Kiss1 Knockout Research Articles

The testosterone-dependent and independent transcriptional networks in the hypothalamus of Gpr54 and Kiss1 knockout male mice are not fully equivalent

BackgroundHumans and mice with loss of function mutations in GPR54 (KISS1R) or kisspeptin do not progress through puberty, caused by a failure to release GnRH. The transcriptional networks regulated by these proteins in the hypothalamus have yet to be explored by genome-wide methods.ResultsWe show here, using 1 million exon mouse arrays (Exon 1.0 Affymetrix) and quantitative polymerase chain reaction (QPCR) validation to analyse microdissected hypothalamic tissue from Gpr54 and Kiss1 knockout mice, the extent of transcriptional regulation in the hypothalamus. The sensitivity to detect important transcript differences in microdissected RNA was confirmed by the observation of counter-regulation of Kiss1 expression in Gpr54 knockouts and confirmed by immunohistochemistry (IHC). Since Gpr54 and Kiss1 knockout animals are effectively pre-pubertal with low testosterone (T) levels, we also determined which of the validated transcripts were T-responsive and which varied according to genotype alone. We observed four types of transcriptional regulation (i) genotype only dependent regulation, (ii) T only dependent regulation, (iii) genotype and T-dependent regulation with interaction between these variables, (iv) genotype and T-dependent regulation with no interaction between these variables. The results implicate for the first time several transcription factors (e.g. Npas4, Esr2), proteases (Klk1b22), and the orphan 10-transmembrane transporter TMEM144 in the biology of GPR54/kisspeptin function in the hypothalamus. We show for the neuronal activity regulated transcription factor NPAS4, that distinct protein over-expression is seen in the hypothalamus and hippocampus in Gpr54 knockout mice. This links for the first time the hypothalamic-gonadal axis with this important regulator of inhibitory synapse formation. Similarly we confirm TMEM144 up-regulation in the hypothalamus by RNA in situ hybridization and western blot.ConclusionsTaken together, global transcriptional profiling shows that loss of GPR54 and kisspeptin are not fully equivalent in the mouse hypothalamus.

Antagonizing Kisspeptins: Physiological Lessons and Pharmacological Challenges

As in many other disciplines of biological sciences, different areas of modern neuroendocrinology have witnessed asynchronous progress during the last years, the field of neurohormonal control of energy homeostasis and body weight probably being the most active in the last decade. In contrast, despite being a very dynamic area in terms of basic and clinical research, the fundamentals of the neuroendocrine control of reproduction seemed to have been well settled by the turn of the century once the nature and mechanisms of action of key regulators of GnRH neurons, ranging from glutamate to nitric oxide and leptin, as well as signals from glial origin, had been (at least partially) deciphered (1). This stable scenario, however, was substantially challenged by the disclosure in late 2003 of the putative reproductive role of kisspeptins, a family of peptides encoded by the KISS1 gene, which were originally defined as metastasis suppressor factors (2, 3). The identification of mutations of the putative kisspeptin receptor,Gprotein-coupled receptor (GPR)54, inhumans with hypogonadotropic hypogonadism unveiled a novel facet of this signaling system and opened up a new research avenue aiming to define the physiological roles of kisspeptins in the control of puberty and fertility. Six years later, the study of kisspeptins has become an active topic on several fronts, with an ever-growing number of publications on different aspects of kisspeptin function (2, 3). Taken together, the experimental studies generated so far strongly suggest that kisspeptin neurons in the forebrain are central for the dynamic regulation of key facets of reproductive maturation and function. The importance of these findings to translational medicine is reinforced by the fact that kisspeptins appear to be among the most potent stimulators of the gonadotropic axis (4), with readily detected effects, even at low doses, in a diversity of species, including humans (5, 6). Despite the universal recognition of the paramount importance of kisspeptins and GPR54 in the neuroendocrine control of reproduction, it is necessary to acknowledge that most of the experimental evidence to date derives from indirect approaches in which expression analyses (mainly at the mRNA level) and/or functional (e.g. hormonal) responses to pharmacological challenges with exogenous kisspeptins have been used to infer the biological roles and physiological relevance of this ligand-receptor system (2). Indeed, these approaches are enormously informative and fully valid and have helped to rapidly decipher important aspects of the biology of kisspeptins. Yet they are also limitations in terms of functional translation. Admittedly, more direct approaches involving functional genomics have also been used to interrogate the kisspeptin system (7–12). But the data published so far come from knockout (KO) mouse models, in which expression of Kiss1 or Gpr54 genes have been permanently deleted, with the potential for compensatory changes or developmental alterations that may impact the observed results. In fact, whereas the reproductive phenotypes of the different Gpr54 and Kiss1 KO mice are qualitatively similar, there are also important differences regarding their severity that suggest caution in interpretation of these results. In this issue of Endocrinology, Pineda et al. (13) provide the first comprehensive validation of protocols of continuous infusion of a peptide analog capable of antagonizing the biological actions of kisspeptins in vivo. Importantly, by central administration of a kisspeptin antagonist, rel-

