Aims: SOX9 is a key transcription factor that regulates many cellular processes such as cell proliferation and cell migration during testicular Sertoli cell differentiation and maintenance. We speculated that SOX9 divides its labour among many target genes. In this study the aim is to investigate the role of a candidate SOX9 target gene we identified called, Tyro3, in testis development.
Methods: Expression and localisation of SOX9 and TYRO3 in a human embryonic carcinoma Sertoli-like cell line, NT2/D1 cells was determined by RT-PCR and immunofluorescence. Cell adhesion and proliferation were performed on an xCELLigence. Mouse XY mouse gonads were dissected from embryos at E11.5 and cultured ex vivo for three days treated with either vehicle (DMSO) or with TYRO3 inhibitor, BMS777607, to determine the role of TYRO3 in mouse gonad development.
Results: We established that SOX9 is required for cell adhesion and proliferation. To determine whether either or both of these processes are mediated via TYRO3, we incubated NT2 cells in the presence of the TYRO3 inhibitor. Increasing concentrations of the inhibitor led to a dose-dependent reduction in cell proliferation but not cell adhesion. TYRO3 is expressed specifically in the Sertoli cells in mouse gonads. In cultured. Incubation with TYRO3 inhibitor, mouse embryonic gonads did not change testis morphology compared to vehicle control. However, the germ cell marker MVH is lost in TYRO3 inhibitor-treated gonad comparing to the DMSO-treated gonad. Cell apoptosis shows no difference between control and TYRO3 inhibitor gonads.
Conclusion: Our study suggests that a key role of SOX9 is through TYRO3 to mediate germ cell survival in the developing testis. Future rescue experiments are aimed at determining whether the role of TYRO3 is the exclusive mediator of SOX9 mediated Sertoli cell proliferation and germ cell survival.