T O P I C S

Gonadotropin-Inhibitory Hormone（GnIH）
Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, and Integrative Brain Science Center at Hiroshima University

Kazuyoshi TSUTSUI

Discovery of GnIH

times on a C-18 reversed-phase column and a cation-exchange column. The mixture of the synthetic and native

Since the molluscan cardioexcitatory neuropeptide Phe-

peptides eluted as a single peak from each column. Thus

Met-Arg-Phe-NH2 (FMRFamide) was found in the ganglia

the isolated native peptide was confirmed as a 12 aminoof the venus clam Macrocallista nimbosa [1], neuropeptides

acid sequence (SIKPSAYLPLRFamide) with RFamide atthat possess the RFamide motif at their C-termini (i.e.,

the C-terminus [2]. This neuropeptide had not been previ-

RFamide peptides) have been characterized in various in-ously reported in vertebrates, although the C-terminal

vertebrates. Subsequently, many immunohistochemical

LPLRFamide was identical to chicken pentapeptide LPLR-

studies that used the antiserum against FMRFamide sug-

Famide peptide [3]. The chicken peptide may be a degradedgested that vertebrate nervous systems possess some un-fragment of the dodecapeptide, as suggested by Dockray etknown neuropeptides similar to FMRFamide. Immunohis-al. [4].

tochemical findings indicated that some of the FMRFamide-

Subsequently, the isolated novel peptide was shown to be

like immunoreactive neurons project to the hypothalamic

located in the quail hypothalamo-hypophysial system and toregion close to the pituitary gland, and thus were predicted

decrease gonadotropin release from cultured anterior pitu-

to play an important role in the regulation of pituitary func-

itary in a dose-dependent manner [2]. We therefore desig-

tion. We therefore looked for a novel RFamide peptide in

nated this novel RFamide peptide as gonadotropin-inhibito-

the avian brain.

ry hormone (GnIH; Fig. 1) [2].

To isolate the RFamide peptide from the brain, Japanesequail (Coturnix japonica) were used and the peptidergic
molecule was probed with a competitive enzyme-linked immunosorbent assay (ELISA), employing the antibody
against the dipeptide, Arg-Phe-NH2 [2]. Acetic acid extracts of quail brain were passed through C-18 reversedphase cartridges, and the retained material was subjected toreversed-phase and cation-exchange high performance liquid chromatography (HPLC). Amino acid sequence analysis of the isolated substance by automated Edman degradation with a gas-phase sequencer revealed the following
sequence: Ser(62)- Ile(252)- Lys(233)- Pro(226)Ser(38)- Ala(194)- Tyr(173)- Leu(148)- Pro(104)Leu(108)- Arg(45)- Phe(52) with the detected amount(pmol) of each amino acid indicated in parentheses. A protonated molecule ion (M+H)+ peak in the fast atom bombardment-MS (FAB-MS) of this peptide at m/z 1389.4 indicated that the peptide is amidated at the C-terminus.
Synthetic and native peptides showed identical retention

Fig. 1. GnIH, a newly discovered hypothalamic neuropeptide, in thequail brain. We isolated a novel hypothalamic dodecapeptide (SIKPSAYLPLRFamide) inhibiting gonadotropin releasein quail [2]. Cell bodies and terminals containing the isolated novel neuropeptide were localized in the paraventricular
nucleus (PVN) and median eminence (ME), respectively[2]. The isolated novel neuropeptide was shown to decrease
gonadotropin release from cultured anterior pituitary in adose-dependent manner [2]. We therefore designated thisnovel hypothalamic neuropeptide as GnIH [2].

Japanese Journal of Reproductive Endocrinology（2005）10：49-53

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Localization of GnIH in the brain

and localization of its transcript, which would provide us
with key information as to the regulation of the mature

The localization of GnIH in the brain is essential to un-

GnIH peptide, along with confirmation of brain area (s) thatderstand its actions. We dissected out the quail brain into

synthesize this novel peptide. A cDNA that encoded theseveral regions, and quantified the concentration of GnIH

GnIH precursor polypeptide was identified in the quail brain

by ELISA using a rabbit polyclonal antibody raised against

using a combination of 3 and 5 rapid amplification of

GnIH [2]. The concentration of GnIH in the diencephalon

cDNA ends (3 /5 RACE) [9]. The deduced GnIH precur-

was much higher than that in the mesencephalon. In con-sor consisted of 173 amino acid residues that encoded one

trast, GnIH concentrations in the cerebrum and cerebellum

GnIH and two putative GnIH-related peptide (GnIH-RP-1

were below the level of detectability. Subsequently, we in-and -RP-2) sequences that included -LPXRF (X=L or Q)

vestigated the precise localization of GnIH in the quail brainat their C-termini. All these peptide sequences wereby immunohistochemistry [2, 5, 6]. Clusters of distinct

flanked by a glycine C-terminal amidation signal and a sin-

GnIH-immunoreactive neurons were found in the paraven-gle basic amino acid on each end as an endoproteolytic site.

