Karen T. Mills

Long-term genital tract changes in female mice treated neonatally with coumestrol

The neonatal mouse model has proven to be an effective system to examine long-term reproductive tract abnormalities resulting from early exposure to estrogens. Newborn C57BL/Crgl mice received 8 x 10(-2) micrograms diethylstilbestrol (DES) or 100 micrograms coumestrol (a plant estrogen) in 0.005 mL dimethyl sulfoxide (DMSO) or DMSO alone or received no treatment for the first 5 days of life. Half of the animals were ovariectomized at 40 days of age. Vaginal lavages were examined for 15 consecutive days before termination at 13 months of age, at which time genital tracts and mammary glands were removed for histological examination. Diethylstilbestrol- and coumestrol-treated animals exhibited ovary-independent persistent vaginal cornification as well as cervico-vaginal pegs and downgrowths, uterine squamous metaplasia, and an enhancement of age-related changes in the ovary including hemorrhagic follicles. In general, neonatal exposure to the naturally occurring plant estrogen, coumestrol, has long-term effects similar to those seen following exposure to natural and synthetic estrogens.

Reproductive abnormalities in female mice exposed neonatally to various doses of coumestrol

Female C57BL/Crgl mice were neonatally exposed to various doses of coumestrol to determine the threshold doses required for the occurrence of reproductive tract abnormalities. Newborn mice received daily subcutaneous injections of 10(-3), 10(-2), 8 X 10(-2), 10(-1), 1, 5, 25, 50, and 100 micrograms coumestrol in 0.005 ml dimethyl sulfoxide (DMSO) or DMSO alone, or received no treatment for the first 5 d of life. Some of the animals were ovariectomized at 40 d of age. Mice were killed at 20-22 mo of age. All neonatal doses of coumestrol advanced vaginal opening before that of controls. At 2 and 20-22 mo of age, doses greater than or equal to 25 micrograms consistently resulted in ovary-independent persistent vaginal cornification as judged by vaginal smears. Intact untreated and DMSO-treated control mice exhibited aging changes in the genital tract, some cervical adenosis and early cervicovaginal pegs and downgrowths, uterine cystic glandular hyperplasia, corpora lutea, and scattered areas of ovarian ceroid deposition. Intact mice receiving neonatal coumestrol exhibited cervicovaginal pegs and downgrowths (at all doses with the exception of 25 and 50 micrograms), cervical adenosis (at doses greater than or equal to 8 X 10(-2) micrograms), uterine squamous metaplasia (significant at doses greater than or equal to 50 micrograms), and a decrease in uterine cystic glandular hyperplasia (significant at doses greater than or equal to 25 micrograms). The levels of 10(-1), 5, and 100 micrograms neonatal coumestrol daily resulted in hemorrhagic follicles. An increase in ovarian ceroid deposition (significant at doses greater than or equal to 5 micrograms) was observed. At 40 d and 20-22 mo of age, corpora lutea were consistently absent from the 100-micrograms-treated animals. Most of the ovariectomized untreated and DMSO-treated control animals showed typical castrate-like morphology of the genital tract, with the majority of the control mice exhibiting uterine cystic glandular hyperplasia. Ovariectomized mice receiving coumestrol neonatally exhibited various degrees of cervicovaginal alterations: pegs and downgrowths (significant at all doses with the exception of 10(-1) micrograms), endometrial collagen deposition (significant at greater than or equal to 25 micrograms), and reduced or absent uterine glands (significant at 10(-3), and 10(-11), and at all doses greater than or equal to 5 micrograms).

Influence of neonatal diethylstilbestrol treatment on androgen and estrogen receptor levels in the mouse anterior prostate, ventral prostate and seminal vesicle

Perinatal exposure to the synthetic estrogen, diethylstilbestrol (DES), affects the structure of both male and female reproductive systems. Changes may also occur in the levels of steroid hormone receptors. Cytosolic and nuclear androgen and estrogen receptor levels (expressed per mg DNA) from the sex accessory glands of male BALB/c mice exposed neonatally to DES were analyzed by exchange assays. Neonatal DES exposure caused significant decreases in: (1) cytosolic androgen and cytosolic and nuclear estrogen receptor levels in the anterior prostate and (2) cytosolic estrogen receptor levels in the ventral prostate. A significant increase was seen in the cytosolic estrogen receptor levels in the seminal vesicle. Significant decreases in cytosolic protein levels occurred in all DES-exposed glands.

Vaginal abnormalities in ovariectomized BALB/cCrgl mice after neonatal exposure to different doses of diethylstilbestrol

Newborn BALB/cCrgl female mice received five daily injections of various doses of diethylstilbestrol (DES), 0.0001-10 micrograms. Mice were killed at 6 days of age or at 4 months after ovariectomy at 40-42 days. Subepithelial nodules of polygonal cells in the upper (Mullerian) vagina during early postnatal life were associated with the later occurrence of ovary-independent persistent stratification with or without cornification in mice treated neonatally with 0.1-10 micrograms DES and thus are a possible predictor of this phenomenon. The thresholds for the induction of ovary-independent epithelial pegs, downgrowths and adenosis (glandular formations) were 0.1 microgram and 0.5 microgram DES/day, respectively.

