Acute and permanent growth effects in the mouse uterus after neonatal treatment with estrogens

Reproductive Toxicology, Vol. 7, pp. 137-153, 1993

0890-6238/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.

Printed in the U.S.A. All rights reserved.

ACUTE AND PERMANENT GROWTH EFFECTS IN THE MOUSE UTERUS AFTER NEONATAL TREATMENT WITH ESTROGENS ANDERS HALLING a n d JOHN-GUNNAR FORSBERG Department of Anatomy, Lund, Sweden Abstract m Acute and late effects of neonatal estrogen treatment were studied in NMRI mice treated with diethylstilbestroi (DES) or estradiol-17~ (E2) on days I to 5 after birth (estrogenized females). The uterine wet weight (UWW) response in 6-day-old females, after 5 daily treatments with DES, had a peak at a daily dose of 10 -z pg DES and declined with higher doses. Females (26-day-old) treated with DES or E 2 neonatally had a reduced U W W response to a challenge with DES; on a dose basis, DES was more effective neonataily than E 2 . A single injection with DES or E 2 in the neonatal period stimulated mitotic activity in the uterine horn epithelium; the U W W response to a 24-h DES pulse increased from day 2 to 6 after birth, but the uterine epithelial mitotic rate response decreased. Epidermal growth factor (EGF) was a more potent stimulator of mitotic activity than DES or E 2 . DES inhibited mitotic activity in the uterine cervical epithelium; EGF protected from this DES effect. In adult estrogenized females, EGF-induced uterine stimulation of 3H-thymidine incorporation subsided more rapidly than in control females; uterine epithelium did not respond to EGF in vitro. Uterine stroma of adult estrogenized females is postulated to house a population of cells under nonovarian proliferation control while the uterine epithelium may be under influence of an ovary-dependent proliferation inhibiting factor that is gradually lost under culture conditions. Key Words: uterus; diethylstilbestrol; estradiol; epidermal growth factor; proliferation; mouse.

INTRODUCTION

increased estrogen receptor level has been reported from the neonatal mouse uterus after estrogen treatment (8-10). The aim of the present study was to investigate the weight and proliferative responses of the neonatal mouse uterus, which is an organ in a state of development and differentiation, to single and repeated doses of natural and synthetic estrogens, and to analyze possible differences in dose-response relationship between the two types of estrogens. Because earlier studies indicated a difference in proliferative response to estrogen between the epithelium in the uterine horns and uterine cervix of neonatal females (11,12), this difference has been further analyzed with respect to type of estrogen and dose dependency. The neonatally estrogen-treated females were challenged with estrogen in immature life, and the uterine response related to type of estrogen and doses used in neonatal life. A major interest was to study possible disturbances in uterine growthcontrolling mechanisms in adult females after neonatal estrogen treatment, including the consequences of a possibly disturbed interaction between estrogen and epidermal growth factor (EGF), which has a physiologic role in estrogen-induced uterine growth (13-15). The goal was to get a comprehensive view of the disrupting effects of neonatal estrogen treatment on later uterine growth characteristics.

In earlier studies on neonatal rat uterine response to estrogen, a full response was not seen until 1 to 2 weeks after birth (1). The "induced protein" (IP) of Gorski appeared in 10-day old rats; at 5 days estradiol (0.2/~g) failed to stimulate RNA synthesis and amino acid incorporation (2,3). By increasing the estradiol dose, a near maximal IP response was seen in 5-day old rats but a full response was not obtained until after a further few days (1). A dissociation has been described between the genomic response to estrogens (RNA and protein synthesis), which appears in 10-day old rats, and the nongenomic responses (uterine eosinophilia and edema), which appear in 15-day old animals (4). According to DeSombre (5), a significant uterine weight response (UVW) to natural or synthetic estrogens appears in rats around day 13 after birth. However, repeated doses of estradiol induced a full response (6) and later diethylstilbestrol (DES) and ethinyl estradiol were found to be more potent than estradiol in increasing uterine weight in perinatal rats, which was ascribed to estradiol being bound to a-fetoprotein (7). An Address correspondence to Dr. J.-G. Forsberg, Department of Anatomy, Biskopsgatan 7, S-223 62 Lund, Sweden. Received 27 August 1992; Revision received 15 October 1992; Accepted 17 October 1992. 137

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Volume 7, Number 2, 1993

MATERIALS A N D METHODS

e x p e r i m e n t s related to estrogen effects in immature females, dimethylsulfoxide (DMSO; E. Merck, Darmstadt, FRG) was used instead of ethanol. F r o m the stock solution, lower concentrations were prepared by further dilution with olive oil. The final dose to be injected was dissolved in 0.025 m L vehicle. In initial d o s e - r e s p o n s e experiments, the D E S dose range was 10 -~° to 5/xg and the E 2 was 10 -2 to 50 /xg. Based on the results from these experiments, daily doses of 10 -2/xg D E S or 5 ~xg D E S or E 2 were used in the majority of the following analyses except in one case in which the lower dose was 10 -6/xg D E S . The exact doses used are given in connection with presentation of the results from each type of e x p e r i m e n t and are also included in Figure 1. To study the specificity of the neonatal U W W response, one group of females was treated with daily doses of 50/xg P for the first 5 days after birth, and killed on day 6. The same dose of P was also used in combination with 10 -2/xg or 5 txg D E S to study the effect of P on the estrogen response. The females were given separate injections of P and DES at different locations. All treatments were given subcutaneously.

Animals

The animals used belong to a closed randombred colony of N M R I mice, originally obtained from Charles River Wiga, Sulzfeld, F R G . The animals were fed a standard pellet diet and given tap w a t e r ad libitum; they were kept in a controlled e n v i r o n m e n t (temperature, humidity) with a 12-h light/12-h dark schedule. S e r u m f r o m females was regularly checked for antibodies to m o u s e hepatitis virus and always found to be negative. The females were allowed to give birth to their litters in separate cages. Within 24 h after birth, a sex diagnosis was made and litters were culled to 8 female pups. All the female pups in one and the same litter were e x p o s e d to only one and the same type o f treatment. Treatment

of females

Diethylstilbestrol (DES), estradiol-17fl (E2), and p r o g e s t e r o n e (P) were obtained from Sigma Chemical Co., St. Louis, MO. A stock solution was p r e p a r e d by dissolving the drugs in a minimal a m o u n t of ethanol and diluting with olive oil. In

uterine weight in mg/ bodywei ]hi in gram ± SO

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Fig. 1. Effect on uterine wet weight (related to bodyweight) of treatment with different doses of DES, estradiol-17/3 (E2), and progesterone (P) on days 1 to 5 after birth, females killed on day 6. N = number of females studied per group. Within each treatment group, statistical comparisons were made between a certain dose group and the group next to the left. The groups "10 -1° /zg DES," "10 -2 /zg E2," and "50 /~g P" were compared with the group "olive oil." Significance levels: *0.05 > P > 0.01; **0.01 > P > 0.001; ***P < 0.001. ns-nonsignificant.

