146-65 million years ago


Therian mammals evolved live birth, a subdivided cloaca, descended testes, and a Y chromosome which determines maleness.

Therians evolved live birth although in marsupials, the young are as small when born alive as those that hatch from the eggs of monotreme mammals.
Female therians developed modifications of the oviduct and the ancestral estrus cycle was similar to that of eutherians with its cyclical growth and loss of the uterine endometrium. In male therians, the testes descended into the pelvis and the penis was no longer located in the cloaca. The cloaca was reduced.

A duplication of a SOX gene, probably the ancestor of SOX3, gave rise to a gene named SRY which is not only expressed in the testis, but is the testis determining factor located on the Y chromosome of therian mammals (placentals and marsupials). To date, the SRY gene has not been identified in monotremes. It is present in marsupials, although it has not been demonstrated to function in sex determination. Genes other than SRY can determine gender, given that some placental mammals lack SRY and that the majority human XY females do not have mutations in the SRY gene.
There are genes present on both the Y chromosomes of marsupials and placental mammals, indicating that the ancestor of therian mammals possessed a Y chromosome. The Y chromosome of placental mammals has undergone some reorganization since the split from the marsupial lineages given that there are genes on the marsupial (and monotreme) Y chromosome which have been lost in placentals (and are found only on the X) and that some genes which are autosomal in marsupials are located on the X and Y chromosomes in placental mammals (ZFY/X, AMELY/X, STS/STSP, and pseudoautosomal genes).
Unlike therian mammals, the monotreme genome does not seem to undergo imprinting and there is no evidence of X inactivation. In marsupials, some imprinting is known and the X inactivation does occur, although its mechanism is not as complex as that observed in placental mammals.
Orphan nuclear factor SF1 has a similar pattern of expression in marsupials and placental mammals. It is involved in the development of the adrenal glands, anterior pituitary, and hypothalamus. It also regulates cytochrome 450 genes which are involved in steroid metabolism in ovaries and testes.