Hormone Transcription Research Articles

Co-localization of three gonadotropin-releasing hormone transcripts in larval, parasitic, and adult sea lamprey brains

RNA expression of lamprey gonadotropin-releasing hormone (lGnRH)-I, -II, and -III was demonstrated in the brains of larval, parasitic phase and adult sea lampreys, Petromyzon marinus, using a highly sensitive triple-label in situ hybridization technique. In female larval lampreys, lGnRH-I and-II were co-expressed in the same neurons throughout the olfactory bulbs, preoptic area (POA), and rhombencephalon (hindbrain); lGnRH-I, -II and -III were triple co-expressed in the hypothalamus and in the paranuclear region of neuronal somas in the rhombencephalon. In female parasitic phase lampreys, lGnRH-I and -II were co-expressed in the POA, thalamus, and preoptico-neurohypophyseal tract (PNT); lGnRH-III was minimally triple co-expressed with lGnRH-I and -II in the hypothalamus. In adult female lampreys, lGnRH-I and -III were co-expressed in the hypothalamus; lGnRH-I was also expressed in the neurohypophysis (NH). In adult male lampreys, lGnRH-I and-III were co-expressed in the primordial hippocampus, POA, thalamus, hypothalamus, NH, and PNT; lGnRH-I was also expressed in the epithalamus. In summary, we provide the first study using in situ hybridization of all three lGnRHs (lGnRH-I, -II, and -III) at three major life stages (larval, parasitic, and adult) of lampreys, which strongly supports previous immunohistological studies and suggests that lGnRH-I and -II are the predominant lGnRHs in larval and parasitic phase lampreys, and that lGnRH-I and -III are the predominant lGnRHs in adult female and male lampreys. Therefore, our results show that lGnRH-I, -II, and -III have different localization and co-expression in the development and sexual maturation of lampreys, which may suggest unique physiological roles at each life stage and sex in the developing and mature lamprey brain.

Simulated herbivory in chickpea causes rapid changes in defense pathways and hormonal transcription networks of JA/ethylene/GA/auxin within minutes of wounding

Chickpea (C. arietinum L.) is an important pulse crop in Asian and African countries that suffers significant yield losses due to attacks by insects like H. armigera. To obtain insights into early responses of chickpea to insect attack, a transcriptomic analysis of chickpea leaves just 20 minutes after simulated herbivory was performed, using oral secretions of H. armigera coupled with mechanical wounding. Expression profiles revealed differential regulation of 8.4% of the total leaf transcriptome with 1334 genes up-regulated and 501 down-regulated upon wounding at log2-fold change (|FC| ≤ −1 and ≥1) and FDR value ≤ 0.05. In silico analysis showed the activation of defenses through up-regulation of genes of the phenylpropanoid pathway, pathogenesis, oxidases and CYTP450 besides differential regulation of kinases, phosphatases and transcription factors of the WRKY, MYB, ERFs, bZIP families. A substantial change in the regulation of hormonal networks was observed with up-regulation of JA and ethylene pathways and suppression of growth associated hormone pathways like GA and auxin within 20 minutes of wounding. Secondary qPCR comparison of selected genes showed that oral secretions often increased differential expression relative to mechanical damage alone. The studies provide new insights into early wound responses in chickpea.

Dynamic Regulation of Hypothalamic DMXL2, KISS1, and RFRP Expression During Postnatal Development in Non-Human Primates

