GnRH Cells Research Articles

The relationship between gonadotropin releasing hormone and ovulation inducing factor/nerve growth factor receptors in the hypothalamus of the llama

BackgroundA molecule identical to nerve growth factor, with ovulation-inducing properties has been discovered in the seminal plasma of South American camelids (ovulation-inducing factor/nerve growth factor; OIF/NGF). We hypothesize that the ovulatory effect of OIF/NGF is initiated at the level of the hypothalamus, presumably by GnRH neurons. The objective of the present study was to determine the structural relationship between GnRH neurons and neurons expressing high- and low-affinity receptors for NGF (i.e., TrkA and p75, respectively) in the hypothalamus.MethodsMature llamas (n = 4) were euthanized and their hypothalamic tissue was fixed, sectioned, and processed for immunohistochemistry on free-floating sections. Ten equidistant sections per brain were double stained for immunofluorescence detection of TrkA and GnRH, or p75 and GnRH.ResultsCells immunoreactive to TrkA were detected in most hypothalamic areas, but the majority of cells were detected in the diagonal band of Broca (part of the ventral forebrain) and the supraoptic nuclei and periventricular area. The number of cells immunoreactive to p75 was highest in the diagonal band of Broca and lateral preoptic areas and least in more caudal areas of the hypothalamus (p < 0.05) in a pattern similar to that of TrkA. A low proportion of GnRH neurons were immunoreactive to TrkA (2.5% of total GnRH cells), and no co-localization between GnRH and p75 was detected. GnRH neuron fibers were detected only occasionally in proximity to TrkA immunopositive neurons.ConclusionsResults do not support the hypothesis that the effect of OIF/NGF is driven by a direct interaction with GnRH neurons, but rather provide rationale for the hypothesis that interneurons exist in the hypothalamus that mediate OIF/NGF-induced ovulation.

Evidence That Dynorphin Acts Upon KNDy and GnRH Neurons During GnRH Pulse Termination in the Ewe.

A subpopulation of neurons located within the arcuate nucleus, colocalizing kisspeptin, neurokinin B, and dynorphin (Dyn; termed KNDy neurons), represents key mediators of pulsatile GnRH secretion. The KNDy model of GnRH pulse generation proposes that Dyn terminates each pulse. However, it is unknown where and when during a pulse that Dyn is released to inhibit GnRH secretion. Dyn acts via the κ opioid receptor (KOR), and KOR is present in KNDy and GnRH neurons in sheep. KOR, similar to other G protein-coupled receptors, are internalized after exposure to ligand, and thus internalization can be used as a marker of endogenous Dyn release. Thus, we hypothesized that KOR will be internalized at pulse termination in both KNDy and GnRH neurons. To test this hypothesis, GnRH pulses were induced in gonad-intact anestrous ewes by injection of neurokinin B (NKB) into the third ventricle and animals were euthanized at times of either pulse onset or termination. NKB injections produced increased internalization of KOR within KNDy neurons during both pulse onset and termination. In contrast, KOR internalization into GnRH neurons was seen only during pulse termination, and only in GnRH neurons within the mediobasal hypothalamus (MBH). Overall, our results indicate that Dyn is released onto KNDy cells at the time of pulse onset, and continues to be released during the duration of the pulse. In contrast, Dyn is released onto MBH GnRH neurons only at pulse termination and thus actions of Dyn upon KNDy and GnRH cell bodies may be critical for pulse termination.

Knockout of the Gnrh genes in zebrafish: effects on reproduction and potential compensation by reproductive and feeding-related neuropeptides.

