Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Biol Reprod ; 101(1): 63-75, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31004472

RESUMO

Growth differentiation factor 8 (GDF8), also known as myostatin, is a member of the transforming growth factor-ß (TGF-ß) family and has been identified as a strong physiological regulator of muscle differentiation. Recently, the functional role of GDF8 in reproductive organs has received increased interest following its detection in the human placenta and uterus. To investigate the effects of GDF8 during porcine oocyte in vitro maturation (IVM), we assessed the quality of matured oocytes. Furthermore, we investigated the specific gene transcription and protein activation levels in oocytes and cumulus cells after IVM and subsequent embryonic development after in vitro fertilization and parthenogenetic activation. Prior to these experiments, the concentration of GDF8 in porcine follicular fluid was determined. During the entire IVM period, 1.3 ng/mL GDF8 and its signaling inhibitor SB431542 (SB) at 5 µM were added as control, SB, SB + GDF8, and GDF8 groups, respectively. Our results demonstrate that supplementation with GDF8 during porcine oocyte IVM enhanced both meiotic and cytoplasmic maturation, with altered transcriptional patterns, via activation of Sma- and Mad-related protein 2/3 (SMAD2/3). Using the pharmacological inhibitor SB431542, we demonstrated that inhibition of GDF8-induced Smad2/3 signaling reduces matured oocyte quality. In conclusion, for the first time, we demonstrated paracrine factor GDF8 in porcine follicular fluid in vivo. Furthermore, we showed that GDF8 supplementation improved mature oocyte quality by regulating p38 mitogen-activated protein kinase phosphorylation and intracellular glutathione and reactive oxygen species levels during porcine IVM.

2.
Theriogenology ; 129: 70-76, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30825707

RESUMO

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-ß family and a physiological regulator. According to recent studies, GDF8 can be detected in follicular fluid and the uterus, suggesting that GDF8 may affect preimplantation embryonic development and act in a paracrine manner to improve the success of late-blastocyst implantation in vivo. We investigated the effect of GDF8 supplementation during in vitro culture (IVC) of porcine embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA) on cleavage, blastocyst formation rate, and total cell number and analysed gene transcription levels and cell linage specification in the resulting blastocysts. First, the concentration of GDF8 in porcine oviductal fluid was determined to be 139.8 pg/mL. Then, 0, 0.2, 2, or 20 ng/mL GDF8 was added to embryos throughout the entire IVC period. Our results showed that supplementation with GDF8 during porcine preimplantation embryo IVC enhanced blastocyst formation and total cell number and altered the transcriptional patterns of genes that regulate pluripotency and cavitation. Furthermore, using differential immunostaining, we demonstrated that supplementation with GDF8 enhanced the expression of the genuine inner cell mass (ICM) marker SOX2 and the ICM/trophectoderm ratio, improving IVF blastocyst quality. In conclusion, for the first time, we demonstrated the presence of the in vivo oviductal factor GDF8 in oviductal fluid. Furthermore, we found that GDF8 supplementation at 0.2 ng/mL increased the blastocyst total cell number and ICM/trophectoderm ratio by inducing the transcription of genes involved in developmental competence and the expression of genuine ICM marker SOX2 during porcine IVF embryo development in vitro.


Assuntos
Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Miostatina/farmacologia , Suínos/embriologia , Animais , Biomarcadores/metabolismo , Linhagem da Célula , Técnicas de Cultura Embrionária/métodos , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Maturação in Vitro de Oócitos/veterinária , Suínos/metabolismo
3.
J Cell Mol Med ; 23(3): 2052-2063, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30609263

RESUMO

Prior to transplantation, preclinical study of safety and efficacy of neural progenitor cells (NPCs) is needed. Therefore, it is important to generate an efficient in vitro platform for neural cell differentiation in large animal models such as pigs. In this study, porcine-induced pluripotent stem cells (iPSCs) were seeded at high cell density to a neural induction medium containing the dual Sma- and Mad-related protein (SMAD) inhibitors, a TGF-ß inhibitor and BMP4 inhibitor. The dSMADi-derived NPCs showed NPC markers such as PLAG1, NESTIN and VIMENTIN and higher mRNA expression of Sox1 compared to the control. The mRNA expression of HOXB4 was found to significantly increase in the retinoic acid-treated group. NPCs propagated in vitro and generated neurospheres that are capable of further differentiation in neurons and glial cells. Gliobalstoma-cultured medium including injury-related cytokines treated porcine iPSC-NPCs survive well in vitro and showed more neuronal marker expression compared to standard control medium. Collectively, the present study developed an efficient method for production of neural commitment of porcine iPSCs into NPCs.

