Feedback Loops Link Odor and Pheromone Signaling with Reproduction

Kisspeptin Mediates the Photoperiodic Control of Reproduction in Hamsters

Gonadotropin-releasing hormone (GnRH) is exclusively expressed in a unique population of hypothalamic neurons that controls reproductive function. GnRH gene expression is highly dynamic. Its transcriptional activity is regulated in a complex spatiotemporal manner during embryonic development and postnatal life. Although a variety of transcription factors have been identified as regulators of GnRH transcription, most are promiscuous in their DNA-binding requirements, and none are solely expressed in GnRH neurons. Their specific activity is probably determined by interactions with distinct cofactors. Here we find that the Groucho-related gene (GRG) family of co-repressors is expressed in a model cell line for the GnRH neuron and co-expresses with GnRH during prenatal development. GRG proteins associate in vivo with the GnRH promoter. Furthermore, GRG proteins interact with two regulators of GnRH transcription, the homeodomain proteins MSX1 and OCT1. Co-transfection experiments indicate that GRG proteins regulate GnRH promoter activity. The long GRG forms enhance MSX1 repression and counteract OCT1 activation of the GnRH gene. In contrast, the short form, GRG5, has a dominant-negative effect on MSX1-dependent repression. Taken together, these data suggest that the dynamic switch between activation and repression of GnRH transcription is mediated by recruitment of the GRG co-regulators.

Gonadotropin-releasing hormone (GnRH) is the central regulator of reproductive function. Expression of the GnRH gene is confined to a rare population of neurons scattered throughout the hypothalamus. Restricted expression of the rat GnRH gene is driven by a multicomponent enhancer and an evolutionarily conserved promoter. Oct-1, a ubiquitous POU homeodomain transcription factor, was identified as an essential factor regulating GnRH transcription in the GT1-7 hypothalamic neuronal cell line. In this study, we conducted a two-hybrid interaction screen in yeast using a GT1-7 cDNA library to search for specific Oct-1 cofactors. Using this approach, we isolated Pbx1b, a TALE homeodomain transcription factor that specifically associates with Oct-1. We show that heterodimers containing Pbx/Prep1 or Pbx/Meis1 TALE homeodomain proteins bind to four functional elements within the GnRH regulatory region, each in close proximity to an Oct-1-binding site. Cotransfection experiments indicate that TALE proteins are essential for GnRH promoter activity in the GT1-7 cells. Moreover, Pbx1 and Oct-1, as well as Prep1 and Oct-1, form functional complexes that enhance GnRH gene expression. Finally, Pbx1 is expressed in GnRH neurons in embryonic as well as mature mice, suggesting that the associations between TALE homeodomain proteins and Oct-1 regulate neuron-specific expression of the GnRH gene in vivo.

Sexually Dimorphic Patterns of Episomal rAAV Genome Persistence in the Adult Mouse Liver and Correlation With Hepatocellular Proliferation

Background: The gonadotropin releasing hormone (GnRH) neurons play a pivotal role in the central regulation of fertility. Kisspeptin binds to the G-protein coupled receptor 54 (GPR54) and GPR54 has been shown to be essential for puberty and subsequent fertility in humans. The recent in vivo studies have proved that kisspeptin is an extremely potent activator of GnRH neurons. However, the precise mechanism of action of kisspeptin on the GnRH neurons and the age-dependent kisspeptin effects are not yet fully understood. In this study, we investigated the effects of kisspeptin on the GnRH neurons over the developmental stages in male and female mice. Methods: Young ( P35) GnRH-GFP transgenic mice expressing green fluorescent protein were used in this study. Acute coronal brain slices containing the preoptic area were prepared, and the identified GnRH neurons were recorded using the gramicidin perforated-patch clamp technique. Results: In young mice, GnRH neurons were excited by bath application of kisspeptin in 36% (13/36) in male, 17% (4/23) in female tested neurons. In adult mice, GnRH neurons were excited in the majority (30/40, 75%) in male, (21/31, 68%) in female neurons tested. However, there was no significant difference between the effects of kisspeptin in male and female mice. In addition, we tested kisspeptin effects in diestrus, proestrus and estrus animals. There were no significant differences of kisspeptin effects over the estrous cycle. Kisspeptin failed to induce excitatory effects on GnRH neurons (6/7, 86%) neurons) by pretreatment of U73122, a protein lipase C (PLC) inhibitor and kisspeptin-induced excitatory effects were decreased by U73122 application (n = 2). Conclusion: These results demonstrated that kisspeptin-induced membrane excitability was increased after puberty and this supports a previous suggestion that GPR54 is essential for puberty and subsequent fertility. (J Korean Endocr Soc 23:302~309, 2008)

