Introduction
The switch that turns on pubertal development remains one of the most elusive triggers in science. One successful approach to uncovering pubertal mechanisms has been the study of the genetic causes of delayed puberty and hypogonadotropic hypogonadism. The discovery of kisspeptin and its receptor, which are critical stimulatory factors for GnRH release, brings us one step closer to understanding the events that initiate puberty. Three experts present their perspectives on kisspeptin.
Clinical Practitioner Perspective
By Ana Claudia Latronico, M.D., Ph.D
Dr. Latronico is professor of medicine and chief of the Endocrinology and Metabolism Division, University of São Paulo Medical School, Brazil.
In a family of eight children, five siblings failed to enter puberty or showed only partial signs of puberty in their late teens. In another case, an eight-year-old girl presented with central precocious puberty without neurologic symptoms or exposure to sex steroids. As described in more detail below, each of these individuals was found to have mutations in the kisspeptin receptor/ligand system that affected the activation of puberty.
Kisspeptin-1 and its cognate receptor are required for normal function of the hypothalamic-pituitary-gonadal axis and play an important role in the physiologic regulation of puberty. Evidence indicates that kisspeptin acts directly on hypothalamic GnRH neurons, stimulating GnRH secretion and consequently gonadodotropin release from the anterior pituitary.
Defects in the kisspeptin system cause hypogonadotropic hypogonadism
The failure of gonadal function secondary to deficient pituitary gonadotropin secretion results in a clinical syndrome defined as isolated hypogonadotropic hypogonadism. The biological hallmark of this disorder is a decreased level of sex steroids associated with low or normal levels of FSH and LH without other pituitary hormonal deficiencies. Loss-of-function mutations in the KISS1R gene were first described in consanguineous families with isolated hypogonadotropic hypogonadism without olfaction abnormalities.2,3 Since then, other inactivating mutations have been described in KISS1R in patients with sporadic and familial normosmic isolated hypogonadotropic hypogonadism.4,5 To date, the frequency of mutations in KISS1R as a cause of this autosomal recessive disorder is relatively low. Further, the types of mutations have been unique, including point mutations, partial deletions and insertions. Very recently, a homozygous inactivating mutation in KISS1 gene was also identified in a large consanguineous family. This mutation results in failure of pubertal progression, confirming that kisspeptin signaling is a critical element in the human hypothalamic-pituitary-gonadal axis.
Kisspeptin system in central precocious puberty
Precocious puberty is characterized by the development of secondary sexual characteristics before the age of eight years in girls and nine years in boys. The premature activation of GnRH secretion leading to central precocious puberty has remarkable female gender predominance and more than 90 percent of the affected girls have the idiopathic form. The relevance of the kisspeptin system in human puberty onset was highlighted by the identification of activating mutations of the KISS1R and KISS1 genes in children with central precocious puberty.7-9 A unique, heterozygous, nonconstitutively activating mutation of the KISS1R gene was associated with a central precocious puberty phenotype. In vitro studies demonstrated that this mutation leads to sustained activation of intracellular signaling pathways in response to kisspeptin.
Kisspeptins as potential therapeutic tools
Central or peripheral administration of the kisspeptin peptides has been demonstrated to stimulate gonadotropin release in several mammalian species, including humans.10 Kisspeptin peptides stimulate gonadotropin release in men, and in women during the preovulatory phase of the menstrual cycle, but fail to stimulate gonadotropin release in women during the follicular phase.10 The sexually dimorphic responses of healthy men and women to kisspeptin administration have important clinical implications for the potential of kisspeptin to treat disorders of reproduction such as infertility. Taken together, potential agonists and antagonists of kisspeptin may have potential for pharmacological intervention in human reproductive diseases and facilitate understanding of the physiological roles of the kisspeptin system.
Clinical Researcher Perspective
By Stephanie Seminara, M.D.
Dr. Seminara is a doctor of reproductive endocrinology at Massachusetts General Hospital, in Boston.
