Recombinant Porcine Growth Hormone Research Articles

Simultaneous detection of recombinant growth hormones in equine plasma by liquid chromatography/high-resolution tandem mass spectrometry for doping control

Growth hormone (GH), or somatotropin, is a protein that may enhance physical performance and facilitate growth and wound healing. For this reason, growth hormones and their recombinant analogues are prohibited in human sports by the World Anti-Doping Agency (WADA) and in horseracing under Article 6 of the International Agreement on Breeding, Racing and Wagering published by the International Federation of Horse Racing Authorities (IFHA). Identifying the illicit use of GHs in both human athletes and racehorses is challenging, especially when differentiating between endogenous and exogenous GHs, and between analogues of GH from different species. This paper describes a multiplexed mass spectrometric method for the simultaneous detection of three recombinant GHs, namely recombinant equine GH (reGH), recombinant human GH (rhGH) and recombinant porcine GH (rpGH), in equine plasma. Recombinant chicken GH (rcGH) was used as an internal standard. The recombinant GHs were extracted from equine plasma by automated C4 solid-phase extraction after ammonium sulfate precipitation, and then cleaned up by chloroform/methanol precipitation before trypsination. Proteotypic peptides were analyzed by liquid chromatography/high-resolution tandem mass spectrometry (LC–MS/HRMS). The limits of detection were estimated to be approximately 0.5ng/mL for reGH, 2.5ng/mL for rhGH and 1.25ng/mL for rpGH. Confirmation at 1ng/mL for reGH and 5ng/mL each for rhGH and rpGH was successfully achieved by comparing the retention times and relative abundances of three major product-ions of the respective standards in accordance with industry criteria published by the Association of Official Racing Chemists. The developed method requires less plasma (2mL) and has a shorter turnaround time as compared with other published mass spectrometric methods, and demonstrates good precision and reproducibility. To our knowledge, this is the first reported method for the simultaneous detection of different recombinant GHs (reGH, rhGH and rpGH) at low ng/mL level in horse plasma samples.

A Quantitative Model of Thermal Stabilization and Destabilization of Proteins by Ligands

Equilibrium binding ligands usually increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. High-throughput screening for the discovery of drug-like compounds uses an assay based on thermal stabilization. The mathematical description of this stabilization is well developed, and the method is widely applicable to the characterization of ligand-protein binding equilibrium. However, numerous cases have been experimentally observed where equilibrium binding ligands destabilize proteins, i.e., diminish protein melting temperature by an amount proportional to the concentration and affinity of the ligand. Here, we present a thermodynamic model that describes ligand binding to the native and unfolded (denatured) protein states explaining the combined stabilization and destabilization effects. The model also explains nonsaturation and saturation effects on the protein melting temperature when the ligand concentration significantly exceeds the protein concentration. Several examples of the applicability of the model are presented, including specific sulfonamide binding to recombinant hCAII, peptide and ANS binding to the Polo-box domain of Plk1, and zinc ion binding to the recombinant porcine growth hormone. The same ligands may stabilize and destabilize different proteins, and the same proteins may be stabilized and destabilized by different ligands.

Muscle type-specific responses of myoD and calpain 3 expression to recombinant porcine growth hormone in the pig

Sixteen castrated male Large White × Landrace pigs were employed to investigate the muscle type-specific changes of gene expression in response to recombinant porcine growth hormone (rpGH) administration. Pigs were injected intramuscularly with rpGH (4 mg/day, n = 8) or saline (n = 8) for 28 days. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the mRNA abundance of genes related to muscle growth in longissimus dorsi (LD) and semitendinosus (ST) muscles. Myofibre-type composition was characterised by the ratio of the expression of myosin heavy chain (MyHC) 1, 2a or 2b relative to 2x. The results showed that the relative myofibre-type composition of neither LD nor ST was affected by rpGH administration. rpGH administration did not induce significant changes in the abundances of myostatin and myogenin mRNA in both types of muscle. MyoD and calpain 3 mRNA were significantly increased after rpGH treatment in ST muscle, whereas the difference was not significant in LD muscle. A tendency of down-regulation was observed for PGC-1α mRNA expression in ST muscle of rpGH-treated group (P = 0.16). These results suggest that myoD, calpain 3 and probably PGC-1α may be involved in the mechanism of exogenous GH action on skeletal muscle growth; rpGH up-regulates mRNA expression of myoD and calpain 3 in a muscle type-specific manner, being more remarkable in ST than in LD, whereas no influences of rpGH on the mRNA expression of myostatin and myogenin were detected.

