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1.
J Korean Med Sci ; 38(49): e377, 2023 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-38111280

RESUMO

BACKGROUND: Hormone replacement therapy (HRT) is used to relieve menopause symptoms, but has been reported to be associated with coronary heart disease and cancers in women. However, a link between HRT and psoriasis has yet to be established. The aim of this study was to determine the association between HRT and the risk of psoriasis. METHODS: We executed a nationwide population-based study. A total of 1,130,741 post-menopause women were enrolled in the national health care insurance database based on the enrollment criteria. The study population was classified into four groups based on the duration of the HRT, and the risk of psoriasis was analyzed. RESULTS: The incidence rates of psoriasis per 1,000 person-years were 3.36 and 4.09 in the no history of HRT and ≥ 5 years of HRT, respectively. After adjustment for age, smoking, alcohol intake, regular exercise, body mass index, diabetes mellitus, hypertension, and dyslipidemia, the most prolonged duration of the HRT group (≥ 5 years) exhibited significantly increased risk of developing psoriasis (hazard ratio, 1.22; 95% confidence interval, 1.16-1.29). CONCLUSION: We propose that HRT in post-menopausal women is associated with an increased likelihood of psoriasis development.


Assuntos
Terapia de Reposição Hormonal , Menopausa , Humanos , Feminino , Pré-Escolar , Estudos de Coortes , Terapia de Reposição Hormonal/efeitos adversos , Pós-Menopausa , Fumar
2.
Int J Mol Sci ; 23(12)2022 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-35743012

RESUMO

Oocyte in vitro maturation (IVM) is the most important first step in in vitro embryo production. One prerequisite for the success of IVM in oocytes is to provide a rich culture microenvironment that meets the nutritional needs of developing oocytes. We applied different equine amniotic fluid mesenchymal stem cell conditioned medium (eAFMSC-CM) from passages 7, 18, and 27 to porcine oocytes during IVM to determine its effects on oocyte development and subsequent embryo development, specifically. The eAFMSC-CM from passage 7 (eAFMSC-CMp7) has a considerable impact on 9 genes: BAX, BCL2, SOD2, NRF2, TNFAIP6, PTGS2, HAS2, Cx37, and Cx43, which are associated with cumulus cell mediated oocyte maturation. GSH levels and distribution of mitochondrial and cortical granules were significantly increased in oocytes incubated with eAFMSC-CMp7. In addition, catalase and superoxide dismutase activities were high after IVM 44 h with eAFMSC-CMp7. After in vitro fertilization, blastocyst quality was significantly increased in the eAFMSC-CMp7 group compared to control. Lastly, the antioxidant effect of eAFMSC-CMp7 substantially regulated the expression of apoptosis, pluripotency related genes and decreased autophagy activity in blastocysts. Taken together, this study demonstrated that the eAFMSC-CMp7 enhanced the cytoplasmic maturation of oocytes and subsequent embryonic development by generating high antioxidant activity.


Assuntos
Líquido Amniótico , Células-Tronco Mesenquimais , Animais , Blastocisto/metabolismo , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Células do Cúmulo/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Fertilização in vitro , Cavalos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/metabolismo , Gravidez , Suínos
3.
Int J Mol Sci ; 23(5)2022 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-35270040

RESUMO

Dystrophinopathy is caused by mutations in the dystrophin gene, which lead to progressive muscle degeneration, necrosis, and finally, death. Recently, golden retrievers have been suggested as a useful animal model for studying human dystrophinopathy, but the model has limitations due to difficulty in maintaining the genetic background using conventional breeding. In this study, we successfully generated a dystrophin mutant dog using the CRISPR/Cas9 system and somatic cell nuclear transfer. The dystrophin mutant dog displayed phenotypes such as elevated serum creatine kinase, dystrophin deficiency, skeletal muscle defects, an abnormal electrocardiogram, and avoidance of ambulation. These results indicate that donor cells with CRISPR/Cas9 for a specific gene combined with the somatic cell nuclear transfer technique can efficiently produce a dystrophin mutant dog, which will help in the successful development of gene therapy drugs for dogs and humans.