Gonadotropin-releasing hormone (GNRH) is known as a pivotal upstream regulator of reproduction in vertebrates. However, reproduction is not compromised in the hypophysiotropic Gnrh3 knockout line in zebrafish (gnrh3-/-). In order to determine if Gnrh2, the only other Gnrh isoform in zebrafish brains, is compensating for the loss of Gnrh3, we generated a double Gnrh knockout zebrafish line. Surprisingly, the loss of both Gnrh isoforms resulted in no major impact on reproduction, indicating that a compensatory response, outside of the Gnrh system, was evoked. A plethora of factors acting along the reproductive hypothalamus-pituitary axis were evaluated as possible compensators based on neuroanatomical and differential gene expression studies. In addition, we also examined the involvement of feeding factors in the brain as potential compensators for Gnrh2, which has known anorexigenic effects. We found that the double knockout fish exhibited upregulation of several genes in the brain, specifically gonadotropin-inhibitory hormone (gnih), secretogranin 2 (scg2), tachykinin 3a (tac3a), and pituitary adenylate cyclase-activating peptide 1 (pacap1), and downregulation of agouti-related peptide 1 (agrp1), indicating the compensation occurs outside of Gnrh cells and therefore is a noncell autonomous response to the loss of Gnrh. While the differential expression of gnih and agrp1 in the double knockout line was confined to the periventricular nucleus and hypothalamus, respectively, the upregulation of scg2 corresponded with a broader neuronal redistribution in the lateral hypothalamus and hindbrain. In conclusion, our results demonstrate the existence of a redundant reproductive regulatory system that comes into play when Gnrh2 and Gnrh3 are lost.

Effects of Selective Deletion of Tyrosine Hydroxylase from Kisspeptin Cells on Puberty and Reproduction in Male and Female Mice.

The neuropeptide kisspeptin, encoded by Kiss1, regulates reproduction by stimulating GnRH secretion. Kiss1-syntheizing neurons reside primarily in the hypothalamic anteroventral periventricular (AVPV/PeN) and arcuate (ARC) nuclei. AVPV/PeN Kiss1 neurons are sexually dimorphic, with females expressing more Kiss1 than males, and participate in estradiol (E2)-induced positive feedback control of GnRH secretion. In mice, most AVPV/PeN Kiss1 cells coexpress tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis (in this case, dopamine). Dopamine treatment can inhibit GnRH neurons, but the function of dopamine signaling arising specifically from AVPV/PeN Kiss1 cells is unknown. We generated a novel TH flox mouse and used Cre-Lox technology to selectively ablate TH specifically from Kiss1 cells. We then examined the effects of selective TH knock-out on puberty and reproduction in both sexes. In control mice, 90% of AVPV/PeN Kiss1 neurons coexpressed TH, whereas in mice lacking TH exclusively in Kiss1 cells (termed Kiss THKOs), TH was successfully absent from virtually all Kiss1 cells. Despite this absence of TH, both female and male Kiss THKOs displayed normal body weights, puberty onset, and basal gonadotropin levels in adulthood, although testosterone (T) was significantly elevated in adult male Kiss THKOs. The E2-induced LH surge was unaffected in Kiss THKO females, and neuronal activation status of kisspeptin and GnRH cells was also normal. Supporting this, fertility and fecundity were normal in Kiss THKOs of both sexes. Thus, despite high colocalization of TH and Kiss1 in the AVPV/PeN, dopamine produced in these cells is not required for puberty or reproduction, and its function remains unknown.

Stress Increases Gonadotropin Inhibitory Hormone Cell Activity and Input to GnRH Cells in Ewes.

Stress reduces GnRH and gonadotropin secretion in sheep, but the central mechanism for this suppressive effect is unknown. Gonadotropin-inhibitory hormone (GnIH) negatively regulates GnRH neurons and gonadotropes. Here, we measured activity of GnIH neurons and contact of GnIH fibers on GnRH neurons during either chronic "pseudostress" or acute stress in sheep. We also measured GnIH secretion into hypophysial portal blood during pseudostress and acute stress. The pseudostress was daily im injections (0.5 mg) of Synacthen Depot (adrenocorticotropin) or vehicle for 4 weeks, which increased the GnIH cell number and gene expression/cell in the hypothalamus, measured by in situ hybridization. Double label immunohistochemistry showed that Synacthen Depot treatment increased the percentage of GnRH cells in close contact with GnIH fibers but did not alter GnIH levels in hypophysial portal blood. Acute stress protocols were either sequential audiovisual predator stress, followed by insulin-induced hypoglycemia, or a single challenge with lipopolysaccharide (iv). Both of these acute stressors activated a c-Fos response in GnIH cells and increased the contacts of GnIH fibers to GnRH cells. Neither acute stress protocol increased GnIH secretion into hypophysial portal blood. These data show that chronic pseudostress and acute stressors increase the function of GnIH cells as well as the degree to which GnIH cells may provide input to GnRH cells. Thus, GnIH cells may provide a central mechanism whereby stress compromises reproduction. Neither chronic pseudostress nor acute stress elevates secretion of GnIH into portal blood, but stress effects mediated by GnIH cells are directed towards GnRH cell bodies.

