Human Reproduction Update, Vol.10, No.1 pp.67-77, 2004
© European Society of Human Reproduction and Embryology 2004; all rights reserved
Neurobiological mechanisms of puberty in higher primates
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA 1 To whom correspondence should be addressed. e-mail: plant1{at}pitt.edu
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Abstract |
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 TOP Abstract IntroductionRole of hypothalamic GnRHA non-gonadal control system...The nature of the...Genomics and the neurobiological...Mechanisms timing the delay...Genetics and gonadarcheConclusionsAcknowledgementsReferences |
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Puberty in humans is comprised of two developmental processes; namely, gonadarche and adrenarche. Of the two, gonadarche is fundamentally the most important, and this review examines the neurobiological mechanisms that first prevent, and later trigger, progression into this critically important phase of human development when the ability to first reproduce is established. The review draws extensively upon results obtained by studies of the rhesus monkey (Macaca mulatta), a representative higher primate which, like man, exhibits a postnatal pattern in activity of the hypothalamic–pituitary–gonadal axis that is characterized by a prolonged period of relative quiescence from late infancy until the initiation of the pubertal process. The proximate cause of the prepubertal quiescence in this neuroendocrine axis is the arrest or restraint of the pulsatile mode of hypothalamic GnRH release by a neurobiological brake that holds in check release of this decapeptide, without seeming to down-regulate the transcriptional activity of the gene encoding GnRH (GnRH-I). Thus, if neurogenomes control the onset of gonadarche, they must reside upstream from that of the GnRH neuron. The genetic and physiological factors (with a particular emphasis on leptin) that time the application and duration of the prepubertal brake on GnRH release are also considered.
Key words: 
-aminobutyric acid/gonadarche/GnRH/leptin/neuropeptide Y
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Introduction |
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TOPAbstractIntroductionRole of hypothalamic GnRHA non-gonadal control system...The nature of the...Genomics and the neurobiological...Mechanisms timing the delay...Genetics and gonadarcheConclusionsAcknowledgementsReferences |
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Puberty is the phase of development during which the potential to reproduce is first realized. In our own species, puberty is typically initiated at the end of the first, or the beginning of the second, decade of life, and its onset is recognized by maturation of the genitalia, development of secondary sexual characteristics, acceleration in growth, changes in affect and, in the female, by the occurrence of menstruation. Paediatric endocrinologists generally consider that human puberty is the result of two independent physiological processes, namely gonadarche and adrenarche (Witchel and Plant, 2003
). Gonadarche refers to the activation of the ovary and testis at the end of the prepubertal phase of development and leads to the dramatic increase in gonadal steroid production and the completion of gametogenesis. The unfolding of gonadarche is manifest by thelarche and menarche in girls, and by testicular enlargement and virilization in boys. Adrenarche, on the other hand, refers to the maturation of the adrenal cortex that leads to increased secretion of adrenal androgens, namely dehydroepiandrosterone, dehydroepiandrosterone sulphate and androstenedione, and is manifest by the appearance of sexual hair, a process termed pubarche. It is interesting to note that adrenarche appears to be peculiar to man and the Great Apes (see Plant, 1994), and the absence of adrenarche in humans does not appear to prevent fertility nor markedly to influence the timing and tempo of gonadarche (Sklar et al., 1980; Grumbach, 2002). Thus, from a biological perspective it would seem reasonable to argue that adrenarche should be viewed as a corollary of the pubertal process rather than as an integral and fundamental component of this important developmental event. From a clinical perspective, however, the impact of pubertal disorders of adrenal androgen secretion on development of the neuroendocrine axis governing the ovary should not be overlooked. This is particularly relevant to the emerging relationship between hyperandrogenism and polycystic ovarian syndrome (Ibàñez et al., 2000). With the foregoing argument in mind, the present review examines our current understanding of the physiological, genomic and genetic mechanisms that underlie gonadarche and that regulate the timing of this process in higher primates. |
Role of hypothalamic GnRH |
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TOPAbstractIntroductionRole of hypothalamic GnRHA non-gonadal control system...The nature of the...Genomics and the neurobiological...Mechanisms timing the delay...Genetics and gonadarcheConclusionsAcknowledgementsReferences |
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That an intermittent hypophysiotrophic gonadotrophin-releasing stimulus is obligatory for operation of the pituitary–gonadal axis in the adult was established by the classic work of Knobil and his colleagues in the 1970s (Belchetz et al., 1978
). This group was able to restore gonadotrophin secretion in hypothalamic-lesioned monkeys with an intravenous infusion of synthetic GnRH (mammalian GnRH, GnRH-I) only when the decapeptide was administered in an intermittent mode. Spontaneous pulsatile release of GnRH by the brain is generated by a diffusely distributed hypothalamic network of several hundred neurons that express the GnRH-I gene (Silverman et al., 1994). Although it is now generally recognized that the mechanism responsible for pulsatility in this neuronal network, the so-called GnRH pulse generator, is intrinsic to the GnRH neurons themselves (Wetsel et al., 1992; Terasawa et al., 1999, 2002), the ability of rat retrochiasmatic hypothalamic explants (where GnRH axons have been severed from their cell bodies) to sustain pulsatile GnRH release (Prunelle et al., 1997) is difficult to explain.