The neurobiological mechanism of puberty onset in primates is currently only partly understood. A recent study reported an important role of Dmx-like 2 (DMXL2), a gene encoding rabconnectin-3α vesicular protein, in human subjects with mental retardation and neuroendocrine impairment of reproduction. To further characterize the potential role of DMXL2 in the regulation of reproduction, we analyzed the expression of DMXL2 in hypothalami of newborn, infantile, juvenile, pubertal, and postpubertal female and male common marmoset monkeys. Additionally, as the relative hypothalamic levels of gonadotropin-inhibitory hormone (GnIH) transcript during postnatal development are unknown in primates, we also quantified messenger RNA (mRNA) levels of RFRP, a gene encoding GnIH. Moreover, the transcript levels of kisspeptin, a well-known regulator of the hypothalamic neurohormonal axis controlling reproduction, were also checked. Transcript and protein levels of DMXL2 and Kiss1 transcript levels increase from the newborn to the infantile and from the juvenile (prepubertal) to the pubertal and the postpubertal period. We also noted a clear upsurge in RFRP transcript levels in the prepubertal period. In conclusion, the hypothalamic expressions of Kiss1 and DMXL2 mRNA increase during infantile, pubertal, and adult stages compared to newborn and juvenile stages in common marmoset monkeys. In contrast, the expression of RFRP mRNA upsurges in juvenile monkeys. Further mechanistic studies are needed to characterize the potential inhibitory role of the GnIH-GPR147 signaling in the prepubertal period and the role of DMXL2 in the molecular cascade regulating the neuroendocrine reproductive axis in primates.

In vivo Functional Consequences of Human THRA Variants Expressed in the Zebrafish.

Heterozygous mutations in the thyroid hormone receptor alpha (THRA) gene cause resistance to thyroid hormone alpha (RTHα), a disease characterized by variable manifestations reminiscent of untreated congenital hypothyroidism but a raised triiodothyronine/thyroxine ratio and normal thyrotropin levels. It was recently described that zebrafish embryos expressing a dominant negative (DN) form of thraa recapitulate the key features of RTHα, and that zebrafish and human receptors are functionally interchangeable. This study expressed several human thyroid hormone receptor alpha (hTRα) variants in zebrafish embryos and analyzed the resulting phenotypes. All hTRα-injected embryos showed variable defects, including cerebral and cardiac edema likely caused by an aberrant looping during heart development, anemia, and an incomplete formation of the vascular network. Moreover, the hTRα-injected embryos presented severe defects of motorneurons and craniofacial development, thus affecting their autonomous feeding and swimming behaviors. Surprisingly, expression of all hTRα mutants had no detectable effect on thyrotropin beta and thyrotropin-releasing hormone transcripts, indicating that their DN action is limited on the thyroid hormone reception beta 2 targets at the hypothalamic/pituitary level in vivo. As previously described in vitro, treatment with high triiodothyronine doses can efficiently revert the observed defects only in embryos injected with missense hTRα variants. Injection of human THRA variants in zebrafish embryos causes tissue-specific defects recapitulating most of the RTHα clinical and biochemical manifestations. The described manipulation of zebrafish embryos represents a novel in vivo model to screen the functional consequences of THRA variants and the rescue potential of new therapeutic compounds.

Time-dependent PPARγ Modulation of HIF-1α Signaling in Hypoxic Pulmonary Artery Smooth Muscle Cells

BackgroundPathogenesis of pulmonary hypertension is complex and involves activation of the transcription factor, hypoxia-inducible factor-1 (HIF-1) that shifts cellular metabolism from aerobic respiration to glycolysis, in part, by increasing the expression of its downstream target pyruvate dehydrogenase kinase-1 (PDK-1), thereby promoting a proliferative, apoptosis-resistant phenotype in pulmonary vascular cells. Activation of the nuclear hormone transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), attenuates pulmonary hypertension and pulmonary artery smooth muscle cell (PASMC) proliferation. In the current study, we determined whether PPARγ inhibits HIF-1α and PDK-1 expression in human PASMCs. MethodsHPASMCs were exposed to normoxia (21% O2) or hypoxia (1% O2) for 2-72 hours ± treatment with the PPARγ-ligand, rosiglitazone (RSG, 10μM). ResultsCompared to normoxia, HIF-1α mRNA levels were elevated in HPASMC at 2 hours hypoxia and reduced to baseline levels by 24-72 hours. HIF-1α protein levels increased following 4 and 8 hours of hypoxia and returned to baseline levels by 24 and 72 hours. PDK-1 protein levels increased following 24 hours hypoxia and remained elevated by 72 hours. RSG treatment at the onset of hypoxia attenuated HIF-1α protein and PDK-1 mRNA and protein levels at 4, 8 and 24 hours of hypoxia, respectively. However, RSG treatment during final 24 hours of 72-hour hypoxia, an intervention that inhibits HPASMC proliferation, failed to prevent hypoxia-induced PDK-1 expression. ConclusionHypoxia causes transient activation of HPASMC HIF-1α that is attenuated by RSG treatment initiated at hypoxia onset. These findings provide novel evidence that PPARγ modulates fundamental and acute cellular responses to hypoxia through both HIF-1-dependent and HIF-1-independent mechanisms.