4.
BMC Vet Res ; 14(1): 331, 2018 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-30404643

RESUMO

BACKGROUND: The porcine brain is gyrencephalic with similar gray and white matter composition and size more comparable to the human rather than the rodent brain; however, there is lack of information about neural progenitor cells derived from this model. RESULTS: Here, we isolated GFAP-positive porcine neural stem cells (NSCs) from the brain explant of a transgenic piglet, with expression of CreERT2 under the control of the GFAP promoter (pGFAP-CreERT2). The isolated pGFAP-CreERT2 NSCs showed self-renewal and expression of representative NSC markers such as Nestin and Sox2. Pharmacological inhibition studies revealed that Notch1 signaling is necessary to maintain NSC identity, whereas serum treatment induced cell differentiation into reactive astrocytes and neurons. CONCLUSIONS: Collectively, these results indicate that GFAP promoter-driven porcine CreERT2 NSCs would be a useful tool to study neurogenesis of the porcine adult central nervous system and furthers our understanding of its potential clinical application in the future. ᅟ.


Assuntos
Proteína Glial Fibrilar Ácida/metabolismo , Células-Tronco Neurais/fisiologia , Suínos/anatomia & histologia , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Astrócitos/metabolismo , Diferenciação Celular , Suínos/genética
5.
Theriogenology ; 120: 147-156, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-30121547

RESUMO

Current research suggests that supplementing in vitro culture (IVC) media with vascular endothelial growth factor (VEGF) may have beneficial effects on the development of porcine embryos in vitro. However, the molecular signaling mechanisms underlying this effect are unclear. Therefore, we aimed to investigate the effects of VEGF on molecular signaling events during in vitro embryonic development of porcine embryos. Porcine oocytes matured in vitro were fertilized, and the resultant zygotes were cultured with 5 ng/mL of VEGF supplemented with or without fetal bovine serum from day 4 till day 7. Without VEGF and/or FBS served as the control group. Real-time quantitative PCR was used to detect expression patterns of apoptosis- and oxidative stress-related genes in day 7 blastocysts (BLs). Early-stage apoptosis was detected by annexin-V assays in day 2 and day 7 embryos. We found that the addition of VEGF throughout the culture period with or without FBS supplementation significantly improved embryo survival and development. Supplementation with VEGF in the IVC medium significantly increased early BL formation (p < 0.05), although addition of FBS on day 4 significantly increased hatched BL formation (p < 0.05) regardless of VEGF supplementation. However, supplementation of media with both VEGF and FBS increased the formation of expanded BLs synergistically. The average total cell numbers per BL were significantly (p < 0.05) higher in embryos supplemented with VEGF and FBS than in those supplemented with either VEGF or FBS alone. We also found that accumulation of reactive oxygen species in VEGF-treated embryos was significantly lower (p < 0.05) than that in untreated embryos. The mRNA levels of caspase-3 were significantly lower (p < 0.05), and those of Bcl-2 and Nrf-2 were significantly higher (p < 0.05) in embryos grown in VEGF-supplemented media than in embryos grown in non-supplemented media. Furthermore, on day 2, the numbers of viable embryos (44.06 ±â€¯3.94%) and blastomeres (67.18 ±â€¯3.60%) were significantly higher (p < 0.05), and the numbers of early apoptotic embryos (55.94 ±â€¯3.94) and blastomeres (23.23 ±â€¯4.22) were significantly lower (p < 0.05) in VEGF-treated BLs than in controls. Furthermore, the numbers of early apoptotic cells in BLs on day 7 were also significantly lower (p < 0.05) in VEGF-treated BLs than in controls. Overall, our results indicate that supplementing IVC media with VEGF during in vitro culture of porcine embryos increases their developmental potential.


Assuntos
Blastocisto/efeitos dos fármacos , Fertilização In Vitro/veterinária , Suínos/embriologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Apoptose , Blastocisto/fisiologia , Meios de Cultura , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Estresse Oxidativo , Transdução de Sinais
6.
Theriogenology ; 113: 197-207, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29554602

RESUMO

Lysophosphatidic acid (LPA) is a phospholipid-derived signaling molecule with biological activities, such as stimulating cell proliferation, differentiation and migration. In the present study, we examined the effect of LPA on porcine oocytes during in vitro maturation (IVM) and subsequent embryonic development following parthenogenetic activation (PA) and in vitro fertilization (IVF). During IVM, the maturation medium was supplemented with various concentrations of LPA (0, 10, 30, and 60 µM). After 42 h of IVM, the 30 µM LPA-treated group showed a significant (P <0.05) increase in nuclear maturation and intracellular glutathione (GSH) levels compared with the other groups. The 30 µM LPA-treated group exhibited a significant decrease in intracellular reactive oxygen species (ROS) levels compared with the other groups. In PA, the 30 µM LPA-treated group had significantly higher cleavage (CL) and blastocyst (BL) rates compared with those of the other LPA-treated groups. In IVF, the 30 µM LPA-treated group had significantly higher CL and BL rates than the other LPA-treated groups. The expression of the developmental competence gene (proliferating cell nuclear antigen, PCNA) in the oocytes and cumulus cells of the individuals in the 30 µM LPA-treated group was significantly increased compared with the control group. In addition, the specific expression of urokinase Plasminogen Activator (uPA) and uPA Receptor (uPAR) in cumulus cells was significantly increased in the 30 µM LPA-treated group. The western blotting results revealed that LPA improves the activities of p38 mitogen-activated protein kinase (MAPK) and epidermal growth factor (EGF) by enhanced phosphorylation. In conclusion, treatment with 30 µM LPA during IVM promotes enhances the EGF-EGFR signaling pathway, resulting in cumulus cell expansion. And then, this treatment improves the developmental potential of PA and IVF porcine embryos by enhancing nuclear and cytoplasmic maturation and reducing ROS.


