REVIEW ARTICLE published: 28 November 2012 doi: 10.3389/fendo.2012.00148 Gonadotropin-inhibitory hormone action in the brain and pituitary

Takayoshi Ubuka,You Lee Son,YasukoTobari and KazuyoshiTsutsui* Laboratory of Integrative Brain Sciences, Department of Biology, Center for Medical Life Science, Waseda University, Tokyo, Japan

Edited by: Gonadotropin-inhibitory hormone (GnIH) was first identified in the Japanese quail as a Hubert Vaudry, University of Rouen, hypothalamic inhibitor of gonadotropin secretion. Subsequent studies have France shown that GnIH is present in the brains of birds including songbirds, and mammals includ- Reviewed by: ing humans. The identified avian and mammalian GnIH universally possess an Lance Kriegsfeld, University of California, USA LPXRFamide (X = L or Q) motif at their C-termini. Mammalian GnIH peptides are also José A. Muñoz-Cueto, University of designated as RFamide-related peptides from their structures.The for GnIH is the Cadiz, Spain G protein-coupled receptor 147 (GPR147), which is thought to be coupled to Gαi protein. *Correspondence: Cell bodies of GnIH neurons are located in the paraventricular nucleus (PVN) in birds and Kazuyoshi Tsutsui, Laboratory of the dorsomedial hypothalamic area (DMH) in mammals. GnIH neurons in the PVN or DMH Integrative Brain Sciences, Department of Biology, Center for project to the median eminence to control anterior pituitary function. GPR147 is expressed Medical Life Science, Waseda in the gonadotropes and GnIH suppresses synthesis and release of gonadotropins. It was University, 2-2 Wakamatsu-cho, further shown in immortalized mouse gonadotrope cell line (LβT2 cells) that GnIH inhibits Shinjuku-ku, Tokyo 162-8480, Japan. e-mail: [email protected] gonadotropin-releasing hormone (GnRH) induced gonadotropin subunit gene transcriptions by inhibiting adenylate cyclase/cAMP/PKA-dependent ERK pathway. GnIH neurons also project to GnRH neurons in the preoptic area, and GnRH neurons express GPR147 in birds and mammals. Accordingly, GnIH may inhibit gonadotropin synthesis and release by decreasing the activity of GnRH neurons as well as directly acting on the gonadotropes. GnIH also inhibits reproductive behavior possibly by acting within the brain. GnIH expres- sion is regulated by a nocturnal hormone melatonin and stress in birds and mammals. Accordingly, GnIH may play a role in translating environmental information to inhibit repro- ductive physiology and behavior of birds and mammals. Finally, GnIH has therapeutic potential in the treatment of reproductive cycle and hormone-dependent diseases, such as precocious puberty, endometriosis, uterine fibroids, and prostatic and breast cancers.

Keywords: gonadotropin-inhibitory hormone, gonadotropin-releasing hormone, gonadotropins, reproductive behavior, melatonin, stress, GPR147, RFamide-related

INTRODUCTION progress of GnIH research investigating its function in the brain The decapeptide gonadotropin-releasing hormone (GnRH) is and pituitary of birds and mammals. We also briefly review the primary factor responsible for the hypothalamic control of recent progresses in the study of GnIH peptides in fish and gonadotropin secretion. GnRH was first isolated from mammals humans. (Matsuo et al., 1971; Burgus et al., 1972) and subsequently from birds (King and Millar, 1982; Miyamoto et al., 1982, 1984) and DISCOVERY OF GnIH IN BIRDS other vertebrates (for reviews, see Millar, 2003, 2005). Gonadal sex Gonadotropin-inhibitory hormone was first discovered in the steroids and inhibin can modulate gonadotropin secretion. How- brain of Japanese quail, Coturnix japonica, while searching a novel ever, no hypothalamic neuropeptide inhibitor of gonadotropin RFamide peptide in birds. RFamide peptides, which have an Arg- secretion was known in vertebrates, although dopamine has been Phe-NH2 motif at its C-terminus, were first isolated in invertebrate reported as an inhibitor of gonadotropin secretion in several species in the late 1970s. The first RFamide peptide, Phe-Met-Arg- fish groups. In 2000, a previously unidentified hypothalamic Phe-NH2 (FMRFamide), was a cardioexcitatory molecule isolated neuropeptide was shown to inhibit gonadotropin release from from the ganglia of the venus clam Macrocallista nimbosa (Price the cultured quail anterior pituitary gland and it was named and Greenberg, 1977). After the discovery of FMRFamide pep- gonadotropin-inhibitory hormone (GnIH; Tsutsui et al., 2000). tide, numerous RFamide peptides that act as neurotransmitters, Although it is now known that GnIH and its receptor are also neuromodulators, and peripheral hormones have been identi- expressed in the gonads of birds (Bentley et al., 2008; Maddi- fied in various invertebrates, including cnidarians, , neni et al., 2008; McGuire and Bentley, 2010; McGuire et al., , molluscs, and arthropods. Subsequent immunohisto- 2011) and mammals (Zhao et al., 2010; Singh et al., 2011a,b; Li chemical studies in vertebrates suggested the presence of RFamide et al., 2012) including humans (Oishi et al., 2012), this review peptides in the central nervous system. It was revealed that some of highlights the discovery of GnIH in the quail brain and the the FMRFamide-immunoreactive (-ir) neurons projected close to

