ME T ABOL I SM C L IN I CA L A N D E XP E RI ME N TAL 86 ( 20 1 8 ) 3 – 1 7

Available online at www.sciencedirect.com

Metabolism
www.metabolismjournal.com

Reproductive Physiology

The hypothalamus-pituitary-gonad axis:

Tales of mice and men

Athina Kaprara a,⁎, Ilpo T. Huhtaniemi b

a
Unit of Reproductive Endocrinology, Medical School, Aristotle University of Thessaloniki, Greece
b
Imperial College, London, UK

A R T I C LE I N FO AB S T R A C T

Article history: Reproduction is controlled by the hypothalamic-pituitary-gonadal (HPG) axis.

Received 17 July 2017 Gonadotropin-releasing hormone (GnRH) neurons play a central role in this axis through
Accepted 29 November 2017 production of GnRH, which binds to a membrane receptor on pituitary gonadotrophs and
stimulates the biosynthesis and secretion of follicle-stimulating hormone (FSH) and
Keywords: luteinizing hormone (LH). Multiple factors affect GnRH neuron migration, GnRH gene
GnRH expression, GnRH pulse generator, GnRH secretion, GnRH receptor expression, and
Hypothalamus–pituitary–gonadal gonadotropin synthesis and release. Among them anosmin is involved in the guidance of
axis the GnRH neuron migration, and a loss-of-function mutation in its gene leads to a failure of
GnRH receptors their migration from the olfactory placode to the hypothalamus, with consequent anosmic
GnRH pulse generator hypogonadotropic hypogonadism (Kallmann syndrome). There are also cases of
hypogonadotropic hypogonadim with normal sense of smell, due to mutations of other
genes. Another protein, kisspeptin plays a crucial role in the regulation of GnRH pulse
generator and the pubertal development. GnRH is the main hypothalamic regulator of the
release of gonadotropins. Finally, FSH and LH are the essential hormonal regulators of
testicular functions, acting through their receptors in Sertoli and Leydig cells, respectively.
The main features of the male HPG axis will be described in this review.
© 2017 Elsevier Inc. All rights reserved.

Abbreviations: HPG, hypothalamic-pituitary-gonadal; GnRH, gonadotropin-releasing hormone; FSH, follicle-stimulating hormone; LH,
luteinizing hormone; CNS, central nervous system; HH, hypogonadotropic hypogonadism; PSA-NCAM, polysialic acid form of neural
adhesion molecule; NELF, nasal embryonic LHRH factor; GABA, gamma-aminobutyric acid; FGF8, fibroblast growth factor 8; FGFR1,
fibroblast growth factor receptor 1; PROK2, prokineticin 2; HGF, hepatocyte growth factor; KS, Kallmann syndrome; aa, amino acid;
DREAM, downstream regulatory element antagonist modulator; Dlx2, distalless homeobox 2; PKC, protein kinase C; RARa, retinoic acid
receptor-a; RXRa, retinoid X receptor-a; ER, estrogen receptor; KiSS1R, kisspeptin receptors; DHEAS, dehydroepiandrosterone sulfate; IGF-
1, insulin-like growth factor 1; MAPK, mitogen-activated protein kinase; Egr-1, early growth response-1; NPY, neuropeptide Y; GPR54, G-
protein coupled receptor 54; IP3, inositol-trisphosphate; ERK, extracellular signal–regulated kinase; JNK, c-Jun N-terminal kinase; AC,
adenylyl cyclase; cAMP, cyclic adenosine-3′:5′-monophosphate; PKA, protein kinase A; CREB, cAMP response element-binding protein;
EGFR, epidermal growth factor receptor; GPCR, G protein-coupled receptor; GnIH, gonadotropin-inhibitory hormone; LRR, leucine-rich
repeats; hCG, human chorionic gonadotropin; DSD, disorder of sexual development; TGF-β, transforming growth factor-β.
⁎ Corresponding author at: Unit of Reproductive Endocrinology, Medical School, Aristotle University of Thessaloniki, Papageorgiou
General Hospital, Ring Road, Municipality of Pavlos Meloas Area of N. Efkarpia, Thessaloniki 56403, Greece.
E-mail addresses: athkap@yahoo.gr (A. Kaprara), ilpo.huhtaniemi@imperial.ac.uk (I.T. Huhtaniemi).

https://doi.org/10.1016/j.metabol.2017.11.018
0026-0495/© 2017 Elsevier Inc. All rights reserved.
4 ME TA BOL I SM C LI N I CA L A ND E XP E RI ME N TAL 86 ( 20 1 8 ) 3 – 1 7

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2. Embryonic Development and Anatomical Distribution of GnRH Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3. GnRH and GnRH Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.1. Regulation of GnRH Gene Expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.2. GnRH Pulse Generator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4. Kisspeptin, Leptin, Neurokinin B and Dynrophin Regulating GnRH Production . . . . . . . . . . . . . . . . . . . . . . . 7
4.1. GnRH Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.2. Gonadotropin-Inhibitory Hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
4.3. Gonadotropins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
. Conflicts
Conflicts of Interest
of Interest. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1. Introduction neurons to migrate properly results in hypogonadotropic