Assuntos
Distrofina , Distrofia Muscular de Duchenne , Animais , Sistemas CRISPR-Cas/genética , Cães , Distrofina/genética , Distrofina/metabolismo , Edição de Genes , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Técnicas de Transferência Nuclear
4.
BMC Biotechnol ; 21(1): 1, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33413301

RESUMO

BACKGROUND: Small animals that show a deficiency in klotho exhibit extremely shortened life span with multiple aging-like phenotypes. However, limited information is available on the function of klotho in large animals such as pigs. RESULTS: In an attempt to produce klotho knockout pigs, an sgRNA specific for klotho (targeting exon 3) was designed and Cas9-sgRNA ribonucleoproteins were transfected into porcine fibroblasts. Transfected fibroblasts were cultured for one to 2 days and then directly used for nuclear transfer without selection. The cloned embryos were cultured in vitro for 7 days and analyzed to detect modifications of the klotho gene by both T7E1 and deep sequencing analysis. Modification succeeded in 13 of 20 blastocysts (65%), 8 of which (40.0%) were monoallelic modifications and 5 (25.0%) were biallelic modifications. Based on high mutation rates in blastocysts, we transferred the cloned embryos to 5 recipient pigs; 1 recipient was pregnant and 16 fetuses were recovered at Day 28 post transfer. Of the 16 fetuses, 9 were resorbing and 7 were viable. Four of 9 (44.4%) resorbing fetuses and 3 of the 7 (42.9%) viable fetuses had monoallelic modifications. Thus, 3 klotho monoallelic knockout cell lines were established by primary culture. A total of 2088 cloned embryos reconstructed with 2 frame-shifted cell lines were transferred to 11 synchronized recipients. Of the recipients, 7 of 11 eleven (63.6%) became pregnant. However, none of the pregnancies was maintained to term. To discover why klotho monoallelic knockout fetuses were aborted, expression of aging- and apoptosis-related genes and klotho protein in placentas from klotho monoallelic knockout and wild-type fetuses was investigated. Placentas from klotho monoallelic knockout fetuses showed negatively changed expression of aging- and apoptosis-related genes with lower relative expression of klotho protein. These results indicated that the reason why klotho monoallelic knockout fetuses were not maintained to term was possibly due to decreased klotho expression in placentas, negatively affecting aging- and apoptosis-related genes. CONCLUSIONS: Klotho monoallelic knockout porcine fetal fibroblasts were successfully established. However, pigs carrying klotho monoallelic knockout fetuses failed to maintain full-term pregnancy and a decrease in klotho expression in placenta likely leads to pregnancy loss.


Assuntos
Feto/metabolismo , Glucuronidase/genética , Glucuronidase/metabolismo , Envelhecimento/fisiologia , Animais , Blastocisto , Sistemas CRISPR-Cas , Linhagem Celular , Clonagem de Organismos , Feminino , Desenvolvimento Fetal , Fibroblastos/metabolismo , Edição de Genes , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Proteínas Klotho , Técnicas de Transferência Nuclear , Placenta , Gravidez , Suínos
5.
Int J Mol Sci ; 22(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299380

RESUMO

Artificial activation of oocytes is an important step for successful parthenogenesis and somatic cell nuclear transfer (SCNT). Here, we investigated the initiation of DNA synthesis and in vivo development of canine PA embryos and cloned embryos produced by treatment with 1.9 mM 6-dimethylaminopurine (6-DMAP) for different lengths of time. For experiments, oocytes for parthenogenesis and SCNT oocytes were cultured for 4 min in 10 µM calcium ionophore, and then divided into 2 groups: (1) culture for 2 h in 6-DMAP (DMAP-2h group); (2) culture for 4 h in DMAP (DMAP-4h group). DNA synthesis was clearly detected in all parthenogenetic (PA) embryos and cloned embryos incorporated BrdU 4 h after activation in DMAP-2h and DMAP-4h groups. In vivo development of canine parthenogenetic fetuses was observed after embryo transfer and the implantation rates of PA embryos in DMAP-2h were 34%, which was significantly higher than those in DMAP-4h (6.5%, p < 0.05). However, in SCNT, there was no significant difference in pregnancy rate (DMAP-2h: 41.6% vs. DMAP-4h: 33.3%) and implantation rates (DMAP-2h: 4.94% vs. DMAP-4h: 3.19%) between DMAP-2h and DMAP-4h. In conclusion, the use of DMAP-2h for canine oocyte activation may be ideal for the in vivo development of PA zygotes, but it was not more effective in in vivo development of canine reconstructed SCNT oocytes. The present study demonstrated that DMAP-2h treatment on activation of canine parthenogenesis and SCNT could effectively induce the onset of DNA synthesis during the first cell cycle.


