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The Freemartin Cattle and Clinical Transplantation: From the Ancients to Modern Day

Lovasik, Brendan P. MD1,2

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doi: 10.1097/TP.0000000000003103
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Abstract

The year 2019 marks the anniversary of 2 important landmarks in transplantation: the 150th anniversary of the first successful skin allograft by Jacques-Louis Reverdin in 18691 and the 65th anniversary of the first successful human-to-human organ transplant by Joseph Murray in 1954. A lesser-known milestone in immunology deserves celebration: the 240th anniversary of the publication of John Hunter’s Account of the Free Martin article, a remarkable treatise on comparative anatomy in the 18th century and one of the first formal studies in the Western scientific canon to critically analyze the freemartin.2 Freemartin cattle, the case in which the female twin of a mixed-sex twin pair is sterile, have a unique role in our contemporary understanding of transplant immunology, and the study of this phenomenon would have far-reaching implications on future understanding of allograft acceptance and acquired tolerance (Table 1).

TABLE 1.
TABLE 1.:
Timeline of milestones in transplantation and tolerance pertaining to the freemartin condition

THE CLASSICAL ORIGINS OF FREEMARTIN CATTLE

The freemartin condition has been known since antiquity—the ancient Romans called the freemartin a “taura,” that is, a “female bull,” implying some recognition of the masculine characteristics of the freemartin; descriptions of the taura are seen in the 2nd century BC writings of Varro.3-5 The earliest noted use of “freemartin” in the English lexicon dates from 1681; a widely accepted meaning of the freemartin name comes from terms Farrow, referring to a sterile cow without milk in the Scottish and Northern English dialects, and Mart, the term applied to cattle killed for food close to the feast of Martinmas (November 11) so that the meat could be prepared for winter provisions; nonbreeding animals, including the sterile freemartin, were preferentially selected for slaughter.4-6 The first anatomical description of freemartin anatomy makes its in an unpublished correspondence between the Italian anatomists Antonio Maria Valsalva (1666–1723) and Giorgio Baglivi (1668–1707); in a letter dated Bologna, May 10, 1692, Valsalva describes his dissection of a freemartin.3

JOHN HUNTER’S FREEMARTINS: A SCIENTIFIC APPROACH

It was not for nearly a century later that Hunter published his Free Martin article. John Hunter (1728–1793) is renowned today as one of the foremost anatomists and surgeons in Western medicine. In his 1779 Free Martin article, published in the Philosophical Transactions of the Royal Society of London, Hunter2 demonstrates his anatomical prowess and leads a fascinating phenotypic and descriptive analysis of the freemartin condition. Hunter describes, “It is a known fact, and, I believe is understood to be universal, that when a cow brings forth 2 calves, and that 1 of them is a bull-calf, and the other a cow to appearance, the cow-calf is unfit for propagation; but the bull-calf becomes a very proper bull […] This cow-calf is called in this country a free-martin; and this singularity is just as well known among the farmers as either cow or bull.” Hunter then depicts his dissections of several freemartin cattle, including fascinating and highly detailed descriptions and drawings of Hunter’s observations of the reproductive anatomy, demonstrating the presence of male internal reproductive organs in an externally female phenotypic animal (Figure 1). While his article stops short of exploring the cause of the freemartin condition, Hunter’s focus on the comparative anatomy of freemartin cattle demonstrate the first formal studies that would foreshadow the importance of the freemartin condition to immune tolerance and human organ and tissue transplantation centuries later. Though the immunologic implications of the freemartin phenomenon would not be realized until centuries after his death, perhaps Hunter’s most influential contribution to modern surgical practice is the development of the surgeon-scientist model—his emphasis on combining applied surgical anatomy and physiology with scientific observation, inquiry and experimentation has become the model for future generations of surgeon-scientists. While Hunter’s individual contribution to the field of transplant immunology is modest, Joseph Murray’s Nobel Laureate lecture specifically credits Hunter and his freemartin studies as a foundational influence in the realization of human transplantation.7

