RESUMO
Probiotics are used in pigs as nutritional supplements to improve health and induce the development of muscle and adipose tissue for enhancing growth performance and harvesting quality meat. In this study, we investigated the effects of Bacillus-based probiotic supplementation on the physiological and biochemical changes in Jeju native pigs (JNPs), including growth performance, backfat layers, blood parameters, serum IgG levels, myogenic and adipogenic markers, and expression of inflammatory markers. Average daily gain and feed efficiency were higher in the Bacillus diet group than in the basal diet group, while backfat thickness was lower in the Bacillus diet group than in the basal diet group. Blood biochemical parameters and hematological profiles were not altered significantly by Bacillus-based probiotic supplementation. Serum IgG concentration increased in the Bacillus diet group compared to the basal diet group. The Bacillus diet group showed increased adipogenic and myogenic markers expression in the longissimus dorsi muscle and adipose tissues. Overall, the data suggest that the Bacillus-based probiotics-supplemented diet regulates myogenesis and adipogenesis in JNPs and improves growth performance. We postulate that this may be due to the changes in the gut microbiota of pigs due to probiotic supplementation.
Assuntos
Bacillus , Animais , Suínos , Adipogenia , Suplementos Nutricionais , Dieta/veterinária , Imunoglobulina G , Ração Animal/análiseRESUMO
Although allogenic meniscus grafting can be immunologically safe, it causes immune rejection due to an imbalanced tissue supply between donor and recipient. Pigs are anatomically and physiologically similar to adult humans and are, therefore, considered to be advantageous xenotransplantation models. However, immune rejection caused by genetic difference damages the donor tissue and can sometimes cause sudden death. Immune rejection is caused by genes; porcine GGTA1, CMAH, and B4GLANT2 are the most common. In this study, we evaluated immune cells infiltrating the pig meniscus transplanted subcutaneously into BALB/c mice bred for three weeks. We compared the biocompatibility of normal Jeju native black pig (JNP) meniscus with that of triple knockout (TKO) JNP meniscus (α-gal epitope, N-glycolylneuraminic acid (Neu5Gc), and Sd (a) epitope knockout using CRISPR-Cas 9). Mast cells, eosinophils, neutrophils, and macrophages were found to have infiltrated the transplant boundary in the sham (without transplantation), normal (normal JNP), and test (TKO JNP) samples after immunohistochemical analysis. When compared to normal and sham groups, TKO was lower. Cytokine levels did not differ significantly between normal and test groups. Because chronic rejection can occur after meniscus transplantation associated with immune cell infiltration, we propose studies with multiple genetic editing to prevent immune rejection.
Assuntos
Imunidade Inata , Menisco , Animais , Humanos , Camundongos , Animais Geneticamente Modificados , Citocinas/genética , Epitopos , Galactosiltransferases/genética , Técnicas de Inativação de Genes , Rejeição de Enxerto , Menisco/transplante , Camundongos Knockout , Suínos , Transplante HeterólogoRESUMO
The two-pore domain K+ (K2P) channel, which is involved in setting the resting membrane potential in neurons, is an essential target for receptor agonists. Activation of the γ-aminobutyric acid (GABA) receptors (GABAAR and GABABR) reduces cellular excitability through Cl- influx and K+ efflux in neurons. Relatively little is known about the link between GABAAR and the K+ channel. The present study was performed to identify the effect of GABAR agonists on K2P channel expression and activity in the neuroblastic B35 cells that maintain glutamic acid decarboxylase (GAD) activity and express GABA. TASK and TREK/TRAAK mRNA were expressed in B35 cells with a high level of TREK-2 and TRAAK. In addition, TREK/TRAAK proteins were detected in the GABAergic neurons obtained from GABA transgenic mice. Furthermore, TREK-2 mRNA and protein expression levels were markedly upregulated in B35 cells by GABAAR and GABABR agonists. In particular, muscimol, a GABAAR agonist, significantly increased TREK-2 expression and activity, but the effect was reduced in the presence of the GABAAR antagonist bicuculine or TREK-2 inhibitor norfluoxetine. In the whole-cell and single-channel patch configurations, muscimol increased TREK-2 activity, but the muscimol effect disappeared in the N-terminal deletion mutant. These results indicate that muscimol directly induces TREK-2 activation through the N-terminus and suggest that muscimol can reduce cellular excitability by activating the TREK-2 channel and by inducing Cl- influx in GABAergic neurons.