The hypothalamic–pituitary–gonadal (HPG) axis is the humoral
component of an intercommunicating neural and endocrine
system that functions in the regulation of fertility. In 1937, a
medical student at Cambridge, Geoffrey Harris, was the first one
to provide evidence that the pituitary–gonadal axis is under
control of the central nervous system (CNS) [1]. The isolation of
a hypothalamic substance that, through the hypophysial portal
circulation, regulates the synthesis and secretion of gonado-
tropins was attained later by two different groups, of Andrew
Schally and Roger Guillemin, from porcine and ovine brain,
respectively. The active molecule was a decapeptide, now
referred to as gonadotropin-releasing hormone (GnRH). Two
years earlier, in 1969, the group of Ernest Knobil had provided
evidence for an episodic manner of luteinizing hormone (LH)
secretion, something that was empirically demonstrated in
ovariectomized ewes by Clarke and Cummins in 1982 [2,3]. The
purpose of this review is to review the role of GnRH in
regulation of the HPG axis in the male.
2. Embryonic Development and Anatomical
Distribution of GnRH Neurons
GnRH neurons comprise a unique neuronal population that
originates outside the CNS. In mice, during development, GnRH
neurons migrate from the medial olfactory placode of the
developing nasal cavity across the nasal septum, together with
vomeronasal axons, and enter into the forebrain with the
nervus terminalis, arching into the septal-preoptic area and
hypothalamus. The exact final location differs among species
[4,5]. From the hypothalamus, processes are extended to the
median eminence, and GnRH is released into capillaries of the
hypophysial portal system in order to reach the anterior
pituitary gland and to modulate its gonadotropin synthesis
and secretion. A typical GnRH neuron in the adult hypothala-
mus has two dendritic projections which extend a distance of
2–3 mm from their cell body [6]. In humans, there are 1000–1500
GnRH neurons and their co-location with multiple central
regulators allows the interaction between GnRH and other
neuroendocrine and metabolic inputs [7]. Failure of GnRH
hypogonadism (HH) and disrupts sexual maturation in mice
and men [8].
Multiple factors play a critical role in the guidance of the GnRH
neuron migration process. Among them, there are adhesion
molecules, such as anosmin, polysialic acid form of neural
adhesion molecule (PSA-NCAM) [9] and cell surface
glycoconjugate [10]. Furthermore, gamma-aminobutyric acid
(GABA) [11], cholecystokinin [12], fibroblast growth factor 8
(FGF8) [13], prokineticin 2 (PROK2) [14–16] and transcription
factors, such as Ebf2 modulate GnRH neuronal migration [17].
Moreover, there are guidance cues, such as ephrins [18] and nasal
embryonic LHRH factor (NELF) [19]. There are factors that guide
GnRH neurons toward the forebrain and factors that guide GnRH
neurons through the cribriform plate to the hypothalamus. The
first category includes semaphorins, plexins [8] and reelin [20].
The second category includes hepatocyte growth factor (HGF)
[21], Axl, Tyro3, and chemokine attractants [8].
Anosmin is the product of KAL1 gene, which is located on the
Xp22.3 region; it was cloned in 1991 [22]. It consists of 680 amino
acids and has a molecular weight of 85–100 kDa [23]. It is
expressed extracellularly with high-affinity binding to cell
membrane heparin sulfate proteoglycans, and its best-
characterized mechanism of action is its interaction with
fibroblast growth factor receptor 1 (FGFR1) [24]. Anosmin is
involved in normal GnRH migration and a failure of this
migration leads to Kallmann syndrome (KS), which is a combi-
nation of HH and deficient sensation of smell [25]. Analysis of
patients with KS showed loss-of-function mutation in KAL1
(frameshifts, deletions and premature terminations) [26].
The NELF gene, located on chromosome 9q34.3, encodes
the nasal embryonic LHRH factor. It is detected in the
developing olfactory and GnRH migratory pathway. NELF
mutations can lead to both KS and normosmic HH [19].
The FGF8 gene, located on chromosome 10q24, encodes
FGF8. The FGFR1 gene, located on 8p11.22-p11.23, encodes
FGFR1. FGF8 and its receptor FGFR1 affect the migration of
GnRH neurons. FGF8 and FGFR1 mutations can lead to both KS
and normosmic HH. Moreover, FGF8 and FGFR1 loss-of-
function mutations can cause cleft lip or palate, whereas
gain-of-function mutations can lead to craniosynostosis. The
FGF17 gene, located on chromosome 8p2.3, encodes FGF17
 ME T ABOL I SM C L IN I CA L A N D E XP E RI ME N TAL 86 ( 20 1 8 ) 3 – 1 7 5

