GH mRNA Research Articles

Mild Maternal Undernutrition Results in a Premature Neonatal Leptin Surge that Promotes Resistance in Male Offspring to a High Fat Diet

Malnutrition alters leptin signaling, resulting in dysregulation of pituitary somatotropes that may be sex-specific. We reported sex differences in acute fasting responses with reductions in serum leptin in males, but not females (1). Maternal undernutrition can also alter the neonatal leptin surge by blunting with 50% food deprivation (2), causing a premature surge with 30% food deprivation (3), and resulting in metabolic dysfunction in adulthood (2,3). We developed a milder undernutrition model to relate more closely to society’s nutritional challenges and to test the hypothesis that a shift in the neonatal leptin surge would result in sex-specific metabolic changes. Compared to paired ad libitum (Fed) dams, we studied pups from undernourished dams that were calorically restricted by 20% (CR20) from embryonic day 15 until postnatal day (PND) 21.We tested 216 offspring from 11 Fed dams and 13 undernourished dams (CR20), detecting a leptin surge in control fed progeny at PND11. CR20 offspring of both sexes had an early surge (PND8) that was 62% (P<0.0001) higher compared to offspring of Fed dams and was maintained at high levels until PND11. Interestingly, GH levels at PND1 were 354% (P<0.0001) higher in the CR20 progeny compared to controls. By PND16, serum IGF-1 levels in underfed pups were lower than levels in control pups (111364±71 vs 244145±135 pg/ml; P=0.0277). CR20 male weights were 13.1% lower (P<0.0001) and lengths were 8.4% shorter (P=0.0002) than controls by 8 weeks of age and did not recover. CR20 female weights were lower by 11% (P=0.0013) and lengths were shorter up to 3 weeks of age.At 3 months of age, offspring were exposed to a 45% HFD for 16 weeks, testing 54 pups from 3 dams per nutrient status. Fed mice from both sexes responded to the HFD with an average weight gain of 12.3g (P<0.0001) in females and 12.6g (P<0.0001) in males. Females from CR20 dams also gained weight (8.74g, P<0.0001) on the HFD, but was significantly lower than females from Fed dams (P=0.0362). Surprisingly, male progeny from CR20 dams did not respond to weight gain by HF feeding when compared to control fed males, appearing to be protected from impact.Sex-specific changes in pituitary Gh, Ghrhr, and Ghsr mRNA levels were as follows. Among CR20 males exposed to HFD, Ghsr mRNA increased by 64% (P=0.0154) when compared to control fed males. Among CR20 females, Gh expression was unchanged with HFD but Ghrhr expression was blunted by 54.7% (P=0.0363). However, Gh mRNA was reduced by 34% (P= 0.0265) when compared to control females on the HFD. Collectively, these data show that mild undernutrition causes a prematurely high leptin surge and sex-specific differences in growth and responses to a HFD, including a potential resistance to a HFD in underfed males.

The Complex World of Regulation of Pituitary Growth Hormone Secretion: The Role of Ghrelin, Klotho, and Nesfatins in It.

The classic concept of how pituitary GH is regulated by somatostatin and GHRH has changed in recent years, following the discovery of peripheral hormones involved in the regulation of energy homeostasis and mineral homeostasis. These hormones are ghrelin, nesfatins, and klotho. Ghrelin is an orexigenic hormone, released primarily by the gastric mucosa, although it is widely expressed in many different tissues, including the central nervous system and the pituitary. To be active, ghrelin must bind to an n-octanoyl group (n = 8, generally) on serine 3, forming acyl ghrelin which can then bind and activate a G-protein-coupled receptor leading to phospholipase C activation that induces the formation of inositol 1,4,5-triphosphate and diacylglycerol that produce an increase in cytosolic calcium that allows the release of GH. In addition to its direct action on somatotrophs, ghrelin co-localizes with GHRH in several neurons, facilitating its release by inhibiting somatostatin, and acts synergistically with GHRH stimulating the synthesis and secretion of pituitary GH. Gastric ghrelin production declines with age, as does GH. Klotho is an anti-aging agent, produced mainly in the kidneys, whose soluble circulating form directly induces GH secretion through the activation of ERK1/2 and inhibits the inhibitory effect that IGF-I exerts on GH. Children and adults with untreated GH-deficiency show reduced plasma levels of klotho, but treatment with GH restores them to normal values. Deletions or mutations of the Klotho gene affect GH production. Nesfatins 1 and 2 are satiety hormones, they inhibit food intake. They have been found in GH3 cell cultures where they significantly reduce the expression of gh mRNA and that of pituitary-specific positive transcription factor 1, consequently acting as inhibitors of GH production. This is a consequence of the down-regulation of the cAMP/PKA/CREB signaling pathway. Interestingly, nesfatins eliminate the strong positive effect that ghrelin has on GH synthesis and secretion. Throughout this review, we will attempt to broadly analyze the role of these hormones in the complex world of GH regulation, a world in which these hormones already play a very important role.

