Porcine Growth Hormone Research Articles

The effect of heat stress on the cellular behavior, intracellular signaling profile of porcine growth hormone (pGH) in swine testicular cells.

At present, heat stress caused by the thermal environment is the main factor that endangers the reproductive function of animals. Growth hormone (GH) is a polypeptide hormone, the biological function of reproductive organs has been reported, and it has many important physiological functions in the body. However, so far, the behavior and signal transduction of GH in testicular cells under heat stress are still unclear. To this end, in the current work, we use a swine testicular cell line (ST) as an in vitro model to explore the cell behavior and intracellular signaling profile of porcine growth hormone (pGH) under heat stress; the results showed that when cells were under heat stress, pGH and GHR were basically not internalized, and a large number of them accumulated on the cell membrane. In addition, we also studied the effect of pGH on the JAK2-STATs signaling pathway and IGF-1 expression under heat stress, we found that the ability of pGH to activate the JAK-STATs signaling pathway and IGF-1 under heat stress was greatly reduced (p < 0.05). In conclusion, our research shows that when cells undergo heat stress, the internalization of pGH and GHR were inhibited, and the activation of the JAK2-STATs signaling pathway and IGF-1 expression were reduced; this lays a solid foundation for further research on the effect of pGH on swine testicular tissue under thermal environment.

The Cellular Behavior, Intracellular Signaling Profile and Nuclear-Targeted Potential Functions of Porcine Growth Hormone (pGH) in Swine Testicular Cells.

Porcine growth hormone (pGH) has many important biological functions and roles, and the biological activity of pGH is closely related with its cell behavior and characteristics. However, so far, the behavior of pGH in swine testicular cell remains unclear. For this, in the current work, the swine testicular cell line (ST) was used as an in vitro model, and CLSM (Confocal laser scanning microscope), IFA (Indirect immunofluorescence assay), FCM (Flow cytometry) and WB (Western-blotting) were used to explore the pGH's cell behivior and function, and the results showed that pGH and GHR could internalize into ST cell and transported to the nucleus. Furthermore, we studied the internalization kinetics of pGH and GHR on ST cell, and found that pGH and GHR internalizes into ST cell in a time-dependent manner. More importantly, we also investigated the potential molecular functions of pGH-GHR after it entered into the cell nuclei. The results indicated that nuclear-localized GHR could participate in cell proliferation by regulating the signal intensity of STAT5. In summary, our current research shows that the nuclear-localized pGH-GHR participates in the cell proliferation of ST cell, which lays a solid foundation for further research on the regulatory effect of pGH on testicular tissue.

Effects of growth hormone and cortisol administration on plasma insulin-like growth factor binding proteins in juveniles of three subspecies of masu salmon (Oncorhynchus masou)

In this study, we examined the effects of porcine growth hormone (GH) and cortisol on plasma insulin-like growth factor binding proteins (IGFBPs) in juveniles of three subspecies of Oncorhynchus masou (masu, amago, and Biwa salmon). Ligand blotting using digoxigenin-labeled human IGF-I was used to detect and semi-quantify three major circulating IGFBP bands at 41, 28, and 22 kDa, corresponding to IGFBP-2b, −1a, and -1b, respectively. GH increased plasma IGFBP-2b concentration in masu and Biwa salmon but suppressed it in amago salmon. Plasma IGFBP-2b levels were increased by cortisol in the three subspecies. Cortisol induced plasma IGFBP-1a in the three subspecies, whereas GH had a suppressive effect in masu and Biwa salmon. Sham and cortisol injections increased plasma IGFBP-1b levels after 1 day in masu and amago salmon, suggesting that IGFBP-1b is induced following exposure to stressors via cortisol. Increased IGFBP-1b levels were restored to basal levels when co-injected with GH in Biwa salmon, and the same trend was seen in masu and amago salmon. However, the suppressive effect of GH disappeared 2 days after injection in the three subspecies. Despite some differences among subspecies, the findings suggest that cortisol is a primary inducer of plasma IGFBP-1b; however, GH counteracts it in the short term. Therefore, GH has the potential to modulate the degree of increase in circulating IGFBP-1b levels during acute stress.