tricular nucleus (PVN) in the hypothalamus. In addition to

We also cloned a cDNA that encoded GnIH in the brain of

the PVN, some scattered small cells were immunoreactive

Gambelʼs white-crowned sparrow [8]. The deduced spar-in the septal area. In contrast to the highly-localized clus-

row GnIH precursor also consisted of 173 amino acid resi-

ters of cell bodies, GnIH-containing fibers were widely dis-dues, encoding one sparrow GnIH and two sparrow GnIH-

tributed in the diencephalic and mesencephalic regions par-related peptides (sparrow GnIH-RP-1 and GnIH-RP-2)

ticularly in the ventral paleostriatum, septal area, preoptic

that included -LPXRFamide (X=L or Q) at their C-termi-

area, hypothalamus, and optic tectum. The most prominent

ni. Although the homology of sparrow and quail GnIH pre-fibers were seen in the median eminence of the hypo-

cursors was approximately 66％, the C-terminal structures

thalamus, and in the dorsal motor nucleus of the vagus inof GnIH, GnIH-RP-1 and GnIH-RP-2 were all identical in

the medulla oblongata.

two species [8, 9]. Subsequently, a cDNA encoding GnIH

We further investigated GnIH localization in the brain of
sparrows, seasonally-breeding avian species [7, 8]. Dense

and GnIH-RPs was also reported in the chicken from agene database.

populations of GnIH-immunoreactive neurons were also

In situ hybridization further revealed the cellular local-

found in the PVN of these birds. The PVN was the only lo-

ization of GnIH mRNA solely in the PVN of quail and spar-

cation where immunoreactive neurons were located [7, 8].

row hypothalami [5, 8]. As already described, immunohisto-

Thus the presence of GnIH in the PVN appears to be a con-chemical analysis using the quail and sparrow also showed

served property among several avian species. In addition, athat quail and sparrow GnIH-immunoreactive cell bodies

widespread distribution of GnIH-containing fibers was also

and terminals were localized in the PVN and median emi-

found in the brain of seasonally-breeding sparrows.

nence, respectively. Thus only the PVN expresses GnIH

Interestingly, GnIH-containing fibers were further ob-

and, in birds, the immunoreactive peptide found in fibers inserved in extremely close proximity to GnRH neurons in

multiple brain areas including the median eminence appears

the preoptic area (POA) in birds [5, 7]. It is therefore plau-

to originate from the PVN only [5, 8].

sible that GnIH may act at the level of the hypothalamus toregulate gonadotropin release as well as at the pituitary.

GnIH precursor polypeptide
We further examined the precursor polypeptide for GnIH

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Japanese Journal of Reproductive Endocrinology Vol.10 2005

GnIH function on gonadotropin release
In view of the immunohistochemical finding indicatingthat GnIH-immunoreactive neurons project to the medianeminence close to the pituitary, we analyzed the effect of

T O P I C S

the isolated SIKPSAYLPLRFamide, GnIH, on the release of

the putative GnIH receptor cDNA specifically bound toluteinizing hormone (LH), follicle-stimulating hormone

GnIH and GnIH-RPs in a concentration-dependent manner(FSH) and prolactin (PRL) using cultured quail anterior[11]. Scatchard plot analysis of the binding showed that thepituitaries [2]. GnIH significantly inhibited LH release, af-

identified GnIH receptor possessed a single class of high af-

ter 100-min incubation. The inhibitory effect on LH releasefinity binding sites (Kd =0.752 nM). Southern blotting

was dose-dependent and its threshold concentration ranged

analysis of reverse-transcriptase-mediated PCR products

between 10−9and 10−8

M. A possible similar tendency forrevealed the expression of GnIH receptor mRNA in the pi-

GnIH to inhibit FSH release was also detected. However,tuitary and several brain regions including diencephalon in

there was no effect of GnIH on PRL release. Based on

the quail [11]. These results indicate that GnIH acts direct-

these results of this novel RFamide peptide isolated fromly on the pituitary via GnIH receptor to inhibit gonadotropin

the quail brain, we therefore named it GnIH [2].

release (Fig. 2). GnIH may also act on the hypothalamus to

We further showed that GnIH was effective in inhibiting
circulating LH in vivo. When administered intraperitoneallyto quail via osmotic pumps, GnIH significantly reduced

inhibit GnRH release.

