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.     

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.     

Uterine gland formation in mice is a continuous process, requiring the ovary after puberty, but not after parturition

Uterine gland formation occurs postnatally in an ovary- and steroid-independent manner in many species, including humans. Uterine glands secrete substances that are essential for embryo survival. Disruption of gland development during the postnatal period prevents gland formation, resulting in infertility. Interestingly, stabilization of beta-catenin (CTNNB1) in the uterine stroma causes a delay in gland formation rather than a complete absence of uterine glands. Thus, to determine if a critical postnatal window for gland development exists in mice, we tested the effects of extending the endocrine environment of pregnancy on uterine gland formation by treating neonatal mice with estradiol, progesterone, or oil for 5 days. One uterine horn was removed before puberty, and the other was collected at maturity. Some mice were also ovariectomized before puberty. The hormone-treated mice exhibited a delay in uterine gland formation. Hormone-treatment increased the abundance of uterine CTNNB1 and estrogen receptor alpha (ESR1) before puberty, indicating possible mechanisms for delayed gland formation. Despite having fewer glands, progesterone-treated mice were fertile, suggesting that a threshold number of glands is required for pregnancy. Mice that were ovariectomized before puberty did not undergo further uterine growth or gland development. Finally, to establish the role of the ovary in postpartum uterine gland regeneration, mice were either ovariectomized or given a sham surgery after parturition, and uteri were evaluated 1 wk later. We found that the ovary is not required for uterine growth or gland development following parturition. Thus, uterine gland development occurs continuously in mice and requires the ovary after puberty, but not after parturition.     

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.     

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 estradiol and progesterone on phosphatidylinositol metabolism in the uterine epithelium of the mouse

The effect of estradiol and progesterone on uterine phosphatidylinositol (PtdIns) metabolism was examined in whole uteri and separated uterine luminal epithelium of ovariectomized mice. Incorporation of [3H]myo-inositol in vitro, into inositol-containing phospholipids extracted from whole uteri, increased in mice injected with estradiol, with maximal incorporation at 9-12 h. The breakdown of PtdIns to inositol polyphosphates was also stimulated in whole uteri by estrogen, with an abrupt increase between 6 and 9 h. Comparable increases in both processes occurred in the uterine epithelium after estrogen stimulation and were inhibited by progesterone pretreatment which by itself had little or no effect. These results suggest that PtdIns metabolism is involved in the stimulation of uterine epithelial cell proliferation by estrogens, and its inhibition by progesterone.     

Specific IgA and IgG antibodies in the secretions of the female reproductive tract: effects of immunization and estradiol on expression of this response in vivo

Uterine and vaginal secretions collected from intact adult female rats were analyzed to determine whether immunization at sites distal to the reproductive tract had any effect on the presence of specific IgA and IgG antibodies in genital tract secretions. Peyer's patch and i.p. immunization and boost with sheep red blood cells (SRBC) stimulated the appearance of specific IgA antibodies in uterine and vaginal secretions of uterine-ligated animals. IgG antibodies were also induced in uterine but not in vaginal secretions. In contrast, subcutaneous immunization and boost elicited a weak IgA uterine and IgG vaginal response. To establish the role of estradiol in regulating the presence of specific antibodies in the female genital tract, ovariectomized rats received primary and/or secondary Peyer's patch immunizations with hormone treatment. Administration of estradiol daily for 3 days before sacrifice resulted in a significant accumulation of IgA and IgG antibodies to SRBC in uterine secretions. In the absence of estradiol, antibody content was negligible. Vaginal antibody levels were also clearly influenced by estradiol. In contrast to the uterus, however, specific IgA and IgG antibodies were present in the vaginal secretions of saline-injected immunized animals and were markedly inhibited in animals treated with estradiol. These results indicate that antibodies in genital tract secretions can be induced by immunization of the Peyer's patches and that their presence in uterine secretions is clearly dependent on estradiol. Further, they indicate that gut-derived specific antibodies enter the vagina in the absence of hormone stimulation and that estradiol exerts an inhibitory effect on their presence in vaginal secretions.     

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.     

Regulation of estrogen-dependent and estrogen-independent levels of progesterone receptor in hamster vagina and uterus

