Vaginal Keratinization Research Articles

Early Vaginal Opening in Juvenile Female Rats Given BRAF-Inhibitor Dabrafenib Is Not Associated with Early Physiologic Sexual Maturation.

Dabrafenib (DAB), an inhibitor of BRAF kinase activity, is approved for metastatic melanoma with a BRAF V600E mutation. In support of pediatric cancer development, a nonclinical juvenile rat toxicity study was conducted in which females had early vaginal opening (VO). It was hypothesized that the early VO was not indicative of sexual maturation, but a result of a local effect on the vagina. An investigative study was conducted that mimicked the definitive study design, with rats given DAB or vehicle orally from Postnatal Day (PND) 7 to 35 and with necropsy subsets just before VO, at the first and second estrus, along with age-matched controls. Histopathology was performed on reproductive tissues, including immunohistochemistry for BRAF expression. VO occurred earlier in DAB females than in controls (PND 27.2 vs. 31.5); however, the timing of the first estrus was unaffected (PND 34.0 vs. 33.0). DAB-treated females evaluated just before VO (PND 22.0) had mostly immature reproductive tracts with no evidence of ovulation, similar to age-matched controls; however, DAB-treated females had keratinized and histologically open vaginas. Also, there was raised skin around the urogenital area, which correlated with hyperplasia/keratosis of the vulvar skin and keratinization of the distal vagina. BRAF expression (evaluated in controls) was localized to these tissues. Thus, early VO in rats given DAB likely represents a local effect accelerating vaginal keratinization to become open and not accelerated sexual maturation.

Neonatal injections of methoxychlor decrease adult rat female reproductive behavior

Methoxychlor (MXC), a commonly used pesticide, has been labeled as an endocrine disruptor. To evaluate the impact of neonatal exposure to MXC on female reproduction, female Sprague-Dawley rats were given subcutaneous injections on postnatal days 1, 3, and 5. The injections contained 1.0mg MXC, 2.0mg MXC, 10μg 17β-estradiol benzoate (positive control), or sesame oil (vehicle). The injections of MXC had no effect on anogenital distance or day of vaginal opening. Treatment with either 2.0mg MXC or estradiol significantly increased the total number of days with vaginal keratinization. Treatment with MXC had no effect on ability to exhibit a mating response as an adult female, although the high dose MXC (2.0) and the positive control (estradiol) animals demonstrated a decrease in degree of receptivity, a decrease in proceptive behavior and an increase in rejection behavior. These data suggest that higher doses of MXC given directly to pups during the neonatal period can act as an estrogen and alter aspects of the nervous system, impacting adult reproductive characteristics.

Uterine carcinoma in mice treated neonatally with tamoxifen.

The induction of preneoplastic and neoplastic lesions by the widely used antiestrogen Tamoxifen was studied in female mice. Outbred CD-1 mice were treated with Tamoxifen (1, 2, 5, 10, 25 or 50 microg/pup/day) for the first 5 days after birth. At 14-17 months, reproductive tract tissues were examined for pathological changes. In the ovary, corpora lutea were lacking while cysts were quite common in Tamoxifen-exposed mice at all doses; cystadenomas were seen in two mice. Structural malformations and epithelial hyperplasia of the oviduct were seen in 100% of the treated mice. Malformations of the uterus, cervix, and vagina were also seen. Excessive vaginal keratinization was not a common feature although vaginal adenosis was observed more often after Tamoxifen treatment than previously reported after similar treatment with diethylstilbestrol (DES). The most striking histological features, however, were seen in the uterus. One hundred percent of the Tamoxifen-treated mice at all doses exhibited uterine hypoplasia with focal areas of basal cell hyperplasia in the lining endometrium. Progressive cellular atypias were seen in the lining endometrium ranging from atypical hyperplasia to uterine adenocarcinoma; the highest incidence of uterine adenocarcinoma was 7/14 (50%) observed in the Tamoxifen 10 microg/pup/day dose group. No similar tumors were observed in corresponding control mice. The induction of atypical uterine hyperplasia and adenocarcinoma combined with other abnormalities observed in genital tract structure following neonatal treatment with Tamoxifen suggests the developing reproductive tract is exquisitely sensitive to perturbation by compounds with hormonal activity. These studies provide the basis for future investigation into the mechanisms of Tamoxifen's carcinogenic effects in experimental animals, and to the risk benefit analysis for the prophylactic use of Tamoxifen in healthy women who are at risk of developing breast cancer.

