Demonstration of nuclear 3,5,3'-triiodothyronine receptor proteins in gonadotrophs and corticotrophs in rat anterior pituitary by double immunohistochemical staining.

Previously we raised an antiserum (4BII) against nuclear T3 receptor (NT3R), which recognizes c-erb A/NT3R alpha 1 and beta, but not alpha 2. An immunohistochemical study using 4BII revealed that among the tissues examined, the cerebral cortex, anterior pituitary, and thyroid gland exhibited strong immunostaining in the nuclei. In the present study we examined the localization of NT3R proteins in individual cell types of rat anterior pituitary, using the technique of double immunostaining with 4BII and antibodies against pituitary hormones. The cryostat sections and paraffin sections of rat pituitary were incubated with 4BII at 4 C overnight. The NT3R proteins were visualized as brown in the nuclei by avidin-biotin peroxidase staining. The control sections incubated with an antiserum which had previously absorbed with c-erb A peptide or an inactive antiserum showed very poor nuclear staining under the same condition. After being washed with 0.1 M glycine-HCl buffer (pH 2.2) for 2 h to remove 4BII, the sections were incubated with antiserum against individual anterior pituitary hormones at 4 C overnight. Using the method of alkali phosphatase/naphthol/Fast blue staining, the peptide hormones in the cytosol were stained blue. Double staining with 4BII and anti-FSH beta or anti-LH beta anti-serum clearly demonstrated that gonadotrophs contained the NT3R proteins. Similarly, the NT3R proteins in corticotrophs were demonstrated by the immunostaining with 4BII and anti-ACTH antiserum. As expected, the NT3R proteins were present in somatotrophs, thyrotrophs, and lactotrophs. On the other hand, folliculo-stellate cells, which are nonhormone secreting cells and are identified by their S-100 protein immunoreactivity, were stained by 4BII only faintly. The present study demonstrated the existence of NT3R proteins not only in somatotrophs, thyrotrophs and lactotrophs but also gonadotrophs and corticotrophs, suggesting some action of thyroid hormone through the receptor in these cells.