GnIH and GnRH expressions in the central nervous system and pituitary of Indian major carp, Labeo rohita during ontogeny: An immunocytochemical study
Introduction
Past studies on fish have shown that several neuropeptides are localized in the preoptic area (POA) and pituitary, which control the secretion of pituitary hormones, other physiological aspects related to the regulation of gonadal development, reproduction, response to stress etc., in fish. The roles of neuropeptides have been well documented by Norris and Lopez (2011). Since the discovery of gonadotropin-releasing hormone (GnRH), a hypothalamic neuropeptide, that regulates the reproductive axis in the brain of mammals at the beginning of 1970s (Burgus et al., 1972, Matsuo et al., 1971), several other GnRHs have been identified in the brain of non-mammalian vertebrates (King and Millar, 1982, Miyamoto et al., 1982, Miyamoto et al., 1984, Sherwood et al., 1986). In fish, several investigators have demonstrated that GnRH has been extensively localized in the brain by using immunocytochemical method (Kah et al., 2007, Palevitch et al., 2009, Parhar et al., 2001). Based on extensive studies in vertebrates, it was generally believed that GnRH was the sole hypothalamic neuropeptide regulating gonadotropin release, and that no other neuropeptide had a direct influence on the reproductive axis.
In 2000, Tsutsui and colleagues discovered a novel hypothalamic neuropeptide that actively inhibits gonadotropin release in Japanese quail Coturnix japonica and termed it gonadotropin-inhibitory hormone (GnIH; Tsutsui et al., 2000). Soon after, several neuropeptides closely related to quail GnIH have been identified in a variety of vertebrates from agnathans to humans (for reviews, see Tsutsui, 2009, Tsutsui and Ukena, 2006, Tsutsui et al., 2006, Tsutsui et al., 2007, Tsutsui et al., 2010a, Tsutsui et al., 2010b, Tsutsui et al., 2012, Tsutsui et al., 2013, Ukena and Tsutsui, 2005). The discovery of GnIH has given a new dimension to the regulation of gonadotropin secretion by the hypothalamus in vertebrates. We now know that GnIH regulates reproduction by inhibiting gonadotropin synthesis and release via action on the GnRH system and the anterior pituitary gland in birds and mammals (for reviews, see Tsutsui, 2009, Tsutsui et al., 2006, Tsutsui et al., 2007, Tsutsui et al., 2010a, Tsutsui et al., 2010b, Tsutsui et al., 2012, Tsutsui et al., 2013).
GnIHs identified in most representative species of gnathostomes have an LPXRFamide (X = L or Q) motif at the C-terminus (for reviews, see Tsutsui, 2009, Tsutsui and Ukena, 2006, Tsutsui et al., 2006, Tsutsui et al., 2007, Tsutsui et al., 2010a, Tsutsui et al., 2010b, Tsutsui et al., 2012, Tsutsui et al., 2013, Ukena and Tsutsui, 2005). These group of peptides which all shares the C-terminal motif are also known as RFamide related peptides (RFRPs). In fish, a cDNA that encodes three RFRPs (gfLPXRFa-1, -2 and -3) was cloned from the brain of goldfish Carassius auratus, even if only gfLPXRFa-3 was identified as a mature peptide (Sawada et al., 2002). The gfLPXRFa peptides were shown to have both stimulatory and inhibitory effects on gonadotropin synthesis and release in goldfish and sockeye salmon Oncorhynchus nerka (Amano et al., 2006, Moussavi et al., 2012, Moussavi et al., 2013, Qi et al., 2013, Zhang et al., 2010). The cDNA encoding the precursor of GnIH peptide was also cloned in grass puffer Takifugu niphobles (Shahjahan et al., 2011).
The localization of LPXRFamide neuron in fish has been shown in the brain of sockeye salmon by immunohistochemistry (Amano et al., 2006) and in goldfish by both immunohistochemistry and in situ hybridization (Sawada et al., 2002). Neurons synthesizing LPXRFamide were commonly localized in the nucleus posterioris periventricularis (NPPv) of the hypothalamus. Unlike goldfish (Sawada et al., 2002), immunoreactive cell bodies were not detected in the nervus terminalis of the sockey salmon (Amano et al., 2006). In both species, however, the fibers projected to a wide range of brain regions and the pituitary.
In fish, a number of studies have shown GnRH in the brain during development (Biju et al., 2005, Halpern-Sebold and Schreibman, 1983, Pandolfi et al., 2002), but no GnIH localization has been investigated. In view of the lack of studies on GnIH expression during development, we examined the distribution pattern of GnIH immunoreactivity and compared it with GnRH expression in the central nervous system (CNS) and pituitary during development of the fresh water teleost fish, Labeo rohita.
Section snippets
Materials and methods
In this study, hatchlings (10–15 mm), frys (15–35 mm), semi-fingerlings (35–65 mm), fingerlings (65–100 mm) and adults (350–370 mm) of Labeo rohita were used. Specimens were anesthetized with ethyl-m-aminobenzoate methanesulphonate (MS-222; Himedia, India) and the brain including the olfactory system, spinal cord and pituitary gland were removed and fixed in Bouin’s fixative overnight. The tissues were cryoprotected in 30% sucrose solution and then 12-μm thick sections were cut on cryotome (Shandon,
Results
The overall neuroanatomical distribution of GnIH and GnRH immunoreactivities in the olfactory system, brain and pituitary of L. rohita is summarized in Table 1.
Fish olfactory system consists of olfactory epithelium (OE), olfactory nerve and olfactory bulb (OB) situated rostral to the telencephalon. For both GnIH and GnRH, we noticed variation in the intensity of immunolabeling from hatchling to fingerling stages. OE and OB were intensely labeled in the early compared to the late developmental
Discussion
In the present study, we localized GnIH-ir and GnRH-ir cells and fibers in the CNS and pituitary of the teleost fish L. rohita during development and in the adult. We are for the first time demonstrating particularly the ontogenetic localization of GnIH in the olfactory system, brain and pituitary. Furthermore, we also studied GnRH expression in the CNS for comparison. Our GnIH study results on adults differ significantly with available studies and show species specificity.
Earlier
Acknowledgments
This study was supported by Grants from DBT [BT/PR4688/AAQ/03/585/2012], UGC [41-22/2012 (SR)], DST [SR/SO/AS/-09/2006] Govt. of India to A.G.J. and by CSIR, senior research fellowship to S.B. A donation of Axio Imager A2 (Zeiss) microscope by Alexander von Humboldt Foundation, Germany to A.G.J. used for the photomicrography is acknowledged.
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