Assuntos
Células do Cúmulo/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Lisofosfolipídeos/farmacologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Suínos , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Células do Cúmulo/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa , Partenogênese/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética
7.
J Vet Sci ; 19(3): 434-445, 2018 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-29284207

RESUMO

Transgenic (TG) pigs are important in biomedical research and are used in disease modeling, pharmaceutical toxicity testing, and regenerative medicine. In this study, we constructed two vector systems by using the promoter of the pig glial fibrillary acidic protein (pGFAP) gene, which is an astrocyte cell marker. We established donor TG fibroblasts with pGFAP-CreERT2/LCMV-EGFPLoxP and evaluated the effect of the transgenes on TG-somatic cell nuclear transfer (SCNT) embryo development. Cleavage rates were not significantly different between control and transgene-donor groups. Embryo transfer was performed thrice just before ovulation of the surrogate sows. One sow delivered 5 TG piglets at 115 days after pregnancy. Polymerase chain reaction (PCR) analysis with genomic DNA isolated from skin tissues of TG pigs revealed that all 5 TG pigs had the transgenes. EGFP expression in all organs tested was confirmed by immunofluorescence staining and PCR. Real-time PCR analysis showed that pGFAP promoter-driven Cre fused to the mutated human ligand-binding domain of the estrogen receptor (CreERT2) mRNA was highly expressed in the cerebrum. Semi-nested PCR analysis revealed that CreERT2-mediated recombination was induced in cerebrum and cerebellum but not in skin. Thus, we successfully generated a TG pig with a 4-hydroxytamoxifen (TM)-inducible pGFAP-CreERT2/EGFPLoxP recombination system via SCNT.


Assuntos
Doenças do Sistema Nervoso Central , Modelos Animais de Doenças , Transferência Embrionária/veterinária , Fibroblastos/transplante , Técnicas de Transferência Nuclear/veterinária , Suínos , Animais , Animais Geneticamente Modificados
8.
Biol Proced Online ; 19: 13, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29075153

RESUMO

BACKGROUND: Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for transgenic animal production using genetically modified somatic cells (GMSCs). However, there are several limitations preventing successful transgenic animal generation by SCNT, such as obtaining proper somatic donor cells with a sufficiently long life span and proliferative capacity for generating GMSCs. Here, we established simian virus 40 large T antigen (SV40LT)-mediated lifespan-extended canine fibroblast cells (SV40LT-K9 cells) and evaluated their potential as nuclei donors for SCNT, based on cellular integrity and SCNT embryo development. RESULTS: SV40LT did not cause canine cell transformation, based on cell morphology and proliferation rate. No anchorage-independent growth in vitro and tumorigenicity in vivo were observed. After SCNT with SV40LT-K9 cells, embryos were transferred into surrogate dogs. All dogs failed to become pregnant. Most embryos did not proceed past the 8-cell stage and only one surrogate showed an implantation trace in its oviduct, indicating that the cells rarely developed into blastocysts. Because of the absence of an in vitro maturation method for canine embryos, we performed identical experiments using porcine fibroblast cells. Similarly, SV40LT did not transform porcine fibroblast cells (SV40LT-Pig cells). During in vitro development of SV40LT-Pig cell-driven SCNT embryos, their blastocyst formation rate was clearly lower than those of normal cells. Karyotyping analysis revealed that both SV40LT-K9 and SV40LT-Pig cells had aberrant chromosomal statuses. CONCLUSIONS: Although lifespan-extended canine and porcine cells via SV40LT exhibit no apparent transforming changes, they are inappropriate for use as nuclei donors for SCNT because of their aneuploidy.

9.
J Reprod Dev ; 63(6): 581-590, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-28993559

RESUMO

Compared with the in vivo environment, porcine in vitro embryo-culture systems are suboptimal, as they induce oxidative stress via the accumulation of reactive oxygen species (ROS). High ROS levels during early embryonic development cause negative effects, such as apoptosis. In this study, we examined the effects of the antioxidant carboxyethylgermanium sesquioxide (Ge-132) during in vitro culture (IVC) on embryonic development in porcine in vitro fertilization (IVF) embryos. Zygotes were treated with different concentrations of Ge-132 (0, 100, 200 and 400 µg/ml). All of the Ge-132 treatment groups displayed greater total cell numbers after IVC (98.1, 98.5 and 103.4, respectively) compared with the control group (73.9). The 200 µg/ml Ge-132 treatment group exhibited significantly increased intracellular GSH levels compared with the control group, whereas the ROS generation levels decreased in Ge-132 dose-dependent manner (P < 0.05). The mRNA expression levels of the KEAP1 gene and proapoptotic genes BAX and CASPASE3 were lower in the Ge-132 treated blastocysts compared with the control group (P < 0.05). The percentages of apoptotic and necrotic cells in the Ge-132 treated embryos on day 2 (48 h) were significantly lower than the untreated embryos (9.1 vs. 17.1% and 0 vs. 2.7%, respectively). In the day 7 blastocysts, the percentages of apoptotic cells in 200 µg/ml Ge-132 treated group were lower compared to controls (1.6 vs. 2.5%). More KEAP1 protein was found to be localized in cytoplasm of the 200 µg/ml Ge-132 treated blastocysts, whereas KEAP1 protein was predominantly nuclei in the control blastocysts. These results indicate that the developmental competence of embryos cultured under Ge-132 treatment may be associated with KEAP1 signaling cascades involved in oxidative stress and apoptosis during porcine preimplantation embryo development.


