Mitogen activated protein kinases (MAPK) are a family of ancient, highly conserved signalling molecules that control intracellular signal transduction. The role of MAPKs in cell adhesion, apoptosis, proliferation and survival is well studied, but their role in sex determination is as yet unclear.
Using our unique and well-studied marsupial model the tammar wallaby, we sought to identify the role of the MAPK pathway in gonadal sex determination. MAPKs may regulate granulosa and Sertoli cell fate and disturbing the MAPK cascade may cause an imbalance between testis- and ovary-promoting genes leading to sex reversal. Using our gonadal explant culture system we tested the effect of inhibitors of the MAPK pathway on undifferentiated tammar gonads to determine whether inhibiting MAPK affects gonadal morphology, Sertoli and granulosa cell fate and leads to a sex-reversed phenotype. Undifferentiated tammar gonads were cultured in the presence or absence of the ERK1/2 inhibitor (U0126) or an inhibitor of p38 (SB202190) in 1.5% agar moulds in media containing DMEM/10% FBS/ Penicillin-Streptomycin at 37oC in Carbonox mix (95% O2, 5% CO2). Immunohistochemistry was used to determine the localisation of key sexual differentiation markers SOX9, AMH and the germ cell marker SSEA-1 in order to identify any signs of sex reversal. Preliminary qPCR results show that inhibition of p38 leads to a significant reduction in SOX9 (p < 0.05) and AMH (p < 0.05) in treated male gonads when compared to controls. In addition, there are morphological effects after culture with inhibitors. In particular, SOX9 translocation to the nucleus is inhibited in male gonads, and they have a female-like surface epithelium, demonstrating that interference of the MAPK pathway has affected the normal sexual differentiation and leads to at least partial gonadal sex reversal. Collectively, these data suggest that the MAPK pathway is a key regulator of mammalian gonadal differentiation.