Cell cycle regulation in mammalian oocytes

Abstract

An unusual feature of mammalian female germ cells is that they are arrested in meiotic prophase, equivalent to mitotic G2-phase, for an extended period of time. In this thesis I have investigated two aspects of this arrest. First, I examined whether cohesin replenishment is required for the maintenance of chromosome cohesion during protracted meiotic prophase arrest. Nipbl, an evolutionarily conserved protein, is a component of protein complex called kollerin, whose activity in loading cohesin onto chromosomes is necessary for accurate chromosome segregation during mitosis. However, until now its function in mammalian meiosis was unknown. I have showed that Nipbl is present on meiotic chromosomes throughout meiotic prophase in mouse spermatocytes and oocytes and it accumulates at chromosomal axes where it co-localises with cohesin. I employed conditional knockout strategy to inactivate Nipbl gene in mouse oocytes arrested in meiotic prophase. Although functional Nipbl transcripts were efficiently depleted, these oocytes underwent meiotic maturation with unaffected chiasmata and cohesion. Surprisingly, Nipbl-deleted eggs were fertile and the loading of mitotic cohesin containing Rad21 was unaffected in fertilized eggs. Aditionally, these eggs could develop into blastocysts upon parthenogenetic activation, however harbouring a high proportion of cells with misaligned chromosomes. These results suggest that Nipbl is very stable in the oocyte.

In the second project we conceived that the maintenance of the cell cycle arrest in primordial oocytes is an important aspect of follicular survival. Previously proposed involvement of the anaphase promoting complex/cyclosome (APC/C), a cell cycle ubiquitin ligase complex in down-regulating the cyclin-dependent kinase activity in fully-grown oocyte led me to inactivate APC/C in dormant oocytes using conditional knockout system. I found that upon APC/C inactivation, primordial follicles were completely depleted before adulthood, within 5 weeks of birth, suggesting that the APC/C activity is required for the survival of primordial oocytes. These results propose the presence of previously unknown mechanism involving APC/C, essential for primordial follicle survival.