The aim of our study was to measure globotriaosylceramide (Gb(3)) and lyso-Gb(3) levels by tandem mass spectrometry in the urine and kidney in Fabry (gla knockout) mice and wild-type controls. We found that urine Gb(3) of male and female Fabry mice was higher than wild-type mice of the same sex but also significantly higher in male mice compared with females of the same genotype. In kidney tissue, sex and genotype-dependent differences in Gb(3) levels paralleled those in the urine. Isoforms C16, C22:1, and C24OHA were particularly higher in males compared with females in both wild-type and Fabry mice. Similarly, kidney lyso-Gb(3) concentrations were significantly higher in 12-month-old male Fabry mice than in their homozygous female counterparts. However, lyso-Gb(3) was undetectable in wild-type mice of both sexes. α-Galactosidase A activity and mRNA levels in kidney were significantly lower in male wild-type mice compared with female mice. This study shows the sex differences in kidney and urine Gb(3) and kidney lyso-Gb(3) levels in both wild-type and Fabry mice, and it suggests that these male-female differences should be taken into consideration when using murine models for Fabry disease.

A Role for the Androgen Receptor in Collagen Content of the Skin

Supplement of 1% lithocholic acid (LCA) in the diet for 5-9 days resulted in elevated levels of the marker for liver damage aspartate aminotransferase and alkaline phosphatase activities in both farnesoid X receptor (FXR)-null and wild-type female mice. The levels were clearly higher in wild-type mice than in FXR-null mice, despite the diminished expression of a bile salt export pump in the latter. Consistent with liver toxicity marker activities, serum and liver levels of bile acids, particularly LCA and taurolithocholic acid, were clearly higher in wild-type mice than in FXR-null mice after 1% LCA supplement. Marked increases in hepatic sulfating activity for LCA (5.5-fold) and hydroxysteroid sulfotransferase (St) 2a (5.8-fold) were detected in liver of FXR-null mice. A 7.4-fold higher 3alpha-sulfated bile acid concentration was observed in bile of FXR-null mice fed an LCA diet compared with that of wild-type mice. Liver St2a content was inversely correlated with levels of alkaline phosphatase. In contrast, microsomal LCA 6beta-hydroxylation was not increased and was in fact lower in FXR-null mice compared in wild-type mice. Clear decreases in mRNA encoding sodium taurocholate cotransporting polypeptide, organic anion transporting polypeptide 1, and liver-specific organic anion transporter-1 function in bile acid import were detected in LCA-fed mice. These transporter levels are higher in FXR-null mice than wild-type mice after 1% LCA supplement. No obvious changes were detected in the Mrp2, Mrp3, and Mrp4 mRNAs. These results indicate hydroxysteroid sulfotransferase-mediated LCA sulfation as a major pathway for protection against LCA-induced liver damage. Furthermore, Northern blot analysis using FXR-null, pregnane X receptor-null, and FXR-pregnane X receptor double-null mice suggests a repressive role of these nuclear receptors on basal St2a expression.