Hunter’s work attracted the interest of another notable contemporary anatomist: Antonio Scarpa (1752–1832), then professor of anatomy at the University of Modena, visited Hunter in London in the autumn of 1781, where he was intrigued by Hunter’s freemartin observations. He would begin his own freemartin dissections and published his results at the Societa Italiana in 1784.3 Alexander Numan (1780–1852), a Dutch comparative anatomist and veterinarian, published a similar account in the Journal vétérinaire et agricole de Belgique (1844), citing Hunter and Scarpa’s articles.6

LILLIE, OWEN, AND THE PHYSIOLOGICAL BASIS OF THE FREEMARTIN

The fascinating natural cause of the freemartin anomaly was not determined until the 20th century, when zoologist Frank Lillie (1870–1947), Chairman of Zoology at the University of Chicago, observed that bovine dizygotic twins develop a fusion of their placenta during embryonic life (1917) (Figure 2).8-10 He concluded that the sterile freemartin is genetically a female, modified by the sex hormones of the male twin, which circulate in both individuals during fetal life owing to anastomosis of the fetal circulation of the 2 individuals. Lillie’s observations on the freemartin led to concept that gene control causes male gonads to differentiate in the presence of testes producing male hormone, whereas female gonads develop in the absence of hormone, and introduced biologists to nature, origin, and action of sex hormones at a time when almost nothing was known about them; it would not be until 1947 when Alfred Jost would prove the gonadal hormones and their influence on sexual differentiation.11

While Lillie is commonly credited with the establishment of the vascular anastomosis in the freemartin condition, he was not the first to report the existence of a vascular anastomosis in freemartin cattle.12 Following the publication of Lillie’s 1917 paper on the freemartin, Lille received letters from colleagues in Europe directing him to 2 articles published in 1911 and 1916 by Karl Keller and Julius Tandler, 2 anatomists in Vienna.13,14 In these papers, Keller and Tandler established that in twinning cases where the freemartin condition existed, there was a vascular anastomosis between the chorions of the placentas, as demonstrated by injecting dye into the blood vessels of the calf fetuses. After being notified of the existence of these Viennese papers, Lillie attempted to get a copy by writing to the Library of Congress and the US Department of Agriculture. Because of the disruption in the flow of scientific literature between Europe and North America caused by World War I, these journals were not available in the United States. After Lillie read the Tandler and Keller articles, he wrote a Letter to the Editor in Science on August 22, 1919 in which he professes his independent and parallel discovery of the cause of the freemartin phenotype and apologizes for having overlooked the work of Keller and Tandler.15

Building on the knowledge gained by Hunter and Lillie, the discovery of transplant tolerance is credited to Ray Owen, a zoologist and geneticist at the University of Wisconsin who studied the inheritance of red blood cell genotyping in twin cattle. Owen’s discoveries serendipitously occurred in his investigation for a Maryland farmer regarding bovine paternity for a fence-hopping bull.16 Following the completion of his PhD, Owen became involved with genotype testing for in number of American purebred cattle associations, performing bull paternity testing using blood group characteristics to ensure valued lines of inheritance. In cows, dizygotic twins are sometimes the product of superfecundation via insemination by 2 different bulls. In the Maryland case, the first calf was a planned liaison with a Guernsey bull, a purebred Guernsey like the mother. Later the same day, a fence-crashing Hereford bull bred the cow again. The twin offspring, a male and female, were clearly phenotypic half-siblings: 1 was a purebred Guernsey and the other a Hereford-Guernsey hybrid with the dominant white face markings of the Hereford. In Owen’s blood testing, the twins had identical blood groups, and both twins contained blood groups from both fathers.17

