The role of maternally derived epidermal growth factor and the epidermal growth factor receptor during organogenesis in the rat embryo.

Epidermal growth factor (EGF) has been implicated in the control of embryonic development, but although the receptor is expressed from an early stage, there is little evidence of embryonic expression of EGF. In order to investigate the role of maternally derived EGF during organogenesis, rat embryos were explanted at d 9.5 and cultured in serum depleted of low molecular weight molecules (retenate) which was then supplemented with EGF. Serum depleted of low molecular weight molecules by prolonged filtration loses its capacity to support normal embryonic development, possibly due to the loss of growth promoting factors. The addition of EGF to retenate significantly improved embryonic development with a maximal effect at 8 ng/ml. The addition of an analogue of EGF, long EGF, to retenate also caused a significant increase in development, although at higher concentrations a decrease in its effect was observed, possibly due to down regulation of the EGF receptor. Therefore, embryos may be able to utilise maternally derived EGF during organogenesis. To test the effects of inhibiting the EGF receptor during organogenesis, d 9.5 embryos were cultured in the presence of tyrphostin 47, a specific EGF receptor inhibitor. Tyrphostin 47 caused a significant dose-dependent decrease in the development of embryos which was also observed when tyrphostin 47 was injected into the vitelline circulation at d 11.5 to bypass the effects of the yolk sac. These findings suggest that the EGF receptor is essential for normal organogenesis and may play a role in the control of proliferation and differentiation. Although EGF is not expressed in the rat embryo at this stage, maternally derived EGF may be the ligand for the embryonic EGF receptor.

Further evaluation of a teratogenicity screen using an intravitelline injection technique.

A blind trial of 31 compounds was conducted in order to evaluate the intravitelline injection technique as a predictor of developmental toxicity. Rat conceptuses were explanted on day 9 of gestation and cultured until 11.5 days, at which time the compounds were injected into the vitelline circulation of the yolk sac. The embryos were then cultured for a further 24 hr after which time they were assessed on 14 parameters and assigned to one of three categories based on predetermined criteria. The results were then compared with the classifications previously allocated using data derived from in vitro studies. The compounds were examples of non-teratogens, non-cytotoxic teratogens and cytotoxic teratogens. Of the 31 compounds, 23 gave the predicted result, three non-teratogens being false positives and five teratogens having no effect in this system. The results therefore demonstrate the ability of the technique to distinguish between the three categories of compound, displaying a specificity of 75%, a sensitivity of 74% and an overall accuracy of 74.5%. Furthermore, it allowed the differentiation of active and inactive analogues of four paired compounds. This method may have applications as a teratogenicity screen or in the elucidation of the mechanism of action of teratogenic compounds.

Intravitelline injection of rodent conceptuses: An improved in vitro developmental toxicity screen.

Many in vitro developmental toxicity screens are of limited usefulness, either because they lack the cell interactions of a whole embryo or because it is difficult to attribute an effect to the substance itself or a generated metabolite. Intravitelline injection into rat conceptuses during organogenesis bypasses the metabolically active visceral yolk sac and allows accurate delivery of specific drugs or their metabolites directly to the embryo. In a blind trial using 31 coded compounds, an overall accuracy of approximately 74% was achieved and four pairs of active/inactive analogues were distinguished. Additionally, thalidomide was shown to have teratogenic effects at low doses, mainly in the neural tube region.

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.

Intravitelline injection of cultured rat embryos: An improved method for the identification of cytotoxic and non-cytotoxic teratogens.

A preliminary study of a novel developmental toxicity screen has been carried out. The technique involves the direct injection into the vitelline circulation of the 11.5-day rat conceptus, by-passing the metabolically active visceral yolk sac. The evaluation was performed blind using four coded model compounds: sulphanilamide (non-cytotoxic, non-teratogen), retinoic acid (teratogen) and methotrexate and cyclophosphamide (both cytotoxic teratogens). Seven parameters of teratogenicity and cytotoxicity were measured (yolk sac diameter, crown-rump length, somite number, yolk sac protein, yolk sac DNA, embryo protein, embryo DNA) and morphological abnormalities were also noted. The results showed that this technique successfully identified the developmental toxins and, moreover, differentiated between teratogens and cytotoxic teratogens. Additionally, the results show that methotrexate and cyclophosphamide produced an effect without prior exogenous activation as is necessary in other in vitro tests.