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A synthetic progestin altrenogest (ALT) is used to synchronize the estrus cycle of swine for fixed-time artificial insemination (AI) and has been shown to improve follicular development and reproductive performances in post-weaning sows. However, the effects of ALT treatment on reproductive tracts, including the ovaries, oviducts and uterus have not been yet clarified. In this study, we examined the expression of genes involved in endometrial responses in ALT-treated sows. ALT did not significantly alter luteinizing hormone (LH), follicle-stimulating hormone (FSH) and estradiol profiles in blood compared to untreated control. Quantitative RT-polymerase chain reaction (qRT-PCR) analysis showed that the expression of genes encoding galectin-3 (LGALS3) and fibroblast growth factor 9 (FGF9) was upregulated in the reproductive tracts of ALT-treated sows, including the ovaries, oviducts and uteri. Moreover, ALT treatment induced the expression of FGF9 and galectin-3 proteins, and promoted their localization to the luminal epithelium of the oviducts and uterus. Our findings suggest that the enhancement of reproductive performance shown by ALT-treated sows is associated with the upregulation of galectin-3 and FGF9, which are essential for endometrial receptivity, successful implantation, and pregnancy.
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Fator 9 de Crescimento de Fibroblastos , Galectina 3 , Suínos/genética , Acetato de Trembolona , Animais , Feminino , Fator 9 de Crescimento de Fibroblastos/metabolismo , Hormônio Foliculoestimulante , Galectina 3/metabolismo , Inseminação Artificial/veterinária , Ovário/efeitos dos fármacos , Ovário/metabolismo , Oviductos/efeitos dos fármacos , Oviductos/metabolismo , Gravidez , Acetato de Trembolona/análogos & derivados , Acetato de Trembolona/farmacologia , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
OBJECTIVE: We examined the localization and expression of H+ pumping vacuolar ATPase (V-ATPase) and cytokeratin 5 (KRT5) in the epididymis of pigs, expressed in clear and basal cells, respectively, during postnatal development. METHODS: Epididymides were obtained from pigs at 1, 7, 21, 60, 120, and 180 days of age; we observed the localization and expression patterns of V-ATPase and KRT5 in the different regions of these organs, namely, the caput, corpus, and cauda. The differentiation of epididymal epithelial cells was determined by immunofluorescence labeling using cell-type-specific markers and observed using confocal microscopy. RESULTS: At postnatal day 5 (PND5), the localization of clear cells commenced migration from the cauda toward the caput. Although at PND120, goblet-shaped clear cells were detected along the entire length of the epididymis, those labeled for V-ATPase had disappeared from the corpus to cauda and were maintained only in the caput epididymis in adult pigs. In contrast, whereas basal cells labeled for KRT5 were only present in the vas deferens at birth, they were detected in all regions of the epididymis at PND60. These cells were localized at the base of the epithelium; however, no basal cells characterized by luminally extending cell projections were observed in any of the adult epididymides examined. CONCLUSION: The differentiation of clear and basal cells progressively initiates in a retrograde manner from the cauda to the caput epididymis. The cell-type-specific distribution and localization of the epithelial cells play important roles in establishing a unique luminal environment for sperm maturation and storage in the pig epididymis.
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Tissue plasminogen activator (tPA) is a protein involved in the breakdown of blood clots. We have previously produced a human tPA (htPA)-overexpressing transgenic pig using a mammary gland-specific promoter. In this study, we have established a transgenic pig mammary gland cell line that produces recombinant htPA. The mammary gland cells grew well and retained their character over long periods of culture. There was no difference in the extent of apoptosis in transgenic cells compared to wild-type mammary gland cells. In addition, the transgenic mammary gland cells expressed and secreted htPA into the conditioned media at a concentration similar to that in milk. This transgenic cell line represents a simple and ethical method for recombinant htPA production.
