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.     

Development of the vaginal epithelium showing estrogen-independent proliferation and cornification in neonatally androgenized mice.

Female mice of the C57 Black/Tw strain given 5 daily injections with 100 microng testosterone (T) or 5 alpha-dihydrotestosterone (DHT) from the day of birth showed estrogen-independent persistent proliferation and cornification of the vaginal epithelium in adulthood. The vaginal epithelium of the mice was essentially similar to that of the controls in histological structure during or shortly after neonatal injections of the androgens. In T- and DHT-mice aged over 20 days, however, a marked proliferation with or without superficial cornification took place in the epithelium lining the proximal and middle parts of the vagina (Müllerian vagina), while neither proliferation nor cornification occurred in the epithelium of the distal vagina (urogenital sinus vagina). On the second day of postnatal life in mice given a single injection with T on the day of birth, the mitotic activity in the epithelium of the middle vagina was heightened, but it dropped to the control level on the third day and remained low until 20 days. By contrast, the mitotic rates in the epithelium of the rest of the vagina in T-mice and of all parts of the vagina in DHT-mice were approximately the same as in the controls until 20 or 30 days. The mitotic rates in the epithelium of the Müllerian vagina were markedly elevated in T-mice at 20 days of age and DHT-mice at 30 days, and thereafter remained almost unchanged until 60 days of age. These results were different from the findings in mice given neonatal injections with the dose of estradiol-17 beta (E) capable of estrogen-independent vaginal cornification (Iguchi et al., 1976). The present finding seem to indicate that the mechanism involved in the induction of estrogen-independent vaginal changes by neonatal administration of androgen (T, DHT) is different from that following neonatal treatment with estrogen (E), although androgen and estrogen act directly on the vaginal epithelium of neonates.     

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.     

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.     

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.     

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.     

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.     

MITOTIC ACTIVITY OF VAGINAL EPITHELIAL CELLS FOLLOWING NEONATAL INJECTIONS OF DIFFERENT DOSES OF ESTROGEN IN MICE

In C57Black/Tw mice given injections of 1 μg estradiol-17β (E) for 5 days beginning on the day of birth, and killed a few days after the treatment, the vaginal epithelium showed estrogen-dependent proliferation and parakeratosis. In contrast, in the mice treated neonatally with 30 μg E for 5 days, the vaginal epithelium exhibited estrogen-independent proliferation and cornification or parakeratosis. Two peaks occurred in the mitotic rate in vaginal epithelial cells in the proximal and middle vaginae of the 1 μgE-treated mice, at 1 and 5 days of age, respectively, while the first peak was lacking in the distal vagina. The mitotic activity in 1 μgE-treated mice declined to the control level at 60 days. In the 30 μgE-treated animals also, 2 peaks were found in the mitotic rate at 1 and 7 days in both the proximal and middle vaginae. In contrast to the 1 μgE-treated mice, although the rate dropped once at 10 days, it increased again at 20 days and remained high thereafter. The second peak at 7 days of age coincided with the active proliferation of nodules appearing in the 30 μgE-treated mice. In the distal vagina, a peak occurred in the mitotic rate at 7 days without a preceding peak like that observed in the other parts of the vagina following the first injection of E on the day of birth.     

Morphological and biochemical alterations in reproductive tracts of neonatal female mice treated with the pesticide methoxychlor

Effects of estradiol-17 beta and the estrogenicity of different doses of the technical grade pesticide methoxychlor were compared in the vagina, uterus, and oviducts of neonatal mice. Beginning within 24 h of birth, neonates received 10 daily i.p. injections of sesame oil vehicle, 10.0 micrograms estradiol, or 0.05, 0.1, 0.5, or 1.0 mg methoxychlor. Estradiol injections induced precocious vaginal opening, complete vaginal cornification, and increased total reproductive tract weight and its DNA content. In comparison to the controls, the three highest methoxychlor doses also significantly increased the weights of the reproductive tracts and stimulated their development. The two highest doses (0.5 and 1.0 mg) also induced precocious vaginal opening and complete vaginal cornification. In addition, the same two doses produced atypical cells in the uterus and oviducts that may be indicative of early dysplasia; similar atypia were not recorded following estradiol treatments. Total DNA content in various reproductive organs increased with increased methoxychlor dosages. Dose-response changes were observed in the oviduct and uterus but not vagina. In summary, methoxychlor stimulated the development of neonatal female reproductive tracts, even at concentrations not previously reported to be biologically active. Furthermore, the higher doses induced abnormalities that were not seen following estradiol treatment; these abnormalities may represent precursors of pathological changes.     

