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My life with progesterone

      The twists and turns of fate have linked my name so many times with the other ovarian hormone that I still shudder when some senior citizen says, “You must have been awfully young when you worked with Doisy.” The confusion arises because there are 2 Allens whose names have become attached to the 2 ovarian hormones. Edgar Allen (1892-1943) and Edward Adelbert Doisy (1893-1986) announced their discovery of a hormone in the ovarian follicular fluid in 1923.
      • Allen E.
      • Doisy E.A.
      An ovarian hormone: a preliminary report on its localization, extraction, and partial purification, and action in test animals.
      George Washington Corner (1889-1981) and I announced the discovery of a second ovarian hormone in the corpus luteum in 1929.
      • Corner G.W.
      • Allen W.M.
      Physiology of the corpus luteum, II: production of a special uterine reaction (progestational proliferation) by extracts of the corpus luteum.
      The 2 Allens are of separate lineage; the 2 female sex hormones are closely related.
      My part in the progesterone story began in September 1926, when I walked into the Dean's office at the University of Rochester seeking admission to the Medical School. I had no appointment, I had made no formal application, and classes had already commenced. The Dean was kind enough to have me interviewed by the Admissions Committee. The next person I saw was George W. Corner, Professor of Anatomy. After a few more interviews, I returned to the Dean's office to learn my fate. George Hoyt Whipple (1878-1976), the Dean, said that he had made a few phone calls and that I was now a medical student. I recommend this irregular approach to medical school; it attracts attention. Late in my first year Dr Corner invited me to become a fellow in anatomy and do research with him.
      Our first manuscript describing the effects of corpus luteum extracts on the uterus of the immature rabbit was ready for publication in the early summer of 1928, but this manuscript never left Corner's desk. Being cautious he decided, before submitting the manuscript, to make an extract from start to finish all by himself. He reasoned that if he, an anatomist, could do it anyone could. He was astonished to find that his extract was not potent. The bad news came to me by telegram. The next day, I drove to the University (a distance of 18 miles) and we agreed to suppress the manuscript until we could find out why my extracts had been potent and his were not. This dilemma was resolved a few months later when we realized that he had selected much smaller rabbits for testing than I had used. When we tested mature rabbits, his extracts were as potent as mine. The manuscript, which did leave his desk in December, 1928, carried the following summary:
      • The experiment described…show that alcoholic extracts of the corpus luteum, freed from phospholipids, contain a substance which when injected into castrated adult female rabbits induces a characteristic alteration of the endometrium identical with the progestational proliferation previously shown to be due to the presence of corpora lutea in the ovaries.…Extracts of follicular fluid containing large amounts of oestrin do not produce progestational proliferation, nor have extracts from human placenta given positive results. It appears therefore, that extracts of the corpus luteum contain a special hormone which has for one of its functions the preparation of the uterus for reception of the embryos by inducing progestational proliferation of the endometrium.
        • Corner G.W.
        • Allen W.M.
        Physiology of the corpus luteum, II: production of a special uterine reaction (progestational proliferation) by extracts of the corpus luteum.
      We submitted a second manuscript on the same day. The summary stated:
      • These experiments demonstrate that in the presence of progestational proliferation induced by corpus luteum extracts, in rabbits deprived of both ovaries at the 18 hour of pregnancy, the embryos may survive and grow normally and normal implantation may occur, whereas in the absence of progestational proliferation the embryos never survive beyond the fourth day. The evidence is now complete that in the rabbit, the corpus luteum is an organ of internal secretion which has for one of its functions the production of a special state of the uterine mucosa (progestational proliferation) and that in turn the function of the proliferated endometrium is to nourish or protect the free blastocysts and to make possible their implantation.
        • Allen W.M.
        • Corner W.M.
        Physiology of the corpus luteum, III: normal growth and implantation of embryos after very early ablation of the ovaries, and under the influence of extracts of the corpus luteum.
      Neither of these summaries conveys the excitement when the time came to kill the first adult rabbit that had received our crude extract. The rabbit had been mated with 2 fertile bucks 6 days before. About 18 hours after mating we had removed the ovaries taking care not to manipulate the tubes. Rabbits ovulate 10 hours after mating so we presumed that fertilized ova in the 2, 3, or 4 cell stage were in the fallopian tubes when the ovaries were removed. The crude extract had been given subcutaneously each day for 5 days. We knew that normal blastocysts should be in the uterus if the extract contained the corpus luteum hormone. Corner excised the uterus, rather calmly it seemed to me, and injected salt solution into the uterus. There was a delay as the uterus distended. Then suddenly several transparent blastocysts popped out into the receiver. They were 1.5 mm in diameter, just the right size for 6-day rabbit embryos. Neither of us said very much but we both knew that much exciting work lay ahead.