The case may be that a pulsatile GnRH drive is critical for sustained gonadotrophin secretion and if compromised, for example in severe undernutrition and during strenuous exercise training, individuals become hypogonadotrophic (Cameron, 1996; Warren and Stiehl, 1999). This, in turn, leads to a hypogonadal state that in women presents as secondary amenorrhoea and anovulation. Thus, the pituitary–gonadal axis may be viewed as a slave to the hypothalamic GnRH pulse generator, and this analogy should be borne in mind as the role of GnRH in initiating gonadarche is considered.
Parenthetically, it should be noted that a second GnRH (GnRH-II) is also found in the primate hypothalamus (Chen et al., 1998; Latimer et al., 2001), and the gene encoding the GnRH-II receptor is expressed by the anterior pituitary (Millar et al., 2001; Neill et al., 2001). Although intravenous administration of a bolus of GnRH-II to the rhesus macaque is able to elicit an LH discharge (Densmore and Urbanski, 2003), the role of this peptide in regulating the pituitary–gonadal axis in general, and the initiation of gonadarche in particular, is unknown.
Although direct measurement of temporal changes in GnRH concentrations in portal blood during puberty have not been reported for any species of primate, the concept that an increased release in pulsatile GnRH is responsible for gonadarche is well founded. A marked increase in hypothalamic release of this decapeptide at the time of puberty in the female rhesus monkey has been described using the technique of push–pull perfusion (Watanabe and Terasawa, 1989), and the notion that neither pituitary nor gonad are limiting to the onset of gonadarche was graphically underlined by Knobil and his colleagues, who, in the late 1970s, treated premenarcheal female rhesus monkeys with uninterrupted intermittent intravenous infusions of GnRH and elicited premature ovulatory menstrual cycles (Wildt et al., 1980). Cognate observations of the male rhesus monkey have subsequently been made (Plant, 1988; Plant et al., 1989a), and the condition of GnRH-dependent precocious gonadarche in man (Witchel and Plant, 2003) further supports the pivotal role of increased hypothalamic GnRH release in triggering the pubertal activation of the pituitary–gonadal axis.
Interestingly, the pubertal increase in pulsatile GnRH release does not reflect the acquisition by the primate hypothalamus of the capacity to generate such an intermittent mode of secretion. Rather, it represents the re-expression of this capacity, a potential that has been held in a state of check since infancy (Plant, 2001). The ability of the network of GnRH neurons to generate a pulsatile discharge of the decapeptide originates during early fetal development (Jaffe, 1989) at the time when these neurons, which are born in the olfactory bulb, are completing their migration to the mediobasal hypothalamus, where they reside in the brain of the adult primate (Ronnekleiv and Resko, 1990). The cell and molecular biology underlying the guidance systems utilized by migrating GnRH neurons is currently an area of intense investigation (Hardelin, 2001; Tobet et al., 2001; Allen et al., 2002; Wray, 2002).
While some authors have argued that gonadarche may be viewed as a continuum of a developmental process that starts during early fetal development (Delemarre-van de Waal, 2002; Grumbach, 2002), the value of this concept to higher primates is not readily apparent. This is because the mechanism that times gonadarche in these species is one that regulates the delay or prevention of this developmental process (Pescovitz, 2000), which, in the case of man, is capable of manifesting itself many years before the normal age of puberty. Thus, while the hypothalamus of the infant primate generates a robust mode of pulsatile GnRH release, this ‘adult’ mode of activity is soon restrained, albeit not abolished, leading to a relatively hypogonadotrophic state, which guarantees the quiescence of the ovary and testis during childhood. Therefore, the timing of gonadarche in primates is governed by the duration of the interval during which the activity of a ‘mature’ hypothalamic GnRH neuronal network to generate a robust pulsatile pattern of activity is restrained.
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A non-gonadal control system delays gonadarche |
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TOPAbstractIntroductionRole of hypothalamic GnRHA non-gonadal control system...The nature of the...Genomics and the neurobiological...Mechanisms timing the delay...Genetics and gonadarcheConclusionsAcknowledgementsReferences |
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The classic description by Conte et al. (1975
) nearly 30 years ago of the developmental pattern of circulating gonadotrophin concentrations from birth to 20 years of age in patients with gonadal dysgenesis, demonstrated that the restraint imposed on pulsatile GnRH release during childhood (2–10 years of age) was applied in the absence of the gonad. Studies of agonadal monkeys have substantiated the early clinical data (Plant, 1985, 1994; Pohl et al., 1995) and have also demonstrated that the restraint exerted on hypothalamic GnRH release prior to puberty is noticeably more intense in the male than in the female (Figure 1). This important sex difference, which is presumably determined by exposure to testicular testosterone secretion in utero, is probably the reason why idiopathic precocious gonadarche is seen most frequently in girls (Witchel and Plant, 2003), and why delayed gonadarche due to constitutive delay in growth and maturation (CD) is observed more frequently in boys (Sedlmeyer and Palmert, 2002). The hypogonadotrophic state resulting from the non-gonadal prepubertal brake on pulsatile GnRH release is amplified by ovarian and testicular feedback, and particularly by ovarian estradiol inhibition (Conte et al., 1975; Pohl et al., 1995), but this finding does not distract from the conclusion that the pivotal component of quiescence in the reproductive axis of the child is extragonadal.