Reproductive alterations in female C57BLCrgl mice exposed neonatally to zearalenone, an estrogenic mycotoxin

Newborn female mice were injected daily for 5 days with 1 microgram zearalenone (Z, a weakly estrogenic mycotoxin present in cereal grains), resulting in ovary-dependent reproductive tract alterations at 8 months of age. Corpora lutea were absent from 25 of 34 (74%) Z-treated mice, indicating ovarian dysfunction. Fifty-six percent of Z-treated mice had dense collagen deposition in the uterine stroma and lacked uterine glands. Squamous metaplasia of the uterine luminal epithelium was found in 59% of Z-treated mice, and altered vaginal epithelium was found in 32% (2 mice had dysplastic lesions). Ovariectomized Z-treated mice were indistinguishable from ovariectomized controls.

Critical Period for Neonatal Estrogen Exposure in Occurrence of Mammary Gland Abnormalities in Adult Mice

Abstract There exists a critical period for the development of cervicovaginal lesions in both mice and humans exposed neonatally and antenatally to sex hormones. Mammary glands from year-old female BALB/c mice exposed neonatally to 20 μg estradiol for 5 days commencing at 1 day of age showed the most mammary abnormalities, significantly greater than in controls (P < 0.005). The incidence of abnormalities declined when treatment was begun after Day 1. Treatments begun after Day 3 did not result in this structural pattern. Mice ovariectomized after treatment all had inactive mammary glands with no abnormalities. There is a critical exposure period for the later occurrence of mammary gland abnormalities. However, the aberrant secretory state which accompanies these mammary gland alterations may be a consequence of permanent alteration in ovarian function or its endocrine control.

Effect of later treatment with estrogen on reproductive tract lesions in neonatally estrogenized female mice

Continuous ethinyl estradiol treatment following ovariectomy at day 38 of BALB/cfC3H mice treated neonatally with estradiol resulted in a more organized vaginal epithelium than that seen in the absence of postnatal estrogen, 30 days after treatment was initiated. Although the epithelium became increasingly disorganized in all neonatally estrogenized groups with age, the later estrogen treatment reduced the incidence and severity of vaginal epithelial lesions and the incidence of vaginal concretions. Uterine epithelial metaplasia was accelerated by the later estrogen treatment.

Sensitivity of the Vagina and Uterus of Mice Neonatally Exposed to Estrogen or Androgen to Postnatal Treatment with Estrogen or Androgen

Abstract Newborn female BALB/cCrgl mice receiving 5 μg of testosterone or 0.01 μg of diethylstilbestrol daily for the first 5 days of life were examined at various times after secondary exposure to testosterone and 17β -estradiol, respectively. Neonatal administration of testosterone induced squamous stratification associated with constant cornification of the vaginal epithelium in intact mice. Later exposure to testosterone suppressed cornification, resulting in superficial epithelial mucification in almost all mice by 4 months of age. However, at 6 months of age, the incidence of mucification dropped to 58%. Cervicovaginal lesions developed in the groups of mice given neonatal testosterone in combination with later testosterone and sacrificed at 4 and 6 months of age. Continuous vaginal stratification was found in 14% of ovariectomized, neonatally diethylstilbestrol-treated mice at 13 months of age. The incidence of this ovary-independent change increased to 40% at 24 months of age. Postnatal estrogen replacement significantly increased the incidence of squamous stratification in these mice. Neonatal diethylstilbestrol treatment alone induced cervicovaginal lesions in 4.5% of ovariectomized mice at 13 months of age; secondary 17β-estradiol exposure significantly enhanced the development of lesions to 44%. However, at 24 months of age, there was no difference in the incidence of lesions in ovariectomized, neonatally treated mice with or without the secondary 17β-estradiol treatment. These results suggest that the effects of neonatal exposure to a relatively low dose of estrogen, androgen, or related substance may become obvious later in life as a result of later exposure to hormones. [P.S.E.B.M. 1992, Vol 199]

Estrogen and progestin receptors in mouse vaginal epithelium and fibromascular wall

Both sodium molybdate and Percoll density gradient stabilize the hormone-binding capacities of the estrogen and progestin receptors and individually increase the recovery of these receptors in prepared cytosols of the separated mouse vaginal epithelium and fibromuscular wall. Their effects are additive. The concentrations of estrogen receptors are similar in the epithelial and fibromuscular compartments, whereas progestin receptor concentrations are higher in the epithelium.

Altered mammary responsiveness to estradiol and progesterone in mice exposed neonatally to diethylstibestrol

Mammary glands from ovariectomised neonatally diethylstilbestrol (DES)-exposed (0.1 microgram daily for the first 5 days of life) mice seem morphologically indistinguishable from those of ovariectomised controls. However, administration of exogenous hormones reveals a differential response. In DES-exposed mice, estrogen implantation resulted in greater incidence of dilated ducts along with greater incidence of dilated ducts along with greater incidence and severity of terminal ductal hyperplasia and greater severity of cystic alveolar adenosis; combined estrogen and progestin treatment resulted in greater severity of terminal duct hyperplasia and less alveolar formation, and progestin treatment resulted in lower incidence and degree of lateral budding. Thus, mammary sensitivity to sex steroids is altered by early exposure of mice to DES.