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1.
Zool Res ; 42(3): 377-388, 2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-33998185

RESUMO

LIN28A, an RNA-binding protein, plays an important role in porcine induced pluripotent stem cells (piPSCs). However, the molecular mechanism underlying the function of LIN28A in the maintenance of pluripotency in piPSCs remains unclear. Here, we explored the function of LIN28A in piPSCs based on its overexpression and knockdown. We performed total RNA sequencing (RNA-seq) of piPSCs and detected the expression levels of relevant genes by quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis, and immunofluorescence staining. Results indicated that piPSC proliferation ability decreased following LIN28A knockdown. Furthermore, when LIN28A expression in the shLIN28A2 group was lower (by 20%) than that in the negative control knockdown group ( shNC), the pluripotency of piPSCs disappeared and they differentiated into neuroectoderm cells. Results also showed that LIN28A overexpression inhibited the expression of DUSP (dual-specificity phosphatases) family phosphatases and activated the mitogen-activated protein kinase (MAPK) signaling pathway. Thus, LIN28A appears to activate the MAPK signaling pathway to maintain the pluripotency and proliferation ability of piPSCs. Our study provides a new resource for exploring the functions of LIN28A in piPSCs.


Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proliferação de Células , Fosfatases de Especificidade Dupla/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas de Ligação a RNA/genética , Suínos
2.
Zool Res ; 42(1): 14-27, 2021 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-33420764

RESUMO

Double sex and mab-3-related transcription factor 1 (Dmrt1), which is expressed in goat male germline stem cells (mGSCs) and Sertoli cells, is one of the most conserved transcription factors involved in sex determination. In this study, we highlighted the role of Dmrt1 in balancing the innate immune response in goat mGSCs. Dmrt1 recruited promyelocytic leukemia zinc finger (Plzf), also known as zinc finger and BTB domain-containing protein 16 (Zbtb16), to repress the Toll-like receptor 4 (TLR4)-dependent inflammatory signaling pathway and nuclear factor (NF)-κB. Knockdown of Dmrt1 in seminiferous tubules resulted in widespread degeneration of germ and somatic cells, while the expression of proinflammatory factors were significantly enhanced. We also demonstrated that Dmrt1 stimulated proliferation of mGSCs, but repressed apoptosis caused by the immune response. Thus, Dmrt1 is sufficient to reduce inflammation in the testes, thereby establishing the stability of spermatogenesis and the testicular microenvironment.


Assuntos
Células-Tronco Germinativas Adultas/metabolismo , Imunidade Inata/fisiologia , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Cabras , Inflamação/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , NF-kappa B , Túbulos Seminíferos , Células de Sertoli/metabolismo , Receptor 4 Toll-Like/genética , Fatores de Transcrição/genética
3.
Zool Res ; 42(4): 401-405, 2021 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-34047080

RESUMO

Single-cell RNA sequencing (scRNA-seq) is useful for exploring cell heterogeneity. For large animals, however, little is known regarding spermatogonial stem cell (SSC) self-renewal regulation, especially in dairy goats. In this study, we described a high-resolution scRNA-seq atlas derived from a dairy goat. We identified six somatic cell and five spermatogenic cell subtypes. During spermatogenesis, genes with significantly changed expression were mainly enriched in the Notch, TGF-ß, and Hippo signaling pathways as well as the signaling pathway involved in the regulation of stem cell pluripotency. We detected and screened specific candidate marker genes ( TKTL1 and AES) for spermatogonia. Our study provides new insights into goat spermatogenesis and the development of testicular somatic cells.


Assuntos
Cabras/genética , Análise de Sequência de RNA/veterinária , Análise de Célula Única , Testículo/citologia , Animais , Cabras/anatomia & histologia , Masculino , Análise de Sequência de RNA/métodos , Espermatogênese/genética
4.
Cell Prolif ; 52(3): e12591, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896067

RESUMO

OBJECTIVES: To date, many efforts have been made to establish porcine embryonic stem (pES) cells without success. Extraembryonic endoderm (XEN) cells can self-renew and differentiate into the visceral endoderm and parietal endoderm. XEN cells are derived from the primitive endoderm of the inner cell mass of blastocysts and may be an intermediate state in cell reprogramming. MATERIALS AND METHODS: Porcine XEN cells (pXENCs) were generated from porcine pluripotent stem cells (pPSCs) and were characterized by RNA sequencing and immunofluorescence analyses. The developmental potential of pXENCs was investigated in chimeric mouse embryos. RESULTS: Porcine XEN cells derived from porcine pPSCs were successfully expanded in N2B27 medium supplemented with bFGF for least 30 passages. RNA sequencing and immunofluorescence analyses showed that pXENCs expressed the murine and canine XEN markers Gata6, Gata4, Sox17 and Pdgfra but not the pluripotent markers Oct4, Sox2 and TE marker Cdx2. Moreover, these cells contributed to the XEN when injected into four-cell stage mouse embryos. Supplementation with Chir99021 and SB431542 promoted the pluripotency of the pXENCs. CONCLUSIONS: We successfully derived pXENCs and showed that supplementation with Chir99021 and SB431542 confer them with pluripotency. Our results provide a new resource for investigating the reprogramming mechanism of porcine-induced pluripotent stem cells.


Assuntos
Endoderma/citologia , Endoderma/embriologia , Suínos/embriologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Técnicas de Cocultura , Cães , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Expressão Gênica , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Suínos/genética , Suínos/metabolismo , Quimeras de Transplante
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