Growth Processes Induced by Estrogenic Hormones in the Uterus of the Mouse'

Home , Transitional epithelium, Uterine epithelium, Uterine gland

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 definite as in the guinea-pig. Also there exists in the mouse a transition zone between cervix proper and uterus, in which the tissue has essentially the structure 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 material, 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 hormones. 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 dissolved 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 direct 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 proliferate 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 epithelium 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. We find, then, a change of the epithelium consisting of one layer of cylindrical cells into an epithelium made up of several layers of cylindrical or cuboidal cells. Neither of these changes, however, proceeds to actual keratinization, an observation which is in accord with the usual absence of keratinization in the uterine end of the cervix. Such a metaplasia is not limited to the cervical end of the uterus; it may be found elsewhere, as for instance near the tubal end, or it may occur irregularly in patches. As to the relative frequency of the regenerative process and of true metaplasia, it is difficult to make a definite statement. Particularly does this apply to regions of squamous epithelium which may be found in various places in association with ulceration affecting wide areas of the uterus in mice which have received weekly injections of 100 or more rat units of estrogen in oil. In other cases, also, the differentiation between these two types may be difficult. 416 LEO LOEB, V. SUNTZEFF, AND E. L. BURNS On the whole, it is very probable that the regenerative origin of the squamous epithelium is the more frequent one. Squamous epithelium was observed in the uterus in the following groups. (A) Antong 177 non-injected (control) mice, areas with squamous epithelium occurred in only 2 animals, both older than twenty months. There was no infection in the uterus of either of these. In a mouse belonging to strain A, twenty-three months old. the epithelium of the surface consisted for the most part of three layers which joined the cervical epithelium. This change was presumably due to regeneration. The connective tissue beneath the epithelium was markedly hyaline. In a twenty-one-month-old New Buffalo mouse several cystic glands were lined with squamous epithelium; this is probably an instance of true metaphsia. (B) Among 63 mice injected with 1-30 rat units of estrogen squamous epithelium was found in 1, a sixteen-month-old mouse belonging to strain A, injected with 10 rat units of estrogen for twelve months. There was marked hyalinization of the connective tissue, but no infection. (C) Among 25 mice injected with 50 rat units of estrogen squamous epithelium was observed in 5 A strain animals, aged eight, ten, eleven, twelve and sixteen and a half months. The injections were begun when the mice were about two weeks old. In 3 animals the squamous epithelium did not seem to have any connection with the cervix. In some instances the squamous surface epithelium grew downward and lined the lumen of some gland. (D) Among 161 mice injected, in most cases once weekly, with 100 or more rat units of estrogen (dissolved in oil) squamous epithelium was noted in 35. The distribution among the various strains and age groups was as follows: Strain A, I: 6 mice; 11: 2 mice. Strain C57, I: 9 mice; 11: 1 mouse. Strain D mice: none. Straitt C3H, I: 5 mice. New Bufdo, I: 2 mice; 11: 1 mouse. Old Bufalo: none. CBA, I: 9 mice. In the majority of these cases the uterus had been infected and, as far as it is possible to judge, the covering of the uterus with squamous epithelium was due to a regenerative growth of the cervical epithelium into it. In some of the mice, however, it seems that a true metaplasia had occurred. The extent to which the surface of the uterus was covered with squamous epithelium varied. In some animals the greater part was thus covered. In a number of instances the squamous epithelium grew down from the surface into the glands and thus glandular cysts became lined with it; in some, processes of squamous epithelium were observed forming pearls reaching down into the connective tissue, but apparently without connection with glands. (E) Among 31 mice injected un'th a cornbination of estrogen and extract of anterior pituitary cattle gland or with the extract alone, squamous epithelium was noted in 2 animals, nineteen and a half and sixteen months old, belonging to the A strain, and in one nineteen- month-old mouse of the C57 strain. These 3 animals had received injections of 10 or 30 rat units of estrogen for variable lengths of time. No infection was present. In 2 of the animals the squamous epithelium was probably derived from the cervical epithelium. In the third the greater part of the uterine epithelium was lined with cylindrical epithelium, but in places there were two layers of cylindrical epithelium in which mitoses occurred; these areas were not connected with the cervix and were probably due to a metaplasia. (F) Among 37 mice injected with a combination of estrogen and corpus luteum extract or Proluton the occurrence of squamous