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the pituitary gland, suggesting a role in the regulation of pituitary Siberian hamster RFRP-1 (Figure 1). Up until now, bovine RFRP-1 function in vertebrates. (Fukusumi et al., 2001) and -3 (Yoshida et al., 2003), rat RFRP- Tsutsui et al. (2000) have isolated a novel RFamide peptide 3(Ukena et al., 2002), Siberian hamster RFRP-1 and -3 (Ubuka from 500 brains of the Japanese quail by high-performance liq- et al., 2012a), macaque RFRP-3 (Ubuka et al., 2009b), and human uid chromatography (HPLC) and a competitive enzyme-linked RFRP-1 and -3 (Ubuka et al., 2009c) are identified as mature pep- immunosorbent assay using an antibody raised against the dipep- tides in mammals (Figure 1; for reviews, see Tsutsui and Ukena, tide Arg-Phe-NH2. The isolated peptide had a novel dodecapep- 2006; Tsutsui et al., 2007, 2010a,b, 2012; Tsutsui, 2009; Tsutsui and tide structure, SIKPSAYLPLRFamide. Its C-terminus was identical Ubuka, 2012). to chicken LPLRFamide that was reported to be the first RFamide peptide isolated in vertebrates (Dockray et al.,1983), which is likely GnIH RECEPTOR to be a degraded fragment of the dodecapeptide. The isolated pep- Bonini et al. (2000) have identified two G protein-coupled recep- tide was localized in the hypothalamo-hypophyseal system, and tor (GPCR) for neuropeptide FF (NPFF), and designated them as shown to decrease gonadotropin release from cultured quail ante- NPFF1 (identical to GPR147) and NPFF2 (identical to GPR74). rior pituitary glands (Tsutsui et al., 2000). This RFamide peptide Hinuma et al. (2000) have also reported a specific receptor for was therefore named GnIH (Tsutsui et al., 2000). RFRP and named it OT7T022, which was identical to GPR147. Following the isolation of GnIH in the quail brain, the pre- The binding affinities for GPR147 and GPR74 and the efficacies on cursor polypeptide for GnIH was determined (Satake et al., 2001). signal transduction pathway were examined, using various analogs A cDNA that encoded GnIH precursor polypeptide was identi- of RFRPs and NPFF. RFRPs showed a higher affinity for GPR147, fied by a combination of 3 and 5 rapid amplification of cDNA whereas NPFF had potent agonistic activity for GPR74 (Bonini ends (3/5 RACE; Satake et al., 2001). The GnIH precursor con- et al., 2000; Liu et al., 2001). Taken together, GPR147 (NPFF1, sisted of 173 amino acid residues that encoded one GnIH and OT7T022) was suggested to be the receptor for RFRP (mammalian two GnIH-related peptides (GnIH-RP-1 and GnIH-RP-2) pos- GnIH). Figure 2 shows the predicted two-dimensional structure of sessing an LPXRFamide (X = L or Q) sequence at their C-termini human GPR147 from its nucleotide sequence (AB040104; Ubuka (Figure 1). These peptide sequences were flanked by a glycine et al.,2008b,2009c). It was shown that RFRPs suppress the produc- C-terminal amidation signal and a single basic amino acid on tion of cAMP in ovarian cells of Chinese hamster transfected with each end as an endoproteolytic site (Figure 1). GnIH-RP-2 was GPR147 cDNA, suggesting that GPR147 couples to Gαi protein also identified as a mature peptide by mass spectrometry in quail (Hinuma et al., 2000). (Figure 1; Satake et al., 2001). GnIH was further isolated as mature To elucidate the mode of action of GnIH in birds, Yin et al. peptides in European starlings (Ubuka et al., 2008a) and zebra (2005) have identified GnIH receptor (GnIH-R) in the quail dien- finch (Tobari et al., 2010) within the class of birds (Figure 1; for cephalon and characterized its expression and binding activity. reviews, see Tsutsui and Ukena, 2006; Tsutsui et al., 2007, 2010a,b, First, a cDNA encoding a putative GnIH-R was cloned by a com- 2012; Tsutsui, 2009; Tsutsui and Ubuka, 2012). bination of 3/5 RACE using PCR primers designed from the sequence of the receptor for RFRPs (GPR147). The crude mem- IDENTIFICATION OF GnIH IN MAMMALS brane fraction of COS-7 cells transfected with the putative GnIH-R In mammals, cDNAs that encode GnIH orthologs, LPXRFamide cDNA specifically bound GnIH and GnIH-RPs in a concentration- peptides, have been investigated by a gene database search dependent manner, indicating that GPR147 is GnIH-R (Yin et al., (Hinuma et al., 2000; for a review, see Tsutsui and Ubuka, 2005). GnIH-R also bound with high affinities to GnIH, GnIH- 2012). Mammalian LPXRFamide peptides are also designated as RPs and RFRPs, which have LPXRFamide (X = L or Q) motif RFamide-related peptides (RFRP) from its structure. Although at their C-termini. In contrast, C-terminal non-amidated GnIH human, macaque, and bovine LPXRFamide precursor cDNAs failed to bind the receptor. Accordingly, the C-terminal LPXR- encoded three RFRPs (RFRP-1, -2, and -3), only RFRP-1 and Famide (X = L or Q) motif seems to be critical for its binding RFRP-3 possessed a C-terminal LPXRFamide (X = L or Q) motif, to GnIH-R (Yin et al., 2005; for reviews, see Tsutsui and Ukena, and RFRP-2 had C-terminal RSamide or RLamide sequences 2006; Tsutsui et al., 2007, 2010a,b, 2012; Tsutsui, 2009; Tsutsui and (Figure 1). On the other hand, rodents do not have RFRP-2 Ubuka, 2012). It was suggested that there is no functional differ- sequence in their precursors (see the precursor sequences of rat ence among GnIH and GnIH-RPs because GnIH-R bound GnIH and hamster in Figure 1). Although the positions that encode and GnIH-RPs with similar affinities (Yin et al., 2005). However, GnIH-RP-1/RFRP-1 or GnIH/RFRP-2 in the precursor polypep- further studies are required to investigate if GnIH and GnIH-RPs tides were conserved between birds and mammals (only RFRP-1 work additively or synergistically to achieve their effects on the in rodents), the positions of GnIH-RP-2 and RFRP-3 in their pre- cells that express GnIH-R. cursor polypeptides were different between birds and mammals (Figure 1). DISTRIBUTION OF GnIH CELLS AND FIBERS IN THE BRAIN The LPXRFamide motif at the C-terminus is followed by LOCATION OF GnIH NEUNONS IN THE BRAIN glycine as an amidation signal and arginine or lysine as endopro- The location of GnIH precursor mRNA was first investigated by teolytic basic amino acids in mammals as well as birds (Figure 1). Southern blot analysis of the RT-PCR products of GnIH precur- Endogenous LPXRFamide peptides can also be cleaved at basic sor cDNA. Within the samples from telencephalon, diencephalon, amino acids at their N-termini. However, there were some excep- mesencephalon, and cerebellum, GnIH precursor mRNA was only tions in the cleavage site at the N-terminal, such as that of expressed in the quail diencephalon (Satake et al., 2001). In situ