The Methylcytosine Dioxygenase Ten-Eleven Translocase-2 (tet2) Enables Elevated GnRH Gene Expression and Maintenance of Male Reproductive Function.

Reproduction depends on the establishment and maintenance of elevated GnRH neurosecretion. The elevation of primate GnRH release is accompanied by epigenetic changes. Specifically, cytosine residues within the GnRH gene promoter are actively demethylated, whereas GnRH mRNA levels and peptide release rise. Whether active DNA demethylation has an impact on GnRH neuron development and consequently reproductive function remains unknown. In this study, we investigated whether ten-eleven translocation (tet) enzymes, which initiate the process of active DNA demethylation, influence neuronal function and reproduction. We found that tet2 expression increases with age in the developing mouse preoptic area-hypothalamus and is substantially higher in a mature (GT1-7) than an immature (GN11) GnRH cell line. GnRH mRNA levels and mean GnRH peptide release elevated after overexpression of tet2 in GN11 cells, whereas CRISPR/cas9-mediated knockdown of tet2 in GT1-7 cells led to a significant decline in GnRH expression. Manipulations of tet2 expression altered tet2 genome binding and histone 3 lysine 4 trimethylation abundance at the GnRH promoter. Mice with selective disruption of tet2 in GnRH neurons (GnRH-specific tet2 knockout mice) exhibited no sign of altered pubertal timing in either sex, although plasma LH levels were significantly lower, and fecundity was altered specifically in adult male GnRH-specific tet2 knockout animals, indicating that tet2 may participate in the maintenance GnRH neuronal function. Exposure to bisphenol A, an environmental contaminant that alters GnRH neuron activity, caused a shift in tet2 subcellular localization and a decrease in histone 3 lysine 4 trimethylation abundance at the GnRH promoter. Finally, evaluation of tet2 protein interactions in GT1-7 cells suggests that the influence of tet2 on neuronal function are not limited to nuclear mechanisms but could depend on mitochondrial function, and RNA metabolism. Together, these studies implicate tet2 in the maintenance of GnRH neuronal function and neuroendocrine control of male reproduction.

Galanin Activates G Protein Gated Inwardly Rectifying Potassium Channels and Suppresses Kisspeptin-10 Activation of GnRH Neurons.

GnRH neurons are regulated by hypothalamic kisspeptin neurons. Recently, galanin was identified in a subpopulation of kisspeptin neurons. Although the literature thoroughly describes kisspeptin activation of GnRH neurons, little is known about the effects of galanin on GnRH neurons. This study investigated whether galanin could alter kisspeptin signaling to GnRH neurons. GnRH cells maintained in explants, known to display spontaneous calcium oscillations, and a long-lasting calcium response to kisspeptin-10 (kp-10), were used. First, transcripts for galanin receptors (GalRs) were examined. Only GalR1 was found in GnRH neurons. A series of experiments was then performed to determine the action of galanin on kp-10 activated GnRH neurons. Applied after kp-10 activation, galanin 1-16 (Gal1-16) rapidly suppressed kp-10 activation. Applied with kp-10, Gal1-16 prevented kp-10 activation until its removal. To determine the mechanism by which galanin inhibited kp-10 activation of GnRH neurons, Gal1-16 and galanin were applied to spontaneously active GnRH neurons. Both inhibited GnRH neuronal activity, independent of GnRH neuronal inputs. This inhibition was mimicked by a GalR1 agonist but not by GalR2 or GalR2/3 agonists. Although Gal1-16 inhibition relied on Gi/o signaling, it was independent of cAMP levels but sensitive to blockers of G protein-coupled inwardly rectifying potassium channels. A newly developed bioassay for GnRH detection showed Gal1-16 decreased the kp-10-evoked GnRH secretion below detection threshold. Together, this study shows that galanin is a potent regulator of GnRH neurons, possibly acting as a physiological break to kisspeptin excitation.

