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Developmental Effects of Ethinylestradiol on Reproductive Organs of Female Mice

AKIKO KIRIGAYA1, SHINJI HAYASHI1, TAISEN IGUCHI2,3 and TOMOMI SATO1

1Graduate School of Integrated Science, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027; 2The Graduate University for Advanced Studies and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787; 3CREST, Japan Science and Technology Corporation, 4-1-8 Motomachi, Kawaguchi 332-0012, Japan

Abstract. Reproductive organs in female mice are susceptible endometriosis and the control of bleeding in women with to exposure to estrogenic substances especially during blood dyscrasias (2). On the other hand, administration of development. In the present study, C57BL/6J female mice were EE2 alone or in combination with progestin induced pituitary exposed to the synthetic estrogens ethinylestradiol (EE2) or tumors, malignant tumors of the connective tissue of the diethylstilbestrol (DES), 10-100 or 6.7-67 Ìg/kg bw, uterus, uterine fundus and cervix, and mammary tumors in respectively, in utero from day 10 to 18 of pregnancy, and their rats and mice (3, 4). In humans, EE2 has been reported to effects were analyzed at 30 and 40 days of age. Both EE2 and increase the incidence of breast cancer (5). Constant use of DES reduced the survival rate of fetuses and newborns in a oral contraceptives was associated with twice as great a risk dose-dependent manner. Polyovular follicles (PF) were found of adenocarcinoma of the cervix (6). in the ovaries of all groups at 30 days of age including oil- Neonatal EE2 exposure increased uterine wet weight and injected controls. However, the incidence of PF was induced uterine adenocarcinoma in rats and mice (7, 8). significantly higher in the 50 Ìg/kg EE2- and 33.3 Ìg/kg DES- Adult EE2 exposure induced mammary adenocarcinomas exposed mice than the control. In vaginal epithelia of the in and pituitary tumors in ACI rats (9). Oral administration of utero EE2 exposed, ovariectomized mice, stratification and EE2 to pregnant mice from day 11 to 17 of pregnancy also cornification were encountered even 10 days after ovariectomy. induced malignant transformation of uterine endometrium, Especially, vaginae in the ovariectomized mice given high dose hyperplasia and cornification in vaginal epithelium, of EE2 or DES in utero showed ovary-independent hypertrophic nipples, follicular cell hyperplasia located in proliferation of the epithelium. Thus, it is clear that prenatal the medually region of ovary and gonadal dysgenesis in both exposure to EE2 or DES induces reproductive abnormalities, male and female off-spring (10-14). Thus, the including PF, ovary-independent vaginal epithelial administration of EE2 is related to the occurrence of tumors stratification and cornification. and abnormal development of reproductive organs. In mice treated perinatally with steroid hormones and A synthetic estrogen 17·-ethinylestradiol (EE2) is known as a synthetic estrogens, a variety of abnormalities in the component of oral contraceptives. EE2 has been effectively reproductive tracts were induced, e.g., estrogen-independent used not only for oral contraceptive use, but also as hormone persistent proliferation and cornification of vaginal epithelium replacement therapy for post-menopausal osteoporosis (1) (15-20) and polyovular follicles (PF) with 2-23 oocytes per and a variety of human gynecological disorders, such as follicle in ovaries of immature mice (20, 21). Couse et al. (22) dysfunctional uterine bleeding, persistent anovulation, demonstrated that neonatal diethylstilbestrol (DES) exposure premature ovarian failure, functional ovarian cysts, pelvic to ·ERKO mice, which lacks estrogen receptor · (ER·), pain (including secondary dysmenorrhea), mittelschmerz, induced no morphological defects in female reproductive organs. This result indicates that abnormalities of reproductive organs caused by perinatal DES treatment are mediated through ER·. Since the binding affinities of EE2 to ERs were Correspondence to: Dr. Tomomi Sato, Graduate School of equivalent to that of 17‚-estradiol (E ) in rat uterus (23), EE Integrated Science, Yokohama City University, 22-2 Seto, 2 2 Kanazawa-ku, Yokohama 236-0027, Japan. Tel: +81 45 787 2394, could affect through ERs in induction of reproductive Fax: +81 45 787 2413, e-mail: [email protected] abnormalities in prenatal and postnatal mice. This study was aimed at further examining the effects of Key Words: Ethinylestradiol, vagina, fetus, polyovular follicles. in utero exposure to EE2 on fetal lethality and female

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Table I. Number of off-spring and gender ratio at gestation day 19.

