Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2009) 19 P109

SFEBES2009 Poster Presentations Cytokines and growth factors (6 abstracts)

Quantum dot labelled IGF-I: a novel technique to study IGF-signal transduction in the human placenta

K Forbes , J Aplin & M Westwood

University of Manchester, Manchester, UK.

In humans, the exchange of nutrients and waste between mother and fetus occurs via the outer layer of the placenta (syncytium; ST); this layer is maintained by continuous growth and differentiation of underlying cytotrophoblasts (CT). Pregnancy complications such as intrauterine growth restriction are associated with abnormal CT proliferation and apoptosis; altered levels of maternal insulin-like growth factors (IGFs) have also been reported in these conditions. We have demonstrated that application of IGF at the maternal surface of first trimester placental explants induces activation of (IGF1R)-mediated signalling pathways to directly enhance CT turnover. IGF1R is located both on the syncytial surface and on CTs and hence IGFs could potentially act at either or both of these surfaces to influence CT kinetics. In this study, we have developed methodology to allow exogenous IGF to be tracked both in live and fixed placental tissue.

IGF-I was fluorescently labelled by incubating biotinylated IGF-I with streptavidin-conjugated fluorescent Quantum Dots (QDs; Molecular Probes). Activity of the resulting QD-IGF-I complex was confirmed in NWT3b (mouse fibroblasts over-expressing the IGF1R). Time lapse fluorescence microscopy demonstrated that QD-IGF-I bound to the cell surface, was internalised and became concentrated in perinuclear areas. Western blotting revealed that QD-IGF-I (50 nM; 20 min in serum-free conditions) enhanced IGF1R phosphorylation.

Following optimisation in cells, first trimester placental explant tissue was incubated with QD-IGF (50 nM) for up to 4 days. Visualisation of QD-IGF-I by fluorescence microscopy revealed that the localisation of exogenously delivered IGF changed over time; 30 min after initial exposure it was present only on the ST surface, by 1 h IGF was also present within the syncytium and at 6 h it was present throughout the ST and in the vicinity of the CT surface. IHC analysis revealed that QD-IGF-I enhanced both CT proliferation (Ki67) and survival (M30) (P<0.05; n=4).

This study demonstrates for the first time that IGF activity is retained after conjugation to QDs. QD-IGF-I binds to the syncytial surface and is subsequently transported across the syncytiotrophoblast. These events may precede the activation of IGF1R on cytotrophoblast.

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