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FIGURE 1 |The alignment of GnIH (RFRP) precursor polypeptides in bold. A broken line indicates the putative human RFRP-2 of birds and mammals. The amino acid sequence of GnIH, GnIH-RP-1, sequence. Accession numbers are human (NM_022150), macaque GnIH-RP-2, RFRP-1, RFRP-3 with Gly (G) as an amidation signal and (NM_001130827), bovine (NM_174168), rat (NM_023952), Siberian Arg (R) or Lys (K) as an endoproteolytic basic amino acid at the C- hamster (JF727837), white-crowned sparrow (AB128164), zebra finch termini are shown in bold. Identified mature peptides are underlined. (AB522971), European starling (EF486798) and Japanese quail Endoproteolytic basic amino acid (R or K) at the N-termini are also shown (AB039815). hybridization for GnIH precursor mRNA further showed that (Tsutsui et al., 2000; Ubuka et al., 2003). GnIH expressing cell bod- cells expressing GnIH mRNA were clustered in the paraventric- ies were also clustered in the PVN in other birds (Bentley et al., ular nucleus (PVN) in the hypothalamus (Ukena et al., 2003). 2003; Osugi et al., 2004; Ubuka et al., 2008a). Immunohistochemistry using GnIH antibody has revealed that In mammals, expression of precursor mRNA of RFRP (mam- clusters of GnIH-ir neurons were expressed in the PVN in quail malian GnIH) was only detected in the dorsomedial hypothalamic

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FIGURE 2 |Two-dimensional representation of the receptor (GPR147) for human GnIH (RFRP). The transmembrane region was predicted using SOSUI (Hirokawa et al., 1998). Glycosylation and disulfide bridge sites were predicted by GPCRDB (Horn et al., 1998). The accession number is AB040104. Adapted from Ubuka et al. (2008b). area (DMH) in the mouse and hamster brains by in situ hybridiza- GnIH INNERVATION IN THE BRAIN tion (Kriegsfeld et al., 2006; Ubuka et al., 2012a). In the rat Although a dense population of GnIH neuronal cell bodies was brain, RFRP precursor mRNA was expressed in the periventric- only found in the PVN in quail, GnIH-ir neuronal fibers were ular nucleus (PerVN), and the portion between the dorsomedial widely distributed in the diencephalic and mesencephalic regions nucleus (DMN) and the ventromedial nucleus (VMN) of the (Ukena et al., 2003). Dense networks of GnIH-ir fibers were found hypothalamus (Hinuma et al., 2000; Legagneux et al., 2009). The in the ventral paleostriatum, septal area, preoptic area (POA), majority of RFRP mRNA expressing neuronal cell bodies were median eminence, optic tectum, and the dorsal motor nucleus of localized in the intermediate periventricular nucleus (IPe) of the the vagus. GnIH-ir neuronal fibers were also widely distributed in hypothalamus in the macaque (Ubuka et al., 2009b), and in the the diencephalic and mesencephalic regions in European starlings DMN and PVN in the sheep (Clarke et al., 2008). (Ubuka et al., 2008a) and white-crowned sparrows (Ubuka et al.,