Assuntos
Apoptose/efeitos dos fármacos , Técnicas de Cultura Embrionária , Embrião de Mamíferos/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Embrião de Mamíferos/metabolismo , Suínos
10.
Theriogenology ; 101: 123-134, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28708509

RESUMO

Growth Differentiation Factor 8 (GDF8) is a member of the transforming growth factor-ß (TGF-ß) family and has been identified as a strong physiological regulator. This factor is expressed as a paracrine factor in mural granulosa cells. To investigate the effects of GDF8 on the in vitro maturation (IVM) of porcine oocytes, we assessed the quality of matured oocytes as well as the specific gene transcription and protein activation levels in oocytes and cumulus cells (CCs) after IVM and subsequent embryonic development after in vitro fertilization (IVF) and parthenogenetic activation (PA). Supplemental concentrations (0, 1, 10, and 100 ng/ml) of GDF8 were provided in IVM medium. Supplementation with GDF8 during IVM induced transcription of specific TGF-ß receptor genes, such as ActRIIb and Alk4/5, and the recognition of the GDF8 by these receptors induced phosphorylation of p38 MAPK. Activated p38 MAPK signaling changed oocyte maturation and cumulus expansion-related gene transcription: Nrf2 and Bcl-2 in oocytes and PCNA, Nrf2, Has2, Ptx3, and TNFAIP6 in CCs. The altered gene expression pattern during IVM resulted in a 10% lower level of intracellular ROS in mature oocytes. The improved cytoplasmic maturation led to an increase in the fertilization efficiency and subsequent embryonic developmental competence. The embryonic development showed increases in the blastocyst formation rate and higher transcription levels of POU5F1 and BCL-2 in the blastocysts. The present study suggests that supplementation of GDF8 during IVM synergistically improved the developmental potential of IVF- and PA-derived porcine embryos by reducing the intracellular ROS level in oocytes by altering the transcription of specific genes and increasing the phosphorylation of p38 MAPK during IVM. In conclusion, for the first time, our results demonstrate that GDF8 can act as a paracrine factor to modulate oocyte maturation by regulating p38 MAPK phosphorylation and intracellular ROS level during porcine IVM.


Assuntos
Técnicas de Maturação in Vitro de Oócitos/veterinária , Miostatina/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Sus scrofa , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Blastocisto/fisiologia , Meios de Cultura , Células do Cúmulo/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fertilização In Vitro/métodos , Fertilização In Vitro/veterinária , Expressão Gênica/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/métodos , Miostatina/genética , Oócitos/química , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Partenogênese/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Espécies Reativas de Oxigênio/análise , Proteínas Recombinantes/administração & dosagem , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos
11.
J Reprod Dev ; 62(6): 635-638, 2016 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-27488694

RESUMO

Zinc supplementation (0.8 µg/ml) in in vitro maturation (IVM) medium significantly enhances oocyte quality. In this study, we compared the development of somatic cell nuclear transfer (SCNT) embryos produced from conventional IVM (control) and zinc-supplemented IVM oocytes. A total of 1206 and 890 SCNT embryos were produced using control and zinc-supplemented oocytes, respectively, and then were transferred to 11 and 8 recipients, respectively. Five control recipients and three zinc-supplemented recipients became pregnant. Two live piglets and eight mummies were born from two control recipients, and ten live piglets and six stillborn piglets were born from three zinc-supplemented recipients. The production efficiency significantly increased in the zinc-supplemented group (0.33% vs. 3.02%). This report suggests that zinc supplementation in IVM medium improved the production efficiency of cloned pigs.


Assuntos
Clonagem de Organismos/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Zinco/administração & dosagem , Animais , Clonagem de Organismos/métodos , Feminino , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Transferência Nuclear , Gravidez , Resultado da Gravidez , Suínos
12.
PLoS One ; 11(7): e0160289, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27472781