Maternal N n Fetuses / litter Body weight (g) Female ratio (%) Survival rate (%) treatments alive total

Sesame oil 7 58 8.0±0.44 8.3±0.42 1.3±0.02 50.4±7.50 96.8±3.18 EE2 (Ìg/kg bw) 10 10 71 6.0±0.86 7.1±0.38 1.1±0.02* 57.7±7.48 82.3±10.40 25 10 84 5.5±0.99 8.4±0.40 1.0±0.02* 40.7±9.92 68.0±12.48 50 37 298 2.6±0.45* 8.1±0.31 1.0±0.02* 48.4±7.10 33.1±5.62* 100 4 31 0.0±0.00* 7.8±1.31 n.a. n.a. 0.0±0.00* DES (Ìg/kg bw) 6.7 11 86 3.9±0.87* 7.8±0.55 1.0±0.06* 45.3±11.07 53.8±11.72* 33.3 10 84 3.7±1.20* 8.4±0.37 1.1±0.03* 57.4±12.04 45.4±14.52* 67 18 143 1.6±0.52* 7.9±0.45 1.0±0.02* 50.2±9.13 18.6±6.05*

N; number of treated mice, n; number of off-spring; n.a.; not applicable; *p<0.05 compared with sesame oil control.

reproductive organs in mice. Our results indicate that EE2 weighed at sacrifice. Ovary and uterus were fixed in Bouin’s lower the survival rate of fetuses and induce the abnormal solution, embedded in paraffin and serially sectioned at 8 Ìm development of reproductive organs, including PF in the thickness. Sections were stained with hematoxylin and eosin. The number of polyovular follicles (PF) having more than 2 oocytes per ovary, ovary-independent vaginal epithelial cell proliferation follicle per mouse were counted, as described previously (24). showing increase in 5-bromo-2’-deoxyuridine (BrdU)- labeled cells and stratification of vaginal epithelial cells with BrdU immunohistochemistry. BrdU-labeled cells were detected by the superficial cornification even without estrogenic immnohistochemistry using anti-BrdU antibody. Vaginae were fixed stimulation. in 10% neutral-buffered formalin solution, embedded in paraffin and serially sectioned at 6 Ìm thickness. The sections were Materials and Methods quenched in 0.3% hydrogen peroxide in methanol for 30 min to remove endogenous peroxidase activity, treated with 2N HCl for 20 Animals. C57BL/6J Jcl and ICR/Jcl mice were given a commercial min to denature genomic DNA and 0.1% trypsin at 37ÆC for 30 min diet (CE-2, CLEA, Tokyo, Japan) and tap water ad libitum and to retrieve the antigen. To block non-specific binding, the sections were kept at 24±1.0ÆC under 12 h light / 12 h dark by artificial were incubated with 1% fetal bovine serum albumin (Sigma illumination (lights on 0800-2000). All animals were maintained in Chemical Co.) in PBS, then incubated with anti-BrdU-peroxidase accordance with the NIH Guide for the Care and Use of (POD), Fab fragments (1:15 dilution, Roche Diagnostics GmbH, Laboratory Animals and were approved by our Institutional Mannheim, Germany) for 1 h. Peroxidase visualization was Animal Care Committee. performed using 0.4 mg/ml DAB (Sigma Chemical, Co.) dissolved in 0.05 M Tris/HCl buffer containing 0.68 mg/ml imidazole (Sigma Treatments. Two- to four-month-old female C57BL/6J mice were Chemical, Co.) and 0.1% hydrogen peroxide for 20 min. The mated with males overnight and the day on which a vaginal plug number of BrdU-labeled cells per 200 cells was counted in the 2 was found in the morning was considered as gestational day (GD) layers of basal vaginal epithelial cells and the stromal cells. 0. Pregnant mice were given daily subcutaneous (s.c.) injections of Statistical analysis. Data were expressed as the mean±standard 4 ml/kg body weight (bw) sesame oil as vehicle, EE2 (10, 25, 50 or 100 Ìg/kg bw) (Sigma Chemical Co., St. Louis, MO, USA), or DES error. Parametric variables were analyzed by one-way analysis of (6.7, 33.3 or 67 Ìg/kg bw) (Sigma Chemical Co.) from GD 10 to 18. variance with post-hoc t-test, Dunnett test or Fisher’s exact test. On GD 19, fetuses were removed by cesarean section and body P<0.05 difference was considered significant. weights and gender were recorded. Pups were foster-nursed to untreated ICR/Jcl mothers and weaned at 22 days of age. Results At 30 days, ovariectomy was performed, and ovaries and oviducts were weighed, separately. All mice were killed at 40 days Neonates exposed to EE2 or DES in utero. In the EE2- or and organs were dissected and weighed. A single s.c. injection of DES-exposed groups, the number of fetuses per litter was not BrdU (1 mg/10 g bw) was given 2 h before sacrifice. Materials, different from controls (Table I). The number of alive fetuses unless otherwise mentioned, were obtained from Wako Pure per litter in all groups of EE - or DES-exposed groups except Chemical Industries, Osaka, Japan. 2 10 Ìg/kg and 25 Ìg/kg EE2 groups was significantly less than Histological analysis. Uteri, vagina, liver, spleen, adrenal glands, that of the oil controls. No alive fetus in the 100 Ìg/kg EE2 kidneys and submandibular glands of mice were dissected and group was found. Fetuses from 67 Ìg/kg DES-exposed mice