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2012b). Thus, it was hypothesized that GnIH may participate not mammals (Temple et al., 2003; Barnett et al., 2006). Double-label only in the regulation of pituitary function, but also in behavioral immunocytochemistry showed GnIH axon terminals on GnRH- and autonomic mechanisms in birds. I and GnRH-II neurons in the songbird brain (Bentley et al., GnIH-ir neuronal fibers were also widely distributed in the 2003; Ubuka et al., 2008a). In situ hybridization of starling GnIH- diencephalic and mesencephalic regions in rodents. Dense GnIH- R mRNA combined with GnRH immunocytochemistry further ir fibers were observed in the lateral septal nucleus, medial POA, showed the expression of GnIH-R mRNA in GnRH-I and GnRH-II amygdala, arcuate nucleus (ARC); moderate GnIH-ir fibers were neurons (Ubuka et al., 2008a; Figure 3). observed in the paraventricular thalamic nucleus, the paraven- Double-label immunocytochemistry also showed GnIH axon tricular hypothalamic nucleus, and the central gray in Siberian terminals on GnRH neurons in the Siberian hamster brain (Ubuka hamsters (Ubuka et al., 2012a). Dense GnIH-ir fibers were also et al.,2012a) and GPR147 was expressed in GnRH neurons (Ubuka observed in limbic and hypothalamic structures in rats (John- et al., 2012a). Central administration of GnIH inhibits the release son et al., 2007). Accordingly, it was hypothesized that GnIH of gonadotropin in white-crowned sparrows (Bentley et al., 2006), may also participate in behavioral and autonomic mechanisms Syrian hamsters (Kriegsfeld et al.,2006), rats (Johnson et al.,2007), in mammals. and Siberian hamsters (Ubuka et al., 2012a) in a manner similar to Innervation of GnIH neuronal fibers was intensively investi- peripheral administration of GnIH (Osugi et al., 2004; Kriegsfeld gated in the rhesus macaque brain (Ubuka et al.,2009b). Abundant et al., 2006; Ubuka et al., 2006). Accordingly, GnIH may inhibit GnIH-ir fibers were observed in the nucleus of the stria ter- the secretion of gonadotropins by decreasing the activity of GnRH minalis in the telencephalon; habenular nucleus, PVN of the neurons in addition to directly regulating pituitary gonadotropin thalamus, POA, PVN of the hypothalamus, IPe, ARC of hypotha- secretion (Figure 3). lamus, median eminence and dorsal hypothalamic area in the Direct application of RFRP-3 to GnRH cells in cultured mouse diencephalon; medial region of the superior colliculus, central brain slices decreased firing rate in a subpopulation of cells, gray substance of the midbrain and dorsal raphe nucleus in the further indicating a direct action of RFRP-3 on GnRH neu- midbrain; and parabrachial nucleus in the pons. GnIH-ir fibers rons (Ducret et al., 2009). In addition, RFRP-3 inhibited firing were observed in close proximity to GnRH-I, dopamine, pro- of -activated vGluT2 (vesicular glutamate transporter opiomelanocortin, and GnRH-II neurons in the POA, IPe, ARC 2)-GnRH neurons as well as of kisspeptin-insensitive GnRH neu- of hypothalamus, and central gray substance of midbrain, respec- rons (Wu et al., 2009). More recent data have confirmed a role tively. Qi et al. (2009) have shown that RFRP (mammalian GnIH) for RFRP-3 using an antagonist, RF9, against GnIH-R. Central cells project to neuropeptideY and pro-opiomelanocortin neurons administration of RF9 to rats and mice led to marked increases in the ARC, and melanin-concentrating hormone neurons in gonadotropin concentrations, providing a pronounced role of in the lateral hypothalamic area, as well as orexin cells in the DMN RFRP-3 as a key regulator of the reproductive axis in mammals and corticotrophin-releasing hormone and cells in the (Pineda et al., 2010). PVN, GnRH neurons in the POA in sheep. GnIH neurons might Recently, Rizwan et al. (2012) have investigated (1) whether thus regulate these important neural systems in addition to directly RFRP-3 can directly inhibit LH secretion without inhibiting GnRH regulating pituitary gonadotropin release (Ubuka et al., 2009b; for neurons; (2) whether RFRP-3 neurons project to GnRH neu- reviews, see Tsutsui,2009; Tsutsui et al., 2010a,b, 2012; Tsutsui and rons and rostral periventricular kisspeptin neurons in mice, and Ubuka, 2012; Figure 3). (3) whether GPR147 and GPR74 are expressed by these neu- rons. Intravenous treatment with the GPR147 antagonist RF9 GnIH ACTION IN THE BRAIN increased plasma LH concentrations in castrated male rats but MODULATION OF THE ACTIVITY OF GnRH AND KISSPEPTIN NEURONS was unable to do so in the presence of the GnRH antagonist BY GnIH cetrorelix. Approximately 26% of GnRH neurons of male and Immunohistochemical studies using light and confocal microscopy diestrous female mice were apposed by RFRP-3 fibers, and 19% indicated that GnIH (RFRP)-ir axon terminals are in probable con- of kisspeptin neurons of proestrous female mice were apposed by tact with GnRH neurons in birds (Bentley et al., 2003), rodents RFRP-3 fibers. They further showed that 33% of GnRH neurons (Kriegsfeld et al., 2006; Ubuka et al., 2012a), monkeys (Ubuka and 9–16% of rostral periventricular kisspeptin neurons expressed et al., 2009b), and humans (Ubuka et al., 2009c). Thus, there is GPR147, whereas GPR74 was not expressed in either popula- potential for the direct regulation of the activity of GnRH neurons tion. These data show that RFRP-3 can act on GnRH neurons by GnIH (RFRP) neurons (Figure 3). as well as kisspeptin neurons to modulate reproduction in rodents Ubuka et al. (2008a) investigated the interaction of GnIH neu- (Figure 3). ronal fibers and GnRH neurons in the European starling brain. It is generally accepted that birds possess at least two forms of EFFECT OF CENTRAL ADMINISTRATION OF GnIH ON BEHAVIORS OF GnRH in their brains. One form is GnRH-I which is thought BIRDS AND MAMMALS to be released at the median eminence to stimulate the secretion Central administration of GnIH or RFRP-3 to the third ven- of gonadotropins from the anterior pituitary (King and Millar, tricle of the brain inhibited reproductive behavior of females 1982; Miyamoto et al., 1982; Sharp et al., 1990; Ubuka and Bent- in white-crowned sparrows (Bentley et al., 2006) or of males in ley, 2009, 2011; Ubuka et al., 2009a). The second form of GnRH is rats (Johnson et al., 2007). It was known that GnRH-II enhances GnRH-II (Miyamoto et al.,1984; Millar,2003), which is thought to copulation solicitation in estrogen-primed female white-crowned influence reproductive behaviors in birds (Maney et al., 1997) and sparrows exposed to the song of males (Maney et al., 1997).

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FIGURE 3 | Schematic model of GnIH (RFRP) action in the brain , orexin, melanin-concentrating hormone (MCH), and pituitary. GnIH (RFRP) neurons in the brain project their axons to corticotrophin-releasing hormone (CRH) and oxytocin neurons in the brain. GnRH-I neurons as well as to the median eminence (ME). GnIH receptor GnIH (RFRP) inhibits reproductive behaviors of birds and mammals by (GnIH-R; GPR147) is expressed on GnRH-I neurons as well as gonadotropes. possibly acting within the brain. The expression of GnIH (RFRP) is regulated GnIH may thus inhibit gonadotropin synthesis and release by inhibiting the by melatonin, stress, and estradiol-17β (E2). Expressions of melatonin activity of GnRH-I neurons as well as directly inhibiting the pituitary receptor (Mel-R), glucocorticoid receptor (GC-R), or estrogen receptor α (ERα) gonadotrope. GnIH (RFRP) neurons may also regulate GnRH-I neurons by in GnIH (RFRP) neurons were shown in several species. These mechanisms regulating the activity of kisspeptin (Kiss) neurons that project to GnRH-I of action of GnIH (RFRP) on gonadotropin secretion or regulatory mechanism neurons. There are also reports showing that GnIH (RFRP) neurons project of GnIH (RFRP) expression may vary between species, sexes, and their axons to GnRH-II, dopamine, pro-opiomelanocortin (POMC), developmental stages.

