Gonadal differentiation is a complex process requiring multiple genes and protein interactions. In mice, PTGDS forms a positive feedback loop with Sox9 to maintain its expression in a similar way to that of FGF9. HPGDS initiates SOX9 translocation into the nucleus during testicular development. In contrast, β-catenin, acting as an intracellular transducer in the WNT4 pathway, not only prohibits the expression of the male sexual differentiation genes and formation of the testicular vasculature, but maintains normal ovarian differentiation. Prostaglandin D synthase and β-catenin sequences are conserved between mice and the marsupial tammar wallaby, inferring that prostaglandin D synthase and β-catenin may have a similar role in tammar gonadal development.
The amino acid sequence of β-catenin is highly conserved in mammals, including in the tammar, but PTGDS and HPGDS are only conserved in the critical motifs of the functional domain regions between mice and tammar. Immunohistochemical staining of β-catenin in key gonadal developmental stages (day 25 fetus and days 2, 8 and 16 after birth) showed similar protein localization as in the mouse during gonadal differentiation. It was mainly stained in the membrane of Sertoli cells and germ cells in developing testis, and in both the membrane and cytosol of germ cells and somatic cells in developing ovary. To decipher the functional role of HPGDS and PTGDS in the developing tammar gonad, testes were cultured with the HPGDS specific inhibitor HQL-79 (150uM; Cayman chemical), while ovaries were treated with the PTGDS activator PGD2 (10uM; Cayman chemical). The effects of HPGDS/PTGDS were assessed using immunohistochemistry of the testis cell markers (SOX9 and AMH) and the ovary marker (WNT4 and β-CATENIN). It appears that the role of prostaglandin D synthase and β-catenin in sexual differentiation has been conserved in mammals for at least 150 million years.