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 gonadal sex determination has only recently been discovered. Extracellular signal-regulated kinase (ERK) is a family of MAPK proteins involved in signal transduction. Two isoforms ERK1 and ERK2 are phosphorylated by the upstream MAP3K, MAP3K1. Recent studies show that mutations in MAP3K1 lead to increased phosphorylation of ERK1/2, enhanced binding of a small GTPase RhoA and is associated with sex reversal in humans. How the MAP3K1 mutations and increased phosphorylation of downstream ERKs leads to sex reversal is not clearly understood. Using our unique and well-studied marsupial model, the tammar wallaby, we sought to identify the role of the MAPK pathway in sexual differentiation.
We used a gonadal explant culture system to analyse the effect of an inhibitor of ERK1/2 (U0126) on undifferentiated tammar gonads. Fetal tammar gonads were cultured in the presence or absence of U0126 in 1.5% agar moulds in media containing DMEM/10% FBS/ penicillin-streptomycin at 37oC in Carbonox gas (95% O2, 5% CO2). Immunohistochemistry was used to determine the localisation of testis promoting SOX 9, AMH, SSEA-1 and β-catenin to examine Sertoli cell, germ cell and granulosa cell morphology. In treated testes, SOX9 remained cytoplasmic and was not translocated to the nucleus of the Sertoli cells and showed a sex-reversed phenotype. RT-PCR showed that MAP3K1 and ERK1/2 were expressed in the gonads from before gonadal differentiation to 8 days post-partum in both sexes. Our data suggest that the MAPK pathway is involved in maintaining the balance between male and female pathways in the tammar as it is in mice and humans.