epithelium was noticed in 8 (3 A, 4 C57, and 1 Old Buffalo); in 7 mice injected with lutein (Proluton) alone squamous epithelium was not observed. In all but the one Old Buffalo mouse 100 rat units of estrogen had been injected. In 3 of these cases we have presumably to deal with a metaplasia of the cylindrical epithelium and especially does this apply to the Old Buffalo mouse, where the surface epithelium consisted mostly of two layers, a lower cuboidal and an upper cylindrical layer. In several animals the squamous epithelium had grown down into the glands. The A mice were nine, twelve and fourteen months old; the C57 mice were seven, ten, eleven, and sixteen months old, and the Old Buffalo animal was ten months old. We may conclude from this list that the incidence of squamous epithelium in the uterus increases with an increase in the amount of estrogen experiment- GROWTH PROCESSES INDUCED BY ESTROGENIC HORMONES 417 ally administered. Only twice was it observed in non-injected control mice, and even in these we cannot exclude the effect of estrogen normally discharged during the sexual cycle. It occurred only once among 63 mice injected with relatively small amounts of estrogen ( 1-30 rat units), If, however, we add to this mouse, 3 others injected with a combination of estrogen and extract of anterior pituitary gland and 1 injected with a combination of estrogen and lutein preparations, in which the amounts of estrogen did not exceed 30 rat units, we find an incidence of 4 per cent as compared with 1.1 per cent in the control series. In the large majority of these cases we have to deal with animals belonging to age groups I1 and 111, whereas, as we shall see shortly, in the mice injected with 50 or 100 rat units of estrogen the highest incidence is in age group I. In the group of mice injected with 50 rat units of estrogen squamous epithelium occurred in 20 per cent, and in the group injected with 100 rat units in 21.7 per cent. As to the various strains of mice affected, in the control mice and in the group injected with 1-30 rat units of estrogen the A mice predominated, but this may be due to the fact that in these experiments the A mice were the most numerous. In general, all strains were affected except the D strain. In this strain, also, there was an almost complete absence of infection in the uterus. It is doubtful, however, whether we have here to deal with a strain peculiarity, since the number of D strain mice which received large amounts of estrogen was relatively small. As to the mechanism underlying the formation of squamous epithelium in the uterus, we have, as stated above, to distinguish between the cases in which it is regenerative in character, due to an extension of the squamous epithelium of the cervix, and others in which a real metaplasia takes place. Defects in the lining of the uterine mucosa may be expected whenever infection occurs in the uterus, and this was a common observation in mice receiving 100 or more rat units of estrogen dissolved in oil, usually in weekly injections. As to the actual metaplasia of an ordinary cylindrical epithelium into an epithelium consisting of two or more layers of cylindrical epithelium, or of layers of cuboidal and cylindrical epithelium, or of several layers of cuboidal epithelium, this is presumably due to the stimulating action of estrogen. We know that the hormone exerts such an effect in vagina and cervix. The transformation of a single layer of cylindrical epithelium into squamous epithelium takes place most readily and rapidly in the vagina; it occurs also, but less completely, in the cervix, and in general the readiness with which this transformation occurs diminishes in the direction from vagina to uterus. In the uterus it is not found under ordinary circumstances. Under certain conditions, however, it is seen that here, too, this change can be accomplished, namely, after long- continued injections of estrogen. Selye, Thomson and Collip (4) observed the production of squamous epithelium in the uterus of mice after a single uterine injection of estrone in corn oil. We did not succeed under the conditions of our experiments in obtaining a real metaplasia of the uterine epithelium under similar conditions, In the majority of cases in which we found a substitution of squamous or transitional epithelium for ordinary cylindrical epithelium, the stroma of the uterine mucosa had a hyaline character. It is not certain, however, that this 418 LEO LOEB, V. SUNTZEFF, AND E. L. BURNS factor had any causal connection with the change in the epithelium, inasmuch as in many cases it was present without any alteration in the nature of the surface epithelium, and in other instances the latter occurred without marked hyalinization. We must, furthermore, consider the readiness with which the administration of large doses of estrogen produces the deposit of a hyaline substance in the uterine stroma and the great frequency with which such a deposit takes place. The simultaneous occurrence of these two changes may therefore be expected according to the law of probability. In this connection it may be of interest to note that in patients in whom cancer developed following long-continued treatment with roentgen rays Wolbach observed a hyalinization of the underlying connective tissue as a prominent feature. It is possible that the regenerative stimulus resulting from a