Neonatal estrogen treatment and its consequences for thymus development, serum level of autoantibodies to cardiolipin, and the delayed-type hypersensitivity response.

Eight-week-old female and male NMRI mice treated neonatally with the synthetic estrogen diethylstilbestrol (DES), estradiol-17beta, or tamoxifen displayed an enlarged thymus when compared with controls (approximately 1.5-fold). In control females, either ovariectomy or adrenalectomy increased thymus weight to the level characteristic for DES-treated females, but these endocrine ablations had no significant effect in DES females. The serum estrogen levels were similar in intact DES, ovariectomized DES, and ovariectomized female controls; serum corticosterone was similar in controls and DES females. The expression of the Thy1.2+ marker and the percentages of CD4+CD8+ DP and CD4+ and CD8+ SP cell subsets were similar in thymocyte populations from 8-wk-old controls and DES females; the CD4+ and CD8+ SP subsets were similar in splenocyte populations. The levels of serum immunoglobulin (Ig) G and IgM autoantibodies to cardiolipin showed age-dependent fluctuations but were similar in controls and DES females; however, the IgG autoantibodies in DES females were qualitatively different from those in controls with respect to sensitivity to bovine serum (a source of beta2-glycoprotein I). Contrary to females, DES-treated males had higher levels of autoantibodies than controls. The delayed-type hypersensitivity (DTH) response to oxazolone was similar in controls and DES animals at 8 wk, increased in DES females and males at 6 mo, but was reduced in DES females at 1 yr. Thus, even though adult mice with thymus enlargement after neonatal estrogen treatment do not differ from controls with respect to the expression of the Thy1.2 marker or percentages of CD4+/CD8+ DP or SP subsets in thymus and spleen, qualitative and quantitative differences occur in immune parameters (autoantibodies to cardiolipin) and a T-cell-dependent immune response (DTH).

New approaches for estimating risk from exposure to diethylstilbestrol.

A subgroup from a National Institute of Environmental Health Sciences, workshop concerned with characterizing the effects of endocrine disruptors on human health at environmental exposure levels considered the question, If diethylstilbestrol (DES) were introduced into the market for human use today and likely to result in low-dose exposure of the human fetus, what would be required to assess risk? On the basis of an analysis of the quality of data on human DES exposure, the critical times and doses for inducing genital tract malformations and cancer must be determined. This would be facilitated through analysis of the ontogeny of estrogen receptor expression in the developing human genital tract. Models of low-dose estrogenic effects will have to be developed for human and rodent genital tract development. Mouse models offer many advantages over other potential animal models because of the wealth of the earlier literature, the availability of sensitive end points, the availability of mutant lines, and the possibility of generating genetically engineered model systems. Through multidisciplinary approaches, it should be possible to elucidate the cellular and molecular mechanisms of endocrine disruption elicited by estrogens during development and facilitate an assessment of risk to humans.

Immunohistochemical studies on the expression and estrogen dependency of EGF and its receptor and C-fos proto-oncogene in the uterus and vagina of normal and neonatally estrogen-treated mice.

BACKGROUND: The final target cell response to estrogen is dependent not only on the estrogen receptor, but also on autocrine/paracrine interactions with growth factors (e.g., EGF) and proto-oncogenes (e.g., c-fos). Because neonatal estrogen treatment results in permanent changes in the female mouse genital tract (permanent vaginal cornification, cervical adenosis and tumors, changed growth control mechanisms in uterus), it was of interest to study possible acute and permanent effects of such treatment on distribution and levels of EGF, its receptor (EGF-r), and c-fos and to relate such changes to morphological development and appearance of epithelial abnormalities. METHODS: Immunohistochemical techniques using frozen sections from the uterus and vagina of neonatal and adult (ovariectomized, estradiol-treated) females, treated with olive oil or diethylstilbestrol in neonatal life. RESULTS: A difference in stromal-epithelial distribution of EGF was demonstrated with respect to region studied (uterus, vagina) and age (neonatal, adult). EGF was localized mainly in the uterine stroma but in both vaginal epithelium and stroma (with a different pattern compared to uterus). In neonatal females, EGF occurred in both tissue components in both regions, and the distribution pattern was quite different from that in adult females. The EGF level was increased by estrogen in adult but not in neonatal females. EGF-r and c-fos occurred in both uterine epithelium and stroma and in the vaginal epithelium; levels and distribution pattern were affected by estrogen. Neonatal estrogen treatment increased the levels of uterine EGF and c-fos in adult life. CONCLUSIONS: There are distinct developmental changes in the distribution and estrogen sensitivity of EGF. Only further studies can prove or disprove the association between the earlier reported disturbed growth control mechanisms in the uterus of adult but neonatally estrogen-treated females and the increased levels of uterine EGF and c-fos. The present results do not seem to explain mechanisms involved in the origin of neonatally estrogen-induced cervicovaginal epithelial abnormalities, nor do they explain the earlier described difference in estrogen-induced proliferative response between the uterine cervix and uterus proper.

