• Asian-Australasian Journal of Animal Sciences
• /
• 제31권4호
• /
• pp.499-504
• /
• 2018

Objective: Recent 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. Methods: In 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. Results: The 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. Conclusion: Nuclear localization of pGHR induced by the in vivo pGH treatment suggests new functions and/or novel roles of nuclear pGHR, which deserve further study.

• Asian-Australasian Journal of Animal Sciences
• /
• 제29권10호
• /
• pp.1508-1514
• /
• 2016

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.

• Asian-Australasian Journal of Animal Sciences
• /
• 제28권4호
• /
• pp.573-583
• /
• 2015

B-32 is one of a panel of monoclonal anti-idiotypic antibodies to growth hormone (GH) that we developed. To characterize and identify its potential role as a novel growth hormone receptor (GHR) agonist, we determined that B-32 behaved as a typical $Ab2{\beta}$ based on a series of enzyme-linked immunosorbent assay assays. The results of fluorescence-activated cell sorting, indirect immunofluorescence and competitive receptor binding assays demonstrated that B-32 specifically binds to the GHR expressed on target cells. Next, we examined the resulting signal transduction pathways triggered by this antibody in primary porcine hepatocytes. We found that B-32 can activate the GHR and Janus kinase (2)/signal transducers and activators of transcription (JAK2/STAT5) signalling pathways. The phosphorylation kinetics of JAK2/STAT5 induced by either GH or B-32 were analysed in dose-response and time course experiments. In addition, B32 could also stimulate porcine hepatocytes to secrete insulin-like growth factors-1. Our work indicates that a monoclonal anti-idiotypic antibody to GH (B-32) can serve as a GHR agonist or GH mimic and has application potential in domestic animal (pig) production.

• 한국가축번식학회지
• /
• 제25권4호
• /
• pp.381-388
• /
• 2001

본 연구는 돼지에서 성장호르몬과 결합되는 부위에 변이를 유도하여 결합력이 향상된 성장호르몬 결합단백질을 획득하기 위하여 수행되었다. 돼지의 지방으로부터 얻은 성장호르몬 수용체 RNA 내 성장호르몬 결합단백질 부분을 756 bp의 cDNA로 전향하고 클로닝한 후 site-directed mutagenesis 방법을 이용하여 26과 122번째 아미노산을 변이시켰다. 26번째 아미노산은 성장 호르몬과의 결합에 관련이 있다고 알려져 있는 돼지 성장호르몬 수용체 외막에 존재하는 다섯 군데의 N-linked glycosylation 부위와 가까이 위치한 부분이고, 122번째 아미노산은 소에서의 결합부위로 알려져 있다. 이렇게 변이를 유도한 성장호르몬 결합 단백질을 pET-32(c) 발현벡터에 삽입시키고 과발현시켰고 이를 정제하여 30 kDa의 변이를 유도한 성장호르몬 결합 단백질을 얻었다. 이러한 방법으로 성장호르몬 결합 단백질을 성장기에 있는 세포나 동물에 주입한다면 보다 향상된 성장을 볼 수 있을 것으로 사료된다.

• 한국발생생물학회지:발생과생식
• /
• 제21권2호
• /
• pp.131-138
• /
• 2017

This study was conducted to investigate stimulatory effect of epidermal growth factor (EGF) on nuclear maturation and the expression level of EGF-receptor (EGFR), GM-130 (a marker of Golgi apparatus), transport protein Sec61 subunit beta ($Sec61{\beta}$), and coatomer protein complex subunit gamma 2 (COPG2) in porcine oocytes. The cumulus-oocyte complexes were collected from follicle with 3-6 mm in diameter. They were incubated in medium with/without EGF for 22 h (IVM I) and subsequently incubated hormone-free medium with/without EGF for 22 h (IVM II). Nuclear maturation state was checked by aceto-orcein stain. Protein expression of EGFR, GM-130, $Sec61{\beta}$, and COPG2 were measured by immunofluorescence. In results, nuclear maturation of oocytes in EGF non-treated oocytes were significantly lower than EGF-treated groups at IVM I or IVM II stage (P<0.05), whereas maturational rate in EGF treatment groups at both of IVM stage was higher in among the all treatment groups (P<0.05). EGFR, GM-130, $Sec61{\beta}$ and COPG2 were expressed in the cytoplasm of oocytes. Especially, GM-130 and EGFR were strongly expressed, but $Sec61{\beta}$ and COPG2 were weakly expressed in cortical area of cytoplasm. The protein level of GM-130, $Sec61{\beta}$, and COPG2 were significantly higher in the EGF-treated groups (P<0.05). However EGFR was no difference between non EGF-treated groups and control. In conclusion, EGF plays an important role in the systems for oocyte maturation with endoplasmic reticulum and Golgi apparatus. In addition, the protein levels of $Sec61{\beta}$ and COPG2 could be changed by EGF in the porcine oocytes during maturation.

• Asian-Australasian Journal of Animal Sciences
• /
• 제14권3호
• /
• pp.307-315
• /
• 2001

Litter size has been one of the important economic traits in porcine reproduction. The insulin-like growth factor (IGF) system has been shown to mediate actions of the steroid hormone or to synergize with other endocrine factors so that it consequently plays roles in reproductive processes, including ovulation, implantation, maintenance of pregnancy, and fetal development. However, the effect of the serum IGF system on porcine litter size has not been deeply studied. Therefore, this study was conducted to relate serum IFG-II concentration and IGF binding protein-3 (IGFBP-3) expression with porcine litter size. Moreover, the possible association of those with estrogen receptor (ER) as a candidate gene for litter size was investigated. Swine were separated into two groups showing high and low litter sizes, and sera were collected from sows in the estrous cycle to postnatal growth of their female progeny. Serum IFG-II concentration was measured by radioimmunoassay and IGFBP-3 expression was detected by Western ligand blotting. During the estrous cycle, IGFBP-3 expression in both groups decreased moderately from metestrus to estrus, but IFG-II concentration showed a reverse pattern. Also, IFG-II concentration and IGFBP-3 expression decreased gradually as pregnancy proceeded. Unlike IGFBP-3, IFG-II decreased moderately as newborn pigs grew. Significant differences in serum IFG-II amount between the two groups were detected at 60 (p<0.01), 75, 90, and 105 d (p<0.05) of pregnancy and at 60 (p<0.01), 45, and 105 d (p<0.05) of postnatal growth. Furthermore, based on ER genotypes, a high litter size group with genotypes AB and BB showed lower IFG-II concentration than a low litter size group with a genotype AA during pregnancy. Taken together, the results indicate that the serum IFG-II and IGFBP-3 are correlated with the litter size in pigs.