Neonatal estrogen and uterine response • A. HALLINGand J.-G. FORSBERG Females were treated daily during the whole neonatal period (days 1 to 5 after birth) or for only part of it and killed by decapitation on day 6. In some experiments the females were given a single treatment at various times in neonatal life and killed 24 h later. Finally, females were treated with DES or E 2 neonatally and challenged with DES (10 -2/xg or 5 p.g) on days 21 through 25 or on day 25 only. These females were killed by cervical dislocation on day 26. Control females were treated with vehicle only. Uterine wet weight response to estrogens in neonatal life and in 26-day old females after a challenge to D E S After killing the females they were weighed on a Precisa 300M balance. The abdominal wall was opened and the uterus cut at the uterotubaljunction on either side as well as just below the level where the two uterine horns converge to form the cervix. The uterus was then removed en bloc and weighed on a Mettler balance. The quotient between the uterine weight (in milligrams) and the bodyweight (in grams) was calculated. For some treatment groups (all with N = 30 observations) the distribution of the quotients deviated significantly from a normal distribution with respect to skewness and/or curtosis. To avoid the consequences of these complications, different treatment groups were compared with the nonparametric Mann-Whitney test for unpaired samples. The null hypothesis was rejected at P < 0.05. Studies on the mitotic rate response to estrogens and E G F in the neonatal uterus Litters of 8 neonatal females were treated subcutaneously with 5/zg DES, 10 -2 p.g DES, 5/zg E2, or vehicle only (olive oil) on the day of birth or day 3 or 5 after birth and killed 24 h later. Slow-release cholesterol-based pellets containing receptor grade mouse epidermal growth factor (EGF; from Sigma Chemical Co., St. Louis, MO) were prepared by Innovative Research of America, Toledo, OH, USA (3/zg per pellet, 3 day release). Pellets were divided into quarters, and the quarters of EGF pellets or placebo pellets were implanted subcutaneously on the back of 3- or 5-day-old females. The females were killed 24 h later, which means that they had been exposed to approximately 250 ng released EGF. Finally, 3- and 5-day-old females were treated with both subcutaneous injection of 5/zg DES and EGF implant. Four hours before death the female pups were

139

treated with 0.5 (day 2 of life) or 1 /zg (day 4 or 6) colchicine (Sigma Chemical Co.) in 0.05 mL RPMI 1640 medium (Flow Laboratories, Irvine, Scotland). The genital tract was fixed in Carnoy's solution, embedded in paraffin, and serially sectioned transversly with a section thickness of 6/zm. The sections were stained in hematoxylin. The number of colchicine-arrested epithelial metaphases were counted and related to the total number of epithelial cell nuclei in the uterine horns or the uterine cervix (common cervical canal). From the uterine horns, a total of at least 1000 nuclei were counted from 4 to 8 randomly chosen sections. From the uterine cervix, the number of cell nuclei were counted just above the level of the top of the vaginal fornices, at the top level, and in two randomly chosen sections at a lower level. The efficiency ofcolchicine therapy was assessed from the absence of anaphase or later mitotic stages in the section series. The mitotic rate was expressed as percent metaphases among the total number of cell nuclei counted. For comparisons between the percent values from different groups, Student's t test was used with correction for the number of degrees of freedom when error variances differed. Studies on the response o f the adult uterus to estrogens and E G F Eight-week old females (controls and neonatally DES-treated) were ovariectomized. Two weeks later they were treated subcutaneously with 1/~g E z in olive oil and killed 24 h later, or they received EGF implants. EGF-containing pellets of the same type as described above or placebo pellets were implanted under the kidney capsule (a quarter of a pellet per mouse, 750 ng EGF, 3-day release). Females were killed 24, 48, 72, 96, or 240 h after pellet implantation. After killing the females, the uterus was dissected out and rinsed from fat and connective tissue. It was then placed in 3 mL Minimal Essential Medium (MEM; Flow Laboratories, Irvine, Scotland) containing 3/xCi [methyl-3H]thymidine (specific activity 5.0 Ci/mmol; Amersham Sweden Inc., Solna, Sweden). The tissue was incubated for 4 h (37 °C; 95% air, 5% CO2 atmosphere). After incubation the tissue was washed in 5% trichloroacetic acid (E. Merck Inc., Darmstadt, Germany) for a total of 30 min with three changes. Finally, the tissue was solubilized in liquid scintillation vials (plastic) with 2 mL Soluene (Packard Instruments, Groningen, Netherlands). After addition of 10 mL scintillation fluid (POPOP; Packard), counting of radioactivity took

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place in a LKB Wallac 1214 Rackbeta Counter. For statistical analysis of the results, the DPM per uterus values obtained were transformed to Iogl0 values using the transformation log (x + 1). Group means of transformed values were used for comparisons with Student's t test with correction of the number of degrees of freedom when error variances differed.

Culture of endometrial epithelial cells Uteri were obtained from 8-week-old females treated with different doses of DES (10 -6 , 10 -4 , 10 -2, 1, or 5/xg per day) or vehicle only (controls) for the first 5 days after birth. One group of DEStreated females (5 tzg DES per day for 5 days after birth) and one group of control females (olive oil neonatally) were ovariectomized at 8 weeks and killed two weeks later. Uterine horns, freed from mesentery and opened longitudinally to expose the endometrial surface, from 15 females were pooled in 5 mL Hanks' balanced salt solution (HBSS; Flow Laboratories, Irvine, England). The tissue pieces were then put into 5 mL of Ca 2+- and Mg2+-free physiologic buffered saline (PBS, pH 7.4) containing 0.5% trypsin (Statens Bakteriologiska Laboratorium, Stockholm, Sweden) and then incubated at 4 °C for 1 h, followed by 1 h incubation at 37 °C (16). After incubation, the uteri were transferred to Minimal Essential Medium where D-valine had been substituted for by L-valine (MEM D-valine; Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal calf serum (FCS; Flow Laboratories, Irvine, Scotland). Using a dissecting microscope, the epithelial cells were gently freed from the underlying stroma with a pair of fine glass needles with blunt ends. The epithelial cells were further dispersed into single cells or small clusters by repeated pipetting with a Pasteur pipette. The cell suspension was then diluted to 75 mL with PBS. With this procedure, a suspension of endometrial epithelial cells was obtained and this suspension was seeded on to 60-mm Petri dishes (Nunc Inter Med, Roskilde, Denmark) that previously had been coated with rat tail collagen prepared as described by Strom and Michalopoulus (17). The cell suspension was seeded onto a pool of culture dishes, the size of which was large enough for 3 dishes with the same suspension being studied at every time stage in an experiment. Irrespective of the number of dishes used in an experiment, care was taken to distribute roughly the same amount of cell material to each culture dish. Each experiment was repeated 3 to 4 times. The endometrial cells were cultured at 37 °C in a 95% air, 5% CO 2 atmosphere. The culture medium was MEM D-valine supplemented with 10% FCS, 2