868 Kirigaya et al: Effects of Ethinylestradiol on Female Mice

Table II. Body and reproductive organ weights of C57BL mice at 30 days Table III. Organ weights of C57BL mice at 40 days of age exposed in of age exposed in utero to EE2 or DES. utero to EE2 or DES.

Maternal n Body weight (g) Organ weights Maternal n Body weight (g) Organ weights treatments (mg/20 g body weight) treatment (mg/20 g body weights)

Ovary Oviduct Uterus Vagina

Sesame oil 20 13.4±0.54 9.3±1.65 3.4±0.27 Sesame oil 20 17.1±0.52 9.1±0.99 19.2±4.10 EE2 (Ìg/kg bw) EE2 (Ìg/kg bw) 10 16 13.5±0.47 9.1±0.77 4.5±0.65 10 10 16.1±0.39 5.5±1.30 15.3±3.79 25 6 13.4±1.30 9.7±0.68 4.5±0.96 25 6 16.7±1.19 5.4±1.22 9.4±1.60 50 5 13.4±2.24 9.1±0.80 3.7±1.08 50 3 18.9±1.47 5.7±0.43 13.5±3.21 DES (Ìg/kg bw) DES (Ìg/kg bw) 6.7 6 15.6±0.95 9.5±0.65 13.7±2.23* 6.7 4 17.5±0.76 6.4±0.33 15.5±0.41 33.3 3 15.6±0.40 7.3±0.56 2.5±0.02 33.3 3 17.9±0.12 9.8±1.60 11.4±0.57 67 1 16.4 7.3 ND 67 1 19.2 6.5 8.3

*p<0.05 compared with sesame oil control; ND: no data. *p<0.05 compared with sesame oil control.

