Margaret K. Pratten

Effects of low insulin levels on rat embryonic growth and development

The risk of congenital abnormality in diabetic pregnancy is about four times that for the normal population. Past clinical studies have suggested hyperglycemia and hyperketonemia as the factors responsible for these abnormalities, with no reference to the possible effects of low insulin levels. We examine the effect of hypoinsulinemia on rat embryonic growth and development in culture while normal glucose levels are maintained. With anti-insulin antibody bound to an affinity column containing cyanogen bromide-activated Sepharose 4B beads, insulin was selectively removed from the homologous culture serum eluted down the column. A culture of rat embryos from the early head-fold stage for 50 h in insulin-depleted normoglycemic homologous serum (insulin levels 0.055–0.18 ng/ml) showed retardation of growth and development when compared with control embryos. Adding physiological amounts (10 ng/ml) of insulin back into the insulin-depleted serum subsequently restored growth level to that of control embryos. We conclude that low insulin levels, encountered in newly diagnosed diabetic pregnancy, may be instrumental in increasing the risk of congenital abnormalities.

Processing of fluorescently labelled insulin and insulin-like growth factor-i by the rat visceral yolk sac

Insulin and the structurally related insulin-like growth factor I (IGF-I) are mitogenic peptides which have been implicated in the embryonic development of the rat. In addition to factors produced by the developing embryo itself, it is likely that maternally-derived growth factors play an important role also, with their postulated initial site of action being the extraembryonic membranes, which surround the embryo throughout gestation. We have examined the processing of these potential regulatory factors by the visceral yolk sac on the 17th day of gestation, using fluorescently-labelled ligands and fluorescence microscopy. Both insulin and IGF-I are rapidly internalized at the yolk sac surface, and appear in the tissue within discrete vesicular structures. Interestingly, in some cases when both labelled proteins are added simultaneously they do not appear to coexist within vesicles. Instead, insulin appears to remain within vesicles close to the apical surface of the yolk sac whereas IGF-I appears to penetrate the tissue more deeply, being readily transported to the internal face of the epithelium. It appears, therefore, that there is some difference in the sorting mechanisms of these related proteins, although the physiological significance of this observation is not clear.

Differences in Binding Characteristics of Rat and Human Transferrin by Rat Visceral Yolk Sac Placenta

Previous work has shown that human serum supplemented with rat transferrin can support the normal growth of cultured rat conceptuses, but that supplementation with human transferrin has no such effect. Such results strongly suggest an hypothesis of species-specificity. This potential specificity was therefore investigated by comparing uptake, binding, competition and vectorial transport of both rat and human transferrin using two well-established systems, those of 17.5 day rat visceral yolk sacs and anembryonic yolk sacs in culture. The results of these investigations show that human transferrin displays a lower rate of uptake and lower binding affinity for the rat transferrin receptor than does rat transferrin. Human transferrin competes poorly with rat transferrin for receptor occupancy unless present in 20-fold excess. Both molecules are taken up by receptor-mediated endocytosis and are processed in a similar manner. Anembryonic yolk sac experiments show that a greater proportion of intact rat transferrin is transported to the exocoelomic fluid than is intact human transferrin. Binding analyses show a difference in binding affinities of the two molecules for the rat transferrin receptor and also that human transferrin exhibits negative cooperativity in its binding. This evidence strongly supports an hypothesis of species-specificity in the binding of transferrin to the transferrin receptor in the rat visceral yolk sac.

The "giant" yolk sac: a model for studying transport across extra-embryonic membranes.

For over a decade it has been possible to maintain in culture post-implantation rat embryos removed between day 9.5 and day 11.5 of gestation, such that growth and differentiation are identical to those observed in vitro (New 1978). In order to prolong the culture period it is necessary to culture embryos in hyperbaric oxygen concentrations and under somewhat unphysiological conditions (New 1978). During these stages of gestation, before the formation of the chorioallantoic placenta, the major nutritional requirements of the embryo are supplied by the uptake, digestion and transport functions of the visceral yolk sac (Freeman et al. 1981). A technique has recently been developed for the culture of the visceral yolk sac as a closed vesicle from the 9.5th day of gestation until term: the “giant” yolk sac. Morphological studies indicate the presence of tight junctions and functional similarities to the in vivo yolk sac (Dunton et al. 1986). This system has been used to study vectorial transport of materials from the external milieu to the embryonic environment.