysis demonstrated that NOBOX mRNA is abundant in germinal vesicle and metaphase II stage oocytes, as well as from pronuclear to 8-cell stage embryos, but barely detectable in embryos collected at the morula and blastocyst stages, suggesting that NOBOX might be a maternal effect gene. Collectively, our results demonstrate that bovine NOBOX is specifically expressed in oocytes and may play a role in early embryonic development in addition to its known function in folliculogenesis. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNA) are key regulators of gene expression in vertebrate development; however, little information exists about their expression in pre-implantation mammalian embryos. We studied the expression of miRNA in bovine cloned and IVF-elongated embryos using microarrays and quantitative RT-PCR (qRT-PCR). Day-7 IVF or handmade cloned blastocysts from an adult cell line that previously yielded live cloned calves were transferred to synchronized heifers and recovered at Day 17 as elongated embryos. MicroRNAs were purified using miRVANA kit+Flash Page fractionator column (Ambion, Austin, TX, USA) coupled to amine reactive dyes CyDye™ or used in qRT-PCR. Arrays were custom-made from 271 mice, human, and rat and 104 predicted miRNAs. After overnight hybridization, slides were scanned and data analyzed. Only miRNAs showing hybridization in 3 out of 6 features per slide were considered to be expressed. MicroRNAs were simultaneously converted to complementary (c)DNA and elongated using miRNA-specific DNA-oligo with 5′ overhang and primer extension by reverse transcription, followed by SYBR Green qPCR with another miRNA-specific primer and an excess of universal forward and reverse primers. The qPCR were normalized against 5S rRNA. Data were analyzed using Kruskal-Wallis nonparametric test for P-values < 0.05. Cloned (n= 48) and IVF (n= 28) blastocysts were transferred into 10 and 4 surrogate cattle, and 42 elongated embryos were recovered; 13 of these with embryonic disc were used for miRNA extraction and analysis. In experiment 1, miRNA profiling (1 slide) from 4 cloned embryos and donor cells yielded 34 and 22 miRNAs expressed in the embryos and cells, respectively. In experiment 2 (differential expression; 3 replicates), 39 miRNAs were expressed in cloned embryos and 32 in IVF embryos. Fifteen of these miRNAs were differentially expressed: 7 were up-regulated in the cloned elongated embryos and 8 in the elongated IVF embryos. The profiles of miRNA expression of cloned embryos, IVF embryos, and the donor cells were compared and reprogramming patterns analyzed. Reprogramming means similar expression among cloned and IVF embryos, but different from the somatic cells. Overall, 50 miRNAs were subjected to nuclear reprogramming; 31 (62%) were reprogrammed correctly, 10 aberrantly (20%), and 9 were not reprogrammed (18%). Most of the aberrantly reprogrammed miRNAs (90%) were switched on after nucleus transfer. Some of the reprogrammed miRNAs clustered in the same genomic location. Based on microarray data, 3 miRNAs over-expressed in the IVF embryos (let7-b, miR-200c, and miR-24), and 1 equally expressed (miR-16) and 2 over-expressed in the cloned embryos (miR-21 and miR-103) were analyzed by qRT-PCR. Expression of all the assayed miRNAs except miR-200c correlated adequately with microarray data (Pearson coefficient of correlation R2= 0.9; P= 0.01). Here we present the first description of miRNA expression and reprogramming in bovine elongated cloned embryos. This can have profound implications for the understanding of nuclear reprogramming in somatic cloning. [ABSTRACT FROM AUTHOR]

