scholarly journals GH-IGF Axis in Sparus aurata: Possible Applications to Genetic Selection

2000 ◽  
Author(s):  
Bruria Funkenstein ◽  
Cunming Duan
Keyword(s):  
Growth Rate ◽  
Cellular Localization ◽  
Sparus Aurata ◽  
Gonadal Development ◽  
Developmental Expression ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Gh Receptor ◽  
Igf I ◽  
Igf Receptor

Many factors affect growth rate in fish: environmental, nutritional, genetics and endogenous (physiological) factors. Endogenous control of growth is very complex and many hormone systems are involved. Nevertheless, it is well accepted that growth hormone (GH) plays a major role in stimulating somatic growth. Although it is now clear that most, if not all, components of the GH-IGF axis exist in fish, we are still far from understanding how fish grow. In our project we used as the experimental system a marine fish, the gilthead sea bream (Sparus aurata), which inhabits lagoons along the Mediterranean and Atlantic coasts of Europe, and represents one of the most important fish species used in the mariculture industry in the Mediterranean region, including Israel. Production of Sparus is rapidly growing, however, in order for this production to stay competitive, the farming of this fish species has to intensify and become more efficient. One drawback, still, in Sparus extensive culture is that it grows relatively slow. In addition, it is now clear that growth and reproduction are physiological interrelated processes that affect each other. In particular sexual maturation (puberty) is known to be closely related to growth rate in fish as it is in mammals, indicating interactions between the somatotropic and gonadotropic axes. The goal of our project was to try to identify the rate-limiting components(s) in Sparus aurata GH-IGF system which might explain its slow growth by studying the ontogeny of growth-related genes: GH, GH receptor, IGF-I, IGF-II, IGF receptor, IGF-binding proteins (IGFBPs) and Pit-1 during early stages of development of Sparus aurata larvae from slow and fast growing lines. Our project was a continuation of a previous BARD project and could be divided into five major parts: i) obtaining additional tools to those obtained in the previous project that are necessary to carry out the developmental study; ii) the developmental expression of growth-related genes and their cellular localization; iii) tissue-specific expression and effect of GH on expression of growth-related genes; iv) possible relationship between GH gene structure, growth rate and genetic selection; v) the possible role of the IGF system in gonadal development. The major findings of our research can be summarized as follows: 1) The cDNAs (complete or partial) coding for Sparus IGFBP-2, GH receptor and Pit-1 were cloned. Sequence comparison reveals that the primary structure of IGFBP-2 protein is 43-49% identical to that of zebrafish and other vertebrates. Intensive efforts resulted in cloning a fragment of 138 nucleotides, coding for 46 amino acids in the proximal end of the intracellular domain of GH receptor. This is the first fish GH receptor cDNA that had been cloned to date. The cloned fragment will enable us to complete the GH - receptor cloning. 2) IGF-I, IGF-II, IGFBP-2, and IGF receptor transcripts were detected by RT-PCR method throughout development in unfertilized eggs, embryos, and larvae suggesting that these mRNAs are products of both the maternal and the embryonic genomes. Preliminary RT-PCR analysis suggest that GH receptor transcript is present in post-hatching larvae already on day 1. 3) IGF-1R transcripts were detected in all tissues tested by RT-PCR with highest levels in gill cartilage, skin, kidney, heart, pyloric caeca, and brain. Northern blot analysis detected IGF receptor only in gonads, brain and gill cartilage but not in muscle; GH increased slightly brain and gill cartilage IGF-1R mRNA levels. 4) IGFBP-2 transcript were detected only in liver and gonads, when analyzed by Northern blots; RT-PCR analysis revealed expression in all tissues studied, with the highest levels found in liver, skin, gonad and pyloric caeca. 5) Expression of IGF-I, IGF-II, IGF-1R and IGFBP-2 was analyzed during gonadal development. High levels of IGF-I and IGFBP-2 expression were found in bisexual young gonads, which decreased during gonadal development. Regardless of maturational stage, IGF-II levels were higher than those of IGF-L 6) The GH gene was cloned and its structure was characterized. It contains minisatellites of tandem repeats in the first and third introns that result in high level of genetic polymorphism. 7) Analysis of the presence of IGF-I and two types of IGF receptor by immunohistochemistry revealed tissue- and stage-specific expression during larval development. Immunohistochemistry also showed that IGF-I and its receptors are present in both testicular and ovarian cells. Although at this stage we are not able to pinpoint which is the rate-limiting step causing the slow growth of Sparus aurata, our project (together with the previous BARD) yielded a great number of experimental tools both DNA probes and antibodies that will enable further studies on the factors regulating growth in Sparus aurata. Our expression studies and cellular localization shed new light on the tissue and developmental expression of growth-related genes in fish.

