Papanicolaou's Vaginal Smear

The second classic embryological investigation leading to important, explicitly reproductive research was that of George Papanicolaou at Cornell Medical College in New York City (Stockard and Papanicolaou 1917a,b). Charles Stockard was a zoologist by training who had been sponsored for the position of chair of anatomy at Cornell by Franklin Paine Mall, chair of anatomy at Johns Hopkins. Mall felt that the future of experimental anatomy lay in recruiting from the ranks of more experimental zoology, thereby promoting border crossings (Corner 1960:181). Stockard began working in teratology while a student of Thomas Hunt Morgan at Columbia (Oppenheimer 1984) and was interested in the influence of chemicals upon developing embryos. He began studying effects of alcohol on guinea pig embryos in 1909 (Pauly 1996).

Through sponsorship by Morgan, Papanicolaou joined Stockard in these efforts as an assistant in anatomy at Cornell in 1914. Papanicolaou held a Greek medical degree and a German doctorate in zoology. He had worked on sex determination in Munich, a project he resumed at Cornell using guinea pigs from the same colony that supplied Stockard's research. While historical accounts vary, it seems most likely that Papanicolaou initiated the use of vaginal examination and vaginal cell smears on slides to ascertain the estrus cycle stage in order to obtain ova at precise stages of development for his own work on the problem of sex determination.^[58] He studied cells from the guinea pig vagina throughout the estrus cycle to determine whether stages of estrus could be indicated by the changing composition of those cells over the cycle.

The answer was a clear yes. But the implications of this research for reproductive investigations went far beyond Papanicolaou's initial modest goals. The stages of the estrus cycle could be determined by microscopically examining smears of easily accessible cells, a procedure that could be done on a routine basis without surgery or sacrificing the animal. Moreover, such cells could serve as indicators of changes from normal phasing due to ablation (removal) of hypothesized hormone-producing organs, and/or transplantation of hormone-producing organs, and/or injection of hypothesized hormone extracts into the animal. And the indicator was not expensive to obtain, nor were the animals! Cheap, fast, and accurate are technical research goals rarely achieved simultaneously.

Each of these dimensions of the smear technique—speed, accuracy, and low cost—gave investigators latitude and flexibility. They could "infer what was happening in the internal reproductive organs without inspecting them directly," an extraordinarily powerful advance (Zuckerman 1970:22). Because so little was known about the sequence of events of the estrus and menstrual cycles, not until these events were more precisely determined and cataloged could experimenters guage the alternations effected by ablation or transplantation or injection. Intervention for clinical or other reasons was dependent on histologists then painstakingly determining these gradual changes. The significance of earlier contributions of German and Austrian gynecologists lay precisely on this point. In the 1870s, they had cataloged the effects of female castration during the period when oophorectomy (removal of the ovaries) was a popular treatment for dysmenorrhea and certain neuroses. Later, Emil Knauer (in 1896) and Josef Halban (in 1900) noted that the ovary probably produced some special substance that normally maintained the uterus (Borell 1985:12, Corner 1965). The vaginal smear technique permitted systematic examination of the biological activity of different organs imputed to produce such hormones.

Ironically, having such accurate, cheap, and accessible indicators in laboratory animals was initially viewed as important to the production of laboratory animals in colonies, especially but not exclusively for embryological research!^[59] The other implications of the technique were appreciated quickly, and it became central to many reproductive endocrinological investigations. For example, while developing a colony of mice for a course in embryology, some of the implications of his morphological work drew Edgar Allen into pioneering investigations in reproductive endocrinology. He and Edward Doisy, a biochemist, soon achieved the first isolation of a reproductive hormone, active ovarian follicular extract (Allen and Doisy 1923; Doisy 1932).

The smear method also became a primary means of studying the estrus and menstrual cycles in different animals. Here Papanicolaou's work was followed quickly by Long and Evans's parallel efforts on the rat (1920,

1922), Edgar Allen's on the mouse (1922), Corner's on the monkey and sow (1921b, 1923), Hartman's on the opossum (1923), and Evans and Cole's work on the dog (1931). Agricultural animals studied included the cow, ewe, mare, and sow.^[60] As Evans (1959:vii) later noted, "It appeared, indeed, for a time that the application of the vaginal smear method would be all that was required to segment the stages of the estrous cycle in all animals ... [but] the beautifully distinct changes in the vaginal lochia of small rodents were peculiar for the smaller forms." Only in the dog was the estrogen level high enough to show pronounced vaginal changes. But Papanicolaou, deeply committed to cytology (1933), pursued the vaginal cytological smear as a potential indicator of something in women. He ultimately found that a vaginal smear could indicate potential and actual pathological changes in the cervix and uterus useful for diagnosis of cancerous, precancerous, and other abnormal conditions. The "Pap smear" is now the most widely used cancer screening technology in the world.^[61] Papanicolaou also attempted early pregnancy detection through smears, but the Ascheim-Zondek urine-based test proved more successful (Carmichael 1973).

In addition to the two classic investigations of Lillie and Papanicolaou, the importance of embryological work for the development of reproductive sciences was reflected in the inclusion of a major chapter on embryology in Sex and Internal Secretions (Willier 1932:94–159), the American "bible" of reproductive sciences, first published in 1932.^[62] George Corner, (1961:ix–xii), in his foreword to the third edition, stated: "To the embryologists of Europe and America we owe in large part also the successful analysis of the mammalian reproductive cycle that has been achieved during this half century." That is, like the gene theory and genetics in the United States (Gilbert 1978, 1987, 1991), the American reproductive sciences also have embryological origins, however long ignored.

It can seem surprising or counterintuitive that problems of reproductive physiology and endocrinology were pursued in America in the early twentieth century predominantly not by physiologists but by zoologists and anatomists, most with backgrounds in embryology. Addressing this historical problem, Diana Long (1987) undertook a statistical analysis of the disciplinary affiliations and identities of "sex researchers" publishing in the American Journal of Physiology from 1923 to 1947. While only about one-third identified themselves primarily as physiologists, two-thirds were members of the American Physiological Society. Long argues that sex researchers, regardless of discipline, gained many benefits through associating with the prestigious physiological society and journal. Prestige and legitimacy were important cultural resources and goals for early reproductive scientists whether they considered themselves anatomists, zoologists, physiologists, or endocrinologists.

The freemartin work supported the endocrinological direction of Ameri-

can reproductive sciences. Then, because of its incredible value as an indicator and its ease of use, the vaginal smear led many investigators from different disciplines and professions into this new domain of reproductive research. By 1925 the reproductive sciences were fully initiated as integrated lines of modern scientific work, with emphasis shifting from classical physiological work to an explicit biochemical endocrinological focus.