and its mutations have been described in patients with KS Table 1 – Genes, encoded proteins and phenotypes
and normosmic HH [13]. associated to hypogonadotropic hypogonadism.
The PROK2 gene, located on chromosome 3p21.1, encodes Gene Encoded protein Phenotype
prokineticin-2. The PROK2R2 gene, located on 20p13, encodes
AXL Member of the Tyro3-Axl-Mer (TAM) KS or nHH
prokineticin receptor. PROK2 and its receptor PROK2R2 affect
receptor tyrosine kinase subfamily
GnRH migration. PROK2 and PROKR2 mutations can lead to CCDC141 KS
both KS and normosmic HH. Moreover, PROK2 and PROK2R2 CHD7 Chromodomain helicase DNA binding KS or nHH
loss-of-function mutations can cause obesity, epilepsy, sleep protein 7
disorders and synkinesia [14,15]. DMXL2 nHH
The SEMA3A gene, located on chromosome 7q21.11, DUSP6 Member of the dual specificity protein KS or nHH
phosphatase subfamily
encodes semaphorine 3A. It plays a role in the development
FEZF1 Transcriptional repressor that belongs to KS
of the olfactory system and SEMA3A loss-of-function is the zinc finger double domain protein
involved in KS [27]. family
KS represents approximately 60% of the cases of HH, FGF8 Fibroblast growth factor 8 KS or nHH
whereas the rest 40% are associated with a normal sense of FGF17 Fibroblast growth factor 17 KS or nHH
smell. Variants of > 25 genes are associated with about half of FGFR1 Fibroblast growth factor receptor KS or nHH
FLRT3 Member of the fibronectinleucine rich KS
all cases of HH, whereas the cause of the rest is still unknown.
transmembrane protein family
HH can be inherited in an X-linked, autosomal dominant, or
GNRH1 Preproprotein that is proteolytically nHH
autosomal recessive manner [27–29]. Variants in genes listed processed to generate a peptide that is a
in Table 1 are associated with HH. member of the gonadotropin-releasing
hormone family of peptides
GNRHR Receptor for type 1 gonadotropin- nHH
releasing hormone
3. GnRH and GnRH Genes
HESX1 Conserved homeobox protein that is a KS
transcriptional repressor in the
Mammalian GnRH (termed GnRH-I) is a 10-amino acid neuro-
developing forebrain and pituitary gland
peptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH2) that HS6ST1 Member of the heparan sulfate KS or nHH
was first isolated from porcine hypothalamus. It plays a key biosynthetic enzyme family
role in the function of reproductive axis. It is produced by IL17RD Interleukin-17 receptor KS
hypothalamic neurosecretory cells (GnRH neurons), and KAL1 Anosmin-1 KS
released in a pulsatile manner into the hypothalamo- KISS1 Kisspeptin nHH
KISS1R Kisspeptin receptor nHH
hypophyseal portal circulation, subsequently reaching the
LEP Leptin nHH
anterior pituitary gland, where it stimulates LH and follicle-
LEPR Leptin receptor nHH
stimulating hormone (FSH) synthesis and release [2]. Its NELF Nasal embryonic LHRH factor KS or nHH
prohormone is synthesized as a larger 92–amino acid (aa) NR0B1 nHH
precursor protein which consists of 23-aa signal peptide, the OTUD4 nHH
functional GnRH decapeptide, an amidation⁄proteolytic pro- PCSK1 Member of the subtilisin-like nHH
cessing signal (Gly-Lys-Arg), and a 56-GnRH-associated peptide proproteinconvertase family
PNPLA6 Phospholipase that deacetylates nHH
[30]. GnRH is also found in cells outside the brain, but its role is
intracellular phosphatidylcholine to
not yet well understood. Maybe GnRH-I act as autocrine and/or produce glycerophosphocholine
paracrine regulator in extrapituitary tissues such as placenta, POLR3B RNA polymerase III subunit B nHH
immune system, gonads, and endocrine tumors [31–35]. PROK2 Prokineticin-2 KS or nHH
According to aa sequence, localization and embryonic PROKR2 Prok receptor KS or nHH
origin, numerous GnRHs have been found from vertebrates RNF216 Ring finger protein 216 nHH
SEMA3A Semaphorine 3A KS
to protochordates [7]. Most vertebrates have at least two, and
SEMA3E Semaphorin 3E KS
usually three, forms of GnRH. In humans, in addition to
SEMA7A Semaphorin 7A KS or nHH
GnRH-I a second subtype, called GnRH-II has been found. SOX10 SRY-related HMG-box transcription factor KS
GnRH-II was first identified from chicken hypothalamus SPRY4 Sprouty RTK signaling antagonist 4 KS or nHH
[36,37]. It is a decapeptide that is found in the midbrain region SRA1 Steroid receptor RNA activator 1 nHH
and serves as a neurotransmitter, rather than as a pituitary TAC3 Tachykinin 3 nHH
releasing factor. Nevertheless, it has been shown that GnRH-II TACR3 Tachykinin receptor 3 nHH
WDR11 Member of the WD repeat protein family KS or nHH
can stimulate gonadotropin release in vivo through its
binding to the type I GnRH receptor [38]. GnRH-I and GnRH-II KS = Kallmann syndrome, nHH = normosmic hypogonadotropic
are found in phylogenetically diverse species, from fish to hypogonadism.
mammals, but whereas the expression of GnRH-I is higher in
the brain, GnRH-II is expressed mostly in other organs [39,40].
A third form of GnRH, GnRH-III, has been identified in
neurons of the telencephalon in teleost fish. it is located on chromosome 8p11.2-p21, and is composed of
In mammals, two genes encoding GnRH have been four exons separated by three introns [40]. The second,
identified. The first gene GnRH1 encodes GnRH-I and it was GnRH2, encodes GnRH-II, and it is mapped to chromosome
first cloned by Seeburg and Adelman in 1984 [41]. In humans, 20p13 [37].
6 ME TA BOL I SM C LI N I CA L A ND E XP E RI ME N TAL 86 ( 20 1 8 ) 3 – 1 7
Fig. 1 – Presentation of the GnRH1 gene 5′-region in rat, mouse and human.
The 5′-flanking region of GnRH1 is highly homologous
between human, mouse and rat (Fig. 1).
In the rat GnRH1, a proximal promoter and a distal
enhancer have been found. Both of them are important for