Volume7, Number 2, 1993 mM glutamine (Flow Laboratories, Irvine, Scotland), benzylpenicillin (100 IU/mL), and streptomycin sulfate (20 ~g/mL). MEM D-valine is selective in supporting growth of endometrial epithelial cells in preference to stromal cells (18,19). Culture medium was changed on days 2 and 4. On the day of culture start and on the following 4 days, three culture dishes (from the pool of 15 dishes used for seeding one and the same cell suspension) per day were pulsed with 3 ~Ci [methyl 3H]thymidine (specific activity 5.0 Ci/mmol; Amersham Sweden AB, Solna, Sweden) for 4 h. At the end of the pulse period, medium and floating cells were discarded and the dishes were rinsed twice with PBS. To remove adherent cells, each dish was incubated in 2 mL Ca 2+-, Mg2+-free PBS with 0.5% trypsin (Statens Bakteriologiska Laboratorium, Stockholm, Sweden) and 0.25% EDTA (Sigma Chemical Co., St. Louis, MO) at 37 °C for 20 min. The free cells were washed in PBS and pelleted by centrifugation at 2000 × g (average) for 5 rain. The pellet was stored at - 2 0 °C until further analysis. Each experiment was repeated three times. For studies on the effect of EGF, cultures of uterine epithelial cells from 8-week-old control females and estrogenized females (daily doses of 5/zg DES for the first 5 days after birth) were set up as described above, using the same type of medium. After 24 h, the cells were washed in 3 changes of PBS (pH 7.4) and incubated for 1 h in serum free medium (basal medium: DMEM/F12, 15/raM HEPES, glutamine 2 mM with added antibiotics). The medium was then changed to basal medium supplemented with insulin (I0 p,g/mL)-transferrin (10 /zg/mL)-sodium selenite (10 ng/mL; all from Boehringer Mannheim Scandinavia, Bromma, Sweden). Half the number of culture dishes (5 out of 10) with control cells and cells from estrogenized females were supplemented with EGF (20 ng/mL; Sigma Chemical Co., St. Louis, MO). Finally, 3 /zCi 3H-Tdr were added to the culture dishes, and incubation occurred for 4 h as described above. The further processing of the cells was as described above. The experiments were repeated four times.

Assays of DNA synthesis and counting of radioactivity Following thawing, the cell pellet was digested overnight with 1 mL papain solution (Sigma Chemical Co.; 125/zg/mL) at 60 °C as described by Kim and colleagues (20). The DNA content (in arbitrary units) was measured in duplicates with a minifluorometer (TKO 100, Hoefer Scientific Instruments, San Francisco, CA USA) using the fluorochrom bis-

Neonatal estrogen and uterine response • A. HALLINGand J.-G. FORSBERG benzimid H 33258 (Hoechst 33258; Riedel-de Haen AG, Seeize, Germany). For measuring DNA synthesis, 100/xL in duplicates from the original cell digest was pipetted into liquid scintillation vials together with 3 mL Instagel (Packard Instruments Co. Inc., Downers Grove, IL, USA). Counting of radioactivity took place in a LKB Wallac 1214 Rackbeta Counter. Proliferative activity was expressed as log (DPM per ng DNA) using the transformation formula log (x + 1).

RESULTS

Uterine wet weight Control animals. The uterine wet weight (UWW) in a group of 6-day-old untreated females was compared with that in a group of similarly aged females treated with vehicle for the first 5 days after birth. There was no significant difference in UWW or body weight between these two groups, thus indicating that handling of the neonatal females and treatment with vehicle did not affect body weight or UWW of the females. Females treated with various daily doses of DES (10 -I° to 5 tzg) or E 2 (10 -2 to 50 txg) in olive oil for 5 days after birth and killed on day 6. The results are demonstrated in Figure 1. The daily doses of 10 -t° and 10 -8/zg had no effect on UWW. With a daily dose of 10 -6 t~g DES, the UWW was significantly increased above the level in controls (0.01 > P > 0.001) and above the 10 -8/zg level (0.05 > P > 0.01). Daily doses of DES in the range l0 -6 to 10 -2/zg resulted in a progressively increased UWW, and the responses were significantly increased at each 10fold increase of dose. The 10-2-/zg dose resulted in maximum response, which means a 235% weight increase compared with controls. A 10-fold higher dose (10-~/zg) had a less stimulatory effect than 10-2 /zg, and this reduction was significant. The response was still more diminished with higher doses (1 and 5/zg per day); the l-p~g response was significantly lower than the 10-2-/zg maximum response, and 5 /zg had a significantly lesser effect than 1/zg. After treatment with 5/zg DES, the response was 148% of the control level and intermediate between that seen with 10 -4 and 10 -3/zg DES on the ascending limb of the dose-response curve. With E2, the response increased progressively with daily doses from 10 -2 to 50/zg per day. The 10-2-/zg response was intermediate between that

141

seen with 10 -6 and 10 -4 /~g DES; the 5/zg E z response was similar to that obtained with 10 -3 /zg DES. Even with 50/xg E 2, the UWW response did not reach the same high level as seen with 10 -2/zg DES. Daily doses of 50/~g P for 5 days after birth had no effect on the UWW. Cumulative UWW response in 6-day-old females after treatment for different periods (1 to 5 days) with a low (10 -2 txg) or a high (5 txg) daily dose of DES. The results are shown in Figure 2. Six-dayold females treated with a single dose of 10 -z/xg DES on the day of birth had a UWW not different from that in controls. Treatment with the same dose on days 1 and 2 resulted in a significantly increased UWW at 6 days. A prolongation of the treatment period from 2 days to 5 days further increased the response. With the high DES dose (5/zg), the results were different. A single treatment on the day of birth significantly increased UWW in 6-day-old females, and the response was further increased after treatments on both day 1 and day 2. Additional treatments after day 2 did not further increase the response. After 2 and 3 treatments, a plateau level was reached from which the response declined when treatment also included days 4 and 5. The 24-h effect of a single DES dose (10 -2 or 5 Ixg) at different neonatal ages. The results are shown in Figure 3. A single dose of 10 -2/~g on the day of birth slightly but significantly increased the UWW approximately 7% above the control level on day 2. With the high dose (5 /zg), the UWW was again slightly but significantly increased (approximately 13%). When similar 24-h experiments were repeated on each of the following days until day 5, progressively increasing responses were noted and the 5-~g responses were not statistically different from the 10-2-/zg responses up to and including day 5. The UWW in 6-day-old females after treatment with 10 -2 /zg DES on day 5 was approximately 143% of the control value; the corresponding response with the 5-/zg dose was 155%. This difference in dose-related response was significant. Effect of progesterone (P) on DES-induced UWW response. A daily dose of 50/zg P for 5 days after birth in combination with 10 -2/zg DES completely inhibited the DES response in 6-day-old females (data not shown). With the same dose of P in combination with 6 /zg DES, the UWW response was approximately 26% of that with the same dose of DES alone.

