261 PHENOTYPIC CHARACTERISTICS AND TISSUE-SPECIFIC IGF2R/IGF2 EXPRESSION PARTITION BOVINE FETAL OVERGROWTH ASSOCIATED WITH IN VITRO FERTILIZATION AND SOMATIC CELL NUCLEAR TRANSFER CLONING

Abstract

Large offspring syndrome (LOS) in ruminants refers to various poorly defined organ pathologies that are associated with fetal overgrowth and are encountered after a range of embryo manipulations (Rhind et al. 2003 Nat. Rev. Genet. 4, 855–864). We have explored the effects of somatic cell nuclear transfer (NT) and in vitro fertilization (IVF) on phenotype and relative expression levels of 2 imprinted genes important for fetal growth, insulin-like growth factor 2 (IGF2) and its receptor (IGF2R). Viable bovine fetuses were recovered near the end of the first trimester of pregnancy, and skeletal muscle, liver, and lung were sampled for real-time RT-PCR analyses. We compared NT-fetuses (n = 23), IVF-fetuses (n = 24), and fetuses generated by artificial insemination (controls, n = 24) in order to separate abnormalities specific to cloning from effects of in vitro gamete and embryo manipulation. Nuclear transfer and IVF-fetuses, both derived from embryos cultured with 10% estrous cow serum, demonstrated significant fetal overgrowth. The increase in body weight relative to controls was similar for both groups (+22%, P < 0.001, and +19%, P < 0.001, respectively), but further analyses clearly separated the NT phenotype from the IVF phenotype. The NT-fetuses were characterized by a shorter crown-rump length but larger thorax circumference, which consequently produced a significantly reduced fetus length-to-thorax circumference ratio in comparison with IVF-fetuses and controls (-9% each, P < 0.0001). Absolute liver weight was significantly increased in NT- and IVF-fetuses (+62%, P < 0.0001, and +20%, P < 0.0001, respectively), but relative liver weight was increased only in NT-fetuses (+30%, P < 0.0001). Heart (P< 0.0001) and kidney (P = 0.0003) mass also showed disproportionate organomegaly in NT-fetuses only, but relative lung weight (NT, P = 0.263; IVF, P = 0.317) was not affected by either embryo technique. Transcript abundance for IGF2 and IGF2R genes were strongly correlated in muscle (r = 0.835, P < 0.0001), liver (r = 0.922, P < 0.0001), and lung (r = 0.772, P < 0.0001). The IGF2 and IGF2R transcript levels in muscle tissue from NT-fetuses were markedly reduced in comparison with both IVF-fetuses (-31%, P < 0.0001, and -41%, P < 0.0001, respectively) and controls (-31%, P < 0.0001, and -41%, P < 0.0001, respectively). In liver tissue, however, transcript levels for NT-fetuses were similar to those of controls, and IVF-fetuses showed markedly elevated, albeit non-significant, IGF2 (+86%, P = 0.0591) and IGF2R (+54%, P = 0.1305) mRNA levels relative to controls. Our data demonstrate that seemingly similar syndromes caused by NT or IVF procedures can be clearly partitioned with respect to phenotype and IGF2/IGF2R expression.