Association of growth hormone and insulin-like growth factor I genotype with body weight, dominance of body weight, and mRNA expression in Korat slow-growing chickens.

ObjectiveGrowth hormone (GH) and insulin-like growth factor I (IGF-I) play a critical role in animal growth rates. We aimed to investigate the effect of GH and IGF-I genotypes on body weight (BW), dominance, and gene expression in slow-growing chickens at different ages.MethodsA total of 613 Korat chickens (KRs) were bred and divided into three groups by genotype – A1A1, A1A3, and A3A3 for GH and AA, AC, and CC for IGF-I. Chickens were weighed every two weeks, and liver and breast muscle tissues were collected at 10 weeks of age. Genetic parameters of KRs were estimated using ASReml software. The GH and IGF-I mRNA levels were measured by quantitative polymerase chain reaction. Significant differences between traits were analyzed using the generalized linear model.ResultsA significant effect of GH genotypes on BW was found at most ages, and the A1A1 genotype had the highest value of BW. Compared with the A3A3 genotype, the A1A1 and A1A3 genotypes showed a higher dominance effect at 0 and 2 weeks, and genotype A1A1 had the highest value of dominance at 8 weeks of age. A difference in GH mRNA levels between genotypes was detected in breast muscle at 6 weeks and in the liver tissue at 2 weeks. In the case of IGF-I gene, the AA genotype had the highest BW at the beginning of life. Significant differences in BW dominance were found at 2 weeks. However, IGF-I mRNA levels were not different among genotypes in both breast muscles and liver tissues.ConclusionOur results revealed that GH and IGF-I influence growth, but may not be involved in heterosis. GH can be used as a marker gene in selection programs for growth because the homozygous genotype (A1A1) had the highest BW at all ages. The IGF-I is not a useful marker gene for selection programs.

Aflatoxicosis Dysregulates the Physiological Responses to Crowding Densities in the Marine Teleost Gilthead Seabream (Sparus aurata).

Simple SummaryA fungal toxin, aflatoxin B1 (AFB1), undermines growth and stress axes of gilthead seabream (Sparus aurata) with depletion of somatic carbohydrate and lipid reservoirs. The present study assessed the physiological consequences of high stocking density versus low stocking density in seabream juveniles, which had previously been fed with AFB1 supplementation. These stressors are likely to converge by inferring animal welfare and economic profitability in the food animal industry. Interestingly, AFB1 seems to cause physiological and molecular dysfunction in response to overcrowding densities. Our results might be relevant to elucidate a potential risk for fish farming that is often overlooked.Several studies in fish have shown that aflatoxin B1 (AFB1) causes a disparity of species-dependent physiological disorders without compromising survival. We studied the effect of dietary administration of AFB1 (2 mg AFB1 kg−1 diet) in gilthead seabream (Sparus aurata) juveniles in combination with a challenge by stocking density (4 vs. 40 g L−1). The experimental period duration was ten days, and the diet with AFB1 was administered to the fish for 85 days prior to the stocking density challenge. Our results indicated an alteration in the carbohydrate and lipid metabolites mobilization in the AFB1 fed group, which was intensified at high stocking density (HSD). The CT group at HSD increased plasma cortisol levels, as expected, whereas the AFB1-HSD group did not. The star mRNA expression, an enzyme involved in cortisol synthesis in the head kidney, presented a ninefold increase in the AFB1 group at low stocking density (LSD) compared to the CT-LSD group. Adenohypophyseal gh mRNA expression increased in the AFB1-HSD but not in the CT-HSD group. Overall, these results confirmed that chronic AFB1 dietary exposure alters the adequate endocrinological physiological cascade response in S. aurata, compromising the expected stress response to an additional stressor, such as overcrowding.

Sex differences in somatotrope response to fasting: biphasic responses in male mice.

Anterior pituitary somatotropes are important metabolic sensors responding to leptin by secreting growth hormone (GH). However, reduced leptin signals caused by fasting have not always correlated with reduced serum GH. Reports show that fasting may stimulate or reduce GH secretion, depending on the species. Mechanisms underlying these distinct somatotrope responses to fasting remain unknown. To define the somatotrope response to decreased leptin signaling we examined markers of somatotrope function over different time periods of fasting. Male mice were fasted for 24 and 48 h, with female mice fasted for 24 h compared to fed controls ad libitum. Body weight and serum glucose were reduced in both males and females, but, unexpectedly, serum leptin was reduced only in males. Furthermore, in males, serum GH levels showed a biphasic response with significant reductions at 24 h followed by a significant rise at 48 h, which coincided with the rise in serum ghrelin levels. In contrast, females showed an increase in serum GH at 24 h. We then explored mechanisms underlying the differential somatotrope responses seen in males and observed that pituitary levels of Gh mRNA increased, with no distinction between acute and prolonged fasting. By contrast, the Ghrhr mRNA (encoding GH releasing hormone receptor) and the Ghsr mRNA (encoding the ghrelin receptor) were both greatly increased at prolonged fasting times coincident with increased serum GH. These findings show sex differences in the somatotrope and adipocyte responses to fasting and support an adaptive role for somatotropes in males in response to multiple metabolic signals.