The biphasic response of porcine tendon to recombinant porcine growth hormone

The purpose of this study was to investigate the effect of recombinant porcine growth hormone (rpGH) administration on the growth and maturational changes of the calcanean tendon in male pigs. Twenty-four orchidectomized crossbred (Duroc X Large White X Landrace) pigs were randomly assigned to 2 months of rpGH-treatment (2mo-rpGH), 3 months of rpGH-treatment (3mo-rpGH), or saline-treated control (Control) groups. Saline or rpGH (10 mg/mL given as a constant dose throughout the experiment) was administered twice weekly via 1 mL intramuscular injections. Following the 2mo-rpGH treatment, tendon concentrations of proteoglycan (uronic acid) significantly decreased, non-reducible collagen cross-link content (HP) significantly increased, and hydroxyproline (Hyp) concentrations remained unchanged, with a concomitant significant increase in tendon DNA concentrations, suggesting an up-regulation of cell proliferation. In the 3mo-rpGH treated animals, a decrease in tendon DNA concentration, an increase in proteoglycan and hydroxyproline concentrations, as well as a decrease in HP cross-links were found, suggesting accretion and differentiation of the extracellular matrix components. These findings support the idea that calcanean tendon responds temporally to rpGH treatment, affecting both cell division and tendon metabolism. Responsiveness of the tendon collagen to rpGH may be influenced by the onset and/or the duration of the exogenous growth hormone treatment.

Homologous growth hormone accelerates bone healing—a biomechanical and histological study

The purpose of this study was to prove whether homologous growth hormone has a beneficial effect in the early phase of bone healing. Therefore the left tibias of 24 Yucatan micropigs were osteotomized and stabilized by plate fixation. The treatment group (12 animals) received 100 μg of recombinant porcine growth hormone (rpGH)/kg body w/day sc, whereas the control pigs (12 animals) received 1 ml sodium chloride as placebo. After a healing period of 4 weeks the animals were sacrificed and destructive torsional testing was performed. For histological evaluation 6-μm serial slices of the tibiae were stained with von Kossa. The total area of callus formation (CA) and the mineralized bone area (BA) were quantified by image analysis. The fraction of mineralized bone tissue within the callus area, the bone density (BD), was calculated as follows: BD = (BA/CA) × 100. Torsional failure load was 91% higher and torsional stiffness 61% higher in the treatment group than in the control group (P < 0.05). The histomorphometric measurements revealed an advance for the CA (GH: 127.6 ± 38.9 mm2; placebo: 75.9 ± 50.7 mm2; P < 0.005) as well as for the BA (GH: 89.3 ± 25.8 mm2; placebo: 55.9 ± 38.5 mm2; P < 0.001) for the GH-treated animals in comparison to the control animals. The BD was similar in both groups (GH: 70.6 ± 8.4%; placebo: 74.0 ± 6.24%; P = 0.28). These data indicate that administration of homologous GH stimulates callus formation and ossification in the early phase of bone healing, which consequently results in an increased mechanical strength and stiffness.

Tissue-specific effect of recombinant porcine growth hormone on expression of growth-related genes in the pig

Tissue-specific effect of recombinant porcine growth hormone on expression of growth-related genes in the pig

Systemic application of growth hormone enhances the early healing phase of osteochondral defects—a preliminary study in micropigs

