Dicer1 expression in preimplantation mouse embryos: Involvement of Oct3/4 transcription at the blastocyst stage

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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.

Section snippets

Materials and methods

Generation of mouse embryos. To obtain oocytes or fertilized embryos, 5-week-old B6D2 F1 female mice were superovulated by intraperitoneal injections of 5 IU pregnant mare serum gonadotropin (PMSG, Sigma, St. Louis, MO), followed by 5 IU gonadotropin (hCG, Sigma) 48 h later. Experiments were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals. Germinal vesicle (GV) stage oocytes were collected 45 h after PMSG injection from the ovary by slicing. The

Expression of Dicer1 in mouse oocytes and embryos

The relative abundance of Dicer1 transcripts was established by RT-PCR using the 2-ddCt method. Ten oocytes/embryos per treatment group were analyzed four times with three replicates (Fig. 1). Samples were normalized using rabbit Globin mRNA as an external reference (Fig. 1A). To normalize the RT-PCR reaction efficiency and quantify Dicer1 mRNA, H2a was applied as an internal standard (Fig. 1B). Following normalization to the Globin mRNA (Fig. 1A), the Dicer1 transcript level was elevated in GV

Discussion

In the present study, we determined the expression patterns of Dicer1 and its possible role in mouse preimplantation development. We initially demonstrated the presence of Dicer1 mRNA in mouse preimplantation embryos using quantitative real-time RT-PCR and immunocytochemistry. Specifically, real-time RT-PCR results revealed elevated expression of Dicer1 transcripts in GV oocytes and lower expression during oocyte maturation, which was further reduced up until the 2-cell stage embryo. The

Acknowledgments

This study was financially supported by Korean Ministry of Science and Technology (NRL to N.H. Kim) and The Ministry of Agriculture and Forestry (Bio-Organ Production Project).

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