A morphologist's approach to the vagina.

The author reviews basic facts on vaginal histology and ultrastructure with respect to passage of different substances and drugs through the epithelium. Particular interest is given to the action mechanism of topically applied intravaginal estrogen, its binding to receptors in different vaginal cell types, and possible local metabolic routes for estrogen. Differences in estrogen sensitivity between uterus and vagina are discussed as well as the background for the high sensitivity in the vagina.

The different responses of the female mouse thymus to estrogen after treatment of neonatal, prepubertal, and adult animals.

Contrary to the common description of estrogen-induced thymus atrophy we have observed a thymus enlargement after treatment of neonatal female mice with estrogen. We now describe an age-dependent difference in the estrogen response (enlargement, atrophy) as well as mechanisms relevant to the response. Groups of female NMRI mice were treated with estrogen (diethylstilbestrol, DES) at different 5-day periods in prepubertal (day 1-5, day 6-10, day 30-34) or postpubertal life (days 48-52). All the treatment groups showed a reduced thymus weight 4 days after the last treatment but later responses differed. Neonatal DES treatment resulted in an ovary-independent thymus enlargement 8 weeks after the treatment when the cortical part was relatively larger than in controls; treatment on days 30-34 was followed by a rebound type of regeneration; the acute weight reduction after treatment on days 48-52 was normalized 16 days later. Neonatal DES treatment transiently depressed the number of thymic S phase cells 4 days after the treatment while apoptosis was similar in controls and DES females. The estrogen receptor pattern was not affected by DES. The number of white blood cells was temporarily depressed while the bone marrow cellularity was still reduced in 8-week-old females. Neonatal treatment with an LH-releasing hormone antagonist reduced thymus weight at 8 weeks but had no effect on the DES-induced enlargement. The delayed-type hypersensitivity response developed differently in controls and DES females. The thymus enlargement after neonatal estrogen treatment could be the result of an increased immigration of precursor cells into the thymus and/or a defect maturation/emigration mechanism. Further studies on different cell subsets are necessary to explain the mechanism behind the thymus enlargement.

A morphologist's approach to the vagina--age-related changes and estrogen sensitivity.

This paper reviews basic facts on vagina histology and ultrastructure with respect to passage of different substances and drugs through the epithelium. Particular interest is devoted to the action mechanism of topically-applied vaginal estrogen, its binding to receptors in different vaginal cell types, and possible local metabolic routes. Differences in estrogen sensitivity between uterus and vagina are discussed as well as the background for the high vaginal sensitivity.

Estrogen effects on lymphoid tissue in neonatal and adult female mice.

Outbred female mice of the NMRI strain were treated with daily doses of diethylstilbestrol (DES; 0.1-10 micrograms), estradiol-17 beta (E2; 5 micrograms), testosterone (T; 5 or 25 micrograms), progesterone (P; 100 micrograms), corticosterone (CC; 10 or 50 micrograms), or olive oil (controls) for the first 5 days after birth. Animals were killed on day 6 after birth, at 2, 4, and 8 weeks or at 4 and 6 months. Only CC resulted in reduced thymus index weight (IW, mg organ weight/g body weight) on day 6 while the estrogen-induced reduced IW was secondary to a reduced body weight. Four-week-old and older estrogen-treated females had increased thymus IW (1.8-fold at 8 weeks after 5 micrograms DES/day neonatally) which was still seen at 6 months. Estrogen-induced thymus enlargement also occurred in 8-week-old inbred NMRI females and in females belonging to the BALB/c strain. Thymus IW in 8-week old T-, P- and CC-treated females was as in controls. The leukocyte counts in blood and bone marrow were lower in DES females than in controls. The responses (thymus IW, peripheral blood leukocyte count) of 8-week-old females to ovariectomy and challenge with DES were different in DES females and in control females. The mitogen response of spleen lymphocytes and thymocytes was similar in controls and DES females. An anomaly in the hypothalamic-pituitary-gonad-thymus axis of adult, neonatally estrogen-treated females might explain the thymus enlargement.

Factors of importance for decreased early embryo survival in female mice treated neonatally with diethylstilboestrol.

