Reprod Dom Anim 45, e495–e502 (2010); doi: 10.1111/j.1439-0531.2009.01355.
x
ISSN 0936-6768
Review Article
Neuroendocrine, Metabolic and Genomic Cues Signalling the Onset of Puberty in
Females
CA Meza-Herrera1, A Gonzalez-Bulnes2, RT Kridli3, M Mellado4, CF Arechiga-Flores5, H Salinas6 and JM Luginbuhl7
1
Universidad Autónoma Chapingo, Unidad Regional Universitaria de Zonas Áridas, Bermejillo, Durango, Me´xico; 2INIA, Departamento de
Reproducción, Madrid, Spain; 3Jordan University of Science and Technology, Irbid, Jordan; 4Universidad Autonoma Agraria Antonio Narro,
Mexico; 5Universidad Autonoma de Zacatecas, Mexico; 6Instituto Nacional de Investigaciones Forestales Agricolas y Pecuarias, Mexico; 7North
Carolina State University. Raleigh, NC, USA
Contents
Puberty is the result of a dynamic interaction between genetic
factors and environmental cues, all of which lead to the
attainment of reproductive capacity. Thus, significant changes
in hormone secretion occur from the pre-pubertal to the
pubertal stage. The objective of this review is to provide an
update of some endocrine, physiological, metabolic and
genetic concepts involved in the establishment of the hypo-
thalamic-hypophyseal-gonadal axis function promoting the
onset of the reproductive function during puberty. To achieve
this purpose, basic aspects of the function of the hypotha-
lamic-hypophyseal-gonadal axis, the control of the axis by
neurotransmitters and the interaction between reproductive
function and metabolic status will be considered. Finally, the
role of the novel kisspeptin system and the GPR54 receptor as
modulators of puberty will be considered, in addition to the
hierarchical expression of the main genes acting as regulators
of the onset of puberty.
Introduction
The transit from pre-puberty to the pubertal state
includes significant changes in the function of the
hypothalamic-hypophyseal-gonadal axis. Prior to pub-
erty, secretion of the gonadotrophin releasing hormone
(GnRH) is low. As puberty approaches, the hypotha-
lamic gonadostat is still depressed, whilst the amplitude
of GnRH pulses increases. The concentrations of
gonadotrophic hormones, follicle stimulating hormone
(FSH) and luteinizing hormone (LH) gradually increase
during puberty, thus stimulating growth and maturation
of follicles as well as oestrogen production by the
ovaries (Huffman et al. 1987; Apter 1997; Apter and
Hermanson 2002; Teresawa 2005; Messinis 2006; Mahdi
and Khallili 2008).
Immediately prior to puberty, the highly efficient
negative feedback system from the ovarian steroids is
particularly active. However, as puberty approaches, the
positive feedback signal from the ovarian steroids is
activated and becomes highly efficient, usually observing
the establishment of normal cyclicity at the end of that
stage. For this reason, the complete transition from
prepubertal anoestrus into puberty is an indispensable
prerequisite for the establishment of reproductive func-
tion (Teresawa 2005; Clarkson and Herbison 2006;
Messinis 2006; Ojeda et al. 2006a).
The objective of this review is to actualize some
endocrine, physiological, metabolic and genetic
 2009 Blackwell Verlag GmbH
concepts, which are involved in the establishment of
the hypothalamic-hypophyseal-gonadal axis function
that promotes the onset of the reproductive function
during puberty. To achieve that purpose, some basic
aspects of the function of the hypothalamic-hypophy-
seal-gonadal axis, the control of the axis by neurotrans-
mitters and the interaction between reproductive
function and metabolic status will be considered.
Finally, the role of the novel kisspeptin system and the
GPR54 receptor as modulators of puberty will be
considered, aside from the hierarchical expression of
the main genes acting as regulators of the onset of
puberty: OCT-2, TTF-1 and EAP-1.
Basic Functional Aspects of the Hypothalamic-
Hypophyseal-Gonadal Axis
The onset of puberty is a centrally-regulated process,
and the detailed mechanisms of its function are still
unknown. It is recognized, yet, that increases in the
activity of the GnRH pulse generator and then in the
pulsatile secretions of gonadotrophins FSH and LH
take place. During puberty, the pulsatile secretions of
gonadotrophins are increased during the day, promoting
a normal gonadal development and function because of
the activation of the GnRH pulse generator (Teresawa
2005; Clarkson and Herbison 2006; Messinis 2006;
Ojeda et al. 2006a).