Because of the putative contact of GnIH neurons with GnRH-II RNA interference (RNAi) of the GnIH gene on the behavior of neurons in white-crowned sparrows (Bentley et al., 2003), Bent- white-crowned sparrows, a highly social songbird species. Admin- ley et al. (2006) investigated the effect of GnIH administration istration of small interfering RNA against GnIH precursor mRNA on copulation solicitation in females of this species. A centrally- into the third ventricle of male and female birds reduced resting administered physiological dose of GnIH inhibited copulation time, spontaneous production of complex vocalizations, and stim- solicitation in estrogen-primed female white-crowned sparrows ulated brief agonistic vocalizations. GnIH RNAi further enhanced exposed to the song of males. Johnson et al. (2007) investigated song production of short duration in male birds when they were the effect of central administration of RFRP-3 on reproductive challenged by playbacks of novel male songs. These behaviors behaviors of male rats. Behavioral tests indicated that RFRP-3 resembled those of breeding birds during territorial defense. The dose-dependently suppressed all facets of male sexual behavior. In overall results suggest that GnIH gene silencing induces arousal. contrast, immunoneutralization of RFRP in the rat brain increased In addition, the activities of male and female birds were negatively male sexual behaviors. These results suggest that GnIH and correlated with GnIH mRNA expression in the PVN. Density of RFRP inhibit reproductive behavior by inhibiting GnRH neuronal GnIH neuronal fibers in the ventral tegmental area was decreased activities or by acting directly within the brain (Figure 3). by GnIH RNAi treatment in female birds, and the number of To identify the mechanism of GnIH neurons in the regula- GnRH neurons that received close appositions of GnIH neu- tion of behavior, Ubuka et al. (2012b) investigated the effect of ronal fiber terminals was negatively correlated with the activity of

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male birds. In summary, GnIH may decrease arousal level result- Rizwan et al. (2009) have injected a retrograde tracer Fluoro-Gold ing in the inhibition of specific motivated behavior, such as in intraperitoneally in rats. The majority of GnRH neurons were reproductive contexts (Ubuka et al., 2012b). labeled but essentially no RFRP neurons were labeled. In con- Central administrations of GnIH or RFRP-3 can also stimu- trast, intracerebral injections of Fluoro-Gold into the rostral POA late feeding behavior in chicken or rats (Tachibana et al., 2005; resulted in the labeling of 75 ± 5% of RFRP cell bodies. These Johnson et al., 2007; Murakami et al., 2008). There is a recent observations suggested that RFRP-3 is not a hypophysiotropic neu- report showing that central administration of RFRP-3 can stim- roendocrine hormone in rats (Rizwan et al., 2009). However, there ulate adrenocorticotropic hormone and oxytocin release, and are also studies indicating that RFRP-3 can act directly to inhibit induce anxiety behavior in rats (Kaewwongse et al., 2011). The fact gonadotropin release from the pituitary of rats (Murakami et al., that RFRP-ir fibers project to various neurons in the brain, such 2008). More extensive studies, analyzing peptide release into the as dopamine and/or pro-opiomelanocortin neurons in the rat, hypophyseal portal blood, fiber projections by retrograde labeling, sheep, and macaque (Qi et al., 2009; Ubuka et al., 2009b; Figure 3) etc., are needed to elucidate the hypophysiotropic action of GnIH suggests multiple functions of GnIH or RFRP in the brain. in various animals (for reviews, see Tsutsui et al., 2007, 2010a,b, 2012; Tsutsui, 2009; Tsutsui and Ubuka, 2012). GnIH ACTION IN THE PITUITARY Since GPR147 couples to Gαi to inhibit adenylyl cyclase (AC; Dense population of GnIH-ir fibers at the median eminence (ME) Hinuma et al., 2000), GPR147 (GnIH-R) activation may reduce in quail (Tsutsui et al., 2000; Ukena et al., 2003) as well as in other intracellular cAMP levels, and reduce the activities of cAMP- birds (Bentley et al., 2003; Osugi et al., 2004; Ubuka et al., 2008a), dependent protein kinase (PKA) and mitogen-activated protein suggested a direct action of GnIH in the regulation of pituitary kinase (MAPK) signaling cascade. On the other hand, GnRH function in birds (Figure 3). The fact that GnIH inhibits synthesis receptor (GnRH-R) couples with Gαq/11 to activate phospholipase and/or release of gonadotropins from cultured quail and chicken C (PLC), which results in the production of inositol tri-phosphate anterior pituitary gland provides strong support for this function (IP3) and diacylglycerol (DAG). In turn, IP3 and DAG increase (Tsutsui et al., 2000; Ciccone et al., 2004). Peripheral administra- intracellular Ca2+ and activate the protein kinase C (PKC) path- tion of GnIH also inhibits gonadotropin synthesis and/or release way and MAPK signaling. GnRH-R was also reported to be in birds (Osugi et al.,2004; Ubuka et al.,2006). In mammals, abun- coupled to Gαs to stimulate AC/cAMP/PKA pathway. A recent dant RFRP-ir fibers were observed in the ME of sheep (Clarke et al., study using immortalized mouse gonadotrope cell line (LβT2 cells) 2008), macaque (Ubuka et al., 2009b), and humans (Ubuka et al., has demonstrated that the inhibitory action of mouse RFRPs on 2009c). As GnIH in birds, RFRP-3 inhibits gonadotropin synthe- gonadotropin gene expression is mediated by an inhibition of sis and/or release from cultured pituitaries in sheep (Sari et al., AC/cAMP/PKA-dependent extracellular signal-regulated kinase 2009) and cattle (Kadokawa et al., 2009). Peripheral administra- (ERK) pathway (Son et al., 2012). In the sheep, RFRP-3 can inhibit tion of RFRP-3 also inhibits gonadotropin release in sheep (Clarke both GnRH-induced intracellular Ca2+ increase and ERK phos- et al., 2008), rats (Murakami et al., 2008), and cattle (Kadokawa phorylation, impacting GnRH-induced gonadotropin release and et al., 2009). It was further shown that GnIH-R (GPR147) mRNA synthesis (Sari et al., 2009). In the chicken, activation of GnRH-R is expressed in gonadotropes in the human pituitary (Ubuka can activate AC and stimulate cAMP responsive element (CRE) et al., 2009c). Taken together, it is likely that GnIH and RFRP- binding protein. GnIH may partly inhibit this GnRH-induced 3 directly act on the pituitary to inhibit gonadotropin secretion CRE activation, thus impacting gene transcription (Shimizu and from the pituitary at least in these avian and mammalian species Bédécarrats, 2010). (Figure 3). Recently, Smith et al. (2012) measured the concentration of REGULATION OF GnIH EXPRESSION RFRP-3 in hypophyseal portal blood in ewes during the non- EFFECT OF MELATONIN breeding (anestrous) season and during the luteal and follicular Identification of the regulatory mechanisms governing GnIH phases of the estrous cycle in the breeding season. Pulsatile RFRP- expression is important in understanding the physiological role 3 secretion was observed in the portal blood of all animals. Mean of the GnIH system. Photoperiodic mammals rely on the annual RFRP-3 pulse amplitude and pulse frequency were higher dur- cycle of changes in nocturnal secretion of melatonin to drive their ing the non-breeding season. RFRP-3 was virtually undetectable reproductive responses (Bronson, 1990). In contrast, a dogma has in peripheral blood plasma. To determine the role of secreted existed that birds do not use seasonal changes in melatonin secre- RFRP-3, Smith et al. (2012) further examined its effects on GnRH- tion to time their reproductive effort, and a role for melatonin stimulated LH secretion in hypothalamo–pituitary-disconnected in birds has remained enigmatic (Wilson, 1991; Juss et al., 1993). ewes, and a significant reduction in the LH response to GnRH was Despite the accepted dogma, there is strong evidence that mela- observed by RFRP-3 administration. These data show that RFRP- tonin is involved in the regulation of several seasonal processes, 3 is secreted into portal blood to act on pituitary gonadotropes, including gonadal activity and gonadotropin secretion (Ohta et al., reducing the action of GnRH in sheep (Smith et al., 2012). 1989; Guyomarc’h et al., 2001; Rozenboim et al., 2002). Ubuka On the contrary it was suggested that GnIH or RFRP-3 may not et al. (2005) hypothesized that melatonin may be involved in the act directly on the pituitary in some birds and rodents, because induction of GnIH expression, thus influencing gonadal activity. there are relatively few or no GnIH (RFRP)-ir fibers in the ME of The action of melatonin on the expression of GnIH was stud- Rufous-winged sparrows (Small et al., 2007), hamsters (Kriegsfeld ied in quail, a highly photoperiodic bird species. Because the et al., 2006; Ubuka et al., 2012a), and rats (Rizwan et al., 2009). pineal gland and eyes are the major sources of melatonin in the

