Research paperImmunohistochemical detection of corticotropin-releasing hormone (CRH) in the brain and pituitary of the hagfish, Eptatretus burgeri
Introduction
The hagfish is commonly thought to represent one of the earliest evolutionary branches among vertebrates (Forey and Janvier, 1993). Hagfishes are included in the class Agnatha with lampreys. In hagfishes and lampreys, the adenohypophysis (AH) is a relatively thin and flat tissue embedded in the connective tissue beneath the neurohypophysis (NH), and the NH and the AH are completely separated (see Nozaki, 2013). Although the AH of the lamprey is divided into the rostral pars distalis (RPD), proximal pars distalis (PPD), and pars intermedia (PI), as in teleost fishes, differentiation of the pars distalis (PD) and PI is not observed in hagfish. Furthermore, while some neuropeptide hormones, such as gonadotropin-releasing hormone (GnRH) and corticotropin-releasing hormone (CRH), are known to exist in lampreys from biochemical study or genome analysis (see Freamat and Sower, 2013, Roberts et al., 2014), no information is available for hagfish. Thus, little is known about the neuroendocrine system of hagfish.
In teleosts, physiological phenomena are regulated by the hypothalamo-pituitary system. For example, gonadal maturation is primarily regulated by the hypothalamo-pituitary-gonadal (HPG) axis. GnRH stimulates the secretion of pituitary gonadotropins (GTHs), and GTHs stimulate the secretion of steroid hormones from the gonads (see Okubo and Nagahama, 2008). In the Pacific hagfish Eptatretus stouti, although GnRH has not been identified biochemically, immunohistochemical localization of GnRH was reported using the antibody raised against salmon GnRH (sGnRH, now called GnRH3). GnRH-immunoreactive (ir) cell bodies were observed in the preoptic region and the infundibular hypothalamus. The axons of GnRH-ir cell bodies in the preoptic region terminate in the NH, and GnRH-ir cell bodies in the infundibular hypothalamus appear to send fibers throughout the brain (Braun et al., 1995). Recently, a functional glycoprotein hormone (GPH) which is thought to be a GTH, has been identified in the brown hagfish Eptatretus atami; an intense immunoreaction to anti-hagfish GPHβ antibody was observed in the cells of the AH, and this hormone induced estradiol and testosterone release in vitro (Uchida et al., 2010). Moreover, it is suggested that estradiol and testosterone are involved in the regulation of pituitary GTH in juvenile brown hagfish (Nozaki et al., 2013). Taking into consideration the distribution of GnRH in the Pacific hagfish Eptatretus stouti, it is suggested that GnRH-GTH-steroid axis exists in the hagfish.
CRH is the key activator of the hypothalamo-pituitary-adrenal (HPA) axis. CRH peptides of all examined species consist of 41 amino acid residues with an amidated C-terminus. The C-terminal region is of particular importance to physiological activity. Human, mouse, and rat CRH have an identical amino acid sequence (Amano, 2015). Stress stimulates the synthesis of CRH in the hypothalamus, which in turn stimulates the synthesis of the proopiomelanocortin (POMC) and cleavage of POMC to adrenocorticotropic hormone (ACTH) in the pituitary (see Pankhurst, 2011). In teleosts, CRH-ir cell bodies in the nucleus preopticus project directly to ACTH cells in the RPD and to α-melanocyte-stimulating hormone (α-MSH) cells in the PI of the pituitary (see Flik et al., 2006). In the Atlantic hagfish Myxine glutinosa, the inshore hagfish Eptatretus burgeri, and the brown hagfish Eptatretus atami, ACTH was detected immunohistochemically in the pituitary using the antibody raised against lamprey ACTH (Nozaki et al., 2005, Nozaki et al., 2007). In the pioneer study of plasma steroids of the Pacific hagfish Eptatretus stouti, it is suggested that the major glucocorticoid in the plasma is corticosterone and that the cortisol levels are detectable by multiple injection of porcine ACTH (Weisbart et al., 1980). Considering that secretion of ACTH is regulated by CRH in vertebrates including teleosts (see Flik et al., 2006), and that corticosteroids exist in the plasma in the hagfish, it is reasonable to speculate that CRH is also present in the hagfish. However, no information is available as to the existence of CRH in the hagfish. Therefore, in this study, we investigated the immunohistochemical localization of CRH in the brain and pituitary of the inshore hagfish Eptatretus burgeri in order to get better comprehension of the neuroendocrine system of the hagfish.
Section snippets
Experimental fish
The hagfish Eptatretus burgeri of both sexes collected in Sagami Bay were purchased commercially. Fish were sampled just after arrival at the laboratory. The experiment was performed following the guidelines of the animal care committee of Kitasato University.
Immunohistochemistry
The fish (body weight 180–400 g) were anesthetized with crushed ice. The brains were excised, fixed with Bouin’s fluid for 24 h at 4 °C, rinsed in cold 70% ethanol, dehydrated through a graded series of ethanol concentrations, and embedded in
Immunohistochemistry
The distribution of CRH-ir cell bodies and fibers in the brain and pituitary is summarized in Fig. 1, Fig. 2. CRH-ir cell bodies were detected in the preopticohypothalamic area (Figs. 1A, 3A, B) and in the medulla oblongata (Figs. 1B, 3E, F). CRH-ir fibers from the preopticohypothalamic area projected to the dorsal wall of the NH, whereas no CRH-ir fibers were detected in the ventral wall of the NH (Fig. 3A, C, D). No CRH-ir cell bodies or fibers were observed when the anti-CRH antibody was
Discussion
The distribution of CRH-ir cell bodies and fibers in the brain and pituitary of the hagfish Eptatretus burgeri was clarified by immunohistochemistry to obtain better understanding of the neuroendocrine system of hagfish. The specificity of the antibody raised against human/mouse/rat CRH was demonstrated by a TR-FIA and absorption test. CRH-ir cell bodies were detected in two brain regions; the preopticohypothalamic area (PO, POp, and Hyinf) and the medulla oblongata (nucleus “A” of Kusunoki et
Acknowledgments
This study was supported in part by Grants-in-Aid for Scientific Research (B) (No. 23380115) from the Ministry of Education, Culture, Sports, Science and Technology, Japan to M. A.
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