RESUMO
Primordial germ cells (PGCs) are responsible for generating all germ cells. Therefore, they are essential targets to be used as a tool for the production of germline chimeras. The labeling and route of PGCs were evaluated during the initial embryonic development of Pseudopimelodus mangurus, using whole-mount in situ hybridization (WISH) and mRNA microinjection in zygotes. A specific antisense RNA probe constituted by a partial coding region from P. mangurus nanos3 mRNA was synthesized for the WISH method. RNA microinjection was performed using the GFP gene reporter regulated by translation regulatory P. mangurus buc and nanos3 3'UTR sequences, germline-specific markers used to describe in vivo migration of PGCs. Nanos3 and buc gene expression was evaluated in tissues for male and female adults and initial development phases and larvae from the first to seventh days post-hatching. The results from the WISH technique indicated the origin of PGCs in P. mangurus from the aggregations of nanos3 mRNA in the cleavage grooves and the signals obtained from nanos3 probes corresponded topographically to the migratory patterns of the PGCs reported for other fish species. Diffuse signals were observed in all blastomeres until the 16-cell stage, which could be related to the two sequences of the nanos3 3'UTR observed in the P. mangurus unfertilized egg transcriptome. Microinjection was not successful using GFP-Dr-nanos1 3'UTR mRNA and GFP-Pm-buc 3'UTR mRNA and allowed the identification of potential PGCs with less than 2% efficiency only and after hatching using GFP-Pm-nanos3 3'UTR. Nanos3 and buc gene expression was reported in the female gonads and from fertilized eggs until the blastula phase. These results provide information about the PGC migration of P. mangurus and the possible use of PGCs for the future generation of germline chimeras to be applied in the conservation efforts of Neotropical Siluriformes species. This study can contribute to establishing genetic banks, manipulating organisms, and assisting in biotechnologies such as transplanting germ cells in fish.
Assuntos
Peixes-Gato , Feminino , Masculino , Animais , Regiões 3' não Traduzidas , Peixes-Gato/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Células Germinativas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Antissenso/metabolismoRESUMO
Resumen: Introducción: El Sangrado Menstrual Excesivo (SME) es un problema frecuente en la adolescencia. La prevalencia de trastornos hereditarios de la coagulación (THC) como causa del SME no está bien establecida y la participación de defectos de la vía fibrinolítica ha sido poco explorada. Objetivo: Determinar la prevalencia de THC y defectos de la fibrinólisis en adolescentes con SME. Pacientes y Método: Se incluyeron 93 adolescentes, edad 11 a 18 años. Los antecedentes personales y familiares de sangra do se obtuvieron con un cuestionario estandarizado. Se controló exámenes: tiempo de protrom- bina (TP), tiempo de tromboplastina parcial activada (TTPa), estudio del factor Von Willebrand, recuento y función plaquetaria. Los pacientes que no fueron diagnosticados como THC, se evaluaron adicionalmente con el tiempo de lisis del coágulo. Resultados: 41 pacientes (44%) fueron diagnos ticados como THC: Enfermedad de Von Willebrand n = 28, defectos de la función plaquetaria n = 8, hemofilia leve n = 5. Se confirmó disminución del tiempo de lisis del coágulo en 31 pacientes. El 54% de pacientes diagnosticado como THC, tuvo SME como la primera manifestación hemorrágica. Conclusión: Estos resultados apoyan la necesidad de evaluación de la coagulación, incluyendo la vía fibrinolítica, en el estudio de adolescentes con SME.
Abstract: Introduction: Heavy Menstrual Bleeding (EMB) is a frequent problem in adolescence. The prevalence of inherited bleeding disorders (IBD) as a cause of EMB is not well established and the involvement of fibri nolytic pathway defects has been poorly explored. Objective: To determine the prevalence of IBD and fibrinolysis defects in adolescents with EMBs. Patients and Method: 93 adolescents (11 to 18 years old) were included. Personal and family history of bleeding were obtained through a standard ized questionnaire. The following lab tests were performed: prothrombin time (PT), activated partial thromboplastin time (aPTT), von Willebrand factor quantification, and platelet count and function. Those patients who were not diagnosed with IBD were further evaluated with clot lysis time assay. Results: 41 patients (44%) were diagnosed as IBD (Von Willebrand disease n = 28, platelet func tion defects n=8, mild hemophilia n = 5. Decreased clot lysis time was found in 31 patients. 54% of patients diagnosed with IBD had EMB as the first hemorrhagic manifestation. Conclusion: These results support the need to evaluate the coagulation process, including the fibrinolytic pathway in the study of adolescents with EMB.