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quail (Underwood et al., 1984), Ubuka et al. (2005) analyzed the applied to sheep. Sheep is a SD breeder that activates its reproduc- effects of pinealectomy (Px) combined with orbital enucleation tive activity in SD and suppresses its reproductive activity in LD. (Ex; Px plus Ex) on the expression of GnIH precursor mRNA It is also understandable that the expression of RFRP is inhibited and GnIH peptide. Subsequently, melatonin was administered by a nocturnal hormone melatonin and SD when the duration of to Px plus Ex birds. Px plus Ex decreased the expression of melatonin secretion is long. Accordingly, it was hypothesized that GnIH precursor mRNA and the content of mature GnIH pep- the increase of RFRP expression may inhibit reproductive activity tide in the hypothalamus. Melatonin administration to Px plus Ex in LD in sheep (Dardente et al., 2008). On the other hand, it was birds caused a dose-dependent increase in the expression of GnIH difficult to understand the inhibitory effect of melatonin or SD on precursor mRNA and the production of mature peptide. They RFRP expression in hamsters, because hamsters are LD breeders also investigated the expression of melatonin receptor in GnIH (Revel et al., 2008; Mason et al., 2010). However, recent report has neurons. In situ hybridization combined with immunocytochem- shown that RFRP may have a stimulatory effect on gonadotropin istry for GnIH revealed that the mRNA of Mel1c, a melatonin secretion in SD in Siberian hamsters (Ubuka et al., 2012a). Long receptor subtype, was expressed in GnIH-ir neurons in the PVN. duration of melatonin secretion in SD may need to decrease RFRP Autoradiography of melatonin receptors further revealed specific expression to inhibit reproductive activities of Siberian hamsters binding of melatonin in the PVN. Accordingly, melatonin appears in SD. Another recent work in male Syrian hamsters suggested to act directly on GnIH neurons through its receptor to induce that GnIH might be excitatory in LD as well (Ancel et al., 2012). expression of GnIH (Ubuka et al., 2005; Figure 3). Long duration of melatonin secretion in SD may decrease RFRP Chowdhury et al. (2010) further investigated the role of mela- expression to inhibit reproductive activities and short duration tonin in the regulation of GnIH release and the negative correlation of melatonin secretion in LD may increase RFRP expression to of GnIH release with LH release in quail. Melatonin administra- stimulate reproductive activities of Syrian hamsters in LD (Ancel tion dose-dependently increased GnIH release from hypothalamic et al., 2012). Further studies are required to understand the role explants in vitro. A clear diurnal change in GnIH release was of melatonin controlling GnIH and RFRP expression in seasonal observed in quail, and this change was negatively correlated breeders. with changes in plasma LH concentrations. GnIH release dur- ing the dark period was greater than that during the light period EFFECT OF STRESS in explants from quail exposed to long-day (LD) photoperiods. Stress leads to reproductive dysfunction in many species, includ- Conversely, plasma LH concentrations decreased during the dark ing rodents and humans. Calisi et al. (2008) hypothesized that period. In contrast to LD, GnIH release increased under short-day stress effects upon reproduction are mediated via the hypotha- (SD) photoperiods, when the duration of nocturnal secretion of lamic GnIH system in birds. They examined the effects of melatonin increases. These results indicate that melatonin may capture-handling stress in the hypothalamus of male and female play a role in stimulating not only GnIH expression but also adult house sparrows. There were more GnIH-positive neurons GnIH release, thus inhibiting plasma LH concentrations in quail in fall birds versus those sampled in spring, and a significant (Figure 3). increase in GnIH-positive neurons was seen in stressed birds A similar, but opposite, action of melatonin on the inhibi- in spring. These data imply an influence of stress upon the tion of the expression of RFRP was shown in Syrian and Siberian GnIH system that changes over the annual cycle of reproduction hamsters, both photoperiodic mammals (Revel et al., 2008; Mason (Figure 3). et al., 2010; Ubuka et al., 2012a). The level of RFRP mRNA and the Kirby et al. (2009) showed that both acute and chronic immobi- number of RFRP-ir cell bodies were reduced in sexually quies- lization stress lead to an up-regulation of the expression of RFRP cent Syrian and Siberian hamsters acclimated to SD photoperiod, in the DMH of adult male rats and that this increase in RFRP compared to sexually active animals maintained under LD pho- is associated with inhibition of downstream HPG activity. They toperiod. The photoperiodic variation of RFRP expression was also showed that adrenalectomy blocks the stress-induced increase abolished in Px hamsters and injections of LD hamsters with in RFRP expression. Immunohistochemistry revealed that 53% melatonin reduced the expression of RFRP down to SD levels, of RFRP cells express receptors for glucocorticoids (GCs), sug- indicating a dependence upon melatonin (Revel et al.,2008; Ubuka gesting that adrenal GCs can mediate the stress effect through et al., 2012a). There are also reports showing that the expression direct action on RFRP cells. These data show that stress-induced of RFRP precursor mRNA is regulated by melatonin in sheep increases in adrenal GCs cause an increase in RFRP that con- (Dardente et al., 2008) and rats (Gingerich et al., 2009). These tributes to hypothalamic suppression of reproductive function results demonstrate that the expression of GnIH and RFRP is (Figure 3). photoperiodically modulated via a melatonin-dependent process Papargiris et al. (2011) tested if GnIH (RFRP) mediates the (Figure 3). inhibitory effect of stress on LH secretion in ovariectomized Quail is a LD breeder, which activates its reproductive activity ewes using a psychosocial stressor, isolation/restraint. Isola- in LD and suppresses its reproductive activity in SD. It is under- tion/restraint stress increased plasma cortisol concentrations and standable that the expression of GnIH is stimulated by a nocturnal decreased plasma LH concentrations. However, there was no sig- hormone melatonin and SD when the duration of melatonin secre- nificant effect of stress on RFRP peptide or mRNA levels, with tion is long. Accordingly, it was hypothesized that the increase of no difference between control or stressed ewes. Furthermore, GnIH expression may inhibit reproductive activity in SD in quail there was no difference in the number of RFRP-ir cells double- (Ubuka et al., 2005). The opposite but similar thoughts can be labeled for Fos between control and stressed ewes and there was

