Jj. Picard

Morphogenesis and quantification of the development of post-implantation mouse embryos.

This paper describes potential improvements in the quantitative assessment of the differentiation of rodent embryos used for in vitro embryotoxicity studies. A chart of schematic illustrations of the developmental stages of seventeen morphological features observed macroscopically in mouse embryos aged 8-10 days (0-30 somites) has been drawn up. The chart is based on the morphological scoring system proposed by Brown and Fabro (1981) for rat embryos and complements the original descriptions. Some intermediate stages have been added to the scoring system. The original and the modified scoring systems were applied to 310 mouse embryos in 31 groups of ten embryos, each with 0 to 30 somites. The modified score is consistently about 25% higher than the original score. The correlation of both the original and the modified scores with the number of somites is best expressed by an asymmetric sigmoid. The chart and the modified scoring system could also be used, with minor adaptations, to assess rat embryos at corresponding developmental stages.

Interlaboratory evaluation of three culture media for postimplantation rodent embryos

The first aim of the study was to compare the ability of rat serum, human serum, and a mixture of human and rat serum (4:1) to support in vitro development of rodent postimplantation embryos. The comparison was made in three laboratories using rat embryos and in one laboratory using mouse embryos. Batches of sera, initial developmental stage, duration of culture, and endpoints were identical in the laboratories. The second aim of the study was to evaluate if other variables that could not be standardized would significantly influence the results of the laboratories. No reproducible difference was observed among the culture media or among the laboratories except that growth and differentiation were slower in the laboratory using mouse embryos. Further experiments are needed to exclude small differences in performance of the media.

Developmental table of the early mouse post-implantation embryo.

The developmental tables of early somite mouse embryos that are presently available from the literature give clear and useful descriptions of the differentiation at successive stages. However, they provide no easy access to the correlation between the growth of the embryo and its differentiation. In the present study, quantitative data concerning normal mouse embryonic development as well as the major developmental events occurring between 0 and 30 pairs of somites were established. Measurements of growth (crown-rump length, head length, absorbancy at 280 nm) and differentiation parameters (morphological score) of 168 to 310 explanted mouse embryos were recorded for each developmental stage (number of pairs of somites). A short description of the major events occurring at the corresponding stages is also presented. The table is more detailed than those presently available and provides a rapid and practical overview of the timing of the appearance of developmental events and differentiation in correlation to the progressive growth of the embryo. It could, therefore, be useful for embryologists and toxicologists. In addition, the development of 67 post-implantation mouse embryos cultured in vitro was compared with the reference table established from in vivo embryos. Our results confirm and extend previous reports showing that embryos cultured in vitro grow and differentiate at a pace very similar to that of embryos developed in vivo.

Defects in the development of branchial nerves and ganglia induced by in vitro exposure of mouse embryos to mercuric chloride.

The embryotoxic and dysmorphogenic effects of mercuric chloride (HgCl2) have been studied in mouse embryos cultured in vitro. In addition, the alterations induced in the developing branchial nerves and ganglia were analyzed. Mouse embryos with 6-8 pairs of somites were exposed for 26 hr to increasing concentrations (0, 12.5, 25, 50 microM) of HgCl2. After this period, a first set of embryos was removed and a second set of embryos transferred to culture medium without HgCl2 and remained in culture for an additional 22 hr. Both sets of embryos were examined for (1) survival, (2) presence of external dysmorphogenesis, (3) growth, and (4) differentiation. Dose-related alterations of these parameters were observed. The main target was the cephalic neural tube (mainly the forebrain), but several other systems were also affected (e.g., the turning of the embryos, the optic system). The 48-hr cultured embryos were immunostained using a monoclonal antineurofilament antibody to visualize defects in the development of branchial nerves and ganglia. HgCl2 induced a pronounced retardation in the differentiation of ganglion/nerve V and a slight retardation in the differentiation of ganglia/nerves VII and IX. The ganglia/nerves VIII and X were not retarded. In addition, hight percentages of abnormalities of ganglion/nerve V and fusions between ganglia/nerves IX and X were observed in these embryos. Disorganized fibers between ganglia/nerves VII-VIII and IX and between ganglia/nerves IX and X were also more frequently observed. At the highest concentration, asymmetric defects were induced by HgCl2 with a more pronounced effect observed on the right side of the embryos. These results demonstrate the usefulness of this approach in evaluating the susceptibility of the developing branchial nerves to the adverse effects of developmental toxicants.

Effects of retinoic acid, auranofin and mercuric chloride on plasminogen activator activity in post-implantation cultured mouse embryos.

Plasminogen activator (PA) activity has been suggested to be an important determinant of cell migration and tissue modelling during organogenesis. We have postulated that in the developing embryo, any abnormal modulation of this enzymatic activity may lead to the production of teratogenic effects. In the present study, we investigated the effect of teratogenic doses (inducing about 50% of malformed embryos) of retinoic acid, auranofin and mercuric chloride on PA activity in post-implantation cultured mouse embryos. At the end of the culture period, PA activities of malformed and normal embryos in the same treatment group were compared. PA activities in compound-exposed embryos were also compared with those in untreated controls. The design of the present experiment allowed the identification of the effect of drugs on PA activity and its possible relation with induced malformations. An increased PA activity was observed in malformed embryos treated with mercuric chloride. PA activity was slightly increased in both groups (normal and malformed) exposed to retinoic acid. No effect of auranofin was observed on embryo PA activity. In conclusion, the data do not confirm but they also do not contradict the hypothesis that abnormal levels of PA activity lead to dysmorphogenesis.