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Glândulas Mamárias Animais/metabolismo , Ativador de Plasminogênio Tecidual/biossíntese , Animais , Animais Geneticamente Modificados , Linhagem Celular , Células Cultivadas , Feminino , Humanos , Leite/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Suínos/genética , Ativador de Plasminogênio Tecidual/genéticaRESUMO
We demonstrated that ETV4 is a transcriptional activator of the NANOG gene in human embryonic carcinoma NCCIT cells. The endogenous expression of NANOG and ETV4 in naïve cells was significantly down-regulated upon differentiation and by shRNA-mediated knockdown of ETV4. NANOG transcription was significantly upregulated by ETV4 overexpression. A putative ETS binding site (EBS) is present in the region (-285 to -138) of the proximal promoter. Site-directed mutagenesis of the putative EBS (-196AGGATT-191) abolished NANOG promoter activity and ETV4 interacted with this putative EBS both in vivo and in vitro. Our data provide the molecular details of ETV4-mediated NANOG gene expression.
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Proteínas E1A de Adenovirus/metabolismo , Células-Tronco de Carcinoma Embrionário/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína Homeobox Nanog/genética , Proteínas Proto-Oncogênicas/metabolismo , Humanos , Proteína Homeobox Nanog/metabolismo , Proteínas Proto-Oncogênicas c-ets , Células Tumorais CultivadasRESUMO
Esculetin, a coumarin derivative, is a phenolic compound isolated from Artemisia capillaris, Citrus limonia, and Euphorbia lathyris. Although it has been reported to have anti-inflammatory, anti-oxidant, and anti-proliferative activities in several human cancers, its anti-proliferative activity against non-small-cell lung carcinoma (NSCLC) and the molecular mechanisms involved have not been adequately elucidated. In this study, we used two NSCLC cell lines (NCI-H358 and NCI-H1299) to investigate the anti-proliferative activity and apoptotic effect of esculetin. Our data showed that esculetin-treated cells exhibited reduced proliferation and apoptotic cell morphologies. Intriguingly, the transcription factor specificity protein 1 (Sp1) was significantly suppressed by esculetin in a dose- and time-dependent manner. Furthermore, the levels of p27 and p21, two key regulators of the cell cycle, were up-regulated by the esculetin-mediated down-regulation of Sp1; the level of a third cell-cycle regulator, survivin, was decreased, resulting in caspase-dependent apoptosis. Therefore, we conclude that esculetin could be a potent anti-proliferative agent in patients with NSCLC.
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Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Fator de Transcrição Sp1/metabolismo , Umbeliferonas/farmacologia , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologiaRESUMO
Growth and differentiation factor 3 (GDF3), a mammalian-specific transforming growth factor ß ligand, and OCT4, one of key stem cell transcription factors, are expressed in testicular germ cell tumors (TGCTs) as well as pluripotent stem cells. To understand the molecular mechanism by which OCT4 and GDF3 function in tumorigenesis as well as stemness, we investigated the transcriptional regulation of GDF3 mediated by OCT4 in human embryonic carcinoma (EC) NCCIT cells, which are pluripotent stem cells of TGCTs. GDF3 and OCT4 was highly expressed in undifferentiated NCCIT cells and then significantly decreased upon retinoic acid-induced differentiation in a time-dependent manner. Moreover, GDF3 expression was reduced by short hairpin RNA-mediated knockdown of OCT4 and increased by OCT4 overexpression, suggesting that GDF3 and OCT4 have a functional relationship in pluripotent stem cells. A promoter-reporter assay revealed that the GDF3 promoter (-1721-Luc) activity was significantly activated by OCT4 in a dose-dependent manner. Moreover, the minimal promoter (-183-Luc) was sufficient for OCT4-mediated transcriptional activation and provided a potential binding site for the direct interaction with OCT4. Collectively, this study provides the evidence about the regulatory mechanism of GDF3 mediated by OCT4 in pluripotent EC cells.