142

Reproductive Toxicology

Volume7, Number 2, 1993

uterine weight in m g / bod~eight in gram _*SD

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Fig. 2. Cumulative effect on uterine wet weight (related to body weight) in 6-day-old females after treatment with a low (10 -2/~g) or a high (5/xg) dose of DES for an increasing number of days, starting on the day of birth, x 1 = treatment on the day of birth (day 1) only; x 2 = treatment on days 1 and 2, etc. For statistical analysis, the groups "10 -2 t~g x 1" and "5 txg x 1" were each compared with the group "controls olive oil x 5." Further, comparisons were made within each dose group between the effect of a certain treatment period and the nearest shorter one. Thus "5 tzg x 2" was compared with "5 tzg x 1," etc. For significance levels, see Figure 1.

The U W W response after treatment with different doses o f DES in neonatal life and later challenge with DES in immature life. The results are summarized in Figure 4. Earlier e x p e r i m e n t s on U W W in controls and in females treated with 10 -2 or 5 tzg D E S for 5 days and killed on day 6 were repeated, but now using olive oil/DMSO as vehicle instead of olive oil/ethanol. There were no differences in U W W r e s p o n s e b e t w e e n the previous results and the present ones (data not shown). U W W was studied in 26-day-old females that were neonatally untreated, vehicle treated (controls), or D E S treated (daily doses of 10 -2 or 5/xg) in neonatal life. The U W W did not differ b e t w e e n untreated and control females but it was reduced to a p p r o x i m a t e l y 84% of the value in controls after t r e a t m e n t with the low D E S dose and to approximately 45% with the high dose (Figure 4). Controls and neonatally D E S - t r e a t e d females were later challenged with 10 -2 or 5 ~g D E S for 1 day (treatment day 25) or 5 days (treatment days 21 to 25) before being killed on day 26. Challenge of controls with 10 -2/xg D E S had no effect, while a 1day e x p o s u r e to 5 tzg D E S resulted in a 2.3-fold and

a 5-day e x p o s u r e in a 5.6-fold U W W increase (Figure 4). In neonatally D E S - t r e a t e d females (5/xg D E S x 5) a challenge with 5/zg for 1 or 5 days resulted in a 2.4-fold and 4.8-fold U W W increase, respectively. The m e a n values for U W W in the same females were, h o w e v e r , only 41% and 38% of the values from controls challenged in a similar way (Figure 4). Challenge with the 5-/zg dose for 1 or 5 days after neonatal t r e a t m e n t with 10 -2/xg D E S for 5 days resulted in a 2.1-fold and 3.6-fold U W W increase, respectively (Figure 4). The final U W W was then 77% and 54%, respectively, of the U W W in females treated with D E S in neonatal life only. Thus, e v e n the low neonatal D E S dose induced changes that affected a later response to an estrogen challenge. A single treatment with 5 ~g D E S on the day of birth and later challenge with the same dose on days 21 to 25 resulted in the same U W W response as in neonatally vehicle-treated controls. H o w e v e r , challenge on day 25 only resulted in a U W W significantly lower than that in controls. T r e a t m e n t with 5 tzg E 2 daily for 5 days after birth did not affect U W W of 26-day-old females (Fig-

Neonatal estrogen and uterine response • A. HALLING and J.-G. FORSBERG

143

uterine weight in mg / bodywei ~ht in gram .* SD .o-

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Fig. 3. The 24-h effect on uterine wet weight (related to body weight) of a single treatment with a low (10 -2/~g) or high (5 t~g) dose of DES. 1 --* 2 = females treated on the day of birth and killed on day 2; 2 --~ 3 = females treated on day 2 and killed on day 3, etc. Within each stage group (for example, "1 --~ 2"), comparisons were made between the "10 -2 /~g DES" dose and "olive oil" and between "5 ~g DES" and " 1 0 - 2 / ~ g DES." For significance levels, see Figure 1.

ure 5). When such females were challenged with 5 /~g DES on days 21 to 25 or on day 25 only, the responses were reduced and similar to those seen in females treated with a single dose of 5/~g DES on the day of birth (Figure 5).

Effects o f estrogen and EGF on uterine and cervical epithelial mitotic rate in neonatal females On day 2 after birth, the epithelium in the uterine cervix was of a pseudostratified columnar type, and the mitotic rate was higher in this epithelium than in that of the uterine horns (0.025 > P > 0.01 ; Figure 6). A 24-hour exposure to 5 t~g DES resulted in a significantly increased mitotic rate in the uterine epithelium (about 2.6-fold increase) while in the cervical region it was significantly reduced (to about 35% of the control value). A 5-/.~g dose of E 2 had a stimulatory effect similar to that of 5 ~g DES on the proliferative activity in the uterine epithelium, but had no effect in the cervical epithelium. When females were treated on day 3 and killed on day 4, no significant difference was found in mi-

totic rate between the uterine epithelium and cervical epithelium in vehicle-treated females (Figure 7). Both the low (10 -2/~g) and high (5 t~g) DES dose significantly stimulated uterine epithelial proliferation with no significant difference between the two doses (mean stimulation approximately 2.2-fold). In the cervical epithelium, the situation was different. While the low DES dose had no effect, the high 5/.~g dose reduced the mitotic rate to approximately 24% of the control value. E2 had a stimulatory effect on uterine mitotic rate that was similar to that of DES, but no effect was seen in the cervical epithelium. E G F strongly increased the mitotic rate in the uterine epithelium Of 4-day-old females, and this effect was more pronounced than that obtained with either dose of DES (Figure 8). A combined treatment with 5 ~g DES and E G F did not result in any significant difference from the result obtained with E G F only. In the cervical epithelium, E G F alone had no effect (Figure 8). After a combined treatment with 5 /~g DES and E G F , the mitotic rate level was not