Effects of 17β-Estradiol on growth-related genes expression in female and male spotted scat (Scatophagus argus).

Growth hormone (GH) is the most important endocrine factor to regulate somatic growth. Spotted scat (Scatophagus argus) is a famous marine aquaculture species in China with a typical sexual growth dimorphism in which females grow faster and larger than males. In this study, gh messenger RNA (gh mRNA) and GH protein expression were examined in the pituitary glands of female and male spotted scat. Based on qPCR analysis, gh mRNA was mainly expressed in the pituitary gland, and weakly in the gonads and hypothalamus. Furthermore, gh mRNA expression in the pituitary gland was significantly higher in females at stages II-IV than in males at stages III-V. In addition, gh mRNA was highly expressed in the ovary and testis during mature development stages. In this study, spotted scat GH polyclonal antibody was produced. Western blot analysis showed that the molecular weight of spotted scat GH was about 21 KDa. Immunohistochemistry (IHC) in pituitary glands showed that GH was mainly expressed in the proximal pars distal (PPD) and a few cells were distributed in the rostral pairs distal (RPD). After injecting 17β-Estradiol (E2) in vivo, gh mRNA expression was significantly up-regulated in the pituitary gland, whereas igf1 and ghr1 mRNA levels were down-regulated in the liver, which might regulate gh mRNA expression in the pituitary gland. These results provide valuable insight into the molecular mechanisms of E2 regulating gh expression in spotted scat.

Association of GH polymorphisms with growth traits in buffaloes

Members of the somatotrophic axis, especially GH and IGF1, are essential for growth. The association between GH polymorphisms and growth traits was numerously studied in cattle; however, no data are available for such association studies in buffalo. Therefore, this study was conducted to screen for polymorphisms in the GH gene and to study their putative association with growth traits in 200 Egyptian buffaloes. Polymerase chain reaction single-strand conformation polymorphism and sequencing were applied to look for polymorphisms in 3 loci spanning all exons and introns of buffalo GH. The C (MspI+) >T (MspI-) SNP in intron3, which is well known in cattle, was not detected in the examined buffaloes. However, 2 missense mutations were detected in exon5: one previously detected p.Leu153Val SNP, with very low frequencies for the mutant (Val) allele and one novel p.Asn174His SNP. At weaning age, the p.Leu153Val SNP was significantly associated with weaning body weight and gain with the positive effect of the wild allele (Leu) and higher GH serum, mRNA, and protein levels in animals with Leu/Asn and Leu/His haplotypes. At yearling age, the 2 SNPs associated with yearling weight and gain with positive effect for the mutant (Val and His) alleles with increased GH, and IGF1 serum, mRNA, and protein and GHR mRNA and protein levels in animals with Val/Asn haplotype. Therefore, the selection of Egyptian buffaloes with the Val/Asn haplotype could improve the growth traits of Egyptian buffaloes at yearling age which is the target age for perfect growing.

Molecular identification, tissue distribution and in vitro functional analysis of growth hormone and its receptors in red-spotted grouper (Epinephelus akaara).

Red-spotted grouper (Epinephelus akaara) is one of the high economic value grouper species, however, the knowledge regarding its growth is limited. In this study, full-length cDNAs of growth hormone (gh) and its receptors (ghr1 and ghr2) were cloned from the pituitary and liver of red-spotted grouper, respectively. Tissue distribution analysis showed that gh mRNA was predominantly expressed in the pituitary. ghr1 mRNA was highly expressed in the liver, muscle, fat and gonad, while ghr2 mRNA expression was ubiquitously high in the peripheral tissues. However, the mRNA expression of both ghr isoforms was relatively low in the central nervous system. Secretory recombinant grouper GH (rgGH) was expressed in yeast Pichia pastoris and verified. HEK293T cells transiently transfected with the GHR isoforms were used to elucidate the receptor-mediated signaling pathways related to growth regulation. rgGH activated rapid phosphorylation of Janus kinase 2, signal transducer and activator of transcription 5 (STAT5) and extracellular signal-regulated protein kinase 1/2 through GHR1, but only STAT5 was phosphorylated via GHR2. rgGH strongly activated STAT5 phosphorylation and significantly stimulated ghr1, ghr2 and insulin-like growth factor (igf1, igf2) mRNA expression in primary cultured hepatocytes. Data showed that the recombinant protein rgGH played effects on igf1/2 mRNA expression via GHR-mediated signaling pathways. Our findings provide essential information about GH and GHRs characteristics in red-spotted grouper.

Metabolic signalling to somatotrophs: Transcriptional and post-transcriptional mediators.