gene transcription. The promoter is within 173 bp upstream
of the transcription start site and the 300 bp enhancer is
located at − 1863 to −1571 bp relative to the transcription start
site.
The previous key regions are highly conserved between rat
and mouse. In the mouse GnRH1, enhancer elements are
located between − 5500 and −2100 bp, whereas a 400 bp region
(− 2100 to − 1700 bp) is found to be a critical enhancer region
for the GnRH gene in mouse brain [40].
In humans, the promoter is located at − 551 to + 1 bp region
of the GnRH1 gene. It was also found that the region between
− 3832 and + 8 bp gives high levels of expression of GnRH genes
in Gt1–7 neuronal cells. The − 3036 to − 2923 bp, − 2766 to −
2539 bp and −1775 to − 1552 bp fragments are similar to
sequences in the rat GnRH promoter. Moreover, the region
between − 343 and + 8 bp has marked homology to the region
between − 332 and + 96 bp of the rat GnRH [40,42,43] (Fig. 1).
3.1. Regulation of GnRH Gene Expression
GnRH1 gene is expressed in a pulsatile manner in the
hypothalamus, and this is closely associated with the
pulsatile manner of its secretion. A specific region between
− 2012 and − 1597 bp was found to be responsible for this
pulsatility [43]. The involvement of Ca2+ and a Ca2 + − binding
protein, downstream regulatory element antagonist modula-
tor (DREAM), in GnRH1 pulsatility has also been found, playing
a possible role in the communication between cytoplasm and
nucleus [44]. A variety of hormones and second messengers
regulate GnRH gene expression. In the rat, the promoter
region contains two Octamer-binding transcription factor-1
binding sites that play a critical role in the regulation of rat
GnRH1 transcription [45]. Within the enhancer, two POU (Pit-1,
Oct-1/2 and Unc-86) homeoprotein Octamer-binding tran-
scription factor-1-binding sites were also identified [46]. Two
other homeodomain proteins, Msx1 and distalless homeobox
2 (Dlx2), the first of which is a repressor and the second an
activator, have been found to be responsible for GnRH1 gene
regulation. Other studies have revealed that GnRH1 gene
regulation is also mediated through the protein kinase C (PKC)
signaling pathway [40]. Studies in rats revealed that GnRH1
gene expression is also under autoregulation, through an
ultrashort loop feedback mechanism [47].
The effect of steroids on GnRH neurons is a contentious
issue. Since Shivers et al. [48] showed a lack of estrogen
receptor (ER) immunoreactivity in GnRH neurons, a direct role
of estradiol on the GnRH neuron has been dismissed. Τhere
are studies that suggest a direct effect of estrogen at the level
of GnRH neurons [49–52]. Immunoreactive studies [53] have
demonstrated that ERs are highly expressed in the hypothal-
amus, not in GnRH neurons but in other neurons, afferent to
GnRH neurons [54,55], such as kisspeptin neurons. The last
ones express ERα, while GnRH neurons do not [56]. It is likely
that estrogen binds to ERα on kisspeptin neurons, and
subsequently inhibits kisspeptin and GnRH release. Indeed,
in gonadectomised animals and humans, kisspeptins, GnRH
and gonadotropin levels increased, whereas sex steroid levels
declined. Moreover, antagonism of kisspeptin receptors
(KiSS1R) in rodents prevented the rise of gonadotropins [57].
Ιn the rat, low (picomolar) estrogen concentrations through
ERα exert negative feedback on GnRH release. On the other
hand, higher (nanomolar) estradiol concentrations acting
through ERβ, have the opposite effect, and evoke a positive
regulation of GnRH secretion [58]. Although most studies have
focused on the act of estradiol, it is also evident that
progesterone functions together with estradiol to regulate
kisspeptin, because both receptors are needed for positive
feedback of the LH surge, and both have been indentified in
kisspeptin neurons, but neither are found in GnRH neurons
[59]. Testosterone exerts negative feedback effects on the
release of GnRH from the hypothalamus. Immunohistochem-
ical analyses in rats demonstrated that hypothalamic GnRH
neurons do not express the androgen receptor, thus, the
effects of testosterone may be mediated by higher-level
afferent neuronal populations, such as kisspeptin neurons
[60], or through its conversion to estradiol. It was found that
glucocorticoids repress GnRH1 gene expression and release
[61], and dehydroepiandrosterone sulfate (DHEAS) inhibits
GnRH gene expression in male rats [62].
Concerning other physiological regulators, it is known that
starvation inhibits the normal function of the reproductive
axis. It was found that insulin and insulin-like growth factor 1
(IGF-1) stimulate GnRH gene expression by inducing mitogen-
activated protein kinase (MAPK) pathway. Moreover, insulin-
 ME T ABOL I SM C L IN I CA L A N D E XP E RI ME N TAL 86 ( 20 1 8 ) 3 – 1 7
dependent stimulation of the GnRH gene is mediated by a
transcription factor, early growth response-1 (Egr-1) [63,64].
Melatonin downregulates GnRH mRNA levels [65], and nitric
oxide suppress the expression of the GnRH gene [66]. About
retinoic acid it was found that all-trans-retinoic acid stimu-
lates GnRH1 gene expression, whereas 9-cis-retinoic acid
represses it [67]. Finally, it has been shown that GABA induces
rat GnRH gene expression in mature rat GnRH neurons,
through hyperpolarization of the neurons, but in embryonic
rat GnRH neurons, GABA reduce rat GnRH gene expression,
through depolarization of the neurons [68].
3.2. GnRH Pulse Generator
GnRH pulse generator is activated in the fetus after the
completion of GnRH neuron migration to the hypothalamus.
Hypothalamic secretion of GnRH increases in early postnatal
life, also leading to temporary activation of gonadal steroido-
genesis, especially in the male. During childhood, gonads can
