Altered activation/detoxication enzymology following neonatal diethylstilbestrol treatment

The effects of neonatal exposure to diethylstilbestrol (DES) on hepatic activation/detoxication enzyme levels in the adult rat were investigated. Neonatal exposure of male rats to DES (DES males) decreased the endogenous levels of UDP-glucuronyltransferase as compared to control males. Female rats exposed neonatally to DES (DES females) had higher endogenous epoxide hydrolase and glutathione transferase activity levels than control females. Adult animals treated neonatally with DES also had altered metabolic potential following exposure to 3-methylcholanthrene and phenobarbital. The DES males treated in adulthood with 3-methylcholanthrene had higher benzo(a)pyrene hydroxylase activities and lower UDP-glucuronyltransferase activity levels than did control males treated in adulthood with 3-methylcholanthrene. The DES males exposed in adulthood to phenobarbital had reduced cytochrome P-450 and glutathione transferase activity levels as compared with respective controls. The DES females treated in adulthood with 3-methylcholanthrene had lower benzo(a)pyrene hydroxylase and epoxide hydrolase activity levels than control females receiving 3-methylcholanthrene. The DES females challenged in adulthood with phenobarbital also had decreased benzo(a)pyrene hydroxylase, epoxide hydrolase, UDP- glucuronyltransferase, and glutathione transferase activity levels as compared with respective controls. Our results demonstrated that neonatal exposure to DES changed the endogenous levels of specific hepatic enzymes and altered the metabolic response of these adult animals to a carcinogen and a drug.     

Induction of sex reversal in Oreochromis mossambicus by diethylstilbestrol

The efficacy of the synthetic estrogen, diethylstilbestrol (DES), for sex-reversal in the Java tilapia, Oreochromis mossambicus was investigated. Fry of 8–10 mm total length were fed diets containing 25, 50 and 100 ppm of DES for 30 days in plastic pools; this was followed by 45–65 days rearing in fertilized cement cisterns where hormone-free diet was given. DES at 50 and 100 ppm induced 100% sex-reversal; DES at 25 ppm resulted in only a slightly larger proportion of females to males. The untreated control group had a higher proportion of males than females. No intersex or sterile individuals were observed among the steroid-treated fish. The present investigation demonstrates that 50 ppm DES administered for 30 days is sufficient to induce a 100% sex-reversal in O. mossambicus.     

Injection of diethylstilbestrol on the first day of incubation affects morphology of sex glands and reproductive behavior of Japanese quail

Japanese quail eggs were injected with DES (0.9-1,000 micrograms) dissolved in 50-microliter of corn oil on day 1 of incubation. Higher doses of DES (250-1,000 micrograms) reduced hatchability to 37-33% compared to 61% for corn oil-injected controls. Lower doses of DES (0.9-125 micrograms) had no effects on hatchability. In a second study, eggs were injected with 0.9 or 1.9 micrograms of DES and the survivors were assessed up to 12 weeks posthatch. DES did not affect hatchability, but did increase mortality during the first 4 weeks posthatch. Females were affected more than males. At 10 days of age, open-field activity of some birds was examined. The acquisition and reversal of a visual discrimination task was studied at 6 weeks of age. DES had no effect on these measurements. Ten females from each group were chosen randomly to determine egg production over a single 28-day period beginning at 6 weeks of age. Exposure to DES blocked egg production in these birds. The oviduct weights of 12-week-old females were decreased by 50%, but ovarian weights were not affected. Testicular weights were not affected. In a third study reproductive behaviors and social-dominance behaviors of males were markedly attenuated in birds exposed to 0.48 or 1.9 micrograms DES in ovo.     

Effects of prenatal exposure to diethylstilbestrol (DES) on hemispheric laterality and spatial ability in human males.

