Drs. Andrew J Watson and Andy Babwah Department of Obstetrics and Gynaecology

1. Drs. Andrew J Watson and Andy Babwah Department of Obstetrics and Gynaecology 5th Fl VRL Children’s Health Research Institute Puberty and kisspeptin-GPR54 signaling: Have we found the Trigger?

2. 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 129:675-683. Shahab M et al., 2005. Increased hypothalamic GPR54 signaling: a potential mechanism for initiation of puberty in primates. PNAS. 102:2129-2134.

3. Gonadotrophins throughout a lifetime

4. Growth Rates

5. Variability of Events

6. Same age but very different phenotypes

7. Puberty- Mechanisms of Action • How does the pituitary increase its output of gonadotropin hormones at the time of puberty?

8. 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.

9. The diurnal pattern of LH/FSH secretion changes

10. 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.

11. Richter and Terasawa Trends in Endo and Met 12: 353 2001

12. 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.

13. Triggers con’t • 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.

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

15. 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.

16. Leptin Con’t • 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????

17. 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

18. KiSS-1 sequence and cleavage products resulting in various kisspeptins

19. 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 femtomol=1 x 10 -15 mol Endocrinology (2004) 145:4073-4077

20. 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) 145:4073-4077 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

21. Kisspeptin-expressing neurons are localized in discrete regionsof the forebrain. KiSS-1 mRNA is expressed in cells that residein the anteroventral periventricular nucleus (AVPV), the periventricularnucleus (PeN), the anterodorsal preoptic nucleus (ADP) and thearcuate 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) 145:4073-4077

22. 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) 481 314–329 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) 146:4431-4436

23. If kisspeptin neurons communicate directly with GnRH neurons, then GnRH neurons should express GPR54. J. Neurosci. (2005) 25:11349-11356 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

24. 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.

26. RT–PCR analysis reveal that total hypothalamic content of KiSS-1 mRNA increases significantly after gonadectomy and decreases with sex steroid replacement Profile of expression of KiSS-1, GPR54, and GnRH genes in rat hypothalamus in adult (75 d old) rats, 2 wk after ORX with or without T replacement. Co: control amplification of S-11 ribosomal protein mRNA served as an internal control GnRH Endocrinology (2004) 145:4565-4574 2 wk later orchidectomy (ORX) of adult animals causes significant increase in hypothalamic KiSS-1mRNA levels. ORX associated with amoderate rise in GPR54 mRNA expression. 2 wk after ORX,serum LH levels rises significantly but hypothalamic steady state levels of GnRH mRNA remained unaffected. 2 wk after ORX, T replacement blunted the rise in LH levels and blockedthe increase in hypothalamic KiSS-1 mRNA levels. Similarly, T supplementation blocked the moderate increase inGPR54 mRNA after ORX and partially suppressed hypothalamic GnRHmRNA levels. Steroids regulate kisspeptins

27. 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 steroidreceptors. In male mice, more than 60% of KiSS-1 neurons inthe Arc express AR and about 90% express the ERa. In the femalemouse, nearly all KiSS-1 neurons express ERa, and approximately30% express ERß Thus, KiSS-1neurons are direct targets for the action of sex steroids inboth the male and female mouse. Endocrinology (2005) 146:3686-3692 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.

28. 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) 145:4565-4574

29. Does kisspeptin-GPR54-GnRH signaling trigger puberty? The electrophysiologic response of GnRH neurons to kisspeptinsappears to change dramatically over the course of puberty. Gramicidin-perforated patch recordings from brain slice preparations of male GnRH-GFP transgenic mice : 30% of GnRH neurons responded to kisspeptin administrationin prepubertal mouse whereas 90% ofGnRH neurons from adult mice responded to the same dose of kisspeptins In addition, the excitatory effectof kisspeptin appears to directly activate GnRH neurons becausethe response remains in the presence of tetrodotoxin Also, central injectionsof lower kisspeptin doses (10–100 fmol) stimulateLH in adult, but not prepubertal, male mice. Thus, it appears in the mouse, GnRH neurons become developmentallyactivated 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-perforated patch recordings were used to assess the electrophysiologic response of GnRH neurons to central kisspeptin administration (red bar). J. Neurosci. (2005) 25:11349-11356

30. 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.