Gene Expression of Dnmt1 Isoforms in Porcine Oocytes, Embryos, and Somatic Cells

Abstract

In the mouse, the dynamics of genomic methylation and the initial events of gametic imprinting are controlled by the activity of an oocyte isoform of the DNA methyltransferase-1 (Dnmt1o) enzyme. The objectives of this study were to identify the alternative splicing variants of Dnmt1 in porcine oocytes and determine the gene expression pattern of the different Dnmt1 isoforms during embryo development. A rapid amplification of cDNA ends (RACE ) system was used to amplify the 5' cDNA end of Dnmt1 isoforms in porcine oocytes. RNA levels of the Dnmt1 isoforms were analyzed in porcine oocytes and embryos. DNMT1 protein expression of oocytes and somatic cells were analyzed by western blot and immunostaining. Two new Dnmt1o RNA isoforms were identified--Dnmt1o1 and Dnmt1o2. The previously reported somatic Dnmt1 isoform (Dnmt1s) was expressed at low but constant levels in oocytes and embryos from the two-cell to the blastocyst stage. Abundant RNA levels of Dnmt1o1 and Dnmt1o2 were detected in oocytes and embryos from the two- to the eight- to 16-cell stage. Levels of these Dnmt1o transcripts were low at the morula and blastocyst stages. Although Dnmt1s was present in all the somatic cell types analyzed, Dnmt1o1 and Dnmt1o2 were not detected in any somatic tissues. As predicted by the RNA sequence and verified by western blot analysis, Dnmt1o1 and Dnmt1o2 RNAs translate one DNMT1o enzyme. Western blot analysis confirmed that both the oocyte and the somatic forms of DNMT1 protein are present in porcine oocytes and early embryos, whereas somatic cells produce only DNMT1s protein. DNMT1o is localized mainly in the nuclei of oocytes and early embryos, whereas DNMT1s is expressed in the ooplasm cortex of oocytes and cytoplasm of early embryos.