Effects of insulin-like growth factor-1 and its analogues on igf binding protein gene expression in preimplantation bovine embryos

Abstract

It is well proved that all components of the insulin-like growth factor system (IGF-I and -I/ and their receptors (IGF-R), IGF binding proteins (IGFBPs)) are already expressed in the early bovine embryo. Supplementation of culture medium with IGF-I has been shown to improve the in vitro development while synthetic analogues with lower affmity for IGFBPs but higher biological activity do not have an embryotrophic effect (Boxhammer et al., Reprod Dom Anim, Suppl 5:138, 1998). To clarify this phenomenon we investigated the transcription levels of IGFBPs as well as of IGF-I-R to characterize potential interactions with IGF-I and analogous peptides (LR 3and Des(1-3)-IGF-I). Embryos were produced in vitro from slaughterhouse ovaries employing standard protocols for maturation (TCM199 + 10% estrus cow serum + 0.1% FSH) and fertilization (1 x 106 sperm/ml in modified TALP + heparin). Culture of the presumptive zygotes was performed in chemically defined Synthetic Oviduct Fluid supplemented with amino acids, 3 mg/ml polyvinyl alcohol and 100 ng/ml recombinant human IGF-I, LR 3or Des(1-3)-IGF-I, respectively, and pools of 12 embryos were collected at day 8 of development. For semi-quantitative RT-PCR total RNA was extracted by using RNeasyTMspin columns (RNeasyTMkit, Qiagen, FRG). RNA was eluted in distilled H20 and reverse transcribed using 20 IU M-MuLV reverse transcriptase (MBI Fermentas, FRG), 1 mM dNTPs, and 3 ~M oligo(dT)17 primers at 37°C for 1 h followed by denaturation at 95°C for 10 min. Hot start PCR (94°C for 5 min before adding 1 IU Taq DNA polymerase (PAN Systems, FRG)) was employed before 35 cycles were run with 30 sec at 94°C for denaturation, 1 rain at 62°C (IGFBP-2) or 65°C (IGFBP3, -5 and IGF-I-R) for annealing of primers and 1 rain at 72°C for extension. After 12 cycles 0.1 ~tM bovine [3-actin 5'and 3'primers were added to 'the reaction tubes containing already 0.5 ~tM of the IGFBP-specific primers, 0.2 mM dNTPs and 2.5 mM MgC12. Following electrophoretic separation each specific fragment was quantified by digital imaging in relation to the housekeeping gene [3-actin used as an internal standard uniformly expressed in all groups.