Anti-estrogenic effect of semicarbazide in female zebrafish (Danio rerio) and its potential mechanisms

Semicarbazide (SMC), a member of the hydrazine family, has various toxic effects and has been detected in organisms, aquatic environments, and food. SMC exposure inhibited the transcription of hepatic vitellogenin and estrogen receptors in female zebrafish (Danio rerio), suggesting that it had anti-estrogenic properties. In order to elucidate the mechanisms underlying these, we exposed female zebrafish to SMC and used enzyme-linked immunosorbent assays to examine plasma 17β-estradiol (E2) and testosterone (T) levels. Gonad histology was analyzed and the mRNA expression of genes involved in the reproductive axis, the gamma-aminobutyric acid (GABA) shunt, and leptin was quantified by real-time PCR. Zebrafish were exposed to 1, 10, 100, or 1000μg/L SMC in a semi-static system for 96hours or 28 days. Plasma E2 levels were significantly decreased and ovarian maturation was inhibited by SMC, suggesting that its anti-estrogenic effect was exerted by reducing endogenous E2 levels. This was likely due to the SMC-mediated inhibition of cytochrome P450 (CYP) 19A mRNA levels, because this enzyme catalyzes the conversion of T to E2 in the gonads. In addition, down-regulation of the mRNA expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, steroidogenic acute regulatory protein, CYP17, and 17beta-hydroxysteroid dehydrogenase was observed; this was predicted to reduce T concentrations and further contribute to the reduced E2 levels. SMC-induced changes in the expression of these steroidogenic genes correlated with decreased transcription of gonadotropic hormones (follicle-stimulating hormone and luteinizing hormone) and significantly elevated leptin expression. Furthermore, SMC also altered expression of the key enzyme in gamma-aminobutyric acid (GABA) synthesis, GABA receptors, and salmon gonadotropin-releasing hormone, thus affecting gonadotropin expression. Overall, SMC acted at multiple sites related to reproduction to reduce plasma E2 levels, consequently exerting an anti-estrogenic effect in female zebrafish. These effects were observed at environmentally relevant concentrations and highlight the importance of controlling SMC contamination.

Silencing of Gonad-Inhibiting Hormone Transcripts in Litopenaeus vannamei Females by use of the RNA Interference Technology.

The method usually employed to stimulate gonadal maturation and spawning of captive shrimp involves unilateral eyestalk ablation, which results in the removal of the endocrine complex responsible for gonad-inhibiting hormone (GIH) synthesis and release. In the present study, RNAi technology was used to inhibit transcripts of GIH in Litopenaeus vannamei females. The effect of gene silencing on gonad development was assessed by analyzing the expression of GIH and vitellogenin, respectively, in the eyestalk and ovaries of L. vannamei females, following ablation or injection with dsRNA-GIH, dsRNA-IGSF4D (non-related dsRNA), or saline solution. Histological analyses were performed to determine the stage of gonadal development and to assess the diameter of oocytes throughout the experimental procedure. Only oocytes at pre-vitellogenesis and primary vitellogenesis stages were identified in females injected with dsRNA-GIH, dsRNA-IGSF4D, or saline solution. Oocytes at all developmental stages were observed in eyestalk-ablated females, with predominance of later stages, such as secondary vitellogenesis and mature oocytes. Despite achieving 64, 73, and 71% knockdown of eyestalk GIH mRNA levels by 15, 30, and 37 days post-injection (dpi), respectively, in dsRNA-GIH-injected females, the expected increase in ovary vitellogenin mRNA expression was only observed on the 37th dpi. This is the first report of the use of RNAi technology to develop an alternative method to eyestalk ablation in captive L. vannamei shrimps.