JAK2-STAT5 signaling is insensitive to porcine growth hormone (pGH) in hepatocytes of neonatal pig

ABSTRACT Porcine growth hormone (pGH) is most important hormone which is involved in the growth and development of pig. However, a series of studies have indicated that neonatal pig is insensitive to pGH; the reason for this phenomenon is still not fully understood. In this work, we try to investigate this issue from the angle of intracellular signaling induced by pGH. In the present study, porcine hepatocytes from neonatal pig were used as a model, and confocal laser scanning microscopy (CLSM), Western blot, co-immunoprecipitation and colocalization assay were used to study pGH’s signaling properties in hepatocytes of neonatal pig and explore the possible mechanism(s) for why intracellular signaling is insensitive to pGH. The results indicated that Janus kinase 2 and signal transducers and activators of transcription 5/3/1 (JAK2-STATs) signaling are not activated. We further investigated the possible mechanism(s) by which JAK2-STATs’ signaling is not activated by pGH and growth hormone receptor (GHR) and found that the negative regulatory molecules of JAK2-STATs signaling may be associated with this phenomenon in the hepatocytes of neonatal pig. In addition, we also explored pGH’s biology in hepatocytes from neonatal pig, it can be found that pGH/GHR could translocate into the cell nucleus, which implies that pGH/GHR may exhibit physiological roles based on their nuclear localization. We found that pGH could not trigger intracellular signaling in the hepatocytes of neonatal pigs, but not young pigs, which provides an important explanation for why the growth of neonatal pig is GH independent.

Co-expressing GroEL\u2013GroES, Ssa1\u2013Sis1 and Bip\u2013PDI chaperones for enhanced intracellular production and partial-wall breaking improved stability of porcine growth hormone

Porcine growth hormone (pGH) is a class of peptide hormones secreted from the pituitary gland, which can significantly improve growth and feed utilization of pigs. However, it is unstable and volatile in vitro. It needs to be encapsulated in liposomes when feeding livestock, whose high cost greatly limits its application in pig industry. Therefore we attempted to express pGH as intracellular soluble protein in Pichia pastoris and feed these yeasts with partial wall-breaking for swine, which could release directly pGH in intestine tract in case of being degraded in intestinal tract with low cost. In order to improve the intracellular soluble expression of pGH protein in Pichia pastoris and stability in vitro, we optimized the pGH gene, and screened molecular chaperones from E. coli and Pichia pastoris respectively for co-expressing with pGH. In addition, we had also explored conditions of mechanical crushing and fermentation. The results showed that the expression of intracellular soluble pGH protein was significantly increased after gene optimized and co-expressed with Ssa1–Sis1 chaperone from Pichia pastoris. Meanwhile, the optimal conditions of partial wall-breaking and fermentation of Pichia pastoris were confirmed, the data showed that the intracellular expression of the optimized pGH protein co-expressed with Ssa1–Sis1 could reach 340 mg/L with optimal conditions of partial wall-breaking and fermentation. Animal experiments verified that the optimized pGH protein co-expression with Ssa1–Sis1 had the best promoting effects on the growth of piglets. Our study demonstrated that Ssa1–Sis1 could enhance the intracellular soluble expression of pGH protein in Pichia pastoris and that partial wall-breaking of yeast could prevent pGH from degradation in vitro, release targetedly in the intestine and play its biological function effectively. Our study could provide a new idea to cut the cost effectively, establishing a theoretical basis for the clinic application of unstable substances in vitro.

Zinc ions increase GH signaling ability through regulation of available plasma membrane-localized GHR.

It is well known that zinc ion (Zn2+ ) can regulate the biological activity of growth hormone (GH). However, until now, the mechanism by which Zn2+ regulates GH biological activity remains unclear. In the current study, we first performed molecular docking between Zn2+ and porcine GH (pGH) using computational biology. We then explored the effect of Zn2+ on the GH signaling ability in the cell model expressing porcine growth hormone receptor (GHR). It was found that the phosphorylation levels of Janus kinase 2, signal transducers and activators of transcription 5/3/1, and GHR increased significantly under Zn2+ treatment, indicating that Zn2+ can enhance the signaling ability of GH/GHR. On this basis, we further explored how Zn2+ regulates the biological activity of GH/GHR. The results showed that downregulation and turnover of GHR changed under Zn2+ /pGH treatment. Zn2+ enhanced the membrane residence time of pGH/GHR and delayed GHR downregulation. Further investigation showed that the internalization dynamic of pGH/GHR was changed by Zn2+ , which prolonged the residence time of pGH/GHR in the cell membrane. These factors acted together to upregulate the signaling of GH/GHR. This study lays a foundation for further exploration of the biological effects of Zn2+ on GH.