144

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Fig. 4. Effect on uterine wet weight (related to body weight) in 6-day- or 26-day-old females after various types of DES treatment in the neonatal period (10 2/xg or 5/xg on days 1 to 5 or 5 txg on the day of birth [day 1] only). Groups of 26-day-old females were challenged with the low or the high dose of DES on day 25 or on days 21 to 25. In 6-day-old females, statistical comparison was made between the groups "10-2 p.g DES" and "5/xg DES"; in 26-day-old, unchallenged females, the effect of neonatal treatment with 10 -2 txg DES was compared with that of olive oil only, and the effect of this DES dose was compared with that of 5/xg DES. The effects of challenge with 5 p.g DES on day 25 only or on days 21 to 25 in neonatafiy olive oil treated females were compared with the effects of similar challenges in females treated with 10-2 Ixg DES neonatally. The effects in the latter type of neonatally treated females were compared with the effects of 5-p.g challenges in females treated with daily doses of 5 txg DES neonatally. Finally, the effects of challenges in females treated with 5 txg DES on the day of birth only were compared with those in neonatally olive oil treated females. For further information, see text. For significance levels, see Figure 1.

different from that in controls. The mitotic rate inhibition obtained with 5 P,g DES alone was thus not apparent when females were treated with both DES and EGF. In 6-day-old females the transformation of the pseudostratified columnar epithelium into an epithelium consisting of a superficial zone and a basal zone had progressed from the vaginal region into the uterine cervix. Because of this, the mitotic rate calculations were done only in the uterine epithe-

lium. The mitotic rate in uterine epithelium from control females was similar to that in 4-day-old controls. A 24-h pulse of I0 2 or 5 /~g DES resulted in a similar and significant increase of mitotic rate (approximately 1.4-fold); 5 /~g E 2 had a nonsignificant effect. In contrast to this, E G F resulted in a 3.8fold increase of mitotic rate compared with controls (Figure 9). The result from a combined treatment with both DES and E G F was not different from that seen with E G F alone.

Neonatal estrogen and uterine response • A. HALLING and J.-G. FORSBERG

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21-25

21-25

Fig. 5. A comparison of responses to a challenge with DES on day 25 or days 21 to 25 after treatment with 5 t~g DES or E2 neonatally. All females were killed on day 26. Neonat. tr. = neonatal treatment; oo = olive oil; 5DES x 1 = 5 /~g DES on the day of birth; 5E,_ x 5 = 5 ~g estradiol-17fl daily for 5 days after birth; 5DES = 5 t~g DES. The groups representing treatment with olive oil or DES neonatally are the same as those included in Figure 4. Statistical comparisons between linked groups, ns-nonsignificant.

Effect of E 2 and EGF on UWW and ~H-thymidine (3H-Tdr) incorporation in uteri from control and neonatally DES (5 I~g per day) treated (estrogenized) adult females The 3H-Tdr incorporation per uterus was higher in ovariectomized estrogenized females than in ovariectomized controls (Figure 10), and this in spite of the uterine weight in estrogenized females being only approximately 50% of that in controls (data not shown). A 24-h E2 pulse stimulated incorporation activity in both ovariectomized controls and ovariectomized estrogenized females. The final incorporation level reached w a s higher in controls than in estrogenized females (0.05 > P > 0.01; Figure 9). Even after E 2 treatment, the uterine weight in estrogenized females was only approximately 50% of that in controls (data not shown); the Ez-induced weight increase was 1.8-fold in both groups of females. Kidney implants with E G F in ovariectomized animals resulted in a p r o n o u n c e d U W W increase in both controls and estrogenized females (Figure 11).

5 /zg

5 #g

DES

E2

C

5 /~g

5 /~g

DES

UTERUS

E2

CERVIX

Fig. 6. Mitotic rate on day 2 after birth in the epithelium of the uterine horns and uterine cervix after treatment with a single dose of 5/~g DES or estradiol-17fl (Ez) on the day of birth. C -- controls. For significance levels, see Figure I. Mitotic r a t e in % ± SD 10

N=5

8

ns

6

2

!

4

! i

o c

10

-2

5

5

D E S DES E 2 UTERUS

C

10

-2

5

D E S DES

5

E2

CERVIX

Fig. 7. Mitotic rate in the epithelium of the uterine horns and uterine cervix after treatment with a low (10-2-/.tg) or high (5-p~g) dose of DES or 5 p.g estradiol-17fl (E2) on day 3 of life, females killed on day 4. C = controls. For significance levels, see Figure 1.

146

Reproductive Toxicology Mitotic rate in % ±

SD

Volume 7. N u m b e r 2, 1993 log DPM/uterus ± SD

***

N

12

ns

=

8

N=5

10

ns

ns

C

-2 10 5 EGF DES DES

EGF 5DES

C

5 EGF EGF DES 5DES C E R V I X

U T E R U S

Fig. 8. Mitotic rate in the epithelium of the uterine horns and uterine cervix after treatment with DES (10 -2 o r 5 p.g) or EGF or a combination of DES and EGF on day 3 of life, females killed on day 4. C = controls; 10 -2 DES = 10 -2/.Lg DES on day 3; 5 DES = 5/~g DES on day 3. For significance levels, see Figure 1. Mitotic rate in % ± SD

C

C E2

DES

DES E z

Fig. 10. Incorporation of 3H-thyroid±he in uteri from 10week old ovariectomized females, controls, or treated with daily doses of 5 p.g DES on days 1 to 5 after birth (estrogenized females). One group of ovariectomized controls and ovariectomized estrogenized females were treated subcutaneously with 1 /xg estradiol-17/3 (E2) in olive oil 24 h before being killed. C = controls; C E2 = estradiol-treated controls; DES = estrogenized females; DES E 2 -- estradiol-treated estrogenized females. For significance levels, see Figure 1.

N = 5

16 14 12 10 8 6

4 2 0 C

10-ZDEs

5DES

5E a

EGF

5DES EGF

U T E R U S

Fig. 9. Mitotic rate in the uterine epithelium of 6-day-old females after treatment with 10 -2 or 5 tzg DES, 5/xg E2, EGF, or a combination of EGF and 5 p.g DES on day 5. For significance levels, see Figure 1.