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The nature of the neurobiological brake delaying gonadarche |
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TOPAbstractIntroductionRole of hypothalamic GnRHA non-gonadal control system...The nature of the...Genomics and the neurobiological...Mechanisms timing the delay...Genetics and gonadarcheConclusionsAcknowledgementsReferences |
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Paradigms
Before discussing progress on this front, it is important to comment briefly on the experimental paradigms that have been employed to examine this issue. Generally, studies of mechanisms of puberty have been conducted in the gonadally intact situation. Intuitively, this approach would seem reasonable, but on closer inspection it becomes clear that such a paradigm is not always the most appropriate, particularly when the neurobiological basis of the onset of puberty is being examined. This is because the resurgence of GnRH pulsatility that activates gonadarche results concomitantly in a dramatic and progressive increase in circulating concentrations of sex steroids, which are capable of remodelling both molecular and structural components of the hypothalamus and other regions of the central nervous system (Witkin et al., 1991; Witkin, 1996; Plant, 2001). Thus, in the intact situation, secondary and tertiary actions of increasing steroid hormone levels in the brain will, at best, confound the interpretation of any primary developmental changes in microstructure and/or gene expression observed in association with the resurgence of GnRH pulsatility. Thus, studies conducted in this laboratory to probe the molecular biology of the brake on pulsatile GnRH release have employed an agonadal paradigm.
GnRH
The first gene that we examined in the context of the brake was, naturally, GnRH-I. Surprisingly, in the agonadal male, hypothalamic GnRH-I mRNA levels show only a modest increase in association with the pubertal resurgence in GnRH pulsatility (El Majdoubi et al., 2000a). Moreover, in intact animals the pubertal increase in GnRH-I gene expression is greatly dampened (Vician et al., 1991; Ma et al., 1994), presumably as a result of imposing gonadal steroid feedback (El Majdoubi et al., 1998, 2000b; Krajewski et al., 2003), and is not associated with a corresponding change in the hypothalamic content of GnRH (Fraser et al., 1989; El Majdoubi et al., 2000a). The modest increase in hypothalamic GnRH-I expression together with the constitutively expressed peptide content may be contrasted with the molecular state of the gonadotroph at corresponding stages of development. This pituitary cell, as might be expected, is profoundly down-regulated prior to the resurgence of pulsatile GnRH release (S. J. Winters and T.M.Plant, unpublished observation), while the GnRH neuron at this stage of development is essentially indistinguishable from that in the adult hypothalamus.
Not only is the GnRH-I gene and its peptide expressed at high levels prior to the pubertal resurgence of pulsatile release, but a pattern of peptide secretion similar to that of the pubertal state may be elicited with remarkable ease from the hypothalamus of the juvenile by applying a repetitive intermittent chemical stimulation with the glutamate receptor agonist, N-methyl-D-aspartate (Gay and Plant, 1988). Taking these findings together, it may be concluded that transcriptional regulation of GnRH-I probably does not play a critical role in the onset of gonadarche. On the contrary, the molecular machinery for generating an adult pattern of GnRH release is extant in the prepubertal situation, and thus it would seem that genomics of the GnRH neuron do not play a pivotal role in timing the pubertal resurgence of pulsatile GnRH release. Rather, the signal controlling the timing of gonadarche originates upstream from the GnRH neuronal network. Before discussing upstream signals to the network of GnRH-I neurons, it is interesting to note that the spontaneous resurgence in pulsatile GnRH release in the agonadal situation occurs explosively, with the pattern of release increasing from an essentially apulsatile mode to a near-adult circhoral mode over an epoch of 40 days (Suter et al., 1998).
Upstream signals
For the last few years we have been pursuing the hypothesis that a major component of the upstream brake on pulsatile GnRH release is provided by hypothalamic neurons expressing neuropeptide Y (NPY) (Plant and Shahab, 2002). This hypothesis is based on the following findings. First, central administration of NPY arrests GnRH pulsatility in agonadal postpubertal male and female monkeys (Pau et al., 1995; Kaynard et al., 1990; Shahab et al., 2003). Second, ontogenetic changes in NPY gene expression and peptide content in the mediobasal hypothalamus of the agonadal male monkey are inversely related to that of GnRH release at the three major stages of postnatal development; namely, infancy–juvenile development–puberty (El Majdoubi et al., 2000a). Moreover, this pattern in the ontogeny in NPY gene expression is correlated with a structural remodelling of the interactions between NPY axonal varicosities and GnRH perikarya in the hypothalamus of the male rhesus monkey (Plant, 2001; Plant and Shahab, 2002). In the mediobasal hypothalamus of the juvenile, the number of appositions between NPY varicosities and GnRH perikarya are greater than that in the pubertal animal. Although the issue of whether the NPY varicosities are truly synaptic has not been definitively established, ultrastructural analysis has confirmed that there is a decrease in overall synaptic input to the GnRH perikarya at this critical developmental transition (Perera and Plant, 1997).