scholarly journals Primary GH insensitivity '(Laron syndrome) caused by a novel 4 kb deletion encompassing exon 5 of the GH receptor gene: effect of intermittent long-term treatment with recombinant human IGF-I

2004 ◽  
pp. 635-642 ◽  
Author(s):  
A Besson ◽  
S Salemi ◽  
A Eble ◽  
F Joncourt ◽  
S Gallati ◽  
...  
Keyword(s):  
Stop Codon ◽  
Receptor Gene ◽  
Standard Deviation Score ◽  
Pcr Analysis ◽  
Laron Syndrome ◽  
Rt Pcr ◽  
Gh Receptor ◽  
Ghr Gene ◽  
Long Term Treatment ◽  
Igf I

OBJECTIVE: GH insensitivity syndrome (GHIS; Laron syndrome) is clinically characterized by severe postnatal growth failure and very low serum levels of IGF-I despite increased secretion of GH. This mainly autosomal recessive syndrome is clinically indistinguishable from isolated GH deficiency (IGHD). Fifty-one different mutations in the GH receptor (GHR) gene have been discovered, whereas only three deletions causing the disorder have been reported so far. In this report, we describe a consanguineous family from Sri Lanka with a novel deletion of 4097 bp in length encompassing exon 5. SUBJECTS AND METHODS: Parents of normal phenotype presented their second child (boy) to our clinic at the age of 7 months with severe growth retardation and the clinical features of IGHD (58 cm, -6.1 standard deviation score (SDS); 5.7 kg, -3.4 SDS). Assessment, however, revealed GHIS with absent GH-binding protein. Thereafter, the patient received intermittent recombinant human IGF-I (rhIGF-I; 80 microg/kg twice daily) treatment prepubertally for 5.5 years. Genomic DNA was extracted for genetic analysis and each exon was PCR amplified individually. Further, in order to amplify the GHR gene from exon 4 to 6, Expand Long Template PCR (Roche) was carried out. In addition, RNA isolation and RT-PCR were performed. RESULTS: Separate PCRs of each of the exons of the GHR gene revealed that exon 5 in the patient was missing. Thereafter, "Long PCR" from exons 4 to 6 revealed a 4097 bp deletion encompassing exon 5, in a homozygous state in the patient and in a heterozygous state in both parents. RT-PCR analysis revealed an exact absence of exon 5 resulting in a frameshift, leading to a stop codon in exon 6, which predicts a truncated, non-functional GHR protein. CONCLUSION: Fifty-one different mutations within the GHR gene causing GHIS have been reported so far. In contrast, only three deletions within the GHR gene are known. We describe a patient suffering from GHIS caused by a novel 4 kb deletion of the GHR gene encompassing exon 5 and, additionally, we focus on the effect of intermittent rhIGF-I treatment during prepuberty.

scholarly journals Amniotic IGF-I supplementation of growth-restricted fetal sheep alters IGF-I and IGF receptor type 1 mRNA and protein levels in placental and fetal tissues

2005 ◽  
Vol 186 (1) ◽  
pp. 145-155 ◽  
Author(s):  
S Shaikh ◽  
F H Bloomfield ◽  
M K Bauer ◽  
H H Phua ◽  
R S Gilmour ◽  
...  
Keyword(s):  
Late Gestation ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Igf Binding Proteins ◽  
Fetal Sheep ◽  
Hepatic Expression ◽  
Protein Levels ◽  
Igf I ◽  
Igf Receptor