In normal individuals, pituitary somatotrophs optimise body composition by responding to metabolic signals from leptin. To identify mechanisms behind the regulation of somatotrophs by leptin, we used Cre-LoxP technology to delete leptin receptors (LEPR) selectively in somatotrophs and developed populations purified by fluorescence-activated cell sorting (FACS) that contained 99% somatotrophs. FACS-purified, Lepr-null somatotrophs showed reduced levels of growth hormone (GH), growth hormone-releasing hormone receptor (GHRHR), and Pou1f1 proteins and Gh (females) and Ghrhr (both sexes) mRNAs. Pure somatotrophs also expressed thyroid-stimulating hormone (TSH) and prolactin (PRL), both of which were reduced in pure somatotrophs lacking LEPR. This introduced five gene products that were targets of leptin. In the present study, we tested the hypothesis that leptin is both a transcriptional and a post-transcriptional regulator of these gene products. Our tests showed that Pou1f1 and/or the Janus kinase/signal transducer and activator of transcription 3 transcriptional regulatory pathways are implicated in the leptin regulation of Gh or Ghrhr mRNAs. We then focused on potential actions by candidate microRNAs (miRNAs) with consensus binding sites on the 3' UTR of Gh or Ghrhr mRNAs. Somatotroph Lepr-null deletion mutants expressed elevated levels of miRNAs including miR1197-3p (in females), miR103-3p and miR590-3p (both sexes), which bind Gh mRNA, or miRNA-325-3p (elevated in both sexes), which binds Ghrhr mRNA. This elevation indicates repression of translation in the absence of LEPR. In addition, after detecting binding sites for Musashi on Tshb and Prl 3' UTR, we determined that Musashi1 repressed translation of both mRNAs in in vitro fluc assays and that Prl mRNA was enriched in Musashi immunoprecipitation assays. Finally, we tested ghrelin actions to determine whether its nitric oxide-mediated signalling pathways would restore somatotroph functions in deletion mutants. Ghrelin did not restore either GHRH binding or GH secretion in vitro. These studies show an unexpectedly broad role for leptin with respect to maintaining somatotroph functions, including the regulation of PRL and TSH in subsets of somatotrophs that may be progenitor cells.

A combination of rapeseed, cottonseed and peanut meal as a substitute of soybean meal in diets of Yellow River carp Cyprinus carpio var

A feeding trial was conducted to evaluate the results of replacing soybean meal (SBM) with other blend plant protein (BP) (rapeseed meal, cottonseed meal and peanut meal) sources on growth, fish body composition, biochemical parameters, non-specific immune index and gene expression of growth hormone/insulin-like growth factor-1 in Yellow River carp Cyprinus carpio. The results showed that the 600 g/kg replacement with BP in diet did not affect the growth performance of Yellow River carp, but the 800 g/kg SBM replacement diet could depress the growth of fish (p < .05). The 800 g/kg SBM replacement diet significantly impacted the superoxide dismutase, malondialdehyde contents, lysozyme, alanine aminotransferase and aspartate aminotransferase activities (p < .05). Fish fed 800 g/kg SBM replacement diet showed lowest insulin-like growth factor 1 and highest growth hormone level than that of other diets (p < .05). Both gene expression levels of GH and IGF-I in hepatic showed significant difference among dietary treatments (p < .05), and the lowest GH and IGF-I mRNA level in liver were found in fish fed 800 g/kg SBM replacement diet (p < .05). In conclusion, no more than 600 g/kg SBM could be replaced by BP in diet of Yellow River carp without adverse effects on the growth performance. However, 800 g/kg SBM replacement with BP in diet inhibited the growth performance, affected some blood parameters and immune response index, and down-regulated GH and IGF-1 gene expression of liver in Yellow River carp. Further, GH level in serum was negatively correlated with GH mRNA level in liver; meanwhile, serum concentrations of IGF-I were positively correlated with hepatic IGF-I mRNA expressions.

Molecular skin changes in Cushing syndrome and the effects of treatment.

We investigated newly diagnosed patients with endogenous CS for molecular changes in skin by biopsy before and a year after treatment of CS. 26 Patients with CS and 23 healthy controls were included. All the patients were evaluated before and a year after treatment. Skin biopsies were obtained from abdominal region before and a year after treatment in patients with CS and once from healthy volunteers. Total RNA was isolated from the skin biopsy samples and the real-time PCR system was used to determine the expression levels of 23 genes in the skin biopsy. Skin expression levels of HAS 1, 2 and 3 mRNAs were lower and COL1A2, COL2A1, COL3A1 mRNAs were higher in patients with CS than in normal controls. MMP-9, TIMP-1 and elastin mRNA expression levels were similar in two groups. Skin IL-1β mRNA expression level was significantly higher in patients with CS. None of these parameters changed significantly 12months after treatment. Patients with CS showed higher skin GH and HSD11B1 mRNA expressions and lower GHR and IGF-1R mRNA expression compared to control. Expression levels of IGF-1, GR and HSD11B2 mRNA were similar in two groups. None of these parameters changed significantly 12months after treatment. CS is associated with increased expression levels of skin COL1A2, COL2A1, COL3A1 mRNAs (which are correlated with increased expression level of skin GH mRNA). Decreased skin HAS may cause decreased synthesis of HA that contributes to thinning of skin in CS. Increased local inflammatory cytokine and HSD11B1 mRNAs may be related to the acne formation in CS. Treatment of CS was not able to reverse these changes and ongoing changes were detected after treatment.