The effect of treating growing gilts with either Ractopamine or Reporcin on pork characteristics

The pork industry is constantly seeking economical and sustainable strategies that will improve production efficiencies and meat quality. Such successful improvements will be of value even here in Botswana to improve food security. The evaluation of pork characteristics after use of these strategies is important. This study aimed at evaluating the effect of a beta-agonist (BA - Ractopamine) and porcine growth hormone (rpGH - Reporcin) on the pork characteristics of the longissimus dorsi (LD) muscle of adult gilts (starting weights of 85+5kg) treated for 27 days under three treatment groups (15 animals per treatment group). The control group was fed a standard commercial diet ad-libitum, while the beta-agonist (BA) group were also fed ad-libitum the standard commercial diet containing Ractopamine (20mg/kg) and the growth hormone (rpGH) group were fed the commercial diet ad-libitum and administered Reporcin (10mg) intramuscularly every other day until the day before slaughter. No significant differences were observed between treatment groups regarding the gilts’ carcass weights after 27 days of treatment (control – 81.98kg; BA – 86.97kg and rpHG – 84.51kg). However, LD muscle from gilts of the growth hormone (1.569%) and Ractopamine (1.502%) groups had significantly lower lipid content compared to the control group at 1.873% (p &lt;0.001), but no significant differences were observed between groups regarding fatty acid profiles. The two growth promoters can thus be locally adopted for use to improve feed utilization, pork production and to lower lipid content in pork of growing gilts without deleteriously affecting fatty acid composition.

Growth Hormone Improves Nerve Regeneration, Muscle Re-innervation, and Functional Outcomes After Chronic Denervation Injury

This study investigates the efficacy of systemic growth hormone (GH) therapy in ameliorating the deleterious effects of chronic denervation (CD) injury on nerve regeneration and resulting motor function. Using a forelimb CD model, 4 groups of Lewis rats were examined (n = 8 per group): Group-1 (negative control) 8 weeks of median nerve CD followed by ulnar-to-median nerve transfer; Group-2 (experimental) 8 weeks of median nerve CD followed by ulnar-to-median nerve transfer and highly purified lyophilized pituitary porcine GH treatment (0.6 mg/day); Group-3 (positive control) immediate ulnar-to-median nerve transfer without CD; Group-4 (baseline) naïve controls. All animals underwent weekly grip strength testing and were sacrificed 14 weeks following nerve transfer for histomorphometric analysis of median nerve regeneration, flexor digitorum superficialis atrophy, and neuromuscular junction reinnervation. In comparison to untreated controls, GH-treated animals demonstrated enhanced median nerve regeneration as measured by axon density (p < 0.005), axon diameter (p < 0.0001), and myelin thickness (p < 0.0001); improved muscle re-innervation (27.9% vs 38.0% NMJs re-innervated; p < 0.02); reduced muscle atrophy (1146 ± 93.19 µm2 vs 865.2 ± 48.33 µm2; p < 0.02); and greater recovery of motor function (grip strength: p < 0.001). These findings support the hypothesis that GH-therapy enhances axonal regeneration and maintains chronically-denervated muscle to thereby promote motor re-innervation and functional recovery.