Healing of osteochondral defects following trauma remains a significant clinical problem, often leading to osteoarthritis. Growth hormone (GH) has been shown to accelerate formation of bone and cartilage tissue in the growth plates and in cell cultures. To investigate the influence of systemically administered recombinant porcine growth hormone (r-pGH) on the healing of osteochondral defects we performed a histomorphometrical analysis of full-thickness cartilage defects in the femoral condyle of micropigs. Forty-eight mature female Yucatan micropigs were divided into two groups, one receiving a daily injection of r-pGH (100 μg/kg), the other receiving sodium chloride as placebo. A circular 6-mm-diameter full-thickness defect of the cartilage was created, extending 1.5 mm into the subchondral bone. The animals were sacrificed after 4 ( n = 24) and 6 ( n = 24) weeks. The von-Kossa stain was used to visualise the calcified structures; cartilage and the fibrous tissue were marked with a combined Safranin-O/light-green stain. The defect filling and the percentage of bone, cartilage, and fibrous tissue into the defect were evaluated using an image analysis system. Furthermore, histological grading was performed using the modified Wakitani score. After 4 weeks no differences were observed between both groups. The defect filling after 6 weeks with newly formed bone was significantly higher in the r-pGH-treated group. The formation of cartilage and fibrous tissue showed a trend towards better healing in the GH-treated group; however, there was no significant difference. In the r-pGH-treated group, the percentage of total defect filling was significantly higher. The evaluation of the vascularity showed a significantly lower number of vessels in the GH-treated group after 6 weeks. Histomorphological grading revealed a significantly lower total Wakitani score in the GH-treated group, which represents a better healing result compared to the controls. The results of the present study suggest that circulating r-pGH or one of its mediators may accelerate osteochondral defect healing by stimulating the formation of osseous and chondral tissue. The analysis of the vascularity leads to the assumption of an advanced maturation of the osteochondral defects under the influence of GH.

Effect of recombinant growth hormone on expression of growth hormone receptor, insulin-like growth factor mRNA and serum level of leptin in growing pigs

Sixteen Large White x Landrace castrated male pigs were allotted into treatment and control group. The treatment group was injected intramuscularly with recombinant porcine growth hormone (rpGH, 4 mg d(-1)) and the control group with vehicle for 28 days. Animals were slaughtered 4 h after final injection for liver, longissimus dorsi (LD) muscle and blood sampling. Serum concentration of insulin-like growth factor 1 (IGF-I) and leptin were determined by RIA. The total RNA was extracted from tissues to measure the abundance of growth hormone receptor (GHR), IGF-I mRNA by RT-PCR with 18S rRNA internal standard. Results showed that rpGH enhanced the average daily weight gain by 26.1% (P < 0.05), the serum IGF-I concentration by 70.94% (P < 0.01), decreased serum leptin by 34.8% (P < 0.01). The relative abundance of GHR and IGF-I mRNA in liver were increased by 24.45% (P < 0.05) and 45.30% (P < 0.01), respectively, but no difference of GHR (P > 0.05) and IGF-I mRNA (P > 0.05) in LD between GH treated and control group was found. These results suggest that rpGH can up-regulate hepatic GHR and IGF-I gene expression and improve animal growth. However the effect of rpGH on GHR and IGF-I gene expression are tissue-specific.

Recombinant species-specific growth hormone increases hard callus formation in distraction osteogenesis

The effect of growth hormone (GH) on secondary fracture healing and callus formation has been demonstrated in several previously investigated animal models. The aim of this study was to investigate and quantify the effects of GH on bone regenerates in a distraction osteogenesis model. In 20 mature female Yucatan micropigs, the tibia and fibula were osteotomized, stabilized with an external fixator, and distracted at 2 mm/day for 10 days after a 4 day latency period. The regenerates were allowed to consolidate for 10 days. Micropigs in the study group (ten animals) received a daily injection of 100 μg per kilogram body weight of recombinant porcine growth hormone (r-pGH). Micropigs in the control group (ten animals) received sodium chloride as placebo. After killing on day 25, a quantitative histomorphometrical analysis of the formed callus and the adjacent cortical bone was performed and the results of polychrome in vivo labeling were assessed. The regenerates of the r-pGH-treated animals showed a significantly larger callus area but no change in callus structure. We found islands of cartilage tissue in the regenerates of both groups; the calli from the control group exhibited a higher fraction of cartilage compared with the r-pGH group, but this was not significant. Quantification of the fluorescent in vivo labeling revealed that the distraction gap in GH-treated group showed significant ossification even during distraction. These results demonstrate that growth hormone can accelerate the maturation of the regenerate in distraction osteogenesis without changing the callus microstructure. This may prove to be a useful clinical tool for shortening the healing time in limb lengthening and bone segment transport.

