Mammalian Growth Hormone Research Articles

Expression, mitogenic activity and regulation by growth hormone of growth hormone/insulin-like growth factor in Branchiostoma belcheri

The growth hormone (GH)/insulin-like growth factor (IGF) axis is unique to all the vertebrate species but its evolutionary origin is ill-defined. We therefore cloned a cDNA encoding Branchiostoma belcheri IGF (BbIGF). BbIGF was expressed in a tissue-specific manner, with the most abundant expression in the hepatic caecum, the putative liver precursor. The recombinant BbIGF expressed in vitro showed mitogenic activity capable of stimulating cell proliferation in the flounder gill, a characteristic of vertebrate IGF. Quantitative real-time polymerase chain reaction demonstrated that the recombinant rat GH was able to induce a significant up-regulation of BbIGF expression in the hepatic caecum. Moreover, Western blotting revealed the presence of a molecule similar to rat GH receptor in the hepatic caecum. These results suggest that BbIGF expression is inducible by exogenous mammalian GH, suggesting the presence of a GH/IGF axis in B. belcheri. The relationship between BbIFG expression and the origin of the vertebrate liver is discussed.

Sequence of the domestic duck (Anas platyrhynchos) growth hormone‐encoding gene and genetic variation in the promoter region

ABSTRACTThe duck growth hormone encoding gene and its promoter region were amplified by polymerase chain reaction (PCR). A total of 5.25 kb were cloned and sequenced. Duck growth hormone (GH) consists of five exons and four introns and is structurally similar to mammalian and chicken GH gene. Although the distal region of duck GH promoter showed no similarity to chicken and turkey promoters, the proximal region of the promoter contained two putative Pit‐1 binding sequences, and showed similarity to chicken and turkey GH promoters. Genetic variation was detected at five positions of the promoter region. The results of this study indicate that the expression of duck GH is likely regulated in a similar manner to that of chicken GH via enhancer‐type cis‐acting elements and the presence of genetic variation in the duck GH gene may be applicable to marker‐assisted selection.

Mammalian genome projects reveal new growth hormone (GH) sequences: Characterization of the GH-encoding genes of armadillo ( Dasypus novemcinctus), hedgehog ( Erinaceus europaeus), bat ( Myotis lucifugus), hyrax ( Procavia capensis), shrew ( Sorex araneus), ground squirrel ( Spermophilus tridecemlineatus), elephant ( Loxodonta africana), cat ( Felis catus) and opossum ( Monodelphis domestica)

Mammalian growth hormone (GH) sequences have been shown previously to display episodic evolution: the sequence is generally strongly conserved but on at least two occasions during mammalian evolution (on lineages leading to higher primates and ruminants) bursts of rapid evolution occurred. However, the number of mammalian orders studied previously has been relatively limited, and the availability of sequence data via mammalian genome projects provides the potential for extending the range of GH gene sequences examined. Complete or nearly complete GH gene sequences for six mammalian species for which no data were previously available have been extracted from the genome databases— Dasypus novemcinctus (nine-banded armadillo), Erinaceus europaeus (western European hedgehog), Myotis lucifugus (little brown bat), Procavia capensis (cape rock hyrax), Sorex araneus (European shrew), Spermophilus tridecemlineatus (13-lined ground squirrel). In addition incomplete data for several other species have been extended. Examination of the data in detail and comparison with previously available sequences has allowed assessment of the reliability of deduced sequences. Several of the new sequences differ substantially from the consensus sequence previously determined for eutherian GHs, indicating greater variability than previously recognised, and confirming the episodic pattern of evolution. The episodic pattern is not seen for signal sequences, 5′ upstream sequence or synonymous substitutions—it is specific to the mature protein sequence, suggesting that it relates to the hormonal function. The substitutions accumulated during the course of GH evolution have occurred mainly on the side of the hormone facing away from the receptor, in a non-random fashion, and it is suggested that this may reflect interaction of the receptor–bound hormone with other proteins or small ligands.

Molecular evolution of the avian growth hormone gene and comparison with its mammalian counterpart

The molecular evolution of all available avian growth hormone (GH) gene sequences was investigated using both maximum-likelihood and parsimony methods, and the patterns compared to those found in mammals. In contrast to the rapid bursts of evolution observed for mammalian GH, the evolutionary rate of the avian GH mature peptide appears to have been more constant. However several positively selected sites were identified at functionally important positions in the avian signal peptide by the site-specific likelihood method. This implies that sequence variation in the avian GH signal peptide may be adaptive, although more conservative parsimony methods failed to confirm this. Nevertheless, the differing patterns of avian and mammalian GH signal peptide molecular evolution are consistent with the apparently differing roles of GH in controlling growth in these taxonomic groups and support the hypothesis that signal peptide sequence variation may in fact be the basis for increased functional complexity.

