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Estrogen receptor (ER) and ER mRNA expression and cell division induced by neonatal diethylstilbestrol (DES) exposure in male C57BL/Tw mouse genital organs were studied using immunohistochemistry, in situ hybridization, and cell counting at metaphase. A single injection of 3 micrograms DES on the day of birth induced ER in both epithelial (E) and stromal (S) cells of the epididymis and the seminal vesicles, in E cells of the ductus deferens, and in S cells of the ductus efferens and the anterior prostate 24 h after the injection. After 5 daily injections of DES, ER was induced in S cells of the ventral prostrate at 5 days of age. The staining intensity of ER increased in E cells of the epididymis and the ductus efferens at 5 days. A single injection of DES induced ER mRNA in both E and S cells of the ductus efferens, the caput epididymis, and the ductus deferens 4 to 12 h after injection of the newborn mice. A single injection of DES stimulated cell division of both E and S cells in the ductus deferens and the epididymis; cell division of S cells of the ductus efferens, the anterior prostate, and the urethra was stimulated 24 h after the injection. After 5 daily injections of DES, cell division was stimulated significantly in E and S cells of the epididymis, the ductus deferens, and the ventral prostate, and in S cells of the seminal vesicle at 5 days. At 30 days of age, mitotic rates were significantly higher in E cells of the seminal vesicles and the epididymides of mice given 5 daily injections of DES.(ABSTRACT TRUNCATED AT 250 WORDS)

Neonatal treatment of female mice with a synthetic estrogen, diethylstilbestrol (DES), results in adenosis, ovary-independent epithelial proliferation and cornification, and downgrowths in the vagina. Protein synthesis was examined in the vagina of 45-day-old, ovariectomized C57BL/Tw mice which had been given 5 daily injections of 2 micrograms DES or the oil vehicle alone from the day of birth, and in those of age-matched, ovariectomized mice given 3 daily injections of 0.1 microgram DES from 42 days of age. [35S]Methionine-labeled proteins of the vagina were analyzed by two-dimensional polyacrylamide gel electrophoresis. Major changes were observed in 11 proteins of the vagina in both neonatally and postnatally DES-exposed groups. Six of the 11 proteins were increased in expression, but one of the remaining proteins was decreased. In the group of neonatally DES-exposed mice alone, expressions of 4 proteins (MW. 128, 90, 46 and 44 kDa) were markedly increased. These results indicate that neonatal exposure of mice to DES induced ovary-independent, persistent alteration in the protein synthesis of the vagina.

Postpubertal progesterone injections slightly inhibited the proliferation and cornification of the vaginal epithelium induced by neonatal injections of diethylstilbestrol (DES). In vaginae of neonatally DES-exposed mice (DES mice), 9 protein expressions (PEX) appeared newly; 13 PEX decreased; 5 PEX increased compared to those in the controls. In vaginae of DES mice, PEX were altered by postpubertal injections of progesterone (DES-P); 3 PEX disappeared; 11 PEX were reversed to those in the controls. Progesterone injections impaired the proliferation of the vaginal epithelium, reversing 11 PEX compared to those in DES mice. In uteri of DES-P mice, 3 PEX increased, 2 PEX decreased, 2 PEX appeared and 1 PEX disappeared compared to those in DES mice. In conclusion, it was shown that postpubertal injections of progesterone alter the protein expression in the vagina and uterus of DES mice.

