Dual Actions of Mammalian and Piscine Gonadotropin-Inhibitory Hormones, RFamide-Related Peptides and LPXRFamide Peptides, in the Hypothalamic–Pituitary–Gonadal Axis

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that decreases gonadotropin synthesis and release by directly acting on the gonadotrope or by decreasing the activity of gonadotropin-releasing hormone (GnRH) neurons. GnIH is also called RFamide-related peptide in mammals or LPXRFamide peptide in fishes due to its characteristic C-terminal structure. The primary receptor for GnIH is GPR147 that inhibits cAMP production in target cells. Although most of the studies in mammals, birds, and fish have shown the inhibitory action of GnIH in the hypothalamic–pituitary–gonadal (HPG) axis, several in vivo studies in mammals and many in vivo and in vitro studies in fish have shown its stimulatory action. In mouse, although the firing rate of the majority of GnRH neurons is decreased, a small population of GnRH neurons is stimulated by GnIH. In hamsters, GnIH inhibits luteinizing hormone (LH) release in the breeding season when their endogenous LH level is high but stimulates LH release in non-breeding season when their LH level is basal. Besides different effects of GnIH on the HPG axis depending on the reproductive stages in fish, higher concentration or longer duration of GnIH administration can stimulate their HPG axis. These results suggest that GnIH action in the HPG axis is modulated by sex-steroid concentration, the action of neuroestrogen synthesized by the activity of aromatase stimulated by GnIH, estrogen membrane receptor, heteromerization and internalization of GnIH, GnRH, and estrogen membrane receptors. The inhibitory and stimulatory action of GnIH in the HPG axis may have a physiological role to maintain reproductive homeostasis according to developmental and reproductive stages.

The C-terminal of GnIH peptides has an LPXRFamide (LPXRFa, X = L or Q) motif. Therefore, peptides orthologous to GnIH are also called RFamide-related peptide (RFRP) in mammals and LPXRFa peptides in non-mammalian and non-avian vertebrates (3). Most of the studies in mammals, birds, and fish have shown inhibitory effects of GnIH on the hypothalamic-pituitarygonadal (HPG) axis; however, several in vivo and in vitro studies in mammals and fish show its stimulatory effects (3,4). Here, we highlight studies that show stimulatory effects of GnIH on the HPG axis and investigate their physiological or pharmacological mechanisms.
Although the C-terminal LPXRFa structure is key for binding of GnIH to its receptor (22), the N-terminal structure may modify the action of GnIH. Studies are needed to investigate the function of the N-terminal of GnIH and the differential effect of orthologous LPXRFa peptides encoded in the precursor polypeptide ( Table 1).

GniH recePtOr
Yin et al. characterized the binding activity of quail GnIH and GnIH-RPs to a G-protein-coupled receptor (GPCR) GPR147. The membrane fraction of COS-7 cells transfected with quail GPR147 cDNA specifically bound GnIH and GnIH-RPs that have a C-terminal LPXRFa motif with similar affinities (22). Hinuma et al. identified a specific receptor for GnIH (RFRP) in mammals, which was identical to GPR147 and named it OT7T022 (28). In the same year, Bonini et al. reported two GPCRs for neuropeptide FF (NPFF), a neuropeptide that has a PQRFamide (PQRFa) motif at its C-terminal that modulates pain, and designated as NPFF1 (identical to GPR147) and NPFF2 (identical to GPR74) (29). LPXRFa peptide precursor gene and PQRFa peptide precursor gene are thought to have diverged from a common ancestral gene through gene duplication (20,21). GPR147 and GPR74 genes are also paralogous (30). The binding affinities of RFRPs to GPR147 and GPR74 and their signal transduction pathways show their higher affinity to GPR147 than NPFF that has a potent agonistic activity on GPR74 (10,29,31), suggesting that GPR147 (NPFF1, OT7T022) is the primary receptor for GnIH (3). However, this may not apply to teleost fishes as they generally have several subtypes of GPR147 and/ or GPR74 (32).