Phoenixin Activates Immortalized GnRH and Kisspeptin Neurons Through the Novel Receptor GPR173.

Reproductive function is coordinated by kisspeptin (Kiss) and GnRH neurons. Phoenixin-20 amide (PNX) is a recently described peptide found to increase GnRH-stimulated LH secretion in the pituitary. However, the effects of PNX in the hypothalamus, the putative signaling pathways, and PNX receptor have yet to be identified. The mHypoA-GnRH/GFP and mHypoA-Kiss/GFP-3 cell lines represent populations of GnRH and Kiss neurons, respectively. PNX increased GnRH and GnRH receptor (GnRH-R) mRNA expression, as well as GnRH secretion, in the mHypoA-GnRH/GFP cell model. In the mHypoA-Kiss/GFP-3 cell line, PNX increased Kiss1 mRNA expression. CCAAT/enhancer-binding protein (C/EBP)-β, octamer transcription factor-1 (Oct-1), and cAMP response element binding protein (CREB) binding sites are localized to the 5' flanking regions of the GnRH, GnRH-R, and Kiss1 genes. PNX decreased C/EBP-β mRNA expression in both cell models and increased Oct-1 mRNA expression in the mHypoA-GnRH/GFP neurons. PNX increased CREB phosphorylation in both cell models and phospho-ERK1/2 in the mHypoA-GnRH/GFP cell model, whereas inhibiting the cAMP/protein kinase A pathway prevented PNX induction of GnRH and Kiss1 mRNA expression. Importantly, we determined that the G protein-coupled receptor, GPR173, was strongly expressed in both GnRH and kisspeptin cell models and small interfering RNA knockdown of GPR173 prevented the PNX-mediated up-regulation of GnRH, GnRH-R, and Kiss1 mRNA expression and the down-regulation of C/EBP-β mRNA expression. PNX also increased GPR173 mRNA expression in the mHypoA-GnRH/GFP cells. Taken together, these studies are the first to implicate that PNX acts through GPR173 to activate the cAMP/protein kinase A pathway through CREB, and potentially C/EBP-β and/or Oct-1 to increase GnRH, GnRH-R, and Kiss1 gene expression, ultimately having a stimulatory effect on reproductive function.

κ-Opioid Receptor Is Colocalized in GnRH and KNDy Cells in the Female Ovine and Rat Brain.

Kisspeptin-neurokinin B-dynorphin (KNDy) cells of the hypothalamus are a key component in the neuroendocrine regulation of GnRH secretion. Evidence in sheep and other species suggests that dynorphin released by KNDy cells inhibits pulsatile GnRH secretion by acting upon κ-opioid receptors (KOR). However, the precise anatomical location and neurochemical phenotype of KOR-expressing cells in sheep remain unknown. To this end, we determined the distribution of KOR mRNA and protein in the brains of luteal phase ewes, using an ovine specific KOR mRNA probe for in situ hybridization and an antibody whose specificity we confirmed by Western blot analyses and blocking peptide controls. KOR cells were observed in a number of regions, including the preoptic area (POA); anterior hypothalamic area; supraoptic and paraventricular nuclei; ventromedial, dorsomedial, and lateral hypothalamus; and arcuate nucleus. Next, we determined whether KOR is colocalized in KNDy and/or GnRH cells. Dual-label immunofluorescence and confocal analysis of the KNDy population showed a high degree of colocalization, with greater than 90% of these neurons containing KOR. Surprisingly, GnRH cells also showed high levels of colocalization in sheep, ranging from 74.4% to 95.4% for GnRH cells in the POA and medial basal hypothalamus, respectively. Similarly, 97.4% of GnRH neurons in the POA of ovariectomized, steroid-primed female rats also contained immunoreactive KOR protein. These findings suggest that the inhibitory effects of dynorphin on pulsatile GnRH secretion may occur either indirectly by actions upon KOR within the KNDy population and/or directly via the activation of KOR on GnRH cells.