OR16-06 Age-Associated Local GH Promotes Colon Neoplasia

Colon polyp and cancer frequency increase with age, yet little is known about age-related mechanisms underlying development of these neoplasms. Defective DNA damage response and accumulation of unrepaired DNA damage can trigger genomic instability and cellular transformation.Patients with acromegaly have a higher prevalence of colon polyps and, arguably, colon adenocarcinoma, while those with GH signaling deficiency do not develop cancer. We showed that APC+/- mice that all develop colon adenomas exhibit a significant decrease in the number and volume of colon tumors with deletion of the GH transcription factor Prop1. Further, DNA damage response triggered GH expression in colon cells, and GH, in turn, altered DNA damage repair, resulting in DNA damage accumulation and cell transformation both in vitro and in vivo. These findings prompted us to hypothesize that accumulated DNA damage in aging colon induces local GH, suppressing DNA damage repair and creating a milieu consistent with genomic instability favoring neoplastic development.In human colon tissue we now show increased expression of γH2AX, a marker of DNA breaks, associated with increased GH transcription (detected by RNA scope) and translation (assessed by immunohistochemistry) as well as GH induction in both human and murine colon after DNA damaging radiotherapy. In vitro studies support these results, showing GH induction in normal human colon epithelial cells, fibroblasts, and 3D human intestinal organoids after DNA damage. Of note, local GH was secreted in the medium, indicating a paracrine effect. Paracrine/autocrine GH expression in these cellular models resulted in suppression of p53, induction of EMT, and attenuation of DNA damage response, and accumulated unrepaired DNA damage in human colon cells and in human intestinal organoids. In vivo, colon cells infected with lentivirus expressing GH generated more metastases than did cells expressing control vector. Co-culturing of human normal colon fibroblasts expressing GH together with normal human colon cells led to increased motility and accumulation of DNA damage as well as increased proliferation of epithelial cells on a gut-on-a-chip microfluidic device, confirming paracrine GH effects. In an in vitro model of aging, culturing human intestinal organoids for up to 2 months resulted in decreased telomere length and increased GH mRNA and protein expression associated with suppressed DNA damage response evident by decreased phosphorylation of ATM and DNA-PKcs, both kinases involved in DNA repair, and DNA damage accumulation assessed by Comet assay. Suppression of GH in these aging organoids led to increased phospho-p53 and reduced DNA damage. Although somatotroph axis endocrine activity decreases with age, local GH induced in response to age-related DNA damage may trigger a “field change,” creating a milieu favorable for colon neoplastic development.

MON-LB014 Autosomal Dominant Growth Hormone Deficiency Due to a Novel c.178g&amp;gt;A Mutation in the GH1 Gene Is Caused by Alternative Splicing to Produce a Small GH Isoform

Introduction: Growth hormone (GH) plays a vital role in human physiology. Mutations in GH1 cause isolated growth hormone deficiency (GHD). The most frequent cause of familial growth hormone deficiency is Type II autosomal dominant GHD (isolated GHD type II) due to several heterozygous GH1 mutations. These mutations have been shown to (a) produce shorter isoforms of GH that do not bind to growth hormone receptors, (b) cause diminished secretion of GH, or (c) result in misfolded GH protein. Methods: Genomic DNA from patients with familial GHD was enriched for the coding exons using hybrid capture technology, and GH1 was sequenced using Next Generation Sequencing technology. The p.A34T mutant protein was expressed in bacteria, and binding to GHR was studied by surface plasmon resonance technology. Computational prediction of transcription indicated that alternative splicing is likely to produce a shorter GH variant with skipping of exon 3 in GH1. Mammalian cell-based studies incorporating transfection of whole GH1 gene containing exons/introns were used to study transcriptional effects. RNA was isolated from cells transfected with WT and mutant GH1 gene and analyzed by RT PCR using primers in the second and fifth exons of GH1 that could identify all possible isoforms of GH1 mRNA. Results: GHD was identified in three female siblings aged 3.25-6.33 years (Ht SDS -3.21 to -1.13, peak GH 2.9-6.6 ng/mL); their mother had previously been diagnosed with GHD at age 12.33 years (Ht SDS -3.44, GH peak < 2 ng/mL). Sequencing of GH1 identified a novel heterozygous variant (c.178G>A; p.Ala34Thr) not found in the Broad ExAc dataset representing >60,000 children without the severe childhood-onset disease. Functional studies using whole gene transfection showed that the c.178G>A mutation leads to alternate splicing resulting in increased production of the shorter 17.5kD isoform of GH due to exon 3 skipping. Results were confirmed by quantitative RT-PCR as well as GH secretion assays, which showed a lower level of GH production from cells transfected with the GH1 gene containing the c.178G>A mutation. The SPR based receptor binding assay and cell proliferation assay using bacterially expressed proteins showed that once produced, the GH protein with the A34T mutation behaves similar to WT GH protein. All these results confirm that the cause of GHD due to the c.178G>A mutation in GH1 is due to altered transcription leading to the production of the shorter 17.5 kD isoform of GH protein and not due to the amino acid change A34T that is caused by the mutation. Conclusion: The presence of a heterozygous GH1 variant (c.178G>A, p.Ala34Thr) in four individuals with GHD suggests that this is a novel cause of IGHD type II. Production of the smaller 17.5 kD GH isoform results in reduced overall GH secretion and loss of binding to GHR due to competition with the normal GH protein, explaining the dominant-negative phenotype.

