Virchows Arch [Cell Pathol] (1984) 47:247-261

V'wehowsA ch/vB 9 Springer-Verlag 1984

Histochemical and ultrastructural characteristics of the endometrial connective tissue stroma from mice continuously fed diethylstilbestrol Robert J. Wordinger 1, Benjamin Highman 2, James W. Townsend 3, and David L. Greenman 2 i Department of Anatomy, North Texas State University, Texas College of Osteopathic Medicine, Fort Worth, Texas 76107 z Food and Drug Administration and Pathology, Services Project, National Center for Toxicological Research, Jefferson, Arkansas 72079 3 Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72202, USA

Summary. Histochemical and transmission electron microscopy were utilized to examine the endometrial connective tissue stroma of mice continuously fed diethylstilbestrol (DES). Virgin female mice were continuously fed diets containing 0, 320, or 640 ppb DES from 4 weeks of age until moribund. All animals reported on in this study were between 622 to 762 days of age when sacrificed. Light microscopy revealed irregular deposits of homogeneous acidophilic material throughout the connective tissue stroma with frequent accumulations seen immediately beneath the surface epithelial layer and surrounding blood vessels. Histochemical results indicated the presence of collagen and fibrin as components of the acidophilic material. Ultrastructural results revealed a homogenous stroma consisting of short segments of collagen fibrils enmeshed in an amorphous component. Numerous plasma cells were seen in close approximation to macrophages and lymphocytes. Fibroblast cell membranes exhibited micropinocytotic vesicles. Eosinophils were numerous in the stroma and often seen in close approximation to fibroblast projections. Vascular endothelial layers contained numerous micropinocytotic vesicles and marginal flaps. The accumulation of homogeneous material within the connective tissue stroma may represent products from collagen degradation and a subsequent localized immune response as well as plasma components. Key words: Diethylstilbestrol - DES - Endometrium - Connective tissue - Transmission electron microscopy

Introduction Reproductive tract abnormalities in female offspring whose mothers were taking the synthetic estrogen diethylstilbestrol (DES) are well documented. Herbst et al. (1971) were the first to report the association of in utero expoOffprint requests to: R. Wordinger at the above address

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sure to DES and vaginal and cervical clear-cell adenocarcinoma in human offspring at the time of puberty. Bern et al. (1975) showed that DES exposure during the neonatal period is associated with preneoplastic and neoplastic changes in the mouse cervix and vagina. Highman et al. (1977, 1980) reported cervical and vaginal adenosis and cervical and endometrial adenocarcinomas in mice continuously fed diets containing DES or 17-fl-estradiol from 6 weeks of age. Oviductal changes in the mice fed DES include epithelial cells with apical nuclei and dilated rough endoplasmic reticulum (Wordinger et al. 1980a). Uterine lesions have also been correlated with continuous estrogen exposure. Gardner and Allen (1939) reported the accumulation of acidophilic material within the connective tissue stroma of mice receiving continuous injections of 17-fl-estradiol. Dunn and Green (1963) produced the same lesion in adult female mice with a single injection of DES on the day of birth. Highman et al. (1977) reported a similar accumulation in mice continuously fed DES from 6 weeks of age. Very little information is available concerning ultrastructural changes associated with the connective tissue stroma of DES exposed mice. Studies of this type are significant since there is growing evidence that the connective tissue stroma may induce or specify the morphogenetic pattern and cytodifferentiation of epithelial membranes (Cunha and Fujii 1981). Exogenous substances may influence normal development by interfering with stromal functions thus leading to alterations in epithelial morphogenesis, differentiation or growth. The objective of this study was to describe the uterine connective tissue stroma within mice continuously fed DES. Preliminary results of this study have been reported previously (Wordinger et al. 1980 b). Materials and methods Animals. Specific-pathogen-free (SPF) mice of the BALB/cStCrltC3H/NCTR strain produced from cesarean-derived parentage were used in this study. They were selected from a nearly completed and unpublished larger study designed to investigate the long term effects of feeding diethylstilbestrol to mice. Mice were housed in polycarbonate shoebox cages with a filter bonnet. Purina autoclavable meal (5010 M) and water were given ad libitum. The animal room was thermostatically controlled around a set point of 22 ~ C on a 12 h light and 12 h dark cycle. Animals in the larger study were divided into groups fed diets containing 0, 5, 10, 20, 40, 160, 320, 640 parts per billion (ppb) DES starting at about 4 weeks of age. The DES was dissolved in 90% ethanol and mixed into the diet as described by Greenman et al. (1977). Control animals received the same feed without the DES. There were no scheduled serial sacrifices of mice. Necropsies and histopathologic studies were done on animals when they became moribund, died or developed palpable masses (mammary tumors) about I cm in diameter. Tissue preparation in current study. The fifteen animals reported on in this study included 5 fed 320, 5 fed 640 and 5 fed 0 ppb DES. They were removed from the larger study when they became moribund and ranged from 662 to 762 days of age. They were sacrificed by an overdose of ether and the reproductive tract was removed and studied for this report. The right uterine horn was fixed in Bouin's solution for 18 25 h and then washed for 24 h in a saturated lithium carbonate-70% ethanol solution. The tissues were routinely processed with an Autotechnicon on a 4 h schedule, embedded in a paraffin-Paraplast mixture (1:1)