Owen, intrigued by his findings, then followed the Maryland case by designing a novel assay for differential hemolysis and testing hundreds of other dizygotic twin combinations and found that this phenomenon occurred in over 90% of cases. In his landmark Science article, Owen reported his findings that dizygotic twin cows possess 2 distinct blood groups: that of their own and that of their twin.17 Owen recognized that the common intrauterine circulation described by Lillie allows for the exchange of hematopoietic cells during embryonic life and the establishment of a chimeric state.18 Interestingly, these calves did not develop isoantibodies to their twin, suggesting a state of immunological tolerance. Owen’s findings, which he dubbed “erythrocyte mosaicism,” were published in his famous paper “Immunogenetic Consequences of Vascular Anastomoses between Bovine Twins” (1945).17

Owen’s remarkable description caused little stir in the scientific community at the time, but was cited in 1949 by Australians MacFarlane Burnet and Frank Fenner,19 who postulated that during embryonic development, “a process of selfrecognition takes place,” and that during that process, “tolerance is acquired by fetal exposure to ‘nonself’ constituents.”18 These observations eventually formed the famous “self-nonself” hypothesis for immune development and led to the creation of the clonal selection theory.18,19

MEDAWAR AND THE FREEMARTIN: THE BASIS OF IMMUNE TRANSPLANT TOLERANCE

Meanwhile, in Britain, biologists Peter Medawar (Figure 3) and Rupert Billingham approached the freemartin phenomenon using their transplant immunology background. As the Chair of Zoology at the University of Birmingham, Medawar was challenged by a colleague to differentiate monozygotic (identical) from dizygotic (fraternal) twin cows.20 Medawar, having previously studied skin graft rejection in the burn unit at the Glasgow Royal Infirmary for the British War Wounds Committee during World War II, postulated that he could differentiate the zygotic twin types by use of skin grafting. Medawar found that, to his surprise, all cow twins incorporated the foreign tissue without immune rejection despite obvious dizygotic phenotypes.

It was only when Medawar became aware of Owen’s work that he could explain his confounding observations, which Medawar published as his paper “Tolerance to Homografts, Twin Diagnosis, and the Freemartin Condition in Cattle” (1953).21 Subsequent experiments by Billingham, Leslie Brent, and Medawar demonstrated that neonatally acquired transplant tolerance could be achieved in mice by inoculation of embryos of an inbred mouse with a living allogeneic cell suspension from a different adult inbred mouse line.22 Burnet and Medawar shared the Nobel Prize in 1960 for “discovery of acquired immunological tolerance.”

MAKING ORGAN TRANSPLANTATION A CLINICAL REALITY

Though true immune transplant tolerance has yet to be consistently achieved in clinical organ transplantation, these pivotal studies of the freemartin condition and its related effects on immune tolerance laid the groundwork for the transition of transplantation from the experimental to the clinical realm. Despite Medawar’s claim that the “biological force” responsible for rejection would “forever inhibit transplantation from 1 individual to another,” 2 clinician scientists, Joseph E. Murray and John Merrill, persevered in their pursuit of making clinical transplantation a reality through the transplantation of a renal allograft between monozygotic twins in 1954.23 While this syngeneic transplant did not directly address the problems of allograft rejection, it was a clinical foundation for the field of organ transplantation. In 1959, the same Boston team led by Murray and Merrill completed one of the most important cases in the history of transplantation when they transplanted a kidney between dizygotic twins to the first successful breaching of such a genetic barrier.24,25 The following year, a team led by René Küss in Paris performed the first successful renal allotransplant between an unrelated donor/recipient pair.26,27 Thomas et al28 would publish the first report of irradiation and hematopoietic stem cell transplantation as a treatment for leukemia in 1957; while bone marrow transplant would not become clinically widespread for another decade, it represented real progress toward induced human chimerism. Deservedly, Murray and Thomas shared the Nobel Prize in 1990 for their “discoveries concerning organ and cell transplantation in the treatment of human disease.”

