Meeting Abstract
Insulin-like growth factors (IGFs) are key peptides hormones in the Insulin and Insulin-like signaling (IIS) pathway, a pathway required for growth, metabolism, and reproduction. IGFs and other key components of the IIS pathway are highly conserved across vertebrate lineages including reptiles, but there are still gaps in our knowledge about the specific functions of members of the IIS pathway in reptiles. Primary cultures are virtually non-invasive study systems that provide models of naturally functioning cells or tissues from donor organisms, although this method is not widely used in non-model organisms. Here we describe the establishment of primary fibroblast cultures from brown anole (Anolis sagrei) tail tips and their use in experiments to determine the functional roles of IGF1 and IGF2 hormones in cellular growth and proliferation in a reptile system. Species-specific IGF hormones were expressed in a bacterial system and purified for use in experimental treatments. Low-serum treatment media containing brown anole IGF1, brown anole IGF2, green anole (Anolis carolinensis) IGF1, whose amino acid sequence of the binding region differs from brown anole, TGFb as a positive growth control, or media without growth hormones were added to cells. Cellular proliferation was indirectly assessed via MTT assay 24 hours, post exposure to treatment medium. IRS-1 mRNA levels were quantified via qPCR at time points of 2 and 4 hours. Results on the effects of the treatments will be discussed with implications for the use of cell culture in non-model organisms to understand the function and evolution of molecular pathways, such as the IIS.
