Drs. Andrew J Watson and Andy Babwah

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Drs. Andrew J Watson and Andy
Babwah Department of Obstetrics and
Gynaecology 5th Fl VRL Childrens Health Research
Institute
Puberty and kisspeptin-GPR54 signaling Have we
found the Trigger?
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Physiology of Puberty
Objectives - State a definition of puberty and
how its "arrival" is known. - Describe some of
the physical changes that occur during puberty.
- Describe the changes in gonadotropin and sex
steroid secretion during puberty. - Summarize
the current understanding of the mechanisms by
which hormonal changes associated with puberty
are regulated. - Investigate factors which may
trigger the mechanisms described above.
Ebling FJ 2005 The neuroendocrine timing of
puberty. Reprod 129675-683. Shahab M et al.,
2005. Increased hypothalamic GPR54 signaling a
potential mechanism for initiation of puberty in
primates. PNAS. 1022129-2134.
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Gonadotrophins throughout a lifetime
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Growth Rates
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Variability of Events
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Same age but very different phenotypes
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Puberty- Mechanisms of Action

• How does the pituitary increase its output of
  gonadotropin hormones at the time of puberty?

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Control of Gonadotrophin Release
1. Pubertal levels of gonadotrophins are secreted
after birth in primates and humans. -This is
followed by a prolonged suppression of pituitary
activity during juvenile years. 2. The
hypothalamic-pituitary gonadal axis is capable of
functioning in adult fashion during infancy.
-normal pubertal changes can be induced in
monkeys by activation of GnRH neurons and
several syndromes of precocious puberty exist in
humans. 3. The negative feedback effects of
gonadal steroids are greater in children than
adults? -led to the "gonadostat" theory, ie the
decreasing feedback sensitivity permits
increased LH/FSH secretion which then activates
the gonads. -this is incomplete because
children with gonadal dysgenesis also display
increased LH/FSH secretion at the time puberty
should occur.
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The diurnal pattern of LH/FSH secretion changes
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Neural Mechanisms of Pubertal GnRH Release

• hypothalamic-pituitary-gonadal axis is restrained
  or inhibited in early life.
• inhibitory neurotransmitter gamma-amino-butyric
  acid (GABA) (females) and neuropeptide Y (NPY)
  (males) may be responsible for this inhibition of
  GnRH release.
• GABA modulates GnRH release before and during
  puberty in monkeys and at the time of GnRH surge
  in adult rats and sheep.
• GABA levels are much higher in prepubertal
  animals and GABA levels are inversely related to
  GnRH levels in pubertal and cycling animals.
• The onset of puberty should be linked to a
  decline in GABA induced inhibition of GnRH
  release.
• Blockade of GABA receptors with bicuculline
  (receptor antagonist) advances timing of menarche
  in female monkeys.
• NPY receptor blocker also induces LH release in
  juvenile male monkeys. NPY mRNA levels are higher
  in juvenile than pubertal monkeys.

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Richter and Terasawa Trends in Endo and Met 12
353 2001
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Triggers for the onset of puberty
The average age of voice change in J. S. Bach's
choir in Leipzig was 17 years. Now the average is
13.5 years.
The diagram shows that the age at which menarche
is achieved has been steadily decreasing over the
last 100 years in these developed countries.
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Triggers cont

• Changes in nutrition have been suggested.
• Weight range at which the adolescent growth spurt
  begins and at which menarche is observed is
  constant
• Suggests that a critical weight must be attained
  before the growth spurt and activation of the
  hypothalamic-pituitary-gonadal axis can occur.
• body weight (or more correctly a critical
  metabolic mass which is related to body weight)
  is the trigger to pubertal events.
• Early occurrence of puberty is then explained by
  the earlier attainment of the critical weight due
  to improvements in nutrition, health care and
  social living conditions.
• moderately obese girls experience an early
  menarche and malnutrition is associated with
  delayed menarche
• However much controversy surrounds this
  hypothesis.
• assumes a causal relationship between the
  critical metabolic mass and the time of onset of
  puberty. It is extremely difficult to predict the
  age of menarche in an individual from the
  knowledge of weight and weight gain.

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body weight (or more correctly a critical
metabolic mass which is related to body weight)
is the trigger to the pubertal events?
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The Leptin Hypothesis

• So how might the GABA or NPY inhibition be
  overcome to initiate the onset of puberty?
• GnRH release is controlled by glutamate which is
  the predominant excitatory neurotransmitter in
  the hypothalamus.
• glutamate actively controls GnRH release but it
  is insufficient to overcome the GABA inhibition
  on GnRH release.
• research has proposed that Leptin is permissive
  and a controller of these events.
• Leptin is a peptide synthesized in adipose tissue
  and is secreted into the general circulation. Its
  role in triggering puberty is still controversial
  however.
• Leptin administration to prebubertal rats and
  mice can advance the onset of puberty and
  reproductive cyclicity in these species. Also
  mice that lack the gene for Leptin synthesis do
  not cycle and are sterile.