Neonatal female mice of the NMRI strain were treated s.c. with 5 micrograms of the synthetic oestrogen diethylstilboestrol (DES) or vehicle only (olive oil; controls) for the first 5 days after birth. Embryos at the two-cell stage from control females were transferred to the oviducts of control females or DES-treated females (6-8-weeks-old). Immediately after transfer, the oviducts were removed and incubated in vitro for 6 h. After incubation, slightly fewer embryos were recovered from oviducts exposed to DES compared with control oviducts (81% versus 92%; 0.05 > P > 0.01). When the recovered embryos were cultured in vitro, 64% of the embryos from control oviducts reached the blastocyst stage in contrast to only 24% of those from oviducts of DES-treated mice; slightly fewer of the latter showed trophoblastic outgrowth (76% versus 93%; 0.05 > P > 0.01). Oviductal transport and uterine attachment was studied by introduction of blue-stained dextran microspheres into the oviduct. The microspheres appeared earlier in the uteri of DES-treated females than in the uteri of controls. Moreover, the spheres were not captured in the uterus in most DES-treated females, but they were trapped when a piece of Spongostan was placed in the uterine lumen. Scanning electron microscope studies showed differences between controls and DES-treated females in uterine mucosal lining of possible importance for embryo attachment to the surface epithelium. These studies showed that neonatal DES treatment caused changes in the adult reproductive tract as revealed by a detrimental effect upon embryo development, more rapid oviductal transport and loss of embryos from the uterus.

Effects of diethylstilbestrol (DES) medication during pregnancy: report from a symposium at the 10th international congress of ISPOG.

This article is a report based on presentations at the symposium Effects of Diethylstilbestrol (DES) Medication during Pregnancy at the conference Reproductive Life, 10th International Congress of The International Society of Psychosomatic Obstetrics and Gynaecology (ISPOG) in Stockholm, Sweden June 14-17, 1992. The objective of this symposium, chaired by Eylard van Hall and Ingar Palmlund, was to provide the basis for a discussion of how the risks of DES had been evaluated in different countries. In the general discussion following the presentations everybody present expressed concerns that DES might still be given to pregnant women in many parts of the world, called for measures to alert medical professionals world-wide to the hazards of the use of DES, and requested measures to effect an international ban of DES in medication for pregnant women.

Acute and permanent growth effects in the mouse uterus after neonatal treatment with estrogens.

Acute and late effects of neonatal estrogen treatment were studied in NMRI mice treated with diethylstilbestrol (DES) or estradiol-17 beta (E2) on days 1 to 5 after birth (estrogenized females). The uterine wet weight (UWW) response in 6-day-old females, after 5 daily treatments with DES, had a peak at a daily dose of 10(-2) micrograms DES and declined with higher doses. Females (26-day-old) treated with DES or E2 neonatally had a reduced UWW response to a challenge with DES; on a dose basis, DES was more effective neonatally than E2. A single injection with DES or E2 in the neonatal period stimulated mitotic activity in the uterine horn epithelium; the UWW response to a 24-h DES pulse increased from day 2 to 6 after birth, but the uterine epithelial mitotic rate response decreased. Epidermal growth factor (EGF) was a more potent stimulator of mitotic activity than DES or E2. DES inhibited mitotic activity in the uterine cervical epithelium; EGF protected from this DES effect. In adult estrogenized females, EGF-induced uterine stimulation of 3H-thymidine incorporation subsided more rapidly than in control females; uterine epithelium did not respond to EGF in vitro. Uterine stroma of adult estrogenized females is postulated to house a population of cells under nonovarian proliferation control while the uterine epithelium may be under influence of an ovary-dependent proliferation inhibiting factor that is gradually lost under culture conditions.

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.

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.

Effects of neonatal exposure to diethylstilbestrol on early mouse embryo development in vivo and in vitro.

Eight-week-old female mice of the NMRI strain that had been treated neonatally with diethylstilbestrol (DES, 5 micrograms/day for five days) or not (controls) were treated with gonadotropins to induce ovulation and then were artificially inseminated. Ova or young embryos were recovered from the oviducts on the morning after insemination and on Days 2, 3, and 4. In other experiments, ova were obtained from inseminated females on the morning after ovulation and cultured in vitro. In DES-treated females, a few zygotes developed to the 4-cell stage, but no more advanced stages were seen. Under in vitro conditions, zygotes from DES-treated females developed into blastocysts and to the implantation stage, but the incidence of these stages was lower than with zygotes from controls. Our results point to an abnormal oviductal function in DES-treated females that is not compatible with early embryo survival, even though an additional zygote factor contributing to degeneration of early cleavage stages cannot be excluded.

Estrogen effects on chromosome number and sister chromatid exchanges in uterine epithelial cells and kidney cells from neonatal mice.