The classical gonadostat theory states that a decreased
sensitivity to the oestradiol negative feedback must occur
in the hypothalamic-hypohpyseal centres controlling
gonadotropin secretion in order for pubertal onset
(Ramirez and McCann 1963). In this way, a decreased
sensitivity to esteroid feedback allows increases in
gonadotropin secretion, which promotes ovarian follicle
maturation, and ovulation. Yet, certain concepts of the
gonadostat theory may not be suitable for all species. In
sheep, GnRH secretion is markedly suppressed prior
puberty, whereas at the beginning of puberty, the
hypothalamic gonadostat is also depressed and the
amplitude of the GnRH pulses increases (Foster and
Ryan 1979). Thereafter, concentrations of LH and FSH
are gradually increased during puberty, stimulating both
follicular maturation and ovarian oestrogen production.
The decapeptide GnRH is synthesized in the pre-optic
area of the hypothalamus and, in adult animals it is
e496 CA Meza-Herrera, A Gonzalez-Bulnes, RT Kridli, M Mellado, CF Arechiga-Flores, H Salinas and JM Luginbuhl
released in a pulsatile fashion, every 60 min, inside the
portal system which connects to the hypophysis. Then,
GnRH binds to its high affinity receptor (GnRH-R)
located not only on the cellular membrane of the
gonadothrops, but also on the ovary and uterus. In
young animals, the pulse interval is much longer, 90–
120 min. It is the acceleration of the frequency of the
pulse and the increase in the amplitude of the pulse, as
well as the total GnRH concentration that activate
transduction signals to promote both the synthesis and
the intermittent release of LH and FSH, thus generating
the onset of the pubertal process. Once bound to its
receptor, GnRH works through protein kinase-C to
induce synthesis and the secretion of LH and FSH. The
GnRH-R expression is regulated by GnRH itself as well
as by gonadal steroids (Huffman et al. 1987; Apter 1997;
Roth et al. 2001; Apter and Hermanson 2002).
Those neurons releasing GnRH represent the critical
cellular type, which, once activated, induce puberty
(Clarkson and Herbison 2006). For that reason, an
appropriate expression of the GnRH-R in the gonado-
trophs is critical for the signalling and secretion of
gonadotrophins mediating the ulterior sexual develop-
ment (Zapatero-Caballero et al. 2004). The GnRH
neurons are important not only because of their
involvement in the onset of reproductive function, but
also in the development of neuromodulatory functions
in the adult (Whitlock et al. 2006).
The discovery of the ligand activator of the upstream
signal route from GnRH linked to the G-protein
receptor gives greater significance to the central role
the hypothalamus plays in the regulation of puberty
(Clarkson and Herbison 2006; Hughes and Kumanan
2006). The onset of puberty is associated with an
increase in the amplitude and frequency of LH. Later
on, a progressive increase in the LH pulsatility occurs
during the day, while a progressive reduction of the
amplification, previously occurring during the night, is
observed. Prepubertal FSH concentrations are relatively
high, and a continuous process of follicular development
and atresia occurs, while relatively high concentrations
of oestradiol are observed. Only after this initial increase
in LH is the ovarian steroidogenesis activated, leading
to increases in oestrogen secretion. Thereafter, both
follicular development and maturation occur under
FSH stimulation (Suttie et al. 1991; Apter 1997).
The Control of Puberty by Modulation of
Different Neurotransmitter Systems
As previously mentioned, GnRH is the primary mes-
senger involved in both sexual maturation and onset of
puberty. The activity of this neuronal system is, in turn,
controlled by different neurotransmitter systems. The
onset of puberty includes changes in a prepubertal
secretion type of gonadotrophins characterized by a low
activity of the GnRH neurons, to later promote an
increase in the amplitude and frequency of GnRH
pulses. At the beginning of puberty, several neurotrans-
mitter systems are involved in the change of GnRH
secretion pattern, which act through quantitative and
qualitative modifications upon the GnRH secretion
pattern (Ebling et al. 1989; Dhandapani and Brann
2000; Mahesh and Brann 2005; Parent et al. 2005;
Clarkson and Herbison 2006; Ojeda et al. 2006a,b).
Not only serotonin (5-HT) and the gamma amino
butyric acid (GABA), but also the catecholamines (CA)
display qualitative differences upon GnRH and LH
secretion, mainly at the beginning of the prepubertal
stage than in late puberty and the adult stage (Moguil-
evsky and Wuttke 2001). Similarly, it has been estab-
lished that the main trans-synaptic excitatory event
stimulating the onset of puberty is an increase in the
glutamatergic neurotransmission because it is the main
way of trans-synaptic communication in the hypothal-
amus. This is a complex process controlled by a plethora
of genes required for the synthesis, transport and release
of glutamate, as well as the expression of several
receptors, both ionotropics and metabotropics which
mediate the actions of glutamate (Ebling et al. 1989;
Dhandapani and Brann 2000; Mahesh and Brann 2005;
Parent et al. 2005; Clarkson and Herbison 2006; Ojeda
et al. 2006a,b).