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and sectioned at 5 p,m on a rotary microtome. The following staining procedures were used: (1) hematoxylin and eosin, (2) periodic acid-Schiff(PAS) with and without a diastase preincubation, (3)Movat's pentachrome stain (Movat 1955), (4)high iron diamine (Spicer 1965), (5) Congo red for amyloid (Luna 1968), (6) Masson's trichrome (Masson 1929), (7) phosphotungstic acid hematoxylin (Puchtler et al. 1963), (8)alcian blue (pH 2.5)-PAS (Spicer et al. 1967), (9) Weigert's fibrin (Lillie and Fullmer 1976), (10) elastic Van Giesons (Mallory 1961) and (11) the Ledrum-Acid Picro Mallory Method (Culling 1957). The left uterine horn was processed for electron microscopy by overnight fixation in 4 ~ C cacodylate-buffered 4% glutaraldehyde at pH 7.3. Tissues were subsequently washed in 0.2 M cacodylate buffer three (3) times for 10 min each. During the buffer washes, the temperature was gradually raised from 4 ~ C to 23 ~ C (room temperature). The tissues were trimmed and post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer at pH 7.3. Following osmium treatment the tissues were rinsed twice for 10 min each in deionized water. Dehydration and clearing were performed in a Reichert EM Processor using ethanol and acetone respectively. Infiltration was performed using mixtures of acetone and Epon-Araldite embedding mixture at room temperature. The tissues were then placed in labelled embedding molds filled with fresh Epon-Araldite and polymerized by heat. Embedded tissues were sectioned on a LKB or a Reichert OmU3 ultramicrotome using glass or diamond knives. Semi-thin (1 ~tm) sections were affixed to glass slides and stained with 1% toluidine blue. Thin sections (60-100 m) were collected on uncoated copper grids and then stained with uranyl acetate and lead citrate (Sato 1968). Grids were then examined by means of a Philips EM201 electron microscope.

Results

Light microscopy. The endometrial connective tissue stroma of control animals consisted of numerous collagen fibers and various cellular elements normally associated with this tissue (Fig. 1 a). Numerous fibroblasts as well as occasional plasma cells, mast cells and macrophages were observed. The endometrial connective tissue stroma of DES-treated mice contained irregular deposits of acidophilic material. This material had a homogeneous appearance and was seen to accumulate immediately beneath the surface epithelial layer (Fig. 1 b). The material is characterized by a paucity of cells and fibrous material as well as its eosinophilia (Fig. 1 c-d). Similar material was seen in the myometrium surrounding groups of muscle fibers and blood vessels. The endometrium displayed hyperplastic glands which penetrated the muscularis. Isolated foci of squamous epithelial cell metaplasia were also observed in DES exposed mice. The results of the histochemical procedures are presented in Table 1. The acidophilic, homogeneous material stained intensely with the diastaseperiodic acid-Schiff method but was negative with both Congo red and the high iron diamine methods. A positive reaction for collagen was obtained with Movat's pentachrome, phosphotungstic acid hematoxylin and Masson's trichrome methods. When the alcian blue-periodic acid-Schiff procedure was utilized, the material was Schiff positive but negative for alcianophilia. The material was also negative for elastic fibers but positive for fibrin localization. Electron microscopy. Control animals displayed a normal endometrial connective tissue stroma consisting of numerous collagen fibers in various planes of section as well as fibroblasts (Fig. 2). Occasional mast cells, plasma