It is generally accepted that progesterone action is mediated in target cells through a specific, intracellular receptor protein. Since various progesterone target tissues respond differently to progesterone action, it may be postulated that such differences result from variations in: (1) the physicochemical properties; (2) the regulation, and/or (3) the intracellular response of the progesterone receptor (Rp) complex. A previous report demonstrated similar physicochemical properties of hamster vaginal and uterine Rp [1]. Our objective in this report was to analyze the regulation of estrogen-independent (ID-Rp) and estrogen-dependent (D-Rp) populations of receptor in different tissues of the lower reproductive tract of the golden hamster. In untreated ovariectomized animals, a basal level of (ID-Rp) was detected in endometrium, myometrium and vagina. Thus, each compartment contained a significant quantity of Rp which was maintained in the absence of continued estrogen support. Following 3 days of estradiol (E2) administration, the level of nuclear estrogen receptor increased and was related quantitatively to the amount of cytoplasmic Rp produced in these tissues. Maximal weight and D-Rp responses in endometrium, myometrium and vagina were obtained with 10-100 micrograms E2 per 100 g BW. The weight response of these tissues was due primarily to cellular proliferation in the endometrium; cellular hypertrophy in the myometrium; and cellular proliferation with concomitant nuclear pyknosis in the vagina. Although the morphological response of these tissues to estrogen action is quite different, the present study reveals no differences in the regulation of ID-Rp and D-Rp levels in these particular compartments. Furthermore, our results demonstrate a relationship between DNA content and ID-Rp and D-Rp levels in target tissues of the lower reproductive tract.     

Quantification of an estrogen-dependent cat uterine protein (CUPED) in uterine flushings of estrogen- and progesterone-treated ovariectomized cats by radioimmunoassay

We previously reported the purification of an estrogen-dependent cat uterine protein (CUPED) and the preparation of a specific anti-CUPED serum in rabbits. Here, we describe a specific radioimmunoassay for CUPED using the purified CUPED and anti-CUPED serum that was utilized to quantify CUPED in daily uterine flushings obtained from steroid-treated ovariectomized cats. The radioimmunoassay was sufficiently sensitive to measure 0.1-100 ng CUPED. CUPED levels were low in untreated ovariectomized cats, increased within one day after the onset of treatment with estradiol, and remained elevated as long as estradiol was unopposed by progesterone. The levels of CUPED decreased when progesterone was added to the treatment regimen either 7, 14, or 28 days after the initiation of estradiol treatment. The data indicate that the presence of CUPED in the uterine flushings is dependent on the presence of estradiol and the absence of progesterone, that CUPED appears in the uterine lumen within one day after the onset of treatment with estradiol, and that the levels of CUPED are sharply reduced within one day of administration of progesterone and become nondetectable after three days.     

Oestrogenic activity of cyproterone acetate in female mice

Effects of cyproterone acetate, a synthetic steroidal compound, on the reproductive organs of female mice have been investigated. This agent caused reduction of ovarian weights indicative of suppression of pituitary gonadotrophins. Oestrogenic nature of cyproterone acetate was investigated in intact and ovariectomized animals taking uterine weight, vaginal keratinization and other biochemical oestrogen sensitive parameters. Cyproterone acetate in ovariectomized animals induced vaginal keratinization increase in uterine weight and uterine protein, RNA, glycogen and sialic acid contents. These effects were parallel to the effects of oestradiol dipropionate in ovariectomized animals, thus indicating oestrogenic activity of the compound.     

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.     

Patterns of epithelial expression of Fos protein suggest important role in the transition from viable to cornified cell during keratinization

An antibody directed against the DNA-binding region of c-fos was used to localize the distribution of cells positive for Fos protein in epithelial tissues. The antibody consistently bound to the nuclei of epithelial cells in the late stages of differentiation, just prior to cornification. The epidermis, palate, buccal mucosa, gingiva, tongue, forestomach and vagina in estrus all produced this type of labelling, suggesting a burst of expression immediately before cell death and cornification. The differentiating cells of the hair follicle, including the hair and inner root sheath, were also labelled. Non-keratinized tissues including junctional epithelium, embryonic epidermis and diestrus vaginal epithelium showed little or no Fos labelling. With the onset of keratinization at 18 days gestation or with induction of estrus in ovariectomized mice with estradiol benzoate, the epidermis and vagina expressed Fos protein in the manner typical for keratinized tissues. The Er/Er mutant epidermis, a tissue that is blocked in its ability to keratinize, overexpresses Fos with Fos-positive cells appearing in virtually every cell layer. Gel shift analysis demonstrates the presence of a functional AP-1 complex in epidermal extracts that is recognized by our antibody. Our data suggest that the expression of Fos is intricately related to epithelial cell differentiation, specifically in relation to the process of cornification and cell death.     

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...     

Non-invasive repeated measurement of urinary progesterone, 17beta-estradiol, and testosterone in developing, cycling, pregnant, and postpartum female mice

Excretory samples from adult female mice were collected non-invasively during development, estrous cycling, pregnancy, and postpartum. In initial studies, urinary measures were statistically more dynamic over days than were fecal measures; thus subsequent studies focused on urine. Higher 17beta-estradiol levels were present in isolated females than in those exposed to males. In cycling females, urinary 17beta-estradiol was more variable than were measures of testosterone or progesterone, showing peaks with an approximate 5-day periodicity. When urinary estradiol and progesterone were monitored in conjunction with vaginal smear cell counts, patterns were idiosyncratic; most females showed distinct peaks in urinary steroids, not in clear synchrony with vaginal cell cornification. Levels of progesterone rose markedly during the first 10 days of pregnancy, then declined before birth. Estradiol showed a substantial peak on days 7-8 of gestation in all females measured. Urinary testosterone was not dynamic during pregnancy, but rose in immediate prenatal and postpartum measures. During post-weaning, pre-pubertal development, urinary levels of progesterone remained constant but levels of estradiol rose substantially over time.     