In primates, the LHRH neurons of the female
hypothalamus are active during the neonatal period,
but they subsequently enter into an arrested stage in the
juvenile and prepubertal periods, because of the high
GABA levels in the median eminence (ME). The
observed reduction in GABA levels, as the animal
grows results in an increase in LHRH release in the ME,
generating the onset of the pubertal process. This
reduction in GABA levels seems to allow glutamate
levels in the ME to increase and seems to contribute to
the generation of the pubertal increase of LHRH
(Teresawa 2005).
Both, the administration of 5-hydroxytryptophan, a
serotonin precursor, and the activation of the GABAergic
system, increase the release of GnRH and LH during
prepubertal stages, although such positive effect disap-
pears during late puberty and promotes an inhibitory
action upon the GnRH neurons in adult animals. Like
glutamate, GABA production requires the participation
of different proteins involved in the synthesis, metabo-
lism, transport and release of this neurotransmitter. As
no changes in the mRNA expression of the enzymes
responsible for GABA, GAD65 and GAD67 have been
observed during the sexual development in primates, it
seems that the expression of such genes is not an event
related to the onset of puberty. Yet, by analogy with the
glutamate system, it can be inferred that other possible
checkpoints in GABA metabolism include the vesicular
transport and ⁄ or the expression of GABA receptors.
Concerning the latter, four GABA transporters have
been described: GAT-1, GAT-2, GAT-3 and BGT-1;
GAT-1 is the predominant transporter in the mamma-
lian brain and contains a responsive element to oestro-
gen (Teresawa 2005; Ojeda et al. 2006b). Conversely,
inhibition of CA synthesis (adrenaline and noradrena-
line) by alpha-methyl.p-tyrosine promotes an increase in
LH secretion during the early prepubertal phase and an
inhibitory effect during both the late pubertal phase and
the adult stage. The latter highlights the inhibitory effect
displayed by CA upon the hypothalamus-hypophyseal
axis function to change to a stimulatory effect during
late puberty and adult stage (Moguilevsky and Wuttke
2001).
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Signalling Mechanisms Controlling Puberty
One of the most important orexigenic factors is the
neuropeptide-Y (NPY), a 36-amino acid peptide. It is a
member of the pancreatic polypeptides family and is
very abundant in the CNS, including the hypothalamus,
as well as the peripheral nervous system (Wojcik-
Gladysz and Polkowska 2006). Wankowska et al.
(2002), evaluating the intracerebroventricular infusion
of NPY upon the secretory activity of the hypophyseal
gonadotrophs, reported that NPY can be an important
component in the mechanisms promoting synthesis and
storage of LH in the gonadotrophs, without observable
changes on the LH serum concentrations. The same
authors concluded that NPY does not affect LH release
in prepubertal ewe lambs. Similarly, such effect was LH-
specific without apparent changes in FSH.
On the other hand, opiodergic and neuroexcitatory
amino acid systems display quantitative differences in the
secretion of GnRH-LH during the prepubertal, peri-
pubertal and adult stages. Opioids have a significant
inhibitory effect during early pre-puberty that decreases
during sexual maturation until puberty is reached. In
fact, endogenous opioids are important inhibitory neu-
rotransmitters and control the pulsatile LH release in
peripubertal stages (Ebling et al. 1989). Peptide opioids
as a group do not seem to inhibit the onset of puberty, but
it is possible that their inhibitory tone can be exerted by a
neuronal subset, which selectively utilize for neurotrans-
mission to the B-endorphin, dinorphin or Met ⁄ Leu-
enkephalin (Teresawa 2005; Ojeda et al. 2006b).
On the contrary, excitatory amino acids (EAA)
increase their stimulatory effect upon GnRH-LH secre-
tion during sexual maturation through increases in the
release of aspartate and glutamate, as well as by
increasing the sensibility of the N-methyl-D-aspartate
(NMDA) receptors (Moguilevsky and Wuttke 2001).
According to Mahesh and Brann (2005) and Parent
et al. (2005), EAA in general and glutamate in particular
exert a marked stimulatory effect upon the reproductive
axis, particularly at the time of puberty. In fact,
administration of glutamate and its agonists promote
the pulsatile release of LH in animals previously exposed
to steroids. Conversely, antagonists of the ionotropic
receptors of glutamate inhibit the LH release, and
abolish both the secretion of LH induced by E2 and the
pre-ovulatory LH surge. According to Teresawa (2005),
the pubertal reduction of the GABAergic inhibition is
followed by an increase in the glutamatergic tone.