state of the stroma which is injurious to the epithelium may act as an additional factor in the production of squamous epithelium from Cylindrical epithelium. As to the manner in which metaplasia of the cylindrical epithelium takes place, our knowledge is very incomplete. In one case we noticed as a first change two or three layers of loosely arranged cylindrical cells in which mitosis occurred; in another case a layer of cuboidal cells developed below the layer of cylindrical cells; in still other instances several layers of cuboidal cells formed beneath the cylindrical epithelium papillae, raising the latter up. Sometimes four or five layers of cuboidal cells were seen, and the upper cells, instead of keratinizing, underwent hyalinization, a process which is quite common in the cervix. The hyalinized cuboidal cells had a tendency to dissolve, as was evidenced by the formation of vacuoles. In the end such a vacuolization affected the whole cell, which was transformed into a soft colloid or gelatinous material. These vacuolization and solution processes were especially noticeable in the cuboidal hyaline cells which lay on top of the squamous epithelium; but they occurred, too, in the cuboidal hyaline cells which developed beneath the cylindrical cells and pushed the latter upwards. Hyaline material developed also in the cytoplasm of the cylindrical cells. The epithelial cells which underwent the change into transitional and squamous epithelium apparently acquired thereby a greater mobility, This was manifested in two ways: (1) the squamous epithelium moved beneath the Cylindrical epithelium where it adjoined the latter and raised it up from the underlying connective tissue; (2) where the squamous epithelium directly covered and thus came in immediate contact with relatively soft hyaline material which had been deposited in the mucosa, it moved downward as a connected row of cells into this gelatinous substance, partly traversing it. There is evidently inherent in the squamous epithelium a tendency to move, but under normal conditions extension into the underlying connective tissue is prevented by the firm consistency of the latter. The relatively soft hyaline material which develops in mice injected with large amounts of estrogen does not offer the same degree of resistance, but on the contrary, acting as a foreign body, it may exert a motor stimulus on these cells. The formation of transitional or squamous epithelium may take place not only in the surface epithelium, but also in the glands. In some cases it seems that the squamous surface epithelium grows downward into the glands; but in other instances low squamous epithelium may develop in the glands in places GROWTH PROCESSES INDUCED BY ESTROGENIC HORMONES 419 where cylindrical epithelium covers the surface of the uterus. Only rarely is it merely the gland duct which is lined with squamous epithelium; as a rule, the latter extends through a part or the whole of the body of the gland. Thus it may happen that one part'of a gland fundus is lined with cylindrical and another part with squamous epithelium. In uterine glands which have undergone this change the squamous epithelium may give origin to cuboidal nucleated, hyaline cells which fill the gland lumen. These glands then assume the characteristics of glands, or of precursors of glands, which are often seen in the cervix near the junction with the uterus. The desquamated hyaline cells may also, in certain cases, have a tendency to dissolve and thus give origin to colloid material which quite com- monly distends these cystic uterine glands. There exist thus transition stages between the typical uterine cysts, lined with cylindrical or cuboidal epithelium and filled with a colloid gelatinous material, and the cysts of the uterine cervix filled with proliferated and desquamated hyaline cells, a condition related to and preceding the stages of keratinization. We may conclude, then, that both the epithelium of the uterine surface and of the uterine glands has the potentiality of becoming transformed into squamous epithelium and of producing hyaline cells analogous to the keratin which develops as a rule in the vagina. There is, however, a marked difference in the threshold of stimulation which is required in the uterus on the one hand, and in the vagina and cervix on the other, to accomplish this change. It is lowest in the vagina, highest in the uterus, and intermediate in the cervix. In the uterus the threshold is so high that the transformation occurs very rarely, and usually only under special conditions, in contrast to conditions in the vagina and cervix, where this change is a normal one and can be relatively readily accomplished. The power to proliferate and form squamous epithelium is therefore not yet entirely lost even in the cylindrical epithelium of the uterus. 