Homologous growth hormone accelerates healing of segmental bone defects

The effect of homologous recombinant porcine growth hormone (r-pGH) on secondary fracture healing was investigated in a diaphyseal defect of the tibia in Yucatan micropigs. A 1 cm defect of the tibia was created surgically and stabilized with an AO 3.5 mm DCP plate. The treatment group (12 animals) received 100 μg of r-pGH per kilogram of body weight subcutaneously once per day, whereas the control pigs (12 animals) received 1 mL of sodium chloride as placebo. For evaluation of the GH-axis, serum levels of insulin-like growth factor-I (IGF-I) were sampled every fourth day. The animals were killed 6 weeks after surgery. Quantitative computed tomography (qCT) was performed to determine bone mineral density (BMD) and bone mineral content (BMC) of the defect zone. The torsional stiffness and the torsional failure load were measured by destructive torsional testing of the defect and contralateral tibiae. qCT measurements revealed a significant increase in the BMC of the defect zone in the treatment group compared with controls (GH BMC = 2833 ± 679 mg, placebo BMC = 2215 ± 636 mg; p < 0.05), whereas the BMD values were similar in both groups (GH BMD = 668 ± 60 mg/mm 2, placebo BMD = 629 ± 52 mg/mm 2, p = 0.12). Torsional failure load was 70% higher and torsional stiffness 83% higher in the treatment group than in the control group ( p < 0.05). The mean serum level of IGF-I in the treatment group increased to 382% of the preoperative basal level and decreased to 69% in the control group, and this difference was highly significant ( p < 0.001). Our data indicate that daily administration of recombinant GH leads to an increase of serum IGF-I levels and stimulates secondary fracture healing, resulting in increased mechanical strength and stiffness of the callus.

The effect of growth hormone on insulin-like growth factor I and bone metabolism in distraction osteogenesis.

Limb lengthening in the left tibia of 30 mature female Yucatan micropigs was performed using distraction osteogenesis. A treatment group of 15 animals received recombinant porcine growth hormone (r-pGH) (100 microg/kg/day) while the others served as controls. Serial serum measurements of total insulin-like growth factor I (IGF-I), free IGF-I, IGF binding proteins -1, -2, -3 and -4 (IGFBP-1 to -4) were performed. Bone-specific alkaline phosphatase (bone-ALP) and the serum carboxyl-terminal telopeptide of type I collagen (ICTP) were measured as bone turnover markers. The GH-treated animals showed a significant increase in total IGF-I, free IGF-I and IGFBP-3 after surgery (P<0.001). Similarly, the treated animals showed a significantly higher level of bone-ALP (P<0.001) throughout the experiment compared to the controls. There was a significant correlation between bone-ALP and total IGF-I (r=0.76) in the GH-treated group and an even higher correlation for free IGF-I (r=0.90). There was no difference in the ICTP serum levels between the two groups. These data indicate that the application of species-specific growth hormone results in a stimulation of bone formation in distraction osteogenesis which may be mediated by IGF-I. The stronger correlation between free IGF-I and bone-ALP indicates that the anabolic effect of IGF-I may be regulated through the IGFBPs by binding and inactivating IGF-I.

Acid denaturation of recombinant porcine growth hormone: formation and self-association of folding intermediates.

We have investigated the conformational changes incurred during the acid-induced unfolding and self-association of recombinant porcine growth hormone (pGH). Acidification (pH 8 to pH 2) of pGH resulted in intrinsic fluorescence, UV absorbance, and near-UV CD transitions centered at pH 4.10. At pH 2.0, a red shift in the fluorescence emission maximum of approximately 3 nm and a 15% loss of the far-UV CD signal at 222 nm imply that the protein did not become extensively unfolded. Acidification in the presence of 4 M urea resulted in similar pH-dependent transitions. However, these occurred at a higher pH (approximately 5.2). At pH 2.0 + 4 M urea, an 8 nm red shift in the fluorescence emission maximum suggests that unfolding was greater than in the absence of urea. The presence of a prominent peak centered at 298 nm in the near-UV CD spectrum, which is absent without urea, signifies further differences in the intermediates generated at pH 2. Sedimentation equilibrium experiments in the analytical ultracentrifuge showed that native pGH and the partially unfolded intermediates reversibly self-associate. Self-association was strongly promoted at pH 2 while urea reduced self-association at both pH 8 and pH 2. These results demonstrate that acidification of pGH in the absence or presence of 4 M urea induced the formation of molten globule-like states with measurable differences in conformation. Similarities and differences in these structural conformations with respect to other growth hormones are discussed.

