Occurrence of permanent changes in vaginal and uterine epithelia in mice treated neonatally with progestin, estrogen and aromatizable or non-aromatizable androgens.

Female mice of the C57 Black/Tw strain were injected daily with 100 microng testosterone, 50 microng testosterone propionate (TP), 100 microng 5 alpha-dihydrotestosterone (DHT) or 50 microng 5 alpha-dihydrotestosterone propionate (DHTP), for 10 days from the day of birth. Two other groups of female mice were given neonatal injections with 20 microng estradiol-17 beta and 100 microng progesterone for 10 days, respectively. All mice were ovariectomized at 60 days of age and killed at 90 days. In 100% of neonatally estrogenized or androgenized, ovariectomized mice, the cranial part of the vagina was lined with stratified epithelium with either cornification or parakeratosis or mucification. Stratification only or stratification with superficial squamous metaplasia or cornification took place in the uterine epithelia of 18% of the TP-treated, 75% of the DHT-treated and 50% of the DHTP-treated, ovariectomized mice. In contrast, neonatally estrogenized, ovariectomized mice did not show the estrogen-independent, persistent uterine changes. Neonatal progesterone treatment failed to induce the permanent changes in the vaginal and uterine epithelia.     

Additional effects of postpuberal estrogen injections on the vaginal epithelium in neonatally estrogenized mice

In the ovariectomized mice given 10 injections of 100 micrograms 17 beta-estradiol at intervals of 2 weeks from 60 days of age, the vaginal epithelium was atrophic when killed more than 2 months after the last injection. If mice given 3 daily injections of 20 micrograms 17 beta-estradiol from the day of birth were similarly treated with estradiol after postpuberal ovariectomy, the vaginal epithelium was stratified and hyperplastic at autopsy performed more than 2 months later. These changes in the epithelium persisted for at least 30 days after transplantation of the vaginae to normal ovariectomized hosts. Neonatal treatments only did not produce such persistent vaginal changes. In view of these results, additional effects of neonatal and postpuberal injections of estrogen on the vaginal epithelium are evident. However, effects of such neonatal and postpuberal injections of estrogen might be transient on the uterine epithelium, since abnormal proliferation was not observed in it.     

Effect of neonatal androgenization on positive feedback in female mice

Exposure of female mice to androgens within 5 days of birth impairs fertility. Such treatment in rats results in a post-pubertal acyclic state of persistent vaginal cornification and in an inability, when ovariectomized, to show normal positive feedback on luteinizing hormone (LH) release in response to steroid challenge. In the present study, we explored whether neonatally androgenized mice demonstrate positive feedback. Female mice were administered 100 micrograms of testosterone propionate (TP) on either Day 1 (TP1) or Day 5 (TP5) after birth, or vehicle on Day 1 (SO1). Androgen-treated mice had a statistically significant advance in onset of vaginal opening as compared with vehicle-treated mice. All mice that received TP entered constant vaginal estrus, whereas those given vehicle showed variable cytology. All mice were ovariectomized at 7 wk of age and received Silastic capsules containing a priming dose of 17 beta-estradiol. When all mice were challenged 1 wk later with sequential administration of estradiol benzoate and progesterone, a significant increase in plasma LH level was present only in the vehicle-treated mice. We conclude that neonatal androgenization defeminizes the neuroendocrine mechanisms controlling gonadotropin release.     

Persistent vaginal cornification in mice treated with estrogen prenatally.

Twelve of 14 female mice of the ICR strain which had received a single injection of 50 mug estradiol-17beta on day 17 of fetal life exhibited irreversible cornification or stratification of the vaginal epithelium which persisted after ovariectomy until sacrifice performed 42-48 days later. Eight of the 12 mice had corpora lutea in their ovaries removed at 3-5 months of age. A similar injection of estradiol on day 15 of fetal life induced irreversible cornification or stratification of the vaginal epithelium in 6 of 12 females and only one of the 6 had corpora lutea in its ovaries when removed at 3-5 months. Mice given the same dose of estradiol on the day of birth or at 3 days of postnatal age invariably had ovaries bearing follicles of varying sizes and hypertrophied interstitial tissue but no corpora lutea. Changes in the vaginal epithelium in these animals were less remarkable as compared to that in prenatally treated mice.     