We have previously reported that chronic intra-amniotic supplementation of the late gestation growth-restricted (IUGR) ovine fetus with IGF-I (20 μg/day) increased gut growth but reduced liver weight and circulating IGF-I concentrations. Here we report mRNA and protein levels of IGF-I, the type 1 IGF receptor (IGF-1R) and IGF-binding proteins (IGFBP)-1, -2 and -3 in fetal gut, liver, muscle and placenta from fetuses in that earlier study in an attempt to explain these contrasting results. mRNA and protein were extracted from tissues obtained at post mortem at 131 days of gestation (term, 145 days) from three groups of fetuses (control, IUGR+saline and IUGR+IGF-I, n=9 per group). Control fetuses were unembolised and untreated. In the IUGR groups, growth restriction was induced from 113 to 120 days by placental embolisation; from 120 to 130 days fetuses were treated with daily intra-amniotic injections of either saline or 20 μg IGF-I. mRNA was measured by RT-PCR or real-time RT-PCR, and protein by Western blot. In liver, muscle and placenta, IGF-I mRNA and protein levels were reduced by between 8 and 30% in IGF-I-treated fetuses compared with saline-treated fetuses and controls with no change in IGF-1R mRNA or protein levels. In contrast, in the gut, IGF-I mRNA and protein levels were not significantly altered with IGF-I treatment, but IGF-1R levels were increased, especially in the jejunum. Immunolocalisation demonstrated that IGF-1R expression was confined to the luminal aspect of the gut. mRNA levels of all three IGFBPs were reduced in the gut of IGF-I-treated fetuses, but hepatic expression was significantly increased. These data demonstrated tissue-specific regulation of IGF-I, IGF-1R and IGFBPs-1, -2 and -3 in response to intra-amniotic IGF-I supplementation, though the underlying mechanisms remain obscure.

Developmental expression of the prolactin receptor gene in rat gonads

1995 ◽  
Vol 147 (3) ◽  
pp. 497-505 ◽  
Author(s):  
F-P Zhang ◽  
A Rannikko ◽  
J Toppari ◽  
A Bartke ◽  
I Huhtaniemi
Keyword(s):  
Gene Expression ◽  
Short Form ◽  
Receptor Gene ◽  
Prolactin Receptor ◽  
Gonadal Development ◽  
Developmental Expression ◽  
Fetal Life ◽  
Rt Pcr ◽  
Rat Testis ◽  
And Function

Abstract The prolactin receptor (PRLR) is a member of the cytokine/prolactin/GH receptor family, and it is widely expressed in various mammalian tissues. Expression of the two different forms of PRLR, differing in the length of their cytoplasmic domains, was studied in rat gonads during fetal and postnatal development. The two forms of PRLR mRNA were analyzed by reverse transcription (RT)-PCR using primer pairs specific for the different forms. The specificity of the cDNA species generated by RT-PCR was verified by Southern hybridization using nested 32P-labeled oligonucleotides. The results indicated that both forms of PRLR mRNA are expressed in the rat testis and ovary, which is in agreement with previous reports. The onset of expression of the two PRLR forms occurs on day 14·5 of fetal life in rat testis. In the ovary, the long form of PRLR mRNA appears 1 day before the short form, i.e. these forms begin to be expressed on fetal days 14·5 and 15·5 respectively. In situ hybridization with antisense cRNA probes specific to each form of the PRLR mRNAs demonstrated specific hybridization of both forms, localized in Leydig cells from day 18·5 of fetal life and at the postnatal ages studied. Compared with our previous findings concerning the ontogeny of LH receptor gene expression, PRLR gene expression starts earlier in development and exhibits no sexual dimorphism. The presence of two forms of PRLR mRNA in the fetal gonads suggest that they might play differential roles in gonadal development and function. Journal of Endocrinology (1995) 147, 497–505

scholarly journals Growth hormone stimulates osteoprotegerin expression and secretion in human osteoblast-like cells

2007 ◽  
Vol 192 (3) ◽  
pp. 639-645 ◽  
Author(s):  
E Mrak ◽  
I Villa ◽  
R Lanzi ◽  
M Losa ◽  
F Guidobono ◽  
...  
Keyword(s):  
Janus Kinase ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Human Osteoblast ◽  
Rt Pcr ◽  
Concentration Dependent Manner ◽  
Gh Receptor ◽  
Igf I ◽  
Intracellular Pathway ◽  
Stimulation Of