Female mice of the NMRI strain were treated with a high dose (5 micrograms) or a low dose (10(-2) micrograms) of the synthetic estrogen diethylstilbestrol (DES) or estradiol-17 beta (E2) 48 and 24 hr, 24 hr, or 12 hr before death on day 3 after birth. Cultures of epithelium from the uterine cervix and uterine horns as well as of kidney cells were set up, and, after a culture period of 3 days, cells were studied for chromosome number and sister chromatid exchanges. Cervical cells from control females had a distinct peak of tetraploid cells, which was depressed after DES treatment but less so after E2 treatment. The tetraploid peak was lower in uterine horn epithelium and was less influenced by DES. No indications were obtained for an estrogen-induced aneuploid cell population in the uterine horn epithelium or cervical epithelium. The incidence of cells with a high number of sister chromatid exchanges (HFCECs) was only about one-fifth in uterine horn epithelium from control females compared with cervical epithelium from the same females. In the cervical epithelium, DES at both dose levels and E2 at the high dose induced an increased and similar incidence of HFCECs (from about 12.5% in controls to about 35%), which was not related to estrogen exposure time. In uterine horn epithelium, the incidence of HFCECs increased from 2.5% in controls to 8.5% after DES treatment. It is postulated that there is a more pronounced genomic plasticity in the cervical epithelium compared with the uterine horn epithelium. The estrogens had no effect on chromosome number or HFCECs in kidney cells.

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.

Pregnancy and delivery characteristics of women whose infants develop child cancer. A study based on registry information.

Data on all cancers among children born 1973-1984 were extracted from the Swedish Cancer Registry and linked to data in the Medical Birth Registry (1,268 cancer cases). For each case, two controls were selected, matched for maternal age, parity, county, and month of birth. A marginally increased risk of childhood malignancy was seen in the offspring of teenage mothers and at parity 1 and 4+. There was an excess among cases of the diagnosis physiological icterus. No statistically significant associations were found between childhood malignancy and infant sex, twinning, birthweight, non-chromosomal malformations, maternal smoking, or complications during pregnancy.

Steroid synthesis in ovarian homogenates from immature mice treated with diethylstilboestrol in neonatal life.

Female mice of the NMRI strain were treated with the synthetic oestrogen diethylstilboestrol (DES) for the first 5 days after birth. Pools of ovaries were removed from groups of 6-, 12-, 21-, 28- and 56-day-old females. An homogenate of an ovarian pool was incubated for 1 h in the presence of [3H]pregnenolone. Synthesized steroids were extracted and separated in a two-dimensional thin-layer chromatography system. Homogeneity of tentative steroids was verified with recrystallization to constant specific activity. Synthesis of [3H]progesterone and [3H]testosterone was demonstrated at 6 days, [3H]androstenedione at 12 days, [3H]17 alpha-hydroxyprogesterone at 21 days, and [3H]oestradiol-17 beta at 28 days. Up to 28 days (21 days for progesterone), the synthetic activity was lower in homogenates of DES-exposed ovaries than in control homogenates. After 28 days, values for recovered [3H]progesterone, [3H]androstenedione and [3H]oestradiol-17 beta were higher in DES homogenates than in control homogenates while the reverse was true for [3H]17 alpha-hydroxyprogesterone and [3H]testosterone. The results are compatible with an early and direct DES inhibitory effect on ovarian steroidogenesis and, later in immature life, a DES-induced disruption of the normal FSH-LH stimulation of ovarian development.

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.

Genotoxic effects of estrogens in epithelial cells from the neonatal mouse uterine cervix: modifications by metabolic modifiers.

Epithelium from the neonatal mouse uterine cervix and uppermost part of vagina was cultured in vitro. The culture medium was supplemented with 17 beta-estradiol (E2; 10(-6)-10(-5) M) or diethylstilbestrol (DES; 10(-8)-10(-5) M) alone or in combination with different metabolic modifiers (alpha-naphthoflavone, beta-naphthoflavone, phenobarbital, metyrapone, indomethacin) with postulated activating or inhibitory effects on DES metabolizing enzymes (cytochrome P-448- and P-450-dependent microsomal monooxidases, prostaglandin cyclooxygenase). E2 at 10(-5) M and DES at 10(-6) and 10(-5) M concentrations increased the incidence of cells with a high number of sister chromatid exchanges (high-frequency chromatid exchange cells, HFCEC). Indomethacin partially depressed DES-induced HFCEC, whereas the incidence was increased by alpha-naphthoflavone, which may be a result of stimulation of the fetal type of P-448-dependent enzyme activity or of DES increasing the metabolic activation of alpha-naphthoflavone. Phenobarbital and beta-naphthoflavone did not affect the incidence of DES-induced HFCEC. Metyrapone alone induced the highest incidence of HFCEC observed in this study, and this effect was inactivated by phenobarbital and/or DES. The mechanisms behind these results are discussed. This study shows that E2 and DES have a genotoxic effect (sister chromatid exchanges) in vitro in epithelial cells from the same target organ as in which epithelial aberrations occur after in vivo estrogen treatment in the neonatal period. The difference in incidence of tetraploid cells between stroma and epithelium is stressed (less than 5% vs. 16-47% depending on experimental group).