RESUMEN
Knockout of the MSTN gene is linked to the enlarged tongue, and it causes suckling difficulty in animals. The suckling difficulty has a severe effect on animal mortality. Thus, special care was required to ensure their survivability. Here, it is critical to promptly ascertain the genotype of all pigs after birth. The main objective of the present study was to develop the restriction enzyme-mediated PCR-RFLP assay for MSTN mutant pig genotyping. To accomplish this, conserved oligonucleotide primer and restriction site were deduced according to the mutated sequence of the MSTN mutant pigs. PCR amplification yielded a 176 bp band for all homozygous MSTN mutant (MSTN-/-), heterozygous MSTN mutant (MSTN+/-) and wild-type (WT) pigs. However, MSTN+/- samples produced two fragments with 176 and 87 bp, and WT samples produced one fragment with 87 bp after being digested by BstNI. MSTN-/- samples were not digested by BstNI and yielded a 176 bp band. Thus, we were able to determine the genotype of all pigs using BstNI restriction enzyme-mediated PCR-RFLP method. Overall, the present study reported a simple and fast PCR-RFLP genotyping method for MSTN mutant pig breeding. The present study may contribute to the establishment of commercial breeding systems and the production of double muscle pigs.
Asunto(s)
Miostatina , Animales , Porcinos/genética , Polimorfismo de Longitud del Fragmento de Restricción , Reacción en Cadena de la Polimerasa , Genotipo , Heterocigoto , Secuencia de Bases , Miostatina/genéticaRESUMEN
Myostatin (MSTN), a negative regulator of skeletal muscle mass, is not well known in extraocular muscles (EOMs). EOMs are specialized skeletal muscles. Hence, in this study, the effect of MSTN on the superior rectus (SR) and superior oblique (SO) of 2-month-old MSTN knockout (MSTN-/-) and wild-type (WT) pigs of the same genotype was investigated. SR (P < 0.01) and SO (P < 0.001) fiber cross-sectional areas of MSTN-/- pigs were significantly larger than those of WT pigs. Compared with WT pigs, MSTN-/- SO displayed a decrease in type I fibers (WT: 27.24%, MSTN-/-: 10.32%, P < 0.001). Type IIb fibers were higher in MSTN-/- pigs than in WT pigs (WT: 30.38%, MSTN-/-: 62.24%, P < 0.001). The trend in SR was the same as that in SO, although the trend in SO was greater than that in SR. The expression of myogenic differentiation factor (MyoD) and myogenic (MyoG) showed a significant increase in MSTN-/- SO (about 2.5-fold and 2-fold, respectively at the gene expression level, about 1.5-fold at the protein level) compared with WT pigs. MSTN plays an important role in the development of EOMs and regulates the muscle fiber type by modulating the gene expression of MyoD and MyoG in pigs.
Asunto(s)
Miostatina , Músculos Oculomotores , Animales , Porcinos/genética , Músculos Oculomotores/metabolismo , Técnicas de Inactivación de Genes , Miostatina/genética , Miostatina/metabolismo , Músculo Esquelético/metabolismo , Fibras Musculares Esqueléticas/metabolismoRESUMEN
Cytidine monophosphate-Nacetylneuraminic acid (Neu5Ac) hydroxylase (CMAH) and glycoprotein, alpha1, 3-galactosyltransferase (GGTA1) double knockout (DKO) pig models were produced to reduce immune reaction for xenotransplantation. However, the role of Neu5Gc and α-Gal in pigs has not been fully elucidated and it is necessary to consider the after-effect of inactivation of GGTA1 and CMAH in pigs. Hematological profiles of DKO pigs were analyzed through complete blood count (CBC). Histology of liver and spleen of DKO were investigated, and lectin blotting and mass spectrometry (MS) were performed to explore glycosylation changes in red blood cell (RBC) membranes of DKO pigs. DKO pigs showed common clinical signs such as weakness (100%), dyspnea (90%) and constipation (65%). DKO pigs revealed a significant decrease in RBC, hemoglobin (HGB) and hematocrit (HGB), and an increase in white blood cell (WBC), lymphocyte (LYM), monocyte (MON), and erythrocyte mean corpuscular volume (MCV). DKO piglets showed swollen liver and spleen, and exhibited raised deposition of hemosiderin and severe bleeding. Lectin assay and MS proved variations in glycosylation on RBC membranes. GGTA1/CMAH DKO pigs developed pathological features which are similar to anemic symptoms, and the variations in glycosylation on RBC membranes of DKO pigs may be attributed to the pathologies observed.