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Leptin Cont

• Since Leptin production in children is directly
  related to their levels of adipose tissue this
  hypothesis fits with the metabolic data that
  suggest a certain critical mass must be obtained
  before puberty is initiated.
• Although it simply remains an intriguing
  hypothesis at this point in time I believe that
  it is very worthwhile to follow progress in this
  area as it might lead to a cohesive explanation
  of the physiological events that control the
  onset of puberty in mammals.
• But if not Leptin ..then what????

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1999 GPR54 first identified as an orphan G
protein-coupled receptor in rat 2001 Natural
ligand for GPR54 discovered, a 54-amino-acid
product of a gene called Kiss1 1996 Kiss1 was
originally identified as a human metastasis
suppressor gene that suppresses metastases of
melanomas and breast carcinomas 2003
investigators discovered that inactivating
mutations of GPR54 are linked with a failure to
progress through puberty and HH
(hypogonadotrophic hypogonadism) in humans
2003 Above observations corroborated in mice
bearing targeted deletions of GPR54.Mutant mice
displayed single major phenotypic anomaly
reproductive dysfunction 2003 studies concluded
kisspeptin-GPR54 signalling essential to initiate
gonadotropin (LH/FSH) secretion at puberty and
support reproductive function in the adult
Kisspeptin/GPR54 signaling
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KiSS-1 sequence and cleavage products resulting
in various kisspeptins
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Kisspeptins stimulate gonadotropin-releasing
hormone (GnRH) (synthesis and/or
secretion?) 2004-extraordinarily low doses of
kisspeptin (1 fmol), injected into the lateral
ventricle of the mouse, elicits rapid and robust
LH/FSH secretion Similar
observations (but with higher doses of
kisspeptin) reported in rat (2004 2005), sheep
(2005), monkey (2005 2006) and human male
(2005)
Effects of different doses of kisspeptin-54
(ranging from 0-5 nmol delivered ICV) on serum
levels of LH, measured 30 min after a bolus
injection
1 femtomol1 x 10 -15 mol
Endocrinology (2004) 1454073-4077
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In vivo, kisspeptin-stimulated LH/FSH release is
dependent on the release of GnRH and does not
reflect a direct action of kisspeptin on the
pituitary
Endocrinology (2004) 1454073-4077
The effects of kisspeptin-54 (50 pmol delivered
ICV) or its vehicle alone, coupled with
pretreatment with a GnRH antagonist, acyline (50
mg, sc), or its vehicle alone
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Kisspeptin-expressing neurons are localized in
discrete regions of the forebrain. KiSS-1 mRNA
is expressed in cells that reside in the
anteroventral periventricular nucleus (AVPV), the
periventricular nucleus (PeN), the anterodorsal
preoptic nucleus (ADP) and the arcuate nucleus
(Arc)
Localization of KiSS-1 mRNA in the forebrain of
the mouse. Each panel depicts a hypothalamic
section. Panel A is most rostral and shows
KiSS-1 mRNA localization (red dots) in the
anteroventral periventricular nucleus (AVPV),
periventricular nucleus (PeN) and anterodorsal
preoptic area (ADP). Panel B is more caudal and
shows KiSS-1 mRNA localization in the arcuate
nucleus (Arc).
(Gottsch et al. 2004, Smith et al. 2005a, b)
Distribution of KiSS-1 mRNA expressing cells in
the hypothalamus. Clusters of white dots
indicate KiSS-1 mRNA-expressing cells. KiSS-1
mRNA-containing cells were observed in the AVPV
(A), PeN (B), anterodorsal preoptic nucleus (C),
medial amygdala (D), and ARC (E and F). 3V,
Third ventricle AC, anterior commissure opt,
optic tract OX, optic chiasm.
Endocrinology (2004) 1454073-4077
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Kisspeptin appears to have direct access to GnRH
neurons
Kisspeptin neurons reside in nuclei such as the
arcuate nucleus and anteroventral periventricular
nucleus and send projections (projections only,
not cell bodies) to the medial preoptic area (E,
F), where there is an abundance of GnRH cell
bodies and, kisspeptin fibers appear in close
approximation to GnRH neurons (D)
Photomicrographs of sections through the rat
forebrain labeled with kisspeptin
antiserum. Kisspeptin mmunoreactive fibers
(projections only, not cell bodies) at the level
of the optic chiasm (OX) are seen in the medial
preoptic nucleus (MPO), medial preoptic area
(MPA), and lateral preoptic area (LPO E,F),
regions in which GnRH neurons are diffusely
distributed F represents higher magnifications
of the boxed areas in E
J Comp Neurol (2005) 481 314329
Metastin-immunoreactive fibers were in close
apposition with GnRH cell bodies in the preoptic
area. (D fibers-brown (punctate), metastin
cell bodies-blue (looks violet), GnRH).
Endocrinology (2005) 1464431-4436
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If kisspeptin neurons communicate directly with
