Reproductive Toxicology, Vol. 5, pp. 265-271, 1991 0890-6238/91 $3.00 + .00 Printed in the U.S.A. Copyright © 1991 Pergamon Press plc

• Abstracts

Validation studies: definitions, problems and practice. Schmid, Beat P. Dept. of Toxicology and Kinetics, Zyma S.A., CH - 1260 Nyon, Switzerland.

Numerous in vitro systems have been developed during the last decade in several areas of toxicology. To derive the optimal value from these systems, they have to be fully and properly validated. The ultimate aim of the validation process is to obtain reliable and relevant methods that can be used for specific purposes in toxicity testing. The major steps in this process can be formally divided into the phases: test develop- ment, intralaboratory assessment, interlaboratory assessment, test database development, evaluation, and acceptance. These general considerations of course, also hold true for teratoge- nicity testing. Using the rat whole embryo culture technique as an example, the system's stages of validation will be high- lighted. Also, the system's application in the 3 following areas will be presented: i) screening purposes (eg, imidazoles), ii) use as adjunct test (eg, pesticides), and iii) use as replacement test (eg, combination products).

Differential yolk sac and facial development in cultured C57BL/6 mouse embryos. Asada S, Nemoto Y,* Osumi- Yamashita N, Eto K. Tokyo Medical and Dental University, Tokyo 113; *Japan Women's University, Tokyo 112.

The organogenesis-stage mouse embryo culture system used in our laboratory uses the yolk sac placenta as the main organ of oxygen transfer. For further development of the embryo in culture after late day 10 of gestation (plug days), the yolk sac is opened and amnion is removed so that more oxygen is supplied to the embryo by direct exposure to the flowing medium. Cultured C57BL mouse embryos with the open yolk sac (OYS) at the stage of 9 tall somites and below showed facial malformations (cleft lip) corresponding the stage of OYS, but no cleft lip was shown in the embryos with OYS at more than 10 tail somites, although there were no significant differences in the protein content and somite num- bers of cultured embryos. In addition, presumptive adhesive epithelial cells, which usually appeared in the fusion area of facial prominences, were not observed in embryos with OYS of 9 tall somites and below by SEM.

These results suggest that amniotic fluid containing a product of yolk sac or culture medium (rat serum) might have a critical role in the differentiation of epithelial cells of facial prominences.

A maternal serum protein localizes at the site of neurula- tion in the mouse embryo. Copp A J, Estibeiro JP, Brook FA. Imperial Cancer Research Fund, Developmental Biology Unit, Department of Zoology, South Parks Road, Oxford OX1 3PS.

In order to study the molecular basis of mammalian neurulation, we screened 8.5- to 11.5-day mouse embryos for proteins associated with neural tube closure. Using 2-dimen- sional SDS-polyacrylamide gel electrophoresis, combined with silver staining, we identified a protein (Mr 88 kD, pI 6.8) that is absent before the onset of neurulation, present during closure of the neural tube, and disappears soon after neurula- tion is complete. We attempted to label this protein by culturing intact mouse embryos in rat serum containing either 35S-methionine or a mixture of 14C-amino acids. In both cases the 88 kD protein failed to label, although other proteins showed good isotope incorporation. This result suggested that the 88 kD protein may originate outside the embryo, and we confirmed this by the following culture experiments. We found that the rat version of the 88 kD protein migrates to a slightly different position on gels than the mouse version of the protein. Mouse embryos after 24-h culture in rat serum contain the rat protein, whereas rat embryos cultured in mouse serum contain the mouse protein. We conclude that a serum protein enters the embryo and localizes at the site of neural tube closure in rodent embryos. It is possible that the 88 kD protein plays a significant role in the neurulation process.

Cerebral vascularization during rat embryo culture. Fuki- ishi Y, Fujimoto M, Hasegawa Y, Asada S,* Yamashita N,* Eto K.* Shionogi Res. Labs., Shionogi & Co. Ltd., Osaka, Japan; and *Inst. Stomatognathic Science, Facl. Dent., Tokyo IVied. Dent. Univ., Tokyo, Japan.

Cerebral vascularization was investigated histologically in the telencephalic region of normal in vivo and in vitro rat embryos, and in the presence of the anti-angiogenetic factor, protamine. In normal in vivo embryos, the earliest penetration of the capillary endothelial ceils into the neuroepithelium occurred at the 3-tall somite (TS) stage on day 11 of preg- nancy. Penetration became profound vertically, and horizontal vascularization was observed in in vivo embryos with more than 11 TS on day 12. After 20- to 24-h culture, embryos cultured from day 11 showed normal morphologic develop- ment with respect to somite number, craniofacial develop- ment, and vascularization of the telencephalic neuroepithe- lium. At start of culture at 11 day, protamine was injected into the amniotic cavity, or into the telencephalic region (neural tube lumen or juxtatelencephalic mesenchyme), or added to the culture medium. Brain angiogenesis was inhibited by protamine injection into the anmiotic cavity, but not by injection into the embryonic structure, suggesting that access of protamine to its site of action (endothelial cells) depends on the route of administration. These results indicate that vascu- larization in the telencephalic region progresses normally in cultured rat embryos and that the culture system provides a suitable means of analyzing brain angiogenesis.

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