Characterization of the first growth hormone gene sequence for a passerine bird—the pied flycatcher (Ficedula hypoleuca)

While the growth hormone (GH) gene has been characterized in a broad range of vertebrates, surprisingly little is known about this gene in birds. In order to extend knowledge of the GH gene in avian species and non-domestic species, the pied flycatcher (Ficedula hypoleuca) GH gene has been sequenced in this study. The overall average pairwise sequence divergence level was 0.08 among all available avian sequences and 0.27 among other taxa. However, the overall genetic organization of the gene is quite conserved. The similarity of the GH gene sequence of pied flycatchers with those of chicken and duck suggests that the rapid bursts of molecular evolution observed in mammalian and fish GH have not occurred during the divergence of passerine and non-passerine birds.

Growth hormone transcription factor ZN-16 genomic coding regions are composed of a single exon and are evolutionarily conserved in mammals

The structure of the gene encoding ZN-16, a transcription factor that binds to the mammalian growth hormone promoter in tandem with Pit-1, was determined in order to elucidate the exon–intron organization of the 16 zinc finger domains of the protein. Southern hybridization of mouse genomic DNA showed fragments with sizes identical to those predicted from mouse ZN-16 cDNA for two different probes covering the 2200 aa coding frame. Mouse genome database sequences also showed no introns in the zn-16 coding regions on chromosome 4. Analysis of human zn-16 by Southern hybridization and genomic database sequence analysis also indicated a single exon for the human protein coding sequences. BLASTP query of available genomic databases with critical zinc finger residues from mouse ZN-16 identified highly similar canine, bovine, and chimpanzee genomic sequences that encode proteins. Phylogenetic analysis of these mammalian proteins resulted in relationships as would be expected in species spanning rodents to humans. All six independent zn-16 sequences show a single exon coding region with no introns, a similarity ruling out the possibility that these genomic sequences are pseudogenes. Thus, mammalian zn-16 has a compact single exon structure encoding a very large protein (2200–3000 aa), the conservation of which may have functional implications such as the importance of posttranscriptional modifications.

Characterization of the 5' flanking region of the growth hormone gene of the marine teleost, gilthead sea bream Sparus aurata: analysis of a polymorphic microsatellite in the proximal promoter

The 5′ flanking region (1712 bp) of the growth hormone (GH) gene of the gilthead sea bream Sparus aurata was cloned, sequenced and characterized. It contains a consensus sequence for TATA box and several Pit-1 binding site motifs. Consensus sequences related to cyclic AMP response element, glucocortiocoid response element, and several other transcription factors were identified by comparison to consensus sequences in fish or mammalian GH genes. The promoter contains two microsatellites (di- and tri-nucleotides repeats) with additional upstream microsatellites (tri-and tetranucleotides), 10-mer tandem repeat, and two long inverted repeats. Analysis of the proximal dinucleotide microsatellite by polymerase chain reaction revealed polymorphism among individuals from a hatchery population and an association of alleles 250 and 254 with growth performance. Segregation analysis of this marker in one family showed Mendelian inheritance. These results suggest that the microsatellite in the promoter region (termed saGHpCA) might be considered as a candidate genetic marker for broodstock management and growth selection programs of Sparus aurata.

Lack of growth enhancement by exogenous growth hormone treatment in yellow perch ( Perca flavescens) in four separate experiments