Ojeda et al. (2006a) reported that the onset of puberty
requires a reciprocal communication neuron-glia which
involves EAA, mainly glutamate and aspartate. In
addition, some growth factors and the coordinated
action of a group of neuromodulators, all of which
representing a higher level of control governing the
pubertal process, are also necessary. Dhandapani and
Brann (2000) as well as Mahesh and Brann (2005)
mentioned that both the neuromodulator glutamate as
well as nitric oxide play an essential role in the
establishment of the pre-ovulatory surge of GnRH in
mammals. Respectively, it has also been reported that
glutamate, NMDA and kainate stimulate GnRH secre-
tion in immature mammals, whereas NMDA-R stimu-
lation generates a precocious puberty in rats and
monkeys.
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e497
Puberty is characterized by an increase in the sensi-
bility of GnRH to glutamate and an increase in the
activity of the enzyme glutaminase in the hypothalamus.
The secretion of GnRH by glutamatergic and GAB-
Aergic neurons seems to occur in an independent way in
the peripubertal stage. In addition, the activation in the
transynaptic glutamatergic regulation of GnRH is
required, as well as a coordinated activity of glutama-
tergic neurons and astroglial cells, which also display an
important active role during puberty (Parent et al.
2005).
Glutamate generates different actions in the hypotha-
lamic mechanism of puberty as has been demonstrated
in different animal models (Parent et al. 2005). The pre-
optic area has been implicated as the primary site of
action of NMDA, whereas NMDA agonists seem to act
mainly at the level of the ME and the arquate nuclei
(Mahesh and Brann 2005). Participation of the kainate
receptor in the induction of an oestradiol-dependent
precocious puberty suggests that this receptor can be
involved in the activation of GnRH secretion mediated
by oestradiol, which has been observed during puberty.
In addition, and in parallel to the increase in glutamate
release in the hypothalamus during the puberty process,
DL-amino methyl propionic acid receptors are also
increased and play an important role as mediators of
the regulatory effects of glutamate during the establish-
ment of puberty (Zamorano et al. 1998).
As previously mentioned, glutamate has been
involved in critical processes of the establishment of
puberty, hormonal pulsatility and sexual behaviour.
Glutamate can promote many of these effects through
activation of the release of the gaseous neurotransmitter
nitric oxide. This neurotransmitter stimulates the GnRH
release through activation of the enzyme guanylate
cyclase, which in turn promotes an increase in the
production of cGMP and the posterior release of GnRH
(Dhandapani and Brann 2000).
Little is known about the transcriptional control of
the genes which codify the enzymes involved in the
synthesis, metabolism and transport of glutamate
(Ojeda et al. 2006b). Yet, the importance of these
homeostatic systems has been recently evaluated
through the use of proteomic quantitative techniques.
In fact Roth et al. (2006), analysing some of the proteins
involved in the synthesis and degradation of glutamate
in the astroglial cells, reported that the enzyme gluta-
mate dehydrogenase catalyses the synthesis of gluta-
mate. They indicated that the highest content of
glutamate in the hypothalamus was observed during
proestrus, while the concentration of the enzyme gluta-
mine synthase that catalyses the metabolism from
glutamate to glutamine decreased during proestrus. An
increased production of glutamate and LHRH was
observed in the hypothalamus prior to the pre-ovulatory
LH surge. The latter suggests that an increase in the
excitatory effects generated by the glutamatergic signals
of glial origin upon the neuronal LHRH networks is an
event, which contributes to the activation in the pulsatile
pubertal secretion of LHRH. The transcriptional regu-
lation of the expression of the vesicular transporters of
glutamate can represent a very important checkpoint of
glutamate homeostasis, because the vesicular transporters
e498 CA Meza-Herrera, A Gonzalez-Bulnes, RT Kridli, M Mellado, CF Arechiga-Flores, H Salinas and JM Luginbuhl
of glutamate are extremely important in the minute-to-
minute control of the release of such neurotransmitter
(Ojeda et al. 2006b).
Modulation of Neuroendocrine Systems and
Metabolic Status
Metabolic status and nutrient availability are some of
the main environmental factors required for the estab-
lishment of reproductive function. The deficit of nutri-
ents in several animal species promotes disturbances in
gonadotrophic hormone secretion both in immature and
adult mammals. It has been proposed that decrease in
the pulsatile secretion of GnRH generates a reduction in
the synthesis and release of LH. In addition, such a
scenario has been further proposed to be one of the
main etiologic factors to cause nutritionally induced
suppression of the hypophyseal-gonadal function
(Wojcik-Gladysz and Polkowska 2006).