Ten males exposed to diethylstilbestrol (DES), a nonsteroidal synthetic estrogen, during gestation were compared to their matched, unexposed brothers on measures of brain hemispheric specialization for processing nonlinguistic spatial information and cognitive abilities. DES exposure was associated with reduced hemispheric laterality and lowered spatial ability. These data provide direct evidence of a relationship between brain laterality, spatial cognitive ability, and prenatal exposure to hormones in human males. Further, the implications of these findings for understanding sexual differentiation of the human brain are discussed.     

Apoptosis in the epididymal epithelium of adult male golden hamster exposed to diethylstilbestrol.

Apoptosis in the testis and prostate exposed to disrupters of endocrine function, including diethylstilbestrol (DES), during neonatal or postnatal periods has repeatedly been demonstrated, but not in the mature epididymis. We investigated the effects of DES, a potent and synthetic estrogen, on apoptosis in the adult. Adult male golden hamsters received an SC injection of DES and were then sacrificed to collect epididymides after 1, 4, or 7 days of treatment. A significant decrease in epididymal weight and an increase in apoptotic cells were shown on the first day after DES injection. Flow cytometry showed that DES treatment (1 mg/kg) for 1, 4, or 7 days induced significant apoptosis both in the caput and the cauda epididymides. Greater numbers of apoptotic cells were detected in the caput than in the cauda at a fixed time after DES treatment. Serum levels of testosterone decreased markedly within 24 hr after DES administration, reaching undetectable levels of 0.1 ng/ml at 4 days and thereafter. These results indicate that DES administration can increase epididymal apoptosis with a decrease in serum testosterone levels. Because DES used to be injected into domestic animals, adult males also have a chance to take this substance through food. Our study indicates that exposure to DES in adults is as toxic as that in the perinatal period.     

Use of diethylstilbestrol and ethynylestradiol to feminize Nile tilapia Oreochromis niloticus (L.) in an outdoor environment

Two synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE), were orally administered to 8.7 mm gonadally undifferentiated Oreochromis niloticus fry for a period of 28 days in an outdoor setting. Diethylstilbestrol was administered at doses of 100 mg, 200 mg, and 400 mg per kg diet. Ethynylestradiol was administered at 50 mg, 100 mg, and 200 mg per kg diet. One group received a non-hormone-treated feed. Hormone treatments produced significantly more (P < 0.05) than 50% females indicating that genotypic male fish were sex-reversed to phenotypic females. No rate of estrogen administration resulted in a 100% female population. Ethynylestradiol (EE) treatments resulted in 58–65% females, 32–35% males, and 3–9% hermaphrodites. Diethylstilbestrol (DES) treatments resulted in 60–80% females, 13–37% males, and 1–7% hermaphrodites. The DES 400 treatment was the most effective in altering phenotypic sex: 80% females, 13% males, 7% hermaphrodites.     

Limb reduction defects in the first generation and deafness in the second generation of intrauterine exposed fetuses to diethylstilbestrol

Maternal treatment with diethylstilbestrol (DES) during pregnancy can produce vaginal adenocarcinoma and other abnormalities of the vagina in her daughters when they reach adolescence or adulthood, miscarriages and absence of full term infants. Concerning malformations in newborns whose mothers were treated with DES, clitoromegaly and malformations of the uterus were reported in females and genital lesions in males. However, the frequencies of major congenital anomalies were not greater than expected. We report three cases of limb reduction defects (LRD) in the first generation of children whose mothers were treated with DES during pregnancy, and two children (one male, one female) with deafness in the second generation after intrauterine exposure to DES. The LRD were not associated with other congenital anomalies. The malformed children with LRD were born between 1965 and 1973. The deafness was also isolated. The two mothers who have no hearing problems and who are healthy were exposed in utero to DES in 1963 and 1965, respectively. Their children were born in 1989 and 1994, respectively. In conclusion, the association of LRD and hearing loss with intrauterine exposure to DES could be coincidental. However, some hypothesis may explain these associations. Congenital hearing loss in the second generation may suggest a transgenerational effect.     