Assuntos
Adenina/análogos & derivados , Replicação do DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Adenina/farmacologia , Animais , Clonagem de Organismos/métodos , Cães , Transferência Embrionária/métodos , Feminino , Técnicas de Transferência Nuclear , Oócitos/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Gravidez
6.
J Cell Physiol ; 235(2): 1386-1404, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31338842

RESUMO

Current studies indicate that application of oviduct cells (OCs) in in vitro system create microenvironment similar to the in vivo conditions by releasing multiple growth factors which has beneficial effects on the development of cumulus-oocyte complexes and embryos. In particular, recent evidence with a coculture system indicates that there is a reciprocal relationship between canine OCs and cumulus cells and that oviductal secretions can promote changes in cellular protein/gene expression. Despite the fact that OCs respond to cumulus cells, a clear understanding of the mechanism by which the components released from OCs that play a role in modulating the biological function of cumulus cells is still elusive. Therefore, we hypothesized that exosomes derived from OCs (OC-Exo), which efficiently mediate cellular communication by transferring their molecular cargo to recipient cells, could be key modulators of the cross-talk with cumulus cells. We aimed to characterize OC-Exo and decipher their physiological effects on cumulus cells via the epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) pathway, which is one of the prerequisite pathways for cell development. Exposure of OC-Exo improved physiological cumulus cell condition including cell concentration, viability, and proliferation rate could reduce the accumulation of reactive oxygen species and the apoptotic rate. Moreover, exosomes could enhance the messenger RNA transcript and protein levels related to EGFR signaling in cumulus cells. The present study provides the first evidence that OC-Exo effectively enhance the physiological condition of cumulus cells exposed to GW4869 or Gefitinib via the EGFR/MAPK signaling pathway and this could be the primary mediators of molecular interactions among cumulus cells and shedding light on the role of exosomes in cumulus cells might permit improvement of oocyte and embryo development in vitro.


Assuntos
Células do Cúmulo/metabolismo , Receptores ErbB/metabolismo , Exossomos/metabolismo , Tubas Uterinas/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Animais , Comunicação Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Cães , Tubas Uterinas/citologia , Feminino
7.
Reproduction ; 160(4): 613-625, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32698140

RESUMO

Oviduct cells produce a favorable environment for the development of gametes by generating multiple growth factors. Particularly, in canine species, immature oocytes undergo serial maturation processes in the oviduct, while the other mammals already possess matured oocytes in ovulatory follicles. However, little is known about the potential effect exhibited by the components released from canine oviduct cells (OCs) for modulating the biological function of oocytes. Recently, exosomes are regarded as promising extracellular vesicles because they represent considerable data for molecular cargo. Therefore, we first investigated the effect of canine oviductal exosomes (OC-Exo) on oocyte development via EGFR/MAPK pathway. Our results showed that OC-Exo labeled with PHK67 are successfully incorporated with cumulus cells and oocytes during IVM. Also, OC-Exo markedly increased the proportion of cumulus-oocyte complexes (COCs) exhibiting cumulus expansion as well as cumulus cell proliferation and maturation rate of oocytes (P < 0.05). Furthermore, gene expression patterns related with EGFR/MAPK pathway including EGFR, PKA, TACE/ADAM17, MAPK1/3, MAPK14, PTGS2, TNFAIP6, GDF9, and BMP15 were positively modified in COCs cultured with OC-Exo (P < 0.05). In addition, OC-Exo significantly up-regulated the protein expression levels of p-EGFR, p-MAPK1/3, GDF9 and BMP15 in COCs (P < 0.05). Consequently, the current study provides a model for understanding the roles of OC-Exo as bioactive molecules for canine oocyte maturation via EGFR/MAPK pathway, which would open a new avenue for the application of exosomes to improve assisted reproductive technology in mammals, including humans.


