Cortisol-Induced Malformation in an In Vitro Developmental Model

Top of pageAbstract Extract: Morphologic effects of cortisol upon development were studied in an in vitro model in which populations of free mesenchymal cells, derived from embryonic mouse limb bud, form a heterogeneous tissue, and in five to six days develop characteristic cartilaginous and muscular elements (fig. 2). Development takes place in microwells with bases of a cellulose ester filter. Cortisol was incorporated into the culture medium at the concentrations shown in table I at various stages in culture (first to fourth day) and for variable periods of time (two hours to six days). The initial cell population density on the filter membrane was varied (table II). Cortisol concentrations of 1.4 × 10-5 M and 7.0 × 10-5 M for up to six days had no visible effects on formation of a tissue syncytium or subsequent differentiation. Concentrations of 1.1 × 10-3 M for longer than 48 hours led to death of the bulk of cells. Intermediate concentrations or shorter exposures were compatible with tissue viability but were associated with morphologic abnormalities. These abnormalities largely resulted from interference with cell aggregation, chondrogenesis and myogenesis. The formation of compact cellular aggregates was impaired by exposure to cortisol concentrations between 1.4 × 10-4 M and 1.1 × 10-3 M. Varying degrees of inhibition were seen, extending from moderate limitation of compact aggregate size, alteration in shape and loss of distinct aggregate margins to total substitution of loose clusters of cells for compact aggregates. The severity of effects was directly related to cortisol concentration and duration of exposure and inversely related to initial cell population density (table III). Later stages in the development of compact aggregates were inhibited when exposure began at some time after the first day. Neither inhibition of cell movement on the filter surface nor restriction in cell population by cortisol appeared to be major factors interfering with aggregation. When exposure was discontinued prior to three days, compact aggregation resumed. Chondrogenesis was totally inhibited by cortisol concentrations of 1.1 × 10-3 M for more than one day and partially inhibited by concentrations of 7.0 × 10-4 M and 3.5 × 10-4 M. In the latter instances, the effects varied with stage of exposure. Exposures for the first two or three days of culture resulted in fewer cartilage centers, confined mainly to the membrane center. Similar exposures during the last three days of culture restricted the size of cartilage centers but did not affect distribution on the filter surface; such exposures were associated with diminished intercellular metachromasia upon staining with toluidine blue. With exposure for the entire culture period, chondrogenesis was uncommon even at such lower concentrations. Myogenesis: was inhibited at concentrations of 2.0 × 10-4 M or greater. Exposure in the first two days of culture impaired bipolar transformation of myoblasts. Exposure at a later time inhibited multinucleated myoblast and myotube development and restricted the number of myogenic cells. Speculation: The present investigation demonstrates that structural anomalies can be produced by a glucocorticoid in an in vitro developmental model and their evolution observed. The model is suitable for studying features of abnormal development because of the simplified morphogenesis inherent in the system. It offers opportunities for varying not only the conditions of exposure but also the cell source. The degree of relevance of the model to the in vivo situation, however, remains to be identified. Keywords: Chondrogenesis; cortisol; mesenchymal cells; myogenesis; teratogenesis