As previously mentioned, GnRH neurons are reason-
ably mature at birth; yet, as the growth process starts,
pulsatile release of GnRH is suppressed. In fact, in
ruminants, there is an increase in gonadotrophic activity
in the early stages after birth, which generates an
increase in the development of antral follicles on the
ovary. Endocrine regulation of this stimulation of the
ovarian follicular system is not fully understood
(Rawlings et al. 2003).
This initial endocrine activity seems to be arrested by
the suppression of negative feedback mechanisms until
females reach a critical live weight (lipostatic theory),
which can allow the onset of puberty, initiate oestrous
cycles and later activate the reproductive function.
Respectively, there is evidence that antral follicles and
genital tubular development occur in a parallel fashion.
Similarly, the early increase in the number and size of
antral follicles can partially be due to changes in both
the secretion pattern and the potency of FSH, whereas
the increased development in the follicular population is
probably caused by increases in the frequency of the
GnRH pulse generator, and in turn by LH and FSH.
The latter occurrs once a critical live weight or certain
percentage of body fat is acquired (Rawlings et al.
2003).
Low protein levels in the diet promote not only a
reduction in live weight but also a decrease in average
daily gain, exerting an inhibitory effect upon the
synthesis and release of LH. Yet, this effect was not
observed when considering FSH. For this reason,
protein concentration in the diet can be an important
modulator of those neuronal processes promoting the
increase in LH levels during puberty (Polkowska et al.
2003). Similarly, severe undernutrition can prevent
ovulation in ewe lambs by abolishing the function of
the GnRH system, which governs the production and
secretion of LH with a high frequency pattern (Foster
and Olster 1985).
Even though the close relationship between live
weight and fertility has been recognized, the peripheral
signals and the neuroendocrine networks responsible for
such phenomenon have only been recently identified.
One key event in this field was the cloning of the
adipocyte derivative hormone, leptin, which has shown
to play a central role as a regulatory signal in the
integration of the energetic homeostasis and reproduc-
tive function (Fernandez-Fernandez et al. 2006).
Leptin, the product of the OB gene, has been related
to the onset of puberty in several animal models. The
onset of puberty differs among genus, and such differ-
ence can be due to the sexual differences observed in the
mechanisms of sexual organization regarding the feed-
back systems of steroids (Foster et al. 2006). In prepu-
bertal females, leptin concentrations increase slowly
with age and body fat reserves. Besides the increase of
fat tissue reserves during puberty, there are also
increases in leptin. In general, plasmatic concentrations
of leptin are significantly correlated with the adipose
mass level both in males and females. Leptin is bound to
a high affinity binding protein similar to the soluble
receptor of leptin (Apter and Hermanson 2002; Apter
2003).
According to Carbone et al. (2005), exogenous
administration of leptin generated a significant increase
of LH levels in adult animals as well as of the glutamate
levels in the hypothalamus. This hypothalamic excit-
atory amino acid has been involved in the neurotrans-
mitter activation of the NMDA-R. These results
demonstrate that leptin stimulates the reproductive axis
during a determined period of sexual maturation and
that the NMDA-R is involved in the facilitatory
action of leptin upon the gonadal axis during sexual
maturation.
Administration of exogenous leptin increases the
release of GnRH in the hypothalamus during prepu-
bertal and peripubertal stages. In peripubertal females,
such increase is accompanied by a significant decrease in
the hypothalamic release of GABA and an increase in
the release of aspartate. The latter suggests that in this
phase of sexual maturation, leptin exerts a stimulatory
effect upon GnRH, inducing the release of EAA, and
reducing the release of inhibitory amino acids such as
GABA, all of them involved in the control of GnRH
release. In prepubertal stages, yet, the stimulatory effect
of leptin upon GnRH seems to be related to the
stimulatory action of GABA, which in this stage of
development increases the release of GnRH (Reynoso
et al. 2003).
According to Ponzo et al. (2005), exogenous admin-
istration of leptin to prepubertal and peripubertal
animals increased the release of hypothalamic GnRH,
plasma LH concentrations as well as the release of
glutamate. An increase in aspartate levels was also
observed, but only in peripubertal animals treated with
leptin, without changes in GABA levels between treat-
ments. The latter demonstrates that leptin promotes
increases in the hypothalamic-hypophyseal function,
leading to serum increases of GnRH and LH, and that
the excitatory amino acid neurotransmitter system
seems to be involved in these changes.
Both leptin and ghrelin exert important roles in the
regulation of intake behaviour, metabolic status and
reproduction by acting upon the CNS and other
reproductive organs. As markers of the nutritional
status, these hormones can act upon the CNS to
initiate the complex process of puberty and to maintain
a normal reproductive function in the adult stage.