Sensitivity and specificity of bioassay of estrogenicity on mammary gland and uterus of female mice

Young intact (18 days of age) and adult ovariectomized (OV-X, ovariectomized between 21 to 24 days of age) C3H/Di mice were used to measure the estrogenicity on the basis of the growth response of mammary epithelial structures and weight of the uterus. The percentage area of the mammary fat pad occupied by mammary epithelial structures was progressively increased by 17beta estradiol from dose 0.001 microg.d(-1). The maximum effective dose of estradiol was 0.01 microg.d(-1) and the dose 10 microg.d(-1) of estradiol decreased mammary size to control levels (inverted-U-shaped dose-response curve). Progesterone alone progressively stimulated mammary growth in young intact females from dose 125 microg.d(-1), in adult OV-X animals from dose 1000 microg.d(-1). Both in young intact and adult OV-X animals, uterine weight progressively increased during estradiol treatment. Progesterone alone had no effect on uterine weight in young intact animals; in adult OV-X animals, uterine weight was increased starting from dose 250 microg.d(-1). Progesterone acted synergistically with estradiol to produce higher mammary growth than that in females treated with estradiol alone. The effects of a combination of estradiol plus progesterone in the mammary gland were mimicked by norethindrone acetate and inhibited by cortisol in both young intact and adult OV-X animals. Testosterone inhibited estradiol plus progesterone stimulated growth of mammary gland only in OV-X animals, but stimulated uterine weights in both young intact and adult OV-X animals. Spleen weight and size of mammary lymph nodes were not affected by estradiol, progesterone, norethindrone acetate or testosterone, but were decreased by cortisol. Cortisol also decreased the percent area of the mammary fat pad occupied by mammary epithelial structures, but had no effect on weight of the uterus. These results show that bioassay of estrogenicity in females is not specific. Mammary and uterine growth is stimulated not only by estrogens but also by progesterone and testosterone, respectively.     

Observations on the cervix uteri of the squirrel monkey

The squirrel monkey uterine cervix was studied macroscopically and microscopically in intact and ovariectomized monkeys. The effect in ovariectomized monkeys of estradiol dipropionate or progesterone of both given after estrogen priming was studied by PAS staining. The lower portion of the cervix was dilated to form a vestibule into which projected fibromuscular colliculi which arose from the isthmic end of the cervix. The stratified squamous epithelium of the vagina was continuous through the external os with a similar epithelium lining the vestibule and covering the external surfaces of the colliculi. The transitional zone between the stratified epithelium and columnar cells was variable. The colliculi were covered internally with mucosal folds of columnar epithelium continuous with those of the endocervical canal. Glycogen concentration in the smooth muscle did not fluctuate markedly, irrespective of the hormones used. Glycogen granules were more numerous in the stratified squamous epithelium. Malt-diastase-resistant material appeared to be more abundant in the columnar epithelium and glandular lumina when the monkeys received both hormones than when they received solely estrogen or progesterone.     

Effect of sex hormones on the content of cyclic nucleotides in the rat denervated uterus

Estradiol dipropionate increased the amount of cGMP and to less extent that of cAMP in the uterine tissues of ovariectomized rats. The combined effect of estradiol and progesterone promoted rise only in the cAMP level. Surgical and pharmacological denervation of both sympathetic and parasympathetic nerve influences in uterus suppressed the effect of the above hormones. Thus, neuromediator processes play an essential role in realization of cAMP- and cGMP-dependent effects in the general program of the sex hormone action in the above organ.     

The immunocytochemical localization of a cat uterine protein that is estrogen dependent (CUPED)

An estrogen-dependent polypeptide (CUPED), which was purified from uterine flushings of estrogen-treated cats, was localized in endometrial epithelial cells of cats using the peroxidase-antiperoxidase immunocytochemical staining procedure. Epithelial cells from animals treated with estradiol for 4, 7, or 14 days and estrogen-primed animals treated with progesterone for 2 days showed positive immunostaining. Staining was absent in untreated ovariectomized animals and in estrogen-primed animals treated with progesterone for 4 days. Specific cytoplasmic staining was confined to apical secretory granules in nonciliated cells of deep uterine glands. Staining was also commonly observed in the lumen of deep glands. Immunostaining was absent in the cells of the surface epithelium, stroma, and myometrium. In addition, other organs such as the oviduct, kidney, liver, pancreas, and lung showed no evidence of specific immunocytochemical staining. Therefore, the estrogen-dependent polypeptide obtained from uterine flushings of estrogen-treated ovariectomized cats is a uterine-specific secretory product that is packaged in apical cytoplasmic granules of uterine epithelial gland cells before being released into the uterine lumen.     

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.