MON-712 Restoration of Growth and Fertility in Zebrafish (Danio Rerio) Model with PROP1 Knockout Generated by CRISPR/Cas9 Genomic Editing

Introduction: Hypopituitarism is defined as the deficiency of one or more pituitary hormones and can occur due to pathogenic allelic variants in transcription factors involved in pituitary development. PROP1 gene is responsible for progenitor cell migration from the marginal zone to the anterior lobe, and its terminal differentiation into corticotropes and gonadotropes cell lines besides somatotropes, lactotropes and thyrotropes due to POU1F1 (also known as PIT1) activation. In humans, mutations in the PROP1 gene are the most common cause of congenital hypopituitarism with GH, TSH, LH/FSH, and progressive ACTH deficiencies. A dwarf phenotype with short stature, pituitary hormone deficiency, and infertility has been described in humans and Ames mice lineage harboring mutations in the PROP1/Prop1 gene. Another valuable animal model used in basic research is the zebrafish (Danio rerio) due to a high homology in neuroendocrine functioning. To test the potential of this model, in our previous study, a 32bp insertion carrying a stop codon was directed into the second exon of prop1 with CRISPR/Cas9, establishing a homozygous mutant strain (prop1mut). Objective: To characterize the phenotype and expression patterns of transcription factors and hormones in the zebrafish prop1mut lineage. Methods: prop1, pit1, and gh1 mRNA levels were analyzed during embryonic development at 24 and 72 hours post-fertilization (hpf). RNA from 30 pooled embryos was extracted using DirectZol RNA Miniprep. cDNA was synthesized from 1ug of total RNA using High-Capacity cDNA Reverse Transcription Kit and qPCR was performed using SYBR Green PCR Master Mix. Gene expression was normalized to ef1a and the prop1mut group was compared with the control wild type group (WT). Animals were kept in the tanks at a density of 15 animals/liter and images were acquired at 13 and 20 days post fertilization (dpf) after brief anesthetization using a stereomicroscope and measured in ImageJ software to determine the larval standard length from nose to the end of the spinal cord. Results: At 24 and 72hpf, prop1mut embryos expressed the altered prop1 mRNA at similar levels to the prop1 expression observed in WT. Lower pit1 expression in prop1mut embryos was observed at both periods (p<0.01). Albeit in low levels, similar gh1 expression was observed in both lineages at 24hpf, and prop1mut embryos presented lower gh1 expression at 72hpf (p<0.001). prop1mut larvae presented a significant decrease in size at 13dpf (p<0.001) but not at 20dpf. Conclusion: In this study, the prop1mut zebrafish model exhibited a dwarf phenotype during larval development associated with diminished pit1 and gh1 expression during the embryonic stage. Additionally, in the juvenile stage, the development rate in prop1mut animals was restored, presenting similar standard lengths observed in WT animals.

