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Abstract: The microRNA (miRNA) genes mir-127 and mir-136 are located near two CpG islands in the imprinted mouse retrotransposon-like gene Rtl1, a key gene involved in placenta formation. These miRNAs appear to be involved in regulating the imprinting of Rtl1. To obtain insights into the epigenetic reprogramming of cloned embryos, we compared the expression levels of mir-127 and mir-136 in fertilized mouse embryos, parthenotes, androgenotes and cloned embryos developing in vitro. We also examined the DNA methylation status of the promoter regions of Rtl1 and mir-127 in these embryos. Our data showed that mir-127 and mir-136 were highly expressed in parthenotes, but rarely expressed in androgenotes. Interestingly, the expression levels of mir-127 and mir-136 in parthenotes were almost twice that seen in the fertilized embryos, but were much lower in the cloned embryos. The Rtl1 promoter region was hyper-methylated in blastocyst stage parthenotes (75.0%), moderately methylated (32.4%) in the fertilized embryos and methylated to a much lower extent (∼10%) in the cloned embryos. Conversely, the promoter region of mir-127 was hypo-methylated in parthenogenetically activated embryos (0.4%), moderately methylated (30.0%) in fertilized embryos and heavily methylated in cloned blastocysts (63–70%). These data support a role for mir-127 and mir-136 in the epigenetic reprogramming of the Rtl1 imprinting process. Analysis of the aberrant epigenetic reprogramming of mir-127 and Rtl1 in cloned embryos may help to explain the nuclear reprogramming procedures that occur in donor cells following somatic cell nuclear transfer (SCNT). [Copyright &y& Elsevier]

Abstract: To gain insights into the roles the paternal genome and chromosome number play in pre-implantation development, we cultured fertilized embryos and diploid and haploid parthenotes (DPs and HPs, respectively), and compared their development and gene expression patterns. The DPs and fertilized embryos did not differ in developmental ability but HPs development was slower and characterized by impaired compaction and blastocoel formation. Microarray analysis revealed that fertilized blastocysts expressed several genes at higher levels than DP blastocysts; these included the Y-chromosome-specific gene eukaryotic translation initiation factor 2, subunit 3, structural gene Y-linked (Eif2s3y) and the imprinting gene U2 small nuclear ribonucleoprotein auxiliary factor 1, related sequence 1 (U2af1-rs1). We also found that when DPs and HPs were both harvested at 44 and 58h of culture, they differed in the expression of 38 and 665 genes, respectively. However, when DPs and HPs were harvested at the midpoints of 4-cell stage (44 and 49h, respectively), no differences in expression was observed. Similarly, when the DPs and HPs were harvested when they became blastocysts (102 and 138h, respectively), only 15 genes showed disparate expression. These results suggest that while transcripts needed for early development are delayed in HPs, it does progress sufficiently for the generation of the various developmental stages despite the lack of genetic components. [Copyright &y& Elsevier]

Abstract: The aim of this study was to determine the effects of IGF-I on the viability and development of porcine presumptive diploid parthenotes developing in vitro. In vitro derived 2-cell parthenotes were cultured to blastocysts with or without exogenous IGF-I in the presence or absence of BSA. The addition of 10 or 100ng/ml IGF-I into NCSU 23 medium did not enhance the development of 2-cell parthenotes to the blastocyst stage. However, IGF-I increased cell numbers (P < 0.001) in the presence of BSA. Similarly, IGF-I alone did not affect apoptosis in the blastocysts but when combined with BSA, it reduced apoptosis (P < 0.05). Real time reverse-transcription polymerase chain reaction revealed that IGF-I significantly decreased the mRNA expression (pro-apoptotic gene Fas or Bak and anti-apoptotic gene Bcl-xL) ratios of both Fas/Bcl-xL (P < 0.01) and Bak/Bcl-xL (P < 0.01) in the presence of 0.4% BSA. These data suggest that IGF-I synergies with BSA to act as a mitogen and apoptotic survival factor in porcine preimplantation parthenotes. [Copyright &y& Elsevier]

The aim of this study was to determine the effects of mouse granulocyte–macrophage colony-stimulating factor (mGM-CSF) on development of porcine parthenotes and nuclear transferred embryos, and on their expression of implantation-related genes. In the presence of bovine serum albumin, mGM-CSF did not increase the percentage of oocytes that developed to the blastocyst stage and at day 7 did not increase cell numbers of embryos. Addition of 2 ng/ml GM-CSF to protein-free culture medium significantly increased the compaction and blastocoel formation of 1- to 2-cell parthenotes developing in vitro. However, total cell numbers were not increased when they were cultured in the presence of GM-CSF. Semi-quantitative reverse transcriptase polymerase chain reaction revealed that mGM-CSF enhances mRNA expression of the leukemia inhibitory factor receptor, but does not influence interleukin-6 or sodium/glucose co-transporter protein gene expression in blastocyst stage parthenotes. These results suggest that mGM-CSF may enhance viability of porcine embryos developing in vitro in a defined culture medium. [Copyright &y& Elsevier]

Abstract: High mobility group box 1 (HMGB1) is considered a component of chromatin and membranes with a role in a variety of biologically important processes. The aim of this study was to determine the effects of HMGB1 on the viability and development of porcine diploid parthenotes cultured in vitro. In vitro derived 4-cell parthenotes were cultured to blastocysts, with or without recombinant HMGB1, in the presence or absence of BSA. The addition of 1, 10, 100 or 1000ng/mL HMGB1 into NCSU 23 medium containing 0.4% BSA did not enhance the development of 4-cell parthenotes to the blastocyst stage and did not change the total number of nuclei in the blastocysts. However, addition of 10 or 100ng/mL HMGB1 into NCSU 23 medium in the absence of BSA increased (P <0.05) both the development rate of parthenotes to the blastocyst stage and total cell numbers. When cultured in NCSU23 medium supplemented with 10 or 100ng/mL HMGB1 and without BSA, apoptosis in parthenotes at the blastocyst stage was decreased (P <0.05). Based on real time RT-PCR, the addition of HMGB1 to the culture medium in the absence of BSA decreased mRNA expression of pro-apoptotic genes Bak (P <0.005) or Caspase3 (Casp3, P <0.01), but not Bcl-xL (Bcl2l2). In conclusion, we inferred that recombinant HMGB1 in the culture medium in the absence of BSA prevented apoptosis of porcine parthenotes and enhanced porcine embryo viability. [Copyright &y& Elsevier]