Postpubertal injections of a synthetic estrogen, diethylstilbestrol (-DES), and an anti-estrogen, tamoxifen (-Tx), stimulate proliferation of vaginal and uterine epithelial cells of ovariectomized (OVX) adult mice. In vaginae of two groups of OVX mice treated neonatally with DES (DES-) and oil vehicle alone (Oil-), postpubertal injections of DES altered all and 25 out of 37 protein expressions examined, except for keratin polypeptides, respectively. Twenty-one of the DES- and Tx-altered protein expressions showed the same behavior, suggesting that Tx acts as an estrogen agonist on these proteins. Both neonatally Oil- and DES-treated OVX mice given postpubertal injections of DES (Oil-DES and DES-DES) and Tx (Oil-Tx and DES-Tx) showed similar histological changes in the vagina. Epithelial proliferation and superficial cornification were observed in vaginae of five groups of Oil-DES, Oil-Tx, DES-DES, DES-Tx and DES-Oil (given oil postpubertally) OVX mice. In these groups, 13 proteins showed the same behavior, implying that these proteins are related to proliferation and cornification of mouse vagina. The histology of the uterus in DES-Oil OVX mice closely resembled that in Oil-Oil OVX mice; in protein expressions of the uterus, 5 proteins in DES-DES and 7 proteins in DES-Tx OVX mice were different from those in Oil-DES and Oil-Tx OVX mice, respectively. These findings indicate that neonatal DES treatment alters the behavior of the vagina and uterus in response to postpubertally administered DES and Tx.

The distribution of the estrogen receptor (ER) was investigated immunohistochemically in the genital tract of 0 (day of birth)--to 50-day-old C57BL/Tw female mice given 5 daily injections of 2 micrograms diethylstilbestrol (DES) from the day of birth using an anti-ER monoclonal antibody. In the oviduct, cervix and vagina of control mice, both epithelial and stromal cell nuclei showed positive ER immunoreaction at 0 day. Nuclei of uterine stromal cells also showed strong ER immunostaining even at 0 day. In contrast, the ER in uterine epithelial cells began to appear by day 5. The number of positive epithelial cells and staining intensity gradually increased until 10 days. In neonatally DES-exposed mice, ER appeared in uterine epithelial cells 24 h after the injection. Polygonal cells appearing in the vaginal epithelium of DES-exposed 3-day-old mice showed weak ER staining. Neonatal DES exposure reduced staining intensity of columnar vaginal epithelial cells and uterine stromal cells at 3 days. Vaginal epithelial cells undergoing ovary-independent stratification and cornification in neonatally DES-exposed mice and normal estrous mice showed ER immunoreaction only in the basal cells.

Neonatal treatment of female mice with diethylstilbestrol (DES) results in genital tract abnormalities including ovary-independent vaginal proliferation and cornification. Protein profiles were examined in vagina and uterus from 45-day-old, ovariectomized C57BL/Tw mice which had been given 5 daily injections of 2 micrograms DES or oil vehicle alone from the day of birth, and in those from 45-day-old, ovariectomized mice given 3 daily injections of 0.1 microgram DES from 42 days of age. Proteins extracted were analyzed by two-dimensional polyacrylamide gel electrophoresis. In the vagina, expression of 37 non-keratin proteins was altered by postpubertal injections of DES as compared with the controls. In the neonatally DES-exposed vagina, expression of 26 of 37 proteins was altered as compared with controls. In the uterus, expression of 22 proteins was altered in the postpubertally DES-exposed group as compared with that in the control; however, the protein expression pattern of the neonatally DES-exposed group closely resembled that of the control except for one protein (no. 23, pI = 6.1, MW = 39.5 kDa) which was specifically increased in the neonatally DES-exposed group. By immunoblot analysis, 6 keratin polypeptides (49.5, 50, 52, 53, 57 and 58 kDa) were identified in vaginae of ovariectomized mice exposed neonatally and postpubertally to DES and of the controls. These results indicate that neonatal DES exposure induces organ specific alterations in the synthesis of proteins in mouse vagina and uterus.

