Cytological Differences in the Localization of Glucocorticoid Receptor-Like Imnunoreactivity in the Normal and Transplanted Pituitary Pars Intermedia

Glucocorticoid receptor-like immunoreactivity (GCRI) was found in the normal pituitary pars intermedia (PI) when immunohistochemistry was used. Since in previous studies we described two kinds of cells in the denervated (grafted) PI, i.e., “light cells” (overactive cells which do not contain detectable melanocyte stimulating hormone) and “dark cells” (hypoactive cells which contain the hormone), it was decided to investigate whether different patterns of distribution of the receptors could be detected in the grafted gland when compared with the intact PI. Intact glands showed the receptors located in the nucleus. In transplanted glands, it was observed that light cells showed receptors in both the nuclei and the cytoplasm; on the other hand, dark cells displayed them in the nuclei only, as is the case in all cells of the normal PI. We had previously interpreted dark cells as dopamine-indifferent, whereas light cells were considered dopamine-sensitive. The changes in the distribution of GCR after denervation by grafting, which only affected the light cells, support the view of other authors that GCR of. the pars intermedia are under the influence of dopamine and reinforce our opinion that dark cells are dopamine-indifferent


INTRODUCTION
So far, only two groups have investigated by immunohistochemistry the presence of glucocorticoid receptors (GCR) in the normal pars intermedia (PI). Each group concluded with opposing results, i.e., Antakly et al. could not dete _rmJne the existence of GCR /1,2/ whereas Bertini et al. found them/4/; however, authors agree that decentralization of the PI induces the development of such receptors due to the absence of dopaminergic innervation [3,9/. In previous studies/6/we described two kinds of cells in the denervated (grafted) PI, i.e., "light cells" (overactive cells which do not contain detectable melanocyte-stimulating hormone [MSH] and "dark cells" (hypoactive cells which contain the hormone). Thus it was decided to investigate whether different patterns of distribution of the receptors could be detected in the grafted gland when compared with the intact PI.

MATERIALS AND METHODS
Normal. adult male and female Sprague-Dawley rats were used. Twenty normal pituitaries and nine neurointermediate lobes, which were decentralized by gaffing them beneath the kidney capsule for 20 days, were studied. Glands were fixed in Elftman's fluid/5/for 3 h and embedded in paraffin. Normal glands were inves-VOL. 3 To test the specificity of the immunohistochemical reaction for GCR the following controls were performed: (a) one of the steps of the immunohistochemical reaction was omitted and, (b) unspecific mouse ascitic fluid was used instead of the primary antibodies.

RESULTS
By using this antiserum the presence of GCRI in normal rats could be determined in every nucleus of the PI cells (Fig. 1). Cytoplasmic staining was not observed. In grafted glands, nuclei preserved their staining property; nevertheless, a marked difference could be seen after comparing light and dark cell cytoplasm. As described in our previous paper /6/, MSHstaining made it possible to differentiate cells containing granules of the hormone (dark cells) from cells devoid of granules (light cells) (Fig. 3).
While light cells showed the presence of GCRI in their cytoplasm after grafting, dark cells remained with a GCRI pattern similar to that of non-transplanted glands (Fig. 2). Controls did not stain at all.
A section from the pituitary of a normal rat stained for GCR. In the pars intermedla (Pl), nuclei, but not cytoplasm, appear positively stained. In the pars distalis (PD) the reaction is stronger and cells displaying either positive (P) or negative (arrow heads) stained cytoplasm are depicted, x 150 DISCUSSION Using the same antiserum as the one used by Bertini et al./4/, we corroborated their findings, namely that the PI actually contains GCRI. As expected, and according to a current view in the field of GCR/11/, they were present in the cell nucleus. Our observations and those of Bertini et al. /4/ are not in accordance with the ones of Antakly et aL/1,2/, who could not determine the existence of the receptors in the normal PI. The reason for the discrepancy might be that the monoclonal antibodies used by us were more sensitive and specific for GCR. Furthermore, we did not prepare cryostat sections since paraffin sections were suitable for the immunostaining; presumably, we succeeded in the staining either because we used Elftman's fixative/5/or because the concentration at which the first antibody was used was relatively high. When Bertini et al. /4/ investigated GCRI with the electron microscope, they observed that some of the receptors were located in the cytoplasm of the normal PI as well. In our light microscope specimens we could not observe such a localization in the normal gland (indeed, electron microscopy should be more sensitive for the reaction products), but we did in light cells of the grafted PI, thus indicating that there was an appreciable increase of receptors in these cells after denervation of the gland. Whether this increase expresses a modification in the wellknown traffic of receptors from the cytoplasm to the nucleus, or corresponds to an increase in receptor synthesis has not yet been ascertained.
We have previously published/6/that, after cessation of dopamine innervation, cells of the PI differentiate into two kinds, i.e., light cells, which were deprived of MSH storage and showed signs of hyperactivation, and dark cells, containing MSH-secretory granules. Light cells were interpreted as dopamine sensitive, whereas dark cells were considered dopamine indifferent; furthermore, we have suggested /7/ that light cells in the grafts secrete ACqT-/ or AC"I analogs instead of MSH; on the other hand, we thought that dark cells possess little or no secretory activity. The development of GCRI in the cytoplasm of the dopamine sensitive light cells speaks in favor of a dependence between dopamine and GCRI in the PI, as published by other authors, who, however, did not detect two cell kinds in the graft/3,9/; moreover, the present observations would reinforce our previous opinion/6/that dark cells, herein described as not showing appreciable changes in the distribution of GCRI after denervation, are dopamine indifferent.