The effect of exogenous growth hormone (GH) treatment on the growth of juvenile yellow perch ( Perca flavescens) was investigated in four experiments. In the first two experiments, juvenile yellow perch were reared at either 13 °C or 21 °C, and injected weekly with bovine GH (bGH) at 0.1, 1.0 or 10.0 μg/g body weight for 84 days. No significant growth enhancement in GH-treated fish was measured in fish in either of the experiments. In the third experiment, juvenile yellow perch were treated with estradiol-17β (E 2, 15 μg/g of diet), bGH (1.0 μg/g body weight) injected weekly or both hormones for 70 days at 21 °C. E 2 alone stimulated growth, but no further growth stimulation occurred in the E 2 + bGH-treated fish. In addition, no growth enhancement was found in fish treated with bGH alone. We measured no difference in serum insulin-like growth factor-I (IGF-I) levels between the treatment groups at 12 and 24 h after the final injection of GH; however, a drop in IGF-I levels after 24 h was observed. In a fourth study, the effect of recombinant yellow perch GH (rypGH, 0.2 or 1.0 μg/g body weight) injected weekly was evaluated in yellow perch juveniles. The fish were reared for 42 days at 18 °C. Neither GH dosages improved growth compared to control-injected and non-injected fish. Taken together, the lack of effect of mammalian GH or rypGH in our experiments suggests (1) low binding affinity between these hormones and the GH receptor in yellow perch, (2) that the endogenous GH levels were already at biologically maximal levels or (3) that other endocrine factors are needed in order for GH to promote yellow perch growth. The reduction in IGF-I levels 24 h after handling suggests a negative effect of handling stress on the GH-IGF-I axis in yellow perch.

Growth hormone and prolactin--molecular and functional evolution.

Growth hormone, prolactin, the fish hormone, somatolactin, and related mammalian placental hormones, including placental lactogen, form a family of polypeptide hormones that share a common tertiary structure. They produce their biological effects by interacting with and dimerizing specific single transmembrane-domain receptors. The receptors belong to a superfamily of cytokine receptors with no intrinsic tyrosine kinase, which use the Jak-Stat cascade as a major signalling pathway. Hormones and receptors are thought to have arisen as a result of gene duplication and subsequent divergence early in vertebrate evolution. Mammalian growth hormone and prolactin show a slow basal evolutionary rate of change, but with episodes of accelerated evolution. These occurred for growth hormone during the evolution of the primates and artiodactyls and for prolactin in lineages leading to rodents, elephants, ruminants, and man. Placental lactogen has probably evolved independently on three occasions, from prolactin in rodents and ruminants and from growth hormone in man. Receptor sequences also show variable rates of evolution, corresponding partly, but not completely, with changes in the ligand. A principal biological role of growth hormone, the control of postnatal growth, has remained quite consistent throughout vertebrate evolution and is largely mediated by insulin-like growth factors. Prolactin has many and diverse roles. In relation to lactation, the relative roles of growth hormone and prolactin vary between species. Correlation between the molecular and functional evolution of these hormones is very incomplete, and it is likely that many important functional adaptations involved changes in regulatory elements, for example, altering tissue of origin or posttranscriptional processing, rather than change of the structures of the proteins themselves.

Molecular Evolution of Growth Hormone (GH) in Cetartiodactyla: Cloning and Characterization of the Gene Encoding GH from a Primitive Ruminant, the Chevrotain (Tragulus javanicus)

In mammals the sequence of pituitary growth hormone (GH) is generally strongly conserved, indicating a slow basal rate of molecular evolution. However, on two occasions, during the evolution of primates and that of cetartiodactyls, the rate of evolution has increased dramatically (25 to 50-fold) so that the sequences of human and ruminant GHs differ markedly from those of other mammalian GHs. To define further the burst of GH evolution that occurred in cetartiodactyls, the GH gene of the chevrotain (Tragulus javanicus) has been cloned and characterized by use of genomic DNA and a polymerase chain reaction technique. Two very similar gene sequences, which probably reflect allelic variation, were isolated. The deduced sequence for the mature chevrotain GH differs from that of the bovine or red deer GH at only two to three residues, and phylogenetic analysis shows that the burst of rapid evolution of GH that occurred in the Cetartiodactyla must have been completed before the divergence of the Tragulidae and the advanced ruminants (Pecora). The rate of evolution during this burst must therefore have been greater than previously estimated. In other aspects (including signal sequence, 5′ upstream sequence, and synonymous substitutions in the coding sequence), the chevrotain GH gene differs considerably from the GH genes of other ruminants and here there is no evidence for the period of accelerated evolution that is seen for GH itself.

Episodic evolution of growth hormone in primates and emergence of the species specificity of human growth hormone receptor.

