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The influence of ovarian follicular niche on oocyte development

Paixão, João Vitor Puttini

Biblioteca Digital de Teses e Dissertações da USP; Universidade de São Paulo; Faculdade de Zootecnia e Engenharia de Alimentos 2023-08-24

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  • Título:
    The influence of ovarian follicular niche on oocyte development
  • Autor: Paixão, João Vitor Puttini
  • Orientador: Meirelles, Flávio Vieira
  • Materias: Rna-Seq; Oócito; Gsk3Β Exossomos; Bovino; Exosomes; Oocyte; Rna-Seq; Bovine
  • Notas: Dissertação (Mestrado)
  • Descripción: Exosomes (Exos), small extracellular vesicles, present in follicular fluid (FF), act in intercellular communication, carrying bioactive contents, such as mRNAs and lnc-RNAs, contributing to oocyte maturation. Hence, it is speculated that Exos present in the fluid of follicles containing good quality or poor quality oocytes are different in their RNA content. The first aim of this work was to comprehend, in detail, the relation between the content of polyadenylated RNAs of Exos originating from the FF and the oocyte/embryo development. To this end, the Exos from the FF were collected and isolated from 3 to 6 mm diameter follicles using a retrospective bovine model previously described. To track its competence, FF and oocyte were individually recovered through the dissection of follicles from randomly selected slaughterhouse`s ovaries. Gametes were matured, parthenogenetically activated and cultured individually for 7 days. Based on developmental records, oocytes and their respective follicular fluids were classified and separated in three groups, accordingly to developmental competence: NCLEAV Group (Incompetent Mature): Oocytes matured in vitro that did not cleave after parthenogenetic activation; CLEAV Group (Incompetent Cleaved): Oocytes cleaved after parthenogenetic activation that blocked the development at 3rd or 4th cell cycle; BLAST Group (Blastocysts): Oocytes that developed to the blastocyst stage. Differential gene expression, exclusive and HUB genes analyses of the transcriptomic data were performed, and transcriptional differences among the more competent (BLAST) and less competent (CLEAV and NCLEAV) groups (p-adjust.<0.1) were observed. Interestingly, several gene transcripts found significantly altered among these groups were related to signaling pathways associated with cell proliferation and meiosis modulation such as WNT, PI3K-Akt and Hippo signaling pathways. Due to its key function in all pathways, GSK3 (Glycogen Synthase Kinase 3) was selected as a candidate protein that potentially regulates these signaling pathways and the oocyte competence. To test this, oocytes were matured under 3 conditions: 1) CTL (control); 2) DKK1 treatment that indirectly stimulates GSK3β through DKK-1 (Dickkopf related protein 1) inhibition of the WNT signaling pathway; 3) CHIR treatment that directly inhibits GSK3β through the chemical probe CHIR99021. Polar body extrusion rates were observed, mature oocytes were parthenogenetically activated and in vitro cultured. The CHIR group presented a higher maturation rate compared with the others but no differences were observed in cleavage and Blastocyst development. To further understand the inhibition effect β-catenin was localized by immunocytochemistry and a increased nuclear localization was observed in CHIR group again. In conclusion the retrospective model identified crucial genes and pathways present in FF-derived Exos that are associated with oocyte competence. Modulation of GSK3β in COCs increased the extrusion rate of the 1st polar body while the other rates did not show statistical changes. However, the characteristics of the modification induced by GSK3β inhibition were similar to those observed in vivo in human oocytes.
  • DOI: 10.11606/D.74.2023.tde-28022024-153212
  • Editor: Biblioteca Digital de Teses e Dissertações da USP; Universidade de São Paulo; Faculdade de Zootecnia e Engenharia de Alimentos
  • Fecha de creación: 2023-08-24
  • Formato: Adobe PDF
  • Idioma: Inglés
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