Treatment of underfed pigs with GH throughout the second quarter of pregnancy increases fetal growth.

Circulating growth hormone (GH) concentrations increase in pregnancy and administration of GH during early-mid pregnancy increases fetal growth in well-fed pigs. To determine whether increased maternal GH could promote fetal growth when feed availability is restricted, fifteen cross-bred primiparous sows (gilts) were fed at approximately 30% of ad libitum intake, from mating onwards and were injected daily i.m. with recombinant porcine GH (pGH) at doses of 0, 13.4+/-0.3 and 25.6+/-0.5 microg/kg live weight from day 25 to day 51 of pregnancy (term approximately 115 days). Treatment with pGH increased maternal backfat loss between day 25 and day 51 of pregnancy, and increased maternal plasma IGF-I concentrations measured at day 51 of pregnancy. Fetal body weight, length and skull width at day 51 of pregnancy were increased by maternal treatment with pGH. Fetal plasma glucose concentrations were increased and maternal/fetal plasma glucose concentration gradients were decreased by maternal pGH treatment at 13.4, but not 25.6 microg/kg.day. Fetal plasma concentrations of urea were decreased by both levels of pGH treatment. Overall, fetal weight was negatively correlated with fetal plasma concentrations of urea, positively correlated with maternal plasma alpha-amino nitrogen concentrations and unrelated to glucose concentrations in either maternal or fetal plasma. This suggests that the availability of amino acids, not glucose, limits fetal growth in the first half of pregnancy in underfed gilts, and that maternal GH treatment may improve amino acid delivery to the fetus.

Exogenous growth hormone induces somatotrophic gene expression in neonatal liver and skeletal muscle.

The extent to which the local somatotrophic axis is functional in extrahepatic tissues in the neonate is unclear. We therefore determined the expression of growth hormone (GH) receptor (GHR), and insulin-like growth factors I and II (IGF-I and IGF-II) mRNA in liver and skeletal muscle (longissimus) of neonatal pigs given daily intramuscular injections of either recombinant porcine GH (1 mg/kg body wt; n = 6) or saline (n = 5) for 7 days. Exogenous GH increased plasma concentrations of GH 30-fold and IGF-I threefold. Abundances of specific mRNA in liver and muscle were measured by RNase protection assays (values are arbitrary density units). In liver, GH treatment increased GHR (6.0 vs. 9.7; P < 0.01) and IGF-I (5.2 vs. 49.0; P < 0.001) but not IGF-II (19.5 vs. 17.2) mRNA. In muscle, GH treatment increased IGF-I mRNA (13.3 vs. 22.8; P < 0.05) but not GHR (8.3 vs. 9.5) or IGF-II (16.1 vs. 16.9). These results demonstrate that exogenous GH can induce local somatotrophic function predominantly in liver but also in muscle of newborn pigs. Our novel finding on the selective increase in muscle IGF-I but not GHR gene expression suggests differences in posttranscriptional regulation and/or intracellular signaling mechanisms.

Effects of a synthetic growth hormone-releasing peptide on growth and carcass traits of pigs