Asunto(s)
Técnicas de Inactivación de Genes , Animales , Porcinos , Trasplante Heterólogo/métodosRESUMEN
Myostatin (MSTN), a member of the TGF-ß superfamily, negatively regulates muscle growth. MSTN inhibition has been known to cause a double-muscled phenotype in skeletal muscle and fibrosis reduction in the heart. However, the role of MSTN in the cardiac extracellular matrix (ECM) needs more studies in various species of animal models to draw more objective conclusions. The main objective of the present study was to investigate whether loss of MSTN affects the cardiac extracellular matrix in pigs. Three MSTN knockouts (MSTN-/-) and three wild type (WT) male pigs were generated by crossing MSTN ± heterozygous gilts and boars. Cardiac ECM and underlying mechanisms were determined post-mortem. The role of MSTN on collagen expression was investigated by treating cardiac fibroblasts with active MSTN protein in vitro. MSTN protein was detected in WT hearts, while no expression was detected in MSTN-/- hearts. The heart-to-body weight ratio was significantly decreased in MSTN-/- pigs. The morphometric analyses, including picrosirius red staining, immunofluorescent staining, and ultra-structural thickness examination of the endomysium, revealed a significant reduction of connective tissue content in MSTN-/- hearts compared to WT. Hydroxyproline, type I collagen (Col1A), and p-Smad3/Smad3 levels were significantly lower in MSTN-/- hearts in vivo. On the contrary, cardiac fibroblasts treated with exogenous MSTN protein overexpressed Col1A and activated Smad and AKT signaling pathways in vitro. The present study suggests that inhibition of MSTN decreases cardiac extracellular matrix.
Asunto(s)
Miostatina , Proteínas Proto-Oncogénicas c-akt , Animales , Colágeno Tipo I/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Femenino , Hidroxiprolina/metabolismo , Masculino , Músculo Esquelético/metabolismo , Miostatina/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Porcinos/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Obesity is associated with increased serum fibrinogen level. Myostatin (MSTN), a strong inhibitor of skeletal muscle growth, is recognized as a potential target for obesity. However, the effect of MSTN inhibition on fibrinogen is not largely known. The objective of the present study was to explore fibrinogen levels after MSTN inhibition. Fibrinogen levels and the fibrin clot structure of MSTN homozygous knockout (KO) and wild-type (WT) pigs (n = 4 in each group) were investigated. The protein expression of fibrinogen in the serum and liver of KO pigs decreased greatly (1.6-fold loss for serum and 2.5-fold loss for liver). KO pigs showed significantly decreased gene expression of fibrinogen chains: FGA (fibrinogen-α; 11-fold), FGB (fibrinogen-ß; 8-fold) and FGG (fibrinogen-γ; 7.4-fold). The basal transcriptional regulators of fibrinogen, HNF1 (hepatocyte nuclear factor 1) and CEBP-α (CCAAT/Enhancing-binding protein-alpha) were remarkably down-regulated after interruption of MSTN expression by siRNA (small interfering RNA) in cultured hepatocytes (about 2- and 4-fold, respectively). Compared with WT pigs, KO pigs displayed altered fibrin clot structure with thinner fibers, decreased turbidity and increased permeability. The findings indicate that the inhibition of MSTN could affect fibrinogen levels and the fibrin clot structure.