showed very low survival rate, i.e., 19%. The body weight of Table IV. Vaginal epithelial stratification and cornification in 40-day-old ovariectomized mice exposed in utero to EE or DES. alive fetuses exposed to EE2 or DES was significantly less 2 than controls (Table I). Treatments n Mucification Stratification Cornification Gender ratios of alive fetuses did not show any difference (%) (%) (%) among experimental groups (Table I). Several dead fetuses exhibited cessation of development and rotted, their gender Sesame oil 8 0 0 0 could not be determined (data not shown). EE2 (Ìg/kg bw) 10 9 4 (44) 1 (11) 1 (11) 25 3 3 (100)* 0 0 Histological analysis of ovary and oviduct in 30-day-old mice 50 2 1 (50) 1 (50) 1 (50) exposed to EE2 or DES in utero. Only one out of the 143 DES (Ìg/kg bw) fetuses in the 67 Ìg/kg DES-exposed group survived until 30 6.7 4 2 (50) 2 (50) 0 days of age. Body and ovarian weights of EE -exposed or 33.3 3 3 (100)* 0 0 2 67 1 1 (100) 0 0 DES-exposed mice at 30 days showed no difference as compared to those of controls (Table II). While the weight *p<0.05 vs. oil controls (Fisher’s exact test). of oviduct in all EE2-exposed groups was not different, the oviducts of the 6.7 Ìg/kg DES-exposed mice were significantly heavier than those of controls. However, no histological changes were observed in the oviducts of the 6.7 of all EE2- or DES-exposed ovariectomized mice were not Ìg/kg DES-exposed mice. different from controls (Table III). In ovary, a gland-like structure was observed in a 50 Ìg/kg Four mice exposed to 10 Ìg/kg EE2 in utero showed EE2-exposed mouse (Figure 1A). In addition, mesonephric mucification of vaginal epithelium. The vaginal epithelium remnants were also observed both in EE2-exposed or DES- of one mouse exposed to 10 Ìg/kg EE2 showed superficial exposed mice (Figure 1B). PF were found in all mice cornification. In 25 Ìg/kg EE2-exposed mouse vagina, only examined (Figures 1 and 2). The total number of PF and mucification was found but not stratification or the incidence of PF in the 50 Ìg/kg EE2- and 33.3 Ìg/kg cornification in the epithelium. One mouse exposed to 50 DES-exposed mice were significantly larger than those of oil Ìg/kg EE2 showed stratification and cornification of the controls. Fifty percent of the 50 Ìg/kg EE2-exposed group vaginal epithelium. Fifty percent of the 6.7 Ìg/kg DES- showed PF of 3-6 oocytes. Neither corpus luteum nor exposed mice showed stratified vaginal epithelium (Figure hypertrophy of the ovarian interstitial tissue was found in 3 and Table IV). The BrdU-labeling index was high in the either the EE2- or the DES-exposed groups. vaginal epithelium of 50 Ìg/kg EE2- and 33.3 Ìg/kg DES- exposed mice. BrdU labeling index in the stromal cells of Histological analysis of uterus and vagina in 40-day-old mice EE2- and DES-exposed mice was not different from exposed to EE2 or DES in utero. Vaginal and uterine weights controls (Figure 4).

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Figure 1. Histology of ovary in 50 Ìg/kg EE2- (A, C, D) and 10 Ìg/kg EE2- (B) exposed 30-day-old C57BL6. Gland-like structure (A), mesonephric remnants (B), polyovular follicles containing 2 oocytes (C) and 4 oocytes (D). Bar = 50 Ìm.

Figure 2. Incidence of polyovular follicles (left) and number of polyovular follicles containing 2 oocytes, 2-3 oocytes or 5-6 oocytes (right) in C57BL6 ● mice at 30 days of age exposed in utero to oil, EE2 or DES. Values in columns indicate the number of mice examined. n.s., no survival, , one sample, *p<0.05 vs. oil controls (Dunnett test).

870 Kirigaya et al: Effects of Ethinylestradiol on Female Mice

Figure 3. BrdU-labeled cells in the vagina of 40-days-old ovariectomized mice. Animals were exposed in utero to oil (A), 25 Ìg/kg EE2 (B) and 50 Ìg/kg EE2 (C). Arrowheads indicate typical BrdU-labeled cells. Bar = 50 Ìm.