Growth hormone (GH) evolution is very conservative among mammals, except for primates and ruminant artiodactyls. In fact, most known mammalian GH sequences differ from the inferred ancestral mammalian sequence by only a few amino acids. In contrast, the human GH sequence differs from the inferred ancestral sequence by 59 amino acids. However, it is not known when this rapid evolution of GH occurred during primate evolution or whether it was due to positive selection. Also, human growth hormone receptor (GHR) displays species specificity; i.e., it can interact only with human (or rhesus monkey) GH, not with nonprimate GHS: The species specificity of human GHR is largely due to the Leu-->Arg change at position 43, and it has been hypothesized that this change must have been preceded by the His-->Asp change at position 171 of GH. Is this hypothesis true? And when did these changes occur? To address the above issues, we sequenced GH and GHR genes in prosimians and simians. Our data supported the above hypothesis and revealed that the species specificity of human GHR actually emerged in the common ancestor of Old World primates, but the transitional phase still persists in New World monkeys. Our data showed that the rapid evolution of primate GH occurred during a relatively short period (in the common ancestor of higher primates) and that the rate of change was especially high at functionally important sites, suggesting positive selection. However, the nonsynonymous rate/synonymous rate ratio at these sites was <1, so relaxation of purifying selection might have played a role in the rapid evolution of the GH gene in simians, possibly as a result of multiple gene duplications. Similar to GH, GHR displayed an accelerated rate of evolution in primates. Our data revealed proportionally more amino acid replacements at the functionally important sites in both GH and GHR in simians but, surprisingly, showed few coincidental replacements of amino acids forming the same intermolecular contacts between the two proteins.

Mammalian Growth Hormone‐Releasing Hormone Indirectly Stimulates Sex Steroid Production in Tilapia

Abstract.— Fish possess a growth hormone similar to mammalian growth hormone, and the presence of a growth hormone‐releasing hormone‐like material in the brain has been demonstrated in several teleost species. We investigated the effect of a mammalian growth hormone‐releasing hormone on serum estradiol‐17β and testosterone concentrations in tilapia Oreochromis mossambicus and a hybrid O. niloticus×O aureus because growth hormone has been implicated in the regulation of gonadal functions in teleosts. Three hours after injecting the releasing hormone, male and female serum testosterone concentrations significantly increased compared to controls, while serum estradiol‐17β concentrations increased significantly only in females. These increases were commensurate with respective serum steroid concentrations in fish injected with gonadotropin releasing‐hormone. Studies utilizing hypophysectomized fish resulted in non‐detectable levels of serum sex steroids in fish treated with the growth hormone‐releasing hormone; however, significant increases occurred in fish treated with human chorionic gonadotropin. Similarly, gonadal tissue incubated with growth hormone releasing hormone had non‐detectable levels of sex steroids in the media, whereas gonads incubated with human chorionic gonadotropin had significant increases in sex steroid concentrations. These studies suggest that growth hormone‐releasing hormone acts at the level of the pituitary. Although mammalian growth hormones have been shown to increase serum gonadotropic hormone concentrations, this study provides evidence that a mammalian growth hormone‐releasing hormone has the ability to indirectly increase sex steroid levels in fish.

Molecular cloning and analysis of the partial sequence ofRhinopithecus roxellanae growth hormone gene

Growth hormone gene (GH) ofRhinopithecus roxellanae was amplified by PCR based on the sequences of the reported mammalian growth hormone gene for the first time. The amplified fragment was about 1.8 kb. It was cloned and its upper stream was sequenced. This sequencing region consists of a 5′flanking regulatory region, exon I and part of exon II, intron I of growth hormone gene. Comparing the corresponding sequences of growth hormone gene betweenRhinopithecus roxellanae and the porcine, we concluded that the homology reached 81% in the region, and there was high conservation in the 5′flanking sequence. The kinds of amino acids of exon I and exon II for about 90% were the same to those in pig. Many mutations occurred in the degenerate site of the triplet code. In the nucleotides of intron I, there were only 72% homologies with those in pig. It means that introns and 3′flanking sequence maybe play an important part in growth hormone gene regulation of the different animals.

Overexpression of Xenopus laevis growth hormone stimulates growth of tadpoles and frogs.

The role of growth hormone (GH) in amphibian metamorphosis is ambiguous based on experiments in which mammalian GH was administered to tadpoles and frogs. We have reexamined the effects of GH by producing transgenic Xenopus laevis that overexpress the cDNA encoding X. laevis GH. These transgenic tadpoles take the same length of time to reach metamorphosis as control tadpoles, but the transgenic tadpoles are twice as large. After metamorphosis, the transgenic frogs grow at a greatly accelerated rate and develop skeletal abnormalities reminiscent of acromegaly. The transgenic frogs are larger than mature frogs in a few months and die in about 1 year. At as early as 10 months of age, the males have mature sperm. We conclude that the growth-promoting effects of GH in this amphibian closely resemble those described for mammals. Although excess GH increases the size of the tadpole, it does not alter the developmental programs involved in metamorphosis.

