Analysis of fetal germ cell development in the mouse: implications for testis cancer

© 2010 Dr. Denise Catherine Miles.Germ cells are the only cells that are capable of transferring an individuals genetic information onto the next generation. In the mouse the germ cell lineage is specified from embryonic day (E)6. The proliferating primordial germ cells enter the bipotential gonad at E10.5. Their commitment to spermatogenesis or oogenesis relies upon the micro-environment provided by the somatic cells of the developing testis or ovary. Commitment of the primordial germ cells to spermatogenesis in males is closely associated with their entry into mitotic arrest, whilst commitment of germ cells to oogenesis in females is followed by their immediate entry into meiosis. At the outset of this study the exact timing of these processes with reference to the identity of the cell cycle regulators involved remained to be defined. Work presented in this thesis used flow cytometry to precisely define the progression of male germ cell into mitotic arrest. The data presented establishes that male germ cells enter mitotic arrest between E13.0 and E14.5. Furthermore, mitotic arrest of male germ cells was accompanied by upregulation of the G1-S phase cell cycle inhibitors p27KIP1, p15INK4B and p16INK4A and the activation of Retinoblastoma strongly indicating that male germ cells enter mitotic arrest through this pathway. Analysis of female germ cells found that entry into meiosis occurred between E13.0 and E15.5, involves the activation of the ATM/CHK2 G2/M arrest pathway, suppression of Cyclin B1/2 and concomitant upregulation of Cyclin B3. As the process of male germ cell mitotic arrest occurs in response to a male specific factor, produced by the gonadal somatic cells, this thesis analysed the potential role of GDNF-RET signalling during the time of male germ cell mitotic arrest. Analysis of Ret -/- mice revealed apoptosis of a subset of the male germ cell population at E15.5. Furthermore, these apoptotic germ cells abnormally express the proliferation marker KI-67 and the cell cycle inhibitor p27KIP1. As analysis Gdnf -/- mice revealed no germ cell apoptosis, it was concluded that RET, but not GDNF signalling, is required for fetal germ cell survival. Further examination of male specific signalling pathways in the developing testis included the functional analysis of Activin/Nodal/TGF Beta signalling. Inhibition of Activin/Nodal/TGF Beta signalling or TGF Beta signalling in ex vivo cultured male gonads resulted in ovotestis formation. Specifically, male gonads lost cord structure and somatic cells aberrantly expressed the female marker FOXL2. Analysis of the germ cell population revealed a portion of germ cells had entered meiosis. These results indicate a novel function for the Activin/Nodal/TGF Beta pathway in male gonads during sex determination. The work presented in this thesis reveals for the first time the cell cycle regulators involved in male and female germ cell mitotic arrest and meiosis respectively. This thesis also provides evidence that RET signalling is required for male germ cell development and that Activin/Nodal/TGF Beta signalling is required for male sex determination. Further studies into germ cell development and the processes outlined in this thesis will be important for identifying underlying causes of infertility and germ cell cancers