Assuntos
Receptores ErbB/metabolismo , Exossomos/fisiologia , Sistema de Sinalização das MAP Quinases , Oócitos/citologia , Oogênese , Oviductos/fisiologia , Animais , Cães , Receptores ErbB/genética , Feminino , Oócitos/metabolismo , Transdução de Sinais
8.
J Cell Physiol ; 234(4): 4030-4043, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30252133

RESUMO

It has become increasingly recognized that coculture has a beneficial effect on the in vitro maturation (IVM) of oocytes and embryo development in many species. However, these effects of coculture on IVM have been documented only for their positive conditioning roles without any evidence on the precise mechanisms underlying the action of coculture systems on the development of cumulus oocyte complexes (COCs). It has been suggested that the epidermal growth factor receptor (EGFR) signaling pathway is important for development of COCs, mediated by several epidermal growth factor (EGF)-like proteins with downstream mitogen-activated protein kinase 1/3 signaling. Therefore, we hypothesized that canine oviduct cells (OCs) in a coculture system, which shows improvement of oocyte quality in several species, are associated with EGFR signaling by exposure to progesterone (P4; imitating its production before ovulation and its continuous increase while oocytes reside in the oviduct to complete maturation in dogs). We designed three experimental groups: control, OCs coculture exposed to P4, and OCs coculture without exposure to P4. The result showed that the OCs coculture exposed to P4 strongly expressed EGF-like proteins and significantly improved COCs and subsequent embryo development. Furthermore, the expression of EGFR-related genes in cumulus cells and GDF9 and BMP15 in oocytes was upregulated in the P4-treated group. This study provides the first evidence that OCs exposed to P4 can induce strong expression of EGF-like proteins, and OCs effectively mediate improved porcine COCs development and subsequent embryo development by altering EGFR signaling related mRNA expression.


Assuntos
Blastocisto/fisiologia , Comunicação Celular , Células do Cúmulo/metabolismo , Família de Proteínas EGF/metabolismo , Receptores ErbB/metabolismo , Técnicas de Maturação in Vitro de Oócitos , Oviductos/metabolismo , Animais , Células Cultivadas , Técnicas de Cocultura , Cães , Família de Proteínas EGF/genética , Receptores ErbB/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Ligantes , Oviductos/citologia , Oviductos/efeitos dos fármacos , Partenogênese , Progesterona/farmacologia , Transdução de Sinais , Sus scrofa
9.
Transgenic Res ; 28(1): 91-102, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30552552

RESUMO

Herein, we successfully generated transgenic pigs expressing both soluble human tumor necrosis factor receptor I IgG1-Fc (shTNFRI-Fc) and human hemagglutinin (HA)-tagged-human heme oxygenase-1 (hHO-1) without Gal epitope. Healthy cloned pigs were produced by somatic cell nuclear transfer (SCNT) using the genetically modified cells. The genetic disruption of the GGTA1 genes and absence of expression of BS-IB4 lectin in tail-derived fibroblast of the SCNT-generated piglets were successfully confirmed. The expression of shTNFRI-Fc and HAhHO-1 was fully identified with protective effect against oxidative stress and apoptosis stimulation. Antibody-mediated complement-dependent cytotoxicity assay for examining the immuno-reactivity of transgenically derived pig cells showed that pigs lacking GGTA1 with the expression of double genes reduce the humoral barrier to xenotransplantation, more than pigs simply expressing double genes and the wild type. Through this approach, rapid production of a pig strain deficient in various genes may be expected to be applicable for xenotransplantation research without extensive breeding protocols.