Somatotrope Responses to Acute and Prolonged Loss of Leptin Signaling

In mice and humans, fasting results in a rise of serum growth hormone (GH). In mice, this coincides with increased Gh and growth hormone releasing hormone receptor (Ghrhr) mRNA. Fasting also results in increased serum ghrelin and decreased serum leptin. We have shown that pituitary somatotropes are dependent on circulating leptin to maintain sufficient stores of GH and GHRHR proteins. We hypothesized that the GH rise seen in fasted mice with reduced serum leptin was due to ghrelin‐stimulated pathways. However, a review of the literature revealed a study by Steyn et al. (2011) which demonstrated an acute decline in GH pulses in male mice after 12–18 h of fasting. This study suggests that somatotropes may have time‐dependent responses, with shorter fasting responses driven by the absence of leptin signals.Our fasting study was composed of three groups of male mice: 1) mice fasted for 6, 12, 24, or 48 hours, 2) mice fasted for 24 and 48 hours with 10% glucose in the drinking water, and 3) mice fed ad libitum. Body weight and serum glucose were significantly reduced with all fasting times. After 6 h of fasting, serum leptin levels were reduced from 9625.63 pg/ml (fed controls) to 1798.15 pg/mL with a further reduction to 774.17 pg/ml after 12 h, which was maintained following 24 and 48 h fasting. Ghrelin levels were unchanged with 6, 12 and 24 h fasting but significantly increased by 55.4% with 48 h fasting. Serum GH was unchanged with 6 and 12 h fasting, but was significantly reduced from 7765 pg/ml (fed) to 2253 pg/ml after the 24 h fast (p=0.03 ANOVA). In the 48 h fasted mice, serum GH was significantly increased over fed mice, from 1828 pg/ml (fed) to 10569 pg/ml (p=0.01 ANOVA). There were significant increases in pituitary Ghrhr mRNA after 24 and 48 h fasting and Gh mRNA was increased after 6 and 48 h of fasting. However, GH and GHRHR content were unchanged.This study has uncovered time‐dependent differential responses to fasting by somatotropes in male mice. The reduced serum GH after 24 h correlated well with the reduction in serum leptin. Similarly, the prolonged fasting (48 h) and elevated serum GH correlated well with the rise in ghrelin. The high Gh and Ghrhr mRNA levels corresponds with rising ghrelin levels. Reduced serum GH at 24 h indicates that somatotropes remain dependent on leptin signals for GH secretion. However, with normal GH or GHRHR content, this points to a defect in a signaling pathway needed for secretion that has yet to be determined.Support or Funding InformationNIH grants 5 R01 HD087057‐03 (GVC and AM), 5 R01 HD 093461‐01 (AM and GVC), and 5 R01 DK113776‐02 (GVC and AM). NIGMS grants P20 GM103425 and P30GM11070 (PI Dr. Edgar Garcia‐Rill). NSF funded Southern Regional Education Board Dissertation Award (TKM).

Isolation of Growth Hormone-Releasing Hormone and Its Receptor Genes from Scatophagus argus and Their Expression Analyses

The teleost Scatophagus argus is a species whose females grows faster than males. Growth hormone (gh) mRNA abundance in females pituitary is higher than that in males; however the mechanism underlining such differential is still unknown. Growth hormone (GH) is tightly associated with GH-releasing hormone (Ghrh) in vertebrates. In this study, Ghrh gene (ghrh) and its receptor gene, ghrhr, were isolated from S. argus. Tissue expression analysis showed that ghrh and ghrhr were mainly expressed in hypothalamus while ghrhr was expressed in pituitary and gh was predominantly expressed in pituitary. Twenty cultured S. argus individuals were used to compare ghrh, ghrhr and gh mRNA abundances, 120 g and 181 g average weight for male (n=11) and female (n = 9), respectively. Real-time PCR indicated that the ghrh and ghrhr mRNA abundances in male hypothalamus were significantly higher than those in female hypothalamus while that of gh mRNA abundance was significantly higher in female pituitary than in male pituitary. The ghrh and ghrhr mRNA abundances were significantly up-regulated in female hypothalamus 3h after injection of 0.1 mg kg−1 body weight Ghrh while pituitary ghrhr and gh mRNA abundances were not affected. In female hypothalamus, ghrh and ghrhr mRNA abundances were not affected at 6h post-injection of 4mg kg−1 body weight 17α-methyltes-tosterone (17α-MT) or 17β-Estradiol (E2). In female pituitary, ghrhr mRNA abundance was down-regulated by 17α-MT while that of gh mRNA abundance was up-regulated by E2. Our findings indicated that E2, rather than Ghrh, plays an important role in up-regulating the expression of gh in female S. argus, which should aid to understand the sexual dimorphism of teleost growth.

29P - Characterisation of growth hormone signal transduction in primary melanoma cell lines

BackgroundGrowth hormone (GH) signalling is initiated by binding of GH to the GH receptor (GHR) and activation of Janus kinase 2, which in turn, promotes phosphorylation of STAT5, PI3K/AKT and MAPK. High levels of GHR expression have been reported in melanoma. This project aims to characterise GH signalling in primary melanoma cell lines and identify cell lines which respond to GH and GHR inhibition, with a view to testing in preclinical xenograft studies. MethodsOver 24 cell lines established at the Auckland Cancer Society Research Centre (ACSRC) in New Zealand were tested for response to GH, prolactin (PRL) and a GHR antagonist, by assessing STAT5 phosphorylation by western blot. mRNA expression was assessed by Nanostring PlexSet. Endogenous protein was assessed by ELISA. ResultsFrom the panel of melanoma cell lines, 62% responded to GH stimulation and GHR inhibition. mRNA expression analysis demonstrated that the IGF1 receptor and GHR were highly expressed across cell lines. Most of the cell lines expressing GHR mRNA responded to GH. GH1 mRNA was expressed at very low levels in a subset of cell lines, and ELISA analysis confirmed the presence of endogenous GH protein. siRNA-mediated knockdown of the GHR reduced GHR mRNA expression and STAT5 phosphorylation by GH. GH increased cell viability in a subset of melanoma cell lines. A vemurafenib-resistant melanoma cell line and B16/F10 mouse cell line were also sensitive to GH and GHR inhibition. Exon analysis will be performed for GH and related genes. ConclusionsIn this study, we have demonstrated GH stimulates signal transduction and promotes enhance of viability of primary melanoma cell lines, and that GHR blockade prevents this effect, indicating that blocking GHR signalling might be a useful strategy to treat melanoma. The next step is to understand whether GHR inhibition plays a role in regulating tumour growth in vivo. Editorial acknowledgementLi Family PhD Scholarship. Legal entity responsible for the studyPeter Shepherd. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Roles of differential expression of miR-543-5p in GH regulation in rat anterior pituitary cells and GH3 cells.