AVENUES IN TRANSPLANT TOLERANCE: A CONTEMPORARY PERSPECTIVE

The contemporary pursuit of clinically applicable, donor-specific tolerance reflects 2 critical issues in organ transplantation: the deleterious effects of lifelong conventional immunosuppression and the high rates of late allograft failure due to chronic rejection.29 Recent progress in has allowed via chimerism induction. Chimerism, a concept which itself has classical roots with the Chimera as a monstrous, fire-breathing hybrid creature first mentioned in Homer’s Iliad, describes a state in which donor hematopoietic cells are present in the recipient, thereby inducing central tolerance via clonal deletion of alloreactive T-cells in the thymus.30 The achievement of clinical tolerance regimens in through renal transplant recipients at Massachusetts General Hospital, Northwestern, and Stanford was recently and elegantly reviewed by Messner et al30 in this journal. Another exciting and promising application of in vivo tolerance is the generation of whole organs via “blastocyst complementation,” in which donor pluripotent stem cells are delivered to a recipient host at the preimplantation stage, thus using their chimera-forming ability to form a donor-derived organ in vivo.31-33 This organ, which would be entirely donor derived, matures in the chimeric host and is then available for syngeneic transplant into the stem cell donor. These innovative approaches targeting chimerism in both the organ donor and recipient represent the next generation of immunologic tolerance and progress toward clinical application.

FREEMARTIN CATTLE AND IMMUNE TOLERANCE: THE LEGACY CONTINUES

The legacy of freemartin cattle in the understanding of acquired tolerance and transplant immunology represents generations of scientific inquiry guided by careful observation and occasional serendipity—chance favoring the prepared mind—from classical antiquity, to late-modern anatomists Hunter and Scarpa, to midcentury geneticists Lillie and Owen, to the origins of contemporary immunology with Medawar. The evolution and clinical success of organ transplantation in the last half-century is one of medicine’s most fascinating stories, and the present-day immunologists and surgeons exploring transplantation and immune tolerance owe much to the intriguing history of the freemartin, several millennia in the making.

FIGURE 1.
FIGURE 1.:
Illustration of Mr Wright’s Freemartin and dissected gonadal structures of Hunter’s freemartins, demonstrating an intersex phenotype. Reproduced with permission from Ref. 34
FIGURE 2.
FIGURE 2.:
Monochorionic fetal circulation of twin calves as identified by Frank Lillie, 1917. Described are the twins’ shared (1) transverse arterial trunk and (2) venous placental anastomoses. Reproduced with permission from Ref. 9
FIGURE 3.
FIGURE 3.:
Peter Medawar. Reproduced with permission from Ref. 35