Diethylstilbestrol-induced perinatal lethality in the rat. I. Relationship to reduced maternal weight gain

An analysis was performed to determine the mechanism of depressed maternal weight gain and its effect on perinatal lethality following prenatal exposure to diethylstilbestrol (DES). Pregnant Sprague-Dawley rats were dosed orally by gavage with DES or corn oil (control) during various intervals of gestation. The maternal weight-gain patterns of control and treated dams and the number of live offspring were recorded. The amounts of feed and water intake and feces and urine output in pregnant dams were measured, and metabolic rate and thyroid hormone levels were also determined. DES (at 45 micrograms/kg/day) was embryo- and fetolethal during implantation and parturition, and there was an accompanying decline in maternal weight. Growth of adult males, nonpregnant females, and weanlings of both sexes was also depressed. During pregnancy, the net intake of feed and water was not altered by the drug, but maternal serum thyroxine and metabolic rate were significantly elevated. Reduced metabolic efficiency, then, is the likely mechanism for weight depression. Reduction of maternal weight gain during pregnancy by DES is a diagnostic indicator of fetolethality, but is probably not causally related to it.     

Effect of in utero exposure to diethylstilbestrol on lumbar and femoral bone, articular cartilage, and the intervertebral disc in male and female adult mice progeny with and without swimming exercise

Introduction

Developmental exposure to estrogens has been shown to affect the musculoskeletal system. Furthermore, recent studies have shown that environmental exposure to estrogen-like compounds is much higher than originally anticipated. The aim of this study was to determine the effects of diethylstilbestrol (DES), a well-known estrogen agonist, on articular cartilage, intervertebral disc (IVD), and bone phenotype.

Methods

C57Bl/6 pregnant mice were dosed orally with vehicle (peanut oil) or 0.1, 1.0, and 10 μg/kg/day of DES on gestational days 11 to 14. Male and female pups were allowed to mature without further treatment until 3 months of age, when swim and sedentary groups were formed. After euthanasia, bone mineral density (BMD), bone mineral content (BMC), bone area (BA), and trabecular bone area (TBA) of the lumbar vertebrae and femur were measured by using a PIXImus Bone Densitometer System. Intervertebral disc proteoglycan was measured with the DMMB assay. Histologic analysis of proteoglycan for IVD and articular cartilage was performed with safranin O staining, and degeneration parameters were scored.

Results

The lumbar BMC was significantly increased in female swimmers at both the highest and lowest dose of DES, whereas the femoral BMC was increased only at the highest. The males, conversely, showed a decreased BMC at the highest dose of DES for both lumbar and femoral bone. The female swim group had an increased BA at the highest dose of DES, whereas the male counterpart showed a decreased BA for femoral bone. The TBA showed a similar pattern. Proteoglycan analysis of lumbar IVDs showed a decrease at the lowest doses but a significant increase at the highest doses for both males and females. Histologic examination showed morphologic changes of the IVD and articular cartilage for all doses of DES.

Conclusions

DES significantly affected the musculoskeletal system of adult mice. Results suggest that environmental estrogen contaminants can have a detrimental effect on the developmental lumbar bone growth and mineralization in mice. Further studies measuring the impact of environmental estrogen mimics, such as bisphenol A, are then warranted.     