To date, five NPY receptors have been cloned (Michel et al., 1998), and all are G-protein-coupled receptors that are linked to inhibitory pathways, which, upon activation, lead to membrane hyperpolarization (Sun et al., 1998). The hypothalamic NPY receptors that underlie the ability of NPY to arrest pulsatile GnRH release remain to be fully elucidated. In the postpubertal situation, the inhibition of hypothalamic GnRH release by centrally administered NPY is mediated, at least in part, by the NPY Y1 receptor, but the pharmacological data supporting a role of this NPY receptor in mediating the prepubertal brake on pulsatile GnRH release are tenuous (Shahab et al., 2003). In another approach to test the hypothesis that increased hypothalamic NPY signalling is a major component of the brake that is exerted on pulsatile GnRH release prior to puberty, we and others have looked for loss of function mutations in the NPY Y1 receptor in children with GnRH-dependent precocious gonadarche (Barker-Gibb et al., 2002a; Freitas et al., 2003). In a total of 35 patients, a heterozygous substitution of T for G at nucleotide 1330 of exon 3 of the gene encoding the Y1 receptor was found in one girl with precocity. Although this mutation results in the substitution of lysine by threonine at position 374 in the carboxy terminus of the receptor, the mother of this subject was found to have the same heterozygous mutation, yet had apparently normal pubertal development (Freitas et al., 2003).
Before leaving the role of NPY in the control of GnRH secretion, it should be noted that NPY also exerts a stimulatory action on GnRH release in the monkey (Woeller et al., 1991; Gore et al., 1993; Pau et al., 1995). For a discussion of this conundrum, the reader is referred to an earlier review by this laboratory (Plant, 2001).
A considerable body of evidence obtained primarily for studies of the female is available to support the idea that -aminobutyric acid (GABA) is also a major component of the neurobiological brake that is imposed on GnRH release prior to puberty (Terasawa and Fernandez, 2001). Using hypothalamic perfusion, release of GABA in the median eminence of the female rhesus monkey declined as pulsatile GnRH release increased at the onset of gonadarche (Mitsushima et al., 1994). Moreover, acute inactivation of the GABAA receptor or reduction in GABA tone in this region of the hypothalamus in the prepubertal female monkey with bicuculline or antisense oligodeoxynucleotide for the mRNA encoding the GABA-synthesizing enzyme, glutamic acid decarboxylase 67 (GAD 67), respectively elicited an immediate discharge of GnRH (Mitsushima et al., 1994, 1996). Furthermore, chronic repetitive administration of bicuculline into the base of the third cerebroventricle at 
15 months of age in pregonadarcheal monkeys leads to premature menarche and precocious first ovulation (Keen et al., 1999). Although changes in hypothalamic expression of GAD 65 and 67 have not been reported during puberty in the female rhesus monkey, in the male of this species they appear unremarkable at this stage of development (Urbanski et al., 1998; El Majdoubi et al., 2000a).
Additional support for a role of GABA comes from clinical observations. Bourguignon et al. (1997) reported that administration of loreclezole, a GABA agonist, to an 11 month old girl with epilepsy and precocious gonadarche decreased LH secretion and led to a regression in pubertal development. Pubertal delay has also been observed in epileptic boys treated with valproic acid, a drug with GABAergic activity (El-Khayat et al., 2003). It should be noted, however, that the pharmacology of valproic acid is complex, and others have reported that this drug and other antiepileptic agents do not invariable delay sexual maturation (Rättyä et al., 1999).
GABA receptors, including the GABAA receptor, are coupled to Cl– channels, and it is generally recognized that GABA activation of these channels in neurons in the adult brain leads to an inward Cl– current that hyperpolarizes the membrane (Kaila, 1994). Indeed, it is generally recognized that GABA is the major inhibitory neurotransmitter in the hypothalamus (Decavel and van den Pol, 1990). Thus, until recently it had been assumed that the site of action of GABA was directly on the GnRH neuron of the prepubertal monkey. In this regard, in the rodent brain, GABA synapses have been reported on GnRH neurons (Leranth et al., 1985), which express GABAA receptor subunits (Jung et al., 1998; Pape et al., 2001).