respond to exogenous gonadotropin stimulation but remain
normally quiescent because the pulsatile GnRH release is
suppressed until its re-awakening at the onset of puberty. The
mechanism responsible for the quiescent infantile and
prepubertal period is still not fully understood [3].
GnRH secretion follows two distinct modes: pulsatile and
surge [69]. The surge mode occurs only in females. Pulsatile
mode refers to episodic release of GnRH, where there are
distinct pulses of GnRH secretion into the portal circulation,
with undetectable GnRH concentrations during inter-pulse
intervals. It was first demonstrated in ovariectomised rhesus
monkeys, and subsequently in humans [70].
GnRH neurons co-ordinate their activity, and show intrin-
sic electrical activity, that depends on intracellular signaling
and mechanisms. Episodic multi-unit electrical activity at the
medial basal hypothalamus suggests that the ‘GnRH pulse
generator’ is anatomically located there [71]. Functionally, the
‘GnRH pulse generator’ relies on complex relations between
neurons that contain norepinephrine, dopamine, serotonin,
GABA, glutamate, neuropeptide Y (NPY) and galanin. Gluta-
mate and norepinephrine stimulate the reproductive axis,
whereas GABA inhibits it. Moreover, other elements, such as
the kisspeptin-neurokinin B-opioid pathway, have a crucial
role in the regulation of GnRH pulsatility [72].
4. Kisspeptin, Leptin, Neurokinin B and
Dynrophin Regulating GnRH Production
Kisspeptin is encoded by KiSS1, which was first discovered in
Hershey in 1996 as a suppressor of metastasis in human
malignant melanoma, and took its name after the famous
chocolates ‘Kisses’ produced in the town [73]. The KiSS1 gene
is located on chromosome 1q32, including four exons, of
which the first two are not translated [74], and it encodes a
precursor 145 amino acid peptide, which is cleaved to a 54
amino acid peptide. This peptide can be further cleaved to 14,
13 and 10 amino-acid peptides, all sharing the C-terminal
sequence of Arg-Phe-NH2 [75]. The orphan G-protein coupled
receptor 54 (GPR54) was identified, in 2001, as the receptor for
kisspeptins. It was first described in the rat brain [76]. It is
7
mapped to human chromosome 19p13.3 and has five exons,
encoding a 398-amino acid protein with seven trans-
membrane domains [77]. The KiSS1R is found throughout
the body, such as to brain, pituitary gland, placenta, gonads,
gastrointestinal tract, liver, and vascular system [78]. After
binding the ligand, KiSS1R activates phospholipase C and
recruits intracellular messengers, inositol triphosphate and
diacylglycerol, which in turn lead to the release of calcium
and PKC activation [79].
In the human hypothalamus, kisspeptin neurons are
localized in the rostral preoptic area and infundibular
nucleus. While the localization in the infundibular/arcuate
nucleus is preserved in all species, the population in the
rostral area is species specific [80]. Studies in humans and
animals have shown the stimulating role of kisspeptin on
gonadotropin secretion [81–83]. Kisspeptin axons compose
pericapillary plexuses in the human infundibular stalk, where
the GnRH is secreted [80], and GnRH neurons express KiSS1R
[84]. Some studies support that, beyond the indirect stimula-
tion which kisspeptin evokes in the secretion of gonadotro-
pins through the action on GnRH, there is a directly
stimulating action on the pituitary gonadotrophs to release
FSH and LH. This was based on the expression of kisspeptin
gene and peptide in gonadotrophs [85]. The stimulatory effect
of kisspeptin on FSH is less marked and less consistent than
on LH in humans and rodents [86,87].
Hypothalamic expression of kisspeptin and its receptor is
increased at puberty in rodents and primates [88,89]. The role
of kisspeptin in the reproductive system of humans became
apparent from patients with pubertal disorders. In patients
with HH and impaired pubertal development, inactivating
point mutations and deletions in KiSS1R were identified
[90,91]. Missense mutations in KISS1 gene have also been
reported in three unrelated children with central precocious
puberty. These mutant peptides demonstrated more resis-
tance to in vitro degradation but normal affinity to KiSS1R,
thus suggesting increased bioavailability as the mechanism of
precocious puberty [92]. A correlation was found between
energy status and kisspeptin system. There is a possibility
that decreased kisspeptin secretion is a putative mechanism
for HH in obese and diabetic patients, because the low levels
of hypothalamic kisspeptin mRNA, gonadotropins and sex
steroids found in the diabetic rat model was corrected by the
administration of kisspeptin [93,94]. Moreover, a disruption in
pubertal development in states of negative energy balance
has been observed and kisspeptin mRNA and gonadotropin
secretion is reduced in mice, rats and monkeys during fasting
[95–97]. A putative mechanism is through leptin.
Leptin is a product of adipose tissue and it was first
isolated in 1994 [98]. It is coded by the lep gene, which is
expressed in adipocytes. Leptin acts via its receptors LepR,
whose long isoform mediates most of its actions. Leptin levels
are higher in the obese people and they are correlated with fat
mass. Leptin decreases appetite and increases energy expen-
diture. Moreover, abnormal levels of leptin have been
associated with HH and fertility problems in males and
rodents [99]. Leptin stimulates the HPG axis, through the
increase of the expression of hypothalamic GnRH and the
stimulation on LH pulsatility [100]. This stimulatory effect is
not a direct one because leptin receptor is not present in
8 ME TA BOL I SM C LI N I CA L A ND E XP E RI ME N TAL 86 ( 20 1 8 ) 3 – 1 7