      The chemical maneuvers leading to the isolation of the hormone progressed rapidly. Within a year I had separated all the known lipids without loss of the hormone.
      • Allen W.M.
      Physiology of the corpus luteum, V: the preparation and some chemical properties of progestin, a hormone of the corpus luteum which produces progestational proliferation.
      By distillation at low pressures our precious oil was separated into 3 fractions, one of which was notably enriched.
      • Allen W.M.
      The preparation of purified progestin.
      When I asked for money to purchase a high-vacuum apparatus, I was told, “Why not? Research is only pouring good money down expensive sinks.” In our case we poured good money down an ordinary sink but we got a good product. I was able to sublime white waxy material from which fractional crystallization I obtained the crystalline hormone.
      When in 1930 we decided to name the hormone progestin,
      • Allen W.M.
      Physiology of the corpus luteum, V: the preparation and some chemical properties of progestin, a hormone of the corpus luteum which produces progestational proliferation.
      Corner considered the advisability of a patent. We disposed of the matter very simply on 2 points. A naturally occurring hormone could not be patented, although the method of producing it could be. The second point revealed Corner's sterling character. He maintained that physicians should not personally benefit from a discovery that might be of benefit to the sick. Our decision not to patent either the name of progestin or the process was easy, as we had received no financial support from drug manufacturers.
      The isolation of the hormone from the waxy material obtained by high-vacuum distillation was a laborious and exasperating experience. However, the month of May 1933 was a glorious month. On May 5, I had the crystalline corpus luteum hormone. On May 18, my daughter, Lucille, was born. My friends gave me double congratulations and I was sitting on top of the world. June was a different story. A second batch of crystals, even though potent, was as different from the first as day is from night. My friends made the obvious suggestion that the real hormone was lurking as a tiny contaminant in my crystals. On July 1, I became an intern in obstetrics and gynecology, wondering what the relationship between the 2 types of crystals might be. Subsequently, by collaboration with a biochemist at Columbia University in New York City, we determined that both crystalline forms of the hormone had the same composition (C21H30O2), that both oxygens were ketonic, and independently we guessed the correct structural formula for the hormone. This required no brilliant deductions. The structural formula of pregnanediol (C21H36O2) was known from previous work by Adolph F. J. Butenandt.
      • Butenandt A.
      Über das Pregnandiol, einen neuen Sterinabkömmling aus Schwangeren-Harn.
      It was a simple matter to foresee the relationship between pregnanediol, our inactive oxyketone, and the hormone. Corner and I realized, of course, that there would be competition in the race for the isolation of the hormone. At least 4 groups, in addition to ourselves, entered the race. The crystalline hormone was announced in the summer of 1934 by Butenandt and Westphal,
      • Butenandt A.
      • Westphal U.
      Zur Isolierung und Characterisierung des Corpus-luteum-Hormons.
      Slotta, Ruschig and Fels,
      • Slotta K.
      • Ruschig H.
      • Fels E.
      Reindarstellung der Hormone aus dem Corpus Luteum.
      Hartmann and Wettstein,
      • Hartmann M.
      • Wettstein A.
      Ein krystallisiertes Hormon aus Corpus luteum.
      and Wintersteiner and Allen.
      • Wintersteiner O.
      • Allen W.M.
      Crystalline progestin.
      In a few months Butenandt and Schmidt had converted pregnanediol to progesterone,
      • Butenandt A.
      • Schmidt J.
      Überführung des Pregnandiols im Corpus-luteum-Hormon.
      and Fernholz
      • Fernholz E.
      Zur Syntheses des Corpus-luteum-Hormons.
      had synthesized the hormone from stigmasterol. These endeavors established the structural formula in record time. What had started as progestin in Corner's laboratory in 1929 had now become an “international hormone.” In July 1935, I was an official delegate to a special conference of the Health Organization of the League of Nations, which was charged with setting up International Standards for some of the sex hormones, including the new hormone of the corpus luteum.
      In so far as the corpus luteum hormone was concerned, the following recommendations (among others) were made:
      • 1.
        That the Conference recommend the adoption, for common use in scientific literature, of the name progesterone for the pure crystalline progestational hormone.
      • 2.
        That an international standard preparation of the progestational hormone be accepted, and that this standard shall consist of the pure, crystalline progesterone, crystallized in the form of needles…
      • 3.
        That the international unit for the progestational hormone shall be the specific progestational activity of 1 milligramme of the international standard preparation.