In contrast to the inhibitory action of GABA in the adult brain, the action of GABA on many neuronal systems in the developing embryonic brain is excitatory (Cherubini et al., 1991). The switch in the electrophysiological action of GABA on developing neurons, which, in the rat, generally occurs around postnatal days 7–14 (Cherubini et al., 1991), appears to be related to an increased expression of a potassium chloride co-transporter, KCC2 (Rivera et al., 1999; Ganguly et al., 2001). Under physiological conditions, KCC2 extrudes K+ and Cl– and thereby maintains intracellular Cl– at concentrations lower than those predicted by the Nernst equilibrium. Thus, GABAA receptor activation in neurons expressing KCC2 will lead to an inward Cl– current and hyperpolarization. Whether GnRH neurons exhibit this developmental switch in expression of KCC2, and therefore respond to GABA with inhibition in early development and with excitation later, is currently a controversial issue. Two studies, both utilizing transgenic mice expressing either ß-galactosidase or green fluorescent protein under the control of the GnRH-I promoter, in order to visualize GnRH neurons in acute hypothalamic slices, have addressed this issue (DeFazio et al., 2002; Han et al., 2002). Both studies monitored electrophysiological activity using the gramicidin perforated patch technique, which enables membrane responses of a neuron to be recorded without changing the intracellular Cl– concentration and, therefore, the resting potential of the cell. In one study, µmol/l concentrations of GABA were added to the medium bathing the slice, and in the other, GABA at mmol/l concentrations was applied as a very brief ‘puff’ to the immediate vicinity of a GnRH-I neuron within the slice being studied. In the former study, the classic switch in GABA action was noted, while in the latter, GABA was shown to be excitatory at all stages of development. This issue is further confounded by the finding that expression of KCC2 in GnRH-I neurons in the hypothalamus of the adult mouse is heterogeneous (Leupen et al., 2003), indicating that subpopulations of GnRH neurons may respond differently to direct GABA inputs.
While the switch in GABA action in the central nervous system (CNS) of rodents occurs postnatally (Cherubini et al., 1991), in primates it is to be expected that this developmental event would occur prenatally. Thus, if GnRH-I neurons in higher primates manifest this developmental switch, the action of increased GABA release during prepubertal development to restrain pulsatile GnRH release could be accounted for by a direct action of the neurotransmitter on GnRH-I neurons. Conversely, if GnRH-I neurons in the postnatal primate brain remain ‘embryonic’ and are excited by GABA, then an indirect action of GABA on inhibitory afferents of the GnRH-I network, such as NPY, would have to be invoked.
In addition to NPY and GABA, glial-derived growth factors, such as transforming growth factor 
(TGF-) have been implicated in the control of the pubertal resurgence of pulsatile GnRH-I secretion. The action of TGF- on GnRH neurons is stimulatory and indirect, involving autocrine/paracrine release of prostaglandin E2 from neighbouring astrocytes (Ojeda et al., 2003). Release of GnRH is then effected by the prostaglandin. Although most studies of the glial regulation of GnRH-I release at the time of puberty have been conducted in the rat (Ojeda et al., 2003), TGF- gene expression in the hypothalamus has been shown to be dramatically increased in the pubertal female rhesus monkey (Ma et al., 1994) and marginally elevated in the agonadal male monkey at a comparable stage of development (El Majdoubi et al., 2000a). Additional circumstantial evidence for this view is provided by the finding that harmatomas removed from patients with GnRH-dependent precocious gonadarche have, on occasion, been found to be rich in astrocytes containing TGF-, but devoid of neurons expressing GnRH-I (Jung and Ojeda, 2002).
Although attempts have been made to develop integrated models of the neurobiological brake that is exerted upon pulsatile GnRH-I release from the primate hypothalamus prior to puberty, they have not been entirely satisfactory (Plant, 2001; Terasawa and Fernandez, 2001) for the following reason. The idea that hypothalamic NPY neurons comprise an essential component of the brake has been developed from studies of the male rhesus monkey, while the GABA hypothesis has been evolved from studies of the female rhesus monkey. Caution is needed in extrapolating findings in the male to the female and vice versa, since the duration and intensity of the neurobiological brake exerted on GnRH release during prepubertal development is greater in the male than in the female (Figure 1; Pohl et al., 1995). Whether this sex difference is purely quantitative or represents fundamental differences in the neurobiology is unknown. Another issue that remains to be clarified is whether the neurobiological mechanism underlying the resurgence of pulsatile GnRH release at the time of gonadarche is a simple reversal of that responsible in late infancy for inhibition of the same network (El Majdoubi et al., 2000c).
