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The pure chromophobes isolated from adenohypophyseal cells of rats were successfully cultured by the roller tube method in a serum-free NCTC-109 medium supplemented with nucleic acids and l-thyroxine. Upon light and electron microscopy, some chromophobes cultured in the control media proliferated by mitosis and others differentiated into immature ambiguous cells. The assay of ACTH, GH, and prolactin showed that these control explants produced a small amount of ACTH and GH, but a large amount of prolactin. When cultured in a CRF (corticotrophin releasing factor) medium, most chromophobes differentiated continuously into acidophils, but not into basophils. Most cells cultured in a CRF medium accumulated granules of various sizes. Since the incorporation of 3H-thymidine into DNA of chromophobes and ambiguous cells reached a maximum at an early stage of their culture, these two cells were assumed to be the maternal cells with the proliferating activity. When the chromophobes were kept in the media containing CRF and colchicine, their cell division was arrested at metaphase. With evident decrease in number of the chromophobes and ambiguous cells, many cells of the explants turned into acidophilic cells providing protein synthesizing ability. Thus the virtual transformation of the chromophobes into acidophils was induced. The explants in a CRF-colchicine medium produced a large amount of ACTH exclusively, and produced neither GH nor prolactin irrespective of the different sizes of granules. During the time course of CRF culture the 3H-thymidine incorporation into nucelar DNA was low when the ACTH content of explants was high, and vice versa. As CRF content of the media was increased, the peak of volumetric percentage of acidophils in the explants appeared earlier. The chromophobes may be dynamic rather than static in a mode of transformation into acidophils following the addition of CRF in to the medium. The cells which were differentiating on the acidophil axis could be, therefore, regarded as ACTH producers in reference to bioassay data.

Effect of CRF on the Morphological and Functional Differentiation of the Cultured Chromophobes Isolated from Rat Anterior Pituitaries