Regulation of differentiation and keratin 10 expression by all-trans retinoic acid during the estrous cycle in the rat vaginal epithelium.

In rodents, the vaginal epithelium shows cyclic changes with an alternating pattern of keratinization under estrogen control and mucification under progesterone control. Retinoids are powerful regulators of cell differentiation, an excess of retinoids suppressing the keratinizing differentiation of keratinocytes. Here, we have examined the vaginal epithelium during the estrous cycle and compare the effects of retinoids on both types of hormonally induced differentiation, i.e. keratinization and mucification. All-trans retinoic acid was administered either by daily injections during the estrous cycle or by a single injection before the estrogen rise; these two protocols gave similar results. Retinoic acid suppressed estrogen-induced vaginal keratinization and cytokeratin K10 expression (a biochemical marker of terminal differentiation). Progesterone-induced mucification was not impaired; however, retinoic acid impeded mucous cell desquamation, suggesting an effect of retinoic acid on cell adhesiveness. Retinoic acid induced the appearance of apoptotic-like cells, as revealed by immunocytochemical staining of DNA fragmentation.

Effects of hormonal therapies and dietary soy phytoestrogens on vaginal cytology in surgically postmenopausal macaques

To evaluate the effects of conjugated equine estrogens, medroxyprogesterone acetate (MPA), conjugated equine estrogens combined with MPA, tamoxifen, and soybean estrogens on vaginal cytology in surgically postmenopausal cynomolgus macaques (Macaca fascicularis). Randomized long-term experimental trial. Cytologic samples were taken from animals in two long-term randomized studies of the effects of hormonal and dietary effects on atherosclerosis. Surgically postmenopausal cynomolgus macaques. Conjugated equine estrogens at the doses given do not exert an estrogenic effect on the with MPA, tamoxifen, and soybean estrogens were given via the diet, at doses scaled from those given to women. Vaginal cytologic maturation index. Conjugated equine estrogens elicited a marked maturation effect, which was antagonized partially by the addition of MPA. Tamoxifen produced a lesser estrogenic response. The cytologic pattern in animals given soybean estrogens or MPA alone did not differ from that of controls. Soybean estrogens at the doses given do not exert an estrogenic effect on the vagina of macaques. Conjugated equine estrogens are potent inducers of vaginal keratinization in this model; tamoxifen has a lesser effect. Medroxyprogesterone acetate partially antagonizes the effects of conjugated equine estrogens, and has no effect when given alone. The results support the possibility that soybean estrogens may be a "tissue-selective" estrogen with minimal effects on the reproductive tract.

Vaginal keratinization during the estrous cycle in rats: a model for evaluating retinoid activity.

A model is described for evaluating the activity of a retinoid based on its effect on the keratinization of the vaginal epithelium that occurs on estrus (day 4) of a 4-day cycle in female rats. This keratinization process is dependent on the endogenous estradiol (E2) secreted between the evening of diestrus 2 (day 2) and that of proestrus (day 3). Various doses of all-transretinoic acid (tRA) were injected at different time points during the estrous cycle and the vaginal keratinization was assessed by microscope examination of unstained native or Papanicolaou-stained smear preparations. Additionally, the preovulatory E2 secretion was measured and ovaries were histologically examined. A single injection of 10 mg/kg tRA either on diestrus 2 (evening) or on proestrus (early morning) was able to induce a complete inhibition of the vaginal keratinization in more than 80% of the cases. This can be considered as a direct effect on the vaginal epithelial differentiation since neither the E2 secretion nor the ovulatory process were affected. The inhibition of vaginal keratinization can be used as a rapid and convenient in vivo model for screening retinoid candidates with antikeratinizing activity.