Plasma testosterone levels and ovarian testosterone content in adult mice treated with diethylstilbestrol neonatally

Neonatal female NMRI mice (n = 16) were treated with 5 micrograms diethylstilbestrol (DES) per day, for the first 5 days after birth and killed postpubertally. Control females (n = 52) were injected with vehicle only and killed in different stages of the estrous cycle. The plasma testosterone level was significantly lower in DES females than in control females in any of the estrous phases. Ovariectomy (n = 5), adrenalectomy (n = 5) or a combination of both ablations (n = 3) did not affect the plasma testosterone in DES treated females while it was significantly reduced in control females (ovariectomy n = 5; adrenalectomy n = 9); most effective was the combination ovariectomy-adrenalectomy (n = 7). Ovarian homogenates from DES treated females (n = 10) had a significantly lower testosterone content than homogenates from control females in any phase of the estrous cycle (6-10 females per phase), which held true on both a per ovary basis and when related to ovarian weight. After a 2 h incubation in vitro, the testosterone levels had increased significantly in DES homogenates (n = 6) and to a lower extent in homogenates from control females in estrus (n = 9). No similar effect was found in homogenates from diestrus (n = 10) or proestrus (n = 9) females. The results are discussed in relation to the special ovarian morphology of adult but neonatally DES treated females and also with respect to endocrine control mechanisms.     

Complications of pregnancy in mice exposed prenatally to DES

Women exposed prenatally to diethylstilbestrol (DES) develop a variety of reproductive tract anomalies. Most of these anomalies have been replicated in strain CD-1 mice after similar DES exposure. Recently, impaired reproductive performance in DES-exposed women has been reported. To see whether the mouse model also replicates this defect, a study of reproduction was performed. Pregnant CD-1 mice were injected with DES and their female offspring were raised to breeding age. The latter were then exposed continuously to untreated males for a maximum of 4 months. Among 74 mated mice, 34 became pregnant and 11 of these pregnancies ended in abortion or stillbirth. Other anomalies encountered were: two fetuses with compressed heads, one of which seemed blocked from delivery by a vaginal adenocarcinoma; two uterine tumors, one of which was a teratocarcinoma; two teratomas located in uterine lumina; and two uteri containing placentas without embryos. Since the frequency of successful pregnancies in the DES-exposed mice was reduced below control levels to a degree similar to that reported for DES-exposed women, the validity of the mouse model has been confirmed for this characteristic.     

Mechanism of genotoxicity of diethylstilbestrol in vivo

Diethylstilbestrol (DES) is a carcinogen in humans and rodents which has eluded mechanistic clarification of its carcinogenic action. In vitro and in vivo, binding of DES to DNA has been found previously, but covalent DNA adducts could not be identified. In this study, the nature of binding was investigated by 32P-postlabeling, a rapid and highly sensitive assay for covalent DNA damage, to distinguish between a genotoxic or epigenetic mechanism of carcinogenesis by DES. A unique and distinct DNA adduct pattern was observed in kidney, liver, uterus (or testes) of female (or male, respectively) Syrian hamsters treated with a single injection of DES (200 mg/kg body weight). This set of DNA adducts closely matched patterns generated in vitro by reaction of diethylstilbestrol-4',4'-quinone with DNA or 2'-deoxyguanosine 3'-monophosphate. The major and several minor DES-DNA adducts in vivo had identical chromatographic mobilities in 11 different solvent systems with corresponding adducts obtained in vitro. The major adduct spot, generated in vitro by reaction of diethylstilbestrol-4',4'-quinone and DNA, was chemically unstable (half-life at 37 degrees C: 4-5 days). The persistence in vivo of these DNA modifications was low (biological half-life: 14 h) presumably because of chemical instability in concert with DNA repair. After injection of identical dosages of DES, adduct concentrations were 4-6-fold higher in females than in males. These results demonstrate that DES is capable of covalently modifying DNA. Moreover, diethylstilbestrol-4',4"-quinone is the major reactive metabolic intermediate responsible for the genotoxic activity of DES. Tumors are expected to arise only in rapidly dividing cells due to the short biological lifetimes of DES-DNA adducts.     

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.     

