The regulation of reproductive function is dependent on the proper coordination between the three axes of reproduction: the hypothalamus, anterior pituitary, and the gonads [hypothalamic-pituitary-gonadal (HPG) axis]. Gonadotropin-releasing hormone (GnRH) is a key regulator of this axis and thus of reproductive function and behavior. GnRH has 23 known isoforms, which are present in both mammalian and non-mammalian species. The N- and C-terminal sequences of GnRH, pGlu-His-Trp-Ser, and Pro-Gly NH 2 respectively, have been highly conserved throughout evolution reinforcing the essential role GnRH plays in reproduction.
Neurons that synthesize GnRH are widely distributed in the basal forebrain and project to the median eminence where GnRH is released in a pulsatile manner into the hypophyseal portal vessels. Secreted GnRH stimulates the synthesis and secretion of the gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), in the anterior pituitary. These gonadotropins subsequently exert their effects at the gonadal level to regulate the secretion of steroid hormones. In the female, LH and FSH act on the ovary to facilitate follicular maturation and to regulate the secretion of estrogen and progesterone, which act as feedback modulators of the hypothalamic-pituitary axis. Estrogen has been shown to both stimulate and inhibit GnRH release depending on the stage of the estrus cycle. Furthermore, GnRH can regulate its own synthesis and secretion via an autocrine mechanism, which can be influenced by other neuropeptides. Despite significant research clarifying the HPG axis, the integration of signals required for normal function of the GnRH neuroendocrine axis is not completely understood.
In our previous work, we have shown that the metabolite of GnRH, GnRH-(1-5), is biologically active and may serve as another regulatory factor of the HPG axis. It has been established that GnRH neurons originate outside of the central nervous system (CNS) and migrate to its final position in the basal forebrain. Along this migratory pathway, GnRH is cleaved by the zinc metalloendopeptidase EC 3.4.24.15 (abbreviated EP24.15), to generate GnRH-(1-5). Although neuropeptidases have been thought to degrade proteins to cease further peptide action, GnRH-(1-5) has been shown to facilitate reproductive behavior and to increase GnRH mRNA levels. More recently, GnRH-(1-5) was demonstrated to inhibit the migration of GN11 cells, a GnRH secreting cell line, by binding the G protein-coupled receptor (GPCR), GPR173. These biologically active roles of GnRH-(1-5) may implicate the peptide as a regulator of the HPG axis rather than simply a metabolic byproduct.
Initial studies by Schwanzel-Fukuda demonstrated that GnRH neurons originate outside the CNS in the nasal region. Subsequent studies validated these observations showing that GnRH neurons migrate from the nasal placode traversing the cribriform plate to target the basal forebrain. In the developing mouse, GnRH immunoreactivity is detected as early as embryonic day (ED) 10.5 in the vomeronasal region. By ED 15, most GnRH neurons have already migrated along vomeronasal tract to the prepoptic area. A subpopulation of GnRH positive cells are localized in the tectum near the mesencephalic vesicle peaking at ED 15; however, they cease to exist in the adult and their function has not been elucidated. The primary GnRH neuronal population in adulthood is estimated to be near 800 cells distributed throughout the basal forebrain. Figure 1A shows the GnRH neuronal migratory route during different developmental stages in the mouse. At approximately ED 15.5, most GnRH neurons have crossed the cribriform plate into the basal forebrain. Interestingly, GnRH positive cells near the nasal region migrate radially, which is in contrast to their tangential migratory pattern once these neurons are in the CNS (Figure 1B). The proper migration of GnRH neurons during development requires the integration of multiple guidance cues in part mediated through the activation of certain GPCRs including prokineticin receptor-2 (PROKR2), and CXCR4. Additionally, the migratory rate of GnRH neurons seems to be another layer of regulation as there is a brief delay in migration as they begin to traverse the cribriform plate to enter the CNS. This delay may serve to aid in the transition between the nasal region and the CNS to properly respond to a changing microenvironment and migratory trajectory. A more comprehensive overview of the development of the GnRH neuroendocrine system can be found in the reviews in and.