TRANSPLACENTAL EFFECT OF ETHINYL ESTRADIOL ON MOUSE VAGINAL EPITHELIUM *

The effect of prenatal administration of ethinyl estradiol (EE) on the vaginal epithelium of adult mice was examined histologically. The mice were the offspring of JCL/ICR strain mice given orally 0.02 mg/kg body weight/day or 0.01 mg/kg/day of EE dissolved in olive oil from day 11 to day 17 of gestation at a stage when the urogenital sinus has just appeared in the embryos. The control mice were offspring of those fed with the vehicle alone. Autopsies were performed at 10 to 14 weeks of age. Another group of mice exposed to 0.02 mg/kg/day of EE or vehicle alone in utero, were spayed at 16 weeks of age and killed at 32 weeks of age. In the experimental nonspayed mice, hyperplasia of the vaginal epithelium with intense cornification was seen. The epithelium was significantly thicker than in the controls and showed an EE dose-response relation. One of the 16 mice exposed to 0.01 mg/kg/day of EE in utero had cystic or gland-like structures in the stroma and mucus-secreting cells in the surface epithelium consisting of columnar cells. In some experimental spayed mice, vaginal hyperplasia with cornified epithelium and hypertrophy of the ovarian interstitial tissue without corpus luteum were seen. These results indicate that EE can cross fetal membranes and affect undifferentiated cells in the urogenital sinus and/or Müllerian epithelium.     

Sensitivity of the vagina and uterus of mice neonatally exposed to estrogen or androgen to postnatal treatment with estrogen or androgen.

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

Further studies on the differentiation of the epithelium in the mouse vaginal anlage

Summary The authors have studied the occurrence of PAS positive substances during the differentiation of the vaginal epithelium in fetuses and neonatal mice. The material consists of normal mice, mice that have received estradiol injections for the first five days after birth, and mice that have received both estradiol and colchicine injections. The cranial 3/5 of the mouse vaginal epithelium is formed from the pseudostratified columnar müllerian epithelium. This undergoes a differentiation and divides into two zones: a superficial zone and a basal zone. The latter arises from cells migrating basally from the superficial zone. Later the two zones merge and the typical prepuberal vaginal epithelium arises. The results of this investigation point to the cell divisions in the superficial zone being of particular importance for the cell differentiation, even though other possibilities cannot be excluded. The effect of estradiol administration on the epithelium in the vaginal anlage is discussed. The circumstance that estradiol may change the determination of the cells is pointed out.This investigation has been supported by a grant from Maggie Stephens' Stiftelse.     

Age-dependent sensitivity of basal cells in mouse vaginal epithelium towards the diferentiating action of estrogen.

Following ovariectomy, the vaginal epithelium of the mouse is reduced to two layers of cells, the basal layer that constitutes the germinative compartment and surface layer. Under estrogenic influence, this tissue undergoes keratinization at the expense of asal cell only. The initially superficial cells are progressively sloughed, without taking part in the cornification process. We have shown previously that within the first 12 hours after estrogen administration (that is, before any change in mitotic activity is detected) an intermediate layer of cells is formed. Thurs, a migratory process is induced, which leads to a very rapid redistribution to basal cells into two layers...     

Prevention by Vitamin A of the occurrence of permanent vaginal changes in neonatally estrogen-treated mice

Cell and Tissue Research - Ovary-independent (estrogen-independent) irreversible proliferation and cornification of the vaginal epithelium in ovariectomized mice caused by neonatal injections of 20...     

Paracrine regulation of epithelial progesterone receptor by estradiol in the mouse female reproductive tract

Regulation of progesterone receptor (PR) by estradiol-17beta (E(2)) in mouse uterine and vaginal epithelia was studied. In ovariectomized mice, PR expression was low in both vaginal stroma and epithelium, but high in uterine epithelium. E(2) induced PR in vaginal epithelium and stroma, but down-regulated PR in uterine epithelium. Analysis of estrogen receptor alpha (ERalpha) knockout (ERKO) mice showed that ERalpha is essential for E(2)-induced PR expression in both vaginal epithelium and stroma, and for E(2)-induced down-regulation, but not constitutive expression of PR in uterine epithelium. Regulation of PR by E(2) was studied in vaginal and uterine tissue recombinants made with epithelium and stroma from wild-type and ERKO mice. In the vaginal tissue recombinants, PR was induced by E(2) only in wild-type epithelium and/or stroma. Hence, in vagina, E(2) induces PR directly via ERalpha within the tissue. Conversely, E(2) down-regulated epithelial PR only in uterine tissue recombinants constructed with wild-type stroma. Therefore, down-regulation of uterine epithelial PR by E(2) requires stromal, but not epithelial, ERalpha. In vitro, isolated uterine epithelial cells retained a high PR level with or without E(2), which is consistent with an indirect regulation of uterine epithelial PR in vivo. Thus, E(2) down-regulates PR in uterine epithelium through paracrine mechanisms mediated by stromal ERalpha.     

Estrogen-independent growth of mouse vaginal epithelium in organ culture.