Direct Actions of Kisspeptins on GnRH Neurons Permit Attainment of Fertility but are Insufficient to Fully Preserve Gonadotropic Axis Activity

Kisspeptins, ligands of the receptor, Gpr54, are potent stimulators of puberty and fertility. Yet, whether direct kisspeptin actions on GnRH neurons are sufficient for the whole repertoire of their reproductive effects remains debatable. To dissect out direct vs. indirect effects of kisspeptins on GnRH neurons in vivo, we report herein the detailed reproductive/gonadotropic characterization of a Gpr54 null mouse line with selective re-introduction of Gpr54 expression only in GnRH cells (Gpr54−/−Tg; rescued). Despite preserved fertility, adult rescued mice displayed abnormalities in gonadal microstructure, with signs of precocious ageing in females and elevated LH levels with normal-to-low testosterone secretion in males. Gpr54−/−Tg rescued mice showed also altered gonadotropin responses to negative feedback withdrawal, while luteinizing hormone responses to various gonadotropic regulators were variably affected, with partially blunted relative (but not absolute) responses to kisspeptin-10, NMDA and the agonist of tachykinin receptors, NK2R. Our data confirm that direct effects of kisspeptins on GnRH cells are sufficient to attain fertility. Yet, such direct actions appear to be insufficient to completely preserve proper functionality of gonadotropic axis, suggesting a role of kisspeptin signaling outside GnRH cells.

Prenatal Testosterone Treatment Leads to Changes in the Morphology of KNDy Neurons, Their Inputs, and Projections to GnRH Cells in Female Sheep.

Prenatal testosterone (T)-treated ewes display a constellation of reproductive defects that closely mirror those seen in PCOS women, including altered hormonal feedback control of GnRH. Kisspeptin/neurokinin B/dynorphin (KNDy) neurons of the arcuate nucleus (ARC) play a key role in steroid feedback control of GnRH secretion, and prenatal T treatment in sheep causes an imbalance of KNDy peptide expression within the ARC. In the present study, we tested the hypothesis that prenatal T exposure, in addition to altering KNDy peptides, leads to changes in the morphology and synaptic inputs of this population, kisspeptin cells of the preoptic area (POA), and GnRH cells. Prenatal T treatment significantly increased the size of KNDy cell somas, whereas POA kisspeptin, GnRH, agouti-related peptide, and proopiomelanocortin neurons were each unchanged in size. Prenatal T treatment also significantly reduced the total number of synaptic inputs onto KNDy neurons and POA kisspeptin neurons; for KNDy neurons, the decrease was partly due to a decrease in KNDy-KNDy synapses, whereas KNDy inputs to POA kisspeptin cells were unaltered. Finally, prenatal T reduced the total number of inputs to GnRH cells in both the POA and medial basal hypothalamus, and this change was in part due to a decreased number of inputs from KNDy neurons. The hypertrophy of KNDy cells in prenatal T sheep resembles that seen in ARC kisspeptin cells of postmenopausal women, and together with changes in their synaptic inputs and projections to GnRH neurons, may contribute to defects in steroidal control of GnRH observed in this animal model.

Point mutations in KAL1 and the mitochondrial gene MT-tRNA(cys) synergize to produce Kallmann syndrome phenotype.

Kallmann syndrome (KS) is an inherited developmental disorder defined as the association of hypogonadotropic hypogonadism and anosmia or hyposmia. KS has been shown to be a genetically heterogeneous disease with different modes of inheritance. However, variants in any of the causative genes identified so far are only found in approximately one third of KS patients, thus indicating that other genes or pathways remain to be discovered. Here, we report a large Han Chinese family with inherited KS which harbors two novel variants, KAL1 c.146G>T (p.Cys49Phe) and mitochondrial tRNAcys (m.5800A>G). Although two variants can’t exert obvious effects on the migration of GnRH neurons, they show the synergistic effect, which can account for the occurrence of the disorder in this family. Furthermore, the disturbance of the mitochondrial cysteinyl-tRNA pathway can significantly affect the migration of GnRH cells in vitro and in vivo by influencing the chemomigration function of anosmin-1. Our work highlights a new mode of inheritance underlay the genetic etiology of KS and provide valuable clues to understand the disease development.