Assuntos
Animais Geneticamente Modificados/genética , Galactosiltransferases/genética , Heme Oxigenase-1/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Animais , Apoptose/genética , Epitopos/genética , Epitopos/imunologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica/genética , Técnicas de Inativação de Genes , Heme Oxigenase-1/imunologia , Humanos , Técnicas de Transferência Nuclear , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Suínos , Transplante Heterólogo
10.
Artif Organs ; 43(3): 278-287, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30374978

RESUMO

This study introduces an implantable scaffold-free cartilage tissue construct (SF) that is composed of chondrocytes and their self-produced extracellular matrix (ECM). Chondrocytes were grown in vitro for up to 5 weeks and subjected to various assays at different time points (1, 7, 21, and 35 days). For in vivo implantation, full-thickness defects (n = 5) were manually created on the trochlear groove of the both knees of rabbits (16-week old) and 3 week-cultured SF construct was implanted as an allograft for a month. The left knee defects were implanted with 1, 7, and 21 days in vitro cultured scaffold-free engineered cartilages. (group 2, 3, and 4, respectively). The maturity of the engineered cartilages was evaluated by histological, chemical and mechanical assays. The repair of damaged cartilages was also evaluated by gross images and histological observations at 4, 8, and 12 weeks postsurgery. Although defect of groups 1, 2, and 3 were repaired with fibrocartilage tissues, group 4 (21 days) showed hyaline cartilage in the histological observation. In particular, mature matrix and columnar organization of chondrocytes and highly expressed type II collagen were observed only in 21 days in vitro cultured SF cartilage (group 4) at 12 weeks. As a conclusion, cartilage repair with maturation was recapitulated when implanted the 21 day in vitro cultured scaffold-free engineered cartilage. When implanting tissue-engineered cartilage, the maturity of the cartilage tissue along with the cultivation period can affect the cartilage repair.


Assuntos
Doenças das Cartilagens/cirurgia , Cartilagem Articular/cirurgia , Cultura Primária de Células/métodos , Engenharia Tecidual/métodos , Animais , Doenças das Cartilagens/patologia , Cartilagem Articular/citologia , Cartilagem Articular/lesões , Cartilagem Articular/patologia , Condrócitos/transplante , Modelos Animais de Doenças , Matriz Extracelular/transplante , Humanos , Masculino , Coelhos , Resultado do Tratamento
11.
Curr Microbiol ; 76(6): 732-737, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30993398

RESUMO

Several bioprocessing technologies, such as separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), and consolidated bioprocessing (CBP), have been highlighted to produce bio-based fuels and chemicals from lignocellulosic biomass. Successful CBP, an efficient and economical lignocellulosic biorefinery process compared with other processes, requires microorganisms with sufficient cellulolytic activity and biofuel/chemical-producing ability. Here, we report the complete genome of Paenibacillus sp. CAA11, a newly isolated promising microbial host for CBP-producing ethanol and organic acids from cellulose. The genome of Paenibacillus sp. CAA11 comprises one 4,888,410 bp chromosome with a G + C content of 48.68% containing 4418 protein-coding genes, 102 tRNA genes, and 39 rRNA genes. The functionally active cellulase, encoded by CAA_GH5 was identified to belong to glycosyl hydrolase family 5 (GH5) and consisted of a catalytic domain and a cellulose-binding domain 3 (CBM3). When cellulolytic activity of CAA_GH5 was assayed through Congo red method by measuring the size of halo zone, the recombinant Bacillus subtilis RIK1285 expressing CAA_GH5 showed a comparable cellulolytic activity to B. subtilis RIK1285 expressing Cel5, a previously verified powerful bacterial cellulase. This study demonstrates the potential of Paenibacillus sp. CAA11 as a CBP-enabling microbe for cost-effective biofuels/chemicals production from lignocellulosic biomass.