Assuntos
Agonistas de Receptores de GABA-A/farmacologia , Neurônios GABAérgicos/metabolismo , Potenciais da Membrana , Muscimol/farmacologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Receptores de GABA/química , Animais , Células Cultivadas , Neurônios GABAérgicos/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Camundongos , Canais de Potássio de Domínios Poros em Tandem/genética , RatosRESUMO
BACKGROUND: In South Korea, pig-to-nonhuman primate trials of solid organs have only been performed recently, and the results are not sufficiently satisfactory to initiate clinical trials. Since November 2011, we have performed 30 kidney pig-to-nonhuman primate xenotransplantations at Konkuk University Hospital. METHODS: Donor αGal-knockout-based transgenic pigs were obtained from 3 institutes. The knock-in genes were CD39, CD46, CD55, CD73, and thrombomodulin, and 2-4 transgenic modifications with GTKO were done. The recipient animal was the cynomolgus monkey. We used the immunosuppressants anti-CD154, rituximab, anti-thymocyte globulin, tacrolimus, mycophenolate mofetil, and steroids. RESULTS: The mean survival duration of the recipients was 39 days. Except for a few cases for which survival durations were <2 days because of technical failure, 24 grafts survived for >7 days, with an average survival duration of 50 days. Long-term survival was observed 115 days after the removal of the contralateral kidney, which is currently the longest-recorded graft survival in Korea. We confirmed functioning grafts for the surviving transplanted kidneys after the second-look operation, and no signs of hyperacute rejection were observed. CONCLUSIONS: Although our survival results are relatively poor, they are the best-recorded results in South Korea, and the ongoing results are improving. With the support of government funds and the volunteering activities of clinical experts, we aim to further improve our experiments and contribute to the commencement of clinical trials of kidney xenotransplantation in Korea.
Assuntos
Sobrevivência de Enxerto , Rim , Animais , Suínos , Transplante Heterólogo/métodos , Macaca fascicularis , Rim/cirurgia , Animais Geneticamente Modificados , Sobrevivência de Enxerto/genética , República da Coreia , Rejeição de Enxerto/genética , Rejeição de Enxerto/prevenção & controleRESUMO
Numerous studies have suggested that K(+) channels regulate a wide range of physiological processes in mammalian cells. However, little is known about the specific function of K(+) channels in germ cells. In this study, mouse zygotes were cultured in a medium containing K(+) channel blockers to identify the functional role of K(+) channels in mouse embryonic development. Voltage-dependent K(+) channel blockers, such as tetraethylammonium and BaCl(2), had no effect on embryonic development to the blastocyst stage, whereas K(2P) channel blockers, such as quinine, selective serotonin reuptake inhibitors (fluoxetine, paroxetine, and citalopram), gadolinium trichloride, anandamide, ruthenium red, and zinc chloride, significantly decreased blastocyst formation (P<0.05). RT-PCR data showed that members of the K(2P) channel family, specifically KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9, were expressed in mouse oocytes and embryos. In addition, their mRNA expression levels, except Kcnk3, were up-regulated by above ninefold in morula-stage embryos compared with 2-cell stage embryos (2-cells). Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed in the membrane of oocytes, 2-cells, and blastocysts. Each siRNA injection targeted at Kcnk2, Kcnk10, Kcnk4, Kcnk3, and Kcnk9 significantly decreased blastocyst formation by ~38% compared with scrambled siRNA injection (P<0.05). The blockade of K(2P) channels acidified the intracellular pH and depolarized the membrane potential. These results suggest that K(2P) channels could improve mouse embryonic development through the modulation of gating by activators.