Mammalian pituitary growth hormone: applications of free flow electrophoresis.

We have used free flow electrophoresis (FFE) technology to study the electrophoretic behavior of growth hormone (GH) molecules, GH secretory granules and GH cell subpopulations contained in pituitary glands of humans and rodents. GH activities in different electrophoresis fractions were measured by immunoassay or bioassay, viz., measurement of chondrocyte proliferation in the tibial growth plate of the hypophysectomized rat. Using FFE we discovered a peptide in human post mortem pituitary tissue and cryopoor human plasma that is active in the tibial line bioassay, is inactive in a GH immunoassay, and is neither GH nor a GH fragment. This peptide, called tibial peptide, has high anodal mobility and is readily separable from GH by FFE. Its molecular mass is approximately 5 kD. It is particularly rich in glycine. A partial amino acid sequence (residues 9-25) in the middle region of the peptide shows that 9 of the 16 residues are nonpolar. On the basis of results from other FFE experiments, using either GH-containing secretory granules or GH-producing cells, we believe that the peptide is stored within the secretion granule of a subpopulation of GH cells. On the basis of recent information elucidating the role of C peptide contained in the insulin storage granule of the pancreatic cell, we propose that the tibial peptide serves a similar role in the GH cell. Thus, not only may tibial peptide aid in proper alignment of disulfide bonds between GH monomers in the secretory granule, but, like the C peptide, it also appears to have biologic activity in its own right.

Mammalian pituitary growth hormone: Applications of free flow electrophoresis

Mammalian pituitary growth hormone: Applications of free flow electrophoresis

Mammalian pituitary growth hormone: Applications of free flow electrophoresis

Mammalian pituitary growth hormone: Applications of free flow electrophoresis

Evolution of the proximal promoter region of the mammalian growth hormone gene.

The evolutionary relationship between the proximal growth hormone (GH) gene promoter sequences of 12 mammalian species was explored by comparison of their trinucleotide composition and by multiple sequence alignment. Both approaches yielded results that were consistent with the known fossil record-based phylogeny of the analysed sequences, suggesting that the two methods of tree reconstruction might be equally efficient and reliable. The pattern of evolution inferred for the mammalian GH gene promoters was found to vary both temporally and spatially. Thus, two distinct regions devoid of any evolutionary changes exist in primates, but only one of these 'gaps' is also observed in rodents, and neither is seen in ruminants. Furthermore, different evolutionary rates must have prevailed during different periods of evolutionary time and in different lineages, with a dramatic increase in evolutionary rate apparent in primates. Since a similar pattern of discontinuity has been previously noted for the evolution of the GH-coding regions, it may reflect the action of positive selection operating upon the GH gene as a single cohesive unit. Strong evidence for the action of gene conversion between primate GH gene promoters is provided by the fact that the human GH1 and GH2 sequences, which are thought to have diverged before the divergence of Old World monkeys from great apes, are more similar to one another than either is to the rhesus monkey GH2 promoter. Finally, it was noted that a number of nucleotide positions in the GH1 gene promoter that are polymorphic in humans appear to be highly conserved in mammals. This apparent conundrum, which could represent a caveat for the interpretation of phylogenetic footprinting studies, is potentially explicable in terms either of reduced genetic diversity in highly inbred animal species or insufficient population data from non-human species.

CDNA cloning and sequence analysis of bubaline growth hormone.

The cDNA for Bubalus bubalis growth hormone (GH) has been cloned and sequence determined through RT-PCR approach. The nucleotide sequence of bubaline GH cDNA was in a single reading frame coding for a protein of 191 residues comprising a putative signal sequence of 27 amino acids. Homology comparison of the sequence with other mammalian GH cDNAs showed a very high degree of evolutionary conservation. Bubaline GH sequence shared a homology of 99.5%, 99.5%, 98.6%, 87.6% and 61.9% with that of ovine, caprine, bovine, porcine and human, respectively at amino acid level.

Discrimination of mammalian growth hormones by peptide‐mass mapping

Discrimination of mammalian growth hormones by peptide‐mass mapping