More than 80% of chromosomal errors detected in human conceptuses occur as a consequence of chromosomal malsegregation during anaphase of the first meiotic division (meiosis I, MI). It is currently unknown how aneuploidy occurs at such a high rate in human oocytes. Advanced maternal age remains the only well-documented risk factor for maternal meiotic malsegregation, but there is, however, a surprisingly poor understanding of the basic mechanism(s) behind the maternal age effect. Our project will characterize proteins involved in the regulation of the asymmetry of the first division and and setting up of anaphase I onset both in ascidian and mouse oocytes. Therefore it will lead to a deeper understanding of the process that regulates ploidy in chordate oocytes. Hence, this work will ultimately be important for human reproduction and cancer since one of the expected outcomes will be the identification and characterisation of novel proteins involved in genome stability. In addition, the use of ascidian and mouse as model organisms will naturally lead to a better understanding on the evolution of the mechanisms which regulate and maintain ploidy during meiosis in the chordate phylum. The specific aims of this project are first to characterize and analyze the proteome of metaphase I chromosomes, then to determine how chromosome segregation is regulated during meiosis and how the meiotic spindle/chromosomes organize asymmetric divisions. In order to advance our understanding of the mechanism of homologous chromosome segregation we have performed a proteomic analysis of metaphase I chromosomes using ascidian chromosomes as starting material – mammalian species do not provide sufficient quantities of oocytes for such an approach (millions of oocytes are required). Now that we have completed the first round of proteomic analysis and have identified 242 metaphase I candidate proteins, we propose a collaborative project whereby two laboratories [one at the Station Zoologique at Villefranche sur Mer (McDougall, ascidian oocyte laboratory) and one at the UPMC in Paris (Verlhac, mouse oocyte laboratory)] will characterize and analyze the proteomic data-set that we have provided. First a verification of candidate proteins will be performed through the construction of fluorescent fusion constructs (Venus, Rfp1/mCherry) and where applicable, immunolabelling. Functional studies will be performed on selected proteins through a combination of knockdown and overexpression studies (dominant negative and positive). Due to the large data-set that we have generated, we will begin by analysing those candidate proteins that impact upon the ongoing research activities. Those research activities have two main themes: 1.) the mechanism that regulates migration of the chromosomes/spindle to the oocyte cortex prior to extrusion of the first polar body and 2.) the mechanism that regulates the segregation of chromosomes during the meiotic divisions.
Marie Hélène VERLHAC (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE COTE D'AZUR
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B
Help of the ANR 479,332 euros
Beginning and duration of the scientific project: - 48 Months