112 EXPRESSION OF DNA METHYLTRANSFERASES (DNMT) IN PLACENTAL TISSUES DURING EARLY PREGNANCY IN SHEEP

Abstract

Normal placental development is critical for placental function and thus for normal embryonic and fetal growth and development. Many factors, including those from the environment or from the application of assisted reproductive techniques, are known to affect embryonic development. Additionally, altered DNA methylation was reported for fetal and/or maternal placenta from compromised pregnancies, and this may contribute to high embryonic/fetal loss. DNA methylation regulated by DNA methyltransferase (DNMT) plays an important role during embryonic development. However, little is known about the expression of DNMT in placental tissues during early pregnancy in any species. To determine the mRNA expression of DNMT 3a and 3b (developmentally-regulated DNMT) in normal placenta, caruncular (CAR, maternal placenta) tissue and fetal membranes (FM, chorioallantois or fetal placenta) were collected on Days 14, 16, 18, 20, 22, 24, 26, 28, and 30 after natural mating (n = 5–8 ewes day^–1) and on Day 9–11 after oestrus (n = 7; non-pregnant [NP] controls, CAR only), snap-frozen, and then used for quantitative real time RT-PCR. For each tissue, data were analysed statistically by ANOVA with the day of pregnancy as the main effect. In CAR and FM, mRNA expression of DNMT3A and 3b was affected (P < 0.01–0.02) by day of pregnancy. In CAR, expression of DNMT3A was similar in NP controls and on days 14, 16, 18, and 30, was decreased (P < 0.01) ∼2-fold on day 20, and then gradually increased to day 30 of pregnancy. In CAR, expression of DNMT3b was similar in NP controls and on days 14, 16, 18, 24, 26, and 28, but was greater (P < 0.02) by ∼2-fold on days 22 and 30 than in NP controls or on days 24 and 26 of pregnancy. For CAR, regression analysis of DNMT3a mRNA expression demonstrated a cubic pattern (R2 = 0.253; P = 0.01) of expression during early pregnancy. In FM, DNMT3a increased (P < 0.01) ∼2-fold from day 16 to 24–30, but DNMT3b gradually decreased (∼0.5–5-fold; P < 0.01) from day 16 to day 30 of pregnancy. For FM, regression analysis of mRNA expression for DNMT3a demonstrated a linear increase (R2 = 0.301; P < 0.01), but for DNMT3b a cubic pattern (R2 = 0.624; P < 0.01) of expression during early pregnancy. These data indicate that DNMT3a and 3b mRNA are differentially expressed in CAR and FM, and the temporal pattern of expression of DNMT3a and 3b differs between maternal and fetal placental tissues during early pregnancy in sheep. Thus, significant changes in mRNA expression of DNMT3a and 3b in CAR and FM indicate that de novo methylation is present in the placenta during early pregnancy in sheep and may be regulated in part by the level of DNMT expression. These data provide a foundation for determining the basis for altered DNA methylation of placental and embryonic tissues in compromised pregnancies.