Introduction: The MAPK genes are critical for gonadal differentiation in eutherian mammals, but their role in marsupial mammals is unknown. Characterisation and phylogenetic analyses of the tammar wallaby MAPK genes show these genes are highly conserved with their orthologues in mammalian and non-mammalian species. Methods: We cultured sexually indifferent tammar gonads in the presence of p38α and -β MAPK inhibitor, SB202190. Results: SB202190 downregulated SOX9 and AMH levels in XY-treated gonads when compared to controls, similar to the effects of oestrogen on the MAPK pathway in males. In contrast, XX gonads treated with the SB202190 inhibitor showed no change in mRNA expression between the control and treated gonads for any of the markers tested. Conclusions: This study confirms that components of the MAPK pathway drive testis differentiation via the key downstream genes SOX9 and AMH in marsupials as is observed in eutherian mammals.

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