It is presently thought that osteoprotegerin (OPG) is a cytokine involved in the regulation of osteoblast/osteoclast crosstalk and maintenance of bone mass. Recent studies showed that GH replacement therapy in GH-deficient patients was able to induce a significant increase of OPG in the plasma, as well as in the cortical and the trabecular bone. In order to determine whether GH could directly modulate OPG secretion, the effect of GH on human osteoblast-like cells (hOB) in primary culture was studied. After detecting the presence of the mRNA for the GH receptor (GHR) by RT-PCR, hOB were exposed to increasing concentrations of GH, from 0.1 to 25 ng/ml, for 24 h. The results showed that GH exposure was able to stimulate OPG secretion in a concentration-dependent manner. In addition, the OPG mRNA levels were increased, indicating that the hormone has a stimulatory effect on gene expression. The stimulatory effect on OPG expression and production was prevented by exposing the cells to tyrphostin AG490 (10 μM), an inhibitor of Janus kinase 2, which is one of the kinases involved in the intracellular pathway activated by the binding of GH to its receptor. Similar results were obtained when the cells were exposed to a receptor antagonist of GH, pegvisomant at 50 nM. GH exposure neither induced an increase in IGF-I expression nor secretion in hOB. These results suggest that the stimulation of OPG production induced by GH in hOB is specific and receptor mediated and further support the view that GH is able to modulate bone remodeling by directly influencing osteoblast–osteoclast crosstalk.

scholarly journals Identification of multiple dmrt1s in catfish: localization, dimorphic expression pattern, changes during testicular cycle and after methyltestosterone treatment

2009 ◽  
Vol 42 (5) ◽  
pp. 437-448 ◽  
Author(s):  
K Raghuveer ◽  
B Senthilkumaran
Keyword(s):  
Sex Differentiation ◽  
Expression Patterns ◽  
Gonadal Development ◽  
Specific Pattern ◽  
Pcr Analysis ◽  
Adult Males ◽  
Rt Pcr ◽  
Testicular Differentiation ◽  
Elevated Expression ◽  
Post Spawning

The double sex and mab-3 related (DM) transcription factor 1 (dmrt1) plays an important role in testicular differentiation. Here, we report cloning of multiple dmrt1s, a full-length and two alternative spliced forms from adult catfish (Clarias gariepinus) testis, which encode predicted proteins of 287 (dmrt1a), 253 (dmrt1b) and 233 (dmrt1c) amino acid residues respectively. Interestingly, dmrt1c lacks the majority of the DM domain. Multiple dmrt1s (dmrt1a and dmrt1c) were obtained from Clarias batrachus as well. Tissue distribution (transcript and protein) of catfish dmrt1 revealed exclusive expression in testis. Semi-quantitative RT-PCR revealed the presence of multiple dmrt1s with high levels of dmrt1a in adult testis but not in ovary. Real-time RT-PCR analysis during testicular cycle showed higher levels of dmrt1 transcripts in preparatory and pre-spawning when compared with spawning and post-spawning phases. Immunocytochemical and immunofluorescence localization revealed the presence of catfish Dmrt1 protein in spermatogonia and spermatocytes, which indicates plausible role in spermatogenesis. Histological analysis indicated initiation of gonadal sex differentiation in catfish around 40–50 days after hatching. The potential role for dmrt1 in testicular differentiation is evident from its stage-dependent elevated expression in developing testis. Furthermore, dimorphic expressions of dmrt1s were evident at different stages of gonadal development or recrudescence in catfish. Treatment of methyl testosterone (MT) during early stages of gonadal sex differentiation resulted in adult males. Interestingly, we also obtained MT-treated fishes having ova-testis gonads. Analysis of dmrt1, sox9a, foxl2 and cyp19a1 expression patterns in MT-treated gonads revealed tissue-specific pattern. These results together suggest that multiple dmrt1s are testis-specific markers in catfish.

Cell-specific localization of insulin-like growth factor binding protein mRNAs in rat liver

2000 ◽  
Vol 278 (3) ◽  
pp. G447-G457 ◽  
Author(s):  
Ellen M. Zimmermann ◽  
Lina Li ◽  
Eileen C. Hoyt ◽  
Jolanta B. Pucilowska ◽  
Steven Lichtman ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Liver Cell ◽  
Target Cells ◽  
Insulin Like Growth Factor ◽  
Specific Expression ◽  
Isolated Rat Hepatocytes ◽  
Gh Receptor ◽  
Igf I ◽  
Igf Receptor