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Signalling Mechanisms Controlling Puberty
By acting through the brain, these hormones can act as
a connection between the level of metabolic reserves
and the reproductive system to regulate energy require-
ments for a normal reproductive function (Budak et al.
2006).
Leptin, which signals the level of energy storage of the
body, displays an essential role in the metabolic control
of the KiSS1 system, as kisspeptin-neurons co-express
leptin receptors. Leptin is capable of normalizing the
defective expression of the KiSS1 gene in biological
models characterized by an impaired secretion of
gonadotrophins linked to hypoleptinemia stages (Tena-
Sempere 2006a, 2006b). Other factors could play an
important role in the attainment of puberty in mam-
mals. These include environmental as well as genetic
factors and the interaction amongst these (Land 1978).
Environmental factors involve season of birth, nutri-
tional status including body weight, total body fat,
metabolites such as glucose, cholesterol and triglyce-
rides, as well as metabolic hormones (Cheung et al.
1997). In goats, it was recently demonstrated that
puberty was depended upon season of birth, and that
heavier animals born in November also depicted a
higher ovulation rate. Therefore, some benefit could be
expected in breeding goat kids at a higher body weight
to obtain a higher prolificacy at the first kidding
(Zarazaga et al. 2009).
The Kisspeptin-GPR54 System in the
Modulation of GnRH and Puberty
The hypothalamic gene KiSS1 has been considered as
an essential integrator of peripheral cues including the
gonadal steroids as well as the nutritional status
(Tena-Sempere 2006a). In fact, recent data suggest a
prominent role of KiSS1 in the metabolic control of
fertility when considering that expression of the
hypothalamic gene KiSS1 is negatively regulated under
conditions of negative energy balance, whereas admin-
istration of kisspeptin is capable of reversing a
hypogonadotrophic stage observed under scenarios of
undernutrition and metabolic disturbance (Tena-
Sempere 2006b).
Kisspeptin, a peptide made up of 53 amino acids, a
product of the KiSS1 gene and its receptor GPR54
which is linked to G-proteins, have emerged as key
elements in the regulation of GnRH secretion (Gottsch
et al. 2006; Tena-Sempere 2006a, 2006b, 2006c). Kiss-
peptins were originally identified as tumoral metastasis
suppressor peptides bound to the GPR54 receptor
linked to G-proteins (Ojeda et al. 2006b). Yet, muta-
tions in the GPR54 gene have been related to an
absence in the onset of puberty and hypogonadotrophic
hypogonadism (Tena-Sempere 2006c). Furthermore,
the proteolytic cleavage of the KiSS1 primary product
generates the decapeptide kispeptin-10, which is a very
potent agent promoting LH release (Ojeda et al.
2006b).
SynCAM, an element involved in synapse formation,
is an immunoglobulin-like adhesion molecule previously
recognized as a tumour suppressor molecule in lung
cancer. It has been proposed that both SynCAM and
kisspeptin conform a network of genes, which, besides
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e499
their function as suppressors’ molecules of tumoral
metastasis in the brain, also act as integrator elements in
the communication neuron-to-neuron and glia-to-neu-
ron, building a functional unit able to initiate the
puberty process (Ojeda et al. 2006b).
Mutations in the GPR54 receptor expression pro-
duce hypogonadotrophic hypogonadism, an indication
that signalling through this receptor is a prerequisite
for sexual maturation. Central administration of kiss-
peptin stimulates secretion of both GnRH and
gonadotrophins in prepubertal and adult animals.
Similarly, increases in the expression of the KiSS1
and GPR54 genes have been observed during the
pubertal development, whereas activation of GPR54 by
kisspeptin administration is enough to induce the
activation of the gonadotrophic axis in immature
animals (Gottsch et al. 2006; Tena-Sempere 2006a,
2006b).
Neurons expressing kisspeptin are direct targets of
the steroid feedback action, both positive and negative,
which differently regulate the mRNA KiSS1 expression
in several brain areas and have been defined with a
relevant role in the establishment of puberty. A double
site of kisspeptin action in the brain has been
suggested, either in the hypothalamic-hypophyseal
region or the ME, an area located outside the blood
brain barrier. Neurons containing kisspeptin are
located in discreet subsets of the pre-optic area and
the arquate nuclei of the brain, whereas those neurons
containing the GPR54 receptors are more diffusely
distributed and include GnRH neurons as well as the
adenohypophysis. In primates, the KiSS1 and GPR54
mRNA levels significantly increase in the hypothala-
mus at the time of puberty suggesting that an increase
in the GPR54 mediated signal contributes to the
pubertal activation of GnRH secretion (Ojeda et al.