Delayed pubertal onset and prepubertal Kiss1 expression in female mice lacking central oestrogen receptor beta.

Ovarian oestradiol is essential for pubertal maturation and adult physiology of the female reproductive axis. It acts at central and peripheral sites through two main oestrogen receptors (ER) α and β. Here we investigate the role of ERβ on central effects of oestradiol, by generating a mouse line specifically lacking the ERβ gene in neuronal and glial cells. Central ERβ deletion delays the age at vaginal opening and first oestrous and reduces uterine weight without affecting body growth. Analysis of factors necessary for pubertal progression shows reduced levels of Kiss1 transcripts at postnatal (P) day 25 in the preoptic area, but not in the mediobasal hypothalamus (MBH) of mutant females. In agreement with these data, the number of kisspeptin-immunoreactive neurons was decreased by 57-72% in the three subdivisions of the rostral periventricular area of the third ventricle (RP3V), whereas the density of kisspeptin-immunoreactive fibres was unchanged in the arcuate nucleus of mutant mice. These alterations do not involve changes in ERα mRNAs in the preoptic area and protein levels in the RP3V. The number and distribution of GnRH-immunoreactive cells were unaffected, but gonadotropin-releasing hormone (GnRH) transcript levels were higher in the P25 preoptic area of mutants. At adulthood, mutant females have normal oestrous cyclicity, kisspeptin system and exhibit unaltered sexual behaviour. They display, however, reduced ovary weight and increased anxiety-related behaviour during the follicular phase. This argues for the specific involvement of central ERβ in the regulation of pubertal onset in female reproduction, possibly through prepubertal induction of kisspeptin expression in the RP3V.

RNAseq analysis of fast skeletal muscle in restriction-fed transgenic coho salmon (Oncorhynchus kisutch): an experimental model uncoupling the growth hormone and nutritional signals regulating growth.

BackgroundCoho salmon (Oncorhynchus kisutch) transgenic for growth hormone (Gh) express Gh in multiple tissues which results in increased appetite and continuous high growth with satiation feeding. Restricting Gh-transgenics to the same lower ration (TR) as wild-type fish (WT) results in similar growth, but with the recruitment of fewer, larger diameter, muscle skeletal fibres to reach a given body size. In order to better understand the genetic mechanisms behind these different patterns of muscle growth and to investigate how the decoupling of Gh and nutritional signals affects gene regulation we used RNA-seq to compare the fast skeletal muscle transcriptome in TR and WT coho salmon.ResultsIllumina sequencing of individually barcoded libraries from 6 WT and 6 TR coho salmon yielded 704,550,985 paired end reads which were used to construct 323,115 contigs containing 19,093 unique genes of which >10,000 contained >90 % of the coding sequence. Transcripts coding for 31 genes required for myoblast fusion were identified with 22 significantly downregulated in TR relative to WT fish, including 10 (vaspa, cdh15, graf1, crk, crkl, dock1, trio, plekho1a, cdc42a and dock5) associated with signaling through the cell surface protein cadherin. Nineteen out of 44 (43 %) translation initiation factors and 14 of 47 (30 %) protein chaperones were upregulated in TR relative to WT fish.ConclusionsTR coho salmon showed increased growth hormone transcripts and gene expression associated with protein synthesis and folding than WT fish even though net rates of protein accretion were similar. The uncoupling of Gh and amino acid signals likely results in additional costs of transcription associated with protein turnover in TR fish. The predicted reduction in the ionic costs of homeostasis in TR fish associated with increased fibre size were shown to involve multiple pathways regulating myotube fusion, particularly cadherin signaling.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1782-z) contains supplementary material, which is available to authorized users.