GnRH neurons, then GnRH neurons should express
GPR54.
J. Neurosci. (2005) 2511349-11356
Expression of GPR54 mRNA in GnRH neurons across
development A, Representative photomicrographs
of cells coexpressing GPR54 mRNA (reflected by
silver grains, appearing as clusters of white
dots) and GnRH mRNA (labeled with Vector Red) in
juvenile (left) and intact adult (right) male
mice. B, Quantitative analysis of GPR54 mRNA in
GnRH neurons demonstrated that neither the
percentage of double-labeled cells (left) nor
the relative expression of GPR54 mRNA in GnRH
neurons (reflected by the number of silver grains
per GnRH neuron right) differed significantly
between P18 and adult mice
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Kisspeptin appears to act directly on GnRH
neurons to stimulate the secretion of GnRH
Proposed interactions between kisspeptin-secreting
neurons and GnRH neurons. In this model,
KiSS-1 mRNA-expressing neurons, from the arcuate
nucleus (Arc) and the anteroventral
periventricular nucleus (AVPV), make synaptic
contact with GnRH neurons within the preoptic
area (POA). Upon activation of the kisspeptin
receptor GPR54, GnRH neurons are stimulated to
release GnRH into the portal circulation, which
in turn stimulates the release of LH and FSH from
the pituitary.
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Steroids regulate kisspeptins
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Steroids regulate kisspeptins
Are kisspeptin neurons direct or indirect (i.e.,
receiving data from other steroid-sensitive
neurons) targets of gonadal-circulating
steroids? Data show that cells expressing KiSS-1
mRNA also express sex steroid receptors. In male
mice, more than 60 of KiSS-1 neurons in the Arc
express AR and about 90 express the ERa. In the
female mouse, nearly all KiSS-1 neurons express
ERa, and approximately 30 express ERß Thus,
KiSS-1 neurons are direct targets for the action
of sex steroids in both the male and female
mouse.
Endocrinology (2005) 1463686-3692
Representative photomicrographs showing
coexpression of KiSS-1 mRNA with ERa (A) and ERß
(B). KiSS-1 mRNA-expressing cells are fluorescent
with Vector Red substrate, and clusters of silver
grains reflect the presence of ERa (A) or ERß (B)
mRNA. The arrows indicate KiSS-1 neurons that
coexpress either ERa or ERß.
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Does kisspeptin-GPR54-GnRH signalling trigger
puberty?
Animals with disabling mutations and targeted
deletions of GPR54 display HH. Activation of
GnRH neurons is the key event that initiates the
onset of puberty but the nature of this
trigger remains to be identified.
Kisspeptin-GPR54 signaling is a plausible
candidate
If kisspeptins triggers puberty, an increase in
KiSS-1 mRNA and/or GPR54 mRNA expression should
be detectable during this time.
Developmental profile of expression of KiSS-1 and
GPR54 genes in rat hypothalamus throughout
postnatal maturation in the male. In the left
panels, representative RT-PCR assays are
presented of expression levels of KiSS-1 and
GPR54 mRNAs in hypothalamic samples from 1-, 5-,
10-, 15-, 20-, 30-, 45-, and 75-d-old as well as
18-month-old male rats. In the right panels,
semiquantitative values are the mean SEM of at
least three independent experiments.
Endocrinology (2004) 1454565-4574
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Does kisspeptin-GPR54-GnRH signaling trigger
puberty?
The electrophysiologic response of GnRH neurons
to kisspeptins appears to change dramatically
over the course of puberty. Gramicidin-perforat
ed patch recordings from brain slice preparations
of male GnRH-GFP transgenic mice 30 of GnRH
neurons responded to kisspeptin administration in
prepubertal mouse whereas 90 of GnRH neurons
from adult mice responded to the same dose of
kisspeptins In addition, the excitatory effect
of kisspeptin appears to directly activate GnRH
neurons because the response remains in the
presence of tetrodotoxin Also, central
injections of lower kisspeptin doses (10100
fmol) stimulate LH in adult, but not prepubertal,
male mice. Thus, it appears in the mouse, GnRH
neurons become developmentally activated by
kisspeptins over the course of puberty
Possible role of kisspeptin in the onset of
puberty. Recent observations suggest that GnRH
neurons become increasingly responsive to
kisspeptin as a function of pubertal maturation
in the mouse. In this example, gramicidin-perforat
ed patch recordings were used to assess the
electrophysiologic response of GnRH neurons to
central kisspeptin administration (red bar).
J. Neurosci. (2005) 2511349-11356
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Conclusions

• Activation of hypothalamic GPR54 receptor
  signaling before puberty in higher primates
  induces precocious GnRH release
• Kisspeptin must now be considered as a candidate
  for upstream signal that stimulate pulsatile GnRH
  release
• What regulates GPR54 and Kisspeptin??? The next
  step in the pathway.

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