Asunto(s)
Miostatina , Enfermedades de los Porcinos , Animales , Fibrina/genética , Fibrina/metabolismo , Fibrinógeno/genética , Fibrinógeno/metabolismo , Homocigoto , Músculo Esquelético/metabolismo , Miostatina/genética , Obesidad , Porcinos/genéticaRESUMEN
Herein, we investigate the high incidence of umbilical hernia and tippy-toe standing and their underlying changes in gene expression and proliferation in myostatin knockout (MSTN-/-) pigs. Thirty-six male MSTN-/- pigs were generated by somatic cell nuclear transfer (SCNT). These pigs presented a considerably high incidence of tippy-toe standing and umbilical hernia (69.4% and 61.1%, respectively). The tendon to body weight ratio was significantly lower than wild-type pigs (0.202 ± 0.017 vs 0.250 ± 0.004, respectively). The crimp length of the MSTN-/- tendon was significantly longer than that of wild-type pigs. The expression of MSTN and the activin type IIB (ACVR2B) was detected in the tendon and linea alba of MSTN-/- pigs. MSTN treatment significantly increased the phosphorylation of Smad2/3 in both tendon and linea alba fibroblasts. Type I collagen (Col1A) and Scleraxis (Scx) expression levels in the tendon and linea alba of MSTN-/- pigs were significantly lower than those in wild-type in vivo, whereas and cyclin-dependent kinase inhibitor 1 (p21) expression levels were higher. Treatment of tendon and linea alba fibroblasts with recombinant MSTN increased Col1A and Scx and decreased p21 expression in vivo. Moreover, there was a significant increase in fibroblast proliferation after treatment. The results indicated that MSTN regulates collagen expression and proliferation in tendon and linea alba fibroblasts; thus, MSTN deficiency causes collagen-related pathological features in MSTN-/- pigs. Hence, MSTN could be used as a therapeutic target for treating UH and tendon abnormalities.
Asunto(s)
Hernia Umbilical , Miostatina , Animales , Colágeno/genética , Hernia Umbilical/genética , Masculino , Músculo Esquelético , Miostatina/genética , Técnicas de Transferencia Nuclear , Porcinos , Dedos del PieRESUMEN
Few studies exist on homozygous myostatin gene mutant (MSTN-/- ) pigs, especially on their reproductive ability. We have previously shown that semen quality of homozygous MSTN-/- boars is comparable to that of wild type (WT). However, no data exist on the reproductive ability of heterozygous MSTN gene mutant (MSTN+/ - ) sows. The present study highlights showed that the heterozygous MSTN+/ - sows have delayed pubertal age than WT sows (255.80 ± 6.79 versus 191.10 ± 3.42, respectively). The number of services per pregnancy of heterozygous MSTN+/ - sows is significantly higher than that of WT sows (3.33 ± 0.43 versus 1.60 ± 0.25, respectively). Moreover, although heterozygous MSTN+/ - sows have natural reproduction ability, their litter size was significantly lower than that of WT sows (7.75 ± 0.44 versus 14.25 ± 0.60, respectively). Offsprings generated from heterozygous MSTN+/ - sow and homozygous MSTN-/- boar were genotyped with the PCR and sequencing method to detect myostatin mutation and to identify whether the piglets are homozygous MSTN-/- or heterozygous MSTN+/ - . The proportion of homozygous MSTN-/- piglets was significantly lower than that of heterozygous MSTN+/ - piglets (2.50 ± 0.35 versus 5.25 ± 0.60, respectively). Furthermore, none of the sows presented dystocia, and the phenotype of heterozygous MSTN+/ - piglets was normal. However, 10% homozygous MSTN-/- piglets died of dyspnoea within 2 hr after birth, 60% of homozygous MSTN-/- piglets showed large tongues, and 50% had umbilical hernias. In summary, this study for the first time reports the reproduction traits of heterozygous MSTN+/ - sows crossbred with homozygous MSTN-/- boars. This study will pave the way in a new direction for the breeding and development of super lean meat varieties in the future.