Gonadotropin-releasing hormone (GnRH) neurons regulate reproduction through pulsatile GnRH release. Women with polycystic ovary syndrome (PCOS) have persistently elevated luteinizing hormone release frequency, reflecting GnRH release; this exacerbates hyperandrogenemia and disrupted reproductive cycles that are characteristic of this disorder. Clinical evidence suggests that neuroendocrine features of PCOS may manifest peripubertally. Adult mice prenatally exposed to androgens (PNA) mimic several reproductive features of PCOS. GnRH neurons from these mice have increased firing activity and receive increased GABAergic transmission, which is excitatory. When changes emerge during development is unknown. To study the typical postnatal development of GABAergic transmission and the effects of PNA treatment and sex, whole-cell voltage-clamp recordings were made of GABAergic postsynaptic currents (PSCs) in GnRH neurons in brain slices from prepubertal through adult control and PNA female and male mice. GABAergic transmission was present by 1 week of age in females and males and increased in frequency, reaching adult levels at 3 and 4 weeks, respectively. GABAergic PSC frequency was elevated in 3-week-old PNA versus control females. PSC frequency in both controls and PNA mice was activity independent, suggesting that PNA induces changes in synapse organization. PNA also alters the functional response of GnRH neurons to GABA. GABA induced firing in fewer neurons from 3-week-old PNA than control females; membrane potential depolarization induced by GABA was also reduced in cells from PNA mice at this age. PNA thus induces changes during development in the presynaptic organization of the GABAergic network afferent to GnRH neurons as well as the postsynaptic GnRH neuron response, both of which may contribute to adult reproductive dysfunction.SIGNIFICANCE STATEMENT The central neuronal network that regulates reproduction is overactive in polycystic ovary syndrome (PCOS), a leading cause of infertility. Recent evidence of neuroendocrine dysfunction in midpubertal girls suggests that the pathophysiological mechanisms underlying PCOS may arise before pubertal maturation. Prenatal exposure to androgens (PNA) in mice mimics several neuroendocrine features of PCOS. GABAergic transmission to gonadotropin-releasing hormone (GnRH) neurons is important for reproduction and is increased in adult PNA mice. The typical development of this network and when changes with PNA and sex arise relative to puberty are unknown. These studies provide evidence that PNA alters prepubertal development of the GABAergic network afferent to GnRH neurons, including both the presynaptic organization and postsynaptic response. These changes may contribute to reproductive dysfunction in adults.

Previous studies have indicated that female animals are more resistant to carbon tetrachloride (CCl(4))-induced liver fibrosis than male animals, and that estradiol (E(2)) treatment can inhibit CCl(4)-induced animal hepatic fibrosis. The underlying mechanism governing these phenomena, however, has not been fully elucidated. Here we reported the role of estrogen-induced miRNA-29 (miR-29) expression in CCl(4)-induced mouse liver injury. Hepatic miR-29 levels were differentially regulated in female and male mice during CCl(4) treatment. Specifically, the levels of miR-29a and miR-29b expression were significantly decreased in the livers of male, but not female, mice following 4 weeks of CCl(4) treatment. The down-regulation of miR-29a and miR-29b in male mouse livers correlated with the early development of liver fibrosis, as indicated by increased expressions of fibrotic markers in male mice relative to female mice. In addition, E(2) was maintained at a higher level in female mice than in male mice. In contrast to TGF-β1 that decreased miR-29a/b expression in murine hepatoma IAR20 cells and normal hepatocytes, E(2) enhanced the expression of miR-29a/b through suppression of the nuclear factor-κB (NF-κB) signal pathway, which negatively regulates miR-29 expression. Furthermore, both E(2) treatment and intravenous injection of the recombinant adenovirus expressing miR-29a/b markedly increased the miR-29a/b level and attenuated the expression of fibrotic markers in mouse livers during CCl(4) treatment, supporting the protective role of E(2)-induced miR-29 in CCl(4)-induced hepatic injury. In conclusion, our results collectively demonstrate that estrogen can inhibit CCl(4)-induced hepatic injury through the induction of hepatic miR-29.

Insulin-like peptide 3 (INSL3) is a member of the insulin superfamily that plays an important role in mediating testes descent during fetal development. More recently, it has also been demonstrated to initiate oocyte maturation and suppress male germ cell apoptosis. These actions are mediated via a specific G-protein-coupled receptor, LGR8. Little is known regarding the structure and function relationship of INSL3, although it is believed that the principal receptor binding site resides within its B-chain. We subsequently observed that the linear B-chain alone (INSL3B-(1-31)) bound to LGR8 and was able to antagonise INSL3 stimulated cAMP accumulation in HEK-293T cells expressing LGR8. Sequentially N- and C-terminally shortened linear analogs were prepared by solid phase synthesis and subsequent assay showed that the minimum length required for binding was residues 11-27. It was also observed that increased binding affinity correlated with a corresponding increase in alpha-helical content as measured by circular dichroism spectroscopy. Molecular modeling studies suggested that judicious placement of a conformational constraint within this peptide would increase its alpha-helix content and result in increased structural similarity to the B-chain within native INSL3. Consequently, intramolecularly disulfide-linked analogs of the B-chain showed a potentiation of INSL3 antagonistic activity, as well as exhibiting increased proteolytic stability, as assessed in rat serum in vitro. Administration of one of these peptides into the testes of rats resulted in a substantial decrease in testis weight probably due to the inhibition of germ cell survival, suggesting that INSL3 antagonists may have potential as novel contraceptive agents.