11. CYSTICCHANGES IN THE UTERINEGLANDS Normally the uterine gland consists of a duct and a fundus in which the lumen is small, but under certain conditions these glands may become dilated and cystic; the degree of this dilatation varies greatly in different cases. The content of the cysts may also vary to a certain extent; it may be a rather con- centrated colloid material staining deeply red with eosin, or the colloid material may become diluted, probably by taking up fluid which has been supplied by the circulation, and stain lightly, The lining of these cysts may consist either of a single layer of cylindrical or cuboidal epithelium, or of squamous epithelium which, through proliferation and desquamation of the lining epithelial cells, is able to produce hyaline nucleated cells, which fill and distend the cyst cavities. As stated, this latter condition is characteristic of the epithelium of the uterine-cervical glands in the area of transition between the epithelium, on the one hand, of the vaginal and intermediate cervix, which readily becomes changed into squamous epithelium, and the epithelium, on the other hand, of the uterine cervix, which is the beginning of the zone where the surface epithelium is cylindrical. These hyaline cells may readily undergo solution processes and then produce colloid material similar to that which fills the 420 LEO LOEB, V. SUNTZEFF, AND E. L. BURNS cysts lined with one layer of cylindrical or cuboidal epithelium. The colloid material represents, presumably in all cases, a product of transformation of the cytoplasm of the gland cells. The following list compares the incidence of this cystic transformation in the various groups of mice. The Roman numbers designate the age classes. (A) Controls: With cysts: I: 0 mice=O%; 11: 15 mice=28.33%; 111: 18 mice= 64.3%; IIIa: 17 mice==77.3%. Without cysts: I: 96 mice=-100%; 11: 38 mice= 71.7%; 111: 10 mice = 35.7% IIIa: 5 mice = 22.7%. In normal mice the incidence of cystic transformation of the glands rises sharply with increasing age. In the large majority of mice older than twenty months cysts are present, but we have not observed them in mice belonging to age class I. (B) Mice Znjected with 1-30 rat units of Estrogen: With cysts: I: 5 mice=38.5%; 11: 13 mice= 50%; 111: 20 mice=66.6%; IIIa: 1 mouse= 100%. Without cysts: I: 8 mice=61.5% 11: 13 mice= 50%; 111: 4 mice=33.4%; IIIa: 0 mice. In this group of mice, injected with relatively small amounts of estrogen, there is a distinct increase in the frequency with which cysts develop. Cysts may appear even in the first age class, although this occurs in only a minority of the animals. (C) Mice Injected with 50 rut zmits of Estrogen: With cysts: I: 8 mice = 66.6%; 11: 8 mice=80%; 111: 3 mice=100%; IIIa: 1 mouse=100(r. Without cysts: 1: 4 mice =33.4%; 11: 2 mjce=20%; 111: 0 mice. In this group the majority of the mice in the first age group already have cystic glands, and in the third age group all mice are thus affected. Furthermore, in this group the maximum incidence of cystic glands is reached. (D) Mice Injected with 100 or More rat units of Estrogen: With cysts: I: 57 mice= 39.3% 11: 7 mice=53.8%; 111: 0 mice; IIIa: 3 mice=100%. Without cysts: I: 88 mice=60.7%; 11: 6 mice=46.2%; 111: 0 mice. In this group the incidence of cysts is apparently less than in the preceding group. Two facts, however, must be taken into consideration. In the first place the number of mice in group C is relatively small and for this reason accidental factors may have entered. In the second place, in group D ulceration in the uterine wall led in many cases to destruction of the glands. These mice were included among those in which cysts were lacking, and the percentage of the latter was thus increased. We may then conclude that the frequency of cyst formation in mice injected with 100 rat units of estrogen in oil was equal to or pos- sibly greater than in the preceding group. (E) Mice Injected with a Combination of Estrogen and Anterior Pituitary Prepara- tions. With cysts: I: 26.6%; 11: 0%; 111: 55.5%; IIIa: 60%. Without cysts: I: 73.4%; 11: 100%; 111: 44.5%; IIIa: 40%. The number of mice in this group is small, but the results are not unlike those obtained in group B, which received similar quantities of estrogen. There was no development of cysts in the very small group of mice injected with anterior pituitary preparations alone. (F) Mice Injected with a Combination of Estrogen and Lutein Preparations: With cysts: I: 54.5%; 11: 50%; 111: 0%. Without cysts: I: 45.5%; 11: 50%; 111: 100%. In this group also the number of mice was relatively small. Among those injected with lutein preparations alone, cysts occurred in only one mouse, and this animal was above the age of eighteen months. It follows from these data that cystic change of the uterine glands increases with advancing age, that it does occur in older non-injected mice, but that the incidence becomes greater under the influence of estrogen injections and that, on the whole, the larger the doses of estrogen the greater the incidence. The increased frequency of cysts with advancing age in control mice, also, may perhaps be due to the cumulative action of estrogen as age increases. As to the mechanism which underlies cyst formation, this process may be due to occlusion of the ducts, leading to a retention of the products of glandular GROWTH PROCESSES INDUCED BY ESTROGENIC HORMONES 42 1 secretion. It is also possible that estrogen, by stimulating the transformation of the cylindrical epithelium into squamous epithelium and the production of hyaline cells, may bring about cystic dilatation of the glands. Moreover, there is some evidence that the colloid material produced in the cystic glands may represent a modified cytoplasm of the glandular cells which is discharged into the lumen; and it may be possible to assume that estrogen stimulates this process. At present, however, these are merely tentative suggestions.