Asunto(s)
Miostatina/genética , Maduración Sexual/genética , Sus scrofa/genética , Animales , Animales Recién Nacidos , Disnea/mortalidad , Disnea/veterinaria , Femenino , Técnicas de Inactivación de Genes , Genotipo , Hernia Umbilical/genética , Hernia Umbilical/veterinaria , Hibridación Genética , Tamaño de la Camada/genética , Masculino , Mutación , Embarazo , Sus scrofa/crecimiento & desarrollo , Sus scrofa/fisiología , Porcinos , Enfermedades de los Porcinos/genética , Enfermedades de la Lengua/congénito , Enfermedades de la Lengua/veterinariaRESUMEN
Panax ginseng, a functional food, has been widely used as an edible nourishment and medicinal supplement. Ginsenoside Rb1 is a major bioactive ingredient of ginseng, which shows very specific anti-apoptosis and anti-oxidant activities. Methylglyoxal (MGO) is one of intermediate products of glucose metabolism, which is absorbed easily from high sugar foods or carbonated beverages. It may involve in a variety of detrimental processes in vivo. However, it has not been fully explored the effects of ginsenoside Rb1 on MGO-induced oocytes damage. This study found that MGO-induced DNA damage and mitochondrial dysfunction result in the failure of porcine oocytes maturation and low in vitro development capacity of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos. Conversely, Rb1 supplementation recovered the rate of maturation, and improved in vitro development capacity of PA and IVF embryos. Rb1 also provided porcine oocytes a lower level of reactive oxygen species production, higher level of ATP content and mitochondrial membrane potential, and stimulated pluripotency gene expression in blastocysts. The findings of this study reveal ginsenoside Rb1 protects porcine oocyte from the cytotoxicity effects of methylglyoxal and provides novel perspectives for the protection of reproduction system by functional food of ginseng.
Asunto(s)
Desarrollo Embrionario/efectos de los fármacos , Ginsenósidos/farmacología , Oocitos/efectos de los fármacos , Partenogénesis/efectos de los fármacos , Piruvaldehído/toxicidad , Animales , Antioxidantes/metabolismo , Blastocisto/efectos de los fármacos , Blastocisto/metabolismo , Daño del ADN/efectos de los fármacos , Desarrollo Embrionario/genética , Femenino , Fertilización In Vitro , Técnicas de Maduración In Vitro de los Oocitos , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Oogénesis/efectos de los fármacos , Panax/química , Especies Reactivas de Oxígeno/metabolismo , PorcinosRESUMEN
Baicalin, a traditional Chinese medicinal monomer whose chemical structure is known, can be used to treat female infertility. However, the effect of baicalin on embryonic development is unknown. This study investigated the effects of baicalin on in vitro development of parthenogenetically activated (PA) and in vitro fertilized (IVF) pig embryos and the underlying mechanisms involved. Treatment with 0.1 µg/ml baicalin significantly improved (P < 0.05) the in vitro developmental capacity of PA pig embryos by reducing the reactive oxygen species (ROS) levels and apoptosis and increasing the mitochondrial membrane potential (ΔΨm) and ATP level. mRNA and protein expression of sonic hedgehog (SHH) and GLI1, which are related to the SHH signaling pathway, in PA pig embryos at the 2-cell stage, were significantly higher in the baicalin-treated group than in the control group. To confirm that the SHH signaling pathway is involved in the mechanism by which baicalin improves embryonic development, we treated embryos with baicalin in the absence or presence of cyclopamine (Cy), an inhibitor of this pathway. Cy abolished the effects of baicalin on in vitro embryonic development. In conclusion, baicalin improves the in vitro developmental capacity of PA and IVF pig embryos by inhibiting ROS production and apoptosis, regulating mitochondrial activity and activating SHH signaling.