Early development of gonads in Muscovy duck embryos

Characteristics of gonadal development were investigated in Muscovy duck embryos at various embryonic ages.Hematoxylin-Eosin and periodic acid-Schiff staining were used to identify primordial germ cells, oogonia and sustentacular cells.Gene specific primers were designed based on conserved regions of duck antimullerian hormone (AMH), oestrogen receptor-α (ESR-α), doublesex and Mab-3 related transcription factor-1(DMRT1) and W chromosome protein kinase C inhibitor/interacting gene (WPKCI). Quantitative real-time polymerase chain reaction was used to characterise gene expression during gonad development in Muscovy duck embryos.Histology indicated that ovarian and testicular cells of Muscovy duck embryos developed on d 9 and 10.Immunohistochemistry showed that mouse vasa homologue-positive cells as well as Glial cell line-derived neurotrophic factor-positive cells increased significantly more in females than in males between d 9 and 10. AMH and ESR-α expression increased significantly during early development. DMRT1 acts prior to and during testis differentiation whereas WPKCI was expressed actively in the female Muscovy duck embryo before the onset of gonadal differentiation.Gonad development in Muscovy duck embryo was associated with several genes that were expressed before morphological features appear and were gender specific.

Captive maturation studies in Penaeus monodon by GIH silencing using constitutively expressed long hairpin RNA

Captive maturation and spawning in Penaeus monodon are currently induced by unilateral eyestalk ablation, a method that removes the gonad-inhibiting hormone (GIH) secreted from the eyestalk. However, unilateral eyestalk ablation creates physiological complications in the brooder due to the unintended removal of other hormones secreted by the eyestalks. Here we studied the effect of gih silencing by long hairpin RNA on the reproductive physiology of P. monodon. We observed 3–5 fold enhanced expression of the androgenic gland hormone (AGH) transcript in males, but no effect on vitellogenin expression in females. In the destalked animals, however, positive effect on the maturation indicator transcripts was seen in both the sexes, and surpassed the efficiency of the silencing treatment. There were indications of active spermatogenesis in histological sections of both the gih silenced and destalked males, but no changes at histological level in the females. We also observed a significantly higher rate of molting and associated mortality in the gih silenced animals at the end of the experiment. Our results suggest that eyestalk ablation is still the most efficient technique to induce maturation in P. monodon. Although gih silencing could be a potential alternative to eyestalk ablation, further research is needed to enhance its efficiency over eyestalk ablation by using tissue specific and/or inducible promoter for lhRNA expression. It is also evident that females need to be fairly large in size for these interventions to succeed. Statement of relevanceInduction of captive maturation has been a challenge for shrimp industry. The seed production is often hampered by the low abundance and high price of naturally collected broodstock. Collection of natural brooders also has the intrinsic problem of WSSV infection. Also, genetic improvement of this highly preferred species is difficult unless the reproductive cycle is closed. For all these reasons it is important to study the reproductive physiology and endocrinology of this species and ways to manipulate it in order to induce captive maturation. The present study utilizes the RNA interference mechanism to silence the gonad-inhibiting hormone gene to see its effect on maturation.

Prohibitin-2 gene reveals sex-related differences in the salmon louse Caligus rogercresseyi

Prohibitins are evolutionarily conserved proteins present in multiple cellular compartments, and are involved in diverse cellular processes, including steroid hormone transcription and gametogenesis. In the present study, we report for the first time the characterization of the prohibitin-2 (Phb2) gene in the sea lice Caligus rogercresseyi. The CrPhb2 cDNA showed a total length of 1406bp, which contained a predicted open reading frame (ORF) of 894 base pairs (bp) encoding for 298 amino acids. Multiple sequence alignments of prohibitin proteins from other arthropods revealed a high degree of amino acid sequence conservation. In silico Illumina read counts and RT-qPCR analyses showed a sex-dependent differential expression, with mRNA levels exhibiting a 1.7-fold (RT-qPCR) increase in adult females compared with adult males. A total of nine single nucleotide polymorphisms (SNPs) were identified, three were located in the 5′ UTR of the Phb2 messenger and six in the ORF, but no mutations associated with sex were found. These results contribute to expand the present knowledge of the reproduction-related genes in C. rogercresseyi, and may be useful in future experiments aimed at controlling the impacts of sea lice in fish farming.