The hypothalamic hormone GnRH has traditionally been touted as the central driver of pituitary gonadotropin secretion, controlling pulsatile gonadotropin secretion, modulating gonadal steroid feedback, and bringing about full fertility in the adult. Understanding GnRH neuronal regulation is essential to understanding the neurohumoral control of human reproduction. In 2003, two groups discovered that in both the human and the mouse, mutations in GPR54 (encoding the kisspeptin receptor, also known as KISS1R) cause hypogonadotropic hypogonadism. This can be corrected by the administration of exogenous GnRH.1,2 Soon thereafter, several groups began assembling genetic, expression, physiologic, transgenic, knockdown, and electrophysiologic data to characterize the physiology of kisspeptin and its seminal role in modulating GnRH release. Kisspeptin was soon discovered to be a powerful stimulus for GnRHinduced LH secretion, and displaced GnRH from its premier position at the top of the reproductive cascade. Kisspeptin is now recognized as critical in regulating the timing of sexual maturation, gonadotropin secretion by gonadal hormones in adults, and the control of fertility by metabolic and environmental (e.g., photoperiod) cues.
Although the physiologic story of the kisspeptin system began to grow very quickly, the human genetics story took longer to evolve. In 2008, an activating mutation in KISS1R was identified in a girl with central precocious puberty.4 Not only did this observation reinforce the notion that kisspeptin is an important gatekeeper of pubertal function in the human, but it was also the first identification of lossand gain-of-function mutations critical to the hypothalamic control of GnRH leading to opposite reproductive phenotypes.
Identifying mutations in KISS1R, KISS1
Throughout this period, investigators struggled to identify large cohorts of patients carrying kisspeptin pathway mutations. Perhaps because of the importance of the kisspeptin pathway in modulating GnRH secretion or its other biologic roles in placental development and metastasis suppression, the total number of cases of individuals bearing mutations in KISS1R remains relatively small. Moreover, despite the obvious candidacy of the kisspeptin gene itself, the identification of mutations in KISS1 eluded investigators for eight years. However, in 2011, heterozygote mutations in KISS1 were reported in patients with GnRH deficiency, validated by in vitro and animal studies.5 This report was followed by the identification of a homozygote mutation in KISS1 in 2012.
Kisspeptin is now understood to be co-expressed with neurokinin B (NKB) and dynorphin, giving rise to the term KNDy neurons (Kisspeptin-Neurokinin B-Dynorphin). NKB is a member of the substance P-related tachykinin family and its receptor is expressed both on KNDy and GnRH neurons. Dynorphin is an opioid that participates in progesterone-mediated negative feedback control of GnRH release. Loss-of-function mutations in the genes encoding neurokinin B (TAC3) and its receptor (TAC3R) result in hypogonadotrophic hypogonadism and pubertal failure. Although mutations in the dynorphin or its receptor have not yet been reported, the presence of mutations in the other two signaling pathways in GnRH-deficient states demonstrates the importance of this neuropeptide ensemble in the hypothalamic control of reproduction.
These genetic discoveries have come back to the bedside. Exogenous kisspeptin has now been administered to healthy men and women, and patients with reproductive disorders, including those with mutations in the neurokinin B signaling pathway.8-14 Its power as a physiological probe lies in its ability to teach us about the fundamental secretory properties of GnRH neurons in vivo, and studies to date have revealed fascinating insights into GnRH neuronal responsiveness in different sex steroid milieu and GnRH pulse generation.
Basic Researcher Perspective
By Manuel Tena-Sempere, M.D., Ph.D.
Dr. Tena-Sempere is professor of physiology at the University of Cordoba and senior researcher at the Instituto Maimónides de Investigación Biomédicas de Córdoba (IMIBIC) and the National Biomedical Research Center in Obesity and Nutrition (CIBERobn), Cordoba, Spain.
Mechanistic insight: Kisspeptins and the control of GnRH neurons
The source of brain kisspeptins has been actively investigated. In rodents, two prominent hypothalamic populations of Kiss1 neurons exist: one in the arcuate nucleus (ARC) and another in the rostral periventricular area of the third ventricle. In other mammals, including humans, Kiss1 neurons are present in the ARC/infundibular region, but the existence of a population equivalent to the rodent RP3V has yet to be confirmed.1,2 ARC and RP3V Kiss1 neurons are functionally dissimilar: they respond differently to several key regulators, express different sets of co-transmitters, and seem to be differently wired to GnRH neurons.
