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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: Dicer1, an RNAse III enzyme, is a key factor for the production of microRNAs involved in post-transcriptional gene silencing. To elucidate the roles of Dicer1 and the microRNA pathway in early embryo development, we initially evaluated its gene expression in mouse oocytes and embryos in vitro. The transcript levels in GV stage oocytes steadily decreased up to the 2-cell embryo stage, and expression remained stable during morulae and blastocyst formation. DICER1 protein synthesis was additionally observed in mouse oocytes and early embryos. Silencing of mRNA expression by RNA interference (siRNA) did not inhibit development up to the blastocyst stage. Real-time RT-PCR experiments confirmed the decreased expression of selected transcription factors, including POU domain, class 5, transcription factor 1 (Pou5f1), SRY-box containing gene 2 (Sox2), and Nanog homeobox (Nanog). Moreover, POU5F1 protein expression was suppressed by Dicer1 siRNA. The results suggest that Dicer1 gene expression is associated with the levels of transcription factors, Pou5f1, Sox2, and Nanog which possibly regulate differentiation processes at the blastocyst stage. [Copyright &y& Elsevier]