Genetically modified animals have numerous applications ranging from basic research to agriculture production. Cloning by nuclear transfer (NT) has made possible the production of transgenic animals using previously genetically modified cell lineages. Gene expression studies and adequate selection of the nuclei donor cell for NT guarantees the presence of the gene construction in the offspring and the absence of deleterious mutations caused by the random insertion of transgenes in functional areas of the genome. Embryonic development after NT requires a change in the transcriptome of the donor cell from a somatic to an embryonic pattern, causing cloning efficiencies to be low because of incomplete or defective nuclear reprogramming. Therefore, the establishment of methodologies able to increase cloning success is highly desirable. The experiment was designed to test if recloning of transgenic fetal fibroblasts increases cloned blastocyst production and the pregnancy rates of transgenic cloned embryos produced by NT. This study compared the developmental potential of cloned embryos reconstructed with fetal fibroblasts genetically modified by lentivirus random integration (control group) expressing the green fluorescent protein gene (eGFP), with a transgenic fetal fibroblast cell line established from a 30-day transgenic pregnancy (recloning group). Fusion, cleavage (72 h post-activation, hpa), blastocyst production (168 hpa), and 30-day pregnancy rates were analyzed. A total of 1213 embryos were reconstructed; 884 (10 replicates) with random transgene insertion fibroblasts and 329 (4 replicates) with cells derived from the cloned fetus. Results were analyzed by chi-square test at 5% significance. No difference was observed (P> 0.05) between control and recloned groups regarding fusion rate (n= 550, 62.22% and n= 189, 57.25%; respectively) or cleavage rate (n= 383, 69.45% and n= 132, 69.84%, respectively). The recloned group, however, showed a higher blastocyst development rate (P< 0.01) compared with the control group (n= 51, 26.98%, and n= 79, 14.36%, respectively) and a higher 30-day pregnancy rate (n= 6, 15.38% and n= 3, 5.56%, respectively). In conclusion, recloning of transgenic fibroblasts from a successfully established pregnancy augments the efficiency in the production of embryos and pregnancy establishment compared with the control group. We speculate that a second round of NT enhances the nuclear reprogramming of donor cells, and moreover, the use of a transgenic cell lineage already proven to be successfully reprogrammed may indicate that transgene integration is not deleterious in that specific cell lineage, resulting in a good source of donor cells to be used in order to produce a homogeneous bioreactor herd. Financial support: FAPESP, Brazil. [ABSTRACT FROM AUTHOR]

Previous evaluation of milk and meat from clone cattle compared with AI control cows indicated that these products were in the normal range of data, but slight differences were observed in their fatty acid (FA) composition and muscle Δ9-desaturase indexes (Heyman et al.2007 Animal 1, 936-972). It was therefore hypothesized that epigenic modifications induced by the nuclear transfer technology may affect the expression of the 2 genes [stearoyl-coenzyme A desaturase (SCD)-1, SCD5] responsible for Δ9-desaturation in bovines. The aim of the present experiment was to analyze the differences between clones and controls on FA composition and on SCD1 and SCD5 gene expression of the semitendinosus (ST) muscle. Biopsies of ST were taken from 5 clones from 2 different Holstein genotypes and 5 Holstein AI controls at 26 months of age. Each sample was immediately split into 3 aliquots, frozen in liquid nitrogen, and stored at -80°C until analysis. Fatty acid composition was analyzed by gas chromatography after lipid extraction and methylation according to Bas et al.(2005 Meat Sci. 71, 317-326). Total RNA was isolated from 300 mg of muscle tissue and abundance of SCD1 and SCD5 genes transcripts was determined by RT-PCRas described by Bernard et al.