Discussion

The effects of sex hormones are different among the developmental stages of adult, neonate and fetus. The target organs of adult mice stimulated by sex hormones show reversible action. In the reproductive tracts of adult mice, cells are under the control of ovarian steroids. After the depletion of endogenous and exogenous sex hormones, cells of the target organs return to the unstimulated state. However, target organs of mice during the critical period of development can be permanently influenced by sex hormones, resulting in irreversible changes. The present study revealed that the developmental effects of EE2 on the survival rate and the reproductive organs in mouse fetus. The survival rate of off-spring was decreased by EE2 in a dose-dependent manner. Some dead fetuses showed cessation of development and growth, and rotted. Since the total number of fetuses in uterus per litter was similar among experimental groups, abortion was not induced by EE2 during pregnancy. One hundred Ìg/kg EE2 was a fatal dose to the off-spring and 50 Ìg/kg EE2 in utero induced a series of abnormalities in reproductive organs such as PF in the ovary and stratification and cornification in the vaginal epithelium. In the DES-exposed groups, 33.3 and 67 Ìg/kg DES increased both PF incidence and a persistent epithelial cell proliferation of vaginal epithelium. In 16-week-old ICR mice exposed to 20 Ìg/kg bw EE2 in utero, hypertrophy of the ovarian interstitial tissue and lack Figure 4. BrdU-labeling index of vaginal epithelial cells (A) and stromal of corpora lutea were reported (11). In the present study, no cells (B) in 40-day-old ovariectomized mice exposed in utero to oil, EE2 or DES. Values in columns indicate the number of mice examined. n.s., no hypertrophy of the ovarian interstitial tissue was observed in survival, ●, one sample, *p<0.05 vs. oil controls (Dunnett test). any mice at 30 days. The weight of ovary in 30-day-old EE2- or DES-exposed mice was not increased compared to that of control mice, confirming that hypertrophy and/or hyperplasia in 50 Ìg/kg EE2-exposed mouse ovary. Since dilated in ovary was not induced by EE2 or DES exposure until 30 mesonephric remnants were quite common in the younger days of age. On the other hand, a novel structure was found DES-treated animals (25), it may originate from a remnant