Fig. I a. Light micrograph of the endometrial connective tissue stroma from a control animal. Uterine lumen (L), Uterine gland (g) H&E (700 • ). b Light micrograph of the endometrial connective tissue stroma from a DES-treated (640 ppb) mouse. Note large accumulation of amorphous material (AM) below the endometrial surface epithelial layer. Uterine lumen (L) H & E (700 • ). e Light micrograph of the endometrial connective tissue stroma from DEStreated (320 ppb) mouse. Note accumulation of amorphous material (AM) around endometrial glands (arrows) H & E (300 • ). d Higher magnification of the endometrial amorphous material (AM). Note the absence of cells and fibrillar material. H & E (700 • )

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Table 1. Histochemistry of the endometrial connective tissue stroma of DES exposed mice

Procedure

Homogeneous Material

1. Hematoxylin and eosin (H&E) 2. Diastase-periodic acid-Schiff(D-PAS) 3. Diastase-alcian blue-periodic acid-Schiff (D-AB-PAS) 4. High iron diamine (HID) 5. Congo red (CR) 6. Movat's pentachrome (MP) 7. Masson's trichrome 8. Phosphotungstic acid-hematoxylin (PTAH) 9. Weigerts fibrin 10. Elastic van Gieson's 11. Ledrum-acid piero-mallory method (LAPM)

Eosiniphilic Periodate (red) positive Periodate positive but alcianophilic negative Negative Negative Positive (yellow) for collagen Positive (blue) for collagen Positive (reddish-brown) for collagen Black-purple-positive for fibrin Negative for elastic Blue areas positive for collagen Red areas positive for fibrin

cells and macrophages were seen. The ultrastructure of the connective tissue stroma of DES animals was striking in contrast. The acidophilic, homogeneous material was characterized by the virtual absence of collagen fibers and the paucity of connective tissue cellular elements (Fig. 3). Occasional short segments of fibers were observed scattered randomly within the homogeneous matrix. Clear spaces were observed (Fig. 4) which may have housed collagen fibers. Fine aggregates of material were occasionally seen in close approximation to these areas (Fig. 5). The interfibrillar material contained material that appeared finely granular (Fig. 5). Plasma cells were seen associated with the homogeneous material. The morphology of these cells varied, with a large number containing enlarged cisternae filled with material indicative of active cellular synthesis (Fig. 6). Plasma cells were frequently seen in close proximity to macrophages and lymphocytes (Fig. 6) while eosinophils were seen in close approximation to fibroblasts and macrophages, although no direct cell contact was observed. Blood vessels within the connective tissue stroma of DES exposed mice displayed numerous micropinocytotic vesicles on both the luminal and basal borders (Fig. 7). Fibroblasts with long cellular processes also displayed numerous micropinocytotic vesicles along the cell wall. Discussion Highman et al. (1977) reported an accumulation of a homogeneous, acidophilic material within the uterine connective tissue stroma of mice continuously fed DES and referred to this material as hyalin. Gardner and Allen (1939) reported a similar accumulation in mice receiving continuous injections of 17-fl-estradiol. Dunn and Green (1963) produced the same lesion in adult mice with a single injection of DES on the day of birth. Our current histochemical studies indicate that the uterine homogeneous material is composed of collagen but may contain a variety of plasma proteins as

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Fig. 2. Endometrial connective tissue stroma of a control animal showing numberous collagen fibers as well as parts of fibroblasts. (9,000 • )

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Fig. 3. Low magnification electron micrograph of the endometrial connective tissue stroma of a DES-treated (640 ppb) mouse showing amorphous material (AM). Note the presence of only a few collagen fragments (arrows). (9,900 x )

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Fig. 4. Higher magnification of the amorphous material showing clear spaces as well as short collagen fragments (arrows). (20,625 • )

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Fig. 5. Higher magnification of the amorphous material (AM) showing distinct collagen fibers (6'). Note the appearance of aggregates of material which appear to be finely granular. (90,000 x )

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Fig. 6. Low magnification electron micrograph of the connective tissue stroma of a DES-treated (640 ppb) mouse showing numerous plasma cells (PC) in close apposition to macrophages (3//) and lymphocytes (L). Some plasma cells have enlarged cisternae indicative of antibody synthesis. (6,000 x )

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Fig. 7. a Low magnification electron micrograph of an endometrial capillary from a DEStreated (320 ppb) mouse. (13,500 x ). b Higher magnification of an endometrial capillary showing numerous micropinocytotic vesicles (arrows) as well as marginary flaps (45,000 • )