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Genomics and the neurobiological brake |
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TOPAbstractIntroductionRole of hypothalamic GnRHA non-gonadal control system...The nature of the...Genomics and the neurobiological...Mechanisms timing the delay...Genetics and gonadarcheConclusionsAcknowledgementsReferences |
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The proximal step in mediating the function of a genome is the appropriate temporal and cell specific expression of a gene or subset of genes. Thus, if physiological genomics underlie gonadarche, the first condition that must be met is that a programmed change or changes in gene expression in specific neuronal (and/or glial cells) phenotypes precede the resurgence in pulsatile GnRH release. The fact that a gene, for example NPY, is expressed or repressed in a particular set of neurons upstream to the GnRH neurons, however, is in itself insufficient to argue that neurogenomics are playing a fundamental role in regulating the timing of gonadarche. Information in the genome may be expressed as a result of an intrinsic event originating within a subset of neurons or the niche, in which the group of neurons reside. Such control by the genome is most clearly operational during early embryogenesis where gene expression unfolds in a programmed manner leading to particular cell lineages, to organogenesis and to differentiated function, as, for example, has been demonstrated for the anterior pituitary (Dasen and Rosenfeld, 2001
). In the context of the timing of gonadarche, the extent to which intrinsic developmental control of gene expression occurs upstream from the GnRH neuron is unknown. Alternatively, a non-hypothalamic (perhaps even a non-CNS) cue might activate signalling pathways leading to expression of a gene, protein synthesis and changes in the activity of upstream neurons and/or glial components that would result in the downstream resurgence of pulsatile GnRH release. While such a control system is conceptually appealing (see below), since it would provide a mechanism for coordinating attainment of adult stature with the onset of sexual maturation, it should not be viewed as a truly genomic mechanism. |
Mechanisms timing the delay to gonadarche |
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TOPAbstractIntroductionRole of hypothalamic GnRHA non-gonadal control system...The nature of the...Genomics and the neurobiological...Mechanisms timing the delay...Genetics and gonadarcheConclusionsAcknowledgementsReferences |
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The timing of the duration of the neurobiological brake on pulsatile GnRH release during juvenile development is presumably governed by a CNS ‘clock’ or by a control system, termed a ‘somatometer’, that tracks some aspect of somatic growth and development (Plant et al., 1989b
). In the case of the somatometer hypothesis, the attainment of a particular proportion of fat has long be suggested to be a cue for the onset of gonadarche in girls (Frisch and Revelle, 1970; Frisch et al., 1973), and interest in this notion was resurrected with the discovery of leptin, a circulating protein hormone, that is produced by expression of the ob gene in adipocytes, and that plays a major role in the regulation of appetite and food intake (Friedman and Halaas, 1998; Schwartz et al., 2000).
The idea that leptin is necessary, in a permissive sense, for gonadarche to unfold at an appropriate time is supported by several clinical observations. First, primary hypogonadotrophic hypogonadism at 23–34 years of age has been reported in association with morbid obesity in one male and two female subjects with a missense mutation in the leptin gene (Strobel et al., 1998; Ozata et al., 1999). Second, hypogonadotrophism and failure to undergo pubertal development at age 13.5, 19 and 19 years of age respectively has been described in three sisters homozygous for mutations in the leptin receptor gene (Clement et al., 1998). Third, primary amenorrhoea at age 15, 17 and 17 years respectively was reported in three patients with congenital generalized lipodystrophy associated with Seip–Berardinelli syndrome (Oral et al., 2002). Although one of the female subjects with leptin deficiency subsequently entered puberty spontaneously (Ozata et al., 1999), the foregoing clinical observations suggest that leptin signalling is required for normal gonadarche. On the other hand, normal pubertal development was reported in two female subjects with severe lipodystrophy (Andreelli et al., 2000), and in one girl with congenital contractual arachnodactyly (Galler et al., 2001), all of whom were markedly hypoleptinaemic. Interestingly, although the lipodystrophic subjects with normal pubertal development had measurable concentrations of circulating immunoactive leptin (0.7–1.6 ng/ml), they were no greater than those in the subjects with Seip–Berardinelli syndrome (0.8–1.0 ng/ml), who failed to enter puberty. The most parsimonious explanation for these inconsistent findings is that immunoactive leptin concentrations in the blood may not accurately reflect the bioavailability of the circulating hormone.
Additional support for the notion that leptin is necessary for gonadarche to occur is provided by studies of the effects of hormone replacement in a small number of leptin-deficient subjects of pubertal or postpubertal age but exhibiting absent or incomplete gonadarche. In a 9 year old girl with a frameshift mutation in the leptin gene and a pubertal bone age (12.5 years), a pulsatile pattern of LH secretion, indicative of a pubertal mode of GnRH pulse generator activity, was observed after 12 months of replacement with recombinant leptin to correct her morbid obesity (Farooqi et al., 1999). The bone age of this subject may be significant because it is generally recognized that, in normal children, bone age is a better predictor of gonadarche than chronological age (Plant et al., 1989b). Continued leptin replacement to this girl was associated with menarche at 12.1 years of age (bone age of 13.4 years), and 1.5 years later Tanner stage 3 was noted (Farooqi et al., 2002). Leptin replacement was also reported to facilitate menstruation in the women with primary or secondary amenorrhoea in association with congenital general lipodystrophy (Oral et al., 2002).