iNtrAceLLULAr siGNALiNG OF GniH recePtOr
Gonadotropin-inhibitory hormone peptides suppress the production of cAMP by binding to GPR147 on the cells, suggesting that GPR147 couples to Gαi protein that inhibits adenylate cyclase (AC) (28,33). Son et al. investigated the precise mechanism of GnIH cell-signaling pathway in a mouse gonadotrope cell line, LβT2 (34). Mouse RFRPs (mRFRPs) suppress GnRH-induced cAMP signaling. mRFRPs also inhibit GnRH-stimulated extracellular signal-regulated kinase (ERK) phosphorylation and gonadotropin subunit gene transcription by inhibiting the protein kinase A (PKA) pathway. Therefore, mRFRPs function as GnIH to inhibit GnRH-induced gonadotropin subunit gene transcription by inhibiting AC/cAMP/PKA-dependent ERK activation in gonadotropes (34) ( Table 2).
Son et al. further investigated the signal transduction pathway that conveys the inhibitory action of GnIH in GnRH neurons by using a mouse GnRH neuronal cell line, GT1-7 (46). Although GnIH significantly suppressed the stimulatory effect of kisspeptin on GnRH release in hypothalamic culture, GnIH had no inhibitory effect on the protein kinase C (PKC) pathway stimulated by kisspeptin in GnRH neurons. On the other hand, GnIH eliminated the stimulatory effect of vasoactive intestinal polypeptide (VIP) on AC activity, p38 and ERK phosphorylation, and c-Fos mRNA expression in GT1-7. This shows the specific inhibitory mechanism of GnIH action on AC/ cAMP/PKA pathway, and demonstrates a common mechanism of GnIH action in gonadotropes and GnRH neurons (34,46) ( Table 2).
Gonadectomized male rats 10-nmol rat RFRP-3 iv Blood was collected 15-120 min after administration LH concentration was decreased at 60 min. Total LH secretion until 75 min after administration was decreased. FSH concentration was decreased at 60 and 75 min after administration Pineda et al.

stiMULAtOrY eFFects OF GniH ON tHe HPG AXis
An electrophysiological study has shown that RFRP-3 exhibits rapid and repeatable inhibitory effects on the firing of 41% of GnRH neurons in adult mice (48). However, stimulatory effect of RFRP-3 was observed in 12% of GnRH neurons ( Table 2). No stimulatory effect of RFRP-3 on the firing of GnRH neurons was observed in diestrus mice but 18% of GnRH neurons were stimulated by RFRP-3 in proestrus female mice (48).
To understand the physiological roles of GnIH in mammalian reproduction, GnIH precursor cDNA and endogenous mature peptides have been identified in the Siberian hamster brain (7). GnIH mRNA expression and number of GnIH-ir perikarya, fibers that innervate GnRH neurons are higher in long days (LD), breeding season, compared with short days (SD), non-breeding season. Intracerebroventricular (icv) administration of hamster RFRP-1 or RFRP-3 to male Siberian hamster inhibits plasma LH concentration 5 and 30 min after administration in LD but stimulates plasma LH concentration 30 min after administration in SD (7) ( Table 2). It has been also shown that central chronic administration of RFRP-3 to male Syrian hamsters adapted to SD fully restores testicular weight and plasma testosterone concentration (44, 70) ( Table 2).