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indicated by positive localization of fibrin. It may be more accurate to refer to this material as fibrinoid. Fibronoid is also eosinophilic, occurs as homogeneous deposits within connective tissue or blood vessels and contains a variety of plasma proteins including fibrin, albumin, immunoglobulins and complement (Dixon 1961). Of particular interest to this investigation is the report that fibrinoid often appears in foci of immunologic injury and may represent aggreagates of antigen, antibody and complement (Dixon 1961). Fibrinoid may also be associated with increased vascular permeability induced by either inflammation or hemodynamic influences and be derived from plasma proteins (Movat et al. 1960). At the ultrastructural level fibrinoid has been described as containing damaged collagen fibrils associated with finely fibrillar and amorphous material (Johannessen 1980). In this study the presence of plasma cells, lymphocytes and macrophages within the homogeneous material indicates the occurrence of a local immune response. The close apposition and in some cases direct physical contact of this cell population suggests intercellular communication as occurs in the presence of an antigen (Nielsen et al. 1974). The various stages of synthetic activity displayed by the plasma cells is suggestive of antibody synthesis. It thus appears that a currently unidentified antigen has been processed by uterine macrophages, triggering a local immune response. The histochemical results indicate that the homogeneous material is not amyloid nor does it have a significant mucosubstance component. However, the material was positive with various collagen staining procedures. Electron microscopy revealed a homogeneous, amorphous material devoid of collagen fibers. This paradox is best explained by the suggestion that this material is the product of collagen degradation as well as an antigen-antibody complex. Padykula (1976) has reported the extracellular degradation of collagen by a neutral collagenase in the endometrium. /n vitro studies have shown that collagenase attacks the collagen fibril and cleaves the molecule to produce 2 fragments (Gross 1974). The fragments of remaining collagen molecules would denature at body temperature and wandering macrophages could phagocytize these fragments. It is known that the macrophage-lymphocyte-plasma cell interaction is a central event in the initiation and regulation of the immune response (Unanue 1972). The presence and close interaction of these cells within the homogeneous material is suggestive of a local response. The uterine fibrinoid changes described in this study seem to be due primarily to the dietary DES. A preliminary survey (unpublished results) of the larger study from which these mice were obtained, indicates that the onset of these changes was shorter and the incidence and severity greater with increasing doses of DES. For example, 6/66 controls, 65/70 fed 320 ppb DES and 62/70 fed 640 ppb DES demonstrated the changes described in this paper. All 6 controls showing fibrinoid changes were on the experiment over 730 days and the changes were confined largely to the endometrium. In the treated mice, the fibrinoid changes were more extensive and

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severe and involved the perivascular tissue and smooth muscle of the myometrium. The fibrinoid changes were seen as early as 184 days following DES exposure. The moribund condition of many BALB/C mice appears to be due largely to aging and not DES exposure. Again, preliminary unpublished results from the larger experiment indicate that few mice were removed before 456 days. The number surviving 730 days included 24/66 controis, 23/70 fed 320 and 7/70 fed 640 ppb DES. Mammary adenocarcinomas caused removal before 730 days of 0, 2 and 13 mice fed 0, 320 and 640 ppb DES respectively. This may account in part for the relatively few mice fed 640 ppb DES surviving past 730 days. What role might diethylstilbestrol play in collagen degradation? Wira and Sandoe (1977) indicated that the endometrium is capable of a humoral immune response which is under estrogen and progesterone regulation. Progesterone has been shown to be an inhibitor of uterine collagenase activity. (Jeffrey and Koob 1973; Holme and Wolssner 1975). However, 17-fl-estradiol has been implicated in the rapid degradation of endometrial collagen during the post-partum period in the rat (Padykula 1976). During the postpartum period of the rat, progesterone levels decline steadily (Wiest 1970) and 17-fl-estradiol reaches its peak (Yoshimaga 1976). In female mice continuously fed diethylstilbestrol, the ovaries are inactive and few, if any, corpora lutea are seen, indicating minimal circulating levels of progesterone (Highman et al. 1977). The presence of diethylstilbestrol, a powerful estrogen, and low levels of progesterone would favor collagen degradation. It is possible that endometrial connective tissue stromal cells (i.e. macrophages) respond to the continuous presence of an estrogen and/or the lack of progesterone by secreting or activating collagenase. The collagenase would initiate collagen breakdown leading to a homogeneous stroma and a local immune response as described in this study. The exact mechanism by which diethylstilbestrol initiates this response is not clear at the present time. In addition to collagen degradation, the homogeneous stroma material may contain various plasma proteins associated with increased vascular permeability. Blood vessels within the connective tissue stroma of DES treated mice displayed numerous micropinocytotic vesicles within the endothelial cells. Light microscopy also localized fibrinoid material in the wall of many endometrial vessels. Estrogen influence on vascular events has been described previously. Intercellular edema as well as a fluid collection within the uterine lumen is common in rodents (Carrol 1945). Bindon (1969) has described increased capillary permeability leading to intercellular edema. Increased permeability has been associated with the development of vascular fenestrations (Martin et al. 1973) as well as the appearance of pores (Freiderici 1967) and marginal folds (Fawcett 1981). Our studies indicate a high degree of vascular permeability allowing the passages of protein rich fluid into the extravascular spaces. The stromal changes induced by exposure to DES are important since there is evidence that the stroma may induce or specify the morphogenetic pattern and cytodifferentiation of epithelial membranes (Cunha and Fujii