A serum-free vaginal explant culture system was established to investigate the in vitro effect of estrogen on the growth of mouse vaginal epithelium. Vaginal explants were isolated from 40-day-old, ovariectomized BALB/cCrgl mice and cultured in a basal unsupplemented medium or in basal medium plus various doses of 17 beta-estradiol. Explants were processed for histology at the end of culture periods or were given 4-hour pulses of tritiated thymidine at various times and processed for autoradiography. Vaginal epithelium increased 3- to 5-fold in thickness and 2-fold in the number of epithelial cell layers during 72 hours of culture without estrogen; addition of estrogen did not significantly influence epithelial growth. Keratinization of vaginal epithelium occurred within 48 hours of culture in the absence of estrogen, and again addition of estrogen did not accelerate its appearance. Covering the explants with collagen decreased the estrogen-independent growth of vaginal epithelium. Autoradiography showed that ca. 70-90% of basal epithelial cells entered S phase during the initial 4 hours of culture and that this number declined rapidly after 48 hours to ca. 20%. Addition of 1.8 nM 17 beta-estradiol significantly decreased the labelling index of basal cells at 48 hours, but did not affect the labelling index at 24 and 72 hours. Stromal cells were not labelled at any time. Thus, DNA synthesis, cellular proliferation, and differentiation (keratinization) of vaginal epithelium in organ culture occurred without estrogen and were not stimulated by the addition of estrogen.     

Involvement of keratinocyte growth factor (KGF)-KGF receptor signaling in developmental estrogenization syndrome of mouse vagina

Exposure of mice to estrogen or keratinocyte growth factor (KGF) in vivo during the neonatal period results in estrogen-independent persistent proliferation and cornification of the vaginal epithelium when the animals become adults. Here, whether and how KGF-signaling is involved in the effects of estrogen on the neonatal mouse vagina were studied with an in vitro method. Newborn mouse vaginae were cultured for 3 days in serum-free medium containing various combinations of estradiol-17 (E₂), KGF, anti-KGF antibody, KGFR inhibitory peptide and heparin, and then transplanted into ovariectomized host mice for 35 days. The vaginae cultured with 5 g/ml E₂ or 5 g/ml KGF had a cornified thick epithelium, while the epithelium of the vehicle-treated controls stayed thin. The E₂ effect was blocked by concurrent treatment with anti-KGF antibody or KGFR inhibitory peptide. KGF treatment alone at doses less than 500 ng/ml did not induce permanent vaginal changes but such changes did occur in vaginae treated with heparin plus as little as 10 ng/ml KGF. On the other hand, heparin inhibited the permanent vaginal changes induced by estrogen. These results suggest that irreversible vaginal changes are induced by the direct action of KGF on the developing vagina and that the developmental estrogenization syndrome of mouse vagina is caused by intensification of endogenous KGF/KGFR signaling by exogenous estrogen.This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (A) and for Encouragement of Young Scientists from the Ministry of Education Science, Sports and Culture, Japan to M.M.     

Effect of epidermal chalone on vaginal epithelial cell proliferation stimulated by estradiol

Non-inbred and hybrid mice, line C57Bl and CBA in diestrus stage were subjected to ovariectomy and in 4-6 weeks they were given subcutaneously 17-beta-oestradiol (1 or 0.1 mkg per one animal). One hour before the animals were sacrificed, they were given 3H-thymidine intraperitoneally. It has been stated that 15-20 h after the estrogen administration the amount of DNA-synthesizing cells in the vaginal epithelium of these animals is 25 times as great as that in the control animals--castrated mice. When the epidermal chalone is administered locally or intraperitoneally (5 mg per one mouse), 10 min after the hormone injection, there is no inhibiting effect of the chalone on the proliferative processes in the vaginal epithelium stimulated with estrogen. When a single intraperitoneal injection of the epidermal chalone is given (5 mg per one mouse) 1 h before, or when it is given three times (2 mg per one mouse) 8, 4 or 1 h before the hormone is injected, there is a definite inhibiting effect in the proliferative processes. Their degree depends on how long the chalone was in contact with the cells.     

The highly modified membrane of cornified cells in stratified squamous epithelia: A comparison of heterogenous deposits in keratinized and nonkeratinized epithelia

Summary The chemical nature of the thickened plasma membrane of cornified cells in stratified squamous epithelium was investigated in comparison with that in noncornified epithelium. Localizations of transglutaminase, molecular weight 92000 daltons, and detection of epidermal cysteine proteinase inhibitor were effected with a monoclonal antibody and a monospecific rabbit anti-inhibitor immunoglobulin, respectively, directed to the antigens. N-(7-dimethylamino-4-methylcoumarinyl) maleimide was used to demonstrate S-S cross-linking. In all keratinizing epithelia, the enzyme and inhibitor were deposited on membranes of granular cells. S-S bonds were formed in cornification with the appearance of electron-dense material by the inner leaflet. Both enzyme and inhibitors occurred on the corneal epithelium, but S-S linkage and the thickened plasma membrane did not form even at the last stage of maturation. On the other hand, the internal vaginal epithelium in the proestrous stage without keratinization contained the enzyme, but neither inhibitor nor S-S linkage. Both antigens and S-S bonds were detected when keratinization proceeded during estrus. The staining patterns in the epithelium near the vaginal introitus were identical to those in the skin. Cuboidal and simple epithelia exhibited none of those constituents. The findings indicated that heterogenous components contribute to modification of the plasma membrane of cornified cells, but S-S cross-linkages are associated exclusively with formation of the ultrastructurally unique membrane structure. In addition, findings suggested hormonal regulation in the chemical modification of the membrane in estrogen-sensitive internal vaginal epithelium.