Frank Rattray Lillie's research on freemartins from 1914 to 1920 in the US led to the theory that hormones partly caused for sex differentiation in mammals. Although sometimes applied to sheep, goats, and pigs, the term freemartin most often refers to a sterile cow that has external female genitalia and internal male gonads and was born with a normal male twin. Lillie theorized that a freemartin is a genetic female whose process of sexual development from an undifferentiated zygote was suppressed or antagonized by her twin's release of male hormones via their shared blood circulation in utero. Despite publications of similar findings by physician Julius Tandler in Vienna, Austria, in 1910 and physician Karl Keller in Wiesensteig, Germany in 1916 prior to Lillie's research, Lillie often receives credit for the hormonal theory of sex differentiation in the freemartin. Lillie's study of freemartins, and the subsequent research by graduate students in Lillie's laboratory at the University of Chicago in Chicago, Illinois, prompted many embryologists to research sex differentiation and hermaphroditism in mammals.

Eugen Steinach researched sex hormones and their effects on mammals in the late nineteenth and early twentieth centuries in Europe. He experimented on rats by removing their testicles and implanting them elsewhere in their bodies, and he found that the testes interstitial cells produce male sex hormones. He developed the Steinach Rejuvenation Procedure, which he claimed could rejuvenate men by increasing their production of sex hormones. Steinach’s work on female sex hormones and on ovarian extracts led to the development of the first standardized injectable estrogen. Steinach’s research on reproductive hormones helped researchers explain the roles of sex hormones and develop hormone drugs.