The basic route of the nuclear translocation porcine growth hormone (GH)-growth hormone receptor (GHR) complex (pGH/GHR) in porcine hepatocytes

Traditional views suggest that growth hormone and the growth hormone receptor (GH/GHR complex) exert their functions only on the plasma membrane. This paradigm, however, has been challenged by recent new findings that the GH/GHR complex could translocate into cell nuclei where they could still exhibit important physiological functions. We also reported the nuclear localization of porcine GH/GHR and their potential functions in porcine hepatocytes. However, the basic path of pGH/GHR’s nuclear translocation remains unclear. Combining previous research results and our current findings, we proposed two basic routes of pGH/GHR’s nuclear transportation as follows: 1) after pGH binding to GHR, pGH/GHR enters into the cytoplasm though clathrin- or caveolin-mediated endocytosis, then the pGH/GHR complex enters into early endosomes (Rab5-positive), and the endosome carries the GH/GHR complex to the endoplasmic reticulum (ER). After endosome docking on the ER, the endosome starts fission, and the pGH/GHR complex enters into the ER lumen. Then the pGH/GHR complex transports into the cytoplasm, possibly by the ERAD pathway. Subsequently, the pGH/GHR complex interacts with IMPα/β, which, in turn, mediates GH/GHR nuclear localization; 2) pGH binds with the GHR on the cell membrane and, subsequently, pGH/GHR internalizes into the cell and enters into the endosome (this endosome may belong to a class of endosomes called envelope-associated endosomes (NAE)). Then, the endosome carries the pGH/GHR to the nuclear membrane. After docking on the nuclear membrane, the pGH/GHR complex fuses with the nuclear membrane and then enters into the cell nucleus.

Controlling AOX1 promoter strength in Pichia pastoris by manipulating poly (dA:dT) tracts

Alcohol oxidase I (AOX1) promoter is the most popular but strictly-regulated methanol inducible promoter for heterologous protein expression in Pichia pastoris. In recent years, AOX1 promoter libraries have been developed with deletion or insertion methods. The present research manipulated poly (dA:dT) tracts in this promoter to control promoter strength, which hadn’t been tried before. There were 34 variants derived from the native AOX1 promoter constructed. And variants were integrated into the same genomic location and upstream of the same reporter gene porcine growth hormone (pGH). To test the transferability of the results obtained from reporter gene pGH, the variants were connected to reporter gene Lac Z. The resulted promoter library spanned an activity range between 0.25 and 3.5 fold of the wild-type promoter activity. In addition, activities of variants correlated with their predicted nucleosome architecture, which were directed by poly (dA:dT) tracts. The cumulative sum of predicted nucleosome affinity across the region (−820 to −540) was related to promoters strength in single deletion variants on a proportional basis. Overall, the research promotes understanding of the regulatory patterns for AOX1 promoter and suggested that varying promoter expression of engineering nucleosome architecture was also a feasible approach in P. pastoris.

Internalization and Nuclear Localization of Porcine Growth Hormone in Porcine Hepatocytes

Internalization and Nuclear Localization of Porcine Growth Hormone in Porcine Hepatocytes

Porcine growth hormone induces the nuclear localization of porcine growth hormone receptor in vivo.

ObjectiveRecent studies have challenged the traditional paradigm that growth hormone receptor (GHR) displays physiological functions only in the cell membrane. It has been demonstrated that GHR localizes to the cell nucleus and still exhibits important physiological roles. The phenomenon of nuclear localization of growth hormone (GH)-induced GHR has previously been described in vitro. However, until recently, whether GH could induce nuclear localization of GHR in vivo was unclear.MethodsIn the present study, we used pig as an animal model, and porcine growth hormone (pGH) or saline was injected into the inferior vena cava. We subsequently observed the localization of porcine growth hormone receptor (pGHR) using multiple techniques, including, immunoprecipitation and Western-blotting, indirect immunofluorescence assay and electronmicroscopy.ResultsThe results showed that pGH could induce nuclear localization of pGHR. Taken together, the results of the present study provided the first demonstration that pGHR was translocated to cell nuclei under pGH stimulation in vivo.ConclusionNuclear localization of pGHR induced by the in vivo pGH treatment suggests new functions and/or novel roles of nuclear pGHR, which deserve further study.