Growth hormone (GH) is an important hormone released by the pituitary gland that plays a key role in the growth and development of organisms. In our study, TargetScan analysis and the dual luciferase reporter assays were used to predict and screen for miRNAs that might act on the rat Gh1 gene, and we identified miR-543-5p. Then, the GH3 cell line and the primary rat pituitary cells were transfected with miRNA mimic, inhibitor, and siRNA. We detected the Gh1 gene expression and the GH secretion by real-time PCR and ELISAs, respectively, to verify the regulatory effect of miR-543-5p on GH secretion. The results showed that miR-543-5p can inhibit Gh1 mRNA expression and reduce GH secretion. MiR-543-5p inhibitor upregulated Gh1 mRNA expression and increased GH secretion compared with the negative control. In summary, miR-543-5p downregulates Gh1 expression, resulting in a decrease in GH synthesis and secretion, which demonstrates the important role of miRNAs in regulating GH and animal growth and development.

The Involvement of Hormones in Fish: Basic and Applied Aspects of Russian Sturgeon (Acipenser Gueldenstaedtii)

Fish are a very large group, containing a huge variety of over 33,000 species.Scientific knowledge about the interaction between hormones of the somatic axis (SA) and the gonadotropic axis (GA), both of which function in the brain, and the pituitary gonad axis (BPG) that controls growth and reproduction, is vital for the domestication of fish in aquaculture.The Acipenserida family comprises 27 species, some of which have a very high economic value, among them Russian sturgeon (Acipenser gueldenstaedtii) that adapted to growth in aquaculture conditions. Many aspects aimed at improving the adaptation and production of Russian sturgeon (Acipenser gueldenstaedtii) have been studied for quite a long time. Models based on the results of our and others’ studies describing the interaction between GA and SA during oogenesis in Russian sturgeon have been suggested. The mRNA relative level of FSH during vitellogenesis (VTL) was higher in females than in males, affecting VTL secretion of vitellogenin (Vg); however, it was lower in the pre-vitellogenic stage than in VTL. No difference was found in mRNA levels of the luteinizing hormone (LH) in Russian sturgeon during the first four years of growth. During its first five years of growth, the level of GH mRNA was higher in females than in males, but due to the high standard deviation of the mean, the difference was not significant. IGF-I mRNA expression differed between the various tissues.

Effects of high and low temperature on expression of GnIH, GnIH receptor, GH and PRL genes in the male grass puffer during breeding season

Gonadotropin-inhibitory hormone (GnIH) is a multifunctional hypophysiotropic neurohormone and has a stimulatory role in the control of reproduction in the grass puffer. To clarify the neuroendocrine mechanisms underlying the effect of changes in water temperature on reproduction in fish, we previously revealed that, in parallel to gonadal regression, both low and high temperature significantly decreased the expressions of the genes encoding kisspeptin (kiss2), kisspeptin receptor (kiss2r), gonadotropin-releasing hormone 1 (gnrh1) in the brain and gonadotropin (GTH) subunits (fshb and lhb) in the pituitary of sexually mature male grass puffer. In this study, we examined the changes in expression of gnih and GnIH receptor gene (gnihr) in the brain and pituitary along with the genes for growth hormone (gh) and prolactin (prl) in the pituitary of male grass puffer exposed to low temperature (14 °C), normal temperature (21 °C, as initial control) and high temperature (28 °C) conditions for 7 days. The levels of gnih and gnihr mRNAs were significantly decreased in both low and high temperature conditions compared to normal temperature in the brain and pituitary. Similarly, the gh mRNA levels were significantly decreased in both low and high temperature conditions. The prl mRNAs showed no significant changes at high temperature, whereas drastically decreased at low temperature possibly by dysfunctional cold stress. Taken together, the present results suggest that, in addition to the inhibitory effect of temperature changes on the Kiss2/GnRH1/GTH system, the suppression of GnIH/GH system may also be involved in the termination of reproduction by high temperature at the end of breeding season.