111. PENETRATIONOF THE UTERINEGLANDS INTO THE MUSCLELAYERS OF THE UTERUS In a restricted number of mice the uterine glands were found to undergo a marked branching and development in the deeper areas of the mucosa; in others the glands began to penetrate in one or a few places into the inner muscle layer and in some cases they reached also into the outer muscle layer and perforated it. It is possible that in a very few instances they produced conglomerations of ducts in the subperitoneal tissue; but if this actually occurred the proliferation did not exceed an adenomatous proliferation, and in no instance did it assume the characteristics of cancerous growth. As a rule, however, and perhaps in all cases, glandular tissue found in the peritoneal region of the uterine wall consisted of islands of pancreatic tissue which, probably as a result of infectious processes in the uterine wall, became adherent to the latter. The proliferation of the uterine glands into the deeper tissues of the wall took place very slowly and gradually. No mitoses were seen in such glands during or after completion of the process of perforation. In accordance with the slowness of growth, the structure of the glands was perfectly normal and there was no indication of a cancerous or even of an adenomatous transformation. In order to simplify the classification of these changes we shall designate them as follows: u signifies a more marked development of the glands in the deeper areas of the mucosa; b indicates perforation of the inner muscle layer, c a perforation of the outer muscle layer. A partial perforation of the inner muscle layer is indicated by u 3 6, and a partial penetration of the outer muscle layer by b + c. (A) Controls: Strain D, 3 mice = 1.7% of all control mice. 11: 1 mouse (a); 111: 2 mice (1, a+b; 1, b); IIIa: 1 mouse (b). (B) Mice Which Received 1-30 rat 24its of Estrogen: 8 mice= 12.7% of all mice in thisgroup. I: lmouse(b);II: 1mouse(a-+6);III:6mice(1a,4a+b,1c),IIIa:4 mice (1 a, 2 a + b, 1 c). (C) Mice Which Received 50 rat units of Estrogen: 5 mice = 20% of all mice in this group. I: 2 mice (a --* b, b); 11: 2 mice (a b, b); 111: 1 mouse (b). (D) Mice Which Received 100 or More rat itnits of Estrogen: 30 mice = 18.6% of all mice in this group. I: 22 mice (3 a, 1 a -+ 6, 12 b, 6 c); 11: 5 mice (2 b, 3 c); I11 and IIIa: 3 mice (2 b, 1 c). (E) Mice Which Received a Combination of Estrogen and Anterior Pituitary Prepa- rations or Anterior Pituitary Preparations Alone: 1 mouse= 3.2% of all mice in this group (or 4% omitting those receiving anterior pituitary preparations alone. 11: 1 mouse (a). (F) Mice Which Received a Combination of Estrogen and Lutein Preparations or Lutein Preparations Alone: Strain A mice (receiving 100 rat units of estrogen in addition to 422 LEO LOEB, V. SUNTZEFF, AND E. L. BURNS lutein preparations), 4 mice = 9.1% (or 11% omitting those receiving lutein preparations alone). We may conclude that even in control mice a penetration of the glands into the deeper tissues occurs, but it is rare. The frequency increases in mice receiving injections of estrogen, the maximum being reached in the groups injected with 50 and 100 rat units. If we again consider the fact that in a large number of those mice which receive 100 or more rat units of estrogen in oil weekly the glands are very much injured, due to infection, it becomes probable that the actual maximum was reached in this group. The frequency of penetration of the glands into the deeper tissue in the different strains was as follows: Strain A: 12 mice or 7.8% of all A mice; C57: 1 mouse or 1.2%; D: 12 mice or 14.8%; C3H: 19 mice or 29.8%; New Buffalo: 3 mice or 13%; Old Buffalo: 2 mice or 4.6%; CBA: 2 mice or 4.3%. The frequency of this occurrence is therefore greatest in the C3H and D mice. These are also the strains of mice in which spontaneous carcinoma of the mem- mary gland is most prone to develop. The relation between these two types of growth processes may, however, be merely a coincidence.

IV. INFLAMMATORYAND NECROTIZING CHANGES IN THE UTERUSOF MICE In a number of the mice used in this series the uterus was found to be thickened and strongly infiltrated with polymorphonuclear leukocytes, which migrated through the epithelium and filled the enlarged cavity with B pus-like or a harder, cheesy material. Usually great parts of the surface epithelium were destroyed, probably as a result of the penetration of the leukocytes. In some instances in a whole section the uterus was found devoid of the covering epithelium. The necrosis and ulceration of the uterine wall extended deeply into the mucosa up to or even into the inner muscle layer. There was, as a rule, an increase in the amount of fibrous tissue, and in some cases pieces of pancreas were adherent to the outer layer of the uterus. As to the mechanism underlying this condition, it is presumably a bacterial infection which directly or indirectly developed as the result of the estrogen injections. We have, however, not yet demonstrated such an infection in a direct manner. Under the influence of estrogen at the time of heat a marked emigration of polymorphonuclear leukocytes from the blood vessels and a subsequent migration through the connective