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Carcinoma Embrionário/genética , Fator 3 de Diferenciação de Crescimento/genética , Fator 3 de Transcrição de Octâmero/genética , Neoplasias Testiculares/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Masculino , Fator 3 de Transcrição de Octâmero/metabolismo , Transcrição GênicaRESUMO
E26 transformation-specific (ETS) transcription factors play important roles in normal and tumorigenic processes during development, differentiation, homeostasis, proliferation, and apoptosis. To identify critical ETS factor(s) in germ cell-derived cancer cells, we examined the expression patterns of the 27 ETS transcription factors in naive and differentiated NCCIT human embryonic carcinoma cells, which exhibit both pluripotent and tumorigenic characteristics. Overall, expression of ETS factors was relatively low in NCCIT cells. Among the 27 ETS factors, polyomavirus enhancer activator 3 (PEA3) and epithelium-specific ETS transcription factor-1 (ESE-1) exhibited the most significant changes in their expression levels. Western blot analysis confirmed these patterns, revealing reduced levels of PEA3 protein and elevated levels of ESE-1 protein in differentiated cells. PEA3 increased the proportion of cells in S-phase and promoted cell growth, whereas ESE-1 reduced proliferation potential. These data suggest that PEA3 and ESE-1 may play important roles in pluripotent and tumorigenic embryonic carcinoma cells. These findings contribute to our understanding of the functions of oncogenic ETS factors in germ cell-derived stem cells during processes related to tumorigenesis and pluripotency.
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Carcinoma Embrionário/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-ets/biossíntese , Ativação Transcricional/efeitos dos fármacos , Tretinoína/farmacologia , Carcinoma Embrionário/patologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Tamanho Celular , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Humanos , Proteínas Proto-Oncogênicas c-ets/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , TransfecçãoRESUMO
BACKGROUND: Lawsonia intracellularis is the causative agent of proliferative enteropathy and is associated with several outbreaks, causing substantial economic loss to the porcine industry. OBJECTIVES: In this study, we focused on demonstrating the protective effect in the mouse model through the immunological bases of two vaccine strains against porcine proliferative enteritis. METHODS: We used live-attenuated Salmonella Typhimurium (ST) secreting two selected immunogenic LI antigens (Lawsonia autotransporter A epitopes and flagellin [FliC]-peptidoglycan-associated lipoprotein-FliC) as the vaccine carrier. The constructs were cloned into a Salmonella expression vector (pJHL65) and transformed into the ST strain (JOL912). The expression of immunogenic proteins within Salmonella was evaluated via immunoblotting. RESULTS: Immunizing BALB/c mice orally and subcutaneously induced high levels of LI-specific systemic immunoglobulin G and mucosal secretory immunoglobulin A. In immunized mice, there was significant upregulation of interferon-γ and interleukin-4 cytokine mRNA and an increase in the subpopulations of cluster of differentiation (CD) 4+ and CD 8+ T lymphocytes upon splenocytes re-stimulation with LI antigens. We observed significant protection in C57BL/6 mice against challenge with 106.9 times the median tissue culture infectious dose of LI or 2 × 109 colony-forming units of the virulent ST strain. Immunizing mice with either individual vaccine strains or co-mixture inhibited bacterial proliferation, with a marked reduction in the percentage of mice shedding Lawsonia in their feces. CONCLUSIONS: Salmonella-mediated LI gene delivery induces robust humoral and cellular immune reactions, leading to significant protection against LI and salmonellosis.
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Lawsonia (Bactéria) , Doenças dos Roedores , Doenças dos Suínos , Vacinas , Camundongos , Animais , Suínos , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Salmonella typhimurium , Camundongos Endogâmicos BALB C , Doenças dos Suínos/prevenção & controleRESUMO
Early diagnosis of pregnancy is directly related to cost-effective livestock production. We produced a rat monoclonal antibody (mAb) against synthesized porcine early pregnancy factor (pEPF) using conventional hybridoma technology and used it as a tool for the detection of early pregnancy in Duroc sows. The rat pEPF-mAb showed reactivity to uterine tissues of pregnant sows 20 or 30 days post-mating (day 0 defined as the day of mating) and non-pregnant sows (confirmed signs of estrus) in western blotting. Immunohistochemical analysis confirmed that pEPF was located in the stromal and grand epithelial tissues of pregnant sows 20 or 30 days post-mating. In the enzyme-linked immunosorbent assay, pEPF expression in urine and blood showed similar results, with the highest expression observed in pregnant sows 20 days post-mating, whereas there was no significant difference in expression levels between non-pregnant sows and pregnant sows 30 days post-mating. The pEPF-mAb-based pregnancy diagnostic kit can be applied to pig urine samples non-invasively collected at 20 days post-mating with 70 % accuracy. Further improvements to the kit's diagnostic performance may lead to substantial benefits for the swine industry, facilitating more efficient and accurate reproductive management.