Assuntos
Genoma Bacteriano , Paenibacillus/genética , Análise de Sequência de DNA , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Composição de Bases , Biotransformação , Ácidos Carboxílicos/metabolismo , Vermelho Congo/metabolismo , Etanol/metabolismo , Genes Bacterianos , Lignina/genética , Lignina/metabolismo , RNA Ribossômico/genética , RNA de Transferência/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
12.
Mol Reprod Dev ; 85(4): 336-347, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29442425

RESUMO

Human endothelial progenitor cells (EPCs) have been applied to regenerative medicine for their roles in angiogenesis as well as neovascularization, and these angiogenetic functions have beneficial effects on maturation of ovarian follicles. However, little information is available on whether EPCs on culture systems affect oocyte maturation and subsequent embryo development. Therefore, the objective of this study was to investigate the effect of EPC co-culture on porcine oocytes during in vitro maturation (IVM) and subsequent embryo development, and to examine gene expression in cumulus cells, oocytes and blastocysts. The effect of co-culture using EPC on porcine oocyte IVM was investigated. Oocytes were activated using electrical stimulation and embryo developmental competence was estimated. The expression of the genes related to cumulus expansion, oocyte maturation, embryo development, and apoptosis were analyzed. In result, there was a significantly increased maturation rate in EPC group compared with control (p < 0.05). Also, oocytes co-cultured with EPCs exhibited significantly improved blastocyst formation rates (p < 0.05). The expression of mRNAs associated with cumulus expansion and apoptosis in cumulus cells was significantly up-regulated in EPC group. Also, markedly increased levels of GDF9, BMP15, and BCL2 were observed in oocytes from the EPC group. Blastocysts in the co-culture group showed significantly higher SOX2, OCT4, and NANOG levels. In conclusion, co-culturing porcine oocytes with EPCs improves their maturation by regulating genes involved in cumulus cell expansion, oocyte maturation, and apoptosis. Moreover, EPC co-culture during IVM enhanced embryo development as shown by increased blastocyst formation rate and pluripotency-related gene expression.


Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário , Células Progenitoras Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Oócitos/metabolismo , Animais , Blastocisto/citologia , Técnicas de Cocultura , Células Progenitoras Endoteliais/citologia , Humanos , Técnicas de Maturação in Vitro de Oócitos , Oócitos/citologia , Suínos
13.
J Reprod Dev ; 64(3): 277-282, 2018 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-29695650

RESUMO

This study was conducted to investigate whether the treatment of dog to pig interspecies somatic cell nuclear transfer (iSCNT) embryos with a histone deacetylase inhibitor, to improve nuclear reprogramming, can be applied to dog SCNT embryos. The dog to pig iSCNT embryos were cultured in fresh porcine zygote medium-5 (PZM-5) with 0, 1, or 10 µM suberoylanilide hydroxamic acid (SAHA) for 6 h, then transferred to PZM-5 without SAHA. Although there were no significant differences in cleavage rates, the rates of 5-8-cell stage embryo development were significantly higher in the 10 µM group (19.5 ± 0.8%) compared to the 0 µM groups (13.4 ± 0.8%). Acetylation of H3K9 was also significantly higher in embryos beyond the 4-cell stage in the 10 µM group compared to the 0 or 1 µM groups. Treatment with 10 µM SAHA for 6 h was chosen for application to dog SCNT. Dog cloned embryos with 0 or 10 µM SAHA were transferred to recipients. However, there were no significant differences in pregnancy and delivery rates between the two groups. Therefore, it can be concluded that although porcine oocytes support nuclear reprogramming of dog fibroblasts, treatment with a histone deacetylase inhibitor that supports nuclear reprogramming in dog to pig iSCNT embryos was not sufficient for reprogramming in dog SCNT embryos.


Assuntos
Clonagem de Organismos/veterinária , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Animais , Reprogramação Celular/efeitos dos fármacos , Clonagem de Organismos/métodos , Cães , Técnicas de Cultura Embrionária/métodos , Suínos , Vorinostat
14.
Reprod Domest Anim ; 53 Suppl 3: 133-138, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30474338