The physiological role of murine scavenger receptor class B type I (SR-BI) was evaluated by in vivo clearances of human HDL3 and LDL in normal and SR-BI knockout (KO) mice. In normal mice, cholesteryl esters (CEs) were removed faster than proteins, indicating a selective uptake process from both HDL3 and LDL. SR-BI KO mice showed 80% losses of HDL-CE selective uptake and the complete loss of LDL-CE selective uptake in the first phase of clearance. However, the second phase was characterized by an acceleration of CE disappearance in SR-BI KO mice. Thus, SR-BI is the only murine receptor mediating HDL-CE selective uptake, whereas a SR-BI-independent pathway specific to LDL can rescue SR-BI deficiency. The analysis of LDL recovered 3 h after injection in mice from different genotypes revealed that LDLs are significantly depleted in CE (reduction from 19% to 50% of the CE/protein ratios). A smaller LDL size in comparison with that of noninjected LDL was also detectable but was more evident for LDL recovered from normal mice. All LDL preparations migrate faster than noninjected LDL on agarose-barbital gels. Thus, both SR-BI-dependent and -independent pathways lead to substantial changes in LDL.

Event Abstract Back to Event Retrograde endocannabinoid signalling via type-1 cannabinoid receptor (CB1) expressed in neuronal afferents of gonadotropin-releasing hormone (GnRH) neurons of the mouse brain Imre Farkas1*, Levente Deli1, Imre Kalló1, Erik Hrabovszky1, Csaba Fekete1, Suzanne M. Moenter2, Masahiko Watanabe3 and Zsolt Liposits1 1 Institute of Experimental Medicine, Hungarian Academy of Science, Department of Endocrine Neurobiology, Hungary 2 University of Virginia, Neuroscience Graduate Program , Departments of Medicine and Cell Biology, United States 3 Hokkaido University, School of Medicine, Department of Anatomy, Japan Cannabinoids have been shown to alter GnRH secretion and inhibit gonadal functions at the hypothalamic level. Immunocytochemistry demonstrated presence of CB1 in nerve fibers in the medial preoptic area (MPOA) and the organum vasculosum of the lamina terminalis (OVLT). In the present study, light and electron microscopic double labeling experiments demonstrated the apposition of CB1-immunoreactive nerve terminals to the dendrites and perikarya of fusiform GnRH neurons residing in the MPOA-OVLT region. Whole cell clamp recordings - carried out on acute preoptic slices of male GnRH-GFP transgenic mice - revealed GABA-A receptor-mediated miniature postsynaptic currents (mPSCs) in GnRH neurons. The administration of the CB1 agonist WIN55,212 (1 μM) significantly decreased the frequency (49.0 ± 10.79 % of the control, p<0.05, n=10) and increased the amplitude of the mPSCs (120.4 ± 5.44 %, p<0.05). The pre-incubation of slices with the CB1 antagonist AM251 (1 μM; 15 min before applying WIN55,212) prevented the effect of WIN55,212 (105.6 ± 8.08 % of the amplitude and 109.0 ± 20.90 % of the frequency of the AM251-control mPSCs, n=8). When AM251 was applied alone, the frequency of the mPSCs increased (162.8 ± 26.4 % of the control, p<0.05, n=9), however, their amplitude did not change (100.3 ± 8.23 %). The data suggest that endogenous cannabinoids released from GnRH neurons exert local and partly tonical actions on the afferent inputs to GnRH neurons via retrograde signalling utilizing CB1 present in the presynaptic GABAergic elements. Supported by grants from the EU FWP 6 (LSHM-CT-2003-503041) and OTKA (T73002). Conference: 12th Meeting of the Hungarian Neuroscience Society, Budapest, Hungary, 22 Jan - 24 Jan, 2009. Presentation Type: Poster Presentation Topic: Homeostatic regulatory mechanisms Citation: Farkas I, Deli L, Kalló I, Hrabovszky E, Fekete C, Moenter SM, Watanabe M and Liposits Z (2009). Retrograde endocannabinoid signalling via type-1 cannabinoid receptor (CB1) expressed in neuronal afferents of gonadotropin-releasing hormone (GnRH) neurons of the mouse brain. Front. Syst. Neurosci. Conference Abstract: 12th Meeting of the Hungarian Neuroscience Society. doi: 10.3389/conf.neuro.01.2009.04.010 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 25 Feb 2009; Published Online: 25 Feb 2009. * Correspondence: Imre Farkas, Institute of Experimental Medicine, Hungarian Academy of Science, Department of Endocrine Neurobiology, Budapest, Hungary, farkas@koki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Imre Farkas Levente Deli Imre Kalló Erik Hrabovszky Csaba Fekete Suzanne M Moenter Masahiko Watanabe Zsolt Liposits Google Imre Farkas Levente Deli Imre Kalló Erik Hrabovszky Csaba Fekete Suzanne M Moenter Masahiko Watanabe Zsolt Liposits Google Scholar Imre Farkas Levente Deli Imre Kalló Erik Hrabovszky Csaba Fekete Suzanne M Moenter Masahiko Watanabe Zsolt Liposits PubMed Imre Farkas Levente Deli Imre Kalló Erik Hrabovszky Csaba Fekete Suzanne M Moenter Masahiko Watanabe Zsolt Liposits Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Fertility depends on the correct maturation and function of approximately 800 gonadotropin-releasing hormone (GnRH) neurons in the brain. GnRH neurons are at the apex of the hypothalamic-pituitary-gonadal axis that regulates fertility. In adulthood, GnRH neurons are scattered throughout the anterior hypothalamic area and project to the median eminence, where GnRH is released into the portal vasculature to stimulate release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary. LH and FSH then regulate gonadal steroidogenesis and gametogenesis. Absence of GnRH neurons or inappropriate GnRH release leads to infertility. Despite the critical role of GnRH neurons in fertility, we still have a limited understanding of the genes responsible for proper GnRH neuron development and function in adulthood. GnRH neurons originate in the olfactory placode then migrate into the brain. Homeodomain transcription factors expressed within GnRH neurons or along their migratory path are candidate genes for inherited infertility. Using a combined in vitro and in vivo approach, we have identified Ventral Anterior Homeobox 1 (Vax1) as a novel homeodomain transcription factor responsible for GnRH neuron maturation and fertility. GnRH neuron counts in Vax1 knock-out embryos revealed Vax1 to be required for the presence of GnRH-expressing cells at embryonic day 17.5 (E17.5), but not at E13.5. To localize the effects of Vax1 on fertility, we generated Vax1flox mice and crossed them with Gnrhcre mice to specifically delete Vax1 within GnRH neurons. GnRH staining in Vax1flox/flox:GnRHcre mice show a total absence of GnRH expression in the adult. We performed lineage tracing in Vax1flox/flox:GnRHcre:RosaLacZ mice which proved GnRH neurons to be alive, but incapable of expressing GnRH. The absence of GnRH leads to delayed puberty, hypogonadism and complete infertility in both sexes. Finally, using the immortalized model GnRH neuron cell lines, GN11 and GT1-7, we show that VAX1 is a direct regulator of Gnrh1 transcription by binding key ATTA sites within the Gnrh1 promoter. This study identifies VAX1 as a key transcription factor regulating GnRH expression and establishes VAX1 as a novel candidate gene implicated in heritable infertility.