Pregnant ICR/JCL mice were given 4 daily subcutaneous injections of 0.2-2000 micrograms diethylstilbestrol (DES) starting on day 15 of gestation. Offspring of mothers given DES were killed at 1-10 days of age and examined for nodules of enlarged polygonal cells under the epithelium of the Müllerian (upper) vagina. Some offspring were ovariectomised at 30 days and killed at 120 days. The nodules which appeared in the prenatally DES-exposed mice (2-2000 micrograms/day) at 3-7 days were not connected with the epithelium of the sinus vagina and reacted positively to an antibody to epidermal growth factor. Nodule formation may prove to be prodromic of later ovary-independent vaginal changes in the DES-exposed mice. Epithelial stratification (2-2000 micrograms/day) and downgrowths and/or pegs (20-2000 micrograms/day) occurred in vaginae of ovariectomized mice exposed prenatally to DES; however, adenosis-like lesions occurred only in the offspring of mothers given the highest prenatal injections of 2000 micrograms DES. Wolffian remnants and hypospadias (2-2000 micrograms/day) were also encountered in the DES-exposed mice. Ovary-independent stratification of the uterine epithelium (20-2000 micrograms/day) and disorganization of the circular musculature (2-2000 micrograms/day) were also observed in the DES-exposed mice. None of these changes was found in ovariectomised 0.2 micrograms DES-exposed and control mice.

In 35-day-old female ICR/JCL mice given 5 daily injections of 1 microgram diethylstilbestrol (DES) from the day of birth, a significantly higher incidence of polyovular follicles was found in the ovaries than in those of age-matched control mice. Gap junctions of granulosa cells of mature follicles in neonatally DES-exposed mice were larger than those of the controls. When stimulated by gonadotropins (PMSG and hCG) and caged with males, the number of tubal embryos in DES-exposed mice was less, but the rate of fertilization and development was not different compared to the controls. Division of oocytes collected from the ovaries of 40-day-old DES-exposed and control mice after stimulation of gonadotropins was examined 24 to 72 h after in vitro insemination to ascertain whether fertilization had occurred in oocytes from polyovular follicles. Seventy-seven % of oocytes from uniovular follicles of control mice developed up to 8-cell stage embryos following in vitro insemination; 66% of those from similar follicles of DES-exposed mice developed into the same stage. By contrast, only 47% of oocytes from polyovular follicles of DES-exposed mice showed the division up to 8-cell stage 72 h after insemination, indicating a significantly lower fertilization rate compared to the oocytes from uniovular follicles of control and DES-exposed mice. Without insemination, oocytes taken from the same pools in these experiments never divided during the period of manipulation and incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

In 35-day-old C57BL/Tw female mice given daily injections of 1 microgram diethylstilbestrol (DES) for 5 days from the day of birth, a significantly higher incidence of polyovular follicles (PF) were found in the ovaries than in those of age-matched control mice. Ovaries of prepubertal mice treated neonatally with oil or DES (DES mice) showed an enhancement of ovulation and luteinization following a combined treatment with eCG and hCG. Tubal ova in DES mice treated with eCG plus hCG were surrounded by many granulosa cells. Incidence of PF in control mice was not changed by eCG plus hCG treatment. In contrast, PF incidence in DES mice was reduced by prepubertal injections of eCG plus hCG. A high incidence of PF was also found in newborn mouse ovaries transplanted for 30 days into ovariectomized adult hosts given DES injections, but not in ovaries transplanted into intact or ovariectomized DES-untreated hosts. When neonatal ovaries were cultivated in a serum-free medium containing DES for 5 days and then transplanted into ovariectomized hosts, PF were formed in the grafts, but not in DES-unexposed grafts. Oocytes from PF in DES mice were found to have a smaller capacity for fertilization when examined in vitro. The present study also demonstrated that neonatal ovaries exposed to estrogen in vivo or in vitro (which produces PF in prepubertal hosts) are capable of responding to gonadotropins given later, resulting in a reduction of PF incidence, and that exogenous estrogen acts directly on neonatal ovaries to induce PF.

