A Practical Approach, Second Edition=Ronald D. Ho.pdf

IN VITRO METHODS FOR THE STUDY OF MECHANISMS OF DEVELOPMENTAL TOXICOLOGY 677with this approach to elucidate the role of growth factor expression (EGF, TGF-α, and TGF-β) inaltered palatogenesis. 228,229 These studies could not demonstrate that perturbation of growth factorreceptors was the sole cause of the altered palatal growth and differentiation, but did indicate thattheir role is important. The perturbation of neural crest cell migration to the appropriate mesenchymalenvironments has also been investigated, suggesting additional possible mechanisms bywhich palatal development can be altered. 230 Molecular studies are easily conducted by use of thissystem but are not covered in this section.3. Visceral Yolk Sac CulturesThe visceral yolk sac plays a vital role in the nourishment, metabolism, and protection of thedeveloping conceptus, especially during the sensitive period of early organogenesis. The invertedVYS epithelium is structured to have the brush border facing the decidual blood lumen. Thisfacilitates the uptake by pinocytosis and the enzymatic degradation of maternal histiotroph (proteinsand other macromolecules derived from the decidual cell mass and from a transudate of the maternalserum) and the transport of carbohydrates, amino acids, and other nutrients. The rodent VYS isdistinct from those of other mammalian species because it retains its form and function until term.A number of embryotoxins and teratogens have been reported to alter VYS function, and theireffects on this organ have been suggested as central to their mechanisms of toxicity. The teratogentrypan blue was first shown to elicit its deleterious effects via inhibition of pinocytosis and/orproteolysis by the VYS. 231 Since these reports, other agents (e.g., leupeptin, anti-VYS antibody,chloroquine, diazomethyl ketones, E-64) have been shown to elicit their effects through inhibitionof pinocytosis or through inhibition of lysosomal proteolysis. 130–137,232–234Because of its direct contact with the culture environment, a number of direct exposures andmanipulations can be performed on the VYS in whole embryo culture without compromisingconceptal viability and function. Many of the characterizations of VYS function, however, used adetached, free-floating VYS in a roller culture system. These studies were used successfully todetermine the extent of proteolytic and pinocytotic activity in the VYS and to characterize changesin these activities elicited through chemical exposure or anti–yolk sac antibody recognition.114,115,233,235 Similar approaches were taken to evaluate the mechanisms of uptake of othermacromolecules, such as antibodies, ferritin, and enzymes. Relatively few studies have beenconducted to determine specific effects of chemicals on the VYS epithelium, endothelium, or stemcell populations, even though considerable evidence exists to support an extensive role of the VYSin several nutritional, metabolic, and physiologic functions.Organ culture of the VYS can also be accomplished at a level of biological organizationintermediate between whole embryo culture and the isolated VYS. These investigations utilize“giant yolk sacs,” in which conceptuses are explanted at an early somite stage (GD 9 in the rat),and the early or presomite-stage embryos are surgically ablated using dissecting needles. 139 Returnof the severed conceptus to culture results in the continued growth of only the VYS. Such preparationshave been shown to retain numerous vital functional and structural characteristics withoutinterference from the embryo proper or interactions associated with the vitelline circulation. Becauseof its ability to trap and sequester products in the exoceloemic fluid, the closed yolk sac that resultsfrom this procedure may have a number of advantages in studies of mechanism involving directionaltransport across the VYS epithelial barrier. It has not been clearly delineated, however, how a lackof vitelline circulation and possible hydrodynamic factors might affect VYS function or response.Nevertheless, such preparations could be of use in determining the mechanisms and effects ofchemical induction of avascular yolk sacs. 161Organ cultures similar to the examples cited above have been used to investigate probablemechanisms that are specific to the functions of other organ systems. Some examples are brieflymentioned in the following sections.© 2006 by Taylor & Francis Group, LLC