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Assisted reproduction technology (ART) procedures are often impacted by post-ovulatory aging (POA), which can lead to reduced fertilization rates and impaired embryo development. This study used RNA sequencing analysis and experimental validation to study the similarities and differences between in vivo- and vitro-matured porcine oocytes before and after POA. Differentially expressed genes (DEGs) between fresh in vivo-matured oocyte (F_vivo) and aged in vivo-matured oocyte (A_vivo) and DEGs between fresh in vitro-matured oocyte (F_vitro) and aged in vitro-matured oocyte (A_vitro) were intersected to explore the co-effects of POA. It was found that "organelles", especially "mitochondria", were significantly enriched Gene Ontology (GO) terms. The expression of genes related to the "electron transport chain" and "cell redox homeostasis" pathways related to mitochondrial function significantly showed low expression patterns in both A_vivo and A_vitro groups. Weighted correlation network analysis was carried out to explore gene expression modules specific to A_vivo. Trait-module association analysis showed that the red modules were most associated with in vivo aging. There are 959 genes in the red module, mainly enriched in "RNA binding", "mRNA metabolic process", etc., as well as in GO terms, and "spliceosome" and "nucleotide excision repair" pathways. DNAJC7, IK, and DDX18 were at the hub of the gene regulatory network. Subsequently, the functions of DDX18 and DNAJC7 were verified by knocking down their expression at the germinal vesicle (GV) and Metaphase II (MII) stages, respectively. Knockdown at the GV stage caused cell cycle disorders and increase the rate of abnormal spindle. Knockdown at the MII stage resulted in the inefficiency of the antioxidant melatonin, increasing the level of intracellular oxidative stress, and in mitochondrial dysfunction. In summary, POA affects the organelle function of oocytes. A_vivo oocytes have some unique gene expression patterns. These genes may be potential anti-aging targets. This study provides a better understanding of the detailed mechanism of POA and potential strategies for improving the success rates of assisted reproductive technologies in pigs and other mammalian species.
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Cryotolerance of matured bovine oocytes is not fully practical even though a promising vitrification procedure with a ultrarapid cooling rate was applied. The present study was conducted to investigate whether recovery culture of vitrified-warmed bovine oocytes with an inhibitor (Y-27632) of Rho-associated coiled-coil kinase (ROCK) can improve the developmental potential after in vitro fertilization (IVF) and in vitro culture. Immediately after warming, almost all oocytes appeared to be morphological normal. Treatment of the postwarming oocytes with 10 µM Y-27632 for 2 h resulted in the significantly higher oocyte survival rate before IVF as well as higher cleavage rate and blastocyst formation rate. Quality analysis of the resultant blastocysts in terms of total cell number and apoptotic cell ratio also showed the positive effect of the Y-27632 treatment. Time-dependent change in mitochondrial activity of the vitrified-warmed oocytes was not influenced by ROCK inhibition during the period of recovery culture. However, the ability of ooplasm to support single-aster formation was improved by the ROCK inhibition. Thus, inhibition of ROCK activity in vitrified-warmed bovine oocytes during a short-term recovery culture can lead to higher developmental competence, probably due to decreased apoptosis and normalized function of the microtubule-organizing center.