tissue and epithelium take place. As a result of this migration the epithelium undergoes injury. The activity of the leukocytes at this period lasts only a short time and it is soon succeeded by a normal state of the uterine wall in which the integrity of the surface epithelium is restored (5). It is possible that the circulatory changes and the injury of the epithelium, the former produced directly and the latter indirectly by the injections of estrogen, are in some way responsible for the infection which probably occurred in the course of many of these experiments. Furthermore, it is con- ceivable that when the strong action of estrogen becomes continuous, the inflammatory reaction likewise becomes continuous and may lead to the changes which we have mentioned. On the other hand, there exists the possibility that, as the result of a more prolonged congestion and a swelling of the cervical end of the uterus, an occlusion occurs here, accompanied or followed by a bacterial GROWTH PROCESSES INDUCED BY ESTROGENIC HORMONES 423 infection which is responsible for the necrosis and other chronic changes in the wall. In some cases collections of polymorphonuclear leukocytes and the associated changes in the uterine wall were limited to one horn of the uterus, or even to a localized area in one horn, and these observations may be considered as confirmatory evidence in favor of the conclusion that we have to deal in this condition with bacterial processes. In this connection it may perhaps be of interest to point out the fact that during the phase of the sexual cycle in which a large amount of estrogen is liberated, injection of non-pathogenic bacteria into the lumen of the ligated uterus leads to a more severe inflammatory reaction than during other periods of the sexual cycle, and especially during the lutein phase (7). If the inflammatory-necrotizing process is very pronounced in the uterus, a great part of the surface epithelium may be lost, the remaining parts being lined either with cylindrical or with squamous epithelium. However, the epithelial covering may be missing altogether, or may consist entirely of cylindrical epithelium. If the changes are less severe, squamous epithelium may be found in certain places and cylindrical epithelium in others. As far as the glands are concerned, these showed in some cases a marked development in the neighborhood of areas of inflammation or infection. In regions where there was much necrosis the glands were usually also injured or lost altogether, and in a number of instances it could be seen that where a part of the uterine cavity was lined with squamous epithelium and another part with cylindrical epithelium, the glands were preserved only beneath the cylindrical epithelium. This is presumably due to the fact that the squamous epithelium covers those areas in which an ulceration has previously led to a destruction of the glands, and where, secondarily, regeneration of the epithelium has taken place. I The following list indicates the frequency of this inflammatory-necrotizing or infectious process and its incidence in the various strains of mice. (A) Control Mice: 11: 1 breeding D mouse = 0.6% of all the mice of this group. (B) Mice Injected with 1-30 rat units of Estrogen: Strain A, 11: 1 mouse = 3.2% of all the mice of this group. (C) Mice Injected with 50 rat units of Estrogen: Strain A, I: 3 mice= 12% of all the mice of this group. (D) Mice Injected with 100 or More rat units of Estrogen: Strain A, I: 2 mice; C57, I: 8 mice; C3H, I: 20 mice; 11: 1 mouse; New Buffalo, I: 2 mice; 11: 1 mouse; CBA, I: 6 mice= 24.9% of all the mice of this group. (E) Mice Injected with a Combination of Estrogen and Anterior Pituitary Prepara- tions: 0 mice. (P) Mice Injected with a Combitution of Estrogen and Lutein Preparations: C57, I: 3 mice; 11: 2 mice= 13.5%. Altogether we found this inflammatory condition in 51 mice. In non- injected control mice it was found only once, and this was in a mouse belonging to the D strain. Injections of estrogen into mice increased the frequency of this condition and the frequency rose with the injection of increasing quantities of estrogen. As in other cases, estrogen dissolved in oil was much more effective than a watery solution. All the strains subjected to injections of estrogen were affected except the D strain and the Old Buffalo strain. How- ever, in one D control mouse the inflammatory-infectious process was also 424 LEO LOEB, V. SUNTZEFF, AND E. L. BURNS noted. The D and Old Buffalo strains were likewise almost entirely free of squamous epithelium as a covering of the mucosa of the uterus. In this connection an observation may be mentioned which, while not directly bearing on the problem under consideration, may be of some interest, A deposit of blood pigment was found in the uterine wall and especially in the inner muscle in a considerable number of mice which had been breeding. It is probable that hemorrhages occur in all breeding mice during the process of labor, perhaps as a result of the detachment of the placenta, and this sign may enable one frequently to distinguish between mice which have had litters and others which have been virgin for a considerable time following parturition.