Effects of in utero exposure to Bisphenol A or diethylstilbestrol on the adult male reproductive system

The objective of this study was to determine whether in utero exposure to Bisphenol A (BPA) induced reproductive tract abnormalities in the adult male testis. Using the C57/Bl6 mouse, we examined sex‐organ weights, anogenital distance, and testis histopathology in adult males exposed in utero via oral gavage to sesame oil, 50 µg/kg BPA, 1000 µg/kg BPA, or 2 µg/kg diethylstilbestrol (DES) as a positive control from gestational days 10 to 16. No changes in sperm production or germ cell apoptosis were observed in adult testes after exposure to either chemical. Adult mRNA levels of genes associated with sexual maturation and differentiation, GATA4 and ID2, were significantly lower only in DES‐exposed testes. In summary, the data indicate no gross alterations in spermatogenesis after in utero exposure to BPA or DES. At the molecular level, in utero exposure to DES, but not BPA, leads to decreased mRNA expression of genes associated with Sertoli cell differentiation. Birth Defects Res (Part B) 92:526–533, 2011. © 2011 Wiley Periodicals, Inc.     

Diethylstilbestrol-treated adult rats with altered epididymal sperm numbers and sperm motility parameters, but without alterations in sperm production and sperm morphology

In this study, we characterized estrogenic effects of diethylstilbestrol (DES) on reproductive parameters in male rats to identify a minimal dose level that alters epididymal and sperm functions but has little or no effect on sperm production and/or spermatogenesis. Adult rats (five animals/group) received s.c. injections of 0.2 ml of corn oil containing DES at a rate of 1.0 mg, 200 microg, 40 microg, 8 microg, 1.6 microg, or 320 ng x rat(-1) x day(-1) for 12 days. The control group received corn oil only. DES effects were similar in the 8-microg group and higher dose groups and included significant (P < or = 0.05) reductions in 1) absolute and relative weights of the head and body of the epididymis (EP), tail of the EP, and seminal vesicle, 2) numbers of sperm in both regions of the EP, and 3) motility characteristics in sperm collected from the tail of the EP. Conversely, no significant changes were observed in relative testis weight, daily sperm production, spermatogenesis, seminiferous epithelial height in stage VII, and sperm morphology. All of the above parameters in the 1.6-microg group (except seminal vesicle weight) and 320-ng group were comparable to those of controls. Plasma testosterone (T) level was reduced to an almost undetectable level in the > or = 8-microg groups and to a very low level in the 1.6-microg group (0.35 vs. 2.36 ng/ml in controls or 320-ng group), but LH level was unaltered. In a parallel fertility study, males received DES at a rate of 40, 8, or 1.6 microg x rat(-1) x day(-1) for 12 days prior to and 12 days during cohabitation (1:1) with untreated females. Of the 15 females cohabited with treated males (5 females/dose), none in the 40-microg and 8-microg groups and 1 in the 1.6-microg group formed a copulatory plug and delivered 8 pups, in contrast to 5/5 copulatory plugs and 13-15 pups/litter in the controls. DES at a rate of 8 microg x rat(-1) x day(-1) for 12 days reduced EP weights, sperm numbers in the EP, and sperm motility patterns but caused minimal to no alterations in daily sperm production, spermatogenesis, or sperm morphology. Factors other than T, or in addition to lower T, may be responsible for DES-induced reproductive disorders (despite lower T, sperm contents and sperm motility patterns in the EP were normal in the 1.6-microg group). Deficits in EP sperm functions and/or sexual behavior (as evident from absence of copulatory plugs) probably accounted for reduced fertility in treated males.     

The functional importance of the oviduct in neonatally estrogenized mouse females for early embryo survival.