RESUMO

Since the generation of world's first cloned dog, Snuppy, in 2005, somatic cell nuclear transfer (SCNT) in dogs has been widely applied for producing several kinds of dogs with specific objectives. Previous studies have demonstrated that cloned dogs show normal characteristics in growth, blood parameters and behavioural aspect. Also, canine SCNT technique has been applied to propagate working dogs with excellent abilities in fields such as assistance of disabled people, drugs detection and rescue activity. Because dogs have similar habituation properties and share many characteristics including anatomic and physiological aspects with humans, they are also primary candidates for human disease models. Recently, transgenic dogs that express red fluorescent protein gene constitutively and green fluorescent protein gene conditionally have been generated. In addition, transgenic dogs with an overexpression of peroxisome proliferator-activated receptor-alpha in specific muscles were generated to enhance physical performance. In 2017, Snuppy was recloned with markedly increased pregnancy and delivery rates compared to the statistics from when Snuppy was first cloned. Such striking improvements in the cloning of dogs using SCNT procedures suggest that dog cloning could be applied in many fields of biomedical science for human diseases research, and the application of cloning is no longer science fiction.


Assuntos
Clonagem de Organismos/veterinária , Cães , Animais , Animais Geneticamente Modificados , Técnicas de Transferência Nuclear/veterinária
15.
Reprod Fertil Dev ; 30(1): 1-7, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29539298

RESUMO

Dog cloning as a concept is no longer infeasible. Starting with Snuppy, the first cloned dog in the world, somatic cell nuclear transfer (SCNT) has been continuously developed and used for diverse purposes. In this article we summarise the current method for SCNT, the normality of cloned dogs and the application of dog cloning not only for personal reasons, but also for public purposes.


Assuntos
Clonagem de Organismos , Cães , Técnicas de Transferência Nuclear/veterinária , Animais , Clonagem de Organismos/métodos , Clonagem de Organismos/tendências , Clonagem de Organismos/veterinária , Cães/embriologia , Transferência Embrionária/veterinária , Embrião de Mamíferos , Técnicas de Transferência Nuclear/tendências
16.
J Assist Reprod Genet ; 34(7): 929-938, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28386814

RESUMO

PURPOSE: In contrast to most other mammals, canine oocytes are ovulated in an immature state and undergo oocyte maturation within the oviduct during the estrus stage. The aim of the study was to investigate whether oviduct cells from the estrus stage affect the maturation of oocytes and show gene expression patterns related to oocyte maturation. METHODS: We analyzed MAPK1/3, SMAD2/3, and BMP6/15 expression in oviduct cells, cumulus cells, and oocytes from anestrus, estrus, and diestrus stages. Next, we investigated the effect of co-culture with oviduct cells derived from the estrus stage upon in vitro maturation (IVM) of canine oocytes. RESULTS: There was significantly higher MAPK1/3 (1.42 ± 0.02 and 2.23 ± 0.06), SMAD2/3 (0.77 ± 0.03 and 2.39 ± 0.07), and BMP15 (2.21 ± 0.16) expression in oviduct cells at the estrus stage (P < 0.05). In cumulus cells, MAPK1 (1.26 ± 0.07), SMAD2/3 (0.82 ± 0.01, 1.04 ± 0.01), and BMP6 (13.09 ± 0.11) expression was significantly higher in the estrus stage (P < 0.05). In oocytes, significant upregulation of MAPK1/3 (14,960 ± 3121 and 1668 ± 253.4), SMAD3 (774.6 ± 79.62), and BMP6 (8500 ± 895.4) expression was found in the estrus stage (P < 0.05). After 72 h of IVM culture, a significantly higher maturation rate was observed in oocytes co-cultured with oviduct cells (10.0 ± 1.5%) than in the control group (3.2 ± 1.4%). CONCLUSIONS: We demonstrate that oviduct cells at the estrus stage highly expressed MAPK1/3, SMAD2/3, and BMP15. Furthermore, canine oviduct cells from the estrus stage enhance the culture environment for canine oocyte maturation.


