Retrospective model for search and identification of imprinted genes linked to the oocyte competence process

• Grant number: 21/09536-7
• Support Opportunities: Scholarships abroad - Research Internship - Master's degree
• Start date: January 30, 2022
• End date: July 29, 2022
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Flávio Vieira Meirelles
• Grantee: Dewison Ricardo Ambrizi
• Supervisor: Lawrence Charles Smith
• Host Institution: Faculdade de Zootecnia e Engenharia de Alimentos (FZEA). Universidade de São Paulo (USP). Pirassununga , SP, Brazil
• Institution abroad: Université de Montréal à Saint-Hyacinthe, Canada
• Associated to the scholarship: 20/02086-3 - Retrospective model to search and identify transcripts linked to the oocyte competence process, BP.MS

Abstract

Immediately after birth, mammalians possess an oocyte reserve estimated to be around 1 milion of cells.However, less than 1% of this pool will become a fertilizable oocyte. Part of this occurs due to a naturalprocess of selection during oogenesis. During the oocyte's growth phase, they must undergo a process ofcytoplasmic maturation, where they accumulate proteins, metabolites and RNAs essential to supportembryonic development. They also need to undergo the so-called "nuclear or epigenetic maturation",where the chromosomes condense, segregate and reprogram/acquire the epigenetic marks. In this point,the oocyte's DNA is correctly marked with DNA methylation and histone modifications. One of the moststudied imprinted genes, the IGF2R, is controlled by elements sensitive to chromatin methylation and isexpressed when the ICR (imprinting control region) region is methylated in the maternal allele. Oocytesacquire DNA methylation in a size-dependent manner. The acquisition happens as a result of theinteractions with the microenvironment provided by the follicular cells and its fluid. The MII chromatinand the 1st polar body, which contains the counterpart of the oocyte's genetic material and it is extrudedafter 1 st meiosis completion, possess high correlation regarding the epigenetic marks. This observationmakes the 1 st polar body a non-invasive and ideal sample to investigate the oocyte's DNA withoutcompromising its genetic material. In a previous work by our group we demonstrated that the expressionof IGF2R is correlated with embryonic development. Therefore, we hypothesize that the methylationpatterns of the IG2R observed on the chromatin of the first polar body of bovine secondary oocytes arecorrelated with their ability to develop to the blastocyst stage. On this basis, it can be used as an additionalmarker to reinforce our current retrospective model, in which oocyte cytoplasmic biopsies are beinganalyzed by RNA-seq to identify transcripts associated with oocyte's competence. To achieve this goal,we propose to compare the methylation patterns between the first polar body and their respective oocyteand the oocyte's ability to develop parthenogenetically to the blastocyst stage in vitro. For this reason, weare soliciting a BEPE scholarship to join Dr. Lawrence Smith's lab(expert in imprinting gene analysis inbovine cells) to carry out this project. Briefly, the first polar bodies and their respective oocytes will betreated with bisulfite and sequenced to compare methylation levels in the ICR region of IGF2R. Secondly,the respective polar bodies from oocytes that had the biopsies sequenced by RNA-Seq (FAPESP projectcurrently underway) will also be treated with bisulfite to analyze methylation levels in ICR region ofIGF2R and correlate it with their ability to develop until blastocyst stage in cows.