(A) GnRH neurons are born in the nasal placode near the vomeronasal organ (VMO) at approximately embryonic day (ED) 11.5 in the mouse. As development progresses GnRH neurons begin to enter the CNS (dashed lines) at ED12.5 and begin to descend within the basal forebrain region at ED15.5. (B) Most GnRH neurons are within the CNS at ED15.5 however the remaining GnRH neurons in the nasal septum (NS) have a morphologically distinct migratory pattern relative to neurons found near the telencephalon (tel). Arrow indicates descending GnRH neurons of a mouse ED15.5 brain (Wu, TJ unpublished data). LTV, left telencephalic vesicle; OB, olfactory bulb; OE, olfactory epithelium; PIT, pituitary; MV, mesencephalic vesicle; T, tectum.
Defects in the HPG axis lead to the development of various types of hypogonadotropic hypogonadism (HH) where the onset of puberty and fertility are impaired. There are a number of genes implicated in the pathogenesis of HH, including mutations in the genes encoding the GnRH peptide and its receptor. Mutations associated with these genes are generally classified as normosmic HH (nHH) where GnRH signaling is impaired yet olfactory senses remain intact. Other forms of HH exist where patients suffer from reduced olfaction in addition to deficiencies in pubertal development and fertility. This form of HH also called Kallmann Syndrome (KS) is attributed to a defect in GnRH neuronal migration. Specifically, precursor olfaction and GnRH neurons emanating from the olfactory placode remain arrested in the cribriform plate during development, never entering the CNS to reach their target sites. Patients diagnosed with nHH or KS have reduced levels of circulating gonadotrophins, LH, and FSH, which would normally stimulate the secretion of steroid hormones from the testes or ovaries. In turn, these patients suffer from delayed pubertal onset and reduced gonadal function among other pathologies.
Recent studies focusing on the molecular basis for KS implicate genes believed to aid in both the origination and migration of GnRH neurons during development such as FGFR1, FGF8, and KAL1. Disruption of FGFR1 signaling was demonstrated to lead to the development of KS. FGFR1 and its ligand are required for the proper birth and maintenance of GnRH neurons during embryogenesis; knockdown of both genes results in the absence of GnRH neurons. In addition to the complete absence of GnRH neurons, disruption of neuronal migration has also been identified as a cause of KS. Observation of a Kallmann’s fetal brain revealed GnRH and olfactory neurons resided in the nasal cavity and failed to cross the cribriform plate to enter the CNS. This improper migration of GnRH neurons was attributed to the deletion of the KAL1 gene located on the X chromosome at Xp22.3. More recently, in vitro studies indicate the product of KAL1 acts as a chemoattractant for immortalized premigratory GnRH neurons by associating with cell surface heparin sulfate proteoglycans. Interestingly, many signaling cues required for normal GnRH neuronal migration require the activation of GPCRs including PROKR2, which has been linked to the development of KS. Mutations localized in the intracellular loop 1 of PROKR2 have been identified in patients with KS and is attributed to aberrant receptor membrane localization and receptor activity. However, complete identification of all factors contributing to the proper migration of GnRH neurons is still limited since the majority of KS cases have yet to be linked to a genetic defect.
Patients with nHH harboring mutations in genes encoding GnRH or its receptor GnRHR do not have obvious anatomical aberrations of the hypothalamus that would suggest a defect in neuronal migration; yet they continue to suffer from delayed pubertal development. Recently, a frameshift mutation in the GnRH gene was identified in teenage siblings who had intact olfaction but suffered from underdeveloped gonadal function. GnRH treatment in one of the siblings increased circulating LH levels, indicating that the GnRHR was responsive to exogenous GnRH administration. These findings are recapitulated in studies using the hypogonadal (HPG) mouse where the GnRH gene is no longer expressed; however still retain the ability to respond to exogenous GnRH. Furthermore, HPG mice receiving preoptic transplantations containing GnRH neurons induced episodic LH sec