Assuntos
Blastocisto/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Potássio/metabolismo , Zigoto/metabolismo , Animais , Blastocisto/efeitos dos fármacos , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Ativação do Canal Iônico , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos ICR , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/genética , Interferência de RNA , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Zigoto/efeitos dos fármacosRESUMO
Pigs are promising donors of biological materials for xenotransplantation; however, cell surface carbohydrate antigens, including galactose-alpha-1,3-galactose (α-Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd blood group antigens, play a significant role in porcine xenograft rejection. Inactivating swine endogenous genes, including GGTA1, CMAH, and B4GALNT2, decreases the binding ratio of human IgG/IgM in peripheral blood mononuclear cells and erythrocytes and impedes the effectiveness of α-Gal, Neu5Gc, and Sd, thereby successfully preventing hyperacute rejection. Therefore, in this study, an effective transgenic system was developed to target GGTA1, CMAH, and B4GALNT2 using CRISPR-CAS9 and develop triple-knockout pigs. The findings revealed that all three antigens (α-Gal, Neu5Gc, and Sd) were not expressed in the heart, lungs, or liver of the triple-knockout Jeju Native Pigs (JNPs), and poor expression of α-Gal and Neu5G was confirmed in the kidneys. Compared with the kidney, heart, and lung tissues from wild-type JNPs, those from GGTA1/CMAH/ B4GALNT2 knockout-recipient JNPs exhibited reduced human IgM and IgG binding and expression of each immunological rejection component. Hence, reducing the expression of swine xenogeneic antigens identifiable by human immunoglobulins can lessen the immunological rejection against xenotransplantation. The findings support the possibility of employing knockout JNP organs for xenogeneic transplantation to minimize or completely eradicate rejection using multiple gene-editing methods.
RESUMO
We evaluated the dietary effects of multiple probiotics in Jeju native pigs, using basal diet and multi-probiotic Lactobacillus (basal diet with 1% multi-probiotics) treatments (n = 9 each) for 3 months. We analyzed growth performance, feed efficiency, backfat thickness, blood parameters, hematological profiles, adipokines, and immune-related cytokines in pig tissues. Average daily gain, feed intake, feed efficiency, backfat thickness, and body weight were not significantly different between both groups. In Lactobacillus group, total protein (p < 0.08) and bilirubin (p < 0.03) concentrations increased; blood urea nitrogen (p < 0.08), alkaline phosphatase (p < 0.08), and gamma-glutamyltransferase (p < 0.08) activities decreased. Lactobacillus group showed decreased adiponectin (p < 0.05), chemerin (p < 0.05), and visfatin expression in adipose tissues, and increased TLR4 (p < 0.05), MYD88 (p < 0.05), TNF-α (p < 0.001), and IFN-γ (p < 0.001) expression in the liver. Additionally, NOD1 (p < 0.05), NOD2 (p < 0.01), and MYD88 (p < 0.05) mRNA levels in proximal colon tissue upregulated significantly. Colon, longissimus dorsi muscle, fat tissue, and liver histological analyses revealed no significant differences between the groups. Conclusively, Lactobacillus supplementation improved liver function and reduced cholesterol levels. Its application may treat metabolic liver disorders, especially cholesterol-related disorders.
RESUMO
Acetylcholine (ACh) causes early activation events in mouse oocytes, but little is known about its precise role in the early embryonic development of mice. We aimed to determine whether and how ACh is capable of rescuing two-cell block in an in vitro culture system. ACh evoked different transient Ca(2+) patterns showing a higher Ca(2+) peak in the two-cell stage embryos (two-cells) than observed in mature oocytes. In early two-cells subjected to an in vitro two-cell block, xestospongin C (Xes-C), an IP3 receptor antagonist, significantly decreased the level of the ACh-induced Ca(2+) increase. The reduction in the ACh-induced Ca(2+) increase by Xes-C in late two-cells was lower than that in early two-cells. Furthermore, KN62 and KN93, both CaMKII inhibitors, were found to reduce the magnitude of the ACh-induced Ca(2+) increase in early two-cells. The addition of ACh to the culture medium showed an ability to rescue in vitro two-cell block. However, the addition of ACh together with both Xes-C and CaMKII inhibitors or with either inhibitor separately had no effect on the rescue of two-cell block. Long-term exposure of late two-cells to ACh decreased morula and early blastocyst development and ACh had a differential effect on early and late two-cells. These results indicate that ACh likely rescues the in vitro two-cell block through activation of IP3R- and/or CaMKII-dependent signal transduction pathways.
