RESUMEN
The Hippo pathway plays a crucial role in cell proliferation and differentiation during tumorigenesis, tissue homeostasis and early embryogenesis. Scaffold proteins from the ezrin-radixin-moesin (ERM) family, including neurofibromin 2 (NF2; Merlin), regulate the Hippo pathway through cell polarity. However, the mechanisms underlying Hippo pathway regulation via cell polarity in establishing outer cells remain unclear. In this study, we generated artificial Nf2 mutants in the N-terminal FERM domain (L64P) and examined Hippo pathway activity by assessing the subcellular localization of YAP1 in early embryos expressing these mutant mRNAs. The L64P-Nf2 mutant inhibited NF2 localization around the cell membrane, resulting in YAP1 cytoplasmic translocation in the polar cells. L64P-Nf2 expression also disrupted the apical centralization of both large tumor suppressor 2 (LATS2) and ezrin in the polar cells. Furthermore, Lats2 mutants in the FERM binding domain (L83K) inhibited YAP1 nuclear translocation. These findings demonstrate that NF2 subcellular localization mediates cell polarity establishment involving ezrin centralization. This study provides previously unreported insights into how the orchestration of the cell-surface components, including NF2, LATS2 and ezrin, modulates the Hippo pathway during cell polarization.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Polaridad Celular , Proteínas del Citoesqueleto , Vía de Señalización Hippo , Neurofibromina 2 , Proteínas Serina-Treonina Quinasas , Proteínas Supresoras de Tumor , Proteínas Señalizadoras YAP , Neurofibromina 2/metabolismo , Neurofibromina 2/genética , Animales , Ratones , Proteínas Señalizadoras YAP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Transducción de Señal , Embrión de Mamíferos/metabolismo , Mutación/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Transporte de Proteínas , Membrana Celular/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/genéticaRESUMEN
PURPOSE: To assess the effects of light from an integrated optical microscope and evaluate the safety of time-lapse observations using a built-in microscope incubator. METHODS: We prospectively compared the fertilization rate and embryonic morphology after intracytoplasmic sperm injection between embryos cultured with time-lapse observations every 15 min in an incubator with an integrated optical microscope and embryos with intermittent observations (once a day) in conventional incubators. RESULTS: No significant differences were observed in the fertilization rate (57.5% vs. 57.5%) or the rate of excellent-good cleavage embryos (36.0% vs. 36.0%). CONCLUSIONS: These results suggest that time-lapse observations using an incubator with an integrated optical microscope may therefore be safely utilized in clinical practice.
Asunto(s)
Fase de Segmentación del Huevo/efectos de la radiación , Luz/efectos adversos , Fotomicrografía/efectos adversos , Adulto , Fase de Segmentación del Huevo/fisiología , Fase de Segmentación del Huevo/ultraestructura , Técnicas de Cultivo de Embriones/instrumentación , Transferencia de Embrión , Desarrollo Embrionario/efectos de la radiación , Femenino , Fertilización , Humanos , Incubadoras , Masculino , Fotomicrografía/instrumentación , Fotomicrografía/métodos , Embarazo , Resultado del Embarazo , Índice de Embarazo , Seguridad , Inyecciones de Esperma IntracitoplasmáticasRESUMEN
When human blastocysts hatch through the zona pellucida, gaining the ability to adhere to the endometrium, crosstalk between the embryo and the uterus may represent a successful outcome of their synchronized development and differentiation. CD26/dipeptidyl peptidase IV is known as a marker molecule of the implantation phase endometrium. To study the role of CD26 in implantation, 35 human hatched blastocysts were prepared by enzymatic treatment of expanded blastocysts that had been grown on schedule from frozen-thawed surplus embryos at the 2- or 4-cell stage. The blastocysts were placed on CD26-overexpressing or mock-transfected control monolayer cell cultures. The CD26-overexpression caused significantly higher blastocyst adhesion rate (53.3% versus 25.0%, P < 0.05) and significantly larger outgrowth area of trophectoderm (1.7-fold, P < 0.05). The second part of the present study was to show the expression of fibronectin, a CD26 ligand, in human preimplantation embryos, using the same donated resources. Fibronectin mRNA was detected by RT-PCR from the single hatched blastocyst (2/2) and from the single early blastocyst (3/6) but not from the single morula (0/5) samples. An indirect immunofluorescence technique verified the localization of fibronectin on the surface of the blastocyst. These results indicate that the adhesion mechanism by endometrial CD26 and embryonal fibronectin may be involved in human blastocyst implantation.