Frank Rattray Lillie's Study of Freemartins (1914-1920)

By Mary Drago
Published: 2014-03-14
Keywords: sex hormones
Frank Rattray Lillie's Study of Freemartins (1914-1920)

Frank Rattray Lillie's research on freemartins from 1914 to 1920 in the US led to the theory that hormones partly caused 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.

Aristotle, who in the fourth century BCE lived in Athens, Greece, described hermaphroditism in animals; but scholar Marcus Terentius Varro, who lived in Rieti, Italy, for most of his life and died in 27 BCE, first wrote about a sterile cow born with a male twin. In 1779 John Hunter, surgeon to King George III in London, England, detailed the anatomy of three freemartins and he observed that each freemartin was born only with a male twin. Before DNA analysis of chromosomes in the late 1950s, scientists determined the organism's sex by the internal sex organs, which, in the freemartin, are male structures. In 1909 David Berry Hart in Edinburgh, Scotland, and in 1910 Leon Jacob Cole at the University of Wisconsin in Madison, Wisconsin, independently concluded that the freemartin resulted from a single egg division to form twins, and that the freemartin was actually a male whose sex glands were underdeveloped. Between 1910 and 1916, prior to Lillie's study of the freemartin, Tandler and Keller published articles about the freemartin and determined in their analyses, like Lillie, that the freemartin was a female cow. Furthermore, Tandler and Keller identified the shared blood circulation in utero of the twins as a cause of the sex differentiation changes to the freemartin.

Lillie conducted his research on the freemartin at the University of Chicago in Chicago, Illinois, where he chaired the Zoology Department from 1910 to 1931. He first expressed interest in determining the cause of sex differentiation in "The Biological Significance of Sexual Differentiation: A Zoological Point of View" published in 1907. Lillie's research on the freemartin began in 1914 when he received a pair of calf fetuses in their fetal membranes from the manager of Lillie's farm, where he had a herd of pure-bred Holstein-Friesian cattle. Because of the proximity of the Union Stockyards to the University of Chicago and the cooperation of Swift and Company's foreman, Lillie received ninety-seven uteri and their respective ovaries when available, containing twins from 1914 to 1920.

The first issue Lillie addressed in his examination of twin fetuses was whether they arose from a single fertilized egg (monozygotic twins) from two fertilized eggs (diplozygotic twins). If the twins were monozygotic, they would be genetically identical, and the freemartin would therefore be male as it was always born with a male twin. Lillie initially hypothesized that he would find the fetuses in separate fetal membranes (chorions) if they were diplozygotic. After examining twenty-two sets of twins, Lillie determined that twins in cattle share a single chorion.

Lillie noted that the fetus begins to grow in the horns of the uterus. Wrapped in the uterine membrane, the fetus first grows in a downward direction, where it descends through a constricted aperture from the horn to the body of the uterus, and then also grows laterally into the other uterine horn. The twin fetus also grows in that manner; such growth creates the opportunity for the chorions to fuse as they lay next to each other. Lillie then turned his observation to the ovaries, looking for the ductless gland which appears in the ovaries after ovulation (corpus luteum). In thirty-six uteri with both ovaries attached, thirty-five cases had either a corpus luteum in each ovary, or two in one ovary. Lillie concluded that twins were diplozygotic and thus that the freemartin must be female as according to Lillie, mere association of two males in utero was an unlikely cause for the transformation of one male into a freemartin.

To bolster his argument that a freemartin was female, Lillie performed a statistical analysis of his data. According to his analysis, it was statistically more likely that the freemartin was a genetic female than a male. He also observed that a freemartin and her twin did not resemble each other and therefore were not identical twins.

Lillie concluded that the twins were diplozygotic after observing twenty-seven cases, and then he examined the anatomy of the chorion to explain the indeterminate sex characteristics of the freemartin. Through injections of the entire arterial and venous sytems, and careful dissections of the chorions, Lillie determined that the fetal freemartin and its twin shared blood circulation, or anastomosis, in the chorion. Normal females born with a male twin did not exhibit any anastomosis in the chorion.

As the freemartin and her male twin share blood circulation, Lillie postulated that hormones released by the male twin affect the sex differentiation of the female. As the male twin is always fertile and shows no alteration of typical sex characteristics, while the female exhibits varying degrees of male sex characteristics, Lillie theorized that the timing of the anastomosis and flow of the male sex hormone to the female fetus accounted for the variation seen in the freemartin. He examined the gross anatomy of the male and female twins and, with histological examination of the cells by graduate student Catherine Lines Chapin, Lillie established that the testis develops earlier than the ovary. To Lillie, the earlier formation of the testis explained how the transfer of male hormone early in development of the female affects her sexual differentiation while the later release of female hormone does not alter the male.