Epidermal growth factor replaces estrogen in the stimulation of female genital-tract growth and differentiation.

The in vivo studies presented here demonstrate that epidermal growth factor (EGF) is an important autocrine and/or paracrine mediator of estrogen-induced growth and differentiation in mouse uterus and vagina. An antibody specific for EGF significantly inhibited estrogen-induced uterine and vaginal growth, thereby implicating EGF involvement in estrogen action. Furthermore, EGF administered via slow-release pellets in ovariectomized mice acted as a potent uterine and vaginal mitogen as well as an inducer of vaginal keratinization. Experiments with ovariectomized, adrenalectomized, hypophysectomized mice indicated that EGF mitogenesis does not require pituitary or adrenal hormones. Treatment with EGF also mimicked estrogen in the induction of uterine lactoferrin (a major estrogen-inducible secretory protein) mRNA and protein. These data suggest that EGF has estrogen-like effects in the promotion of cell growth in the reproductive tract and that EGF may serve as an important mediator of estrogen action in vivo.

Studies on the mechanism of action of danazol and gestrinone (R2323) in the rat: evidence for a masked estrogen component*

AbstractThe effects of androgen and estrogen receptor antagonists on the action of danazol and gestrinone (R2323) were investigated. The tropic effect of danazol and gestrinone on sex accessory tissues of castrated, immature male rats was inhibited by the antiandrogen ftutamide, whereas the uterotropic action of these steroids in immature female and adult ovariectomized rats was not inhibited by ftutamide. In contrast, the uterotropic effect of danazol was reduced by the antiestrogen, LY156758. The estrogen-sensitive endpoints, vaginal keratinization and uterine progesterone receptor concentration, were enhanced by treatment with a combination of ftutamide and either danazol or gestrinone. These data indicate that danazol and gestrinone have estrogenic activity that is masked by the androgenic component of these drugs.

Interactive responses to diethylstilboestrol in C3H mice

Female C3HeB/FeJ mice were fed diethylstilboestrol (DES) at a dietary concentration of 100 ppb beginning at 6 wk of age. Several oestrogen-sensitive target organs (uterus, vagina, bone and mammary gland) were removed and examined histologically when mice died, became moribund, were killed at scheduled intervals or were removed because of presumptive mammary tumours (palpable mass having a 1-cm diameter). Oestrogen-sensitive histological observations for which there was less than a 100% prevalence and/or severity included uterine fibrosis and glandular hyperplasia, vaginal mucoid stroma and keratinization, mammary adenocarcinomas and osseous trabecular proliferation or marrow fibrosis. Mice from this treated population were grouped according to the specific grade of uterine glandular hyperplasia. Each mouse having a given grade of hyperplasia was matched with a mouse that failed to develop glandular hyperplasia and was fed the 100 ppb DES diet for approximately the same time period. This procedure provided three sets of matched pairs (no hyperplasia v. grade 0; no hyperplasia v. grade 1 and no hyperplasia v. grade 2). Pairwise comparisons were then made in these groups for each of the other oestrogen endpoints. Within each set of matched pairs, mice with glandular hyperplasia had a greater prevalence and/or severity of uterine fibrosis and vaginal mucoid stroma and vaginal keratinization than those without hyperplasia and differences within matched sets were greater as the severity of hyperplasia increased. In contrast, the prevalence of osseous trabecular proliferation or marrow fibrosis were equivalent across all grades of glandular hyperplasia and the prevalence of mammary adenocarcinomas was inversely related to the severity of glandular hyperplasia. These data suggest the existence of a subpopulation of inbred mice that is especially sensitive to DES exposure for uterine and vaginal endpoints and highly insensitive to mammary tumour induction.