Assuntos
Amidas/farmacologia , Criopreservação/métodos , Oócitos/efeitos dos fármacos , Piridinas/farmacologia , Vitrificação , Quinases Associadas a rho/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Blastocisto/citologia , Blastocisto/fisiologia , Bovinos , Centro Organizador dos Microtúbulos/efeitos dos fármacos , Centro Organizador dos Microtúbulos/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Oócitos/citologia , Oócitos/fisiologiaRESUMO
The Rho GTPases are the sub-group of Ras super family and identified in all eukaryotes. The Rho GTPases effect different cellular signaling pathways involved in a number of diseases such as cancer, neurological and cardiovascular disorders. Members of Rho GTPases including RhoA, RhoC and Rac1 play a major role in regulation of apoptosis in different kind of stress conditions. Here we investigated the Rho GTPase activating protein 15 (ArhGAP15) gene knock-down effect on apoptosis induced by ethanol in bovine fibroblast cells. The bovine Fibroblast cells were treated and transfected with two different concentrations (50 and 100 nM) of ArhGAP15 siRNA for 48 h respectively. Both concentrations of siRNA were effective and the results of RT-PCR revealed an efficient knock-down of ArhGAP15 mRNA in fibroblast cells. Further, the normal cells exposed to a 100 mM ethanol concentration showed a reduction in cell viability and induced the ratio of apoptosis related Bax/Bcl-2 proteins compared with ArhGAP15 siRNA transfected ethanol treated cells. Ethanol also increased caspase-3 expression in normal fibroblast cells compared with transfected cells. The ArhGAP15 knock-down cells treated with ethanol decreased Bax/Bcl-2 ratio and lower caspase-3 protein levels in ArhGAP15 knocked-down cells. Our results suggest that apoptosis induced by ethanol involves the activation of Rho GTPase activating protein 15 and silencing of the said gene protects apoptosis.
Assuntos
Apoptose/genética , Etanol/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , RNA Interferente Pequeno/genética , Animais , Apoptose/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Fibroblastos/citologia , RNA Mensageiro/genética , Transfecção/métodos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Y-box binding protein 1 (YBX1) plays important roles in RNA stabilization, translation, transcriptional regulation, and mitophagy. However, its effects on porcine preimplantation embryos remain unclear. In this study, we knocked down YBX1 in the one-cell (1C) stage embryo via small interfering RNA microinjection to determine its function in porcine embryo development. The mRNA level of YBX1 was found to be highly expressed at the four-cell (4C) stage in porcine embryos compared with one-cell (1C) and two-cell (2C) stages. The number of blastocysts was reduced following YBX1 knockdown. Notably, YBX1 knockdown decreased the phosphatase and tensin homolog-induced kinase 1 (PINK1) and parkin RBR E3 ubiquitin protein ligase (PRKN) mRNA levels. YBX1 knockdown also decreased PINK1, active mitochondria, and sirtuin 1 levels, indicating reduced mitophagy and mitochondrial biogenesis. Furthermore, YBX1 knockdown increased the levels of glucose-regulated protein 78 (GRP78) and calnexin, leading to endoplasmic reticulum (ER) stress. Additionally, YBX1 knockdown increased autophagy and apoptosis. In conclusion, knockdown of YBX1 decreases mitochondrial function, while increasing ER stress and autophagy during embryonic development.
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Many studies have been conducted to improve technology for semen cryopreservation in pigs. However, computer-assisted analysis of sperm motility and morphology is insufficient to predict the molecular function of frozen-thawed semen. More accurate expression patterns of boar sperm proteins may be derived using the isobaric tags for relative and absolute quantification (iTRAQ) technique. In this study, the iTRAQ-labeling system was coupled with liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis to identify differentially expressed CM10-fractionated proteins between fresh and frozen-thawed boar semen. A total of 76 protein types were identified to be differentially expressed, among which 9 and 67 proteins showed higher and lower expression in frozen-thawed than in fresh sperm samples, respectively. The classified functions of these proteins included oxidative phosphorylation, mitochondrial inner membrane and matrix, and pyruvate metabolic processes, which are involved in adenosine triphosphate (ATP) synthesis; and sperm flagellum and motile cilium, which are involved in sperm tail structure. These results suggest a possible network of biomarkers associated with survival after the cryopreservation of Duroc boar semen.