Growth hormone-specific induction of the nuclear localization of porcine growth hormone receptor in porcine hepatocytes

The phenomenon of nuclear translocation of growth hormone receptor (GHR) in human, rat, and fish has been reported. To date, this phenomenon has not been described in a domestic animal (such as pig). In addition, the molecular mechanisms of GHR nuclear translocation have not been thoroughly elucidated. To this end, porcine hepatocytes were isolated and used as a cell model. We observed that porcine growth hormone (pGH) can induce porcine GHR's nuclear localization in porcine hepatocytes. Subsequently, the dynamics of pGH-induced pGHR's nuclear localization were analyzed and demonstrated that pGHR's nuclear localization occurs in a time-dependent manner. Next, we explored the mechanism of pGHR nuclear localization using different pGHR ligands, and we demonstrated that pGHR's nuclear translocation is GH(s)-dependent. We also observed that pGHR translocates into cell nuclei in a pGH dimerization-dependent fashion, whereas further experiments indicated that IMPα/β is involved in the nuclear translocation of the pGH-pGHR dimer. The pGH-pGHR dimer may form a pGH-GHR-JAK2 multiple complex in cell nuclei, which would suggest that similar to its function in the cell membrane, the nuclear-localized pGH-pGHR dimer might still have the ability to signal.

Abstract 15: Growth Hormone Enhanced Recovery After Chronic Denervation Injury: Augmenting Axonal Regeneration, Promoting Motor Reinnervation, and Improving Muscle Function

INTRODUCTION: Several experimental studies suggest that growth hormone-based therapies have the potential to accelerate and augment axonal regeneration while simultaneously act directly on muscle and Schwann cells (SCs) to minimize denervation atrophy prior to re-innervation. The purpose of this study was to assess the impact of growth hormone (GH) therapy on preventing the deleterious effects of chronic denervation (CD) injury on nerve regeneration and resulting muscle function. We hypothesized that systemic GH therapy can maintain chronically-denervated muscle and SCs, accelerate axonal regeneration, and improve murine extremity function in the setting of chronic denervation (CD) by preserving muscle mass and promoting motor reinnervation. METHODS: We utilized a newly-developed, rat forelimb CD model to assess the effects of GH therapy on: a) improving nerve regeneration; and b) preventing muscle atrophy and augmenting muscle re-innervation and function. Four groups of rats were examined: (1) Group-1 (n = 8) underwent 8 weeks of median nerve CD injury followed by repair; (2) Group-2 (experimental, n = 8) underwent 8 weeks of median nerve CD followed by repair and treatment with highly purified lyophilized pituitary porcine GH (0.6 mg/day); (3) Group-3 animals (n = 8) underwent no median nerve CD injury; (4) Group-4 animals (n = 8) were naïve controls. All animals underwent weekly muscle functional testing followed by median nerve and flexor muscle harvest for nerve histomorphometry, muscle weight, atrophy, and re-innervation analysis. To enable the assessment of the effects of GH therapy on maintaining denervated muscle and SCs: Group-1 animals also underwent 8 weeks of contralateral sciatic nerve CD; Group-2 animals underwent 8 weeks of sciatic nerve CD (w/ GH treatment as stated above); Group-3 animals underwent 1 week of sciatic nerve CD; and Group-4 animals underwent no sciatic nerve CD. The sciatic nerve and lateral gastrocnemius muscle were harvested after completion of sciatic nerve CD injury time point to assess for SC proliferation/senescence markers (via qPCR) and muscle atrophy (via muscle weight and cross-sectional area muscle staining). RESULTS: Median nerve regeneration was higher in GH-treated animals when compared to untreated controls as measured by axon density (p < 0.005), axon diameter (p < 0.0001), and myelin thickness (p < 0.0001). Furthermore, GH improved muscle re-innervation (27.9% vs 38.0% NMJs re-innervated; p < 0.02) and prevented muscle atrophy (865 ± 48.3 vs 1081 ± 101.4 μM2; p < 0.02). GH-treated rats demonstrated greater functional muscle recovery as compared to untreated controls (hand grip: 1.8 ± 0.3 N vs. 1.0 ± 0.1 N, p=0.001). Lastly, Group 2 animals demonstrated higher expression of SC proliferation and migration markers, c-Jun & erbB-3, and less muscle atrophy (0.21 ± 0.02 g vs. 0.17 ± 0.01 g; P=0.55) when compared to Group 1 at 8 weeks after sciatic nerve CD. CONCLUSION: Systemic GH therapy can improve nerve regeneration and maintain chronically-denervated muscle by promoting re-innervation and subsequent muscle function.