The current study was to determine the effects of growth hormone-releasing peptide ([His1, Lys6]-GHRP) on growth performance and carcass traits in pigs, and to determine whether the results were comparable to those of recombinant porcine growth hormone (r-PGH) treatment. Thirty Landrace and Yorkshire pigs (40–45 kg) were randomly divided into three groups, each group consisted of two boars, five gilts and three barrows. Peptides were administered daily by intramuscular injection with the following schemes and dosages: Group A ([His1, Lys6]-GHRP, 200 μg/day in 0·5 ml saline), Group B (r-PGH, 3 mg/day in 0·6 ml saline), and Group C (sterilized physiological saline, 0·5 ml/day). [His1, Lys6]-GHRP caused significant improvements both in pig growth and carcass traits. The average daily gain (ADG) and feed efficiency of the [His1, Lys6]-GHRP treated group were higher than those of the control, but less than with the r-PGH treatment. The net increase in the ADG by [His1, Lys6]-GHRP was c. 14%, and was 23% by r-PGH (both P&lt;0·01). The feed efficiency was improved 17% by [His1, Lys6]-GHRP (P&lt;0·01) and 20% by r-PGH (P&lt;0·01). On the other hand, the back-fat thickness in both [His1, Lys6]-GHRP and r-PGH treated groups (1·76±0·041 cm and 1·72±0·040 cm, respectively) was considerably less (P&lt;0·05) than that of the control group (1·81±0·040 cm). In addition, the lean cuts percentage and the rib longissimus muscle area were comparable in the [His1, Lys6]-GHRP and r-PGH treatments (lean cuts percentage: 57±2·0% and 57±1·5%, respectively; rib longissimus muscle area: 32·8±0·91 cm2 and 32·9±1·10 cm2, respectively), and both were significantly (P&lt;0·05) greater than those of the control group (lean cuts percentage: 53±1·5%; rib longissimus muscle area: 28·3±1·61 cm2). In conclusion, [His1, Lys6]-GHRP appeared to be a promising tool for controlling carcass fat in pigs.

Recombinant growth hormone accelerates bone regenerate consolidation in distraction osteogenesis

The purpose of the present study was to prove whether homologous GH has a stimulating effect on bone healing. Therefore, left tibiae of 30 micropigs were osteomized and distracted over an external fixator at the rate of 2 mm/day on each of 10 consecutive days. Animals were killed after a healing period of another 10 days. The treatment group received 100 μg of recombinant porcine growth hormone (rpGH) per kilogram of body weight per day. Serial torsional nondestructive biomechanical tests were performed in vivo using a newly developed measurement device. After killing, destructive torsional strength testing of the sites of distraction was performed. To determine the endocrine response to the administration of rpGH, serum levels of insulin-like growth factor-I (IGF-I) were determined. Nondestructive in vivo testing showed that torsional stiffness of the regenerate was significantly higher in the treatment group than in the control group. Final regenerate torsional failure load was 131% higher and ultimate torsional stiffness was 231% higher in the treatment group than in the control group. The mean serum level of IGF-I increased to 440% of preoperative basal level in the treatment group and remained unchanged in the control group. Our data indicate that systemic administration of recombinant homologous growth hormone greatly accelerates ossification of bone regenerate in distraction osteogenesis.

Degradation of recombinant porcine growth hormone in the presence of guanidine hydrochloride

This study was conducted to investigate the effect of guanidine hydrochloride (Gdn HCl) on the rate of chemical degradation of recombinant porcine growth hormone (pGH) at alkaline pH. The protein was incubated in 0.2 M Tris buffer, pH 9 at 37°C containing Gdn HCl at concentrations ranging from 0 to 6 M. The conformation of pGH under the different solution conditions was assessed by circular dichroism spectropolarimetry. Degradation was monitored by reversed phase HPLC (RP-HPLC), size exclusion chromatography (SEC) and SDS-PAGE. An associated form of pGH was populated in the presence of 3 and 4 M Gdn HCl, whereas the protein was completely unfolded in 5 and 6 M Gdn HCl. The apparent degradation rate constant determined by RP-HPLC increased by a factor of 18 in the presence of 3 M Gdn HCl and by a factor of five in the presence of 6 M Gdn HCl relative to that in buffer alone. RP-HPLC and SEC data suggested that the product distribution also differed between buffer and solutions containing Gdn HCl. While the increased rate of degradation for the denatured state was not unexpected, the dramatic increase at an intermediate Gdn HCl concentration was surprising and indicated the importance of considering chemical instability when proteins are exposed to denaturants.

Suppression of somatotroph function induced by growth hormone treatment in neonatal pigs