Lillie published the results of his study of the freemartin in a number of articles, four of which are described here. The first article, "The Theory of the Free-Martin" was published in 1916. It was a preliminary report of the study on forty-one cases of bovine twins. Lillie wrote the article in response to Leon J. Cole's 1916 abstract "Twinning in Cattle, with Special Reference to the Free Martin." Lillie challenged Cole's hypothesis that freemartins are genetically male using the statistical analysis of Cole's cases as well as of his own. Lillie also described his theory of sex hormones' responsibility for the indeterminate sex of the female.

Lillie's second article, "The Free-martin: A Study of the Action of Sex Hormones in the Foetal Life of Cattle," was published in 1917. The article described the study on fifty-seven pairs of twins and Lillie's theory of hormonal action. The article also included illustrations of a few of these cases.

After the 1917 article, Lillie learned about Tandler and Keller's works. He wrote "Tandler and Keller on the Free-Martin," in 1919 to acknowledge their work and to identify its similarities to his own. He said that he hadn't heard of their work because their initial publication was in a journal unknown to US biologists and that World War I had hindered references to their work by other scientists.

Lillie's 1923 article discussed his study on freemartins, "Supplementary Notes on Twins in Cattle." Lillie described the sets of twins numbered fifty-eight to ninety-seven. Lillie asserted that the additional information derived from those cases supported his conclusions from 1917. Lillie also addressed challenges to his theory from other scientists. Lillie included some of the information previously published by Tandler and Keller in another statistical analysis to challenge David Berry Hart in Edinburgh, Scotland, who had said that the freemartin was a male. Lillie countered arguments from Carl Richard Moore, also at the University of Chicago, and from Knud Sand at the University of Copenhagen in Copenhagen, Denmark. Both had concluded that sex gland antagonism did not account for hermaphroditism. Lillie countered this conclusion by noting that timing of the release of the male hormone inhibits the growth of the ovary in the freemartin. Carl Gottfried Hartman at University of Texas in Austin, Texas, had hypothesized that some hermaphrodites in mammals resulted from female sex hormone altering the sex of the male. Lillie stated that Hartman's argument was flawed, as the female hormone is not produced early in the fetal life when male sex differentiation could be altered. Julian Sorell Huxley at King's College London in London, England, had contended in the 1920s through the 1930s, based upon the freemartin, that sex hormones could completely invert the sex of one type into another. In addressing Huxley's argument, Lillie referred to his own study and emphasized that the changes to the freemartin's sexual organs were variable, and never a complete conversion.

DNA analysis later confirmed Lillie's position that the freemartin is genetically female. By the 1960s, however, scientists no longer held that male sex hormones partly caused the freemartin. In 1984 the Anti-Müllerian Hormone (AMH) was purified and ultimately shown to inhibit ovaries from developing.