DISCUSSION In our earlier investigations we have observed certain alterations in the uterus in mice treated with long-continued injections of estrogen, such as ulceration and apparent infection, the occasional transformation of cylindrical surface epithelium into squamous epithelium, and the presence of cystic glands. Other investigators have seen some of these changes and referred to them in their papers. We have mentioned the experiments of Selye, Thomson and Collip (4) in regard to the production of squamous epithelium in the uterus of mice injected with estrogen, either intraperitoneally or into the cavity of the uterus. Lacassagne noted that in mice injected with estrogen a cystic transformation of the glands may occur; he has furthermore referred to the production of squamous epithelium in the uterus. One of our aims, however, has been to study these changes, so far as possible, in a quantitative manner. The large number of mice which served for these investigations made a quantitative statistical treatment possible within certain limits. We found that all the changes named occurred also in mice not treated previously with estrogenic hormone, especially older mice. In these non-injected mice, however, they were rare, except for cystic change of the glands, which was observed somewhat more frequently and with increasing frequency with advancing age. As far as the frequency of these changes in the estrogen-injected mice is concerned, it was greater, on the whole, the larger the effective dose of estrogen. The effectiveness of estrogen was much increased by administering it dissolved in oil instead of in water; in this way absorption was much retarded and the effect was more continuous. In the case of the cystic glandular changes, however, the maximum effect was reached in the group of mice injected daily with 50 rat units of estrogen dissolved in water. It is probable that the high incidence of destructive ulcerative processes in the animals injected with 100 or more rat units of estrogen dissolved in oil accounts for the smaller number with cystic glandular changes in this group. In regard to strain differences, it was found that mice belonging to strain D were almost free from inflammatory-infectious conditions in the uterus and also from the occurrence of squamous epithelium as a covering of the uterine mucosa. On the other hand, the down-growth of the glands into the deeper tissues was observed most often in mice belonging to strains D and C3H. These were also the strains in which spontaneous carcinoma of the mammary GROWTH PROCESSES INDUCED BY ESTROGENIC HORMONES 42 5 gland occurred most frequently. Further observations will be necessary, however, to determine whether this parallelism is more than a coincidence. The principal fact established in these investigations is the lack of effectiveness of injections of estrogen in the production of cancerous or even precancerous conditions in the uterus of mice. In certain instances in which squamous epithelium covered the inner surface of the uterus there was some down-growth of the squamous epithelium into a few glands; there may perhaps have been, also, in a few cases, a metaplasia of the glandular epithelium into squamous epithelium, and in a few instances it appeared as if the squamous epithelium had grown a short way downward into the underlying connective tissue and had here formed some pearls; however, where this happened there had probably been a preceding necrosis, or at least a marked injury of parts of the wall of the uterus. Typical precancerous lesions which were in the process of changing into cancer were found in not a single animal. Like- wise, the down-growth of the glands into the muscle tissue was usually limited to one or a few small areas and was not associated with any marked proliferative activity, as was evidenced by the lack of mitoses and by the normal structure of the glands. In general, then, all the growth processes induced or intensified by injections of estrogen were of a very mild character and very slow, and in most cases mitoses were not seen in areas in which these growth processes occurred. There are two possible explanations for this relative ineffectiveness of estrogen in the uterus of the mouse. It may be assumed (1) that estrogen does not fix itself on the epithelium nor, perhaps, on the underlying connective tissue of the uterus to the same extent as it does in vagina and mammary gland. This, however, is not probable in view of the readiness with which estrogen produces typical uterine changes during the follicular phase of the sexual cycle. A second assumption is (2) that the epithelial structures of the uterus are more resistant to the growth-stimulating action of estrogen than are those of vagina, cervix and mammary gland. The second interpretation is in all probability correct. This is indicated also by the fact that while the epithelium of vagina and cervix undergo marked proliferative changes during estrus, the surface epithelium and glands of the uterus react to estrogen essentially by hyper- trophy. In the mammary gland tissue again not only do proliferative changes occur during the follicular phase of the sexual cycle, but even much more marked growth processes take place during pregnancy. We may therefore assume that the tissues of mammary gland, vagina, and cervix are more re- sponsive to growth stimulation by estrogen than are the uterine surface epithelium and glands. The degree of responsiveness to growth stimulation may be graded approximately in the following order : Mammary gland, vagina, cervix, and uterine epithelial structures. To this gradation of responsiveness to normal stimulation corresponds the readiness with which cancerous changes can be induced in these organs; they occur most readily in the mammary gland and least readily in the uterus. These considerations do not exclude the possibility that if still stronger stimuli were employed, carcinoma-like changes might ultimately be produced experimentally in the uterus of the mouse. The product of growth stimulation and the power of the tissues to react to growth 426 LEO LOEB, V. SUNTZEFF, AND E. L. BURNS stimulation determines the intensity and kind of growth that takes place. The second of these two factors may or may not be of a hereditary nature. If it should be hereditary, this relationship might be expressed in the equation H (heredity) X S (intensity of stimulation) = C (the readiness with which the cancerous transformation occurs). We