Endometrial expression of selected transcripts in postpartum of primiparous Holstein cows with clinical and subclinical endometritis

Postpartum endometritis compromises milk production and fertility in high-producing dairy cows. Infection of the endometrium induces an inflammatory response with secretion of cytokines that lead to polymorphonuclear cells (PMN) influx and bacterial clearance. Considering that only a portion of cows with endometritis is eligible for clinical diagnosis, there is an increasing effort for developing reliable tools and protocols for diagnosis of subclinical endometritis. Recent reports have indicated that primiparous cows are at greater risk of uterine infection and primiparous cows with subclinical endometritis produce less milk compared to healthy cows. In the present study, gene expression profiles were compared for selected cytokine and hormone endometrial transcripts in the postpartum of primiparous Holstein cows with clinical and subclinical endometritis. Cows were classified as healthy (no signs of clinical endometritis), cows with subclinical endometritis (PMN<5% in the cytological sample) and cows with clinical endometritis (PMN>5%). Although, cows with clinical endometritis had greater (P<0.05) relative amounts of mRNA for the IL1A, IL6, IL17A, TNFα, PGES and PGHS2 genes compared to healthy cows; no significant differences were detected between clinical and subclinical endometritis groups. Spearman correlation coefficients were positive between relative amounts of gene expression as indicated by amount of these transcripts and PMN percentages and ranged from 0.74 to 0.93 (P<0.05). Relative amounts of cytokine mRNA suggest similar inflammatory response in the endometrium of cows with subclinical and clinical endometritis. Moreover, differential relative amounts of hormone transcripts suggest dysregulation of the luteolytic mechanism and PG synthases but not ERα in cows with endometritis.

Lysosomes signal the nucleus to control aging

Aging Folick et al. propose a mechanism by which a lysosomal enzyme influences nuclear events that control longevity in the worm (see the Perspective by Shuo and Brunet). Increased expression of the lysosomal acid lipase LIPL-4 increased longevity, and this effect depended on the presence of the lysosomal lipid-binding protein LBP-8. LBP-8 acts as a chaperone that helps carry lipds to the nucleus. The authors identified the fatty acid oleoylethanolamide (OEA) as a potential signaling molecule whose transport to the nucleus could activate nuclear hormone receptors and transcription factors NHR-49 and NHR-80. The transcriptional targets of NHR-49 and NHR-80 in turn regulate longevity. Science , this issue p. [83][1] [1]: /lookup/doi/10.1126/science.1258857

A Juvenile Hormone Transcription Factor Bmdimm-Fibroin H Chain Pathway Is Involved in the Synthesis of Silk Protein in Silkworm, Bombyx mori

The genes responsible for silk biosynthesis are switched on and off at particular times in the silk glands of Bombyx mori. This switch appears to be under the control of endogenous and exogenous hormones. However, the molecular mechanisms by which silk protein synthesis is regulated by the juvenile hormone (JH) are largely unknown. Here, we report a basic helix-loop-helix transcription factor, Bmdimm, its silk gland-specific expression, and its direct involvement in the regulation of fibroin H-chain (fib-H) by binding to an E-box (CAAATG) element of the fib-H gene promoter. Far-Western blots, enzyme-linked immunosorbent assays, and co-immunoprecipitation assays revealed that Bmdimm protein interacted with another basic helix-loop-helix transcription factor, Bmsage. Immunostaining revealed that Bmdimm and Bmsage proteins are co-localized in nuclei. Bmdimm expression was induced in larval silk glands in vivo, in silk glands cultured in vitro, and in B. mori cell lines after treatment with a JH analog. The JH effect on Bmdimm was mediated by the JH-Met-Kr-h1 signaling pathway, and Bmdimm expression did not respond to JH by RNA interference with double-stranded BmKr-h1 RNA. These data suggest that the JH regulatory pathway, the transcription factor Bmdimm, and the targeted fib-H gene contribute to the synthesis of fibroin H-chain protein in B. mori.