Comparative study of blocking effects of various retinoids on the occurrence of permanent proliferation of vaginal epithelium in mice treated neonatally with estrogen

Neonatal injections of 20 micrograms 17 beta-estradiol (E2) induced persistent proliferation and cornification of the vaginal epithelium in adult ovariectomized C57 Black/Tw mice. However, permanent vaginal changes were prevented by various retinoids when, simultaneously with E2 treatment, the animals were given injections of 100 micrograms daily dose of retinol, retinol acetate, retinal or of 200 micrograms daily dose of retinol palmitate (RoP). Neonatal injections of a 100 micrograms daily dose of RoP had no preventive effect on the occurrence of E2-induced permanent vaginal changes. This finding suggests that the preventive effect of RoP is weaker than that of other retinoids showing approximately the same degree of prevention. Combined treatment with E2 plus retinoic acid (even a small dose of 20 micrograms) had such a toxic effect on newborn mice that they died within 7 days after birth, while the animals given neonatal injections of 20 micrograms retinoic acid alone survived until the termination of the experiment.     

Ultrastructural characteristics of the vaginal epithelium of neonatally estrogenized mice in response to subsequent estrogen treatment.

Adult mice which had received 10 daily injections of 20 microng estradiol beginning with the day of birth were in a "persistent-estrous" state, showing ovary-independent proliferation and cornification of the vaginal epithelium. Ultrastructural changes of the vaginal epithelium in neonatally estrogenized mice was examined after a single postpuberal injection of 10 microng estradiol and compared with those seen in normal mice to estrogen. In ovariectomized normal mice, the basal cells were round. The nucleus was polygonal and contained peripheral condensed chromatin. After estradiol treatment, the basal cells became columnar. The nucleus was round to oval, containing dispersed chromatin. In neonatally estrogenized ovariectomized mice, the basal layer of vaginal epithelium consisted of round cells with polygonal nuclei, much as in normal ovariectomized mice. The nucleus occupied a large area of the cytoplasm and contained prominent nucleoli. Intercellular spaces were moderately distended. Late estradiol treatment resulted in distended intercellular spaces and in the appearance of the other cell type along with round cells in the basal layers: the columnar cells containing an oval nucleus with dispersed chromatin, resembled the basal cells in normal ovariectomized mice receiving postpuberal estrogen injection. The intercellular spaces between the columnar cells were narrow compared with those between round cells. However, the nuclei of round cells still had prominent nucleoli and peripheral condensed chromatin regardless of subsequent estrogen treatment. This fact suggests that these nuclei do not respond to estrogen. These results clearly show that the vaginal epithelium of neonatally estrogenized mice with ovary-independent persistent cornification consists of a mixed population of cells.     

Proliferative response of seminal vesicle cells to androgen in mice castrated neonatally and pretreated with estrogen or androgen at adulthood

Seminal vesicle cells of neonatally castrated adult mice show poor response to androgen, compared to those of mice castrated at adulthood; effects of pretreatment with androgen or estrogen at adulthood on androgen-induced proliferation of the seminal vesicle cells were examined in neonatally castrated mice. Male mice castrated at day 0 after birth were pretreated with daily injections of testosterone propionate (TP, 100 micrograms/mouse), 17 beta-estradiol (E2, 5 micrograms/mouse) or vehicle for 20 days starting from day 60; daily TP injections (100 micrograms/mouse) for 30 days were started again from day 110 in all the pretreated mice to examine androgen-induced proliferation by incorporation of 5-[125I]iodo-2'-deoxyuridine into the whole seminal vesicles. Both TP and E2 pretreatments significantly increased the seminal vesicle weight found before TP treatment. However, androgen-induced proliferation of the seminal vesicle found in neonatally castrated mice (poor response; long duration with a low peak on day 3) was changed at least in part to that found in mice castrated at adulthood (good response; short duration with a high peak on day 3) only following the TP pretreatment but not at all following the E2 pretreatment. The E2 pretreatment induced poor androgen-induced proliferation with a low peak on day 7.     

