99 EFFECT OF SUPPRESSION OF BOVINE DNA METHYLTRANSFERASE 1 ON EMBRYONIC DEVELOPMENT AND EXPRESSION OF IMPRINTED GENES

Abstract

DNA methyltransferase 1 (DNMT1) is believed to be involved in DNA methylation, which is a well-characterized epigenetic modulator shown to have essential functions in germ line and embryonic genome imprinting. This study was conducted to investigate the consequences of suppressing and inhibiting DNMT1 on the development, level of apoptosis, and expression of imprinted genes in pre-implantation bovine embryos. In vitro-produced zygotes were categorized into 4 groups; namely, those injected with Smartpool siRNA (SpsiRNA; Dharmacon Inc., Chicago, IL) (n = 800), 5aza-2′-deoxycytidine (5-AZA; Sigma, St. Louis, MO) (n = 864), nuclease-free water (n = 850), and uninjected control (n = 755). The mRNA expression data were generated using RT-PCR based on the relative standard curve method employing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a normalizer. Apoptotic index (API) was calculated by dividing the number of apoptotic cells to total cell number. The proportion of 2-, 4-, 8-cell and 2-, 4-, 8-, and 16-cell embryos was assessed 48 and 72 h, respectively, post-micro injection (pmi), whereas blastocyst rate was assessed at Day 8 pmi. Data on embryonic development and the relative mRNA abundance were analyzed using ANOVA followed by a multiple pair-wise mean comparison using Tukey test. The proportion of 2-, 4-, and 8-cell embryos at 48 h pmi was not significant among treatment groups. However, the proportion of the 8-cell embryos was significantly lower (P < 0.05) in SpsiRNA (16.3 ± 4.5) and 5-AZA injected groups (17.7 ± 4.9) compared with water-injected (26.8 ± 2.9) and uninjected controls (30.7 ± 6.2). The lowest total blastocyst rate (P < 0.05) was observed in the 5-AZA treatment group (16.9 ± 4.9) compared with SpsiRNA (23.4 ± 2.1) and water-injected (24.1 ± 5.3) and uninjected controls (29.4 ± 2.1). Microinjection of SpsiRNA reduced the target mRNA by 80 and 50% in 8-cell and blastocyst stage embryos, respectively, compared with uninjected control, and the protein expression level was also reduced at 8-cell embryos as confirmed by Western blotting. Injection of 5-AZA had no significant effect on mRNA or protein expression. The greatest API (P < 0.05) was found in SpsiRNA (4.2 ± 2.0) and 5-AZA (4.1 ± 1.7) injected groups compared with water-injected (2.8 ± 2.1) and uninjected controls (2.9 ± 2.3). The relative expression study also showed that microinjection of SpsiRNA and 5-AZA increased the expression of IGF2 (by 67 and 55%), IGF2R (29 and 30%), and IGFPB-4 (22 and 24%), respectively, compared with uninjected control, without affecting the expression of both IGF2R and IGFPB-4. In conclusion, suppression of DNMT1 resulted in lower proportion of 8-cell embryos, reduced blastocyst rate, and increased apoptotic index, and affected the expression of some imprinted genes, demonstrating a critical role of this gene in bovine embryonic development.