SUMMARY The anterior pituitary of the rat is used as a model for the study of the effects of freezing or plastic embedding on the maintenance of antigenicity. Rat anterior pituitaries are fixed in 2.5% glutaraldehyde in 0.1 m phosphate buffer pH 7.4. Some of the blocks are post‐fixed before being divided into two lots. One batch is frozen, while the other is dehydrated and embedded. The indirect antibody enzyme method is applied to ultrathin sections obtained by cryoultramicrotomy after freezing or by sectioning after embedding. All six pituitary hormones are detected by both methods. Comparison shows that the morphological characteristics are identical for both techniques, though ultrastructural preservation is better after embedding. Immunoreactivity is found in secretory granules and sometimes in the endoplasmic reticulum. Osmium postfixation may reduce or even abolish antigenicity in plastic‐embedded tissue. After cryoultramicrotomy, however, even after osmium fixation, antibody may be used 1000 times more diluted than after plastic embedding. Embedding preserves ultrastructure and limited antigenicity while the use of cryoultramicrotomy is a far more sensitive technique.

Growth hormone-releasing factor-like immunoreactivity was visualized in monkey pituitary gland by immunocytochemistry on ultrathin sections obtained by cryoultramicrotomy. Antibodies were raised against synthetic human pancreas growth hormone-releasing factor (1–40). Growth hormone-releasing factor-like immunoreactivity was observed in somatotropes (identified by immunocytochemistry) only. The other pituitary cell types were not immunoreactive. In somatotropes, immunoreactivity was observed at the plasma membrane but only very scarcely, in the cytoplasm (cytoplasmic matrix and secretory granules) and in the nucleus. These results (1) provide immunocytological evidence for the presence of growth hormone-releasing factor or of an immunoreactive fragment of its molecule in the pituitary; (2) indicate the presence of this peptide in one particular pituitary cell type; and (3) provide cytological evidence for direct participation of growth hormone-releasing factor in the regulation of the somatotropic function.

The tetradecapeptide, somatostatin (SRIF), is a potent inhibitor on pituitary hormone release, by a direct effect. The immunocytological method was used with the aim of localizing SRIF at the cellular and subcellular levels. Rat pituitaries were fixed and frozen. Ultrathin sections, obtained by cryoultramicrotomy, were incubated with anti-SRIF serum. The antigen-antibody reaction was detected by 4-chloro-1-naphthol. SRIF immunoreactivity was observed in somatotrophs, thyreotrophs and prolactin cells, but not in corticotrophs or gonadotrophs. In immunoreactive cells, SRIF was found in the cytoplasmic matrix, in the secretory granules and in the nucleus distributed primarily in the euchromatin, in the vicinity of the heterochromatin regions. SRIF immunoreactivity was also observed at the plasma membrane. No immunoreactivity was observed when nonimmune serum or anti-SRIF serum incubated with SRIF was used. No modification was observed when anti-SRIF serum incubated with gonadoliberin, thyroliberin or vasopressin was used. These data (1) provide immunocytological evidence for the presence of somatostatin in pituitary gland, and (2) indicate the presence of SRIF peptide in the somatotrophs, thyreotrophs and prolactin cells.

Endogenous SS14- as well as SS28-like immunoreactive materials were detected in both male and female rats by radioimmunoassay and by immunocytochemistry on ultrathin frozen sections. The content of somatostatin-like immunoreactivity was 0.39 +/- 0.08 pg per mg adenohypophysis. Immunoreactivity was localized by immunocytochemistry in three pituitary cell types: somatotrophs, lactotrophs and thyrotrophs, but not in corticotrophs and gonadotrophs. In these three pituitary cell types the SS28- and the SS14-like immunoreactive materials were localized in the cytoplasm and in the nucleus. In the cytoplasm the immunoreactivity was seen in the cytoplasmic matrix and in the secretory granules. In the nucleus it was present mainly in the euchromatin close to the heterochromatin. In somatotrophs and lactotrophs, SS14- and SS28-like immunoreactive materials have been detected at the plasma membrane level. These results suggest that (1) endogenous SS14 and SS28 are present in adenohypophysis in somatotrophs, lactotrophs and thyrotrophs, and (2) the two peptides act on both the cytoplasmic components and the nucleus.

Internalization of 125I-labelled atrial natriuretic peptide ([ 125I]ANP) by rat adrenal glomerulosa cells in vivo was investigated by means of an ultrastructural autoradiographic approach. One to 30 min after IV injection of [125I]ANP, silver grains were found, at the light microscope level, over all glomerulosa cells; coinjection of 20 micrograms of unlabelled ANP inhibited this binding by 64%. At the electron microscope level, the time-course study indicated maximal silver grain densities in plasma membranes 1 min after IV injection; grains were detected in mitochondria (external membranes and matrix) 2 min after injection, with maximal labelling at 15 min. The cytoplasmic matrix was labelled only 30 min after injection. During the time-course, labelling of nuclei, Golgi apparatus, and lysosomes was minimal. The data suggest that after binding to plasma membranes ANP is rapidly internalized and distributed within glomerulosa cells. The association of radioactivity with mitochondria suggests that ANP may have intracellular sites of action complementary to those on plasma membranes.