RESUMO

Pigs provide outstanding models of human genetic diseases due to their striking similarities with human anatomy, physiology and genetics. Although transgenic pigs have been produced using genetically modified somatic cells and nuclear transfer (SCNT), the cloning efficiency was extremely low. Here, we report an improved method to produce diploid cloned embryos from porcine induced pluripotent stem cells (piPSCs), which were synchronized to the G2/M stage using a double blocking method with aphidicolin and nocodazole. The efficiency of this synchronization method on our piPSC lines was first tested. Then, we modified our traditional SCNT protocol to find a workable protocol. In particular, the removal of a 6DMAP treatment post-activation enhanced the extrusion rate of pseudo-second-polar bodies (p2PB) (81.3% vs. 15.8%, based on peak time, 4hpa). Moreover, an immediate activation method yielded significantly more blastocysts than delayed activation (31.3% vs. 16.0%, based on fused embryos). The immunofluorescent results confirmed the effect of the 6DMAP treatment removal, showing remarkable p2PB extrusion during a series of nuclear transfer procedures. The reconstructed embryos from metaphase piPSCs with our modified protocol demonstrated normal morphology at 2-cell, 4-cell and blastocyst stages and a high rate of normal karyotype. This study demonstrated a new and efficient way to produce viable cloned embryos from piPSCs when synchronized to the G2/M phase of the cell cycle, which may lead to opportunities to produce cloned pigs from piPSCs more efficiently.


Assuntos
Clonagem de Organismos , Diploide , Embrião de Mamíferos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Metáfase , Suínos/embriologia , Animais , Blastocisto , Células Cultivadas , Camundongos
13.
Anim Biotechnol ; 27(2): 126-32, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26913554

RESUMO

Comparing the coding and regulatory sequences of genes in different species provides information on whether proteins translated from genes have conserved functions or gene expressions are regulated by analogical mechanisms. Herein, we compared the coding and regulatory sequences of glial fibrillary acidic protein (GFAP) from humans, mice, and pigs. The GFAP gene encodes a class III intermediate filament protein expressed specifically in astrocytes of the central nervous system. On comparing the mRNA, regulatory region (promoter), and protein sequences of GFAP gene in silico, we found that GFAP mRNA 3'-untranslated region (3'-UTR), promoter, and amino acid sequences showed higher similarities between humans and pigs than between humans and mice. In addition, the promoter-luciferase reporter gene assay revealed that the pig GFAP promoter functioned in human astrocytes. Notably, the 1.8-kb promoter fragment upstream from transcription initiation site showed strongest transcriptional activity compared to 5.2-kb DNA fragment or other regions of GFAP promoter. We also found that pig GFAP mRNA and promoter activity increased in pig fibroblasts by human IL-1ß treatment. Taken together, these results suggest that the regulatory mechanisms and functions of pig genes might be more similar to those of humans than mice, indicating that pigs, particularly miniature pigs, are a useful model for studying human biological and pathological events.


Assuntos
Proteína Glial Fibrilar Ácida/genética , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Genética/genética , Animais , Astrócitos , Humanos , Camundongos , RNA Mensageiro/química , RNA Mensageiro/genética , Suínos , Porco Miniatura/genética , Regiões não Traduzidas/genética
14.
J Reprod Dev ; 62(2): 177-85, 2016 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-26821870

RESUMO

The ultrastructure of porcine putative embryonic stem cells and porcine fetal fibroblasts (PFFs) was analyzed by transmission electron microscopy. The aim of this study was to compare the features of organelles in in vitro fertilization (IVF) derived porcine embryonic stem cells (IVF-pESCs) and somatic cell nuclear transfer (SCNT) derived pESCs (SCNT-pESCs). Also, the features of organelles in high-passage IVF-pESCs were compared with those in low-passage cells. The ultrastructure of PFFs showed rare microvilli on the cell surfaces, polygonal or irregular nuclei with one to two reticular-shaped nucleoli and euchromatin, low cytoplasm-to-nucleus ratios, rare ribosomes, rare rough endoplasmic reticulum, elongated mitochondria, rich lysosomes and rich phagocytic vacuoles. IVF-pESCs showed rare microvilli on the cell surfaces, round or irregular nuclei with one to two reticular-shaped nucleoli and euchromatin, low cytoplasm-to-nucleus ratios, rich ribosomes, long stacks of rough endoplasmic reticulum, elongated mitochondria, rare lysosomes and rare autophagic vacuoles. By contrast, SCNT-pESCs showed rich microvilli with various lengths and frequencies on the cell surfaces, polygonal nuclei with one reticular shaped nucleoli and heterochromatin, high cytoplasm-to-nucleus ratios, rare ribosomes, rare rough endoplasmic reticulum, round mitochondria, rich lysosomes and rich phagocytic vacuoles with clear intercellular junctions. Furthermore, high-passage IVF-pESCs showed irregularly shaped colonies, pyknosis and numerous lysosomes associated with autophagic vacuoles showing signs of apoptosis. In conclusion, this study confirms that the ultrastructural characteristics of pESCs differ depending on their origin. These ultrastructural characteristics might be useful in biomedical research using pESCs, leading to new insights regarding regenerative medicine and tissue repair.