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1981). In utero, exogeneous substances may influence normal development by interfering directly or indirectly with stromal functions. Cunha (1976) has shown for example that the cytodifferentiation and morphogenetic pattern of the immature Mullerian epithelium is induced by the stroma associated with it. Also, in adult tissues, exogenous hormones may alter normal stromal-epithelial interactions leading to alterations in epithelial morphogenesis, growth and/or function. The isolated foci of squamous cell metaplasia seen in the DES exposed mice may be a reflection of this type of alteration. It may be significant to note the localization of significant amounts of fibrinoid material immediately beneath the surface epithelial layer although not necessarily in apposition to foci of squamous cell metaplasia. Clearly, more studies are needed to determine what effect, if any, DES has on the stroma-epithelial interation in adult tissue. In conclusion, the acidophilic, homogeneous material seen in the endometrial stroma of DES exposed mice resembles fibrinoid. Histochemistry revealed the presence of collagen and fibrin within this material while electron microscopy demonstrated scattered collagen fibrils and a fine amorphous ground substance. The constituents of this material probably originated from collagen breakdown and a subsequent localized immune response as well as increased vascular permeability leading to the extravascular accumulation of plasma proteins. References Bern HA, Jones LA, Mills KT, Kohrman A, Mori T (1976) Use of the neonatal mouse in studying long-term effects of early exposure to hormones and other agents. J Toxicol Environ Health [Suppl] 1:103-116 Bindon BM (1969) The incorporation of x31I-iodinated human serum albumin in the ovary and uterus before implantation in the mouse. J Endocrinol 45 : 543-548 Carroll WR (1945) Variations in water content of the rat's uterus during continuous estrogenic treatment. Endocrinology 36: 266-271 Culling CEA (1957) Handbook of histopathologieal technique. Butterworth, London Cunha GR (1976) Stromal induction and specification of morphogenesis and cytodifferentiation of the epithelia of the Mullerian ducts and urogenital sinus during the development of the vagina in mice. J Exp Zool 196:361 Dixon F (1961) Mechanism of cell and tissue damage produced by immune reactions, 2nd Internat! Sym Immuno Pathol. Benno, Schwabe and Co, Basel Dunn TB, Green AW (1963) Cysts of the epididymis, cancer of the cervix, granular myoblastomas and other lesions after estrogen injection in newborn mice. J Natl Canc Instit 31:425-455 Freiderici HHR (1967) The early response of uterine capillaries to estrogen stimulation. An electron microscopic study. Lab Invest 17:322-333 Fawcett DW (1981) The cell. WB Saunders Philadelphia Gardner WU, Allen E (1939) Malignant and non-malignant uterine and vaginal lesions in mice receiving estrogens and androgens simultaneously. Yale J Biol Med 12:213-234 Greenman DL, Dooley K, Breeden CR, Gass GJ (1977) Strain differences in the response of the mouse to diethylstilbestrol. J Toxicol Environ Health 3 : 589-597 Gross J (1974) Collagen biology: Structure, degradation and disease. In: The harvey lectures, 1972-1973. Academic Press, New York, pp 351432 Harris ED Jr, Vater CA, Mainardi CL, Werb Z (1978) Cellular control of collagen breakdown in rheumatoid arthritis. Agents Actions 8:36-41 Herbst AL, Alfedder H, Poskanzer DC (1971) Adenocarcinoma of the vagina: Association

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