Vitamin A prevents the irreversible proliferation of vaginal epithelium induced by neonatal injection of keratinocyte growth factor in mice

Exposure of female mice to estrogen during the perinatal period results in estrogen-independent persistent proliferation and cornification of the vaginal epithelium when the animals become adults. However, the occurrence of such irreversible vaginal changes is blocked by concurrent vitamin A treatment. Neonatal exposure to keratinocyte growth factor (KGF), which is a paracrine mediator of epithelial-mesenchymal interactions, also induces the persistent proliferation and cornification of the vaginal epithelium in adult mice. This study was designed to examine whether concurrent administration of vitamin A inhibits the development of the irreversible vaginal changes in mice exposed neonatally to KGF. The vaginal epithelium in ovariectomized 35-day-old mice given 5 microg of KGF for 3 days after birth possessed a significantly larger number of layers and increased thickness as compared to that in control mice. Concurrent injections of 100 IU of vitamin A acetate inhibited the occurrence of the irreversible proliferation of the vaginal epithelium. These changes were equal to the results observed when 20 micro g of estrogen with or without vitamin A acetate was administered for 5 days after birth. Unlike the case of estrogen treatment, the effect of neonatal treatment with KGF seemed to appear after a latent period, since the vaginal epithelium did not show proliferation soon after the treatment. We discuss the inhibitory effect of VA on the irreversible vaginal changes induced by neonatal KGF treatment with reference to endocrine disruption by neonatal estrogen exposure.     

Prepubertal treatment with estrogen or testosterone precipitates the loss of regular estrous cyclicity and normal gonadotropin secretion in adult female rats

To examine the effects of prepubertal steroid environment on subsequent estrous cyclicity and gonadotropin secretion, Silastic implants containing 25, 50 or 100% 17 beta-estradiol (E2;n=34), 50% diethylstilbestrol (DES; n=16) or 50% testosterone (T; n=17) were placed into female rats at 12 days of age and removed on the day of vaginal opening (18-24 days of age). At 80 days of age, the percentages of regularly cycling females in the E2-(three groups combined), DES- and T-implanted groups were 59%, 0% and 59%, respectively. By 110 days of age, the percentages were reduced to 24%, 0% and 0%, and at 140 days of age 6%, 0% and 0%, respectively. Many of these females displayed irregular estrous cycles followed by a persistent estrous (PE) state. By contrast, 89% of the control females (blank implants or no implant) maintained regular cycles up to 140 days of age. At 150 days of age, an i.p. injection of gonadotropin-releasing hormone (GnRH; 100 ng/100 g BW) markedly increased serum luteinizing hormone (LH), but not follicle-stimulating hormone (FSH), in intact PE females treated prepubertally with E2 implants. After the test with GnRH, PE rats were ovariectomized (OVX). Thirty days after OVX, similar GnRH administration significantly increased serum levels of both LH and FSH, but these responses were significantly (P less than 0.01) reduced when compared with those in OVX controls. Progesterone administration to estradiol benzoate-primed, acutely (3 days) OVX, or long-term (43 days) OVX-PE females did not increase LH or FSH release. These results indicate that exposure to exogenous estrogen or T prior to puberty precipitates the decline in estrous cyclicity associated with the loss of gonadotropin surge response, presumably due to an alteration in hypothalamic GnRH release.     

Roles of prepubertal androgen, estrogen or androgen plus prolactin on androgen-induced proliferative response of seminal vesicles in adult mice

Male mice castrated on day 0 after birth were pretreated daily with testosterone propionate (TP, 4 micrograms/g body weight), 17 beta-estradiol (E2, 0.2 micrograms/g body weight) or vehicle for 21 days starting from day 20. In another experiment, male mice were castrated on day 25; two pituitaries from 60-day-old females were immediately grafted under the capsule of the left kidney in one group. The castrated mice with or without grafts were pretreated daily with TP (4 or 20 micrograms/g body weight) for 36 days starting from day 25, and the left kidney was removed on day 60. Daily TP injections (4 micrograms/g body weight) were started again at 30 days after the end of pretreatments to examine androgen-induced proliferation, and incorporation of 5-[125I]iodo-2'-deoxyuridine into the whole seminal vesicles was used as an index of proliferation. In the neonatally castrated mice, both TP and E2 pretreatments given during the prepubertal period significantly increased seminal vesicle weight even long after the end of the pretreatments. However, androgen-induced proliferative response found in the neonatally castrated adult mice (poor response; long duration with a low peak) was changed to that found in mice castrated at adulthood (good response; short duration with a high peak) by the TP pretreatment only but not at all by the E2 pretreatment. In the mice castrated on day 25, a pharmacological dose of TP or TP plus hyperprolactin could not enhance or change the adult castration type of androgen-induced proliferation induced by physiological prepubertal androgens, although both treatments significantly enhanced the prepubertal growth of the seminal vesicles.     

Effects of perinatal exposure to a synthetic estrogen and progestin on mammary tumorigenesis in mice

The effects of perinatal exposure to synthetic estrogens and progestins on mammary tumorigenesis were studied in female C3H/HeN/MTV + mice. Mice were treated neonatally with 0.001 microgram/day diethylstilbestrol (DES), with 15 micrograms/day 17 alpha-hydroxyprogesterone caproate (HPC), or with oil on days 1-5 of life (birth = day 1). As adults, neonatally hormone-treated mice received long-term treatment with a synthetic estrogen and progestin combination or vehicle. Animals were palpated weekly for mammary gland tumors. The effect of treatment on the probability of tumor development was examined. Neonatal treatment with a low dose of DES increased the probability of mammary-gland tumor formation, whereas neonatal treatment with HPC had a slightly protective effect on tumorigenesis. Subsequent treatment of adult mice with synthetic steroids did not affect mammary gland tumorigenesis in neonatally DES-treated or oil-treated animals. There was a significant interaction between the effect of neonatal HPC treatment and subsequent steroid treatment on mammary tumorigenesis but examination of the data indicated that this interaction was due to the protective effect of HPC in the absence of subsequent exposure to synthetic steroids and the probability of tumor appearance in mice treated with both HPC and synthetic steroids as adults did not differ from that of neonatally oil-treated controls.     

Age-related changes in ovarian responsiveness to gonadotropins in normal and neonatally estrogenized mice

Young ovariectomized mice were transplanted with ovaries obtained from either neonatally estrogenized or normal mice at different ages. Cyclic estrus ensued in 71% of the mice receiving ovarian grafts from 3-month-old normal donors. If donors were 12, 15 and 20 months old, cyclic estrus took place in 15, 10 and 0% of the recipients, respectively. By contrast, after transplantation of ovaries from neonatally estrogenized mice, and 3, 12 or 15 months, cyclic estrus occurred in about 42-48% of the recipients regardless of the age of donors. Three of 17 recipients receiving ovarian grafts from 20-month-old neonatally estrogenized donors still showed cyclic estrus. Therefore, in neonatally estrogenized mice, decline of ovarian responsiveness to circulating gonadotropins appears to be inhibited or delayed until at least 15 months of age.     

Diethylstilbestrol-induced cervical and vaginal adenosis using the neonatal mouse model

The relevance of diethylstilbestrol (DES) administration to neonatal mice as a model for human pathology attributed to the use of DES in high-risk pregnancies has been investigated, particularly with respect to cervical and vaginal changes in female offspring. Neonatal DES treatment of mice results in tonic pituitary gonadotropin release and continuous estrogen secretion by the ovary. Studies were designed to determine the effect of this altered ovarian endocrine activity on cervical and vaginal histopathology. Ovariectomy of DES-treated mice, with or without estradiol replacement, did not eliminate the lesions, nor did estrogen and progesterone administered in a regimen intended to mimic estrous cycle changes. Induction of the constant estrus state by neonatal estradiol benzoate or testosterone propionate administration or by exposure to constant light did not produce the type of vaginal or cervical changes seen in DES mice. Thus, altered ovarian function is apparently not required for the vaginal and cervical changes appearing in later life. A role for endogenous (or exogenous) ovarian hormones in the developmental progression toward normality is suggested.     