Asunto(s)
Apoptosis/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Flavonoides/farmacología , Mitocondrias/efectos de los fármacos , Oocitos/efectos de los fármacos , Animales , Células Cultivadas , Técnicas de Cultivo de Embriones , Embrión de Mamíferos , Femenino , Fertilización In Vitro , Proteínas Hedgehog/metabolismo , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/fisiología , Oocitos/citología , Oocitos/fisiología , Oogénesis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Porcinos/embriologíaRESUMEN
Baicalin, a monomer of flavonoids extracted from dried roots of Scutellaria baicalensis, is used to treat female infertility. However, the effect of baicalin on oocyte maturation is unknown. In this study we investigated the effects of baicalin on the IVM of pig oocytes and subsequent embryo development following parthenogenetic activation (PA). We found that 0.1µgmL-1 baicalin significantly (P<0.05) increased the IVM rate of oocytes compared with the non-treatment (control) group by reducing levels of reactive oxygen species (ROS). In addition, the mRNA expression of genes related to nuclear maturation and cumulus cell expansion, mitochondrial membrane potential and ATP content was significantly (P<0.05) higher in baicalin-treated than control oocytes. To determine whether baicalin treatment during IVM of pig oocytes improves subsequent development of PA embryos, we measured the cleavage and blastocyst formation rates, as well as the number of cells per blastocyst. All these parameters were significantly (P<0.05) higher in the baicalin-treated than control group. In conclusion, this study demonstrates that baicalin improves pig oocyte maturation and subsequent embryo development invitro by inhibiting production of ROS and reducing apoptosis in oocytes.
Asunto(s)
Antioxidantes/administración & dosificación , Apoptosis/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Flavonoides/administración & dosificación , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oocitos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Técnicas de Cultivo de Embriones , Desarrollo Embrionario/fisiología , Femenino , Técnicas de Maduración In Vitro de los Oocitos/métodos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Oocitos/metabolismo , Partenogénesis/efectos de los fármacos , Partenogénesis/fisiología , Especies Reactivas de Oxígeno/metabolismo , PorcinosRESUMEN
Myostatin (MSTN) is a member of the transforming growth factor-ß superfamily that negatively regulates skeletal muscle development. A lack of MSTN induces muscle hypertrophy and increases formation of fast-twitch (Type II) muscle fibres. This study investigated muscle development in newborn heterozygous (MSTN+/-) and homozygous (MSTN-/-) MSTN-knockout piglets. Detailed morphological and gene and protein expression analyses were performed of the biceps femoris, semitendinosus and diaphragm of MSTN+/-, MSTN-/- and wild-type (WT) piglets. Haematoxylin-eosin staining revealed that the cross-sectional area of muscle fibres was significantly larger in MSTN-knockout than WT piglets. ATPase staining demonstrated that the percentage of Type IIb and IIa muscle fibres was significantly higher in MSTN-/- and MSTN+/- piglets respectively than in WT piglets. Western blotting showed that protein expression of myosin heavy chain-I was reduced in muscles of MSTN-knockout piglets. Quantitative reverse transcription-polymerase chain reaction revealed that, compared with WT piglets, myogenic differentiation factor (MyoD) mRNA expression in muscles was 1.3- to 2-fold higher in MSTN+/- piglets and 1.8- to 3.5-fold higher MSTN-/- piglets (P<0.05 and P<0.01 respectively). However, expression of myocyte enhancer factor 2C (MEF2C) mRNA in muscles was significantly lower in MSTN+/- than WT piglets (P<0.05). MSTN plays an important role in skeletal muscle development and regulates muscle fibre type by modulating the gene expression of MyoD and MEF2C in newborn piglets.
