Central Mechanism Controlling Pubertal Onset in Mammals: A Triggering Role of Kisspeptin

Pubertal onset is thought to be timed by an increase in pulsatile gonadotropin-releasing hormone (GnRH)/gonadotropin secretion in mammals. The underlying mechanism of pubertal onset in mammals is still an open question. Evidence accumulated in the last 15 years suggests that kisspeptin/neurokinin B/dynorphin A (KNDy) neurons in the hypothalamic arcuate nucleus play a key role in pubertal onset by triggering pulsatile GnRH/gonadotropin secretin in mammals. Specifically, KNDy neurons are now considered a part of GnRH pulse generator, in which neurokinin B facilitates and dynorphin A inhibits, the synchronized discharge of KNDy neurons in autocrine and/or paracrine manners. Kisspeptin serves as a potent secretagogue of GnRH secretion and thus its release is fundamental to pubertal increase in GnRH/gonadotropin secretion in mammals. Proposed mechanisms inhibiting Kiss1 (kisspeptin gene) expression during childhood to juvenile varies from species to species: we envisage that negative feedback action of estrogen plays a key role in the inhibition of Kiss1 expression in KNDy neurons in rodents and sheep, whereas estrogen-independent inhibition of kisspeptin secretion by γ-amino butyric acid or neuropeptide Y are suggested to be responsible for the pre-pubertal suppression of GnRH/gonadotropin secretion in primates. Taken together, the timing of pubertal onset is postulated to be controlled by upstream regulators for kisspeptin biosynthesis and secretion in mammals

Evolutionally Conserved Function of Kisspeptin Neuronal System Is Nonreproductive Regulation as Revealed by Nonmammalian Study

The kisspeptin neuronal system, which consists of a neuropeptide kisspeptin and its receptor Gpr54, is considered in mammals a key factor of reproductive regulation, the so-called hypothalamic–pituitary–gonadal (HPG) axis. However, in nonmammalian vertebrates, especially in teleosts, existence of kisspeptin regulation on the HPG axis is still controversial. In this study, we applied multidisciplinary techniques to a teleost fish, medaka, and examined possible kisspeptin regulation on the HPG axis. First, we generated knockout medaka for kisspeptin-related genes and found that they show normal fertility, gonadal maturation, and expression of gonadotropins. Moreover, the firing activity of GnRH1 neurons recorded by the patch clamp technique was not altered by kisspeptin application. Furthermore, in goldfish, in vivo kisspeptin administration did not show any positive effect on HPG axis regulation. However, as kisspeptin genes are completely conserved among vertebrates except birds, we surmised that kisspeptin should have some important nonreproductive functions in vertebrates. Therefore, to discover novel functions of kisspeptin, we generated a gpr54-1:enhanced green fluorescent protein (EGFP) transgenic medaka, whose gpr54-1–expressing cells are specifically labeled by EGFP. Analysis of neuronal projection of gpr54-1:EGFP–expressing neurons showed that these neurons in the ventrolateral preoptic area project to the pituitary and are probably involved in endocrine regulation other than gonadotropin release. Furthermore, combination of deep sequencing, histological, and electrophysiological analyses revealed various novel neural systems that are under control of kisspeptin neurons—that is, those expressing neuropeptide Yb, cholecystokinin, isotocin, vasotocin, and neuropeptide B. Thus, our new strategy to genetically label receptor-expressing neurons gives insights into various kisspeptin-dependent neuronal systems that may be conserved in vertebrates

Design and synthesis of fluorescent probes for GPR54.

Kisspeptins are neuropeptides that induce the secretion of gonadotropin-releasing hormone via the activation of the cognate receptor, G-protein coupled receptor 54 (GPR54). The kisspeptin-GPR54 axis is associated with the onset of puberty and the maintenance of the reproductive system. In this study, several fluorescent probes have been designed and synthesized for rat GPR54 through the modification of the N-terminus of rat kisspeptins to allow for the visualization of the expression and localization of kisspeptin receptor(s) in living cells and native tissues. The tetramethylrhodamine (TMR) and rhodamine green (RG)-labeled kisspeptins exhibited good binding and agonistic activities towards GPR54, and the results of the application studies demonstrated that these fluorescent probes could be used effectively for the detection of GPR54 receptors in flow cytometry and confocal microscopy experiments

Evolutionally Conserved Function of Kisspeptin Neuronal System Is Nonreproductive Regulation as Revealed by Nonmammalian Study

The kisspeptin neuronal system, which consists of a neuropeptide kisspeptin and its receptor Gpr54, is considered in mammals a key factor of reproductive regulation, the so-called hypothalamic–pituitary–gonadal (HPG) axis. However, in nonmammalian vertebrates, especially in teleosts, existence of kisspeptin regulation on the HPG axis is still controversial. In this study, we applied multidisciplinary techniques to a teleost fish, medaka, and examined possible kisspeptin regulation on the HPG axis. First, we generated knockout medaka for kisspeptin-related genes and found that they show normal fertility, gonadal maturation, and expression of gonadotropins. Moreover, the firing activity of GnRH1 neurons recorded by the patch clamp technique was not altered by kisspeptin application. Furthermore, in goldfish, in vivo kisspeptin administration did not show any positive effect on HPG axis regulation. However, as kisspeptin genes are completely conserved among vertebrates except birds, we surmised that kisspeptin should have some important nonreproductive functions in vertebrates. Therefore, to discover novel functions of kisspeptin, we generated a gpr54-1:enhanced green fluorescent protein (EGFP) transgenic medaka, whose gpr54-1–expressing cells are specifically labeled by EGFP. Analysis of neuronal projection of gpr54-1:EGFP–expressing neurons showed that these neurons in the ventrolateral preoptic area project to the pituitary and are probably involved in endocrine regulation other than gonadotropin release. Furthermore, combination of deep sequencing, histological, and electrophysiological analyses revealed various novel neural systems that are under control of kisspeptin neurons—that is, those expressing neuropeptide Yb, cholecystokinin, isotocin, vasotocin, and neuropeptide B. Thus, our new strategy to genetically label receptor-expressing neurons gives insights into various kisspeptin-dependent neuronal systems that may be conserved in vertebrates