Assuntos
Células-Tronco Embrionárias/ultraestrutura , Fertilização In Vitro/métodos , Técnicas de Transferência Nuclear , Animais , Apoptose , Blastocisto/citologia , Linhagem Celular , Núcleo Celular/ultraestrutura , Técnicas de Cocultura , Citoplasma/ultraestrutura , Células-Tronco Embrionárias/citologia , Retículo Endoplasmático Rugoso/ultraestrutura , Fibroblastos/ultraestrutura , Camundongos , Camundongos Endogâmicos ICR , Microscopia Eletrônica de Transmissão , Microvilosidades/ultraestrutura , Mitocôndrias/ultraestrutura , Fagocitose , Suínos
15.
Theriogenology ; 85(4): 601-16, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26542134

RESUMO

The establishment of porcine embryonic stem cells (ESCs) would have great impact in biomedical studies and preclinical trials through their use in genetic engineering. However, authentic porcine ESCs have not been established until now. In this study, a total of seven putative ESC lines were derived from porcine embryos of various origins, including in vitro fertilization, parthenogenetic activation, and, in particular, induced pluripotent stem (iPS) nuclear transfer (NT) from a donor cell with induced pluripotent stem cells (iPSCs). To characterize these cell lines, several assays including an assessment of intensive alkaline phosphatase activity, karyotyping, embryoid body formation, expression analysis of the pluripotency-associated markers, and the three germ layerassociated markers were performed. Based on quantitative polymerase chain reaction, the expression levels of REX1 and FGFR2 in iPS-NT lines were higher than those of cells of other origins. Additionally, only iPS-NT lines showed multiple aberrant patterns of nuclear foci elucidated by immunofluorescence staining of H3K27me3 as a marker of the state of X chromosome inactivation and a less mature form of mitochondria like naive ESCs, by transmission electron microscopy. Together, these data suggested that established putative porcine ESC lines generally exhibited a primed pluripotent state, like human ESCs. However, iPS-NT lines have especially unique characteristics distinct from other origins because they have more epigenetic instability and naive-like mitochondrial morphology than other putative ESC lines. This is the first study to establish and characterize the iPSC-derived putative ESC lines and compare them with other lines derived from different origins in pigs.


Assuntos
Blastocisto/citologia , Clonagem de Organismos , Células-Tronco Embrionárias/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Suínos/embriologia , Animais , Blastocisto/fisiologia , Proliferação de Células , Células Alimentadoras , Regulação da Expressão Gênica no Desenvolvimento , Cariotipagem , Camundongos , Microscopia Eletrônica de Transmissão , Mitocôndrias/fisiologia , Partenogênese
16.
J Reprod Dev ; 61(6): 549-57, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26370787

RESUMO

Ganglioside is an acidic glycosphingolipid with sialic acids residues. This study was performed to investigate the effect and mechanism of ganglioside GT1b in porcine oocytes in the process of in vitro maturation (IVM) and preimplantation development. Metaphase II (MII) rates were significantly (P < 0.05) different between the control group and the 5 nM GT1b treatment group. Intracellular glutathione (GSH) levels in oocytes matured with 5 nM and 20 nM and GT1b decreased significantly (P < 0.05). The 10 nM group showed a significant (P < 0.05) decrease in intracellular reactive oxygen species (ROS) levels compared with the control group. Subsequently, the level of intracellular Ca(2+) in oocytes treated with different concentrations of GT1b was measured. Intracellular Ca(2+) was significantly (P < 0.05) increased with a higher concentration of GT1b in a dose-dependent manner. Real-time PCR was performed and showed that the expression of bradykinin 2 receptor (B2R) and calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) in cumulus cells was significantly (P < 0.05) decreased in the 20 nM GT1b treatment group. Treatment with 5 nM GT1b significantly (P < 0.05) decreased the expression of CaMKIIδ. In oocytes, treatment with 5 nM GT1b significantly (P < 0.05) decreased CaMKIIγ and POU5F1 (POU domain, class 5, transcription factor 1). However, treatment with 20 nM GT1b significantly (P < 0.05) increased the expression of POU5F1. Finally, embryonic developmental data showed no significant differences in the two experiments (parthenogenesis and in vitro fertilization). In conclusion, the results of the present study indicated that GT1b plays an important role in increasing the nuclear maturation rate and decreasing the intracellular ROS levels during IVM. However, GT1b inhibited maturation of the cytoplasm by maintaining intracellular Ca(2+) in the process of oocyte maturation regardless of the cell cycle stage. Therefore, GT1b is thought to act on another mechanism that controls intracellular Ca(2+).


Assuntos
Desenvolvimento Embrionário/fisiologia , Gangliosídeos/fisiologia , Técnicas de Maturação in Vitro de Oócitos , Suínos/fisiologia , Animais , Cálcio/análise , Feminino , Fertilização In Vitro , Glutationa/análise , RNA Mensageiro/análise , Espécies Reativas de Oxigênio , Reação em Cadeia da Polimerase em Tempo Real , Receptor B2 da Bradicinina/análise
17.
Mol Med Rep ; 12(4): 5973-82, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26238161