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of the medulary portion of gonadal primordia or remnants In ICR mice, 87% of the mice exposed to 20 Ìg/kg EE2 of the fetal menonephric tubules, also called paroophoron. and 12.5% of the mice exposed to 10 Ìg/kg EE2 in utero During embryonic development, cranial mesonephric- (from GD 11 to 17) showed vaginal epithelial cornification, derived tissues showed high 3H-estradiol binding (26), however, ovariectomy reduced the number of mice with suggesting that EE2 might inhibit the regression of cornification and exposed to 20 Ìg/kg EE2 down to 33% mesonephric tubules in females through ERs. (11). In the present study, 11% and 50% of the C57BL mice In the previous study, PF were found in the ovaries of exposed to 10 Ìg/kg EE2 or 50 Ìg/kg EE2, respectively, 30-day-old C57BL/Tw mice neonatally exposed to the oil showed cornification even after ovariectomy. Comparing vehicle and 1 Ìg DES for 5 days at 90 and 100% these results, the C57BL mice seem to be more sensitive to incidences, respectively (26). In the present study, PF were EE2 than the ICR mice. Moreover, 50% of 6.7 Ìg/kg DES- also found in ovaries of all mice prenatally exposed to oil, exposed C57BL mice showed stratified vaginal epithelium; 10, 25, 50 Ìg/kg EE2 and 6.7 Ìg/kg DES. In 50 Ìg/kg EE2- however, ovariectomized ICR mice exposed to 6.7 Ìg/kg exposed mice, a follicle having 6 oocytes was found in DES in utero showed no epithelial stratification in vagina ovary. These results indicate that in perinatal exposure to (35). This result suggests that there is a strain difference in EE2 and DES increases the incidence of PF containing response to synthetic estrogens during fetal stage in mice. three or more oocytes. Ashby et al. (36) reported that B6CBF1 mice were less Ovulation occurred in the ovary of DES-exposed mice in sensitive to DES compared with other strains (CD-1, response to equine and human chorionic gonadotropin (eCG C57Bl6 and Alpk) in the mouse uterotrophic assay. and hCG), resulting in a reduction of PF incidence. Oocytes However, CD-1 was much more resistant to the inhibition from PF in the DES-exposed mice showed a significantly of testes weight by implanted E2 than C17/Jls and C57BL/6J lower in vitro fertilization rate as compared to those from (37). Persistent vaginal cornification was induced by 0.1 Ìg uniovular follicles in control and DES-exposed mice (27, 28). E2 in the RIII strain but not in the BACB/c strain (15). Exogenous estrogen appeared to act directly on neonatal Thus, the sensitivity in response to estrogens is dependent ovary to induce PF formation in vivo and in vitro (27). These on mouse strain. Furthermore, in case of the evaluation of results suggest that both oocytes and follicles in the ovary adverse effects of synthetic chemicals, care must be taken as might be affected by estrogens. Jefferson et al. (29) to which strain of animals should be used. demonstrated that neonatal exposure to genistein induced PF In summary, mouse fetuses exposed in utero to a high in the ovary of ·ERKO mice but not in that of ER‚ dose of EE2 stopped growing and developing, resulting in knockout mice, suggesting that genistein can affect oocytes embryonic lethality. A series of abnormalities, such as PF and/or follicles through ER‚. Indeed, ER‚ was highly in the ovaries and persistent epithelial cell proliferation, expressed in the granulosa cells of rat ovary (30) and mouse stratification and cornification in the vagina were also ovary from day 5 to adult (31). In addition, Lemmen et al. induced in utero exposure to EE2 in the female (32) showed that ER‚ mRNA expression was observed in reproductive organs. both ovary and testis in a 16.5-day fetus. Furthermore, neonatal exposure to genistein inhibited oocyte nest break- Acknowledgements down (33). In mouse ovary, germ cell cysts break down into individual oocytes that become surrounded by somatic This work was partially supported by a Grant-in-Aid for pregranulosa cells to form primordial follicles after birth (34). Scientific Research on Priority Areas (A) (T.I. and T.S.), a It is hypothesized that an inhibition of oocyte nest breakdown Grant-in-Aid for Encouragement of Young Scientists (T.S.) from the Ministry of Education, Culture, Sports, Science and results in an induction of PF. Thus, maternal exposure to EE 2 Technology of Japan, a Grant for Support of the Promotion of could affect ovarian differentiation and induce PF even in Research at Yokohama City University (S.H. and T.S.), a Health fetal ovary via ER‚. Additional studies are necessary to Sciences Research Grant from the Ministry of Health, Labor and clarify the molecular mechanisms in induction of PF by Welfare, Japan (T.I). prenatal exposure to EE2. The numbers of BrdU-labeled cells in ovariectomized References mice exposed to 50 Ìg/kg EE2 in utero slightly increased, suggesting that maternal exposure to EE2 could induce an 1 DeCherney A: Bone-sparing properties of oral contraceptives. ovary-independent proliferation in fetal vagina. Persistent Am J Obstet Gynecol 174: 15-20, 1996. cornification of vaginal epithelium was induced by DES via 2 Kaunitz AM: Oral contraceptive health benefits: perception versus reality. Contraception 59: 29S-33S, 1999. ER· (22). The binding affinities of EE to ERs were 2 3 Poel WE: Pituitary tumors in mice after prolonged feeding of equivalent to that of E2 in rat uterus (23). Thus, synthetic progestins. Science 154: 402-403, 1966. reproductive abnormalities by maternal exposure to EE2 4 IARC monogr. Eval Carcinog Risk Chem Hum may be induced both through ER· and ER‚. Ethynyloestradiol 21: 233-255, 1974.

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