Asunto(s)
Transdiferenciación Celular/genética , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Músculo Esquelético/citología , Miostatina/genética , Porcinos , Animales , Animales Modificados Genéticamente , Animales Recién Nacidos , Clonación de Organismos/veterinaria , Técnicas de Inactivación de Genes , Heterocigoto , Homocigoto , Músculo Esquelético/fisiología , Porcinos/genéticaRESUMEN
BACKGROUND: Myostatin (MSTN) negatively regulates skeletal muscle development; however, its functions in internal organs have not been thoroughly investigated. Here, we compared the morphological, molecular, and biological characteristics of the heart, liver, spleen, lungs, kidneys, and tongue of homozygous MSTN mutant (MSTN-/- ), heterozygous MSTN mutant (MSTN+/- ), and wild-type (WT) piglets. RESULTS: The heart and liver were lighter in MSTN-/- piglets than in MSTN+/- piglets, while the tongue was heavier in MSTN-/- piglets than in WT piglets (P < 0.05). Furthermore, the tongue was longer in MSTN-/- piglets than in WT piglets, and myofibers of the tongue were significantly larger in the former piglets than in the latter ones (P < 0.01). mRNA expression of MSTN in all organs was significantly lower in MSTN-/- and MSTN+/- piglets than in WT piglets (P < 0.05). Meanwhile, mRNA expression of follistatin, which is closely related to MSTN, in the heart and liver was significantly higher in MSTN-/- piglets than in MSTN+/- and WT piglets (P < 0.05). In addition, protein expression of MSTN in the heart, kidneys, and tongue was significantly lower in MSTN-/- piglets than in WT piglets (P < 0.01). CONCLUSION: These results suggest that MSTN is widely expressed and has marked effects in multiple internal organs. Myostatin has crucial functions in regulating internal organ size, especially the tongue. © 2019 Society of Chemical Industry.
Asunto(s)
Estructuras Animales/crecimiento & desarrollo , Animales Modificados Genéticamente/crecimiento & desarrollo , Miostatina/genética , Porcinos/crecimiento & desarrollo , Porcinos/genética , Estructuras Animales/metabolismo , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Folistatina/genética , Folistatina/metabolismo , Mutación , Miostatina/metabolismo , Tamaño de los Órganos , Porcinos/metabolismoRESUMEN
Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5µM RepSox and 50nM LBH589 (RepSox+LBH589) for 24h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P<0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox+LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox+LBH589 group. Moreover, RepSox+LBH589 improved epigenetic reprogramming. In summary, RepSox+LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the invitro development of porcine SCNT embryos.
Asunto(s)
Desarrollo Embrionario/efectos de los fármacos , Técnicas de Transferencia Nuclear , Panobinostat/administración & dosificación , Pirazoles/administración & dosificación , Piridinas/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Blastocisto/citología , Blastocisto/efectos de los fármacos , Blastocisto/metabolismo , Reprogramación Celular/efectos de los fármacos , Desarrollo Embrionario/genética , Epigénesis Genética/efectos de los fármacos , Femenino , Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/administración & dosificación , Proteína Homeótica Nanog/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción SOXB1/genética , Sus scrofaRESUMEN
In this study we examined the effects of JNJ-7706621, a cyclin-dependent kinase inhibitor, on the in vitro growth of pig embryos that had been produced either by parthenogenetic activation (PA) or somatic cell nuclear transfer (SCNT). A significantly higher percentage of PA embryos reached the blastocyst stage by Day 7 after exposure to 10µM JNJ-7706621 for 4h compared with embryos exposed to 5µgmL-1 cytochalasin B for 4h (P<0.05). Similarly, the rate of Tyr15 phosphorylation of the complex of cyclin and p34cdc2 (CDK1) was significantly elevated in the JNJ-7706621-treated embryos compared with embryos exposed to cytochalasin B or non-treated controls (P<0.05). In contrast, Thr161 phosphorylation of CDK1 was significantly lower in the JNJ-7706621-treated group compared with the cytochalasin B-treated as well as the non-treated group (P<0.05). Similarly, the level of M-phase-promoting factor (MPF) in embryos was significantly lower in the JNJ-7706621-treated group compared with the cytochalasin B-treated and non-treated groups (P<0.05). In addition, more SCNT embryos reached the blastocyst stage after treatment with JNJ-7706621 than following exposure to cytochalasin B (P<0.05). In conclusion, these results reveal that exposure to 10µM JNJ-7706621 for 4h improves early development of PA and SCNT porcine embryos by suppressing the activity of CDK1 and a concomitant reduction in the level of MPF.
