Kallmann syndrome (KS) associates congenital hypogonadism due to gonadotropin-releasing hormone (GnRH) deficiency and anosmia. The genetics of KS involves various modes of transmission, including oligogenic inheritance. Here, we report that Nrp1 sema/sema mutant mice that lack a functional semaphorin-binding domain in neuropilin-1, an obligatory coreceptor of semaphorin-3A, have a KS–like phenotype. Pathohistological analysis of these mice indeed showed abnormal development of the peripheral olfactory system and defective embryonic migration of the neuroendocrine GnRH cells to the basal forebrain, which results in increased mortality of newborn mice and reduced fertility in adults. We thus screened 386 KS patients for the presence of mutations in SEMA3A (by Sanger sequencing of all 17 coding exons and flanking splice sites) and identified nonsynonymous mutations in 24 patients, specifically, a frameshifting small deletion (D538fsX31) and seven different missense mutations (R66W, N153S, I400V, V435I, T688A, R730Q, R733H). All the mutations were found in heterozygous state. Seven mutations resulted in impaired secretion of semaphorin-3A by transfected COS-7 cells (D538fsX31, R66W, V435I) or reduced signaling activity of the secreted protein in the GN11 cell line derived from embryonic GnRH cells (N153S, I400V, T688A, R733H), which strongly suggests that these mutations have a pathogenic effect. Notably, mutations in other KS genes had already been identified, in heterozygous state, in five of these patients. Our findings indicate that semaphorin-3A signaling insufficiency contributes to the pathogenesis of KS and further substantiate the oligogenic pattern of inheritance in this developmental disorder.

Although coordinated actions of several areas within the hypothalamus are involved in the secretion of gonadotrophin‐releasing hormone (GnRH), the median eminence of the hypothalamus, where the nerve terminals are located, plays a particularly critical role in the release of GnRH. In adult females, prior to the preovulatory surge of GnRH, the retraction of specialised ependymoglial cells lining the floor of the third ventricle named tanycytes allows for the juxtaposition of GnRH nerve terminals with the adjacent pericapillary space of the pituitary portal vasculature, thus forming direct neurohaemal junctions. These morphological changes occur within a few hours and are reversible. Such remodelling may promote physiological conditions to enhance the central release of GnRH and potentiate oestrogen‐activated GnRH release. This plasticity involves dynamic cell interactions that bring into play tanycytes, astrocytes, vascular endothelial cells and GnRH neurones themselves. The underlying signalling pathways responsible for these structural changes are comprised of highly diffusible gaseous molecules, such as endothelial nitric oxide, and paracrine communication processes involving receptors of the erbB tyrosine kinase family, transforming growth factor beta 1 and eicosanoids, such as prostagladin E2. Some of these molecules, as a result of their ability to diffuse within the median eminence, may also serve as synchronising cues allowing for the occurrence of functionally meaningful episodes of GnRH secretion by coordinating GnRH release from the GnRH neuroendocrine terminals.

Endothelial-cell–derived Sema3A promotes axonal outgrowth and plasticity and thereby regulates neurohormone release in the adult rodent brain in response to the ovarian cycle.

Considerable research has been devoted to the understanding of how nitric oxide (NO) influences brain function. Few studies, however, have addressed how its production is physiologically regulated. Here, we report that protein-protein interactions between neuronal NO synthase (nNOS) and glutamate NMDA receptors via the scaffolding protein postsynaptic density-95 (PSD-95) in the hypothalamic preoptic region of adult female rats is sensitive to cyclic estrogen fluctuation. Coimmunoprecipitation experiments were used to assess the physical association between nNOS and NMDA receptor NR2B subunit in the preoptic region of the hypothalamus. We found that nNOS strongly interacts with NR2B at the onset of the preovulatory surge at proestrus (when estrogen levels are highest) compared with basal-stage diestrous rats. Consistently, estrogen treatment of gonadectomized female rats also increases nNOS/NR2B complex formation. Moreover, endogenous fluctuations in estrogen levels during the estrous cycle coincide with changes in the physical association of nNOS to PSD-95 and the magnitude of NO release in the preoptic region. Finally, temporary and local in vivo suppression of PSD-95 synthesis by using antisense oligodeoxynucleotides leads to inhibition of nNOS activity in the preoptic region and disrupted estrous cyclicity, a process requiring coordinated activation of neurons containing gonadotropin-releasing hormone (the neuropeptide controlling reproductive function). In conclusion, our findings identify a novel steroid-mediated molecular mechanism that enables the adult mammalian brain to control NO release under physiological conditions.