Zebrafish adult-derived hypothalamic neurospheres generate gonadotropin-releasing hormone (GnRH) neurons.

ABSTRACTGonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide essential for fertility in vertebrates. Human male patients lacking GnRH and treated with hormone therapy can remain fertile after cessation of treatment suggesting that new GnRH neurons can be generated during adult life. We used zebrafish to investigate the neurogenic potential of the adult hypothalamus. Previously we have characterized the development of GnRH cells in the zebrafish linking genetic pathways to the differentiation of neuromodulatory and endocrine GnRH cells in specific regions of the brain. Here, we developed a new method to obtain neural progenitors from the adult hypothalamus in vitro. Using this system, we show that neurospheres derived from the adult hypothalamus can be maintained in culture and subsequently differentiate glia and neurons. Importantly, the adult derived progenitors differentiate into neurons containing GnRH and the number of cells is increased through exposure to either testosterone or GnRH, hormones used in therapeutic treatment in humans. Finally, we show in vivo that a neurogenic niche in the hypothalamus contains GnRH positive neurons. Thus, we demonstrated for the first time that neurospheres can be derived from the hypothalamus of the adult zebrafish and that these neural progenitors are capable of producing GnRH containing neurons.

Gonadotropin and kisspeptin gene expression, but not GnRH, are impaired in cFOS deficient mice

cFOS is a pleiotropic transcription factor, which binds to the AP1 site in the promoter of target genes. In the pituitary gonadotropes, cFOS mediates induction of FSHβ and GnRH receptor genes. Herein, we analyzed reproductive function in the cFOS-deficient mice to determine its role in vivo. In the pituitary cFOS is necessary for gonadotropin subunit expression, while TSHβ is unaffected. Additionally, cFOS null animals have the same sex-steroid levels, although gametogenesis is impeded. In the brain, cFOS is not necessary for GnRH neuronal migration, axon targeting, cell number, or mRNA levels. Conversely, cFOS nulls, particularly females, have decreased Kiss1 neuron numbers and lower Kiss1 mRNA levels. Collectively, our novel findings suggest that cFOS plays a cell-specific role at multiple levels of the hypothalamic–pituitary–gonadal axis, affecting gonadotropes but not thyrotropes in the pituitary, and kisspeptin neurons but not GnRH neurons in the hypothalamus, thereby contributing to the overall control of reproduction.

Kisspeptin is a component of the pulse generator for GnRH secretion in female sheep but not the pulse generator.

We tested the hypothesis that kisspeptin cells constitute the "pulse generator" for GnRH secretion. In ewes, we determined whether iv administered kisspeptin elicits a secretory pulse of LH in anaesthetized, sex-steroid suppressed ovariectomized ewes. A response was seen in both anaesthetized and conscious animals, which was not associated with induction of c-Fos labeling in GnRH cells, supporting the notion that kisspeptin acts on the neurosecretory GnRH terminals. Response was lower in the anaesthetized animals, suggesting that some nonkisspeptin elements may be involved in GnRH responses. Microinjection of kisspeptin (100 nmol) into the median eminence of conscious ewes elicited a pulse of LH, indicating that kisspeptin acts at this level to cause GnRH secretion. To determine which cells are activated at the time of GnRH secretion, we blood sampled 18 ewes during the luteal phase of the estrous cycle and harvested brains after 3 hours. Three of these ewes displayed a pulse of LH within 30 minutes of euthanasia. An increase in c-Fos labeling was seen in kisspeptin and glutamate cells of the arcuate nucleus but not in GnRH neurons, preoptic kisspeptin neurons, or preoptic glutamate neurons. Immunohistochemistry in 4 hypothalami showed that 72% of arcuate kisspeptin cells receive glutamatergic input. These data support the concept that the kisspeptin cells of the arcuate nucleus drive pulsatile secretion of GnRH at the level of the median eminence, but this may involve "upstream" input from glutamate cells. We conclude that the pulse generator for GnRH secretion involves more than 1 element.