Asunto(s)
Desarrollo Embrionario/fisiología , Técnicas de Transferencia Nuclear , Partenogénesis/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Triazoles/farmacología , Animales , Blastocisto/efectos de los fármacos , Embrión de Mamíferos , Oocitos/efectos de los fármacos , PorcinosRESUMEN
Abnormal epigenetic modifications are considered a main contributing factor to low cloning efficiency. In the present study, we explored the effects of quisinostat, a novel histone deacetylase inhibitor, on blastocyst formation rate in porcine somatic-cell nuclear transfer (SCNT) embryos, on acetylation of histone H3 lysine 9 (AcH3K9), and on expression of POU5F1 protein and apoptosis-related genes BAX and BCL2. Our results showed that treatment with 10 nM quisinostat for 24 hr significantly improved the development of reconstructed embryos compared to the untreated group (19.0 ± 1.6% vs. 10.2 ± 0.9%; p < 0.05). Quisinostat-treated SCNT embryos also possessed significantly increased AcH3K9 at the pseudo-pronuclear stage (p < 0.05), as well as improved immunostaining intensity for POU5F1 at the blastocyst stage (p < 0.05). While no statistical difference in BAX expression was observed, BCL2 transcript abundance was significantly different in the quisinostat-treated compared to the untreated control group. Of the 457 quisinostat-treated cloned embryos transferred into three surrogates, six fetuses developed from the one sow that became pregnant. These findings suggested that quisinostat can regulate gene expression and epigenetic modification, facilitating nuclear reprogramming, and subsequently improving the developmental competence of pig SCNT embryos and blastocyst quality.
Asunto(s)
Clonación de Organismos , Embrión de Mamíferos/metabolismo , Epigénesis Genética/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Ácidos Hidroxámicos/farmacología , Técnicas de Transferencia Nuclear , Acetilación/efectos de los fármacos , Animales , Embrión de Mamíferos/citología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Porcinos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
OBJECTIVE: To examine the effect of SU9516, a cyclin-dependent kinase inhibitor, on the induction of tetraploid blastocyst formation in porcine embryos by parthenogenetic activation. RESULTS: Karyotype analysis of blastocysts showed that in the SU9516-treatment group 56% were tetraploid, whereas in the cytochalasin B (CB) group 67% were diploid. The level of maturation-promoting factor (MPF) in stimulated embryos treated with 10 µM SU9516 for 4 h was lower than in embryos treated with CB group (103 vs. 131 pg/ml). The mRNA expression levels of Nanog significantly increased in SU9516-treated embryos than CB group. CONCLUSION: SU9516 can induce tetraploid blastocyst formation at high efficiency. SU9516 can significantly influence the in vitro developmental competence of porcine parthenogenetically activated embryos by influencing the level of MPF and the gene related apoptosis and pluripotency.
Asunto(s)
Blastocisto/efectos de los fármacos , Imidazoles/metabolismo , Indoles/metabolismo , Inhibidores de Proteínas Quinasas/metabolismo , Tetraploidía , Animales , Citocalasina B/metabolismo , Cariotipificación , Porcinos/embriologíaRESUMEN
OBJECTIVES: To explore the effects of heterozygous myostatin-knockout (MSNT+/-) on muscle characteristics, specifically fiber-type distribution and expression of myosin heavy chain isoforms in pigs. RESULTS: The fiber cross-sectional area of the semitendinosus and semimembranosus muscles were much larger in MSTN+/- pigs at birth than in wild-type (WT) pigs. MSTN+/- pigs had a higher proportion of fast-type fibers and lower succinate dehydrogenase activity in muscles than WT pigs. The myosin heavy chain IIB mRNA level in both two muscles was ~ threefold higher in MSTN+/- pigs compared with WT pigs. CONCLUSION: MSTN+/- pigs exhibit a disproportionate increase in muscle mass and can have a higher body weight due to fiber hypertrophy, a change in the fiber-type distribution, and alteration of myosin heavy chain isoforms levels, leading to more fast glycolytic fibers.