The acute salinity changes activate the dual pathways of endocrine responses in the brain and pituitary of tilapia

To analyze and compare the stress and osmoregulatory hormones and receptors in pituitary during acute salinity changes, the expression patterns of corticotropin releasing hormone (crh) in hypothalamus, prolactin (prl) releasing peptide (pRrp) in telencephalon and diencephalon, glucocorticoid receptors 2 (gr2), and mineralocorticoid receptor (mr), crh-r, pro-opiomelanocorticotropin (pomc), pRrp, prl, dopamine 2 receptor (d2-r), growth hormone (gh), gh-receptor (gh-r) and insulin-like growth hormone (igf-1) transcripts in pituitary were characterized in euryhaline tilapia. The results indicate that the crh transcripts increased in the hypothalamus and rostral pars distalis of the pituitary after the transfer of fish to SW. Similarly, the pRrp transcripts were more abundant in SW acclimated tilapia forebrain and hypothalamus. The crh-r, gr2 and mr transcripts were more expressed in rostral pars distalis and pars intermedia of pituitary at SW than FW tilapia. The data indicate that the SW acclimation stimulates these transcripts in the specific regions of the brain and pituitary which may be related to the activation of the hypothalamic–pituitary–interrenal (HPI)-axis. The results of dual in situ hybridization reveal that the transcripts of crh-r, gr2 and mr with pomc are highly co-localized in corticotrophs of pituitary. Furthermore, we demonstrate high expression of pRrp in the brain and low expression of pRrp and prl transcripts in the pituitary of SW fish. No crh-r and corticosteroid receptors were co-localized with prl transcripts in the pituitary. The gh-r and igf-1 mRNA levels were significantly increased in SW acclimated tilapia pituitary whereas there was no difference in the gh mRNA levels. The data suggest that the locally produced pRrp and d2-r may control and regulate the expression of prl mRNA in pituitary. Therefore, the dual roles of pRrp are involved in the stress (via brain-pituitary) and osmoregulatory (via pituitary) pathways in tilapia exposed to acute salinity changes.

An acute injection of corticosterone increases thyrotrophin-releasing hormone expression in the paraventricular nucleus of the hypothalamus but interferes with the rapid hypothalamus pituitary thyroid axis response to cold in male rats.

The activity of the hypothalamic-pituitary-thyroid (HPT) axis is rapidly adjusted by energy balance alterations. Glucocorticoids can interfere with this activity, although the timing of this interaction is unknown. In vitro studies indicate that, albeit incubation with either glucocorticoid receptor (GR) agonists or protein kinase A (PKA) activators enhances pro-thyrotrophin-releasing hormone (pro-TRH) transcription, co-incubation with both stimuli reduces this enhancement. In the present study, we used primary cultures of hypothalamic cells to test whether the order of these stimuli alters the cross-talk. We observed that a simultaneous or 1-h prior (but not later) activation of GR is necessary to inhibit the stimulatory effect of PKA activation on pro-TRH expression. We tested these in vitro results in the context of a physiological stimulus on the HPT axis in adult male rats. Cold exposure for 1 h enhanced pro-TRH mRNA expression in neurones of the hypophysiotrophic and rostral subdivisions of the paraventricular nucleus (PVN) of the hypothalamus, thyrotrophin (TSH) serum levels and deiodinase 2 (D2) activity in brown adipose tissue (BAT). An i.p. injection of corticosterone stimulated pro-TRH expression in the PVN of rats kept at ambient temperature, more pronouncedly in hypophysiotrophic neurones that no longer responded to cold exposure. In corticosterone-pretreated rats, the cold-induced increase in pro-TRH expression was detected only in the rostral PVN. Corticosterone blunted the increase in serum TSH levels and D2 activity in BAT produced by cold in vehicle-injected animals. Thus, increased serum corticosterone levels rapidly restrain cold stress-induced activation of TRH hypophysiotrophic neurones, which may contribute to changing energy expenditure. Interestingly, TRH neurones of the rostral PVN responded to both corticosterone and cold exposure with an amplified expression of pro-TRH mRNA, suggesting that these neurones integrate stress and temperature distinctly from the hypophysiotrophic neurones.