Disruption of the Single Copy Gonadotropin-Releasing Hormone Receptor in Mice by Gene Trap: Severe Reduction of Reproductive Organs and Functions in Developing and Adult Mice

Mutations in the GnRH receptor gene (GNRHR) can result in hypogonadotropic hypogonadism in humans. Unlike most mammals, mice lack a second form of GnRH (GnRH2) and a type 2 GnRH receptor. To determine whether the GnRH receptor is critical at all stages of reproduction and whether this receptor has additional physiological functions in developing and adult mice, we have generated mice from an embryonic stem cell line containing a retroviral vector with multiple stop codons inserted into intron 1 of the Gnrhr gene. This gene trap insertion resulted in the disruption of exon 2 and exon 3 of the Gnrhr gene. The insertion also contained a lacZ gene that was used as a reporter for GnRH receptor expression in these mice. This model has a similar phenotype to the clinical syndrome of hypogonadotropic hypogonadism. Null Gnrhr mice had small sexual organs, low levels of FSH, LH, and steroid hormones, failure of sexual maturation, infertility, and inability to respond to exogenous GnRH. However, the defective GnRH receptor did not prevent morula/blastocyst development, implantation, masculinization of fetal male mice, or maintenance of early pregnancy. The phenotype of this null Gnrhr mouse was more severe than models in the literature, including the N-ethyl-N-nitrosourea-induced Gnrhr mutant, the kisspeptin (Kiss1) knockout, and the kisspeptin receptor (Gpr54) knockout. In terms of gonadal morphology, adult gene trap-Gnrhr null mice demonstrate a complete cessation of reproduction and serve as an important model for understanding GnRH/GnRHR physiology.

Distribution of Kisspeptin Neurones in the Adult Female Mouse Brain

Kisspeptin-GPR54 signalling is essential for normal reproductive functioning. However, the distribution of kisspeptin neuronal cell bodies and their projections is not well established. The present study aimed to provide a detailed account of kisspeptin neuroanatomy in the mouse brain. Using a polyclonal rabbit antibody AC566, directed towards the final ten C-terminal amino acids of murine kisspeptin, three populations of kisspeptin-expressing cell bodies were identified in the adult female mouse brain. One exists as a dense periventricular continuum of cells within the rostral part of the third ventricle, another is found within the arcuate nucleus, and another is identified as a low-density group of scattered cells within the dorsomedial nucleus and posterior hypothalamus. Kisspeptin-immunoreactive fibres were abundant within the ventral aspect of the lateral septum and within the hypothalamus running in periventricular and ventral retrochiasmatic pathways. Notable exclusions from the kisspeptin fibre innervation were the suprachiasmatic and ventromedial nuclei. Outside of the hypothalamus, a small number of kisspeptin fibres were identified in the bed nucleus of the stria terminalis, subfornical organ, medial amygdala, paraventricular thalamus, periaqueductal grey and locus coerulus. All kisspeptin cell body and fibre immunoreactivity was absent in brain tissue from Kiss1 knockout mice. These observations provide a map of kisspeptin neurones in the mouse brain and indicate that a limited number of mostly medial hypothalamic and lateral septal brain regions are innervated by the three hypothalamic kisspeptin cell populations; the functions of these projections remain to be established.

Kiss1 −/− Mice Exhibit More Variable Hypogonadism than Gpr54−/− Mice

The G protein-coupled receptor Gpr54 and its ligand metastin (derived from the Kiss1 gene product kisspeptin) are key gatekeepers of sexual maturation. Gpr54 knockout mice demonstrate hypogonadotropic hypogonadism, but until recently, the phenotype of Kiss1 knockout mice was unknown. This report describes the reproductive phenotypes of mice carrying targeted deletions of Kiss1 or Gpr54 on the same genetic background. Both Kiss1 and Gpr54 knockout mice are viable but infertile and have abnormal sexual maturation; the majority of males lack preputial separation, and females have delayed vaginal opening and absence of estrous cycling. Kiss1 and Gpr54 knockout males have significantly smaller testes compared with controls. Gpr54 knockout females have smaller ovaries and uteri than wild-type females. However, Kiss1 knockout females demonstrate two distinct phenotypes: half have markedly reduced gonadal weights similar to those of Gpr54 knockout mice, whereas half exhibit persistent vaginal cornification and have gonadal weights comparable with those of wild-type females. FSH levels in both Kiss1 and Gpr54 knockout males and females are significantly lower than in controls. When injected with mouse metastin 43-52, a Gpr54 agonist, Gpr54 knockout mice fail to increase gonadotropins, whereas Kiss1 knockout mice respond with increased gonadotropin levels. In summary, both Kiss1 and Gpr54 knockout mice have abnormal sexual maturation consistent with hypogonadotropic hypogonadism, although Kiss1 knockout mice appear to be less severely affected than their receptor counterparts. Kiss1 knockout females demonstrate a bimodal phenotypic variability, with some animals having higher gonadal weight, larger vaginal opening, and persistent vaginal cornification.