RESUMO

The present study investigated the effects of zinc deficiency during in vitro maturation (IVM) of porcine oocytes. Zinc deficiency was induced by administering the membrane­permeable zinc chelator N,N,N',N'­tetrakis­(2­pyridylmethyl)­ethylendiamine (TPEN). First, the effects of zinc deficiency during IVM on a TPEN­treated group and a TPEN+zinc-treated group compared with a control group were assessed. The oocyte maturation rates and subsequent embryonic developmental competence of the TPEN+zinc­treated oocytes were similar to those of the control oocytes (metaphase II [MII] rate, 93.0 and 92.7%, respectively, and blastocyst [BL] formation rate, 42.0 and 40.0%, respectively). These results were significantly different from those obtained for the TPEN­treated oocytes (MII rate, 0.61%; BL formation rate, 0%). Although the TPEN­treated oocytes were arrested at metaphase I (MI), the distribution of microtubules was normal. However, microfilament formation was abnormal in the TPEN­treated oocytes. Furthermore, the effect of a temporary zinc deficiency during IVM on oocyte maturation and subsequent embryonic development was assessed. TPEN (10 µM) was added to the IVM medium for 0, 7, 15 or 22 h. The 0 h­treated oocytes showed an 83.9% MII rate, while the 7 h­treated oocytes had a significantly lower MII rate (44.8%). Most of the 15- and 22 h­treated oocytes were arrested at MI (MI rate: 98.0 and 97.2%, respectively; MII rate, 0% in both groups). Reductions in the BL formation were dependent on the TPEN treatment duration (29.3, 9.2, 0, and 0% after 0, 7, 15 and 22 h, respectively). In conclusion, zinc is an essential element for successful oocyte maturation and embryonic development in pigs. Zinc deficiency caused a meiotic block and had lasting effects on early embryonic development.


Assuntos
Oócitos/efeitos dos fármacos , Zinco/deficiência , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Células Cultivadas , Desenvolvimento Embrionário/efeitos dos fármacos , Etilenodiaminas/efeitos adversos , Glutationa/metabolismo , Metáfase , Oócitos/citologia , Espécies Reativas de Oxigênio/metabolismo , Suínos
18.
Theriogenology ; 84(7): 1075-87, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26194698

RESUMO

Granulocyte colony-stimulating factor (G-CSF) is required for proliferation, differentiation, and survival of cells. It is also a biomarker of human oocyte developmental competence for embryo implantation. In humans, the G-CSF concentration peaks during the ovulatory phase of the ovarian cycle. In this study, the expressions of G-CSF and its receptor were analyzed by polymerase chain reaction in granulosa cells (GCs), CL, cumulus cells (CCs), and oocytes. Cumulus-oocyte complexes were aspirated from antral follicles of 1 to 3 mm (small follicles) and 4 to 6 mm (medium follicles). Cumulus-oocyte complexes from two kinds of follicles were matured in protein-free maturation medium supplemented with various concentrations of G-CSF (0, 10, and 100 ng/mL). By real-time polymerase chain reaction, the expressions of G-CSF and its receptor were detected in GCs, CL, CCs, and oocytes. Interestingly, the G-CSF transcript levels were significantly lower in oocytes than in the other cell types, whereas the G-CSF receptor transcript levels in oocytes were similar to those in GCs. After 44 hours of IVM, no differences in the rate of nuclear maturation were detected; however, the intracellular reactive oxygen species levels in oocytes from both groups of follicles matured with 10 ng/mL of human recombinant G-CSF (hrG-CSF) groups were significantly lower (P < 0.05). After parthenogenetic activation, the cleavage rates were significantly (P < 0.05) higher in 100 ng/mL hrG-CSF-treated small (63.3%) follicles than in 0, 10 ng/mL hrG-CSF-treated small (38.6% and 49.0%, respectively) follicles and 0 ng/mL hrG-CSF-treated medium (52.1%) follicles, and the cleavage rates were significantly (P < 0.05) higher in 10 ng/mL hrG-CSF-treated medium (76.3%) follicles than in all other groups. The blastocyst formation rates were significantly (P < 0.05) higher in 100 ng/mL hrG-CSF-treated small (31.2%) follicles than in 0 and 10 ng/mL hrG-CSF small (10.4% and 15.6%, respectively) follicles, and the 10 ng/mL hrG-CSF medium (45.7%) follicle was significantly (P < 0.05) higher than in all other groups. The total cell number in blastocysts from the 10 ng/mL hrG-CSF medium (106.5) follicles was significantly (P < 0.05) increased compared to 0, 10, 100 ng/mL hrG-CSF small (55.0, 73.7 and 59.5, respectively) follicles and 0, 100 ng/mL hrG-CSF-treated medium (82.5 and 93.5, respectively) follicles. After IVF, the blastocysts stage was significantly (P < 0.05) increased in 10 ng/mL hrG-CSF-treated medium (36.4%) follicles. Fertilization efficiency was significantly high in 100 ng/mL of small (29.1%) and 10 ng/mL of medium (44.0%) follicles. We also examined the Bcl2 and ERK2 transcript levels and found that they were significantly higher in the small and medium follicle treatment groups. In conclusion, these results indicate that hrG-CSF improve the viability of porcine embryos.


Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Suínos , Animais , Blastocisto/fisiologia , Células do Cúmulo/química , Feminino , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/análise , Fator Estimulador de Colônias de Granulócitos/genética , Células da Granulosa/química , Humanos , Oócitos/química , RNA Mensageiro/análise , Espécies Reativas de Oxigênio/análise , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Fator Estimulador de Colônias de Granulócitos/análise , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Proteínas Recombinantes
19.
Theriogenology ; 84(4): 531-7, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26047708

RESUMO

This study aimed to investigate the effect of zinc on in vitro development of porcine embryos. We evaluated the effects of zinc on blastocysts formation and investigated gene expression at zinc-deficient and supplemented conditions. Zinc-deficient in vitro condition was induced by 10-µM N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylendiamine (TPEN) (zinc chelator) treatment during IVC. On parthenogenetic activated embryos, this treatment significantly decreased cleavage rate and blastocyst formation compared with the control (0.0% and 0.0% vs. 69.0% and 36.0%, respectively). And time effect of the zinc deficiency exposure is observed. Blastocyst formation rate was significantly decreased as zinc-deficient time increases (54.1%, 31.0%, 9.0%, and 1.2% for zinc deficiency during 0, 3, 5, and 7 hours). However, zinc supplementation during IVC supported in vitro embryonic development. On parthenogenetic activated embryos, supplementation of 0.8 µg/mL of zinc during IVC significantly increased blastocyst formation compared with other groups (43.9%, 57.8%, 67.1%, 51.4%, and 52.6% for zinc supplementation of 0, 0.4, 0.8, 1.2, and 1.6 µg/mL). In vitro-fertilized (IVF) embryos showed similar results. The blastocyst formation rate was significantly higher in the 0.8 µg/mL of zinc-supplemented group than in the other groups (21.3%, 24.1%, 36.1%, 25.9%, and 25.2% for zinc supplementation of 0, 0.4, 0.8, 1.2, and 1.6 µg/mL). PCNA, POU5F1, and Bcl2 messenger RNA expressions were unregulated in IVF-derived blastocysts in the 0.8 µg/mL of zinc-supplemented group compared with the control. These results suggest that zinc is required for embryonic development, and supplementation with adequate zinc concentrations during IVC improved the viability of porcine embryos, possibly by increasing PCNA, POU5F1, and Bcl2 gene expression of embryos.


Assuntos
Técnicas de Cultura Embrionária/veterinária , Suínos/embriologia , Zinco/farmacologia , Animais , Meios de Cultura/química , Etilenodiaminas/toxicidade , Regulação da Expressão Gênica no Desenvolvimento , Partenogênese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
20.
Theriogenology ; 83(2): 294-305, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25442018

RESUMO

In the process of IVM, cumulus-oocyte complexes (COCs) separate from the follicular microenvironment, leading to the loss of endocrine interactions between follicular mural somatic cells and COCs. To restore the microenvironment, a coculture system was established using cumulus-derived somatic cells (CSCs) for IVM. The CSCs were cultured in Dulbecco's modified Eagle's medium for 48 hours with varying numbers of CSCs (0.0, 2.5 × 10(4), 5.0 × 10(4), and 10.0 × 10(4)) and then cultured in tissue culture medium 199 (TCM 199) for 4 hours before adding the oocytes. Cumulus-oocyte complexes from 3- to 6-mm follicles were matured in 500 µL of TCM 199 with eCG and hCG for 22 hours and then cultured in TCM 199 without hormones for 22 hours. After IVM, the group with 2.5 × 10(4) CSCs showed a significant increase in intracellular glutathione levels compared with the control group. In the evaluation of sperm penetration, efficient fertilization was increased in the groups with 2.5 × 10(4) and 5.0 × 10(4) CSCs compared with controls (44.9 and 46.5 vs. 32.1, respectively). The mRNA expression pattern analysis in matured COCs showed a significant upregulation of PCNA, COX-2, Has2, Ptx3, and Nrf2 in the 2.5 × 10(4) CSC group compared with controls. During COC maturation at 0, 11, 22, 33, and 44 hours, the 2.5 × 10(4) and 5.0 × 10(4) CSC groups showed a significantly altered mRNA expression of BMP15 and GDF9. The developmental competence of the matured oocytes in all groups was evaluated after IVF and parthenogenetic activation (PA). After IVF, the 2.5 × 10(4) CSC group showed significantly higher cleavage, blastocyst formation rate, and total cell numbers compared with controls (60.0%, 35.7%, and 127.3 vs. 43.2%, 21.1%, and 89.3, respectively). After PA, the 2.5 × 10(4) CSC group had significantly higher blastocyst formation rate and total cell number than the control group (52.0% and 120.4 vs. 35.4% and 90.9, respectively). In conclusion, these results suggest that the presence of a population of 2.5 × 10(4) CSCs during IVM synergistically improved the developmental potential of IVF- and PA-derived porcine embryos by increasing the intracellular glutathione level via changing of a specific gene expression pattern during oocyte maturation.


Assuntos
Técnicas de Cocultura , Células do Cúmulo/fisiologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , Sus scrofa , Animais , Blastocisto/fisiologia , Desenvolvimento Embrionário , Feminino , Fertilização In Vitro/veterinária , Expressão Gênica , Glutationa/análise , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/química , Partenogênese , RNA Mensageiro/análise , Espécies Reativas de Oxigênio/análise
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA