GROWTH PROCESSES INDUCED BY ESTROGENIC HORMONES IN THE UTERUS OF THE MOUSE' LEO LOEB, V. SUNTZEFF,AND E. L. BURNS (From fhe Laboratory of Rescurch Pathology, Oscar Johnson Institute, Washington University School of Medicine, St. Louis) In former investigations we have studied the growth processes which take place in the mammary gland ( 1j and in the vagina and cervix (2) of mice under the 'influence of estrogenic hormones, and the conditions under which these .processes become cancerous. The investigations recorded here extend this analysis to the uterus of the mouse. By uterus, we understand that part of the vaginal-cervical-uterine tract which begins approximately at the point of bifurcation. In earlier studies on the guinea-pig, it was pointed out (3) that the cervix may be divided into three parts: a vaginal, an intermediate, and a uterine part. The condition is similar in the mouse, although in this animal the division is perhaps not quite so defi- nite as in the guinea-pig. Also there exists in the mouse a transition zone be- tween cervix proper and uterus, in which the tissue has essentially the struc- ture of the uterus but still retains some cervical characteristics, namely, a somewhat greater tendency to the production of low squamous epithelium than is present in the uterus proper. Thus there may be found here an area in which are uterine glands covered by low squamous rather than by cylindrical epithelium; furthermore, there may develop in this area glandular cysts which are filled with hyaline nucleated cells instead of with a diffuse gelatinous ma- terial, such as is usually encountered in uterine cysts. In this transition zone the cysts may be lined entirely or partially with a low squamous epithelium. In accordance with the characteristics of the epithelium of the cervix and vagina and its tendency to assume the squamous-cell type, we find in these areas a greater incidence of precancerous and cancerous changes. In the uterus, on the other hand, as the experiments to be reported in this paper prove, the tendency to produce precancerous or cancerous changes is much less. The uterus and other sex organs were studied in 501 mice, this number in- cluding 177 control mice which were not subjected to treatment with sex hor- mones. The strains used and the ages at which the various groups were autopsied were as follows (I signifies an age of one to eleven months; 11, twelve to seventeen months; 111, eighteen months and over, and IIIa, twenty months and over) : (A) Control Mice Strain A, 55 mice. I: 34 mice; 11: 17 mice; 111: 4 mice (IIIa: 4 mice) Strain C5?,43 mice. I: 33 mice; 11: 7 mice; 111: 3 mice (IIIa: 3 mice) Strain D, 23 mice. I: 9 mice; 11: 10 mice; 111: 4 mice (1IIa: 3 mice) 1 These investigations were carried out with the aid of grants from the International Cancer Research Foundation and from the Jane Coftin Childs Memorial Fund for Medical Research. We are indebted to Dr. Erwin Schwenk of the Schering Corporation for supplying us generously with Progynon B (benzoic acid ester of dehydrofolliculin) which is the estrogenic hormone used in these experiments. 413 414 LEO LOEB, V. SUNTZEFF, AND E. L. BURNS (A) Control Mice (cont.) Strain C3H, 13 mice. I: 7 mice; 11: 5 mice; 111: 1 mouse (IIIa: 1 mouse) Strain New Buffalo, 9 mice. I: 0 mice; 11: 3 mice; 111: 6 mice (IIIa: 3 mice) Strain Old Buffalo, 18 mice. I: 3 mice; 11: 9 mice; 111: 6 mice (IIIa: 5 mice) Strain CBA, 16 mice. I: 10 mice; 11: 2 mice; 111: 4 mice (IIIa: 4 mice) TOTAL,177 mice. I: 96 mice; 11: 53 mice; 111: 28 mice (IIIa: 23 mice) (B) Mice Injected with &.30 rat units of Estrogen (Dissolved in Water, injected Dairy in the Majority of Cases) Strain A, 29 mice. I: 7 mice; 11: 10 mice; 111: 12 mice (IIIa: 1 mouse) Strain C57, 10 mice. I: 3 mice; 11: 0 mice; 111: 7 mice (IIIa: 6 mice) Strain D, 11 mice. I: 3 mice; 11: 6 mice; 111: 2 mice (IIIa: 1 mouse) Strain New Buffalo, 3 mice. I: 0 mice; 11: 1 mouse; 111: 2 mice (IIIa: 2 mice) Strain Old Buffalo, 10 mice. I: 0 mice; 11: 9 mice; 111: 1 mouse (IIIa: 1 mouse) - TOTAL,63 mice. I: 13 mice; 11: 26 mice; 111: 24 mice (IIIa: 11 mice) (Cj Mice Injected with 50 rat units of Estrogen (Dissolved in Water, Injected Daily in the Majority of Cases) Strain A, 9 mice. I: 5 mice; 11: 3 mice; 111: 1 mouse Strain D, 3 mice. I: 1 mouse; 11: 1 mouse; 111: 1 mouse Strain C3H, 10 mice. I: 6 mice; 11: 4 mice; 111: 0 mice Strain New Buffalo, 3 mice. I: 0 mice; 11: 2 mice; 111: 1 mouse (IIIa: 1 mouse) TOTAL,25 mice. I: 12 mice; 11: 10 mice; 111: 3 mice (IIIa: 1 mouse) (D) Mice Injected with 100 or More rat units of Estrogen (Dissolved in Oil, as a Rule Once a Week) Strain A, 45 mice. I: 43 mice; 11: 2 mice; 111: 0 mice Strain C57, 17 mice [13].