(2005 J. Dairy Sci. 88, 1478-1489) and Lengi and Corl (2007 Lipids 42, 499-508). Results are expressed as percentage of total FA for the FA composition and mRNA abundance of SCD1 and SCD5 determined as relative to the abundance of cyclophilin A mRNA. Statistical analyses were performed using the GLM procedure of SAS (SAS Institute, Cary, NC). Single degree of freedom orthogonal contrasts were used to compare effects of cloning (AI controls v.clones) as well as effects of clone genotypes (genotype 1 v.genotype 2). The C14:0, C14:1 cis-9, C16:0, C16:1 cis-9, and C18:0 proportions in ST were not different between clones and controls. However, clones tended to have a lower proportion of C18:1 n- 9 (-3.1% of total FA; P< 0.07) and higher proportions of C18:2 n- 6 (+1.2% of total FA; P< 0.01), C18:3 n- 3 (+0.7% of total FA; P< 0.05) and n- 3 polyunsaturated FA (+1.17% of total FA; P< 0.05) than controls. Ratios of C14 and C16 Δ9-desaturation in ST were not different between clones and controls but a lower C18 Δ9-desaturation ratio for the clones compared with controls was observed (0.76 v.0.79; P< 0.05). The mRNA abundance of SCD1 was lower in clone compared with control cows (3.8 v.8.5; P< 0.05), which could be explained by the higher proportion of n- 3 polyunsaturated FA observed in clones because of the negative effects of these polyunsaturated FA on SCD gene expression. The only difference observed between genotypes was for the C18:0 proportion in muscle (P< 0.05). In conclusion, in our set of animals, cloning decreased the ST muscle gene expression of SCD1 but not of SCD5, which is related to a slight decrease in C18 Δ9-desaturation ratio. [ABSTRACT FROM AUTHOR]

In ruminants after blastocyst formation and before implantation the embryo elongates drastically. This peri-implantation stage is characterized by active proliferation of trophoblastic cells and by changes in gene expression. In cloned embryos inappropriate expression of genes during early stages could be the cause of embryonic losses. The aim of this study was to compare the expression pattern of selected developmentally important genes in Day 7 embryos produced by somatic cell nuclear transfer with gene expression at Day 17 during elongation. IVF embryos at each stage were used for comparison. For analysis we used an RT-PCR approach (conventional + quantitative real-time PCR; qPCR). For nuclear transfer we used handmade cloning with an adult fibroblast cell line that previously yielded live cloned calves. Cells were confluent, non-serum starved in passage 4. Cloned embryos were cultured in SOF medium for 7 days in the well of the well (WOW) system. Semen from a proven bull was used for IVF and the presumptive zygotes were cultured under the same conditions as cloned embryos. At Day 7, embryos (cloned or IVF) were either transferred to recipients or pooled in tens for RNA extraction using NucleoSpin RNA XS Kit (Macherey-Nagel, Düren, Germany), converted to complementary (c)DNA and amplified with specific primers in PCR reactions. Transferred embryos were recovered at elongation stage (Day 17); their RNA was extracted and treated as above. The genes studied were NANOG, FGF4, OCT4, EOMES, IFNtau, and ACTB (internal standard for quantification). Data were analyzed by Kruskal Wallis test using the InfoStat program (Buenos Aires, Argentina). Differences were considered significant at P< 0.05. Development to blastocysts was 65% for clones and 32% for IVF embryos; 19 and 6 elongated embryos were flushed back, respectively. For gene expression analysis we used 5 pools of 10 blastocyts each and 12 (6 IVF; 6 cloned) elongated embryos with embryonic disc. At pre-implantation, we found OCT4, FGF4, IFNtau, and NANOG expressed and their relative levels were quantified (except for NANOG). Expression of OCT4 was higher in the cloned embryos; expression of EOMES was not detected at this stage. At Day 17, all 5 genes were expressed and quantified via qPCR, and interesting differences were found: relative levels of expression of FGF4 decreased in both types of embryos from Days 7 to 17, whereas those for IFNtau, NANOG, and EOMES increased significantly. Expression of OCT4 remained constant in IVF embryos but not in the cloned embryos (higher at Day 7). Our results provide an initial approach to the dynamic changes occurring in the expression of developmentally important genes in the transition from expansion to elongation. The decrease in FGF4 expression as well as the increase in EOMES and NANOG expression is a new finding that could shed light on the mechanisms of trophoblastic differentiation in bovine elongation. [ABSTRACT FROM AUTHOR]