Novel regulators of oocyte differentiation and meiosis

• Autor(a) principal: Jesus, André
• Data de Publicação: 2023
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.1/19696
• topic: Oogénese; Meiose; Oócito; Cariossoma; Drosófila; Domínio/Área Científica::Ciências Médicas::Outras Ciências Médicas

Oogenesis is a complex multicell process shared between several species, by which female gametes are formed. Fundamental for this process is the timely expression of different oogenesis genes and a nuclear division called meiosis. In Drosophila melanogaster both processes are highly regulated, with failures in this regulation leading to fertility impairments. Drosophila oogenesis is a continuous process that starts with an asymmetric division of the germ line stem cells and terminates with meiotic completion during oocyte activation and egg deposition. In Drosophila there is a prolonged arrest in diplotene stage of prophase I, with oocyte chromatin condensation into the karyosome. At this stage the oocyte is transcriptionally quiescent, being all transcription performed by aiding nurse cells. Mutations that specifically abrogate Drosophila karyosome formation led to meiotic chromosome segregation defects, clearly suggesting that formation of this structure is important for meiosis. Also, right before this meiotic progression, the oocyte’s nucleus shows a brief transcription activity for which epigenome regulation is essential. The genes expressed during this transient reactivation are unknown. Regulation of karyosome formation, maintenance and reactivation is poorly known, our aim is to identify different proteins that regulate some or several of these processes. We theorize that at least some of the proteins required for karyosome formation, maintenance and reactivation are likely to co-localize with the karyosome at some stages of oocyte development. The specific aim of this master thesis work was to find if different proteins from a list of candidates were present in the oocyte karyosome. The selection of the candidate proteins was based on the cumulative fulfilment of the following criteria: ii) gene ontology (GO) term that suggest interaction with DNA; ii) high expression during oogenesis measured by mass spectroscopy analysis, and iii) available GFP-tagged version of the protein. The presence of the candidate proteins in the karyosome was analyzed by confocal microscopy in which we search for colocalization of the GFP-tagged versions of the candidate proteins with the oocyte DNA stained with DAPI. For this screen results we observed karyosome co-localization with the proteins SSRP, PIT and CG14230; interesting GFP signal was observed in other structures, namely in the germarium subareas and nucleoplasm and RNA splicing structures in latter stages.