Assuntos
Acetilcolina/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Benzilaminas/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Receptores de Inositol 1,4,5-Trifosfato/efeitos dos fármacos , Compostos Macrocíclicos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Oxazóis/farmacologia , Sulfonamidas/farmacologiaRESUMO
Two-pore domain K(+) (K(2P)) channels that help set the resting membrane potential of excitable and nonexcitable cells are expressed in many kinds of cells and tissues. However, the expression of K(2P) channels has not yet been reported in bovine germ cells. In this study, we demonstrate for the first time that K(2P) channels are expressed in the reproductive organs and germ cells of Korean cattle. RT-PCR data showed that members of the K(2P) channel family, specifically KCNK3, KCNK9, KCNK2, KCNK10, and KCNK4, were expressed in the ovary, testis, oocytes, embryo, and sperm. Out of these channels, KCNK2 and KCNK4 mRNAs were abundantly expressed in the mature oocytes, eight-cell stage embryos, and blastocysts compared with immature oocytes. KCNK4 and KCNK3 were significantly increased in eight-cell stage embryos. Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed at the membrane of oocytes and blastocysts. KCNK10 and KCNK4 were strongly expressed and distributed in oocyte membranes. These channel proteins were also localized to the acrosome sperm cap. In particular, KCNK3 and KCNK4 were strongly localized to the post-acrosomal region of the sperm head and the equatorial band within the sperm head respectively. These results suggest that K(2P) channels might contribute to the background K(+) conductance of germ cells and regulate various physiological processes, such as maturation, fertilization, and development.
Assuntos
Bovinos/metabolismo , Células Germinativas/química , Canais de Potássio de Domínios Poros em Tandem/análise , Reação Acrossômica/fisiologia , Animais , Sequência de Bases , Primers do DNA/genética , Feminino , Masculino , Dados de Sequência Molecular , Oócitos/química , Ovário/química , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Capacitação Espermática/fisiologia , Espermatozoides/química , Espermatozoides/fisiologia , Testículo/químicaRESUMO
The objective of this study was to evaluate the effect of electrical stimulation (EST) on pronuclear formation, chromosomal constitution, and developmental capability among in vitro matured pig oocytes following intracytoplasmic sperm injection (ICSI). After ICSI, the oocytes were randomly distributed and cultured into 3 groups: the EST activated ICSI group, non-activation ICSI group, and in vitro fertilization (IVF) group. The proportion of oocytes in which 2 pronuclei were formed in ICSI groups was significantly higher in the former groups than in the IVF group (96.2 and 93.5 vs. 64.5%, respectively, P<0.05). The cleavage rate was significantly higher in EST activated ICSI group (78.6%) than in the IVF and non-activated ICSI groups (51.8 and 46.0%, respectively, P<0.05), as was the proportion of oocytes that developed to the blastocyst stage at day 7 (18.9 vs. 11.6 and 9.1%, respectively, P<0.05). Diploid blastocysts were observed in 52.4, 63.0, and 65.2% of oocytes in the IVF, activated, and non-activated ICSI groups, respectively. Eight out of 23 gilts (34.8%) were confirmed to be pregnant in activated ICSI groups, but none of these pregnancies were carried to term. These results show that oocyte activation after ICSI is effective in elevating the cleavage rate and blastocyst development, while ensuring normal chromosome composition. Further research is needed to determine the pregnancy maintenance requirements for ICSI-embryos in pigs.
Assuntos
Desenvolvimento Embrionário/fisiologia , Oócitos/fisiologia , Injeções de Esperma Intracitoplásmicas/veterinária , Suínos/fisiologia , Animais , Estimulação Elétrica , Feminino , Masculino , Gravidez , Distribuição AleatóriaRESUMO
IP3-induced Ca2+ release is the primary mechanism that is responsible for acetylcholine (ACh)-induced Ca2+ oscillation. However, other mechanisms remain to explain intracellular Ca2+ elevation. We here report that ACh induces Ca2+ influx via T-type Ca2+ channel by activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII), and the ACh-induced Ca2+ influx facilitates the generation of Ca2+ oscillation in the mouse ovulated oocytes (oocytes(MII)). ACh increased Ca2+ current by 50+/-21%, and produced Ca2+ oscillation. However, the currents and Ca2+ peaks were reduced in Ca2+ -free extracellular medium. ACh failed to activate Ca2+ current and to produce Ca2+ oscillation in oocytes pretreated with KN-93, a CaMKII inhibitor. KN-92, an inactive analogue of KN93, and PKC modulators could not prevent the effect of ACh. These results show that ACh increases T-type Ca2+ current by activation of CaMKII, independent of the PKC pathway, in the mouse oocytes.