The present investigation has examined which subunits of the GABA_A receptor are expressed by gonadotropin-releasing hormone (GnRH) neurons in the juvenile and adult male mouse. Cells of defined morphology, located in the medial septum (MS) and rostral preoptic area (POA), were patch-clamped in the acute brain slice preparation and their cell contents extracted. A reverse transcriptase polymerase chain reaction (RT-PCR) procedure using nested primers was used to establish individual GnRH mRNA-expressing cells which were then evaluated for eleven GABA_A receptor (α1–5, β1–3, γ1–3) subunit transcripts. Single and multiple GABA_A receptor subunit mRNAs were detected in approximately 70% of all GnRH neurons. A range of different subunit mRNAs (α1, α2, α5, β1, β2, β3, γ2) were found in juvenile GnRH neurons, with the α1γ2 and α5γ2 combinations encountered most frequently within individual cells. The expression profile in adult GnRH neurons was more extensive than that detected in juveniles with α1, α2, α3, α5, β1, β2, β3, γ1 and γ2 subunits all being detected. The major difference in subunit profile between GnRH neurons located in the MS and POA involved the β subunits. The principal postnatal developmental change was one of increasing overall subunit heterogeneity in maturing POA GnRH neurons. The profile of GABA_A receptor subunit mRNAs detected in male GnRH neurons was quite different to that reported by us for female GnRH neurons in the mouse using the same RT-PCR approach. Together, these findings indicate that postnatal GnRH neurons are likely to express a range of GABA_A receptor subunit mRNAs in a sexually dimorphic and developmentally-regulated manner.