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Insulin-Like Growth Factor I (IGF-I) and Long R³IGF-I Differently Affect Development and Messenger Ribonucleic Acid Abundance for IGF-Binding Proteins and Type I IGF Receptors in in Vitro Produced Bovine Embryos¹

Institute of Molecular Animal Breeding (K.P., M.S., K.B., D.E., E.W.) and Laboratory of Molecular Biology (G.J.A.), Gene Center, Ludwig Maximilian University, 81377 Munich, Germany; and Institute of Animal Physiology and Genetics, Czech Academy of Sciences (J.M.), 27721 Libechov, Czech Republic

Address all correspondence and requests for reprints to: Prof. Dr. Eckhard Wolf, Institute of Molecular Animal Breeding, Gene Center, Ludwig Maximilian University, Feodor Lynen Strasse 25, 81377 Munich, Germany. E-mail: ewolf{at}lmb.uni-muenchen.de

The insulin-like growth factor (IGF) system is a complex network, including ligands (IGF-I and -II), binding proteins (IGFBP-1 to -6), and receptors, of which the type I IGF receptor (IGF-I-R) is important for transmission of most biological effects of IGFs. As IGFs are secreted in large amounts by the female reproductive tract, it has been hypothesized that maternal IGFs may affect embryonic growth and differentiation in a fine-tuned manner, involving modulation of IGF effects by embryonic IGFBP and IGF-I-R expression. To address this point, we cultured in vitro produced bovine embryos in a chemically defined culture system in the presence (100 ng/ml) of recombinant human IGF-I, long R³IGF-I (LR³), or without IGF supplementation (control). The affinity of LR³ to IGFBPs measured by competition assays and Western ligand blots is at least 3 orders of magnitude lower than that of IGF-I. LR³ was most efficient in stimulating early embryonic cleavage, whereas further development was most potently supported by IGF-I. Total cell numbers of blastocysts were highest in the presence of LR³ (105 ± 4), followed by IGF-I (96 ± 5), and the control group (91 ± 3; P < 0.05). Differential cell staining of blastocysts revealed that these differences were mainly represented by trophectoderm cell numbers. Analysis of messenger RNA (mRNA) expression for IGFBPs and IGF-I-R was performed by RT-real-time PCR, using expression of the nonregulated housekeeping gene glyceraldehyde-3-phosphate dehydrogenase for normalization. Embryonic IGFBP-2 mRNA levels in the LR³ treatment group were 1.7-fold (P < 0.001) and 2.8-fold (P < 0.001) higher than those in the IGF-I and control groups, respectively. IGFBP-5 mRNA levels were about 2-fold (P < 0.001) elevated in both IGF treatment groups, with slightly (P < 0.05) higher levels in IGF-I- than in LR³-treated embryos. Similarly, IGFBP-3 mRNA abundance was increased (P < 0.05) in embryos from the IGF-I vs. the LR³ culture system. IGF-I-R mRNA levels were reduced by IGF-I (80% of control; P < 0.01), but increased by LR³ (1.3-fold vs. control; P < 0.001). These data show that the affinity for IGFBPs of IGF peptides is relevant for their effects on preimplantation embryos and affects different parameters, i.e. development, cell numbers, and mRNA expression for components of the IGF system, in different directions.

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