Exposure to diethylstilbestrol during pregnancy permanently alters the ovary and oviduct

To determine the effects of transplacental exposure to diethylstilbestrol (DES) on the ovary and oviduct of the CD-1 mouse, timed pregnant mice were injected subcutaneously with DES (100 micrograms/kg) on Days 9 through 16 of gestation and female offspring sacrificed from 4 weeks to 10 months of age. Following DES exposure, ovarian alterations such as inflammation, a prominent interstitial compartment composed of medullary tubule-like structures, and intra- and para-ovarian cysts from mesonephric remnants were observed. In addition, there were oviductal abnormalities including malformation. As reported previously, the oviduct was closely adherent and coiled around the ovary in a similar position to that seen in the fetal mouse. This malformation was termed developmental arrest of the oviduct (DAO) and was a consistent finding in female offspring exposed prenatally to DES (100 micrograms/kg). Increased prevalence of salpingitis and microscopic alterations in the oviduct were also observed. Oviductal epithelium was mostly secretory type with basal vacuoles. In some cases, oviductal epithelium was hyperplastic and formed mucosal folds resembling glands which extended through the muscularis (diverticulosis). The extent of the adenomatous mucosal folds and the degree of extension through the muscularis increased with the age of the animal (100% at 10 months). Some characteristics of this abnormality resembled salpingitis isthmica nodosa, a lesion described in women which is associated with ectopic pregnancies and subfertility. Gross and microscopic changes in the oviduct were more consistent than were the changes among other portions of the reproductive tract of DES-treated mice previously reported. Since subfertility has been described in this mouse model as well as in prenatally DES-exposed women, the data presented in this report may help in evaluation of the reported reduced fertility in exposed patients as well as other infertility patients.     

Ovarian reproductive function after exposure to diethylstilbestrol in neonatal life

Inbred and random-bred NMRI mice were treated with diethylstilbestrol (DES, 5 micrograms per day) or vehicle (olive oil) on Days 1-5 after birth. At the age of 8 wk, females were treated with saline or eCG and hCG to induce ovulation. Ova never occurred in the ampulla of the uterine tube of saline-treated, DES-treated females when these mice were not mated. After gonadotropin treatment, ova were found in the ampulla of all olive oil-treated females and in approximately 80% of DES-treated females. The number of ovulated ova was similar in both groups. Twenty percent of gonadotropin-treated, DES-treated females had ova in the ampulla and a vaginal plug after being caged with males but none became pregnant. Ovaries from inbred control or DES-treated females were grafted to the ovarian bursa of control or DES-treated ovariectomized hosts. DES-treated hosts, carrying control or DES-exposed ovaries, never became pregnant. Control females, with control ovaries or DES-exposed ovaries, became pregnant; pregnancy rate and litter size were similar for control mice regardless of whether they were supporting DES-exposed or control ovaries. Oocytes from ovaries exposed neonatally to DES can thus give rise to apparently normal offspring. The results also indicate DES-induced nonovarian disturbances, e.g. tubal and/or endometrial function, both of which are important for fertility. In the grafting experiments, a high mortality rate was found in inbred DES-exposed females caged with males. All deaths were associated with vaginal concrements (vaginal stones) and intestinal complications.     

Superovulation and early embryo development in the adult mouse after prenatal exposure to diethylstilboestrol

Pregnant mice were injected subcutaneously with diethylstilboestrol (DES: 10 micrograms/kg body weight in 0.1 ml corn oil) or corn oil alone on Day 15 or 16 of gestation (Day 1 = day of copulatory plug) and allowed to give birth. Female progeny from control and DES-exposed animals were superovulated with exogenous gonadotrophins at 6-8 weeks of age. In-vivo results indicated that the total number of ovulated ova, 2-cell embryos and blastocysts were significantly increased in DES-exposed progeny but that there was a decline in developmental potential from the ovulated ova stage to the blastocyst stage in these animals. However, there was no significant difference in the in-vitro development of 2-cell embryos to the blastocyst stage between control and DES-exposed animals. These results indicate that the ovaries of mice exposed in utero to DES are capable of responding to exogenous gonadotrophins and that second generation progeny have the potential for normal development to the early postblastocyst stage of embryogenesis. The in-vivo decline in developmental potential may be attributable to reproductive tract abnormalities rather than ova/embryo defects.     

Molecular differentiation of the mouse genital tract: altered protein synthesis following prenatal exposure to diethylstilbestrol

Exposure in utero to the synthetic estrogen diethylstilbestrol (DES) has been associated with the subsequent development of reproductive tract lesions in both women and experimental animals. Using the techniques of organ culture and two-dimensional (2-D) gel electrophoresis, the effects of DES on protein synthetic patterns were studied during fetal and neonatal development of the CD-1 mouse. The protein patterns, analyzed by comparing 2-D fluorograms after [35S] methionine incorporation at different developmental stages, were correlated with the histology at the same age. Several qualitative and quantitative changes in protein synthesis were observed after prenatal DES exposure. A protein, apparent by Day 14 of gestation, with molecular weight approximately 70,000 and pI of 5.8, was observed to be greatly diminished in all reproductive tract tissues exposed to DES during prenatal development. This alteration, induced in utero, persists through the early postnatal differentiation of the genital tract (17 days old). This protein may provide an early marker for alterations in normal reproductive tract function.     

Cytologic evidence of extensive keratotic reaction in women exposed to diethylstilbestrol in utero

Squamous mucous membranes and squamous metaplastic epithelium sometimes undergo hypermaturation with the production of a keratin layer. Anucleated keratotic squamous plaques in smears are generally recognized as cytologic evidence of this altered maturation. This keratotic reaction was quantified in cytologic smears from 191 women exposed in utero to diethylstilbestrol (DES). Keratotic reaction was observed in the vaginal smear in 40% of the cases, in the cervical smear in 26% and in the endocervical smear in 19%; overall, a keratotic reaction was observed in at least one specimen from 48% of the women. These frequencies are higher than those reported in other studies. The observed frequency was age related. The significance of the hyperkeratosis, including its possible relationship to a lower dysplasia rate among DES-exposed women, is unclear. No conclusions can be drawn until more is known about behavioral factors in DES-exposed women.     

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.     

The development of cervical and vaginal adenosis as a result of diethylstilbestrol exposure in utero

Exposure to exogenous hormones during development can result in permanent health problems. In utero exposure to diethylstilbestrol (DES) is probably the most well documented case in human history. DES, an orally active synthetic estrogen, was believed to prevent adverse pregnancy outcome and thus was routinely given to selected pregnant women from the 1940s to the 1960s. It has been estimated that 5 million pregnant women worldwide were prescribed DES during this period. In the early 1970s, vaginal clear cell adenocarcinomas (CCACs) were diagnosed in daughters whose mother took DES during pregnancy (known as DES daughters). Follow-up studies demonstrated that exposure to DES in utero causes a spectrum of congenital anomalies in female reproductive tracts and CCACs. Among those, cervical and vaginal adenoses are most commonly found, which are believed to be the precursors of CCACs. Transformation related protein 63 (TRP63/p63) marks the cell fate decision of Müllerian duct epithelium (MDE) to become squamous epithelium in the cervix and vagina. DES disrupts the TRP63 expression in mice and induces adenosis lesions in the cervix and vagina. This review describes mouse models that can be used to study the development of DES-induced anomalies, focusing on cervical and vaginal adenoses, and discusses their molecular pathogenesis.     

In utero diethylstilbestrol (DES) exposure alters Hox gene expression in the developing müllerian system.

Diethylstilbestrol (DES) was widely used to treat pregnant women through 1971. The reproductive tracts of their female offspring exposed to DES in utero are characterized by anatomic abnormalities. Here we show that DES administered to mice in utero produces changes in the expression pattern of several Hox genes that are involved in patterning of the reproductive tract. DES produces posterior shifts in Hox gene expression and homeotic anterior transformations of the reproductive tract. In human uterine or cervical cell cultures, DES induces HOXA9 or HOXA10 gene expression, respectively, to levels approximately twofold that induced by estradiol. The DES-induced expression is not inhibited by cyclohexamide. Estrogens are novel morphogens that directly regulate the expression pattern of posterior Hox genes in a manner analogous to retinoic acid regulation of anterior Hox genes. Alterations in HOX gene expression are a molecular mechanism by which DES affects reproductive tract development. Changes in Hox gene expression are a potential marker for the effects of in utero drug use that may become apparent only at late stages of development.     

Effects of early exposure to diethylstilbestrol on cellular protein expression by mouse vaginal epithelium and fibromuscular wall

Epithelia and fibromuscular walls were dissociated from the vaginae of ovariectomized BALB/cCrgl mice (ca. 41 days old) exposed neonatally to diethylstilbestrol (DES) or sesame oil and purified by centrifugation through Percoll density gradients. Neonatal exposure to DES caused the vaginal epithelium to become permanently proliferated and partially keratinized; the control epithelium was low cuboidal. The major cellular proteins expressed in each tissue compartment were examined by two-dimensional gel electrophoresis of [35S]methionine-labeled tissues. The epithelia and fibromuscular walls displayed distinctive two-dimensional protein patterns. In the DES-exposed vaginal epithelium, the expression of two proteins (one had a molecular size of 65 kDa and a pl of 6.0, and the other had a molecular size of 38 kDa and a pl of 6.3) was increased, while the expression of three proteins with molecular sizes of 25, 30, and 140 kDa and pls of 5.6, 5.6 and 6.7, respectively, was reduced, relative to the control epithelium. In the DES-exposed vaginal fibromuscular walls, the expression of 9 proteins was increased whereas the levels of 21 specific proteins, distinct from those in the epithelium, were decreased. Thus, long-term tissue-specific alterations in the synthesis of a select number of cellular proteins occur in the DES-exposed vagina.     

The mouse bioassay for the detection of estrogenic activity in rodent diets: I. A standardized method for conducting the mouse bioassay

A standardized procedure was developed for conducting the mouse bioassay for detecting estrogenic activity in rodent diets. Studies were conducted with CD-1 mice to determine the appropriate weaning age and length of bioassay period. Uterine growth curves were generated from mice weaned at 15 days of age and fed a negative control diet until 28 days of age. These mice showed slow regular increases in uterine weights from 15 22 days of age followed by rapid uterine growth in some mice from 24 to 28 days of age. Estrogenic bioassays using female mice weaned at 15 days of age and fed the positive control diets containing 4 or 6 ppb diethylstilbestrol (DES) demonstrated significant (P less than 0.05) increases in uterine weight and in uterus to body weight (U:BW) ratios over those of mice fed the negative control diet without DES for 5, 7 or 9 days after weaning. In contrast, mice weaned at 17 days of age showed significant (P less than 0.05) increases in uterine weight and in U:BW ratios only at 5 days after weaning. Six ppb DES was required in the positive control diet to produce a 1.5 fold increase in the U:BW ratio over those of mice fed the negative control diet. It was concluded that mice should be weaned at 15 days of age and that the bioassay period should be terminated at 7 days, when the mice are 22 days old, for best reproducible results. The criteria for a valid bioassay should include the demonstration of a significant statistical increase in the U:BW ratios of mice fed the DES positive diet over those of mice fed the negative control diet.     

Intrauterine death, fetal malformation, and delayed pregnancy in Ljungan virus-infected mice

BACKGROUND: A picornavirus (Ljunganvirus [LV]) has recently been associated with disease during pregnancy in its natural rodent reservoir and in humans. A study of laboratory mice infected under controlled conditions was therefore undertaken. METHODS: CD-1 female mice were infected gestational day two and subjected to varying regimes of stress. RESULTS: LV infection in combination with stress resulted in uterine resorptions, malformations, and neonatal death. A short delay in time to first pregnancy and births was observed in pairs infected in utero. CONCLUSIONS: LV is found in different species of native animals in both Europe and the United States and human epidemiological evidence connects LV and human reproduction, while the observations here indicate that LV is responsible for reproductive problems in a laboratory mouse model. The current findings suggest that the hypothesis that LV also causes disease in pregnant women and their offspring deserves further study.     

Effect of certain growth factors on proliferation in serum-free collagen gel culture of vaginal epithelial cells from prepuberal mice exposed neonatally to diethylstilbestrol

Neonatal treatment with diethylstilbestrol (DES) induces ovary-independent vaginal epithelial changes in mice. The response of vaginal epithelial cells from intact prepuberal BALB/cCrgl mice treated neonatally with 2 micrograms of DES for 5 days to growth-stimulatory and -inhibitory factors was studied using a serum-free collagen gel culture system that sustains the growth of normal vaginal epithelial cells. Cells from control and DES-exposed mice at 21 days of age showed about a 5-fold increase in number during 10 days in a serum-free medium supplemented with transferrin, bovine serum albumin fraction V, insulin, and epidermal growth factor. Epidermal growth factor and insulin stimulated dose-related proliferation of vaginal epithelial cells from both control and DES-exposed mice; however, cells from DES-exposed mice showed a reduced growth response to epidermal growth factor and an increased growth response to insulin, compared with control cells. Insulin-like growth factor I (1-100 ng/ml) tested in the absence of insulin failed to stimulate cell growth. Transforming growth factor-beta (0.05-5 ng/ml) consistently inhibited cell growth in a dose-dependent manner.     