Regulation of GnIH expression

plasma LH (Ubuka et al., unpublished observation). GnIH

To understand the physiological role of GnIH in avian re-injected simultaneously with GnRH inhibited the surge of

production, we recently, characterized developmental

plasma LH above the baseline in sparrows [8]. Further-changes in GnIH expression in the quail hypothalamo-hy-

more, GnIH injections also decreased breeding levels of LH

pophysial system [6]. Our data indicated that, as appears toin free-living sparrows [8].

be the case for GnRH [12], GnIH begins its function aroundIn addition to the inhibitory effects of GnIH on gonadotropin release, there is evidence that GnIH inhibits gonado-

hatch and acts as a hypothalamic factor to regulate gonadotropin release in quail [6].

tropin biosynthesis in vitro [10]. The suppressive effect of

Until now, a regulatory mechanism (s) governing GnIH

GnIH on gonadotropin mRNA was associated with an inhi-

expression has remained unclear. Although many bird spe-

bition of both LH and FSH release in the chicken [10]and
quail (Ubuka et al., unpublished observation).

Mode of action of GnIH
Identification of the receptor for GnIH is crucial to elucidate the mode of action of GnIH. We therefore identified
the receptor for GnIH in the quail diencephalon and characterized its expression and binding activity [11]. We firstcloned a cDNA encoding a putative GnIH receptor by a
combination of 3 and 5 rapid amplification of cDNA ends
(RACE) using PCR primers designed from the sequence
for the receptor for rat RFamide-related peptide (RFRP),
an orthologous peptide of GnIH. Hydrophobic analysis revealed that the putative GnIH receptor possessed seventransmembrane domains, indicating a new member of the Gprotein-coupled receptor (GPCR) superfamily [11]. The
crude membrane fraction of COS-7 cells transfected with

Fig. 2. The mode of action of GnIH on gonadotropin release. We
identified the receptor for GnIH and characterized its expression and binding activity in quail [11]. The identified GnIH
receptor specifically bound to GnIH [11]. The expression of
GnIH receptor was found in the pituitary and several brain
regions including the hypothalamus [11]. Thus GnIH acts directly on the pituitary via GnIH receptor to inhibit gonadotropin release. GnIH may also act on the hypothalamus to inhibit GnRH release.

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cies are photoperiodic, a dogma has existed that birds do
not use seasonal changes in melatonin secretion to time
their reproductive effort, and a role for melatonin in birds
has remained enigmatic [13, 14]. Despite the accepted dogma, there is strong evidence that melatonin is involved inregulation of several seasonal processes, including gonadal
activity and gonadotropin secretion [15-18]. In light ofthese reports and considering GnIH s inhibitory effects on
gonadotropin secretion [2, 8], we manipulated melatoninlevels in quail by removing sources of melatonin and investigated the action of melatonin on GnIH expression in thequail brain [19]. Pinealectomy combined with orbital enucleation (Px+Ex) decreased the expression of GnIH pre-Fig. 3. The mode of action of melatonin on GnIH expression. Melatonin originating from the pineal gland and eyes induced
GnIH expression in GnIH neurons [19]. Melatonin receptor(Mel1c) was expressed in GnIH neurons [19]. Thus melatonin acts directly on GnIH neurons via its receptor to induce
GnIH expression.

cursor mRNA and the mature peptide GnIH in the diencephalon including the PVN and median eminence. Melatonin administration to Px+Ex birds caused a dosedependent increase in expression of GnIH precursor mRNAand production of mature peptide. The expression of GnIH
was photoperiodically controlled and increased under shortday (SD) photoperiods [19], when the duration of melatonin secretion increases [20, 21]. Interestingly, Mel1c, a melatonin receptor subtype was expressed in GnIH-ir neurons
in the PVN [19]. Melatonin receptor autoradiography further revealed specific binding of melatonin in the PVN [19].
Thus melatonin appears to act directly on GnIH neurons via
its receptor to induce GnIH expression (Fig. 3).
To give our findings a broader perspective, we recently
cloned a homolog of GnIH from the brain of Siberian hamster, a photoperiodic mammal (Inoue et al., unpublisheddata). The expression of the GnIH homolog in hamster hypothalamus was also controlled by melatonin (Inoue et al.,unpublished observation). It is likely that the mammalian
homolog of GnIH transduces photoperiodic information via
changes in the melatonin signal and thus influences the reproductive axis of hamsters as in birds.
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