Permanent proliferation and cornification of the vaginal epithelium occurred in adult ovariectomized (OVX) mice which had received neonatal injections of 5 micrograms diethylstilbestrol (DES). Occurrence of the permanent epithelial proliferation was prevented by injections of 200 IU vitamin A acetate (VA) given simultaneously with neonatal DES-treatment in mice when OVX at 30 days of age, but not in those OVX at 270 days. For the suppression of the permanent vaginal changes, however, more than one month were required after DES plus VA-treatment. In DES plus VA-treated OVX (at 30 days) mice, the vaginal epithelium indicated a lower mitotic rate than in DES-treated OVX mice. Clear cells were found in the degenerating vaginal epithelium in 63% of DES plus VA-treated OVX mice, while in the proliferated epithelium, this type of cells appeared only in 10% of DES-treated OVX mice. In 40% of 180-day-old, DES-treated OVX (at 30 days) mice, permanent stratification took place in the uterine epithelium, while it was inhibited in the age-matched DES plus VA-treated OVX mice. However, the uterine change was not prevented in 310-day-old, DES plus VA-treated mice when OVX at 270 days. This findings suggests that the relatively long-term presence of ovaries nullifys the inhibitory effect by VA on the occurrence of permanent changes in the utero-vaginal epithelium induced by neonatal DES treatment.

Pregnant ICR/JCL mice were given either four daily subcutaneous injections of 20-2000 micrograms diethylstilbestrol (DES)/day or a 4-day intravenous infusion of 20-200 micrograms DES/day starting on Day 15 of gestation. Female mice were injected with 0.01-50 micrograms DES/day for 5 days starting on the day of birth. Females given oil injections during the neonatal period and offspring of mothers given injections of an infusion of respective vehicles alone served as controls when corresponding ages were attained. Incidence of adenosis-like lesions (ADL) in the vaginal epithelium and stratification of the uterine epithelium (UST) were determined in both offspring and neonatally exposed mice at 30 days of age. Injections of 0.1-50 micrograms DES/day during the neonatal period induced ADL in the fornical epithelium of vagina (36-70%), but not UST. A high incidence of ADL (80-88%) was found in the fornical and upper-vaginal epithelia in offspring of mothers given injections of 200-2000 micrograms DES/day, and UST was encountered in 38-70% of these mice. Offspring of mothers given an infusion of 20 micrograms DES/day had low incidences of ADL (11%) and UST (22%), whereas offspring of mothers infused with 200 micrograms DES/day exhibited high incidences of ADL (71%) and UST (86%). In offspring of mothers given injections of 2000 micrograms DES/day, ADL appeared in the fornical and upper-vaginal epithelial as early as 15 days of age. Thus, the present study revealed that ADL and UST occur at a high incidence in infantile mice exposed prenatally to high doses of DES. Previous studies failed to detect such effects perhaps due to the strain of mice, methods, duration, and/or dose of DES used.

The occurrence of polyovular follicles (PF) was examined at 10-34 days of age in the ovaries of BALB/cCrgl female mice given five daily injections of 0.1 microgram diethylstilbestrol (DES), 2 micrograms DES, 100 micrograms progesterone (P), 137 micrograms 17 alpha-hydroxyprogesterone caproate (HPC), 20 micrograms testosterone (T), 20 micrograms 5 alpha-dihydrotestosterone (5 alpha-DHT), or oil vehicle alone starting on the day of birth, and of C57BL/Tw females given five neonatal injections of 1 microgram DES, 20 micrograms 17 beta-estradiol (E2), 50 micrograms 5 alpha-DHT, 50 micrograms 5 beta-DHT, or the vehicle alone. Ovaries of 30-day-old C57BL mice given five daily injections of 1 microgram DES starting at 3-25 days of age were also examined. PF incidence (% of PF per ovary) and PF frequency (% of mice with PF) were significantly greater in BALB/c mice receiving injections of DES, P, HPC, and T than in the controls. In DES-treated mice at 34 days, PF incidence (2-13 oocytes/follicle) was 120-340 times higher than in the controls. BALB/c mice treated with T, P, and HPC showed PF incidence (two to four oocytes/follicle) three- to six-fold higher than in the controls. In 30-day-old C57BL mice treated with T, E2, and DES, PF incidence also increased by two- to 50-fold. 5 alpha-DHT and 5 beta-DHT failed to increase PF incidence. PF incidence was significantly increased only when neonatal DES treatment was begun on days 0 to 3, but was reduced when started at days 10-25.(ABSTRACT TRUNCATED AT 250 WORDS)