The weight gain 24 h after implantation was approximately 2.4-fold in control females and 2.l-fold in estrogenized females. The m e a n uterine weight in E G F - s t i m u l a t e d estrogenized females was then approximately 63% of that in EGF-stimulated controls. F r o m these p e a k values the U W W later decreased almost linearly until the control level was reached 240 h after implantation in controls and at 72 h in estrogenized females. The results from studies on 3H-Tdr incorporation are shown in Figure 12. Variance analysis of the whole material indicated a significant difference in results with respect to different treatment groups and different time intervals after implantation of E G F or cholesterol pellets. The 3H-Tdr incorporation was significantly higher in estrogenized females carrying a placebo implant than in control females with a similar implant at all time stages studied except at 96 h. This exception m a y be a r a n d o m result. The difference was levelled out in E G F - i m p l a n t e d females b y the strong 24-h stimulation of incorporation activity. Using

Neonatal estrogen and uterine response • A. HALLING and J.-G. FORSBERG

147

U t e r i n e w e t w e i g h t (UWW) in nag ± SD -7

40

30

20

10

@

etc. r..~ r,..) ~

,"'~

Fig. 11. Uterine wet weight in 10-week-old ovariectomized control females or estrogenized females (daily doses of 5/xg DES on days i to 5 after birth) killed at various times after implantation of a pellet with 750 ng EGF under the kidney capsule. C = control females implanted with a cholesterol pellet (placebo); C EGF = control females implanted with an EGF pellet; DES = estrogenized females implanted with a placebo pellet; DES EGF = estrogenized females implanted with an EGF pellet. The column labels are similar for all time stages studied. For significant differences, see text. nontransformed values for incorporation activity, EGF resulted in a 14-fold increase in control females and a 5-fold increase in estrogenized females. In estrogenized females the stimulation subsided at 48 h, while in controls a significant (0.05 > P > 0.01) difference from placebo-implanted controls was still seen at 72 h, but not at 96 h. This points to a prolonged EGF effect on 3H-Tdr incorporation in control females compared with estrogenized females, a situation similar to that seen in the studies on uterine weight.

3H-Tdr incorporation in cultured uterine epithelium from 8-week-old, control and estrogenized females The uterine epithelium from control females grew to confluence after 5 days in culture, without contamination with fibroblasts. A summary of the results is shown in Figure 13. Epithelium from 8-week-old females treated with 10 -6 to 5/zg per day of DES in neonatal life had a lag phase that was about one day longer than in

controls before starting to increase 3H-Tdr incorporation. In cultures of control epithelium, the incorporation reached a maximum level on day 3 and remained stable until day 6 only to become significantly lower on day 7. Epithelium from females treated with I0 -6 and 10-4/zg DES reached maximum incorporation level on day 4 and remained stable on day 5 (the last stage studied for these dose levels). With 10 -2/zg DES, incorporation of 3H-Tdr was at maximum on day 5 and remained stable until day 7. Epithelium from females treated with 1 /.tg DES per day neonatally increased incorporation until day 6 when it reached the same level as seen with the lower doses. With 5 /zg DES neonatally, incorporation increased from day 2 through day 6, and it was similar on days 6 and 7. However, the maximum incorporation activity with 5/xg DES was lower than that obtained with the lower doses in neonatal life. Thus, the results demonstrate that by increasing daily doses of DES in neonatal life, the maximum 3HTdr incorporation activity in cultured endometrial

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Reproductive Toxicology

Volume 7, Number 2, 1993

log D P M / u L e r u s ± SD 6

N = 6-7

r 5

4

24 h

48 h

t~

etc.

72 h

96 h

240 h

Fig. 12. Incorporation of 3H-thymidine in uteri from 10-week-old ovariectomized control (C) or estrogenized (DES; daily doses of 5 ~g DES on day 1 to 5 after birth) females implanted under the kidney capsule with a cholesterol (placebo) pellet (C; DES) or a pellet with 750 ng EGF (C EGF; DES EGF). Females were killed at various times (24 h to 240 h) after implantation of a pellet. Column labels similar for all time stages studied. For significant differences, see text.

epithelium from adult females is reached progressively later during the culture period. With the highest dose (5 ~g per day) the incorporation does not reach the same high level as seen with lower doses. In another series of experiments, endometrial cells from adult ovariectomized control females or estrogenized (5 /~g DES per day for 5 days after birth) females were cultured for a total of 9 days and studied for 3H-Tdr incorporation activity at different times during the culture period (Figure 14). A difference in 3H-Tdr incorporation between cells from control females and those from estrogenized females (0.05 > P > 0.01) was seen only at the 3-day stage. This may be a random finding without any biologic significance. The incorporation activity increased from day 1 until day 7, when a plateau level was reached.

The effect of EGF on ~H-Tdr incorporation on cultured uterine epithelium from adult controls and estrogenized females Cells were cultured in serum containing medium for 24 h and after passage in serum free medium for

1 h, the medium was changed to a fresh serum-free one, supplemented or not supplemented with EGF. The culture period in the latter medium was 4 h. The results are demonstrated in Figure 15. Endometrial cells from control females responded to EGF with an increased incorporation of 3H-Tdr (0.05 > P > 0.01) while no effect was seen in cultures from estrogenized females (5/zg DES per day for 5 days after birth). The incorporation activity in cultures of cells from estrogenized females was lower than in cultures of cells from control females ( P < 0.001).

DISCUSSION Results presented in this paper demonstrate a significant uterine wet weight (UWW) response to estrogens in neonatal mice. The lowest daily DES dose with a significant effect on UWW after a 5-day treatment period was 10 -6/zg (I 13% of the value in controls); a maximum response was obtained with 10-2/zg (260% of the value in controls). Higher daily doses resulted in a declining response. Because the

Neonatal estrogen and uterine response • A. HALLING and J.-G. FORSBERG

log(dpm/ng

149

DNA)

+SD

N=9

4.5 ~4.0

r-

! 5.5 Z

3.0 2.5 2.0 1.5 1.0 0.5 Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Fig. 13. Incorporation of 3H-thymidine in primary cell cultures of uterine epithelium from 8-week-old females treated with olive oil (controls) or various daily doses of DES (10 -6 to 5 ~g) for the first 5 days after birth. Cultures were studied daily, from day 1 to day 7 after explantation. [] = controls; [] = 10 -6/~g DES; [] = 10 -4 ~g DES; [] = 10 -2 ~g DES; [] = 1 p.g DES; • = 5/~g DES. N = number of culture dishes studied per group.

daily 10 -2/~g DES dose was the one with the maxi-

mum cumulative uterine stimulating effect in neonatal life and because 5/~g was the higher dose with the most depressed response, these two opposing doses were the most commonly used in the following experiments. Progesterone suppressed the DES response: 94% with the 10 -2 /~g DES dose and 74% with the 5/~g DES dose. In contrast to the dose-related differences in cumulative response, the 24-h response to 5 t~g DES was similar to the 10 -2 ~g dose; the response was age-dependent from day 2 to 6. With E2, the cumulative UWW response on day 6 increased with increasing dose and duration of treatment period. A daily dose of 10 -2 ~g E2 for 5 days resulted in about the same stimulation of UWW as 10 -6/~g DES while 5 p,g E2 had an effect similar to that seen with 10 -3 ~g DES (ascending limb of the dose-response curve) or 5 t~g DES (descending

limb of the dose-response curve). Not even 50/~g E 2 resulted in the same maximum response as 10 -2 /~g DES. An epithelial squamous response to E 2 was seen in the neonatal vagina on day 5 after treatment with 0.1 /~g on days 1 to 4 after birth (21). The difference in efficacy between DES and E 2 could be explained by E 2 but not DES being bound to rodent a-fetoprotein, which is a buffer mechanism for E2 (22,23). Using immunohistochemical methods, estrogen receptor protein has been demonstrated in genital stroma of newborn female mice, but not in the uterine epithelium (8-10). Epithelial receptor protein is not observed until day 8 in NMRI mice (8) or day 4 in CD-1 and BALB/c mice (9,10). Treatment with DES results in a precocious appearance of receptor protein in the uterine epithelium on day 2 after birth (8,9). These receptor studies as well as the present UWW and mitotic rate results indicate that the

