86 DNA METHYLTRANSFERASE (DNMT)1 AND DNMT3a PROTEIN EXPRESSION IN PRE-IMPLANTATION BOVINE EMBRYOS DERIVED IN VIVO AND IN VITRO

Abstract

DNA methylation accounts, beside the modifications of histones, for the major epigenetic reprogramming event during embryonic development and is an important mechanism of transcriptional regulation. DNA methyltransferase 1 (DNMT1) is the main enzyme responsible for maintenance of DNA methylation, whereas DNA methyltransferase 3a (DNMT3a) catalyzes de novo methylation. Recently, it has been shown that in vitro production (IVP) of bovine embryos significantly influences the relative abundance of DNMT1 and DNMT3a transcripts throughout bovine pre-implantation development (Höffmann K et al. 2006 Reprod. Fertil. Dev. 18, 231–232 abst).The objective of the present study was to examine the influence of IVP on the amount and localization of DNMT1 and DNMT3a protein during early pre-implantation development by immunostaining and confocal laser microscopy. In vivo-derived embryos were collected from synchronized and superovulated heifers by transvaginal endoscopic flushing of oviducts (zygotes to 8-cell stages; Besenfelder U et al. 2001 Theriogenology 55, 837–845) or by uterine flushing (16-cell stages to blastocysts). For in vitro production of bovine embryos, standard protocols of IVP were used (Wrenzycki C et al. 2001 Biol. Reprod. 65, 323–331). Zona-free embryos of both origins were wholemount fixed and permeabilized. Nonspecific immunoreactions were blocked. Embryos were stained with primary antibodies at the appropriate dilutions (anti-DNMT1 at 1:50, anti-DNMT3a at 1:25). After washing, the samples were incubated with secondary Alexa 647-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR) at dilutions of 1:100 (DNMT1) or 1:50 (DNMT3a). After additional washing, embryos were incubated in Sytox Green (Molecular Probes) and mounted on slides. Controls were produced using only the secondary antibody or by omission of both antibodies. Embryos were analyzed using a confocal scanning laser microscope (LSM 510, Zeiss, Hamburg, Germany). Fluorescence intensity of individual embryos was assessed on central optical sections using ImageJ software (National Institutes of Health, Bethesda, MD). The amount of DNMT1 protein was increased (P < 0.05) in in vivo-derived embryos at the 4-cell, 10 to 16-cell stage, morulae, and blastocysts compared with their in vitro-derived counterparts. Expression of DNMT3a protein was significantly upregulated in IVP-derived 2-cell embryos, whereas a significant increase was seen in 4-cell embryos and blastocysts of in vivo origin. DNMT1 is localized in the cytoplasm of the pre-implantation embryos of both origins and is translocated to the nucleus at the early morula stage. DNMT3a can be found in the cytoplasm of all pre-implantation stages. For the first time, protein expression patterns of DNMT1 and DNMT3a have been analyzed in in vivo- and in vitro-produced bovine embryos throughout pre-implantation development. These results indicate that IVP alters protein expression of genes related to epigenetic modifications very early in development, even before the embryonic genome has been activated.

We acknowledge HW Schaumann Stiftung for financial support.