Fig. 2 – Regulation of the hypothalamic-pituitary-gonadal axis. Diagram of neuroanatomy of the GnRH pathway showing
neural systems that regulate GnRH secretion and feedback mechanisms. (+): stimulatory, (−): inhibitory, Era: estrogen receptor
a, PR: progesterone receptor, GABA: gamma-aminobutyric acid, GnRH: gonadotropin-releasing hormone, GnIH: gonadotropin-
inhibitory hormone.

GnRH neurons but is present in 40% of kisspeptin neurons in
the mouse arcuate nucleus [101].
In 2009, a role for neurokinin B in hypothalamic regulation
was also demonstrated when it was reported that missense
mutations in TAC3 (which encodes neurokinin B) and TAC3R
(which encodes the neurokinin B receptor) resulted in HH
[102]. Two years earlier, another group had demonstrated
that, in a subset of neurons in the arcuate nucleus of the ewe,
kisspeptin and neurokinin were co-localized, and in addition
a third peptide was co-expressed on the same neurons. This
peptide was called dynorphin, which is an endogenous opioid
[103]. Based on the first letter of each of these peptides, these
neurons called by the acronym KNDy. In contrast to
neurokinin B, that stimulates GnRH release, dynorphin
inhibits GnRH release [104] (Fig. 2).
GnRH pulse frequency influences the ratio of FSH to LH
secretion. A slower pulse frequency leads to a decrease in LH
secretion and to an increase in FSH secretion. In that way, the
ratio of FSH to LH secretion is elevated. The ratio of FSH to
LH secretion is also affected by the extent of FSH and LH
glycosylation, the localization of N-glycans and their
branching. Variations in glycosylation affect the participation
of gonadotropins in various biochemical processes, such as
hormone secretion, its receptor binding and rate of degrada-
tion [105].
4.1. GnRH Receptors
GnRH binds to a membrane receptor on pituitary
gonadotrophs and stimulates the biosynthesis and secretion
of LH and FSH. The GnRH receptor is a member of the
rhodopsin-like G protein-coupled receptor superfamily
(GPCR), which is characterized by the seven-transmembrane
domain structure. An important feature of the mammalian
GnRH receptor is the absence of the cytoplasmic C-terminal
tail [106], which is present in non-mammalian GnRH recep-
tors [107]. The gene of the human GnRH receptor is composed
of three exons separated by two introns, and it has been
identified as a single copy on chromosome 4q21.2. The
human gene has some additional features that do not exist
in other species, such as the location of the transcription start
sites and the presence of TATA boxes [108]. GnRH receptors
and splice variants have been identified in normal human
pituitary and pituitary adenomas [109]. In normal
 ME T ABOL I SM C L IN I CA L A N D E XP E RI ME N TAL 86 ( 20 1 8 ) 3 – 1 7
adenohypophysis the receptor is expressed in gonadotrophs,
thyrotrophs, and somatotrophs [110]. GnRH receptors have
also been identified in the cytotrophoblast and
syncytiotrophoblast cell layers of human placenta, human
corpus luteum, luteinized granulose cells, epithelial ovarian
carcinoma, ovarian cancer cell lines, endometrial carcinoma
cell lines, normal and neoplastic prostatic tissues, prostatic
cancer cell lines, breast carcinoma, melanoma, mammary
cancer lines, liver, heart, skeletal muscle, kidney and periph-
eral blood mononuclear cells [35,106]. Ligand binding assays
for GnRH receptors in human ovary and testis yielded
negative results [111], but a more recent study demonstrate
GnRH and GnRH receptor mRNA in human testis tissue [112].
It is well documented that the expression of GnRH receptor in
the pituitary is regulated by GnRH-I. When a decline in GnRH-I
stimulation to the pituitary is observed, as occurs in multiple
conditions including lactation, undernutrition, or seasonal pe-
riods of reproductive quiescence, the number of GnRH receptors
on pituitary gonadotrophs declines, but subsequent exposure of
the pituitary to GnRH-I induces and restores the levels of
receptors via calcium mobilization. This is known as upregula-
tion or self-priming. This augmentation of GnRH receptor
production is differentially controlled by varying GnRH-I pulse
frequencies and differs among species [113]. In cultured rat
pituitary cells the maximal augmentation of GnRH receptor
production occurs at a frequency of every 30 min [114], and in
human ovarian cancer and peripheral blood mononuclear cells a
biphasic effect of GnRH-I on GnRH receptor expression has been
observed [115,116]. On the other hand, continuous exposure to
GnRH brings about down-regulation of GnRH receptors and,
subsequently, the inhibition in LH and FSH synthesis and
secretion, called desensitization. Clinical applications of the
down-regulation include the therapy for precocious puberty of
hypothalamic origin and endocrine treatment of prostate cancer.
The use of a long-acting GnRH agonist down-regulates pituitary
GnRH receptors, and suppress the premature activation of
