Chromosome Cohesion during Protracted Meiotic Arrest in Oocytes

We are trying to understand the basic mechanisms that make sure eggs receive a single copy of every chromosome. Genetic information, the blue-print of our body, is written on the chromosomes, which is first replicated before cells divide. Mistakes in distributing equal numbers of chromosomes can generate eggs with too many or too few chromosomes, which leads to spontaneous abortion or developmental disorders such as Down Syndrome. The chance of this kind of mistake happens more often when women are over the age of 35. Replicated chromosomes have to be kept stuck to each other until their separation into two daughter cells after fertilisation. From previous studies we know that glue proteins link these replicated chromosomes together. In eggs, the glue proteins have to hold replicated chromosomes for decades, because chromosomes are replicated before the birth of a girl, but the separation of chromosomes is triggered by sperm at the time of fertilisation. It is unknown, how these glue proteins are maintained during this long period of time. In this study, we will use mammalian models to test if the glue protein is added to chromosomes in order to maintain stickiness. We will observe eggs with advanced techniques, for example, detecting proteins on chromosome samples and taking movies of cell division and chromosome separation. Recent studies also discovered that abnormal glue proteins cause another genetic disease known as Cornelia de Lange Syndrome, probably because of irregular gene regulation during the process of reading the blue-print. We will also study the function of the glue proteins in gene regulation by inactivating the glue proteins in eggs.

Our objective is to determine whether cohesin replenishment is required for the maintenance of chromosome cohesion during the protracted meiotic prophase arrest of mammalian oocytes and if so, dissect the underlying molecular mechanism for cohesin turnover. We will take two complementary approaches which are both dependent on genetically modified mouse systems that permit gene deletion and induction in arrested growing oocytes. Firstly, we will inactivate Nipbl (also known as Scc2) that is required for cohesin loading in mitotic cells after pre-meiotic DNA replication in oocytes and investigate its effect on maintenance of cohesion and cohesin s association with chromosomes. Loss or decline of cohesion/cohesin will suggest that cohesin is replenished through Nipbl s activity. Since Nipbl has been suggested to have additional functions apart from cohesin loading, we expect that our study will also elucidate Nipbl s roles in loading of condensin and Smc5/6 complex, chromosome condensation and gene expression in oocytes. In our second approach, we will induce expression of Rec8-N (a version of meiotic kleisin Rec8 that bears mutations at separase cleavage sites) during oocyte growth. Incorporation of Rec8-N into functional cohesin complexes will result in asynchronous and delayed chromosome segregation in meiosis I and this will suggest the existence of a mechanism to generate cohesion during protracted meiotic prophase in oocytes. Alternatively, cohesin is suggested to be a stable complex that does not rely on turnover, leading to an assumption that genes encoding cohesin subunits are not required for the maintenance of cohesion during meiotic arrest and meiotic division. We will inactivate cohesin subunits during the arrest and test if cohesion is maintained. These functional studies will shed light on the chromosome cohesion in arrested oocytes that must be maintained for faithful chromosome segregation.