2006b).
The pulsatile increase in GnRH secretion observed in
the prepubertal stages occurs due to the coordinated
changes in the trans-synaptic communication and to the
interaction between neuron-to-glial cells. As the excit-
atory signals from neurons and glial cells increase, a
reduction in inhibition in the trans-synaptic tone is
observed, generating the pubertal activation of GnRH
secretion. The more prevalent neuronal systems invoked
in this process include glutamate and the peptide
kisspeptin, which exert neurotransmission and neuro-
modulation processes, whereas the most important
inhibitory signal is provided by GABAtergic and
opiatergic neurons (Ojeda et al. 2006b).
It has been proposed that glial cells and GnRH
neurons display a morphological and functional rela-
tionship which depends on growth factors that act
through serine-threonine-kinase receptors as the trans-
forming growth factor (TGFB1). Those growth factors
send their signals through receptors with tyrosine-kinase
activity such as the insulin-like growth factors (IGF-1),
the fibroblast growth factor, members of the epidermal
growth factor family, the TGFalfa and the neuregulins.
A complete discussion on the roles of these growth
factors has been previously presented by Garcia-Segura
and McCarthy (2004), Gill et al. (2004) and Ojeda et al.
(2006a,b).
e500 CA Meza-Herrera, A Gonzalez-Bulnes, RT Kridli, M Mellado, CF Arechiga-Flores, H Salinas and JM Luginbuhl

The Hierarchical Expression of Genes This regulatory mechanism is important to initiate

Modulating the Onset of Puberty:
OCT-2, TTF-1 and EAP-1
The establishment of the nerve function of GnRH
neurons requires the ordered and hierarchical comple-
mentation of a set of genes whose primordial objective is
to establish the required conditions for a productive
interaction between neurons and glial cells. In addition
to residing at the centre of a complex regulatory
network, these genes should maintain a hierarchical
structure in such network to assure that the neuronal
system includes redundancy and combinatory diversity
(Ojeda et al. 2006a,b; Tena-Sempere 2006c).
Ojeda et al. (2006b) proposed a potential role of three
candidate genes as controllers in the hierarchy of genic
expression when controlling the pubertal process: OCT-
2, TTF-1 and EAP-1. The OCT-2 proteins are more
abundant in astrocytes than in cultivated neurons,
suggesting that the OCT-2 gene can be important in
the transregulation of the transcription of astroglial
genes. Both SynCAM and TGFalfa have been identified
as targets of this gene because the promoter of SynCAM
and TGFalfa possess binding sites for the OCT-2 gene.
puberty in females because: (i) The hypothalamic
mRNA levels of OCT-2 are increased during the
prepubertal development in an independent format
from gonadal action, (ii) Blockage in the OCT-2
synthesis reduces TGFalfa and delays the age at first
ovulation and (iii) Hypothalamic lesions inducing sexual
precocity activate both OCT-2 and TGFalfa in the
astrocytes close to the place of injury.
The second candidate gene is the TTF-1 (thyroid
transcription factor-1), which is required for dience-
phalic morphogenesis. After birth, it is expressed in
selected neuron populations and hypothalamic glial
cells, in GnRH neurons, in proopioenkephalinergic
neurons, as well as in the ME. Such gene acts on these
cellular groups to promote specific functions in each
cellular type. Not only DNA arrays but also quantita-
tive PCR analyses in the hypothalamic regions revealed
a pubertal increase in the TTF-1 expression. Elimination
of the TTF-1 gene from the hypothalamic sub-networks,
where it is commonly expressed, generated a delay in
puberty, a disruption in the initial oestrous cyclicity, as
well as a decreased reproductive function. Such

Fig. 1. Proposed interactions between neuroendocrine, metabolic and genomic cues signalling the onset of puberty. Nutritional state may
regulate both leptin, in a long-term fashion while insulin release may be affected in a real-time ⁄ short-term fashion. These two metabolic hormones
may act on appetite and reproductive centres in the hypothalamus, and activate ⁄ deactivate a set of genes (OCT2, TTF1 and EAP1), whose
primordial objective is to establish conditions at hypothalamic level for the establishment of nervous function of GnRH neurons. In addition, the
hypothalamic gene KiSS1, its product kisspeptin and the activation of its G-protein coupled GPR54 receptor have also been involved in the
activation of GnRH neurons. Besides that, a set of neurotransmitters and growth factors have been identified as positive or negative modulators
of glutamatergic neurons co-expressing such neurotransmitters and growth factors, whose actions activate the hypothalamic release of the
hypothalamic decapeptide GnRH. Activation of glutamatergic neurons has been involved as a critical key component in the establishment of
puberty acting via the release of the gaseous neurotransmitter nitric oxide. This neurotransmitter stimulates GnRH release through activation of
the enzyme guanylate cyclase, which in turn promotes an increase in the production of cGMP and the posterior release of GnRH in the portal
circulation, stimulating, in turn, the release of LH and FSH from the pituitary and initiating a complete activation in the communication among
the key elements involved in the onset of puberty: the hypothalamus, the hypophysis and the ovary. Recognition of additional genes and
neurological pathways modulating both the timing of pubertal onset and its tempo are warranted

 2009 Blackwell Verlag GmbH

Signalling Mechanisms Controlling Puberty
deficiencies were accompanied by an increase in the
genic expression of pre-proenkephalin and by a hypo-
thalamic suppression of GnRH as well as in the mRNA
levels of KiSS1.