Student Award for Outstanding Research Winner in the Undergraduate Category for the 2017 Society for Biomaterials Annual Meeting and Exposition, April 5–8, 2017, Minneapolis, Minnesota: Development and characterization of stimuli‐responsive hydrogel microcarriers for oral protein delivery

A family of pH-responsive terpolymers composed of methacrylic acid (MAA), N-vinyl pyrrolidone (NVP), and poly(ethylene glycol) monomethylether monomethacrylate (PEGMMA) have been developed and evaluated for their pH-responsive swelling behavior, protein-loading capabilities, and cytocompatibility. These terpolymer hydrogels, designated as P((MAA-co-NVP)-g-EG), were synthesized with varying PEG chain lengths and monomer feed ratios. The incorporation of MAA into the terpolymer structure was quantified with potentiometric titration. Equilibrium and dynamic swelling studies confirmed the pH-responsive behavior of the hydrogel, with the system remaining collapsed/complexed in acidic pH conditions and swollen/decomplexed in neutral pH conditions. The ability of the hydrogels to partition protein into the swollen network was assessed for two model proteins of varying molecular weight: insulin and porcine growth hormone. Finally, the cytocompatibility of the hydrogels in the presence of two model intestinal cell lines was investigated and confirmed minimal cytotoxicity at and below 2.5 mg/mL. The developed P((MAA-co-NVP)-g-EG) hydrogels exhibit unique properties that could potentially be utilized for drug delivery and separation applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1243-1251, 2017.

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.

Development of a P((MAA-co-NVP)-g-EG) Hydrogel Platform for Oral Protein Delivery: Effects of Hydrogel Composition on Environmental Response and Protein Partitioning.

Hydrogels based upon terpolymers of methacrylic acid, N-vinyl pyrrolidone, and poly(ethylene glycol) are developed and characterized for their ability to respond to changes in environmental pH and to partition protein therapeutics of varying molecular weights and isoelectric points. P((MAA-co-NVP)-g-EG) hydrogels are synthesized with PEG-based cross-linking agents of varying length and incorporation densities. The composition is confirmed using FT-IR spectroscopy and shows peak shifts indicating hydrogen bonding. Scanning electron microscopy reveals microparticles with an irregular, planar morphology. The pH-responsive behavior of the hydrogels is confirmed under equilibrium and dynamic conditions, with the hydrogel collapsed at acidic pH and swollen at neutral pH. The ability of the hydrogels to partition model protein therapeutics at varying pH and ionic strength is evaluated using three model proteins: insulin, porcine growth hormone, and ovalbumin. Finally, the microparticles are evaluated for adverse interactions with two model intestinal cell lines and show minimal cytotoxicity at concentrations below 5 mg mL-1 .

Different intracellular signalling properties induced by human and porcine growth hormone

Growth hormone (GH) is reportedly species-specific. Primate growth hormone can trigger non-primate growth hormone receptor (GHR), but primates GHR cannot be activated by non-primate GH. However, it is also unclear that why primate GH and non-primate GH have different biological activities. Thus, we analysed primate growth hormone (human growth hormone (hGH)) or non-primate GH (porcine growth hormone (pGH))-induced intracellular signalling in 3T3-F442A cells and rat hepatocytes in a dose- and time-dependent manner to explore the different biological activities between them. The results revealed that both hGH and pGH can activate Janus kinase 2 (JAK2), Signal transducers and activators of transcription 1, 3 and 5 (STATs 1, 3 and 5) and extracellular signal-regulated kinase 1/2 (ERK1/2). There were no significant differences in JAK2 or ERK1/2 tyrosine phosphorylation after hGH and pGH treatment, but there were different between hGH and pGH in STAT/1/3/5 tyrosine phosphorylation, and JAK2, STAT/1/3/5 tyrosine phosphorylation was time-dependent and dose-dependent, whereas ERK1/2 was not. Both hGH and pGH demonstrated similar kinetics for STATs 1, 3 and 5 phosphorylation, but the pGH-mediated tyrosine phosphorylation was weaker than that mediated by hGH. Our observations indicated that the levels of main signalling proteins phosphorylation triggered by hGH or pGH were not exactly the same, which may explain the different biological activities showed by primate GH and non-primate GH.