It seems reasonable to conclude from the foregoing discussion that a permissive action of leptin is required for gonadarche. The question now becomes whether leptin also serves as the signal that times the pubertal resurgence of pulsatile GnRH release and is therefore responsible for timing the termination of the delay of gonadarche. For this notion to be valid, two conditions must be satisfied: (i) an elevation in plasma leptin must precede the pubertal resurgence of pulsatile GnRH, and (ii) a premature elevation of circulating leptin would elicit precocious GnRH release. With regard to the first condition, cross-sectional and longitudinal studies of both boys and girls indicate in general that circulating leptin concentrations increase during pubertal development (Blum et al., 1997; Carlsson et al., 1997; Clayton et al., 1997; Garcia-Mayor et al., 1997; Ahmed et al., 1999; Ankarberg-Lindgren et al., 2001). Parenthetically, the pubertal increases in circulating leptin concentrations in man are inversely related to changes in levels of the soluble leptin receptor (Kratzsch et al., 2002; Mann et al., 2003), indicating that the rise in bioavailable leptin at this stage of development may be more robust than that of total immunoactive leptin. The binning of data with respect to Tanner stage, and the paucity of detailed sequential information on both leptin levels and LH pulsatility during transition from Tanner stage 1 to stage 2, however, does not allow the question of whether circulating leptin increases immediately prior to the initiation of gonadarche to be addressed. One exception to this situation is provided by the longitudinal study of eight boys by Mantzoros et al. (1997), who reported that elevations in circulating leptin concentrations occurred prior to nocturnal elevation in testosterone secretion, a robust marker of the onset of gonadarche in boys (Boyar et al., 1974; Judd et al., 1974). As previously noted (Mann and Plant, 2002), however, the leptin levels reported in the latter study were extremely variable, and the seemingly tantalizing increments of circulating levels of leptin were actually rather trivial in many of the subjects.
Studies of the relationship between circulating leptin levels and the onset of gonadarche in other species of higher primate have been restricted to the rhesus monkey, and only the male has been examined. In the male monkey, as in boys, an early marker of the initiation of gonadarche is a nocturnal elevation of testosterone secretion (Plant, 1985). In contrast to boys, however, the longitudinal relationship between the onset of nocturnal testosterone secretion and plasma leptin levels is unremarkable (Figure 2; Plant and Durrant, 1997). Results from cross-sectional studies of the monkey (Urbanski and Pau, 1998; Mann et al., 2000) are also consistent with the view that gonadarche in the male is triggered in the absence of a rise in circulating leptin.
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With regard to the second condition that needs to be met if leptin is to be considered as a candidate for the trigger of gonadarche, data are available for the monkey. In this species, a continuous intravenous infusion of recombinant human leptin to agonadal juvenile males, equivalent in age to 4–5 year old boys, did not trigger precocious activation of GnRH pulse generator activity (Figure 3; Barker-Gibb et al., 2002b
). Also of interest in this regard is the report that moment-to-moment patterns of LH secretion, and FSH and testosterone concentrations, remained prepubertal in a 5 year old leptin-deficient boy during 12 months of replacement treatment with recombinant human leptin. Replacement was calculated to achieve circulating leptin levels that were not >150% of those in normal children of comparable age and gender (Farooqi et al., 2002).
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Two other disorders of pubertal development in man that provide potential paradigms relevant to the role of leptin in timing gonadarche merit comment. First, circulating leptin concentrations were unchanged at the time delayed gonadarche was initiated in boys with CD (Gill et al., 1999
). Second, while circulating leptin levels have been reported in both boys and girls with GnRH-dependent precocious gonadarche (Palmert et al., 1998; Heger et al., 1999), understandably measurements of the hormone prior to manifestations of the pathophysiological condition were not reported.
Taking the foregoing considerations together, the role of leptin in primate gonadarche may be summarized as follows. A threshold concentration of plasma leptin, which appears to be quite low, is needed for this developmental process to unfold (Mann and Plant, 2002): a notion that is consistent with the finding that, in underweight postpubertal women, regular menstruation was associated with leptin levels in the range of 1.9 ng/ml (Köpp et al., 1997). Although this action of the hormone may be viewed as obligatory for gonadarche to unfold, leptin does not provide the signal that times the resurgence of pulsatile GnRH release and, therefore, the cue that terminates the prepubertal state. Rather, leptin allows for the expression of this critical hypothalamic event, which is triggered by a mechanism that remains elusive. This permissive action of leptin on hypothalamic GnRH release at puberty is also necessary for maintenance of gonadal function throughout postpubertal development. Leptin also appears to subserve a similar role in rodents (Cheung et al., 1997, 2001).