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no difference in the cellular expression of RFRP mRNA between adulthood hormonal milieus. They identified two interspersed groups. Accordingly, GnIH (RFRP) may not mediate the effects of subpopulations of RFRP cells (high RFRP-expressing, HE; low stress on LH secretion in ewes or the effect of stress may depend RFRP-expressing, LE), which have unique developmental and on the presence of gonadal sex steroids. steroidal regulation characteristics. The number of LE cells robustly decreased during postnatal development, whereas HE EFFECT OF SEX STEROIDS cell number increased significantly before puberty. In adults, they Estrogen secreted by the ovary feedbacks to the brain and pitu- found that E2 and testosterone moderately repress RFRP expres- itary to regulate gonadotropin secretion (Herbison, 1998; Petersen sion in both HE and LE cells, whereas the non-aromatizable et al., 2003). Wintermantel et al. (2006) found that estrogen posi- androgen dihydrotestosterone has no effect. They determined that tive feedback to generate the preovulatory gonadotropin surge was approximately 25% of RFRP neurons coexpress ERα in each sex, normal in estrogen receptor (ER) β knockout mice, but absent in whereas RFRP cells do not express androgen receptor in either ERα mutant mice. Because GnRH neurons do not express ERα, sex, regardless of hormonal milieu. They detected coexpression estrogen positive feedback upon GnRH neurons must be indirect. of GPR147 but no coexpression of GPR74 in GnRH neurons of RFRP (mammalian GnIH) neuronal system may be involved in both intact and gonadectomized males and females. RFRP-3 may estrogen feedback signaling to GnRH neurons because RFRP neu- thus exert its effects on reproduction either directly, via GPR147 rons in rodents express ERα and respond with c-Fos expression in a subset of GnRH neurons, and/or indirectly, via upstream to an acute administration of estradiol-17β (E2; Kriegsfeld et al., regulators of GnRH (Figure 3). 2006; Figure 3). The role of RFRP-3 in regulating ovulatory function was inves- RECENT PROGRESS IN GnIH STUDIES ON FISH AND tigated in female hamsters (Gibson et al., 2008). The cellular HUMANS activity of RFRP neurons was suppressed at the time of the LH We have described the progress of GnIH research investigating surge, suggesting removal of negative feedback by RFRP-3 at this its function in the brain and pituitary of birds and mammals. time. The SCN, the master circadian clock triggering ovulation in Recently, there were important progresses in the study of GnIH rodents, projects to a large proportion of RFRP neurons, providing peptides in fish and humans as we briefly summarize them below. a mechanism for timing removal of negative drive on the GnRH A cDNA encoding three GnIH orthologs, LPXRFamide pep- system. Activities of the SCN, GnRH, and RFRP neurons were tides, was cloned from the goldfish brain by a combination coordinated with ovulation (Gibson et al., 2008). Li et al. (2012) of 3/5 RACE (Sawada et al., 2002). Mass spectrometric analy- investigated the expression patterns in the reproductive axis of the ses revealed that a tridecapeptide (SGTGLSATLPQRFamide) is female pig across the estrous cycle. The hypothalamic levels of both expressed in the brain as an endogenous ligand. Immunoreactive RFRP and its receptor mRNA were lowest in estrus and peaked cell bodies were restricted to the nucleus posterioris periventric- in the proestrus and diestrus phases. Smith et al. (2010) exam- ularis and the nervus terminalis and immunoreactive fibers were ined Kiss1 and RFRP mRNA throughout the menstrual cycle of a distributed in several brain regions including the nucleus later- female primate, rhesus macaque. Kiss1-expressing cells were found alis tuberis pars posterioris and pituitary (Sawada et al., 2002). in the POA and ARC, and RFRP-expressing cells were located in Amano et al. (2006) analyzed the hypophysiotropic activity of the PVN/DMN. Kiss1 expression in the caudal ARC and POA the three goldfish LPXRFamide peptides (gfLPXRFa-1, -2, and was higher in the late follicular phase of the cycle (just before the -3) in sockeye salmon. gfLPXRFa-ir cell bodies were detected in GnRH/LH surge) than in the luteal phase. RFRP expression was the nucleus posterioris periventricularis of the hypothalamus and also higher in the late follicular phase suggesting that RFRP fine immunoreactive fibers were distributed in various brain regions tunes GnRH/LH surge in primates. There are also reports showing and the pituitary in sockeye salmon. gfLPXRFamide peptides the correlation of RFRP expression and testicular activities of male stimulated the release of FSH, LH, and GH from cultured pitu- mice (Sethi et al., 2010a,b). itary cells (Amano et al., 2006). In contrast, Zhang et al. (2010) Molnár et al. (2011) investigated the possibility that RFRP have identified the orthologous GnIH genes in zebrafish, stick- neurons are involved in estrogen feedback signaling to the repro- leback, medaka, and Takifugu. The zebrafish GnIH precursor ductive axis in mice. They compared the expression of RFRP contained three putative LPXRFamide peptides. Intraperitoneal mRNA of ovariectomized mice, with and without E2 replacement. administration of the mature zebrafish LPXRFa-3 (zfLPXRFa- Subcutaneous administration of E2 via silastic capsules for 4 days 3) significantly reduced the basal serum LH level in goldfish significantly down-regulated RFRP mRNA expression. In ovariec- (Zhang et al., 2010). tomized mice, low levels of ERα immunoreactivity were detectable Moussavi et al. (2012) examined the effects of synthetic in 18.7 ± 3.8% of RFRP neurons, whereas RFRP neurons did not gfLPXRFamide peptides on pituitary LHβ and FSHβ subunit, exhibit ERβ immunoreactivity. The estrogenic down-regulation and gfLPXRFamide peptide receptor (gfLPXRFa-R) mRNA lev- of RFRP expression may contribute to estrogen positive feedback els and LH secretion in goldfish. Intraperitoneal injections of to the reproductive axis. However, whether E2 regulates RFRP gfLPXRFa-3 increased pituitary LHβ and FSHβ mRNA levels at neurons directly or indirectly remains an open question because early to late gonadal recrudescence, but reduced serum LH and ERα immunoreactivity is present only in a subset of RFRP cells pituitary gfLPXRFa-R mRNA levels, respectively, at early to mid- (Figure 3). recrudescence and later stages of recrudescence. Static incubation Poling et al. (2012) examined changes in RFRP neurons in with gfLPXRFa-3 elevated LH secretion from dispersed pituitary mice of both sexes during development and under different cell cultures from prespawning fish, but not at other recrudescent

