Abstract
BackgroundNucleoplasmin 2 (NPM2) is an oocyte-specific nuclear protein essential for nuclear and nucleolar organization and early embryonic development. The aims of this study were to clone the bovine NPM2 gene, determine its temporal expression during oocyte development and early embryogenesis, and evaluate the potential role of miRNA-181a in regulation of its expression.MethodsA 329 bp cDNA fragment was amplified from bovine fetal ovary using primers designed based on the conserved regions of the human and mouse NPM2 cDNA sequences. RACE experiments were performed to obtain the 5' and 3' ends of the bovine NPM2 cDNA. Real time PCR and Western blot analysis were used to examine the expression of bovine NPM2 in oocytes and early embryos. Co-expression of bovine NPM2 and miRNA-181a in Hela cells was performed to determine if expression of bovine NPM2 is regulated by miRNA-181a.ResultsThe bovine NPM2 cDNA is 851 bp in length encoding a protein of 200 amino acids. The protein contains the conserved bipartite nuclear localization sequence and shows 53% and 62% identity with mouse and human NPM2, respectively. Expression of bovine NPM2 mRNA is restricted to ovaries. NPM2 mRNA is abundant in GV and MII stage oocytes, decreases in early cleavage stage embryos, and barely detectable in morula and blastocyst stage embryos. Similarly, expression of NPM2 protein is high in oocytes and early embryos but extremely low in blastocysts. The abundance of NPM2 mRNA is significantly lower in oocytes isolated from persistent versus growing dominant follicles (P < 0.05). A miR-181a binding site in the 3'UTR of the NPM2 transcript was identified. Transfection experiments showed that bovine NPM2 protein expression is reduced in Hela cells expressing miR-181a compared to control cells without miR-181a, indicating that translation of NPM2 is repressed by miR-181a.ConclusionsOur data suggest that expression of bovine NPM2 is temporally regulated during early embryogenesis and miR-181a may play a role in its regulation.
Highlights
Nucleoplasmin 2 (NPM2) is an oocyte-specific nuclear protein essential for nuclear and nucleolar organization and early embryonic development
We report the cloning of bovine NPM2, its mRNA and protein expression during oocyte maturation and early embryonic development and the potential role of miR-181a in regulation of its expression
Results and discussion cDNA cloning and tissue distribution of bovine NPM2 The complete cDNA sequence (851 bp) for bovine NPM2 was obtained by assembly of the sequences from the PCR and RACE fragments (Figure 1A)
Summary
Nucleoplasmin 2 (NPM2) is an oocyte-specific nuclear protein essential for nuclear and nucleolar organization and early embryonic development. Maternal mRNAs that accumulate in the oocyte during oogenesis play important roles during initial stages of embryonic development, before activation of the embryonic genome [1]. Some of the maternal transcripts are oocyte-specific and known as maternal effect genes which are required for the early cleavage events post fertilization [2,3]. NPM2, an oocyte-specific nuclear factor, plays an important role in this process. Knockout of NPM2 in mice reduced initial cleavage of embryos and impaired development to the 2-cell stage, and resulted in fragmentation and asynchrony of further cleavage and death by 50 hr post-fertilization [6]. Microinjection of NPM into bovine oocytes after nuclear transfer resulted in increased viability of embryos and higher rate of pregnancy [9], suggesting a role for NPM in facilitating reprogramming of the somatic nucleus
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