Assuntos
Oócitos/metabolismo , Animais , Técnicas de Cocultura , Células do Cúmulo/metabolismo , Cães , Estro/metabolismo , Feminino , Fertilização in vitro/veterinária , Perfilação da Expressão Gênica/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Sistema de Sinalização das MAP Quinases , Oócitos/crescimento & desenvolvimento , Oviductos/metabolismo , RNA Mensageiro/metabolismo
17.
Biochem Biophys Res Commun ; 479(4): 927-932, 2016 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-27586272

RESUMO

The objective of this study was to determine the ability of spermine to act as an antioxidant in scavenging reactive oxygen species (ROS), maintaining sperm function and decreasing cryocapacitation after cryopreservation. Although motility did not increase with spermine treatment, values for membrane integrity were significantly increased (P < 0.05). Higher percentages of linearity and straightness with a lower amplitude of lateral head displacement (ALH) indicated that spermine inhibits hyperactivation. Concentrations of intracellular and extracellular ROS were decreased in the treatment group (P < 0.05). Higher expression of an anti-apoptotic gene (Bcl-2) and lower expression of a pro-apoptotic gene (Bax), together with decreased expression of the mitochondrial ROS modulator ROMO1, DNA repair due to oxidative damage (OGG1), spermine synthase (SMS), NADPH oxidase associated with motility (NOX5) and spermine amino oxidase (SMOX), all showed that 5.0 mM spermine treatment was beneficial to spermatozoa. Furthermore, the proportion of live spermatozoa with intact acrosomes after thawing in the treatment group was higher than in the control. After incubation in canine capacitating medium, numbers of live capacitated spermatozoa with reacted acrosomes were higher than in the control. Our results indicate that 5.0 mM spermine is an optimal concentration for maintaining sperm function, reducing ROS production, preventing apoptosis and adverse effects of cryocapacitation during canine sperm cryopreservation.


Assuntos
Antioxidantes/farmacologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Espermina/farmacologia , Animais , Antioxidantes/administração & dosagem , Antioxidantes/metabolismo , Criopreservação/métodos , Crioprotetores/administração & dosagem , Crioprotetores/farmacologia , Cães , Expressão Gênica/efeitos dos fármacos , Masculino , Espécies Reativas de Oxigênio/metabolismo , Preservação do Sêmen/métodos , Capacitação Espermática/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/citologia , Espermina/administração & dosagem , Espermina/metabolismo
19.
J Phys Ther Sci ; 28(10): 2894-2897, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27821957

RESUMO

[Purpose] The purpose of the present study was to examine the effects of neurofeedback training on postural changes in the cervical spine and changes in the range of motion of the neck and in the Neck Disability Index in adults with forward head posture. [Subjects and Methods] The subjects of the study were 40 college students with forward head posture, randomly divided into a neurofeedback training group (NFTG, n=20) and a control group (CG, n=20). The neurofeedback training group received six sessions of pottery and archery games, each for two minutes, three times per week for four weeks, using the neurofeedback system. [Results] There were no significant effects within and between groups in terms of the absolute rotation angle, anterior weight bearing, and range of extension and flexion by x-ray imaging. There were significant effects in the neurofeedback training group pre- intervention and post-intervention in Neck Disability Index. There were significant effects between groups in Neck Disability Index. [Conclusion] It is thought that neurofeedback training, a training approach to self-regulate brain waves, enhances concentration and is therefore an effective intervention method to improve neck pain and daily activities.

20.
J Phys Ther Sci ; 28(10): 2938-2941, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27821966

RESUMO

[Purpose] The purpose of the present study was to examine the effects of neurofeedback training on electroencephalogram changes in the cervical spine in adults with forward head posture through x-ray. [Subjects and Methods] The subjects of the study were 40 college students with forward head posture, randomly divided into a neurofeedback training group (NFTG, n=20) and a control group (CG, n=20). The neurofeedback training group performed six sessions of pottery and archery games, each for two minutes, three times per week for four weeks, while using the neurofeedback system. [Results] There were significant effects within and between groups in terms of the Delta wave, the Theta wave, the Alpha wave, the Beta wave, or the sensory motor rhythm. Especially, the Delta wave, Beta wave, and the sensory motor rhythm were showed significant effects between the groups. [Conclusion] It is thought that neurofeedback training, a training approach to self-regulate brain waves, enhances concentration and relaxation without stress, as well as an increase in attention, memory, and verbal cognitive performance. Therefore an effective intervention method to improve neck pain and daily activities.

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