Intact rat conceptuses were cultured from day 9.5 of gestation on. Individual components of the conceptus, including the embryo and the extraembryonic membranes (consisting of yolk sac, amnion, and allantoic placenta), were isolated and examined for insulin receptors at two time points during organogenesis: 1) day 10.4 of gestation (∼10–12 somites) when the yolk sac had become vascularized and just before closure of the anterior neuropore and 2) day 11.6 (∼27–31 somites) when vascularization of the chorioallantoic placenta had been established and the neural tube was closed completely. The studies were designed to provide inferential insights about the possible role of insulin in embryogenesis during different phases of nutrient delivery.
Active insulin degradation occurred with embryo as well as membrane homogenates during incubation at 37°C. Degradation was markedly reduced at 4°C, and binding of 125I-labeled insulin by embryo or membrane homogenates prepared on day 10.4 or 11.6, respectively, of gestation approached equilibrium after a 20-h incubation at this temperature. Values for the specific binding of tracer (0.4 ng/ml) or carrier (10.4 ng/ml) insulin by embryo and membrane homogenates were the same on days 10.4 and 11.6; specific binding was significantly greater with preparations of membranes than embryo at both time points. Full binding curves on day 11.6 showed similar affinities for insulin by embryo and membranes (Ke = 1.2 × 108/M and 4.6 × 108/M, respectively). Specificity of the binding was confirmed by comparison with proinsulin, desoctapeptide insulin, and insulin-like growth factor I and by the demonstration that maximally effective amounts of human antiinsulin receptor antiserum inhibited insulin binding ∼75% in embryos and 92% in membranes on both days. Photoaffinity cross-linking studies confirmed the presence of a major insulin-binding moiety in embryos and membranes similar to that found in rat liver plasma membranes (L) but different in apparent molecular weight (liver plasma membranes > membranes > embryo).
Our studies indicate that specific insulin receptors are present in the embryo and membrane components of the early postimplantation rat conceptus at two different stages in the development of nutrient delivery systems. However, the embryos and extraembryonic membranes display some differences with regard to the number and binding characteristics of their specific insulin receptors at both time points. The functional significance of these differences remains to be elucidated.
