Abstract
Reproduction is a resource intensive process by which animals propagate their genes, hence it must be coordinated carefully with the body’s availability of energy stores to produce viable offspring. The adipocyte-derived hormone leptin relays the peripheral metabolic status of the body to the gonadotropin-releasing hormone (GnRH) neurons to control the hypothalamic- pituitary-gonadal (HPG) axis. This communication is primarily achieved in the hypothalamus, via hormone-responsive neurons operating upstream of GnRH neurons, since GnRH cells do not express leptin receptors (LepR). Nitric oxide synthase-1 (nNOS) neurons co-expressing LepR are suggested to be a direct conduit whereby leptin signals are relayed to the HPG axis. Inhibition of nNOS activity has been shown to cause sporadic ovulation, blunted pre-ovulatory luteinising hormone (LH) levels, and consequent hypothalamic hypogonadism in female mice.
This project initially aimed to elucidate whether nNOS neurons are sufficient to mediate leptin’s permissive actions on reproductive function in vivo, in the absence of leptin signals from any other neurons. Cre-lox technology was adopted to produce transgenic mice that would express LepR in only the nNOS neurons, referred to as the LepR-rescue group. Age at puberty onset, estrous cyclicity, insulin tolerance, and body weights were analysed. Unexpectedly, no significant differences were found between the LepR-rescues and wildtype controls.This appeared to result from Cre-mediated germline excision of the target STOP-flox sequence, which led to global expression of the intact LepR.
Since prior gamma-aminobutyric acid (GABA) neuron-specific LepR knockout experiments suggested that leptin can act via the GABA-ergic neurons but not the glutamatergic neurons to signal GnRH release, this study mapped co-localisation of nNOS neurons that are both GABAergic and leptin-responsive, to raise functional significance for specific hypothalamic regions. Immunohistochemistry (IHC) of both nNOS and phosphorylated signal transducer and activator of transcription 3 (pSTAT3, a marker for leptin signalling) was performed on brain sections of GABA neuron reporter mice (Vgat-Cre X tdTomato). The percentage co-localisation of GABA and leptin-induced pSTAT3 in nNOS neurons was 13% in the arcuate nucleus, 3% in the dorsomedial hypothalamic nuclei and in the medial preoptic area. It is anatomically evident that nNOS neurons form a part of the neuroendocrine network in controlling puberty and fertility.