Social opportunity rapidly regulates expression of CRF and CRF receptors in the brain during social ascent of a teleost fish, Astatotilapia burtoni.

In social animals, hierarchical rank governs food availability, territorial rights and breeding access. Rank order can change rapidly and typically depends on dynamic aggressive interactions. Since the neuromodulator corticotrophin releasing factor (CRF) integrates internal and external cues to regulate the hypothalamic-pituitary adrenal (HPA) axis, we analyzed the CRF system during social encounters related to status. We used a particularly suitable animal model, African cichlid fish, Astatotilapia burtoni, whose social status regulates reproduction. When presented with an opportunity to rise in rank, subordinate A. burtoni males rapidly change coloration, behavior, and their physiology to support a new role as dominant, reproductively active fish. Although changes in gonadotropin-releasing hormone (GnRH1), the key reproductive molecular actor, have been analyzed during social ascent, little is known about the roles of CRF and the HPA axis during transitions. Experimentally enabling males to ascend in social rank, we measured changes in plasma cortisol and the CRF system in specific brain regions 15 minutes after onset of social ascent. Plasma cortisol levels in ascending fish were lower than subordinate conspecifics, but similar to levels in dominant animals. In the preoptic area (POA), where GnRH1 cells are located, and in the pituitary gland, CRF and CRF1 receptor mRNA levels are rapidly down regulated in ascending males compared to subordinates. In the Vc/Vl, a forebrain region where CRF cell bodies are located, mRNA coding for both CRFR1 and CRFR2 receptors is lower in ascending fish compared to stable subordinate conspecifics. The rapid time course of these changes (within minutes) suggests that the CRF system is involved in the physiological changes associated with shifts in social status. Since CRF typically has inhibitory effects on the neuroendocrine reproductive axis in vertebrates, this attenuation of CRF activity may allow rapid activation of the reproductive axis and facilitate the transition to dominance.

Vasoactive Intestinal Peptide Modulation of the Steroid-Induced LH Surge Involves Kisspeptin Signaling in Young but Not in Middle-Aged Female Rats

Age-related LH surge dysfunction in middle-aged rats is characterized, in part, by reduced responsiveness to estradiol (E2)-positive feedback and reduced hypothalamic kisspeptin neurotransmission. Vasoactive intestinal peptide (VIP) neurons in the suprachiasmatic nucleus project to hypothalamic regions that house kisspeptin neurons. Additionally, middle-age females express less VIP mRNA in the suprachiasmatic nucleus on the day of the LH surge and intracerebroventricular (icv) VIP infusion restores LH surges. We tested the hypothesis that icv infusion of VIP modulates the LH surge through effects on the kisspeptin and RFamide-related peptide-3 (RFRP-3; an estradiol-regulated inhibitor of GnRH neurons) neurotransmitter systems. Brains were collected for in situ hybridization analyses from ovariectomized and ovarian hormone-primed young and middle-aged females infused with VIP or saline. The percentage of GnRH and Kiss1 cells coexpressing cfos and total Kiss1 mRNA were reduced in saline-infused middle-aged compared with young females. In young females, VIP reduced the percentage of GnRH and Kiss1 cells coexpressing cfos, suggesting that increased VIP signaling in young females adversely affected the function of Kiss1 and GnRH neurons. In middle-aged females, VIP increased the percentage of GnRH but not Kiss1 neurons coexpressing cfos, suggesting VIP affects LH release in middle-aged females through kisspeptin-independent effects on GnRH neurons. Neither reproductive age nor VIP affected Rfrp cell number, Rfrp mRNA levels per cell, or coexpression of cfos in Rfrp cells. These data suggest that VIP differentially affects activation of GnRH and kisspeptin neurons of female rats in an age-dependent manner.