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We demonstrate the regulation of OCT4 gene expression mediated by liver receptor homolog-1 (LRH-1) in human embryonic carcinoma cells. LRH-1 and OCT4 are co-expressed in undifferentiated NCCIT cells and decreased during retinoic acid-induced differentiation. Dose-dependent overexpression of LRH-1 transactivated the OCT4 promoter activity and its dominant negative form with a deletion of activation function-2 motif reduced the activity even in the presence of LRH-1. The OCT4 promoter contains potent three LRH-1 binding sites; one within conserved region (CR) 1 and two within CR2. Mutagenesis of each binding site affected the decrease in OCT4 promoter activity and the 2nd binding site mutant most significantly reduced the transcriptional activity, compared to that of 1st and 3rd binding site mutants, respectively. Simultaneous disruption of 2nd and 3rd binding sites led to significant down-regulation of the activity even in the presence of 1st binding site-containing CR1. Moreover, mutation of each binding element within native or exogenous minimal promoter-driven CR1 or CR2 also decreased the promoter activity to some different extent, suggesting that three binding elements may be implicated in the induction of the full-activity of OCT4 promoter. In vivo binding assay revealed the 2nd and 3rd binding motifs within CR2 were more enriched than the 1st one within CR1. Taken together, our study indicates that LRH-1 acts as a transcriptional activator in the regulation of OCT4 gene expression through the cooperative interaction with three binding sites directly or/and indirectly.
Assuntos
Carcinoma Embrionário/genética , Regulação Neoplásica da Expressão Gênica , Fator 3 de Transcrição de Octâmero/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Ativação Transcricional , Sítios de Ligação , Carcinoma Embrionário/patologia , Linhagem Celular Tumoral , Humanos , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/genética , Transcrição Gênica , Tretinoína/farmacologiaRESUMO
OBJECTIVE: The objective of this study was to estimate the genetic correlation (rpc) of growth performance between purebred (Duroc and Korean native) and synthetic (WooriHeukDon) pigs using a single-step method. METHODS: Phenotypes of 15,902 pigs with genotyped data from 1,792 pigs from a nucleus farm were used for this study. We estimated the rpc of several performance traits between WooriHeukDon and purebred pigs: day of target weight (DAY), backfat thickness (BF), feed conversion rate (FCR), and residual feed intake (RFI). The variances and covariances of the studied traits were estimated by an animal multi-trait model that applied the Bayesian inference. RESULTS: rpc within traits was lower than 0.1 for DAY and BF, but high for FCR and RFI; in particular, rpc for RFI between Duroc and WooriHeukDon pigs was nearly 1. Comparison between different traits revealed that RFI in Duroc pigs was associated with different traits in WooriHeukDon pigs. However, the most of rpc between different traits were estimated with low or with high standard deviation. CONCLUSION: The results indicated that there were substantial differences in rpc of traits in the synthetic WooriHeukDon pigs, which could be caused by these pigs having a more complex origin than other crossbred pigs. RFI was strongly correlated between Duroc and WooriHeukDon pigs, and these breeds might have similar single nucleotide polymorphism effects that control RFI. RFI is more essential for metabolism than other growth traits and these metabolic characteristics in purebred pigs, such as nutrient utilization, could significantly affect those in synthetic pigs. The findings of this study can be used to elucidate the genetic architecture of crossbred pigs and help develop new breeds with target traits.
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The present study aimed to investigate the characteristics of mPEA15 expressing transgenic pig (TG pig) as a potential model for diabetes. Expression analysis confirmed the ubiquitous expression of mPEA15 in TG pigs at F4. Oral glucose tolerance test results showed that restoration of normal glucose levels was significantly delayed in the TG pigs when compared with that in the wild-type pigs (WT pigs). Primary skeletal muscle cells isolated from TG pigs demonstrated reduced glucose uptake and reduced GLUT4 translocation to the plasma membrane in response to insulin treatment. Combined, these results suggest that mPEA15 expressing pigs has a glucose intolerance and insulin resistance which are known to mediate the pathophysiology of type 2 diabetes mellitus. Thus, mPEA15 transgenic pigs would serve as a promising model for diabetes translational research.
