Brain endothelial cells control fertility through ovarian-steroid-dependent release of semaphorin 3A.

Neuropilin-1 (Nrp1) guides the development of the nervous and vascular systems, but its role in the mature brain remains to be explored. Here we report that the expression of the 65 kDa isoform of Sema3A, the ligand of Nrp1, by adult vascular endothelial cells, is regulated during the ovarian cycle and promotes axonal sprouting in hypothalamic neurons secreting gonadotropin-releasing hormone (GnRH), the neuropeptide controlling reproduction. Both the inhibition of Sema3A/Nrp1 signaling and the conditional deletion of Nrp1 in GnRH neurons counteract Sema3A-induced axonal sprouting. Furthermore, the localized intracerebral infusion of Nrp1- or Sema3A-neutralizing antibodies in vivo disrupts the ovarian cycle. Finally, the selective neutralization of endothelial-cell Sema3A signaling in adult Sema3aloxP/loxP mice by the intravenous injection of the recombinant TAT-Cre protein alters the amplitude of the preovulatory luteinizing hormone surge, likely by perturbing GnRH release into the hypothalamo-hypophyseal portal system. Our results identify a previously unknown function for 65 kDa Sema3A-Nrp1 signaling in the induction of axonal growth, and raise the possibility that endothelial cells actively participate in synaptic plasticity in specific functional domains of the adult central nervous system, thus controlling key physiological functions such as reproduction.

SEMA3A, a gene involved in axonal pathfinding, is mutated in patients with Kallmann syndrome.

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.

Function-related structural plasticity of the GnRH system: a role for neuronal-glial-endothelial interactions.

As the final common pathway for the central control of gonadotropin secretion, GnRH neurons are subjected to numerous regulatory homeostatic and external factors to achieve levels of fertility appropriate to the organism. The GnRH system thus provides an excellent model in which to investigate the complex relationships between neurosecretion, morphological plasticity and the expression of a physiological function. Throughout the reproductive cycle beginning from postnatal sexual development and the onset of puberty to reproductive senescence, and even within the ovarian cycle itself, all levels of the GnRH system undergo morphological plasticity. This structural plasticity within the GnRH system appears crucial to the timely control of reproductive competence within the individual, and as such must have coordinated actions of multiple signals secreted from glial cells, endothelial cells, and GnRH neurons. Thus, the GnRH system must be viewed as a complete neuro-glial-vascular unit that works in concert to maintain the reproductive axis.

Nitric oxide as key mediator of neuron-to-neuron and endothelia-to-glia communication involved in the neuroendocrine control of reproduction.

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.

Estradiol induces physical association of neuronal nitric oxide synthase with NMDA receptor and promotes nitric oxide formation via estrogen receptor activation in primary neuronal cultures.

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.

Coupling of neuronal nitric oxide synthase to NMDA receptors via postsynaptic density-95 depends on estrogen and contributes to the central control of adult female reproduction.

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.

Phenotypic and molecular characterization of proliferating and differentiated GnRH-expressing GnV-3 cells.

GnRH neurons provide the primary driving force upon the neuroendocrine reproductive axis. Here we used GnV-3 cells, a model of conditionally immortalized GnRH-expressing neurons, to perform an analysis of cell cycle and compare the gene expression profile of proliferating cells with differentiated cells. In the proliferation medium, 45 ± 1.5% of GnV-3 cells are in S-phase by FACS analysis. In the differentiation medium, only 9 ± 0.9% of them are in S-phase, and they acquire the characteristic bipolar shape displayed by preoptic GnRH neurons in vivo. In addition, GnV-3 cells in the differentiated state exhibit electrophysiological properties characteristic of neurons. Transcriptomic analysis identified up-regulation of 1931 genes and down-regulation of 1270 genes in cells grown in the differentiation medium compared to cells in the proliferation medium. Subsequent gene ontology study indicated that genes over-expressed in proliferating GnV-3 cells were mainly involved in cell cycle regulations, whereas genes over-expressed in differentiated cells were mainly involved in processes of differentiation, neurogenesis and neuronal morphogenesis. Taken together, these data demonstrate the occurrence of morphological and physiological changes in GnV-3 cells between the proliferating and the differentiated state. Moreover, the genes differentially regulated between these two different states are providing novel pathways potentially important for a better understanding of the physiology of mature GnRH neurons.

Phosphorylation of N-methyl-D-aspartic acid receptor-associated neuronal nitric oxide synthase depends on estrogens and modulates hypothalamic nitric oxide production during the ovarian cycle.

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.