Metabolic Influences on Reproduction: Adiponectin Attenuates GnRH Neuronal Activity in Female Mice

Metabolic dysfunctions are often linked to reproductive abnormalities. Adiponectin (ADP), a peripheral hormone secreted by white adipose tissue, is important in energy homeostasis and appetite regulation. GnRH neurons are integral components of the reproductive axis, controlling synthesis, and release of gonadotropins. This report examined whether ADP can directly act on GnRH neurons. Double-label immunofluorescence on brain sections from adult female revealed that a subpopulation of GnRH neurons express ADP receptor (AdipoR)2. GnRH/AdipoR2+ cells were distributed throughout the forebrain. To determine the influence of ADP on GnRH neuronal activity and the signal transduction pathway of AdipoR2, GnRH neurons maintained in explants were assayed using whole-cell patch clamping and calcium imaging. This mouse model system circumvents the dispersed distribution of GnRH neurons within the forebrain, making analysis of large numbers of GnRH cells possible. Single-cell PCR analysis and immunocytochemistry confirmed the presence of AdipoR2 in GnRH neurons in explants. Functional analysis revealed 20% of the total GnRH population responded to ADP, exhibiting hyperpolarization or decreased calcium oscillations. Perturbation studies revealed that ADP activates AMP kinase via the protein kinase Cζ/liver kinase B1 pathway. The modulation of GnRH neuronal activity by ADP demonstrated in this report directly links energy balance to neurons controlling reproduction.

Glucose responsiveness in a novel adult-derived GnRH cell line, mHypoA-GnRH/GFP: Involvement of AMP-activated protein kinase

Glucose regulates energy homeostasis and reproductive function within the hypothalamus. The underlying mechanisms responsible for glucose regulation of GnRH gene transcription were investigated using a novel murine immortalized, adult-derived hypothalamic cell line, mHypoA-GnRH/GFP. Analysis of GnRH mRNA synthesis and secretion following agonist treatment demonstrated that the mHypoA-GnRH/GFP cell line is a representative model of in vivo GnRH neurons. c-fos mRNA levels, following glucose exposure, indicated that these neurons were responsive to low (0.5mM) and high (5mM) glucose, and high glucose stimulated GnRH mRNA transcription in a metabolism-dependent manner. Glucose inhibited AMPK activity, and was linked to the downstream stimulation of GnRH mRNA levels. The effect was confirmed with an AMPK antagonist, Compound C. Collectively, these findings demonstrate that glucose can directly regulate GnRH transcription, while implicating the AMPK pathway as an essential mediator of nutritional signaling in a novel GnRH neuronal cell model.

Cocaine- and Amphetamine-Regulated Transcript Is a Potent Stimulator of GnRH and Kisspeptin Cells and May Contribute to Negative Energy Balance-induced Reproductive Inhibition in Females

Cocaine- and amphetamine-regulated transcript (CART) is a hypothalamic neuropeptide implicated in both metabolic and reproductive regulation, raising the possibility that CART plays a role in reproductive inhibition during negative metabolic conditions. The current study characterized CART's regulatory influence on GnRH and kisspeptin (Kiss1) cells and determined the sensitivity of different CART populations to negative energy balance. CART fibers made close appositions to 60% of GnRH cells, with the majority of the fibers (>80%) originating from the arcuate nucleus (ARH) CART/pro-opiomelanocortin population. Electrophysiological recordings in GnRH-green fluorescent protein rats demonstrated that CART postsynaptically depolarizes GnRH cells. CART fibers from the ARH were also observed in close contact with Kiss1 cells in the ARH and anteroventral periventricular nucleus (AVPV). Recordings in Kiss1-GFP mice demonstrated CART also postsynaptically depolarizes ARH Kiss1 cells, suggesting CART may act directly and indirectly, via Kiss1 populations, to stimulate GnRH neurons. CART protein and mRNA levels were analyzed in 2 models of negative energy balance: caloric restriction (CR) and lactation. Both CART mRNA levels and the number of CART-immunoreactive cells were suppressed in the ARH during CR but not during lactation. AVPV CART mRNA was suppressed during CR, but not during lactation when there was a dramatic increase in CART-immunoreactive cells. These data suggest differing regulatory signals of CART between the models. In conclusion, both morphological and electrophysiological methods identify CART as a novel and potent stimulator of Kiss1 and GnRH neurons and suppression of CART expression during negative metabolic conditions could contribute to inhibition of the reproductive axis.