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stages. gfLPXRFa-3 suppressed LHβ mRNA levels at early recrude- release in vivo (Clarke et al., 2008; Smith et al., 2012). In view of scence and prespawning but elevated LHβ at mid-recrudescence, its potent inhibition of gonadotropin secretion, human RFRP- and consistently attenuated FSHβ mRNA in a dose-specific man- 3 (human GnIH) has the potential of an alternative or adjunct ner in vitro. These results indicate that the effect of gfLPXRFa-3 therapeutic agent to inhibit endogenous levels of gonadotropins depends on maturational status and administration route in and steroid hormones in humans. Thus, RFRP has therapeutic goldfish (Moussavi et al., 2012). potential in the treatment of hormone-dependent diseases, such Recently, the mature peptide structures of human GnIH pep- as precocious puberty, endometriosis, uterine fibroids, benign tides (human RFRP-1 and RFRP-3) were identified by mass spec- prostatic hyperplasia, and prostatic and breast cancers. trometry (Ubuka et al., 2009c). Because the structure of human RFRP-3 was identical to the structure of sheep RFRP-3, physi- ACKOWLEDGMENTS ological functions of human RFRP-3 were studied in the sheep. This work was supported by Grants-in-Aid for Scientific Research It was shown that sheep/human RFRP-3 inhibits gonadotropin from the Ministry of Education, Science and Culture, Japan synthesis and release in vitro (Sari et al., 2009) and gonadotropin (22132004 and 22227002 to Kazuyoshi Tsutsui).

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FEBS Lett. 512, 255–258. flict of interest. mone (GnIH): biosynthesis, mode precursor mRNA and its seasonal reg- Ukena, K., Ubuka, T., and Tsutsui, K. of action and functional significance. ulation. Gen. Comp. Endocrinol. 162, (2003). Distribution of a novel avian Received: 28 August 2012; paper pending Prog. Neurobiol. 88, 76–88. 301–306. gonadotropin-inhibitory hormone in published: 24 September 2012; accepted:

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11 November 2012; published online: 28 and pituitary. Front. Endocrin. 3:148. Copyright © 2012 Ubuka, Son, Tobari reproduction in other forums, provided November 2012. doi: 10.3389/fendo.2012.00148 and Tsutsui. This is an open-access arti- the original authors and source are cred- Citation: Ubuka T, Son YL, Tobari Y This article was submitted to Frontiers cle distributed under the terms of the ited and subject to any copyright notices and Tsutsui K (2012) Gonadotropin- in Neuroendocrine Science, a specialty of Creative Commons Attribution License, concerning any third-party graphics inhibitory hormone action in the brain Frontiers in Endocrinology. which permits use, distribution and etc.

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