        Report of the second conference on the standardization of sex hormones. Quart Bull Health Organ, League of Nations 1935;4:618-29.

        • Allen W.M.
        • Butenandt A.
        • Corner G.W.
        • Slotta K.H.
        Nomenclature of corpus luteum hormone.
      The Conference ended in a happy note, but we sensed that the shadows of war were slinking across Europe. None of us could have predicted, however, that the ships that had carried me across the Atlantic (The Bremen and the Europe), and the British battleships I had seen as we crossed the Channel, would soon find a watery grave.
      Corner and I both left Rochester in 1940, after 14 years of happy and productive association. He returned to his native Baltimore to become Director of the Carnegie Laboratories alongside the Johns Hopkins Hospital, and I ventured west to become the Chairman of the Department of Obstetrics and Gynecology at Washington University in St Louis.

      References

        • Allen E.
        • Doisy E.A.
        An ovarian hormone: a preliminary report on its localization, extraction, and partial purification, and action in test animals.
        J Am Med Assoc. 1923; 81: 819-821
        • Corner G.W.
        • Allen W.M.
        Physiology of the corpus luteum, II: production of a special uterine reaction (progestational proliferation) by extracts of the corpus luteum.
        Am J Physiol. 1929; 88: 326-399
        • Allen W.M.
        • Corner W.M.
        Physiology of the corpus luteum, III: normal growth and implantation of embryos after very early ablation of the ovaries, and under the influence of extracts of the corpus luteum.
        Am J Physiol. 1929; 88: 340-346
        • Allen W.M.
        Physiology of the corpus luteum, V: the preparation and some chemical properties of progestin, a hormone of the corpus luteum which produces progestational proliferation.
        Am J Physiol. 1930; 92: 174-188
        • Allen W.M.
        The preparation of purified progestin.
        J Biol Chem. 1932; 98: 581-605
        • Butenandt A.
        Über das Pregnandiol, einen neuen Sterinabkömmling aus Schwangeren-Harn.
        Ber Dtsch Chem Ges. 1930; 63: 659-663
        • Butenandt A.
        • Westphal U.
        Zur Isolierung und Characterisierung des Corpus-luteum-Hormons.
        Ber Dtsch Chem Ges. 1934; 67: 1440-1442
        • Slotta K.
        • Ruschig H.
        • Fels E.
        Reindarstellung der Hormone aus dem Corpus Luteum.
        Ber Dtsch Chem Ges. 1934; 67: 1270-1273
        • Hartmann M.
        • Wettstein A.
        Ein krystallisiertes Hormon aus Corpus luteum.
        Helv Chim Acta. 1934; 17: 878-882
        • Wintersteiner O.
        • Allen W.M.
        Crystalline progestin.
        J Biol Chem. 1934; 107: 321-336
        • Butenandt A.
        • Schmidt J.
        Überführung des Pregnandiols im Corpus-luteum-Hormon.
        Ber Dtsch Chem Ges. 1934; 67: 1901-1903
        • Fernholz E.
        Zur Syntheses des Corpus-luteum-Hormons.
        Ber Dtsch Chem Ges. 1934; 67: 1855-1857
      1. Report of the second conference on the standardization of sex hormones. Quart Bull Health Organ, League of Nations 1935;4:618-29.

        • Allen W.M.
        • Butenandt A.
        • Corner G.W.
        • Slotta K.H.
        Nomenclature of corpus luteum hormone.
        Science. 1935; 82: 153

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