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Genetics and gonadarche |
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TOPAbstractIntroductionRole of hypothalamic GnRHA non-gonadal control system...The nature of the...Genomics and the neurobiological...Mechanisms timing the delay...Genetics and gonadarcheConclusionsAcknowledgementsReferences |
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The recognition that genetics play a role in the timing of gonadarche has been recently highlighted (Palmert and Boepple, 2001
). In general, studies that have described age of menarche in monozygotic and dizygotic twins report a greater concordancy in the timing of this developmental index in monozygotic twins than in dizygotic twins (Gedda and Brenci, 1975; Fischbein 1977a,b; Sharma, 1983; Treloar and Martin, 1990; Kaprio et al., 1995). Other indicators of the progression of gonadarche, such as peak height velocity and plasma LH concentrations have also been reported to exhibit greater concordancy in monozygotic twins (Wasada et al., 1978; Sharma, 1983). The impact of genetics in the timing of gonadarche is further indicated by the findings of a high degree of heritability in the age of menarche (Gedda and Brenci, 1975; Fischbein 1977a). Age of menarche also varies with racial/ethnic group, although when evaluating epidemiological data it should be noted that study designs have not been uniform, most have been retrospective and few have evaluated different ethnic populations within the same geographical location and socio-economic niche. Notwithstanding, studies of different ethnic groups living in the USA have revealed that black girls have a significantly earlier age of menarche than their white counterparts (Herman-Giddens et al., 1997; Biro et al., 2001; Sun et al., 2002; Chumlea et al., 2003). Similarly, girls in London of Afro-Caribbean and Indo-Pakistani parentage have earlier age of menarche than white girls of British parentage (Ulijaszek et al., 1991). It should be noted that when a genetic influence is established it remains unclear whether the genotype is directly determining the timing of the application and duration of the neurobiological brake on prepubertal GnRH release (and therefore dictating the delay of gonadarche), or simply governing permissive factors that are required for the resurgence of pulsatile GnRH release to be manifest once the brake has been withdrawn.
Mutations of several genes involved in differentiation and development of the hypothalamic–pituitary–gonadal axis have been reported (Kalantaridou and Chrousos, 2002), and, not surprisingly, such mutations have profound effects on pubertal development. For example, mutations of the KAL-1 gene, which encodes anosmin, an extracellular glycoprotein that appears to be involved in the migration of GnRH neurons from the olfactory bulb to the hypothalamus during fetal development (Hardelin, 2001), may lead to hypogonadotrophic hypogonadism and, therefore, to the absence of gonadarche. Such mutations, however, although providing great insight into the early development of the reproductive axis, contribute little to our understanding of the control system that times the pubertal resurgence of pulsatile GnRH release. Quantal progress on this front is to be anticipated with the identification, should they exist, of specific ‘puberty genes’, i.e. those that directly dictate, during prepubertal development, the timing of the application and removal of the neurobiological brake restraining the activity of the hypothalamic GnRH pulse generator. To date, however, attempts to identify ‘puberty genes’ by screening for mutations in patients with GnRH-dependent precocious gonadarche have not been realized (Barker-Gibb et al., 2002a; Freitas et al., 2003).
Although similar analyses of patients with delayed gonadarche due to CD have not been reported to date, genotyping of individuals with this ‘normal’ variant of pubertal development may be rewarding. Indeed, a recent study of a relatively large number of subjects with CD indicated a genetic predisposition, as reflected by the finding that parents of children with CD recall a later age of menarche and the adolescent growth spurt than those of a control group of children (Sedlmeyer and Palmert, 2002; Sedlmeyer et al., 2002).
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Conclusions |
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TOPAbstractIntroductionRole of hypothalamic GnRHA non-gonadal control system...The nature of the...Genomics and the neurobiological...Mechanisms timing the delay...Genetics and gonadarcheConclusionsAcknowledgementsReferences |
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The fundamental process underlying puberty in higher primates including man is gonadarche, which is triggered by a resurgence in pulsatile GnRH release from the hypothalamus. The potential for robust pulsatile GnRH secretion is extant well before the initiation of puberty, and the mechanism that times gonadarche should therefore be viewed as one that governs the delay, rather than the onset, of this critical stage in the ontogeny of reproductive function. The developmental delay is initiated by a neurobiological brake imposed during infancy on the neuronal network responsible for generating a pulsatile mode of GnRH release. Withdrawal of the brake several years later allows gonadarche to unfold. Interestingly, the pubertal resurgence of pulsatile GnRH release appears to be largely emancipated from transcriptional control of GnRH-I, the gene encoding GnRH. Instead, the brake is comprised of upstream neuronal and perhaps glial signals that regulate release of GnRH synthesized by molecular machinery that, postnatally, does not appear to be subjected to marked developmental regulation. The nature of the upstream cues remains incompletely understood, but NPY and GABA have emerged as prime candidates from studies of the male and female rhesus monkey respectively. Complete resolution of this issue is likely to be confounded by the sex differences in the duration and intensity of the neurobiological brake, particularly if they are reflected in the underlying neurobiology. The pubertal resurgence of GnRH release is also associated with structural remodelling in the hypothalamus. As of yet, specific ‘puberty genes’, i.e. those that time the application of the neurobiological brake, have not been identified, but their existence is suggested because of the high degree of heritability in the age of gonadarche. Whether these genes are expressed in the central nervous system or in somatic systems is also an open question. Lastly, for a resurgence in pulsatile GnRH release to be manifest following removal of the neurobiological brake at the end of the prepubertal phase of development, several permissive factors must be in place. Leptin is one of these.
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Acknowledgements |
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TOPAbstractIntroductionRole of hypothalamic GnRHA non-gonadal control system...The nature of the...Genomics and the neurobiological...Mechanisms timing the delay...Genetics and gonadarcheConclusionsAcknowledgementsReferences |
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Work conducted in the author’s laboratory was supported by NIH grants HD 13254 and HD 08610.
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References |
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