Moussavi et al. investigated the effect of intraperitoneal (ip) administration of goldfish LPXRFa-3 on LHβ and FSHβ subunit mRNA levels in the pituitary and serum LH concentration during gonadal cycle in goldfish (71). Circulating 17β-estradiol (E2) level is very low at early gonadal recrudescence (gr), increasing at mid-gr, very high at mid-late gr, and decreasing at late gr stages. LPXRFa-3 increased LHβ and FSHβ mRNA levels at early to mid-late and late gr, respectively. However, serum LH tAbLe 2 | Continued FiGUre 1 | Schematic diagram of the mechanism of gonadotropin-inhibitory hormone (GnIH) action in the hypothalamic-pituitary-gonadal axis. GnIH neurons act on aromatase and gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus and gonadotrope in the pituitary via GnIH receptor. Aromatase neurons synthesize estradiol-17β (E2) from testosterone (T) in the hypothalamus and E2 can act on GnRH neurons via membrane estrogen receptor (mER). GnIH stimulates K + channel to hyperpolarize GnRH neurons and gonadotrope, and decrease GnRH and luteinizing hormone (LH) release, respectively. E2 stimulates Ca 2+ channel to depolarize GnRH neurons and stimulates GnRH release. GnRH stimulates GnRH receptor and Ca 2+ channel to depolarize gonadotrope and stimulates LH release. Low concentration of E2 inhibits Ca 2+ channel on the gonadotrope and LH release stimulated by GnRH. LH stimulates synthesis and release of E2 and T from ovary and testis, respectively. GnIH and GnRH receptors and GPR30 (mER) belong to Class A G-protein coupled receptor family and may form heteromers to modulate ligand binding affinity and signal transduction. Binding of GnIH, GnRH, and E2 with their receptors can downregulate their cognate receptors by internalization. These complex stimulatory and inhibitory mechanisms may regulate reproductive homeostasis according to developmental and reproductive stages.  Table 3).
Ip administration of grouper GnIH-I, II, and III decreased GnRH1 mRNA level in the hypothalamus (77). However, GnRH3 mRNA level in the hypothalamus was increased by ip administration of GnIH-III. On the other hand, LHβ mRNA level in the pituitary was decreased by GnIH-II (Table 3). Ip administration of lamprey LPXRFa-2 increased GnRH-I and III content in the brain, gonadotropin β mRNA level in the pituitary [(20), Table 3]. A study in European sea bass has shown that intramuscular administration of sea bass GnIH-2 increased GnRH2 and kiss1 receptor mRNA levels in the brain (27). On the other hand, GnIH-1, 2 decreased pituitary LHβ mRNA level and plasma LH level. Plasma FSH level was only decreased by GnIH-1 ( Table 3).

POssibLe MAcHNisM OF tHe stiMULAtOrY eFFects OF GniH ON tHe HPG AXis
The mechanism of GnIH (RFRP-3) effect on the electrophysiological activity of GnRH neurons was studied in transgenic mice having vesicular glutamate transporter 2 (vGluT2)-GnRH neurons (47). GnIH and RFRP-3 produced a non-desensitizing hyperpolarization with IC50 values of 34 and 37 nM, respectively, in vGluT2-GnRH neurons via a direct postsynaptic Ba 2+ -sensitive K + current mechanism (Figure 1, Table 2).
It is known that E2 secreted from the ovary negatively and positively act on the hypothalamus and pituitary to regulate the HPG axis in females. However, it is also known that E2 is synthesized from androgen by aromatase neurons in the hypothalamus (82). Recent studies have shown that E2 synthesized in the brain (neuroestrogen) directly and rapidly act on GnRH neurons via membrane estrogen receptor (mER) to regulate GnRH release (83,84). GPR30 (85,86), ERβ (87,88) or other membrane receptors are thought to transduce the rapid effect of E2 on GnRH GnRH1 mRNA level in the brain was decreased by 1, 2, 4 µg GnIH-1. GnRH2 mRNA level in the brain was decreased by 1, 2, 4 µg GnIH-2. Kiss1 mRNA level in the brain was decreased by 2-µg GnIH-2. Kiss2 