Diethylstilbestrol was continuously infused into pregnant mice through the tail vein. In male fetus exposed to diethylstilbestrol (DES) from days 15 to 19 of gestation, Leydig cells of the testis were found to be increased in number when examined at 3-60 days of age. Mitotic rate in the interstitial tissue was elevated in 0- to 3-day-old mice exposed prenatally to DES. The ratio of the intertubular space to testis size was greater at 3 days of age than at 1 day in prenatally DES-exposed mice, while in the controls, the rate was lower at 3 days than at 1 day. Spermatogenic activity was lower in DES-exposed adult mice than in the controls, as shown by the spermatogenic index.

Postnatal development of uterine abnormalities was examined in the offspring of mice given 4 daily subcutaneous injections of 2,000 micrograms diethylstilbestrol (DES) and of oil vehicle alone from days 15 to 18 of gestation. The prenatally DES-exposed mice (DES mice) exhibited a disorganization of the inner circular musculature of the uterus as early as at 5 days of age. Invaginations of the luminal epithelium into the stroma which began to form in 5-day-old control and 10-day-old DES mice developed into uterine glands at 10 and 15 days, respectively. The number of glands per uterine section was significantly reduced in DES mice as compared with the controls at 10-30 days. Stratification with or without squamous metaplasia (ST) was found in the luminal epithelium of DES mice at 20 days. Five groups each of DES mice and the controls ovariectomized (OVX) at 10 days were given 5 daily subcutaneous injections of 10(-4), 10(-3), 10(-2), 10(-1), and 1 microgram 17 beta-estradiol (E2) starting at 25 days. ST was never observed in OVX mice given prepubertal injections of any E2 dose, whereas in DES OVX mice aged 30 days, ST was produced by prepubertal injections of E2 doses higher than 10(-2) micrograms/day. Uterine responsiveness, weight gain and gland formation were significantly reduced in DES mice given prepubertal E2 injections compared to the controls.

Polyovular follicles (PF) occur in the ovary of 30-day-old offspring of ICR/JCL mice given 4 daily subcutaneous injections of 20-2,000 micrograms diethylstilbestrol (DES)/day from days 15 to 18 of gestation. PF containing 2-9 oocytes per follicle in the prenatally DES-exposed mice are increased 33- to 112-fold as compared to controls. In 5- to 25-day-old offspring of mothers given injections of 2,000 micrograms DES/day, PF are observed 17-65 times more frequent than in controls.

A high incidence of vaginal adenosis-like lesions (ADL, 67%) was found in 30-day-old offspring of ICR/JCL mice given 4 daily subcutaneous injections of 2,000 micrograms diethylstilbestrol (DES) on days 15-18 of gestation. The prenatally DES-exposed mice and the controls ovariectomized at 10 days of age were given 5 daily subcutaneous injections of 10(-4), 10(-3), 10(-2), 10(-1), or 1 micrograms 17 beta-estradiol (E2), respectively, starting at 25 days of age. ADL was never observed in the age-matched controls given prepubertal injections of any E2 dose. In contrast, high incidences of ADL (63-100%) were found in the vaginal fornix and upper vagina of 30-day-old DES mice receiving prepubertal injections of 10(-3)-1 micrograms E2/day, whereas in DES mice given prepubertal injections of the vehicle alone and a lower daily dose of 10(-4) micrograms E2, incidences were nil and 25%, respectively. Mitotic figures were found in the columnar epithelium with ADL, although the rate was lower than in the ADL-freed stratified epithelium. It is suggested, therefore, that an ovarian hormone (probably estrogen) participates in the prepubertal induction of ADL in DES mice.