reproductive axis [117]. On the other hand, the effect of GnRH-II
or its analog significantly inhibits GnRH receptor expression,
regardless of the concentration used [118].
Estradiol suppresses GnRH receptor expression both at the
pituitary and extrapituitary tissues, via an ERα dependent
mechanism [119,120]. Similarly, progesterone inhibits GnRH
receptor expression in the pituitary [121]. Activin stimulates the
synthesis of GnRH receptor in rat pituitary cultures and its
antagonist follistatin decrease the basal expression of the gene
[122]. Finally, it was found that human chorionic gonadotropin
downregulates the GnRH receptor in the rat testis [123], but
increases GnRH receptor gene transcription in choriocarcinoma
JEG-3 cells [124], indicating a tissue specific effect.
Mutations of the GnRH receptor gene lead to idiopathic HH,
which is an autosomal inherited disorder and, in contrast
with KS, is not associated with anosmia [125].
The nature of G protein-coupled signaling mediating GnRH
responses depends on the cellular context. There is evidence that
GnRH receptors can couple to multiple G proteins. In the pituitary
gonadotrophs the primary pathway involves Gq/11 [126]. After
the coupling phospholipase C is activated, and this results in the
formation of diacylglycerols and inositol-trisphosphate (IP3),
which leads to the activation of several PKC isoforms and the
mobilization of intracellular Ca2+, respectively. Increased calcium
9
entry stimulates gonadotropin release [127]. The MAPK plays an
important role in the intracellular signaling, providing a crucial
link for the transmission of signals from the cell surface to the
nucleus, thus affecting gonadotropin transcription. Activation of
extracellular signal–regulated kinase (ERK)-one of the MAPK
cascades - by GnRH in these cells depends mainly on the
phosphorylation of Raf1 by PKC, supported by a pathway
involving c-Src, dynamin, and Ras. On the other hand, the
activation of c-Jun N-terminal kinase (JNK), another MAPK
cascade, involves PKC, c-Src, CDC42/Rac1, and probably MEKK1.
The GnRH receptor can also couple Gs, leading to the activation of
adenylyl cyclase (AC) and a rise in cyclic adenosine
monophosphate (cAMP) levels and the activation of protein
kinase A (PKA) and cAMP response element-binding protein
(CREB) [128,129] (Fig. 3). However, the signaling mechanism of the
GnRH receptor in some human reproductive tract tumors differs
significantly. In them, the transmission of GnRH receptor signal
mainly occurs via Gi, epidermal growth factor receptor (EGFR), c-
Src, and is not dependent on PKC [130,131].
After the activation of GPCR follow their desensitization
and internalization. For many GPCRs phosphorylation within
the C-terminal tail of the receptor facilitates binding to β-
arrestins, leading to arrestin-dependent receptor desensitiza-
tion and internalization [132]. In this process a crucial role is
played by the carboxyl-terminal receptor tail that is absent in
the mammalian GnRH receptor [133]. As a result, they do not
undergo agonist-induced phosphorylation and they are
therefore resistant to receptor desensitization, and internal-
ize slowly. The underlying mechanism is not well known, but
may involve post-receptor adaptive processes such as IP3
receptor down-regulation and desensitization of Ca2+ mobi-
lization in pituitary cells [134].
It must be mentioned that there are two major types of
receptors in vertebrates, but the pattern of distribution is
unusual. Type I GnRH receptors are not restricted to mam-
mals, but can be present in the lobe-finned and cartilaginous
fishes. Skate and coelacanth are the only examples of animals
with both type I and II GnRH receptors [135]. In many
mammalian species, type II GnRH receptor genes are
disrupted or deleted. Humans belong to this category,
possessing a gene encoding type II GnRH receptor but lacking
a functional type II GnRH receptor [136]. The type II GnRH
receptor possesses the carboxyl-terminal tails and shows
rapid desensitization and internalization [134].
4.2. Gonadotropin-Inhibitory Hormone
It seems that GnRH is not the only hypothalamic regulator of
the release of gonadotropins. In 2000 a new hypothalamic
neuropeptide, that inhibits pituitary gonadotropin secretion,
was discovered [137]. It is a decapeptide, now referred to as
gonadotropin-inhibitory hormone (GnIH). It was originally
identified in birds and afterwards in mammals and other
vertebrates [138]. GnIH neurons are located in the
paraventricular nucleus of the hypothalamus [139]. GnIH
inhibits the release and the synthesis of LHβ- and FSHβ-
subunits, by acting through a GPCR (GPR 147) on the pituitary
and on GnRH neurons of birds and mammals [138]. Moreover,
the presence of GnIH and GnIH receptor in the gonads, along
with the expression of them in cells related to steroid
10 ME TA BOL I SM C LI N I CA L A ND E XP E RI ME N TAL 86 ( 20 1 8 ) 3 – 1 7