* I: 16 mice [12]; 8 11: 1 mouse; 111: 0 mice Strain D, 11 mice. I: 9 mice; 11: 0 mice; 111: 2 mice (IIIa: 2 micej Strain C3H, 41 mice [39].3 I: 37 mice [35]; * 11: 4 mice; 111: 0 mice Strain New Buffalo, 9 mice. I: 5 mice; 11: 3 mice; 111: 1 mouse (IIIa: 1 mouse) Strain Old Buffalo, 8 mice. I: 5 mice; 11: 3 mice; 111: 0 mice Strain CBA, 30 mice. I: 30 mice; 11: 0 mice; 111: 0 mice TOTAL,161 mice [155].8 I: 145 mice [139]; 8 11: 13 mice; 111: 3 mice (IIIa: 3 mice) (E) Mice Injected with Estrogen and Acid Extract of Cattle Anterior Pituitary Gland Strain A, 9 mice. I: 6 mice; 11: 1 mouse; 111: 2 mice (IIIa: 1 mouse) Strain C57, 7 mice. I: 2 mice; 11: 0 mice; 111: 5 mice (IIIa: 2 mice) Strain D, 9 mice. I: 7 mice; 11: 0 mice; 111: 2 mice (IIIa: 2 mice) (Eu) Mice Injected with Extruct of Cattle Anterior Pituitary Gland Alone Strain A, 3 mice. I: 1 mouse; 11: 2 mice; 111: 0 mice Strain D, 3 mice. I: 2 mice; 11: 1 mouse; 111: 0 mice TOTAL(E and Ea), 31 mice (25 plus 6). I: 18 mice; 11: 4 mice; 111: 9 mice (IIIa: 5 mice) (F) Mice Injected with Estrogen and Corpus Luteum Extract (Proluton) Strain A, 7 mice. I: 3 mice; 11: 4 mice; 111: 0 mice Strain C57, 8 mice. I: 4 mice; 11: 4 mice; 111: 0 mice Strain D, 15 mice. I: 3 mice; 11: 6 mice; 111: 6 mice (IIIa: 2 mice) Strain Old Buffalo, 7 mice. I: 1 mouse; 11: 6 mice; 111: 0 mice 2 Some were Injected twice a week with 100 rat units, and still others with 100 rat units dis- solved In oil and 50 rat units dissolved in water once a week. 8In a second series in which the effects of hormone injections on the stroma of vagina and uterus were studied, on which we shall report later, the same mice were used, except for a few groups in which, as indicated in square brackets, a few of the mice were omitted. Altogether 495 mice were used in the second series. GROWTH PROCESSES INDUCED BY ESTROGENIC HORMONES 415 (Fa) Mice Injected with Corpus Luteum (Proluton) Alone Strain A, 1 mouse. I: 0 mice; 11: 1 mouse; 111: 0 mice Strain D, 6 mice. I: 6 mice; 11: 0 mice; 111: 0 mice TOTAL(P and Fa), 44 mice (37 plus 7). I: 17 mice; 11: 21 mice; 111: 6 mice (IIIa: 2 mice) Totals: Strain A, 158 mice Strain C57, 85 mice Strain D,81 mice Strain C3H, 64 mice Strain New Buffalo, 24 mice Strain Old Buffalo, 43 mice Strain CBA, 46 mice Altogether 501 mice [495 mice used in the study of the stroma]. The following changes may take place in the uterus of mice under the di- rect or indirect influence of estrogen : (I) The normal one-layered cylindrical epithelium may change into an epithelium which consists of several rows of cylindrical cells, into a partly cylindrical and partly squamous epithelium, or into a typical squamous epithelium; (11) gland ducts may become cystically dilated and filled with colloid material; (111) the uterine glands may prolifer- ate and penetrate into and through the muscle tissue; (IV) infection may take place, in consequence of which the uterine cavity becomes transformed into an abscess and parts of the uterine wall may become necrotic and ulcerated; (V) parts of the pancreas may become adherent to the uterus. We shall discuss the frequency with which these changes occurred in the various groups of mice, as well as their significance. I. CHANGEIN THE CHARACTEROF THE UTERINESURFACE EPITHELIUM Two processes may lead to a change in the character of the surface epi- thelium of the uterus. (1) There may occur'a regenerative growth of the squamous cervical epithelium from the cervix into the adjoining uterus; this is usually due to a preceding ulceration of the uterine mucosa. In certain cases the ingrowth of the cervical squamous epithelium can be followed into the uterus. In other cases there are indications that such an ingrowth may have occurred, although there seems to have been no infection or ulceration. (2) In certain instances a real metaplasia of the uterine surface epithelium may take place.