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Tissue-Specific Effects of In Vitro Fertilization Procedures on Genomic Cytosine Methylation Levels in Overgrown and Normal Sized Bovine Fetuses¹

Institute of Molecular Animal Breeding and Biotechnology,³ Gene Center of the Ludwig-Maximilian University, D-81377 Munich, Germany Department of Analytical Chemistry,⁴ University of Wuppertal, D-42119 Wuppertal, Germany Division of Epigenetics,⁵ Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany Biotechnology Group,⁶ Bavarian State Research Center for Agriculture, D-85586 Grub, Germany Bavarian Research Center for Biology of Reproduction (BFZF),⁷ D-85764 Oberschleissheim, Germany Pediatric Endocrinology Section,⁸ University Hospital, D-72076 Tuebingen, Germany

ABSTRACT

Epigenetic perturbations are assumed to be responsible for phenotypic abnormalities of fetuses and offspring originating from in vitro embryo techniques. We studied 29 viable Day-80 bovine fetuses to assess the effects of two in vitro fertilization protocols (IVF1 and IVF2) on fetal phenotype and genomic cytosine methylation levels in liver, skeletal muscle, and brain. The IVF1 protocol employed 0.01 U/ml of FSH and LH in oocyte maturation medium and 5% estrous cow serum (ECS) in embryo culture medium, whereas the IVF2 protocol employed 0.2 U/ml of FSH and no LH for oocyte maturation and 10% ECS for embryo culture. Comparisons with in vivo–fertilized controls (n = 14) indicated an apparently normal phenotype for IVF1 fetuses (n = 5), but IVF2 fetuses (n = 10) were significantly heavier (19.9%) and longer (4.7%), with increased heart (25.2%) and liver (27.9%) weights, and thus displayed an overgrowth phenotype. A clinicochemical screen of 18 plasma parameters revealed significantly increased levels of insulin-like growth factor 1 (40.8%) and creatinine (37.5%) in IVF2, but not in IVF1, fetuses. Quantification of genomic 5-methylcytosine (5mC) by capillary electrophoresis indicated that both IVF1 and IVF2 fetuses differed from controls. We observed significant DNA hypomethylation in liver and muscle of IVF1 fetuses (–16.1% and –9.3%, respectively) and significant hypermethylation in liver of IVF2 fetuses (+11.2%). The 5mC level of cerebral DNA was not affected by IVF protocol. Our data indicate that bovine IVF procedures can affect fetal genomic 5mC levels in a protocol- and tissue-specific manner and show that hepatic hypermethylation is associated with fetal overgrowth and its correlated endocrine changes.

conceptus, developmental biology, embryo growth factors, in vitro fertilization

¹ Supported by the Institute for Science and Health (St. Louis, MO) pursuant to Task Order Agreement 03–0900–01RFA02 (to O.J.S.) and a grant from the Deutsche Forschungsgemeinschaft (Priority Program Epigenetics; to F.L.).

² Correspondence and current address: Stefan Hiendleder, Department of Animal Science, University of Adelaide, Roseworthy Campus, Roseworthy, SA 5371, Australia. FAX: 61 8 8303 7972; stefan.hiendleder{at}adelaide.edu.au

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