  1. Aristotle. De generatione animalium [On the Generation of Animals]. Trans. Arthur Platt. Oxford: Clarendon Press, 1910. (Accessed March 11, 2014).
  2. Capel, Blanche, and Doug Coveney. "Frank Lillie's Freemartin: Illuminating the Pathway to 21st Century Reproductive Endocrinology." Journal of Experimental Zoology Part A: Comparative Experimental Biology 301A (2004): 853–6.
  3. Chapin, Catherine L. "A Microscopic Study of the Reproductive System of Foetal Free-Martins." Journal of Experimental Zoology 23 (1917): 453–82. (Accessed March 11, 2014).
  4. Cole, Leon J. "Twinning in Cattle, with Special Reference to the Free Martin." Science 43 (1916): 177.
  5. Dreger, Alice Domurat. Hermaphrodites and the Medical Invention of Sex. Cambridge, MA: Harvard University Press, 1998.
  6. Freeman, Gary. "Explaining the Freemartin: Tandler and Keller vs. Lillie and the Question of Priority." Journal of Experimental Zoology Part B: Molecular and Developmental Evolution 308B (2007): 105–12.
  7. Grave, Caswell. "The American Society of Zoologists. II." Science 43 (1916): 176–84.
  8. Hart, David Berry. "The Structure of the Reproductive Organs in the Free-Martin, with a Theory of Significance of the Abnormality." Proceedings of the Royal Society of Edinburgh 30 (1909-1910): 230–41.
  9. Hartman, Carl Gottfried. "Studies in the Reproduction of the monkey Macacus (Pithecus) rhesus, with Special Reference to Menstruation and Pregnancy." Carnegie Contributions to Embryology 134. Washington, D.C.: Carnegie Institute of Washington, 1932.
  10. Hartman, Carl Gottfried. Time of Ovulation in Women: Study on the Fertile Period in the Menstrual Cycle. Baltimore, MD: Williams and Wilkins, 1936.
  11. Hunter, John. "Account of the Free Martin." Philosophical Transactions of the Royal Society of London 69 (1779): 279–93. (Accessed March 10, 2014).
  12. Huxley, Julian Sorell. "Constant Differential Growth-Ratios and their Significance." Nature 114 (1924): 895–6.
  13. Huxley, Julian Sorell. "Chemical Regulation and the Hormone Concept." Biological Reviews 10 (1935): 427–41.
  14. Keller, Karl. "Die Körperform des unfruchtbaren Zwillings beim Rindes. Ein Beitrag zur Kenntnis des asexuellen Form und des Kastraten Typus." [The body shape of the infertile twin cattle. A contribution to the knowledge of the asexual form and of the castrated type.] Jahrbuch für wissenschaftliche und praktische Tierzucht [Yearbook of scientific and practical animal breeding] 11 (1916): 103–164.
  15. Lillie, Frank R. "The Biological Significance of Sexual Differentiation: A Zoological Point of View." Science 25 (1907): 372–6.
  16. Lillie, Frank R. "The Theory of the Free-Martin." Science 43 (1916): 611–3.
  17. Lillie, Frank R. "The Free-Martin: A Study of the Action of Sex Hormones in the Foetal Life of Cattle." The Journal of Experimental Zoology 23 (1917): 371–452.
  18. Lillie, Frank R. "Sex-Determination and Sex-Differentiation in Mammals." Proceedings of the National Academy of Sciences of the United States of America 3 (1917): 464–70. (Accessed March 10, 2014).
  19. Lillie, Frank R. "Tandler and Keller on the Free-Martin." Science 50 (1919): 183–4.
  20. Lillie, Frank R., and K. F. Bascom. "An Early Stage of the Free-Martin and the Parallel History of the Interstitial Cells." Science 55 (1922): 624–25.
  21. Lillie, Frank R. "Supplementary Notes on Twins in Cattle." Biological Bulletin 44 (1923): 47–8. (Accessed March 10, 2014).
  22. Moore, Carl Richard. "On the Superposition of Fertilization on Parthenogenesis." Biological Bulletin 31 (1916): 137–80. (Accessed March 10, 2014).
  23. Moore, Carl Richard. "On the Capacity for Fertilization after the Initiation of Development." Biological Bulletin 33 (1917): 258–95. (Accessed March 10, 2014).
  24. Price, Dorothy. "Mammalian Conception, Sex Differentiation, and Hermaphroditism as Viewed in Historical Perspective." American Zoologist 12 (1972): 179–91.
  25. Sand, Knud. "Experimenteller Hermaphroditismus." [Experimental hermaphroditism.] Pflüger's Archiv für die gesamte Physiologie des Menschen und der Tiere [Pflüger's Archive for the Physiology of Humans and Animals] 173 (1919): 1–7. (Accessed March 12, 2014).
  26. Sand, Knud. "Experiments on the Internal Secretion of the Sexual Glands, Especially on Experimental Hermaphroditism." The Journal of Physiology 53 (1919): 257–63. (Accessed March 12, 2014).
  27. Sand, Knud. "Experiments on the Endocrinology of the Sexual Glands." Endocrinology 7 (1923): 273–301. (Accessed March 12, 2014).
  28. Swett, Walter Whittier, Charles Arthur Matthews, and Roy Ralph Graves. "Early Recognition of the Freemartin Condition in Heifers Twinborn with Bulls." Journal of Agricultural Research 61 (1940): 587–623.
  29. Tandler Julius, and Karl Keller. "Über den Einfluss der Kastration auf den Organismus. IV. Die Körperform der weibichen Frühkastraten des Rindes." [On the Influence of castration on the organism. IV. The body shape of the female freemartin of cattle.] Archiv für Entwicklungsmechanik der Organismen [Archive for the Developmental Mechanics of Organisms] 31 (1910): 209–306.
  30. Tandler Julius, and Karl Keller. "Über das Verhalten des Chorions bei Verschiedengeslechhticher Zwillingsgravidität des Rindes und über die Morphologie Des Genitales des weiblichen Tiere welche einer solchen Graviditätenstammen." [About the behavior of the chorion at various connections in twin pregnancy in cattle and on the morphology of the female genitalia of animals from such a pregnancy.] Deutsche tierärztliche Wochenschrift [German veterinary weekly] 19 (1911): 148–149.
  31. Varro, Marcus Terentius. Rerum Rusticarum Libri Tres [On Agriculture in Three Books]. Trans. Lloyd Storr-Best. London: G. Bell and Sons, 1912. (Accessed March 11, 2014).
  32. Watterson, Ray Leighton. "The Striking Influence of the Leadership, Research, and Teaching of Frank R. Lillie (1870-1947) in Zoology, Embryology and Other Biological Sciences." American Zoologist 19 (1979): 1275–87.
  33. Willier, Benjamin H., Ross G. Harrison, Henry Bryant Bigelow, and Edward Grant Conklin. "Addresses at the Lillie Memorial Meeting Woods Hole, August 11, 1948." Biological Bulletin 95 (1948): 151–62. (Accessed March 12, 2014).

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Drago, Mary, "Frank Rattray Lillie's Study of Freemartins (1914-1920)". Embryo Project Encyclopedia (2014-03-14). ISSN: 1940-5030

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Arizona State University. School of Life Sciences. Center for Biology and Society. Embryo Project Encyclopedia.


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Freemartinism; Hormones; Lillie, Frank Rattray, 1870-1947; Intersexuality in animals; Experiment