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Retrospective model to search and identify transcripts linked to the oocyte competence process

Grant number: 20/02086-3
Support type:Scholarships in Brazil - Master
Effective date (Start): July 01, 2020
Effective date (End): June 30, 2022
Field of knowledge:Agronomical Sciences - Veterinary Medicine - Animal Reproduction
Principal researcher:Flávio Vieira Meirelles
Grantee:Dewison Ricardo Ambrizi
Home Institution: Faculdade de Zootecnia e Engenharia de Alimentos (FZEA). Universidade de São Paulo (USP). Pirassununga , SP, Brazil

Abstract

Oogenesis is the process of formation and differentiation of primordial female germinative cells until the generation of a fertilized egg. This is an event that begins in the prenatal life and ends in the adult life of the female. During the growth and development of the female the oocyte pool is found in Diplotene of Meiosis I, more precisely in the state known as germinal vesicle (GV). In GV, these oocytes are transcriptionally active and store large amounts of mRNAs which are known as maternal RNAs due to their origin. These RNAs are considered part of the oocyte quality and are associated with cytoplasmic maturation. They are important to support early embryonic development, since the genome remains inactive during the first divisions of the zygote. In addition, the epigenetic nuclear maturation is also important for the development of the blastocyst. Thus, imprinted genes are fine tuned to control the expression that occurs either in the maternal or paternal allele. So far, only one study has used a retrospective study model to associate maternal RNAs with individuals who reach the blastocyst stage. This study showed that the IGF2R gene was present only in samples of good quality (developed to blastocyst). However, this study was carried out using the microarray technique which detects only the chosen targets, does not detect pre-miRNAs, lncRNAs, splicing and non-annotated transcripts. In addition, DNA methylation occurs during oocyte growth where the ooplasm provides a microenvironment for the oocyte and 1° CP genome. IGF2R, an imprinting gene, is often found aberrant in individuals with epigenetic syndromes which are due to the induction of the environment in vitro. Therefore, with pertinence of the elucidated facts, the goal of this study is to use a retrospective model through cytoplasmic and polar body biopsies removed individually from oocytes in MII. Maternal transcripts will be analyzed using RNA-Seq for each biopsy. For corpuscle, differential DNA digestion with restriction enzymes for the IGF2R region will be performed. We will use 1° polar body to analyze the oocyte epigenome without destroying it and to verify if the epigenetic maturation occurred in an adequate way for IGF2R in the oocyte genome. The compared groups will be those that manage to reach the blastocyst stage and those whose development is blocked in the 2-16 cell stage, respectively the competent and the non-competent. (AU)