Eight-week-old virgin untreated female mice were induced to ovulate using equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG), and were then caged with males overnight. Females with a vaginal plug on the following morning were killed 24 hours later and 2-cell embryos were flushed from the oviduct. These embryos were transferred to the oviduct of 8-week-old control females, to females of the same age treated with 5 micrograms diethylstilbestrol (DES) sc in olive oil for the first 5 days after birth, or to females treated with 1 microgram estradiol-17 beta for 2 days before and 2 days after transfer (estrogen dominated/ED/females). Two days after transfer, a significantly lower number of embryos were recovered from oviducts of DES females compared to control females and a still lower number from ED females. The recovered embryos were cultured in vitro for 4 days testing trophoblast outgrowth ("implantation stage"). The incidence of embryos reaching this stage after development in DES-exposed oviducts was only half of that for embryos passing control oviducts or ED oviducts. It is concluded that the adult oviductal environment in neonatally DES-treated females significantly decreases early embryo developmental potential. The oviductal factor(s) harmful to the embryo may be related to a persistent and possibly increased level of circulating estrogen level in DES females.     

Effect of perinatal synthetic steroid hormone (allylestrenol, diethylstilbestrol) treatment (hormonal imprinting) on the bone mineralization of the adult male and female rat

Neonatal treatment with allylestrenol or diethylstilbestrol (DES) reduced the bone mineral content (BMC/bw) of the adult (four months old) female rats, without influencing bone mineral density (BMD/bw). In males these neonatal treatments elevated BMC and BMD alike. Ovariectomy alone decreased BMC and BMD alike; however the neonatal hormone treatments did not influence this reduced value. Ovariectomy of two months old animals increased body weight without the influence of neonatal hormone treatments. In adult males, the body weight was reduced significantly by neonatal DES and non-significantly by neonatal allylestrenol treatment. The experiments call attention to the possible human bone-effects of allylestrenol, which was used in the last decades as medication protecting endangered pregnancies.     

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.     

Brugia pahangi: Antibodies and microfilaremias in Lewis rats

Male and female Lewis rats were inoculated subcutaneously in the left groin with 75 infective larvae of Brugia pahangi and microfilaremias were followed for as long as 420 days postinoculation. Patent infections developed in 64% of the female rats and 95% of the male rats. Mean prepatent periods were similar (65.9 and 63.9 days, respectively), but mean microfilaremias in males rose much higher, to a mean of 218 mf/0.25 ml blood at 270 days postinoculation. IgG titers, as measured by enzyme-linked immunosorbent assay (ELISA), to adult worm somatic antigen were higher than those to microfilariae in almost all rats. For both sexes, the most consistently microfilaremic rats had highest titers to these antigens. Granulomas with degenerating microfilaria were present in the spleen of male rats with high microfilaremias (>100–300 mf/0.25 ml blood). Ouchterlony precipitin reactions suggested that most rats with spleen granulomas responded to microfilarial antigen components to which most rats without granulomas did not. Neither spleen granulomas nor antibody responses measured in this study appeared to have protective (microfilaremia-lowering) value. As measured by microfilaremias, the male Lewis rat is not as susceptible as some conventional hosts of B. pahangi, but it does consistently become infected and remains microfilaremic for more than a year. Preferential male susceptibility indicates that this model may be useful for studying this aspect of human lymphatic filariasis.     

Failure to detect a second-generation effect in female mice after neonatal treatment with an estrogen (diethylstilbestrol).

Inbred female mice of the NMRI strain were treated subcutaneously with 5 micrograms diethylstilbestrol (DES) in olive oil or vehicle only for the first 5 days after birth. One group of DES-treated females was killed at the age of 8-12 weeks, and the uterine cervix and adjacent parts of the vagina and uterine horns prepared for histological studies. In all preparations, the cervical epithelial lining contained regions with heterotopic columnar epithelium (HCE) along 69-100% of the length of the common cervical canal. Ovaries from neonatally DES-treated females were grafted to 8-week-old ovariectomized control hosts and these hosts were mated to control males 2 weeks later. The hosts gave birth to normal-sized litters. The female offspring from these litters had a normal cervical epithelial lining and, in turn, gave birth to normal-sized litters. These results indicate that treatment of neonatal female mice with DES does not affect the female germ cells as far as concerns factors associated with the development of HCE or reduced fertility in the next generation.