Nitric oxide (NO) is a peculiar chemical transmitter that freely diffuses through aqueous and lipid environments and plays a role in major aspects of brain function. Within the hypothalamus, NO exerts critical effects upon the gonadotropin-releasing hormone (GnRH) network to maintain fertility. Here, we review recent evidence that NO regulates major aspects of the GnRH neuron physiology. Far more active than once thought, NO powerfully controls GnRH neuronal activity, GnRH release and structural plasticity at the neurohemal junction. In the preoptic region, neuronal nitric oxide synthase (nNOS) activity is tightly regulated by estrogens and is found to be maximal at the proestrus stage. Natural fluctuations of estrogens control both the differential coupling of this Ca²⁺-activated enzyme to glutamate N-methyl-D-aspartic acid receptor channels and phosphorylation-mediated nNOS activation. Furthermore, NO endogenously produced by neurons expressing nNOS acutely and directly suppresses spontaneous firing in GnRH neurons, which suggests that neuronal NO may serve as a synchronizing switch within the preoptic region. At the median eminence, NO is spontaneously released from an endothelial source and follows a pulsatile and cyclic pattern of secretion. Importantly, GnRH release appears to be causally related to endothelial NO release. NO is also highly involved in mediating the dialogue set in motion between vascular endothelial cells and tanycytes that control the direct access of GnRH neurons to the pituitary portal blood during the estrous cycle. Altogether, these data raise the intriguing possibility that the neuroendocrine brain uses NO to coordinate both GnRH neuronal activity and GnRH release at key stages of reproductive physiology.

Within the preoptic region, nitric oxide (NO) production varies during the ovarian cycle and has the ability to impact hypothalamic reproductive function. One mechanism for the regulation of NO release mediated by estrogens during the estrous cycle includes physical association of the calcium-activated neuronal NO synthase (nNOS) enzyme with the glutamate N-methyl-d-aspartate (NMDA) receptor channels via the postsynaptic density 95 scaffolding protein. Here we demonstrate that endogenous variations in estrogens levels during the estrous cycle also coincide with corresponding changes in the state of nNOS Ser1412 phosphorylation, the level of association of this isoform with the NMDA receptor/postsynaptic density 95 complex at the plasma membrane, and the activity of NO synthase (NOS). Neuronal NOS Ser1412 phosphorylation is maximal on the afternoon of proestrus when both the levels of estrogens and the physical association of nNOS with NMDA receptors are highest. Estradiol mimicked these effects in ovariectomized (OVX) rats. In addition, the catalytic activity of NOS in membrane protein extracts from the preoptic region, i.e. independent of any functional protein-protein interactions or cell-cell signaling, was significantly increased in estradiol-treated OVX rats compared with OVX rats. Finally, lambda phosphatase-mediated nNOS dephosphorylation dramatically impaired NOS activity in preoptic region protein extracts, thus demonstrating the important role of phosphorylation in the regulation of NO production in the preoptic region. Taken together, these results yield new insights into the regulation of neuron-derived NO production by gonadal steroids within the preoptic region and raise the possibility that changes in nNOS phosphorylation during fluctuating physiological conditions may be involved in the hypothalamic control of key neuroendocrine functions, such as reproduction.

No abstract

Estrogens and nitric oxide (NO) exert wide‐ranging effects on brain function. Recent evidence suggested that one important mechanism for the regulation of NO production may reside in the differential coupling of the calcium‐activated neuronal NO synthase (nNOS) to glutamate NMDA receptor channels harboring NR2B subunits by the scaffolding protein post‐synaptic density‐95 (PSD‐95), and that estrogens promote the formation of this ternary complex. Here, we demonstrate that 30‐min estradiol‐treatment triggers the production of NO by physically and functionally coupling NMDA receptors to nNOS in primary neurons of the rat preoptic region in vitro. The ability of estradiol to activate neuronal NO signaling in preoptic neurons and to promote changes in protein‐protein interactions is blocked by ICI 182,780, an estrogen receptor antagonist. In addition, blockade of NMDA receptor NR2B subunit activity with ifenprodil or disruption of PSD‐95 synthesis in preoptic neurons by treatment with an anti‐sense oligodeoxynucleotide inhibited the estradiol‐promoted stimulation of NO release in cultured preoptic neurons. Thus, estrogen receptor‐mediated stimulation of the nNOS/PSD‐95/NMDA receptor complex assembly is likely to be a critical component of the signaling process by which estradiol facilitates coupling of glutamatergic fluxes for NO production in neurons.