Fig. 3 – GnRH signaling cascade. AC: adenylylcyclase, cAMP: cyclic adenosine monophosphate, PKA: protein kinase A, CREB:
cAMP response element-binding protein, PLC: phospholipase C, DAG: diacylglycerol, PKC: protein kinase C, JNK: c-Jun N-
terminal kinase, ERK: extracellular signal–regulated kinase.

biosynthesis and gamete maturation, reveals the regulatory
role of GnIH on steroidogenesis and gametogenesis [140]. In
hamsters GnIH was found in seminiferous tubules and GnIH
receptor expression was confined to spermatocytes and
spermatides [141]. In macaque GnIH is expressed in Leydig
cells, Sertoli cells, spermatogonia and spermatocytes [140].
The physiological role of GniH in mammals remains still
largely unresolved. GnIH suppresses LH secretion in juvenile
and prepubertal mice, but it is unlikely to have a major role in
the timing, progression or duration of puberty [142]. It was
found that GnIH could interrupt kisspeptin-induced activa-
tion of GnRH neurons [143]. GnIH may also act as a mediator
of stress-induced reproductive disruption [138] (Fig. 2).
4.3. Gonadotropins
As referred to above GnRH-I stimulates the synthesis and
release of FSH and LH. They are heterodimeric glycoprotein
hormones that consisted of a common α-subunit bound non-
covalently to a hormone-specific β-subunit (FSHβ and LHβ)
[144]. The activation of the α-subunit synthesis is Ca2+
dependent. This process is enhanced by PKC and requires
ERK1/2 and c-SRC [145]. An essential role of JNK has been
found by others [146]. The activation of the β-subunit
synthesis requires the Ca2+, PKC and MAPC signaling path-
ways [147]. α- and β-subunits are synthesized as a precursor,
with a signal peptide preceding the authentic subunit
sequence. In the endoplasmic reticulum proteins undergo
cleavage and N-glycosylation, leading to immature α/β
heterodimers. Along passing the Golgi apparatus their glyco-
sylation and conformation take place, and subsequently they
will be stored in separate granules. GnRH-II is also capable to
stimulate FSH and LH secretion acting through the GnRH type
I receptor [38].
FSH and LH are essential as regulators of ovarian and
testicular function. In men, LH stimulate testosterone pro-
duction of Leydig cells, which in turn exerts sexual and
anabolic actions, and in addition participates in the
 ME T ABOL I SM C L IN I CA L A N D E XP E RI ME N TAL 86 ( 20 1 8 ) 3 – 1 7
maintenance of spermatogenesis through its paracrine action
on Sertoli cells. The latter also forms the target of FSH. Studies
in rats and mice have shown that the role of testosterone is
crucial for spermatogenesis, especially its distal part, and
relatively independent of FSH. Moreover, testosterone is
essential for male genital differentiation and growth [148].
FSH stimulates Sertoli cell proliferation in prepuberty and
defines the size of their population, including the size of the
testes and the quantity of spermatogenesis, and acting as a
factor constraining apoptosis [149]. Hence, the main action of
FSH is, alongside testosterone, the augmentation of the sperm
production [148].
Human LH receptor and FSH receptor are encoded by
single copy genes on the short arm of chromosome 2 and
separated by 200 kb. They belong to the rhodopsin/2 adren-
ergic receptor-like family A of GPCRs [150]. The rat LH receptor
gene is encoded by a 70 kb genomic DNA and contain 11
exons. The FSH receptor is encoded by a 190 kb genomic DNA
and contains 10 exons. Both receptors consist of an N-
terminal extracellular domain that includes a number of
irregular leucine-rich repeats (LRR) where the ligands bind,
the seven transmembrane domain responsible for signal
transduction, and a short intracellular C-terminal tail. The
LH receptor binds both LH and human chorionic gonadotropin
(hCG) with high affinity, whereas the FSH receptor only binds
FSH.
Νaturally occurring activating and inactivating mutations
have been reported in gonadotropin receptor genes. The first
identified mutation was a constitutively activating mutation
of LH receptor in a male patient with precocious puberty [151].
Since then similar mutations have been reported by others
[152]. Asp578His has been found to be responsible for
hyperplasia and Leydig cell adenoma [153]. LH receptor
defects in human 46,XY individuals result in insufficient
testosterone production during the period of fetal sex
differentiation, leading to disorders of sex development
(DSD) characterized by a variety of phenotypes, from com-
plete feminisation of external genitalia, to minor hypospadias
or even just micropenis [154,155]. In knockout mice for LH and
LH receptor, normal prenatal and pre-pubertal sexual differ-
entiation and gonadal development was found, but the male
animals failed to undergo puberty and showed a diminished
sex steroid production [156]. Loss of the FSH receptor function
by knockout leads to decreased numbers of Sertoli and
somewhat reduced spermatogenesis, but the animals remain
fertile [157].
Activin, inhibin and follistatin regulate gonadotroph func-
tion. They are expressed at all levels of the HPG axis, and in
several nonreproductive tissue, suggesting a possible biolog-
ical role in various physiologic functions [158].
Inhibin is a glycoprotein hormone that belongs to the
transforming growth factor-β (TGF-β) superfamily. It is
mainly produced by the Sertoli cells. Inhibin consists of two
disulphide-linked subunits, a common α subunit (20 kDa) and
a β subunit (13 kDa). The latter exists in two forms, βA and βB,
giving rise in two isoforms, inhibin A (αβA) and inhibin B
(αβB). Ιnhibin B is the major form produced in the human
male. Its concentration is high during early postnatal life,
reaching a still detectable level until the beginning of puberty.
During puberty, it again increases and declines with ageing.
11
FSH increases cAMP levels of inhibin in testes. In human adult
inhibin levels are inversely correlated with serum FSH and
positively correlated with Sertoli cell number, sperm count,
sperm concentration, and testicular volume [159].
Activins are dimers of the two β-subunits, βA and βB,
giving rise to three activins, A (βΑβΑ), ΑΒ (βΑβΒ) and B (βBβΒ).
Two other β-subunits were also found (βC and βE) but their
function is still uknown [160]. Activins are gonadotroph cell-
derived ligands which act through a hetero-oligomeric serine/
threonine receptor complex and intracellular Smad proteins
[161].
Inhibin down-regulates FSH release from the anterior
pituitary, whereas activin stimulates it. Inhibin acts primarily
in gonadotrophs, where it reduces the levels of FSHβ mRNA
and FSH protein by blocking activin-stimulated transcription.
Antagonism of activin action by inhibin is caused by
interaction of the inhibin β-subunit with the activin receptor,
that prevents the initiation of the intracellular activin
signaling cascade. Because inhibin has a lower affinity to
activin receptors than activin itself, a collateral protein acting
as a coreceptor is necessary. One such protein, betaglycan,
also known as the type III TGFβ receptor, functions as an
inhibin coreceptor, and potentiates antagonistic action of
inhibin on activin [160,162].
Follistatin is a cysteine-rich, glycosylated, monomeric
protein [163]. It modulates the bioactivity of activin dimers,
being their functional antagonist. Follistatin binds to the
activin subunits, blocking their interactions with receptors.
It has an inhibitory effect on pituitary FSH release. FSH
stimulates the gonadotrophs to produce follistatin, and
follistatin inactivates activin locally [164]. Follistatin expres-
sion is found in gonadal tissue and in pituitary gonadotrophs.
In addition, it is found in non-reproductive tissue suggesting a
paracrine or autocrine role [160] (Fig. 2). Activin A and
follistatin are also expressed in the hypothalamus.
5. Conclusions
In this review, beginning from the embryonic development
and anatomic distribution of GnRH neurons, we follow the
pathways of the HPG axis in various species. The HPG axis
functions as a complex unit in the regulation of reproduction
and fertility. There are numerous studies regarding the
factors that influence the migration of GnRH neurons,
because GnRH plays a particular critical role in the function
of the reproductive axis, and acts as the intermediate factor
between hypothalamus and hypophysis. Several GnRHs have
been found from protochondrates to vertebrates. The pulsa-
tile manner of their secretion is essential for optimal
gonadotropin synthesis and secretion in mice to males.
Kisspeptin, first described in the rat brain, with species-
specific localization in hypothalamus, is a potent upstream
stimulator of GnRH and gonadotropin secretion. GnRHs act
through GnRH receptors, which in mammals differ from the
lower species by their absence of the cytoplasmic c-terminal
tail, which suppresses their desensitization and internaliza-
tion. GnIH is another hypothalamic regulator of gonadotro-
pins, originally identified in birds, and subsequently in
12 ME TA BOL I SM C LI N I CA L A ND E XP E RI ME N TAL 86 ( 20 1 8 ) 3 – 1 7
mammals and other vertebrates. GnRH stimulates the syn-
thesis of FSH and LH, the main tropic regulators of testicular
functions in all species.
Conflicts of Interest
None.
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