196 CHANGES IN METABOLIC EXPRESSION PROFILES IN PLACENTA AND FETAL LIVER FROM BOVINE CLONED CONCEPTI

Abstract

Accelerated fetal growth may be observed in pregnancies from IVP embryos, which can be associated with increased concentration of sugar moieties (glucose, fructose) in the fetal plasma and related fluids (Bertolini et al. 2004 Reproduction 128, 341–354). The aim of this study was to compare the gene expression profiles of 14 key enzymes from seven metabolic pathways, four sugar transporters and nine bioactive molecules in liver and placental tissues obtained from Day-225 bovine concepti. Day-7 bovine in vivo- (control) and in vitro-derived [IVF or NT] blastocysts, produced by established procedures (Ribeiro et al. 2009 Cloning Stem Cells 11, 377–386), were transferred to synchronous recipients. Four control, 4 IVF, and 6 NT pregnancies were terminated on Day 225 of gestation for the comparison of maternal, fetal, and placental traits, and for the collection of tissue samples for molecular analyses by RT-qPCR for transcripts related to fructogenesis (AR, SORD), fructolysis (KHK, DAK, ALDOB), glycolysis (GAPDH, LDHA, LDHB), gluconeogenesis (Pepck, Fbp, G6PC), pentose phosphate pathway or PPP (G6PD), cholesterol (HMGCR) and fatty acid syntheses (ACACA), sugar transporters (SLC2A1, SLC2A2, SLC2A3, SLC2A5), components of the IGF system (IGF1, IGF2, IGF1R, IGF2R), markers for placental function (PL, PAG-1, LOC503858), and apoptotic activity (BAX, BCL2). RNA from placentome and fetal liver samples was extracted using Trizol^® reagent (Invitrogen, Carlsbad, CA, USA), and cDNA synthesis from total RNA were done with the SuperScript^® III First-Strand Synthesis System (Invitrogen). For the RT-qPCR, 1 µL of cDNA, 300 nM of each primer and 1X Power SYBR Green PCR Mastermix (Applied Biosystems, Foster City, CA, USA) were used in the StepOnePlus real-time PCR system (Applied Biosystems), with results normalized using 2^–ΔΔCT method with ribosomal protein S9 (RPS9) as the housekeeping gene. Data were analysed by the GLM procedure (LSM ± s.e.m.), with pairwise comparisons by the Tukey test (Minitab software, Minitab Inc., State College, PA, USA). Placental, fetal liver, and fetal weights were higher in NT concepti (6.9 ± 0.8 kg, 769.9 ± 93.8 g, and 27.2 ± 3.7 kg) than controls (3.2 ± 0.1 kg, 290.3 ± 3.6 g, and 12.5 ± 0.5 kg) and IVF (3.2 ± 0.4 kg, 345.8 ± 30.5 g, and 13.7 ± 1.0 kg) counterparts, respectively. In general, IVF-derived conceptus traits were similar to controls for most parameters evaluated in this study. When compared with controls, gene expression levels in the placenta indicated 2- to 5-fold increased activity in fructogenic, gluconeogenic, and pentose phosphate pathways, sugar transport (SLC2A1, -3 and -5), and bioactive markers (IGF1 and -2, and PL), whereas the gluconeogenic, fructolytic, and fatty acid pathways and SLC2A2 expressions were 3- to 35-fold up-regulated in the fetal liver of clones. These data indicated that a significant difference exists in activity in metabolic pathways and placental function in cloned concepti, suggesting an active glucose synthesis, an increase in fructose synthesis by the placenta, and in fructose catabolism by the fetus, which may be a reflection of an association between changes in metabolic fetal programming and excessive prenatal growth after cloning.