mRNA level in the brain was decreased by 2, 4 µg GnIH-2. Kiss1 receptor mRNA level in the brain was decreased by 2-µg GnIH-2. GnIH mRNA level in the brain was decreased by 1, 2 µg GnIH-2. GnIH receptor mRNA level in the brain was decreased by Immature, mature male and female cinnamon clownfish 100-ng/g bw goldfish LPXRFa-3 ip After 0, 6, 12, and 24-h administration with or without 100ng/g bw sbGnRH brain, pituitary and blood were collected GnIH and GnIH receptor mRNA levels in the brain were increased at 6, 12 and 24 h. a GnIH and GnIH receptor mRNA levels in the brain decreased by sbGnRH were increased at 6, 12 and 24 h. a sbGnRH mRNA level in the brain, plasma GnRH, FSH, LH levels, pituitary GTHα, FSHβ, LHβ mRNA levels were decreased at 6, 12 and 24 h. a sbGnRH mRNA level in the brain, plasma GnRH, FSH, LH levels, pituitary GTHα, FSHβ, LHβ mRNA levels increased by sbGnRH were decreased at 6, 12 and 24 h a Choi et al. Plasma testosterone and 11-ketotestosterone levels were decreased by sbGnIH-1, 2 in November and December (early and mid-spermatogenesis). GnRH2, sbGnIH, sbGnIH receptor, kiss1 receptor mRNA levels in the brain were increased by sbGnIH-2. LHβ mRNA level in the pituitary was decreased by sbGnIH-1 and -2. Plasma FSH level was decreased by sbGnIH-1. Plasma LH level was decreased by sbGnIH-1 and -2 Paullada-Salmerón et al.   release (83,89). E2 stimulates GnRH release by increasing intracellular Ca 2+ concentration (90) and electrophysiological activity of GnRH neurons (91,92). More recently, it has been shown that GnIH neurons terminal on aromatase neurons that express GnIH receptor and increase neuroestrogen concentration in the hypothalamus by stimulating aromatase activity in quail (93,94). Therefore, it is possible that GnIH stimulates the electrophysiological activity of some GnRH neurones (48) by increasing neuroestrogen concentration in the hypothalamus. GnIH may further stimulate LH release that was shown in hamsters (7) by stimulating the activity of aromatase neurons and increasing neuroestrogen concentration in the hypothalamus and stimulating the electrophysiological activity of GnRH neurons and GnRH release (Figure 1). Binding of GnRH with GnRH receptor on gonadotropes results in the activation of intracellular Gαq/11 and phospholipases and generation of the second messengers, inositol 1-, 4-, 5-tris-phosphate, diacylglycerol, and arachidonic acid, which stimulate Ca 2+ mobilization and PKC activity. Ca 2+ mobilization initiates gonadotropin release (Figure 1). PKC activates mitogenactivated protein kinases (MAPKs) such as ERK, jun-N-terminal kinase, and p38 MAPK, which initiate the transcriptional activity of gonadotropin subunit genes (95). GnRH receptor also couples with Gαs to stimulate AC/cAMP/PKA pathway, which was shown in LβT2 cells (96) and rat gonadotropes (97). Because GnIH signaling pathway triggered by Gαi does not interfere with Gαq/11 triggered pathway, GnIH may suppress gonadotropin subunit gene transcription by inhibiting AC/cAMP/PKA pathway stimulated by GnRH receptor and Gαs (34). GnIH may also suppress gonadotropin release by hyperpolarizing gonadotropes by activating K + channel via GnIH receptor [(47), Figure 1].
However, recent studies of GPCR have shown that GPCR not only functions as a monomer or homodimer but also as a heterodimer with different GPCR resulting in modulation of ligand binding affinity, signal transduction, and internalization of the receptors (98,99). It has been shown that Class A GPCRs form homo-and heteromers (100). As GnRH and GnIH receptors, and GPR30 all belong to Class A GPCR family (101), it is possible that they form heteromers in GnRH neurons and/or gonadotropes to modify the action of their ligands. Some of the stimulatory effect of GnIH on the HPG axis may be due to heteromerization of GnIH and GnRH receptor and GPR30 (Figure 1).