Reproductive Toxicology

150

log ( d p m / n g ± SD

Volume 7, Number 2, 1993

DNA) N=

4.0

12

3.5 3.0 2.5 2.0 1.5 4.0

0.5

~

0

~ 4

f

f

f

i 1 5 Day

5"] etc.

Fig. 14. Incorporation of 3H-thymidine at various times during culture of endometrial epithelial cells from 10-week-old ovariectomized control and estrogenized (5 ~g DES per day for 5 days after birth) females. C (D) = controls; DES ( , ) -- estrogenized females. N = number of culture dishes studied per group. *0.05 > P > 0.01 compared to same day control.

mouse uterus already within 24 h after birth is capable of a true receptor-dependent estrogen response. In line with this, treatment of female mice with 5 tzg E2 on day 1 and 2 after birth strongly increased the amount of a specific cell product in the cervicovaginal epithelium on day 3 (24). Uterine growth in mice from birth until day 15 was considered independent of ovarian and adrenal hormones, but it was sensitive to exogenous estradiol administered on the day of birth (25). The uterine epithelial mitotic rate response to a single dose of DES or E 2 was related to neonatal age. The 5 /xg E 2 dose resulted in, compared with controls, a 2.3-fold increase in 2-day old females, a 1.9-fold increase in 4-day females, and a 1.3-fold increase in 6-day females. The corresponding figures for 5/xg DES were 2.6, 2.0, and 1.5 and for 10 -2/zg DES 2.4 (4-day females) and 1.5 (6-day females). The responses to the two DES doses were not significantly different. There were no striking differences in DES dose-related responses for either the epithelial mitotic rate response or the U W W response to a single 24-h DES pulse. H o w e v e r , while the U W W response increased from day 2 to 6 after birth, the mitotic rate response decreased.

While DES induced increased mitotic rate in the uterine epithelium, it was depressed in the untransformed fetal type of pseudostratified columnar cervical epithelium 24 h after a single DES injection on day 1 or 3. This regional difference in acute mitotic rate DES response is in line with earlier studies using continuous treatment with E 2 or DES; the DES mitotic inhibitory effect was observed with daily doses as low as 10 -5/xg (11,12). E 2 has been shown to shorten the duration of the S and Gj phases of the cell cycle in the uterine epithelium, while it lengthens the S and above all the G, phases in the cervical epithelium of neonatal mice (26). The estrogeninduced proliferative inhibition in the columnar cervicovaginal epithelium has been postulated to be an important mechanism for the DES-induced regional restricted epithelial abnormalities in the uterine cervix and upper vagina of both women and mice (27). Treatment with E G F on day 3 resulted in a uterine mitotic rate that was higher than that seen with DES; no further increase was obtained with a combination of E G F and DES. While the E G F effect was striking in the uterine horn epithelium, no effect was seen in the cervical epithelium which, together with the results from the DES studies, points to a

Neonatal estrogen and uterine response • A. HALLINGand J.-G. FORSBERG log (DPM/ng DNA) N = 20

3.0 2.5 2.0

L ns

1.5 1.0 0.5 0.0

i

/ / / / / / / / / /

i

Co EGF

Co C

Co

Co E G F DES

Fig. 15. Effect of EGF on cultured endometrial cells from control females (C) and females treated neonatally with daily doses of 5/zg DES (DES). Co = serum-free medium cocktail; Co EGF = EGF containing medium cocktail; *0.05 > P > 0.01; ns = nonsignificant. N = number of culture dishes studied per group.

regional difference in proliferation control mechanisms. In combination with DES, EGF restored the DES depressed mitotic activity to the control level. Receptors for EGF have been described in the mouse uterine and vaginal epithelium already on day 13 in fetal life, and the receptors in 3- to 17-day old females are capable of binding labelled EGF (28). Estrogen has been demonstrated to regulate acutely synthesis of EGF and the levels of EGF receptor in immature female rats, which may play a role in estrogen-stimulated growth (13-15). The EGF restoration of DES-induced mitotic depression to the control level could be interpreted in different ways. Exogenous EGF might substitute for a regionally restricted low and estrogen-resistant endogenous EGF level or, more likely, EGF could have a nonspecific general growth-promoting effect, masking the DES-induced mitotic inhibition. While the 24-h response to a single DES treatment increased with increasing age, the cumulative response in 6-day-old females declined after a longer than 3-day treatment period with daily doses higher than 10 -2/~g. A daily 5-~g dose for 5 days after birth resulted in a UWW of 26-day-old females that was only 45% of that in controls. The response of these low-weight uteri to a challenge with 5/xg DES on

151

days 21 to 25 was only 38% of that in similarly treated controls. Even 10 -2 /zg DES per day for 5 days, which resulted in maximum response in 6-day-old females, or a single dose of 5 /xg on the day of birth reduced the UWW response to a later DES challenge. Based on results from the 5-day challenge period, a single neonatal 5-/xg dose was somewhat less effective than repeated 10-2-/zg doses. Daily doses of 5 ~g E2 for 5 days after birth did not affect the UWW of 26-day-old females, but had the same depressive effect on a 5-day challenge response as a single dose of 5/zg DES on the day of birth. The uterine hypoplasia in immature and adult rats and mice after neonatal estrogen treatment and the reduced response to an estrogen challenge has been described previously (29-35). In this study, however, we focused on the dose-response pattern in neonatal life and its consequences for the dose-response relationship in immature females. The neonatal doses that induce uterine hypoplasia are the same as those that have a genotoxic effect (36) and result in epithelial aberrations in the uterine cervix (12). A possible common explanation for all these phenomena is that DES, and even E2, may activate enzymes, dependent on dose and treatment period, that could metabolize the estrogens into metabolites of a possible reactive/toxic and less estrogenic nature than DES (37-41). Such metabolites could counteract a receptor-dependent trophic estrogen response and result in genotoxicity, epithelial aberrations, and later hypoplasia. Another possibility is an interference with synthesis/replenishment of the estrogen receptor or involvement of an endogenous inhibitor of estrogen binding to nuclear sites (42,43). Treatment of neonatal rats with DES or E2 reduces the estrogen receptor content in adult rat uteri (34,35,44,45). Sequential estrogen treatment may suppress DNA polymerase activity (46) directly or indirectly via induction of a nuclear protein with inhibiting effects on thymidine kinase, DNA polymerase, and RNA polymerase II (47). Uteri from adult ovariectomized estrogenized females had a higher 3H-Tdr incorporation activity than similarly treated controls, while the reverse was true for UWW. This could point to the presence in estrogenized females of a subpopulation of uterine cells with ovary-independent proliferation. Similar results were reported by Gibson and colleagues (48), who found that proliferation control in estrogenized females (1 p.g E2/g body weight) was not only ovaryindependent, but also independent of peripheral estrogen formation. In in vitro experiments on endometrial epithelial cells, the maximum level of 3H-Tdr incorporation