Kisspeptin projections to GnRH neuronal cell bodies in rodents seem to originate mainly from RP3V, although projections from some ARC Kiss1 neurons may also exist.3 Studies in monkeys have shown kisspeptin fibers in close proximity to GnRH terminals in the median eminence, suggesting a regulatory role on GnRH release. The above evidence is compatible with additional, indirect kisspeptin effects on GnRH neurons1,2, but other evidence points to direct actions at the pituitary level.
Kiss1 neurons as gatekeepers of puberty and ovulation
The absence of puberty in humans and mice with genetic inactivation of Gpr54 or Kiss11,5 fueled mechanistic studies on how kisspeptins participate in the control of mammalian puberty. These analyses documented the complex pattern of activation of Kiss1 neurons, which seems to be essential for the normal timing of puberty. This phenomenon includes not only the increase of kisspeptin tone, but also of the sensitivity to the stimulatory effects of kisspeptins. In addition, there is a rise in the number of kisspeptin neurons and their projections to GnRH neurons during puberty.6 Blockade of Gpr54 or timed ablation of Kiss1 neurons at the infantile-juvenile transition has been shown to disturb puberty onset.7,8 Experimental manipulations known to alter brain sex differentiation have been shown to perturb the development of the hypothalamic Kiss1 system.
Compelling experimental evidence suggests a crucial role of kisspeptins in the generation of the pre-ovulatory surge of gonadotropins, which triggers ovulation. Indeed, Kiss1 neurons in the RP3V are activated in rodents during the pre-ovulatory period, whereas blockade of kisspeptin action abrogates the ovulatory LH surge.7,9 Experimental evidence suggests that the pre-ovulatory rise of estrogen is responsible for the activation of Kiss1 neurons in the RP3V before ovulation, as a major mechanism for its positive feedback effects.
Kiss1 neurons as conduits for sex steroid and metabolic regulation of GnRH neurons
Kiss1 neurons are sensitive to changes in sex steroid milieu and are involved in conveying the negative feedback of sex steroids in the tonic control of gonadotropin secretion.9 ARC Kiss1 neurons, which express ER and androgen receptors, increase Kiss1 expression after sex steroid withdrawal and decrease Kiss1 expression following gonadal steroid replacement. The functional relevance of such changes is illustrated by the finding that the normal gonadotropin increases that occur upon removal of sex steroids are lost in the absence of kisspeptin signaling.
Metabolic cues also appear to use Kiss1 neurons as a conduit for their well-known influence on puberty onset and fertility.10 For example, metabolic stress linked to reproductive failure, including not only persistent negative energy balance but also obesity, blunt the hypothalamic (mainly ARC) expression of Kiss1/kisspeptins, whereas kisspeptin replacement can overcome the gonadotropic defects in some of those conditions.1,10 Leptin was initially suggested to operate as direct metabolic regulator of Kiss1 neurons. Yet this model has been recently challenged by expression and functional genomic data suggesting that leptin actions on Kiss1 neurons might be preferentially indirect.
Kiss1 neurons may also funnel additional regulatory signals onto GnRH neurons, including ageing, stress, and environmental cues. This reinforces the view that Kiss1 neurons are upper-order sensors of a wide array of reproductive regulators, which drive GnRH secretion as result of their capacity to integrate diverse regulatory signals.
Future directions in kisspeptin research
Considerable effort is being invested in elucidating the signals that interact with and/or modulate kisspeptin signaling in the brain. Although interest has focused on kisspeptin-NKB interactions12, many other central and peripheral transmitters are also under investigation.13 Similarly, recent data strongly suggest that, in addition to transcriptional mechanisms, epigenetic and post-transcriptional regulation of Kiss1 pathways is likely to play important roles.1,14 Experimental efforts along these lines will allow an integrated understanding of how kisspeptins operate to control puberty and fertility; a basic knowledge that may enable the refinement of current strategies for clinical care and management of reproductive disorders.
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