A recent study has shown that centrally administered GnIH can decrease plasma LH concentration in ovariectomized (OVX) prepubertal female mice that were treated with E2 but not in OVX mice that were not treated with E2 (43) ( Table 2). E2 can abolish intracellular free Ca 2+ concentration and LH release in ovine pituitary culture induced by GnRH (102). The inhibitory effect of low concentration of E2 on LH release was shown in bovine anterior pituitary mediated by GPR30 expressed on the gonadotrope (103,104). These results suggest the modification of GnIH action by E2 in the hypothalamus and pituitary (Figure 1).
Finally, it is known for a long time that binding of GnRH with GnRH receptors is followed by aggregation, complex formation and internalization (105). Chronic administration of GnRH or antagonist administration can desensitize pituitary gonadotropes, downregulate GnRH receptor and suppress serum LH, FSH and sex-steroid levels (106)(107)(108). It is therefore possible that chronic central administration of GnIH (RFRP-3) to male Syrian hamsters adapted to SD restores testicular weight and plasma testosterone concentration by downregulation of GnIH receptor in the hypothalamus and pituitary (44,70) (Table 2). It is also possible that stimulatory effect of GnIH on the pituitary of fish is due to downregulation of GnIH receptor by chronic administration (79,80), high concentration of GnIH (66,80,81) or antagonistic effect of LPXRFa peptides of different species (66,79) (Table 3). Inhibitory effects of GnIH on the HPG axis are shown when GnIH peptides are tested with relatively low concentrations in a shorter time frame (73)(74)(75)(76) (Tables 2 and 3; Figure 1).
Complex mechanism may be involved in in vivo studies that show stimulatory and inhibitory effects of GnIH on the HPG axis in addition to downregulation of receptors and changes in the number of receptors depending on reproductive and developmental stages and endogenous sex-steroid levels (Tables 2 and 3; Figure 1). It is also important to note that GnIH peptides are produced in gonads (3,109) and it has been shown that they have direct effects on gonadal activates in mammals (110)(111)(112)(113)(114), birds (115)(116)(117) and fishes (118). Most of these studies showed inhibitory effects of GnIH peptides on gonadal activities, but stimulatory activity of GnIH peptides was also shown in mouse ovary (114) and goldfish testis (118). Therefore, in vivo studies that showed effects of GnIH peptides on gonadal activates (Tables 2 and 3) may include direct effects of GnIH peptides on the gonads. cONcLUsiON Gonadotropin-inhibitory hormone orthologous peptides have a characteristic LPXRFamide C-terminal motif in most vertebrate species, which is critical for receptor binding. The primary receptor for GnIH is GPR147 that inhibits cAMP production in target cells. GnIH generally decreases gonadotropin synthesis and release by directly acting on the gonadotrope or by decreasing the activity of GnRH neurons. However, one study shows stimulatory effects of GnIH on the electrophysiological activity of some GnRH neurons in mice (48). Stimulatory effect of GnIH on GnRH neurons in the hypothalamus may be explained by the action of neuroestrogen synthesized in the hypothalamus by the stimulatory action of GnIH on aromatase neurons that terminate on GnRH neurons that express estrogen membrane receptor. GnIH may further stimulate LH release that was shown in hamsters by stimulating the electrophysiological activity of GnRH neurons and GnRH release (7,44). Peripheral sex-steroid levels may also modify the action of GnIH (7,44,71,72). Some of the stimulatory effects of GnIH on the HPG axis may be due to heteromerization of GnIH and GnRH receptors and GPR30 in GnRH neurons and/ or gonadotropes, which modifies ligand binding and signaling transduction mechanism. Stimulatory effect of GnIH on the HPG axis may also be due to internalization of GnIH receptor by high concentration or chronic administration of GnIH or antagonistic effect of the peptides administered (20,66,77,(79)(80)(81). Besides pharmacological effect of administered peptides, the general inhibitory action of GnIH by decreasing cAMP concentration and inducing hyperpolarization in target cells and the additional stimulatory action of GnIH by neuroestrogen synthesis, receptor heteromerization, and internalization may have a physiological role to maintain reproductive homeostasis according to developmental and reproductive stages.