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.     

Proliferative response of seminal vesicle cells to androgen and estrogen in neonatally castrated mice

Proliferation and death of androgen- and estrogen-responsive cells in seminal vesicles were compared between neonatally and adult (on Day 60 after birth) castrated mice. Daily injections of either testosterone propionate (TP) or estradiol-17 beta (E2) were started on Day 90 after birth; the incorporation of 5-[125I]iodo-2'-deoxyuridine ([125I]IdUrd) into the whole seminal vesicles was used as an index for proliferation. Although the peak of [125I]IdUrd uptake was observed 3 days after starting TP injections in both neonatally and adult castrated mice, the peak was lower and the period of proliferation was much longer in the former than in the latter. When TP injections were stopped, the fraction of surviving cells that synthesized DNA on Day 3 of TP injections was much larger in neonatally than adult castrated mice. The difference was attributed to the presence of TP-induced proliferation of fibromuscular cells in the neonatally castrated mice but not in the adult castrated mice; only the fibromuscular cells but not epithelial cells survived after stopping TP injections. Although injections of E2 increased the proliferation of epithelial cells but did not the weight of seminal vesicles in adult castrated mice, the same procedure increased the proliferation of both epithelial and fibromuscular cells and the weight in neonatally castrated mice. The E2-induced fibromuscular cells seemed to survive in the presence or absence of E2. The present results seem to indicate that androgen- and estrogen-induced proliferation of fibromuscular cells is irreversible in seminal vesicles of neonatally castrated mice and that the depletion of androgen in the seminal vesicle during neonatal and prepubertal periods is at least in part compensated by the administration of androgen, even after 90 days of age.     

Influence of neonatal diethylstilbestrol treatment on prolactin receptor levels in the mouse male reproductive system

Neonatal exposure to the synthetic estrogen, diethylstilbestrol, is known to affect the structure of the male reproductive system; thus, changes may also occur in the levels of hormone receptors. Prolactin receptor levels from the reproductive systems of male BALB/c mice exposed neonatally to diethylstilbestrol were analyzed. Neonatal exposure to diethylstilbestrol caused significant decreases (i) in prolactin receptor levels in the seminal vesicle, ductus deferens, and anterior and ventral prostates and (ii) in tissue weight and protein content in reproductive organs other than the ventral prostate.     

Effects of testosterone and estrogen on hepatic levels of cytochromes P450 2C7 and P450 2C11 in the rat.

The effects of exogenous hormone treatment on the expression of cytochromes P450 2C7 and P450 2C11 were studied in neonatally gonadectomized and sham-operated male and female rats. Hepatic levels of cytochrome P450 2C7 were found to be two- to threefold higher in intact adult female versus male rats. Neonatal gonadectomy resulted in a reversal of the relative cytochrome P450 2C7 levels in male and female animals at maturity. Expression of this isozyme was restored in ovariectomized females by estradiol treatment. Furthermore, neonatal and/or pubertal administration of estradiol to intact male rats induced cytochrome P450 2C7 to adult female levels. On the other hand, administration of testosterone at all times examined had no effect in intact female rats, but decreased cytochrome P450 2C7 to normal levels in neonatally castrated males treated during adulthood. Neonatal testosterone treatment also increased hepatic cytochrome P450 2C7 content in both ovariectomized females and intact males. These results indicate that estrogen is required for full expression of cytochrome P450 2C7 while the effect of testosterone is ambiguous. In comparison, neonatal gonadectomy of male rats abolished the adult expression of cytochrome P450 2C11. Normal levels were restored only by treatment with testosterone during adulthood. Neonatal testosterone treatment did not induce cytochrome P450 2C11 levels in gonadectomized rats of either sex. In contrast, neonatal estrogen treatment suppressed cytochrome P450 2C11 expression in intact adult male rats to the same extent as neonatal castration. These results indicate that androgen exposure during the adult, and not the neonatal, phase is essential for full expression of cytochrome P450 2C11.     

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.