过表达西藏小型猪GHR显负性突变体的慢病毒载体构建及其功能验证

目的：构建过表达西藏小型猪GHR基因显负性突变体的慢病毒载体，并对其进行功能验证。方法：首先以pRLG3A为模板，PCR扩增CAG启动子，In-Fusion克隆至Cla I/BamH I双酶切的pCD550A-1载体中，替换其EF1α启动子，获得pCAGGP；然后从西藏小型猪肝脏组织cDNA中PCR扩增GHR基因的显负性突变体片段(dnGHR)；最后将dnGHR片段插入pCAGGP多克隆位点，最终得到过表达猪GHR显负性突变体的慢病毒载体pCdnGGP。对所构建的质粒进行测序和酶切鉴定。将所构建的pCdnGGP载体转染293T细胞，并将包装好的慢病毒感染猪的胚胎成纤维细胞(PEFs)，倒置荧光显微镜检测GFP表达，收集细胞并提取总RNA检测dnGHR基因在293T细胞和PEFs中的表达，以对pCdnGGP进行体外功能验证。结果：测序和酶切证实成功构建了pCdnGGP。pCdnGGP转染293T细胞以及病毒感染PEFs之后，倒置荧光显微镜下可见绿色荧光，RT-PCR检测证实dnGHR在293T细胞和PEFs中均能正常表达。结论：成功构建过表达猪GHR基因显负性突变体的慢病毒载体，为相关后续研究打下了良好基础。 Objective: To generate lentiviral vector harboring the dominant-negative mutant of porcine growth hormone receptor, and in vitro confirm the functionality of the resulting plasmid. Methods: Firstly, CAG promoter was amplified from pRLG3A, and subsequently subcloned into pCD550A-1 at the Cla I/BamH I sites by In-Fusion cloning, designated pCAGGP, as verified by restriction enzyme digestion and sequencing. Secondly, the fragment of dominant-negative mutant of growth hormone receptor (dnGHR) was amplified from liver cDNA of Tibet minipig, and then inserted into pCAGGP at the multiple cloning site (MCS) by In-Fusion cloning to generate pCdnGGP, as verified by restriction enzyme digestion and sequencing. To in vitro confirm the functionality of the resulting plasmid (pCdnGGP), pCdnGGP was transiently transfected into 293T cells to produce lentiviurs LV- CdnGGP, and LV-CdnGGP was used to infect pig embryonic fibrolast (PEFs), followed by EGFP assay under inverted fluorescence microscope and detecting dnGHR transgene expression by RT-PCR. Results: Enzyme digestion and DNA sequencing demonstrated that pCdnGGP was successfully constructed. 293T cells transiently transfected with pCdnGGP and PEFs infected by LV-CdnGGP displayed the green fluorescence under inverted fluorescence microscope and the increased expression of dnGHR. Conclusion: The lentiviral vector harboring pig dnGHR gene was successfully constructed, which will lay a solid foundation for further research.

Preparation and Characterization of an Antibody Antagonist That Targets the Porcine Growth Hormone Receptor.

A series of antagonists specifically targeting growth hormone receptors (GHR) in different species, such as humans, rats, bovines, and mice, have been designed; however, there are currently no antagonists that target the porcine growth hormone (GH). Therefore, in this study, we developed and characterized a porcine GHR (pGHR) antibody antagonist (denoted by AN98) via the hybridoma technique. The results from enzyme-linked immunosorbent assay, fluorescence activated cell sorter, indirect immunoinfluscent assay, and competitive receptor binding analysis showed that AN98 could specifically recognize pGHR, and further experiments indicated that AN98 could effectively inhibit pGH-induced signalling in CHO-pGHR cells and porcine hepatocytes. In addition, AN98 also inhibited GH-induced insulin-like growth factor-1 (IGF-1) secretion in porcine hepatocytes. In summary, these findings indicated that AN98, as a pGHR-specific antagonist, has potential applications in pGH-pGHR-related research on domestic pigs.