Asunto(s)
Técnicas de Inactivación de Genes , Fibras Musculares Esqueléticas/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Miostatina/genética , Animales , Animales Recién Nacidos , Embrión de Mamíferos , Folistatina/metabolismo , Masculino , Fibras Musculares Esqueléticas/química , Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/genética , Técnicas de Transferencia Nuclear , Tamaño de los Órganos , Isoformas de Proteínas , ARN/análisis , ARN/genética , ARN/metabolismo , PorcinosRESUMEN
OBJECTIVE: The aim of this study was to investigate the developmental competence of oocytes parthenogenetically activated by an electric pulse (EP) and treated with anisomycin and to determine whether this method is applicable to somatic cell nuclear transfer (SCNT). RESULTS: Embryos derived from porcine oocytes parthenogenetically activated by an EP and treatment with 0.01 µg/mL anisomycin had a significantly improved in vitro developmental capacity. Furthermore, 66.6% of blastocysts derived from these embryos had a diploid karyotype. The blastocyst formation rate of cloned embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 0.01 µg/mL anisomycin for 4 h. The level of maturation-promoting factor was significantly decreased in oocytes activated by an EP and treated with anisomycin. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR. CONCLUSION: Our results demonstrate that porcine oocyte activation via an EP in combination with anisomycin treatment can lead to a high blastocyst formation rate in parthenogenetic activation and SCNT experiments.
Asunto(s)
Anisomicina/farmacología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Partenogénesis/efectos de los fármacos , Animales , Estimulación Eléctrica , Desarrollo Embrionario , Femenino , Técnicas de Transferencia Nuclear , Oocitos/fisiología , Embarazo , PorcinosRESUMEN
OBJECTIVE: To investigate the effect of the small molecule, RepSox, on the expression of developmentally important genes and the pre-implantation development of rhesus monkey-pig interspecies somatic cell nuclear transfer (iSCNT) embryos. RESULTS: Rhesus monkey cells expressing the monomeric red fluorescent protein 1 which have a normal (42) chromosome complement, were used as donor cells to generate iSCNT embryos. RepSox increased the expression levels of the pluripotency-related genes, Oct4 and Nanog (p < 0.05), but not of Sox2 compared with untreated embryos at the 2-4-cell stage. Expression of the anti-apoptotic gene, Bcl2, and the pro-apoptotic gene Bax was also affected at the 2-4-cell stage. RepSox treatment also increased the immunostaining intensity of Oct4 at the blastocyst stage (p < 0.05). Although the blastocyst developmental rate was higher in the group treated with 25 µM RepSox for 24 h than in the untreated control group (2.4 vs. 1.2%, p > 0.05), this was not significant. CONCLUSION: RepSox can improve the developmental potential of rhesus monkey-pig iSCNT embryos by regulating the expression of pluripotency-related genes.
Asunto(s)
Clonación de Organismos/métodos , Embrión de Mamíferos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Transferencia Nuclear , Pirazoles/farmacología , Piridinas/farmacología , Animales , Células Madre Pluripotentes Inducidas/metabolismo , Macaca mulatta , Proteína Homeótica Nanog/metabolismo , Factor 3 de Transcripción de Unión a Octámeros , Oocitos/metabolismo , PorcinosRESUMEN
We examined the in vitro developmental competence of parthenogenetic activation (PA) oocytes activated by an electric pulse (EP) and treated with various concentrations of AZD5438 for 4 h. Treatment with 10 µM AZD5438 for 4 h significantly improved the blastocyst formation rate of PA oocytes in comparison with 0, 20, or 50 µM AZD5438 treatment (46.4% vs. 34.5%, 32.3%, and 24.0%, respectively; P 0.05). Furthermore, 66.67% of blastocysts derived from these AZD5438-treated PA oocytes had a diploid karyotype. The blastocyst formation rate of PA and somatic cell nuclear transfer (SCNT) embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 10 µM AZD5438 for 4 h (11.11% vs. 13.40%, P > 0.05). In addition, the level of maturation-promoting factor (MPF) was significantly decreased in oocytes activated by an EP and treated with 10 µM AZD5438 for 4 h. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR; however, there were no differences between the AZD5438-treated and non-treated control groups. Our results demonstrate that porcine oocyte activation via an EP in combination with AZD5438 treatment can lead to a high blastocyst formation rate in PA and SCNT experiments.