REFERENCES

1. Ehrenfried A. Reverdin and other methods of skin-grafting—historical. Boston Med Surg J. 1909; 161:911–917
2. Hunter J. An account of the free martin. Philos Trans R Soc Lond B Biol Sci. 1779279–293
3. Belloni L. Valsalva, J. Hunter, and scarpa on the freemartin. J Hist Med Allied Sci. 1952; 7:136–140
4. Forbes TR. The origin of freemartin. Bull Hist Med. 1946; 20:461–466
5. Forbes TR. An early record of a freemartin. J Hist Med Allied Sci. 1979; 34:355–356
6. Hart DB. Numan, the veterinarian and comparative anatomist of Utrecht: a forgotten observer on the free-martin. Edinb Med J. 1912; 8:197–228
7. Murray JE. Human organ transplantation: background and consequences. Science. 1992; 256:1411–1416
8. Lillie FR. The theory of the free-martin. Science. 1916; 43:611–613
9. Lillie FR. The free-martin; a study of the action of sex hormones in the foetal life of cattle. J Exp Zool. 1917; 23:371–452
10. Lillie FR, Bascom KF. An early stage of the free-martin and the parallel history of the interstitial cells. Science. 1922; 55:624–625
11. Jost A. The age factor in the castration of male rabbit fetuses. Proc Soc Exp Biol Med. 1947; 66:302
12. Freeman G. Explaining the freemartin: Tandler and Keller vs. Lillie and the question of priority. J Exp Zool B Mol Dev Evol. 2007; 308:105–112
13. Tandler J, Keller K. Ueber das Verhalten des Chorions bei Veschiedengeslechhticher Zwillingsgravidität des Rindes und ueber die Morphologie Des Genitales des weiblichen Tiere welche einer solchen Graviditätenstammen. Deut Tierärzt Wochenschr. 1911; 19:148–149
14. Keller K, Tandler J. Ueber des Verhalten der Eihäubei der Zwillungsträchtigkeit des Rindes. Untersuchungen ueber die Entstehungsurache der geschlechtlichen Unter-entwicklung von weiblichen Zwillingskalbern welche einen männlichen Kalbe zur Entwicklung gelangen. Wiener Tierärztl Wochenschr. 1916; 3:513–526
15. Lillie FR. Tandler and Keller on the freemartin. Science. 1919; 50:183–184
16. Martin A. Ray Owen and the history of naturally acquired chimerism. Chimerism. 2015; 6:2–7
17. Owen RD. Immunogenetic consequences of vascular anastomoses between bovine twins. Science. 1945; 102:400–401
18. Adams AB, Pearson TC, Larsen CP. Heterologous immunity: an overlooked barrier to tolerance. Immunol Rev. 2003; 196:147–160
19. Burnet FM, Fenner F. The Production of Antibodies. 19492nd edMelbourne, Australia: Macmillan
20. Barker CF, Markmann JF. Historical overview of transplantation. Cold Spring Harb Perspect Med. 2013; 3:a014977
21. Billingham RE, Lampkin GH, Medawar BP, et al. Tolerance to homografts, twin diagnosis, and the freemartin condition in cattle. Heredity. 1952; 6:201–212
22. Billingham RE, Brent L, Medawar PB. Actively acquired tolerance of foreign cells. Nature. 1953; 172:603–606
23. Merrill JP, Murray JE, Harrison JH, et al. Successful homotransplantation of the human kidney between identical twins. J Am Med Assoc. 1956; 160:277–282
24. Merrill JP, Murray JE, Harrison JH, et al. Successful homotransplantation of the kidney between nonidentical twins. N Engl J Med. 1960; 262:1251–1260
25. Starzl TE. The early days of transplantation. JAMA. 1994; 272:1705
26. KUSS R, LEGRAIN M, MATHE G, et al. Homologous human kidney transplantation. Experience with six patients. Postgrad Med J. 1962; 38:528–531
27. Starzl TE, Barker C. The origin of clinical organ transplantation revisited. JAMA. 2009; 301:2041–2043
28. Thomas ED, Lochte HL Jr, Lu WC, et al. Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. N Engl J Med. 1957; 257:491–496
29. Leventhal J, Miller J, Abecassis M, et al. Evolving approaches of hematopoietic stem cell-based therapies to induce tolerance to organ transplants: the long road to tolerance. Clin Pharmacol Ther. 2013; 93:36–45
30. Messner F, Etra JW, Dodd-O JM, et al. Chimerism, transplant tolerance, and beyond. Transplantation. 2019; 103:1556–1567
31. Wu J, Platero-Luengo A, Sakurai M, et al. Interspecies chimerism with mammalian pluripotent stem cells. Cell. 2017; 168:473–486.e15
32. Yamaguchi T, Sato H, Kato-Itoh M, et al. Interspecies organogenesis generates autologous functional islets. Nature. 2017; 542:191–196
33. Freedman BS. Hopes and difficulties for blastocyst complementation. Nephron. 2018; 138:42–47
34. Palmer JF. The Works of John Hunter, with Notes. 1837. London: Cambridge University Press
    35. Simpson E. Medawar’s legacy to cellular immunology and clinical transplantation: a commentary on Billingham, Brent and Medawar (1956) ‘Quantitative studies on tissue transplantation immunity. III. Actively acquired tolerance’. Philos Trans R Soc Lond B Biol Sci. 2015; 370:20140382
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