The third candidate gene is EAP-1 (early at puberty),
which is located as the TTF-1 gene, in chromosome 14
in humans. The hypothalamic levels of EAP-1 increase
during puberty in females, suggesting its involvement in
the control of puberty. The EAP-1 gene codifies a
nuclear protein which is expressed in neuronal subsets
involved in both stimulatory and inhibitory secretion of
GnRH, such as the glutamatergic, GABAergic, pro-
enkephalinergic neurons, the KiSS-1 neurons as well as
the GnRH neurons. As in the case with the TTF-1 gene,
EAP-1 transactivates the promoter in those genes
involved in the onset of puberty (i.e. GnRH), while it
suppresses the expression of those related with inhibi-
tory routes (i.e. pre-proenkephalin). Suppression of the
hypothalamic EAP-1 expression, as that for TTF-1,
delays puberty and disrupts the oestrous cycle, high-
lighting the importance of EAP-1 as a gene of high
hierarchy in the neuron-to-neuron regulation of the
GnRH secretion at puberty (Ojeda et al. 2006b).
Final Considerations
The mechanisms involved in the initiation of puberty are
quite complex. It has been proposed that GnRH
neurons are reasonably mature at birth. As the growth
process starts, yet, the pulsatile release of GnRH is
suppressed. Whereas the most prevailing invoked excit-
atory neuronal systems consider glutamate and the
kisspeptins with their GPR54 receptors for neurotrans-
mission and neuromodulatory processes, the most
important inhibitory systems include GABAergic and
opiatergic neuronal systems. As the animal approaches
puberty, inhibitory signals diminish until they are
removed. This generates an increase in the pulsatile
release of GnRH as a result of increasing neuronal
signals, mainly glutamatergic. The increase in the
pulsatile release of GnRH results in a cascade of events
leading to an increase in the synthesis and release of LH
and FSH, as well as an increase in both steroidogenesis
and gametogenesis.
It is well-established that GnRH pulsatility is signif-
icantly reduced under negative energy balance observed
with suboptimal nutritional scenarios and disturbance
of metabolic conditions. In that respect, the role of some
metabolic hormones such as leptin, ghrelin and insulin
as body reserve markers and adequate energy level is to
be noted. Metabolic hormones act positively on the
central nervous system to increase pulsatility and
frequency of GnRH release. As a plethora of genes are
activated and inactivated in the brain, the series of
complex events determining the tempo of puberty must
be regulated by a certain number of master genes.
Presently, neuroendocrine control of puberty gov-
erned by a network of genes with a high hierarchical
nature has been suggested (OCT-2, TTF-1 & EAP-1).
These genes are highly dominant and closely connected
in the command of genic regulation. In addition, a
partial overlap of a second sub-network of subordinated
genes has been proposed. They are less connected with
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e501
one another than genes of high hierarchy, yet having a
notable redundancy and high diversity in such system.
The existence of a network of hypothalamic genes
located in different but interacting hierarchical levels is
consistent with the proposed idea that the onset of
puberty is genetically determined and neuromodulated
through a polygenic strategy highly hierarchical in the
command of the genic expression (Fig. 1).
Author contributions
CA Meza-Herrera and A Gonzalez-Bulnes, designed the outline and
the structure of the review. CA Meza-Herrera, A Gonzalez-Bulnes,
and RT Kridli made an in-depth search of information, and
substantially contributed to the discussions and the review of the
document. JM Luginbuhl, M Mellado, CF Arechiga-Flores and
H Salinas made specific topic contributions in the development of the
review.
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Submitted: 15 Sep 2008
Author’s address (for correspondence): Dr. Cesar A. Meza-Herrera,
Galeana 585 Poniente, Colonia Centro, Lerdo, Durango, Mexico
35150. E-mail: cmeza2000@hotmail.com
 2009 Blackwell Verlag GmbH