The long-term aim of the proposed research is to elucidate the role that ligands and receptors of the insulin family of peptides play in early mammalian development. Very little is known currently concerning the role of insulin and insulin-like growth factors (IGFs) in the earliest stages of development. It is important to study these stages, since diabetic women are known to suffer from infertility as well as increased incidence of fetal malformations. We will study the mode of uptake, translocation, and cellular processing of IGF-I and -II. Saturating concentrations of colloidal-gold labelled ligands will be used to establish receptor identity. Confirmatory studies will use high-resolution immunocytochemistry to examine the extent to which internalized ligands remain immunologically (and presumably, functionally) intact. In addition, we will examine the possibility that intact IGFs are translocated to the nucleus, as has already been shown for insulin. The effects of IGFs on gene expression and protein biosynthesis in cleavage-stage embryos will be evaluated by SDS-PAGE minigels and high resolution 2-D gel analysis. The relationship between insulin responsiveness and glucose transport will be studied by examining. developmental stages for mRNA transcripts and cellular location of glucose-transporter proteins, using mRNA phenotyping, indirect immunofluorescence, high-resolution immunocytochemistry, and Western blot analysis. To determine whether IGF-binding proteins are synthesized by preimplantation mouse embryos or surrounding reproductive tract tissues, we will use a similar combination of immunological and mRNA phenotyping techniques as those proposed for studies on glucose transporters. Cleavage-stage mouse embryos will be microinjected with antisense RNAs or will be incubated in medum containing antisense oligonucleotides directed against mRNAs encoding insulin and IGF ligands and receptors, as well as against mRNAs encoding glucose transporter proteins. Antisense nucleotides will block expression (translation) of target mRNAs and thereby allow us to evaluate the role of these gene products in the early mammalian embryonic program.
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