Diethylstilbestrol exposure in utero: a paradigm for mechanisms leading to adult disease

The synthetic estrogen diethylstilbestrol (DES) was administered to pregnant women between the 1940s and the mid-1970s and is believed to be responsible for numerous uterine/cervical/vaginal malformations and cancers that appeared after birth and in young adult life. This medical tragedy has served as one of the prototypical examples of a phenomenon known as "endocrine disruption," in which either environmental agents or other compounds disrupt normal hormonal signaling in the body. Whereas DES signals through estrogen receptors, the subsequent molecular targets were largely unknown. We had identified Wnt7a as a target in this pathway and have used genetic analyses of mutant mice to demonstrate that disruption of Wnt7a is the key event leading to the DES phenotypes and cancers. We find that Wnt7a expression is only transiently deregulated in response to DES exposure, leading to the conclusion that critical events during early reproductive tract development results in a permanent change or "reprogramming" in subsequent development.     

Structural and functional changes in ovaries from adult mice treated with diethylstilboestrol in the neonatal period

Ovaries from 8-week-old female NMRI mice in different stages of the oestrous cycle, or from females neonatally treated with the synthetic oestrogen diethylstilboestrol (DES; 5-10(-6) micrograms daily for 5 days), were studied histologically and for the ability to synthesize steroids from [3H]pregnenolone in vitro. Daily doses of 10(-4) micrograms DES or higher resulted in absence of corpora lutea. In ovaries lacking corpora lutea, the interstitial tissue dominated and the cells in this compartment were large with a clear cytoplasm. The steroids synthesized in ovarian homogenates were separated with thin-layer chromatography. The homogeneity of the steroids was checked in recrystallization experiments. Daily doses of 5-10(-4) micrograms DES in the neonatal period resulted in pronounced deviations in the pattern of ovarian steroids synthesized as compared with control ovaries. In DES-exposed ovaries, the synthesis of androstenedione and, above all, progesterone was high while the synthesis of 17 alpha-hydroxyprogesterone and testosterone was reduced compared with controls. These results could argue for a difference in activities of 17 alpha-hydroxylase and 17 beta-ol-dehydrogenase in ovaries from DES-treated females compared with controls. After transplantation of DES-exposed ovaries to ovariectomized control females, the steroid pattern changed to that typical for control ovaries. Control ovaries transplanted to DES-treated females had a steroid pattern similar to that of DES-exposed ovaries.     

Sexual Differentiation of Cognitive Abilities in Women Exposed to Diethylstilbestrol (DES) Prenatally

In nonhuman animals, prenatal exposure to androgens or estrogens enhances development of male-typical characteristics (masculinizes) and impairs development of female-typical characteristics (defeminizes). We investigated the hypothesis that prenatal exposure to the synthetic estrogen, diethylstilbestrol (DES), similarly masculinizes or defeminizes cognitive development in women. Forty-two DES-exposed women and 26 of their unexposed sisters were studied. No group differences were seen for abilities at which females excel on average (verbal fluency, perceptual speed and accuracy, and associative memory), for abilities at which males excel on average (visuospatial abilities), or for abilities that do not show sex differences (vocabulary, nonverbal intelligence). The time of prenatal exposure to DES correlated with visuospatial performance with later exposure associated with better performance. However, the subgroup of women exposed to DES late in gestation did not differ from unexposed women on these measures. Results support the conclusion that prenatal exposure to DES has little or no influence on cognitive development in women. However, they do not preclude other types of early hormonal influences on human cognition, such as prenatal influences of androgen or influences of androgens or estrogens during the early postnatal period.     

Histological assessment of the mouse uterus from birth to puberty for the appearance of LGL-1+ natural killer cells.

