Tissue-specific effects of in vitro fertilization procedures on genomic cytosine methylation levels in overgrown and normal sized bovine fetuses.
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.
Institute of Molecular Animal Breeding and Biotechnology, Gene Center of the Ludwig-Maximilian University, D-81377 Munich, and Pediatric Endocrinology Section, University Hospital, Tuebingen, Germany. stefan.hiendleder@adelaide.edu.au
Stefan
S
Hiendleder
Michaela
M
Wirtz
Cora
C
Mund
Martina
M
Klempt
Horst-Dieter
HD
Reichenbach
Miodrag
M
Stojkovic
Myriam
M
Weppert
Hendrik
H
Wenigerkind
Martin
M
Elmlinger
Frank
F
Lyko
Oliver J
OJ
Schmitz
Eckhard
E
Wolf
D044503:5-Methylcytosine / Q000032:analysis*; D000818:Animals; D001921:Brain / Q000196:embryology; D001923:Brain Chemistry; D002417:Cattle / Q000196:embryology* / Q000235:genetics; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods*; D047109:Fetal Development; D020567:Fetal Weight; D005333:Fetus / Q000737:chemistry; D005640:Follicle Stimulating Hormone / Q000008:administration & dosage / Q000494:pharmacology; D016678:Genome; D008099:Liver / Q000737:chemistry / Q000196:embryology; D008297:Male; D009132:Muscles / Q000737:chemistry / Q000196:embryology; D010641:Phenotype; D011247:Pregnancy
D044503:5-Methylcytosine; D005640:Follicle Stimulating Hormone