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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

Hitoshi Ozawa

Hitoshi Ozawa Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kawaramachi-Hirokoji, Kamigyo-Ku, Kyoto 602-8566, Japan

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Fang Han

Fang Han Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kawaramachi-Hirokoji, Kamigyo-Ku, Kyoto 602-8566, Japan

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Mitsuhiro Kawata

Mitsuhiro Kawata Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kawaramachi-Hirokoji, Kamigyo-Ku, Kyoto 602-8566, Japan

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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.

Article Type:: Research Article

DOI:: https://doi.org/10.1677/joe.1.05656

Volume/Issue:: Volume 183: Issue 3

Free access

Specific expression of an oxytocin-enhanced cyan fluorescent protein fusion transgene in the rat hypothalamus and posterior pituitary

Akiko Katoh

Akiko Katoh Departments of, Physiology, Otorhynolaryngology, Department of Physiology, Section on Neural Gene Expression, Department of Cellular Neurophysiology, Department of Anatomy and Neurobiology, Molecular Neuroendocrinology Research Group, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan
Departments of, Physiology, Otorhynolaryngology, Department of Physiology, Section on Neural Gene Expression, Department of Cellular Neurophysiology, Department of Anatomy and Neurobiology, Molecular Neuroendocrinology Research Group, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan

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Hiroaki Fujihara

Hiroaki Fujihara Departments of, Physiology, Otorhynolaryngology, Department of Physiology, Section on Neural Gene Expression, Department of Cellular Neurophysiology, Department of Anatomy and Neurobiology, Molecular Neuroendocrinology Research Group, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan

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Toyoaki Ohbuchi

Toyoaki Ohbuchi Departments of, Physiology, Otorhynolaryngology, Department of Physiology, Section on Neural Gene Expression, Department of Cellular Neurophysiology, Department of Anatomy and Neurobiology, Molecular Neuroendocrinology Research Group, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan
Departments of, Physiology, Otorhynolaryngology, Department of Physiology, Section on Neural Gene Expression, Department of Cellular Neurophysiology, Department of Anatomy and Neurobiology, Molecular Neuroendocrinology Research Group, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan

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Tatsushi Onaka

Tatsushi Onaka Departments of, Physiology, Otorhynolaryngology, Department of Physiology, Section on Neural Gene Expression, Department of Cellular Neurophysiology, Department of Anatomy and Neurobiology, Molecular Neuroendocrinology Research Group, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan

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W Scott Young III

W Scott Young III Departments of, Physiology, Otorhynolaryngology, Department of Physiology, Section on Neural Gene Expression, Department of Cellular Neurophysiology, Department of Anatomy and Neurobiology, Molecular Neuroendocrinology Research Group, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan

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Govindan Dayanithi

Govindan Dayanithi Departments of, Physiology, Otorhynolaryngology, Department of Physiology, Section on Neural Gene Expression, Department of Cellular Neurophysiology, Department of Anatomy and Neurobiology, Molecular Neuroendocrinology Research Group, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan

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Yuka Yamasaki

Yuka Yamasaki Departments of, Physiology, Otorhynolaryngology, Department of Physiology, Section on Neural Gene Expression, Department of Cellular Neurophysiology, Department of Anatomy and Neurobiology, Molecular Neuroendocrinology Research Group, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan

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Mitsuhiro Kawata

Mitsuhiro Kawata Departments of, Physiology, Otorhynolaryngology, Department of Physiology, Section on Neural Gene Expression, Department of Cellular Neurophysiology, Department of Anatomy and Neurobiology, Molecular Neuroendocrinology Research Group, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan

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Hitoshi Suzuki

Hitoshi Suzuki Departments of, Physiology, Otorhynolaryngology, Department of Physiology, Section on Neural Gene Expression, Department of Cellular Neurophysiology, Department of Anatomy and Neurobiology, Molecular Neuroendocrinology Research Group, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan

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Hiroki Otsubo

Hiroki Otsubo Departments of, Physiology, Otorhynolaryngology, Department of Physiology, Section on Neural Gene Expression, Department of Cellular Neurophysiology, Department of Anatomy and Neurobiology, Molecular Neuroendocrinology Research Group, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan

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Hideaki Suzuki

Hideaki Suzuki Departments of, Physiology, Otorhynolaryngology, Department of Physiology, Section on Neural Gene Expression, Department of Cellular Neurophysiology, Department of Anatomy and Neurobiology, Molecular Neuroendocrinology Research Group, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan

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David Murphy

David Murphy Departments of, Physiology, Otorhynolaryngology, Department of Physiology, Section on Neural Gene Expression, Department of Cellular Neurophysiology, Department of Anatomy and Neurobiology, Molecular Neuroendocrinology Research Group, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan

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Yoichi Ueta

Yoichi Ueta Departments of, Physiology, Otorhynolaryngology, Department of Physiology, Section on Neural Gene Expression, Department of Cellular Neurophysiology, Department of Anatomy and Neurobiology, Molecular Neuroendocrinology Research Group, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan

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We have generated rats bearing an oxytocin (OXT)-enhanced cyan fluorescent protein (eCFP) fusion transgene designed from a murine construct previously shown to be faithfully expressed in transgenic mice. In situ hybridisation histochemistry revealed that the Oxt–eCfp fusion gene was expressed in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) in these rats. The fluorescence emanating from eCFP was observed only in the SON, the PVN, the internal layer of the median eminence and the posterior pituitary (PP). In in vitro preparations, freshly dissociated cells from the SON and axon terminals showed clear eCFP fluorescence. Immunohistochemistry for OXT and arginine vasopressin (AVP) revealed that the eCFP fluorescence co-localises with OXT immunofluorescence, but not with AVP immunofluorescence in the SON and the PVN. Although the expression levels of the Oxt–eCfp fusion gene in the SON and the PVN showed a wide range of variations in transgenic rats, eCFP fluorescence was markedly increased in the SON and the PVN, but decreased in the PP after chronic salt loading. The expression of the Oxt gene was significantly increased in the SON and the PVN after chronic salt loading in both non-transgenic and transgenic rats. Compared with wild-type animals, euhydrated and salt-loaded male and female transgenic rats showed no significant differences in plasma osmolality, sodium concentration and OXT and AVP levels, suggesting that the fusion gene expression did not disturb any physiological processes. These results suggest that our new transgenic rats are a valuable new tool to identify OXT-producing neurones and their terminals.