Exocytosis sensitivity to growth hormone-releasing hormone in subsets of GH cells in rats under different corticosterone conditions. Ultrastructural study using microwave irradiation for fixation and immunocytochemistry

Abstract

Growth hormone (GH) cells in the rat anterior pituitary have been morphologically classified into three subtypes: type I (mature) containing large secretory granules about 350 nm in diameter, type II (intermediate) containing a mixture of large and small granules, and type III (immature) containing small granules about 150 nm in diameter. However, the functional implications of morphological heterogeneity, especially the different sensitivities to growth hormone-releasing hormone (GRH) under different corticosteroid conditions have not been elucidated to date.In the present study, by application of microwave irradiation (MWI) for fixation and immunocytochemistry, new findings of the exocytotic response have been revealed among the subsets of GH cells following adrenalectomy (ADX), corticosterone treatment and/or GRH treatment.The MWI gave effective results for fixation, especially for the permeability of the fixative, and showed good results for immunoelectron microscopy using the protein-A gold method. Moreover, the use of MWI greatly shortened the fixation, processing and immunolabeling times without compromising the quality of ultrastructural preservation and the specificity of labeling.The number of exocytotic figures was low in all subtypes of GH cells in the sham-operated control rats. GRH treatment induced a significant increase in exocytosis in each subtype of GH cells, particularly in type I (mature) and type II (intermediate) GH cells in the control rats. GRH injection to rats for 4 days after ADX also showed an increase in exocytosis, but the degree was significantly less in comparison with the GRH injection in the control group. Corticosterone replacement given to ADX rats induced a clear recovery of the exocytotic response to GRH to the control level. Serum GH content measured by radioimmunoassay correlated with these morphological results.These results suggest that the secretion of GH stimulated by GRH is closely related to corticosteroids, and that the sensitivity to GRH differs among the three subtypes of GH cells.

PICK1 Deficiency Impairs Secretory Vesicle Biogenesis and Leads to Growth Retardation and Decreased Glucose Tolerance

“…Interestingly, in pituitaries from PICK1-deficient mice, the GH staining appeared less intense and less well organized, with no or only very few cells showing the WT pattern (Figure 5A). Electron microscopy confirmed the difference in intracellular structure of the GH-producing cells (identified by their characteristic architecture [30]). In the PICK1-deficient GH cells, we observed a reduction in the number of secretory dense core vesicles (DCVs) compared to WT cells (Figure 5B–C).…”

Macrophage SREBP1 regulates skeletal muscle regeneration

“…Specimens for electron microscopic observation were prepared as described previously with slight modification ( 46 ). Briefly, under deep anesthesia, mice were perfused through the left cardiac ventricle with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) followed by physiological saline.…”

Microwave irradiation for fixation and immunostaining of endothelial cells in situ

“…Recent technological advances in microwave irradiation devices have also allowed greater control of the irradiation level, irradiation time, and intermittent microwave irradiation (Kumada et al 2004, Temel et al 2005, 2006; therefore, microwave irradiation should be applicable for many histological techniques including immunostaining and electron microscopy (Cavuşog lu et al 1998, Kahveci et al 2003, Koga et al 2003, Ozawa et al 2004). We established a method for rapid fixation and immunofluorescence microscopy using intermittent microwave irradiation.…”