The liver is a major source of circulating insulin-like growth factor I (IGF-I), and it also synthesizes several classes of IGF binding proteins (IGFBPs). Synthesis of IGF-I and IGFBPs is regulated by hormones, growth factors, and cytokines. They are nutritionally regulated and expressed in developmentally specific patterns. To gain insight into cellular regulatory mechanisms that determine hepatic synthesis of IGF-I and IGFBPs and to identify potential target cells for IGF-I within the liver, we studied the cellular sites of synthesis of IGF-I, IGF receptor, growth hormone (GH) receptor, and IGFBPs in freshly isolated rat hepatocytes, endothelial cells, and Kupffer cells. We also localized cellular sites of IGFBP synthesis by in situ hybridization histochemistry. Western ligand and immunoblot analyses were used to determine IGFBP secretion by isolated cells. Two IGF-I mRNA subtypes with different 5′ ends (class 1 and class 2) were detected in all isolated liver cell preparations. Type 1 IGF receptor mRNA was detected in endothelial cells, indicating that these cells are a local target for IGF actions in liver. GH receptor was expressed in all cell preparations, consistent with GH regulation of IGF-I and IGFBP synthesis in multiple liver cell types. The IGFBPs expressed striking cell-specific expression. IGFBP-1 was synthesized only in hepatocytes, and IGFBP-3 was expressed in Kupffer and endothelial cells. IGFBP-4 was expressed at high levels in hepatocytes and at low levels in Kupffer and endothelial cells. Cell-specific expression of distinct IGFBPs in the liver provides the potential for cell-specific regulation of hepatic and endocrine actions of IGF-I.

scholarly journals Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

2021 ◽  
Vol 11 (13) ◽  
pp. 5776
Author(s):  
Varvara G. Blinova ◽  
Natalia S. Novachly ◽  
Sofya N. Gippius ◽  
Abdullah Hilal ◽  
Yulia A. Gladilina ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Inflammatory Reactions ◽  
Effector Cells ◽  
Cycle Regulation ◽  
Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

scholarly journals The C Region of Human Insulin-like Growth Factor (IGF) I Is Required for High Affinity Binding to the Type 1 IGF Receptor

1989 ◽  
Vol 264 (19) ◽  
pp. 11004-11008 ◽  
Author(s):  
M L Bayne ◽  
J Applebaum ◽  
D Underwood ◽  
G G Chicchi ◽  
B G Green ◽  
...  
Keyword(s):  
Growth Factor ◽  
Human Insulin ◽  
Affinity Binding ◽  
Insulin Like Growth Factor ◽  
High Affinity Binding ◽  
High Affinity ◽  
Igf I ◽  
Igf Receptor

scholarly journals Role of Innate Inflammation in the Regulation of Tissue Remodeling during Tooth Eruption

2021 ◽  
Vol 9 (1) ◽  
pp. 7
Author(s):  
Yusuke Makino ◽  
Kaoru Fujikawa ◽  
Miwako Matsuki-Fukushima ◽  
Satoshi Inoue ◽  
Masanori Nakamura
Keyword(s):  
Bacterial Infections ◽  
Tooth Movement ◽  
Tooth Eruption ◽  
Intercellular Adhesion Molecule ◽  
Enamel Organ ◽  
Pcr Analysis ◽  
Maturation Stage ◽  
Oral Epithelium ◽  
Rt Pcr ◽  
Inflammatory Reactions

Tooth eruption is characterized by a coordinated complex cascade of cellular and molecular events that promote tooth movement through the eruptive pathway. During tooth eruption, the stratum intermedium structurally changes to the papillary layer with tooth organ development. We previously reported intercellular adhesion molecule-1 (ICAM-1) expression on the papillary layer, which is the origin of the ICAM-1-positive junctional epithelium. ICAM-1 expression is induced by proinflammatory cytokines, including interleukin-1 and tumor necrosis factor. Inflammatory reactions induce tissue degradation. Therefore, this study aimed to examine whether inflammatory reactions are involved in tooth eruption. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed sequential expression of hypoxia-induced factor-1α, interleukin-1β, and chemotactic factors, including keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2), during tooth eruption. Consistent with the RT-PCR results, immunohistochemical analysis revealed KC and MIP-2 expression in the papillary layer cells of the enamel organ from the ameloblast maturation stage. Moreover, there was massive macrophage and neutrophil infiltration in the connective tissue between the tooth organ and oral epithelium during tooth eruption. These findings suggest that inflammatory reactions might be involved in the degradation of tissue overlying the tooth organ. Further, these reactions might be induced by hypoxia in the tissue overlying the tooth organ, which results from decreased capillaries in the tissue. Our findings indicate that bacterial infections are not associated with the eruption process. Therefore, tooth eruption might be regulated by innate inflammatory mechanisms.

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