The evolutionary process of mammalian sex determination genes focusing on marsupial SRYs

Background: Maleness in mammals is genetically determined by the Y chromosome. On the Y chromosome SRY is known as the mammalian male-determining gene. Both placental mammals (Eutheria) and marsupial mammals (Metatheria) have SRY genes. However, only eutherian SRY genes have been empirically examined by functional analyses, and the involvement of marsupial SRY in male gonad development remains speculative. Results: In order to demonstrate that the marsupial SRY gene is similar to the eutherian SRY gene in function, we first examined the sequence differences between marsupial and eutherian SRY genes. Then, using a parsimony method, we identify 7 marsupial-specific ancestral substitutions, 13 eutherian-specific ancestral substitutions, and 4 substitutions that occurred at the stem lineage of therian SRY genes. A literature search and molecular dynamics computational simulations support that the lineage-specific ancestral substitutions might be involved with the functional differentiation between marsupial and eutherian SRY genes. To address the function of the marsupial SRY gene in male determination, we performed luciferase assays on the testis enhancer of Sox9 core (TESCO) using the marsupial SRY. The functional assay shows that marsupial SRY gene can weakly up-regulate the luciferase expression via TESCO. Conclusions: Despite the sequence differences between the marsupial and eutherian SRY genes, our functional assay indicates that the marsupial SRY gene regulates SOX9 as a transcription factor in a similar way to the eutherian SRY gene. Our results suggest that SRY genes obtained the function of male determination in the common ancestor of Theria (placental mammals and marsupials). This suggests that the marsupial SRY gene has a function in male determination, but additional experiments are needed to be conclusive.