Direct regulation of gonadotrophin-releasing hormone (GnRH) transcription by RF-amide-related peptide-3 and kisspeptin in a novel GnRH-secreting cell line, mHypoA-GnRH/GFP.

RF-amide-related peptide-3 [RFRP-3; also often referred to as the mammalian orthologue of the avian gonadotrophin-inhibitory hormone (GnIH)] and kisspeptin have emerged as potent modulators of neuroendocrine function via direct regulation of the reproductive axis in the hypothalamus and pituitary. There are few studies focusing on the direct regulatory effects of RFRP-3 and kisspeptin on gonadotrophin-releasing hormones (GnRH) neurones. We report their effect on GnRH mRNA expression and release in a novel GnRH neuronal cell model, mHypoA-GnRH/GFP, generated from adult-derived GnRH-GFP neurones. The neurones express receptors for both RFRP-3 and kisspeptin, Gpr147 and Gpr54, respectively. Incubation with 100nm RFRP-3 results in attenuation of GnRH mRNA expression by approximately 60%. Conversely, incubation with 10nm of Kiss-10 induced GnRH mRNA expression, whereas the combined effect was an overall repression of GnRH mRNA levels. With transcription inhibitors, the repression of GnRH mRNA levels was linked to a transcriptional mechanism but not mRNA stability. No significant changes in GnRH secretion were observed upon RFRP-3 exposure in these neurones. Our findings suggest that the suppressive signalling of RFRP-3 on GnRH transcription may dominate over kisspeptin induction in the mHypoA-GnRH/GFP GnRH neuronal cell model.

Liver X receptor regulation of thyrotropin-releasing hormone transcription in mouse hypothalamus is dependent on thyroid status.

Reversing the escalating rate of obesity requires increased knowledge of the molecular mechanisms controlling energy balance. Liver X receptors (LXRs) and thyroid hormone receptors (TRs) are key physiological regulators of energetic metabolism. Analysing interactions between these receptors in the periphery has led to a better understanding of the mechanisms involved in metabolic diseases. However, no data is available on such interactions in the brain. We tested the hypothesis that hypothalamic LXR/TR interactions could co-regulate signalling pathways involved in the central regulation of metabolism. Using in vivo gene transfer we show that LXR activation by its synthetic agonist GW3965 represses the transcriptional activity of two key metabolic genes, Thyrotropin-releasing hormone (Trh) and Melanocortin receptor type 4 (Mc4r) in the hypothalamus of euthyroid mice. Interestingly, this repression did not occur in hypothyroid mice but was restored in the case of Trh by thyroid hormone (TH) treatment, highlighting the role of the triiodothyronine (T3) and TRs in this dialogue. Using shLXR to knock-down LXRs in vivo in euthyroid newborn mice, not only abrogated Trh repression but actually increased Trh transcription, revealing a potential inhibitory effect of LXR on the Hypothalamic-Pituitary-Thyroid axis. In vivo chromatin immunoprecipitation (ChIP) revealed LXR to be present on the Trh promoter region in the presence of T3 and that Retinoid X Receptor (RXR), a heterodimerization partner for both TR and LXR, was never recruited simultaneously with LXR. Interactions between the TR and LXR pathways were confirmed by qPCR experiments. T3 treatment of newborn mice induced hypothalamic expression of certain key LXR target genes implicated in metabolism and inflammation. Taken together the results indicate that the crosstalk between LXR and TR signalling in the hypothalamus centres on metabolic and inflammatory pathways.

The Root of the Problem

Restrained growth of a key root tip region involves an interplay between hormonal activation and transcription factor levels.