The effect of recombinant porcine growth hormone (pGH) treatment on pituitary function was evaluated in young pigs. Piglets received intraperitoneal recombinant pGH implants (0.5 mg/d sustained release) or vehicle implants beginning at 3 d of age. Ten piglets were sacrificed at 4 and 6 wk of age (five piglets/treatment group) for the collection of pituitary glands, blood, and liver tissue. Blood samples also were drawn at 3 and 12 d of age. Serum concentrations of GH, prolactin (PRL), thyroid-stimulating hormone (TSH), insulin-like growth factor-1 (IGF-1) and IGF-2 were evaluated. Levels of IGF-1 and IGF-2 mRNA were determined in liver samples. Treatment with GH increased circulating levels of GH and IGF-1 (P < 0.01), but not PRL, TSH, or IGF-2. Hepatic IGF-1, but not IGF-2, mRNA levels were increased by pGH (P < 0.001). Cultured pituitary cells from each animal were challenged with 0.1, 1, and 10 nM GH-releasing hormone (GHRH); 2 mM 8-Br-cAMP; or 100 nM phorbol myristate acetate. The release of GH from cultured pituitary cells was stimulated by all secretagogues (P < 0.001). The secretion of GH, but not PRL or TSH, in culture was inhibited by previous in vivo GH treatment (P < 0.001). Similarly, cellular GH, but not PRL or TSH, content was lower in the GH-implant group (P = 0.005). Cell cultures from 6-wk-old piglets secreted more GH, but not PRL or TSH, than cultures from 4-wk-old piglets (P < 0.05). Likewise, cellular GH, but not PRL or TSH, content was greatest in cultures from 6-wk-old animals (P = 0.002). Piglet growth was not affected by exogenous GH treatment (P = 0.67). These results demonstrate that exogenous pGH treatment selectively down-regulates somatotroph function in young pigs.

Molecular interaction of growth hormone with two monoclonal antibodies recognizing distinct epitopes.

Two monoclonal antibodies (mAb), designated PS-7.6 and PS-11.2, were generated against recombinant porcine growth hormone (pGH) and shown to enhance the hormonal activity in promoting the growth of animals. The mAb were compared and their functional relationship investigated. It was demonstrated by radioimmunoassay that PS-11.2 did not compete, but rather enhanced binding of 125I-pGH tracer to PS-7.6, suggesting that both mAb recognized distinct epitopes and also were additive in their antigen bindings. Surface plasmon resonance analysis using optical BIAcore technology (Pharmacia Biosensor, Piscataway NJ, USA) provided additional data to support this idea because pGH, after being captured by PS-11.2, remained capable of interacting with PS-7.6. An anti-idiotypic mAb was employed and shown to interact with PS-7.6 but not PS-11.2, implying that the differences in the Fab variable regions of these two mAb might contribute to their epitope specificity. Binding kinetics were determined by the BIAcore and the individual affinities of PS-7.6 and PS-11.2 to pGH were 6.8 x 10(-8) and 1.2 x 10(-9) mol/L, respectively. When these mAb were sequentially subjected to the BIAcore, however, their affinities decreased by approximately 100-fold. Therefore, binding of pGH with one mAb significantly impaired a subsequent interaction with another mAb despite the fact that both mAb targeted different epitopes. Hypophysectomized rats were used for functional analysis and pGH was active in promoting growth of these GH-deficient animals. The hormonal effect was further improved by incubating pGH with either PS-7.6 or PS-11.2 prior to administration. However, enhancement by individual mAb was completely abolished when pGH was treated with both mAb together, indicating their unpredictable biological interference with each other. Therefore, the present findings clearly demonstrate that although PS-7.6 and PS-11.2 recognize separate epitopes, their individual interactions with pGH are closely interrelated both immunologically and biologically.

Effect of recombinant vertebrate growth hormones on growth of adult abalone, Haliotis kamtschatkana

Enhancement of cultivar growth through hormone treatment is of interest in aquaculture research owing to its potential for increasing production. In this study, injection of exogenous growth hormones was investigated as a means of enhancing growth in adult abalone, Haliotis kamtschatkana. Fifty individually caged abalone were held in a common aquarium tank with a constant flow of fresh ambient seawater and fed ad libitum on kelp ( Nereocystis leutkeana). The abalone were divided into five groups of ten animals each. Every group had a similar mean weight (78 g) and length (7 cm). Four groups received weekly intramuscular injections (5 μg g −1 body weight) of either (1) recombinant bovine growth hormone, (2) recombinant porcine growth hormone, (3) somatostatin, or (4) bovine serum albumin. The fifth group served as an uninjected control. The abalone were weighed biweekly throughout the 10 week experiment. Water content and gonad index were assessed for each group at the end of the experimental period. There were no significant differences in weight gain, water content, or gonad index among the five groups.