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OCT4 plays a crucial role in pluripotency and self-renewal of embryonic stem cells. OCT4 is also expressed in testicular germ cell tumors (GCTs), suggesting the important function of OCT4 as an oncogenic factor in GCTs. To understand the molecular mechanism of human OCT4 (hOCT4) in tumorigenesis as well as stemness, we identified hOCT4 transactivation domains in human embryonic carcinoma cells. Context analyses of heterologous GAL4 and natural hOCT4 revealed that each N-terminal domain or C-terminal domain independently stimulated transcriptional activity, and that both domains are required for synergistic transactivation by deletion mapping analysis. Dose-dependent overexpression of exogenous hOCT4 significantly decreased the transcriptional activity of the hOCT4 promoter. This inhibition was reversed by the removal of one or both domains. These results suggest that the inhibitory effect of hOCT4 is mediated by transactivation domains, and that the self-regulation of hOCT4 may be mediated via a negative feedback loop in pluripotent cells.
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Regulação Neoplásica da Expressão Gênica , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/metabolismo , Ativação Transcricional , Sítios de Ligação , Linhagem Celular Tumoral , Análise Mutacional de DNA , Humanos , Fator 3 de Transcrição de Octâmero/genética , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Deleção de Sequência , Transcrição GênicaRESUMO
Recently, diabetes mellitus (DM) has shown rapid global increases with about five million deaths annually. Animal models are imperative to understand disease mechanisms and develop diagnostic, preventive, and therapeutic interventions in translational research. Rodent and mini-pig models have been established and widely used for DM research. However, domestic pig models are limited in spite of advantages such as pharmacokinetic and physiopathological availability. This study examines the potential use of domestic pigs expressing recombinant human erythropoietin (rhEPO) as disease and therapeutic response models for DM. We previously generated transgenic pigs (n = 16, EPO Tg) in which rhEPO was expressed and circulated in all organs. Thirty-two pigs, including 16 controls, were fed high fat (HF) diets for 42 weeks. Subsequently, blood samples for chemical and metabolic analysis were collected after fasting for 24â h and glucose loading for oral glucose tolerance tests (OGTTs). We found increased activation of the PI3â K/Akt signaling pathway under hypoxic conditions after rhEPO treatment, and HF diet-inducible-obesity in the EPO Tg and control pigs. OGTTs showed lower fasting glucose levels in the EPO Tg pigs than in controls before and after the HF diet, suggesting that rhEPO may affect glucose concentrations. Insulin and C-peptide concentrations responded slowly to glucose administration and returned to initial levels after 2â h. The blood test results suggest that EPO might affect metabolic and chemical components such as glucose, high-density lipoprotein, glucagon, triglyceride, and free fatty acid. Our findings support the use of rhEPO transgenic domestic pigs as model animals for translational DM research.
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Male germ cell apoptosis has been described in heat-damaged testes by cryptorchidism. In the present study, wild type pig testes were compared with cryptorchid testes via histological and immunohistological analyses. Spermatozoa were not detected in two cryptorchid testes and the diameters of seminiferous tubules were significantly reduced in cryptorchid pig testes compared with wild type pig testes. Cells expressing marker genes for undifferentiated spermatogonia, such as protein gene product 9.5 was significantly decreased in cryptochid pig testes. In addition, the numbers of cells expressing DEAD-box polypeptide 4 (VASA), synaptonemal complex protein 3, protamine, and acrosin (a biomarker of spermatocyte, spermatid, and spermatozoa) were significantly reduced in cryptochid pig testes. However, the number of vimentin-expressing Sertoli cells was not changed or was significantly increased in cryptorchid pig testes. This result indicates that male germ cells are specifically damaged by heat in cryptorchid pig testes and not Sertoli cells. These findings will facilitate the further study of spermatogenesis and the specific mechanisms by which cryptorchidism causes male infertility.