The appearance of natural killer (NK) cells during growth and maturation of the murine uterus was studied by immunohistochemistry, using the monoclonal antibody LGL-1. To determine the contributions of microorganisms in the environment and of T-cell and B-cell regulation to the establishment of a uterine NK cell population, uteri from barrier-raised, flora-defined, random-bred CD-1 mice and from genetically T-cell- and B-cell-deficient SCID mice (genotype C.B-17 scid/scid) were compared to uteri from conventionally raised CD-1 mice. Uteri were studied from birth to the ages at which these mice are normally paired for mating (7-10 wk). Absolute uterine weight and the ratio of uterine weight to body weight increased remarkably between 3 and 5 wk of age in each group of animals. Growth continued beyond Week 5 of age, and in all groups the ratio of uterine weight to body weight was similar at puberty, although both the flora-defined CD-1 and SCID mice were significantly smaller than conventionally reared mice. LGL-1+ cells could not be detected in any of the neonatal uteri examined. LGL-1+ cells were first detected at 2 wk of age in uteri from the conventional and flora-defined CD-1 mice. A significant increase in the number of LGL-1+ NK cells occurred in the CD-1 uterus between Weeks 2 and 3 of age and again between Weeks 5 and 7 of age. Environmental conditions did not alter the frequency of LGL-1+ cells between the two groups of CD-1 mice at any age studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mouse bioassay for the detection of estrogenic activity in rodent diets: II. Comparative estrogenic activity of purified, certified and standard open and closed formula rodent diets

A major source of exogenous estrogenic substances, which may affect laboratory animals, comes from the diet. To test the possibility that commercially available rodent diets may significantly influence uterine weights and uterine:body weight (U:BW) ratios, estrogen bioassays were performed using female CD-1 mice weaned at 15 days of age and assigned randomly to a variety of commercial test diets or to a control diet (Purina #5002) containing 0 or 6 ppb added diethylstilbestrol (DES) for comparison. Mice were housed five per cage and given deionized water and feed ad libitum. Uterine:BW ratios from 15 mice per diet were determined after 3, 5 and 7 days of feeding. Mice fed The American Institute of Nutrition purified diet (AIN-76A) or the Purina #5015 natural ingredient breeder diet had significantly (P less than 0.05) increased U:BW ratios at 3, 5 and 7 days post weaning when compared to the control diet without added DES. This increase in U:BW ratios was similar to the U:BW ratios observed in a natural ingredient maintenance diet (Purina #5002), containing 6 ppb of DES. These results show that significant differences exist in the level of substances which can cause increase in uterine weight in some commercial diets. The diet may be important when performing or comparing certain types of studies, especially those relating to estrogenic substances. A standardized diet with minimal estrogenic activity may be desirable for such studies. It is unclear from the present studies what substances might be responsible for the uterine growth promoting activity in the diets examined.     

MSX2 promotes vaginal epithelial differentiation and wolffian duct regression and dampens the vaginal response to diethylstilbestrol

In utero exposure to diethylstilbestrol (DES) leads to patterning defects in the female reproductive tract (FRT) and a propensity to the development of vaginal adenocarcinomas in humans. In the mouse, DES treatment similarly induces a plethora of FRT developmental defects, including stratification of uterine epithelium and presence of glandular tissue in cervix and vagina. Uterine abnormalities are associated with repression of the homeobox gene Msx2, and DES leads to an altered uterine response in Msx2 mutants including a dilated uterine lumen. Here we investigate the role of Msx2 in normal vaginal development and in FRT response to DES. During vaginal development, Msx2 is required for Tgfbeta2 and Tgfbeta3 expression and for proper vaginal epithelial differentiation. Moreover, Msx2 is involved in caudal Wolffian duct regression by promoting apoptosis. Consistently, neonatal DES exposure represses Msx2 expression in the Wolffian duct epithelium and inhibits its apoptosis and subsequent regression. Intriguingly, although DES treatment also represses Msx2 expression in the vaginal epithelium, a much more severe DES-induced vaginal phenotype was observed in Msx2 mutant mice, including a complete failure of Müllerian vaginal epithelial stratification and a severely dilated vaginal lumen, accompanied by loss of p63 and water channel protein expression. These results demonstrate a critical role for Msx2 in counteracting the effect of DES on FRT patterning and suggest that the response to DES may be highly variable depending on the genotype of an individual.