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seen in control cultures occurred about one day later in cultures of cells from females treated with I0 6 /~g DES neonatally than in the control cultures. With increasing neonatal doses the control level was reached still later; cells from females treated with 5 ~g DES neonatally never reached the control level. In cultures of vaginal epithelial cells from neonatally DES-treated females, the initial proliferation rate was lower than when the cells were from control vaginas; after 10 days in culture, the difference was no longer evident (49). This delay in maximum incorporation activity could be explained by a gradual sorting out of cells incapable of proliferation and overgrowth of an initially small population of normal proliferating cells. Another possibility is a gradual loss of some cellular, possibly ovary-dependent, proliferation-inhibiting factor (47). The latter alternative is supported by our findings of similar incorporation activity in cultures of endometrial epithelial cells from ovariectomized control females and estrogenized females. The discrepancy between the in vivo (higher 3H-Tdr incorporation in uteri from ovariectomized estrogenized females than in control uteri) and in vitro findings (similar incorporation in endometrial epithelial cells from ovariectomized estrogenized and control females; delayed or reduced incorporation maximum in the same cells from intact females) might be explained by different uterine cell populations being affected differently by the neonatal DES treatment. In estrogenized females the uterine stroma could house the postulated subpopulation of non-ovary-dependent proliferating cells, while the endometrial epithelial cells might be under some proliferation-inhibiting influence that is lost under culture conditions or after ovariectomy. EGF stimulated UWW and 3H-Tdr incorporation, but less so in estrogenized females than in controls; the effect subsided more rapidly in estrogenized females than in controls. This points to DESinduced permanent changes in the EGF receptoriigand interaction or in EGF-related mechanisms beyond this interaction. Endometrial epithelial cells from adult estrogenized females did not respond to EGF in serum-free medium, but a slight response was seen in cells from control females. A reduced EGF response in DES-exposed mice has been described previously (50). Thus, neonatal DES treatment has immediate and region-specific effects in the neonatal uterus, associated with later epithelial abnormalities in the uterine cervix (12) and permanent disturbances in growth-controlling mechanisms in the uterine horns.

Volume 7, Number 2, 1993 - - This investigation was supported by grants from the Swedish Medical Research Council (Grant no. B92-12X09056-03A), the Crafoord Foundation, Lund, the Medical Faculty al the University of Lurid, and from the Swedish Society for Medical Research, Department of Pharmacology, Karolinska Institute, Stockholm, Sweden. Acknowledgments

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Neonatal estrogen and uterine response • A. HALLING and J.-G. FORSBERG 20. Kim Y-J, Sah RLY, Doong J-YH, Grodzinsky AJ. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem. 1988;174:168-76. 21. Forsberg J-G. The effect of estradiol- 17/3on the epithelium in the mouse vaginal anlage. Acta Anat (Basel). 1966;63:71-88. 22. Raynaud J-P. Influence of rat estradiol binding plasma protein (EBP) on uterotropic activity. Steroids. 1973;21:249-58. 23. Sheehan DM, Branham WS. Dissociation of estrogeninduced uterine growth and ornithine decarboxylase activity in the postnatal rat. Teratogenesis Carcinog Mutagen. 1987;7:411-22. 24. D6skeland S-O, Kalland T, Forsberg J-G. A study on the estradiol-induced augmentation of a specific cell product in the vaginal epithelium of the neonatal mouse. Devel Growth Differentiation. 1979;21:111-8. 25. Ogasawara Y, Okamoto S, Kitamura Y, Matsumoto K. Proliferative pattern of uterine cells from birth to adulthood in intact, neonatally castrated, and/or adrenalectomized mice, assayed by incorporation of [t-'5I]iododeoxyuridine. Endocrinology. 1983;113:582-7. 26. Eide A. The effect of estradiol on the cell kinetics in the uterine and cervical epithelium of neonatal mice. Cell Tissue Kinet. 1975;8:249-57. 27. Forsberg J-G. Studies on the developmental mechanism of estrogen-induced irreversible changes in the mouse cervicovaginal epithelium. Natl Cancer Inst Monogr. 1979;51:41-56. 28. Bossert NL, Nelson KG, Ross KA, Takahshi T, McLachlan JA. Epidermal growth factor binding and receptor distribution in the mouse reproductive tract during development. Developmental Biol. 1990;142:75-85. 29. Wrenn TR, Wood JR, Bitman J. Oestrogen responses of rats neonatally sterilized with steroids. J Endocrinol. 1969; 45:415-20. 30. Maier DB. Newbold RR, McLachlan JA. Prenatal diethylstilbestrol exposure alters murine uterine respones to prepubertal estrogen stimulation. Endocrinology. 1985;116:1878-86. 31. Ostrander PL, Mills KT, Bern HA. Long-term responses of the moust uterus to neonatal diethylstilbestrol treatment and to later sex hormone exposure. J Natl Cancer Inst. 1985;74:121-35. 32. Iguchi T, Takasugi N. Postnatal development of uterine abnormalities in mice exposed to DES in utero. Biol Neonate. 1987;52:97-103. 33. Branham WS, Zehr DR, Chen J J, Sheehan DM. Alterations in developing rat uterine cell populations after neonatal exposure to estrogens and antiestrogens. Teratology. 1988;38: 271-9. 34. Medlock KL, Sheehan DM, Nelson C J, Branham WS. Effects of postnatal DES treatment on uterine growth, development, and estrogen receptor levels. J Steroid Biochem. 1988;29:527-32. 35. Medlock KL, Branham WS, Sheehan DM. Long-term effects of postnatal exposure to diethylstilbestrol on uterine estrogen receptor and growth. J Steroid Biochem Mol Biol. 1992 ;42:23 -8. 36. Forsberg J-G. Estrogen effects on chromosome number and

37. 38. 39.

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49.

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