have formerly expressed the relation between hereditary and stimulating factors by the same equation. While in no case could carcinomatous or even true precancerous changes be induced in the uterus of the mouse by injection of estrogen alone or of estrogen in combination with lutein or anterior pituitary preparations, still some abnormal proliferative changes were initiated by these means. These consist in the first place in a penetration of glands into the muscle tissue and, secondarily, in a metaplasia of the surface epithelium into squamous epithelium. It is very probable that such a transformation of cylindrical into squamous epithelium requires a greater intensity of proliferative stimulation of the epithelium than the maintenance of the structure of the unilayered cylindrical epithelium, and. that the transition of cylindrical into squamous epithelium needs therefore the action of special growth stimuli-either regenerative or hormonal in nature. In this case, however, we have to contend with the uncertainty as to the frequency with which the formation of squamous epithelium is due to a true metaplasia. As stated above, we have, in all probability, in the majority of cases to deal with a regenerative growth of the squamous epithelium of the cervix into the uterus. Yet there can be little doubt that in a number of instances a true metaplasia did occur. In addition to these growth processes there is the possibility that the cystic changes which take place in the uterine glands in mice with advancing age and under the influence of estrogen may represent a condition related to growth processes. We have found, furthermore, indications that a similar equation may apply to the change which takes place in the cylindrical epithelium when it becomes transformed into transitional and into squamous epithelium. This occurs under the influence of very slight stimulation in the epithelium of vagina and cervix, which is hereditarily sensitized to such a response. How- ever, it may also be produced in the epithelial tissues lining the uterus, but in this case the intensity of stimulation and the time during which the stimuli are active must be very much greater. The same requirement seems to appIy, also, to various other surfaces lined with cylindrical epithelium, where different kinds of intense stimulation, such as pressure and other mechanical and chemical factors, may induce a change of cylindrical into squamous epithelium. These considerations are in agreement with our former observations that under the influence of stimuli which cause cancerous growth, carcinomatous structures originating in the cylindrical epithelium of the uterine cervix may give origin also to structures consisting of squamous epithelium, and that gland- like formations which develop at the border between the cylindrical and squamous epithelium of the cervix may show characteristics intermediate between the epithelial invaginations found in the cervix and the uterine glands. The great resistance which is observed in the uterus of the mouse to cancerous and even to precancerous transformation of the epithelial structures distinguishes it from vagina and cervix, with their tendency to precancerous and even to cancerous changes under the influence of the long-continued ac- GROWTH PROCESSES INDUCED BY ESTROGENIC HORMONES 427 tion of growth stimuli. Even the area which we designated as the uterine cervix resembles in this respect the cervix more closely than it does the uterus, inasmuch as it may participate in the cancerous changes which occur in the other parts of the cervix and in the vagina. This abnormal response of the tissues confirms the conclusion that an intermediate zone between vagina and uterus should be distinguished from the latter because of the difference in responsiveness to growth-stimulating hormones observed in these tissues. SUMMARY Among approximately 500 mice, the majority of which had been injected with various doses of estrogen over different periods of time the microscopic study of the sex organs in no case showed cancerous changes or even true precancerous proliferations. There is reason for assuming that the reactivity of the uterine epithelial structures to growth stimulation is less than that of the corresponding tissues in vagina, cervix, and mammary gland, an assumption which is in accord with the behavior of these tissues during the sexual cycle and during pregnancy. We may therefore conclude that cancerous transformation depends, among other factors, on the product of the intensity of growth stimuli acting on a tissue and the responsiveness of the affected tissue. There are indications that in a similar manner the transformation of cylindrical epithelium into transitional or squamous epithelium also depends upon the product of the intensity of certain effective growth stimuli and an inherited responsiveness of these tissues to these stimuli. In a minority of the mice in these experiments certain changes were observed which represent abnormal but non-cancerous growth processes, namely, a penetration of the uterine glands into or through the musculature of the uterus and a metaplasia of the cylindrical surface epithelium and perhaps also of some glands into transitional or squamous epithelium. In a considerable number of cases, however, the squamous epithelium owed its origin to regenerative processes which led to an extension of the cervix epithelium into the uterus. It is possible that in the cystic transformation of the uterine glands, also, growth stimulation may be involved. All these changes may occur, although with much less frequency, in non-injected control mice, as a rule becoming more frequent with advancing age. Under the influence of estrogen they are produced, on the whole, the more readily the greater the dosage used and the more continuous its action.

REFERENCES

1. LOEB, L., BURNS,E. L., SUNTZEFF,V., AND MOSKOP,M.: Am. J. Cancer 30: 47, 1937. 2. SUNTZEBF,V., BURNS,E. L., MOSKOP,M., AND LOEB, L.: Am. J. Cancer 32: 256, 1938. 3. L~EB,L.: Endocrinology 12: 161, 1928. 4. SELYE,H., THOMSON,D. L., AND COLLIP,J. B.: Nature 135: 65, 1935. 5. LOEB,L.: Biological Bulletin 27: 1, 1914; Proc. SOC.Exper., Biol. & Med. 13: 162, 1916; Surg. Gynec. & Obst. 25: 300, 1917. STOCKARD,C. R., AND PAPANICOLAOU,G. N.: Am. J. Anat. 22: 225, 1917. 6. LOEB,L., BURNS,E. L., SUNTZEFF,V., AND MOSKOP,M.: Proc. SOC.Exper. Biol. & Med. 35: 320, 1936. 7. LINTON,C. S., KIPPEN,A. A., AND LOEB, L.: J. Infect. Dis. 56: 89, 1935.