Neoplastic and nonneoplastic responses to chronic feeding of diethylstilbestrol in C3H mice.

Over 3000 female C3H/Hen-MTV+ mice continuously received graded dietary levels (0, 2.5, 5, 10, 20, 40, 80, 160, 320, and 640 ppb) of diethylstilbestrol (DES) beginning at weaning. Mice were scheduled to be killed after 3 or 26 wk of exposure and were palpated weekly and removed for histological evaluation when subcutaneous masses reached 1 cm diameter. Mammary tumors were more prevalent than in controls only at 320 and 640 ppb DES. However, palpable mammary tumors appeared significantly earlier than in controls in mice fed 80 ppb and above. Mice killed at 3 wk and later showed a dose-response for several nonneoplastic endpoints. At 3 wk, moderate to severe uterine glandular hyperplasia, lack of corpora lutea, and vaginal keratinization were more prevalent than in controls at 80 ppb; cervical adenosis was more prevalent than in controls at 160 ppb and above. Generally, the prevalence of other nonneoplastic responses such as uterine fibrosis, stromal mucoid changes, and bony trabecular proliferation were increased above control levels only later than 3 wk at 160 ppb and greater. This study demonstrated neoplastic and nonneoplastic responses to DES at and above 80 ppb, but gave no clear evidence of either type of response below this level. Conclusions are, (1) dietary levels of DES causing nonneoplastic effects also cause neoplastic effects when fed chronically, and (2) neoplastic levels of DES may be predicted from a 3 wk feeding study in C3H/HeN-MTV+ female mice based on nonneoplastic responses.

Effect of cyproterone acetate on the reproductive system of the female rat

Effects of cyproterone acetate, a synthetic steroidal compound, on the reproductive organs of female rats 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 rats taking uterine weight and vaginal keratinization as an index of oestrogenicity. Cyproterone acetate in ovariectomized animals induced vaginal keratinization and increased the uterine weights. These effects were parallel to the effect of oestradiol dipropionate in ovariectomized animals, thus indicating oestrogenic activity of cyproterone acetate. We may conclude that the above compound caused antifertility effects due to its oestrogenic nature at the dose level of 2 mg/alternate day in rats when the compound was administered subcutaneously.

The Effects of Various Steroids on the Vaginal Histology in the Rat

In an effort to define more clearly the estrogenic properties of some of the progestogens used as constituents in oral contraceptives keratinization of the rat vaginal epithelium was used as an index of estrogenicity and compared to uterine weight responses. After 30-day-old females were ovariectomized 12 days were allowed for uterine and vaginal regression. On Day 13 oral administration began and continued for 14 days. Autopsy was 24 hours after final gavage. The steroids administered were estrogens (ethinyl estradiol mestranol) an androgen (methyl testosterone) a corticoid (hydrocortisone) and progestogens (progesterone lynestrenol norethisterone norethisterone acetate norethynodrel norgestrel and ethynodiol diacetate). Methyl testosterone hydrocortisone norgestrel and progesterone produced no vaginal keratinization at any dose. A positive response was obtained with all other steroids. The most and least potent among these latter compounds were ethinyl estradiol and norethisterone respectively. In the study of uterine weights significant increases were obtained with all of the steroids but some of the compounds required massive doses. The uterine-weight analysis in short was a less precise index of estrogenicity than was keratinization.

Ultrastructure of vaginal keratinization in estrogen treated immature BALB/cCRGL mice.

In 10-day-old Balb/cCrgl mice, the subcutaneous injection of 0.1 μg of estradiol in distilled water per animal per day resulted in the conversion, over a 4 day period, of the original 3 cell layered cuboidal epithelium to a stratified, multilayered, fully keratinized epithelium. By light microscopy, there was development of a prominent stratum germinativum and of a mucinified surface on the 1st day, followed by the sequential formation of a stratum spinosum, a stratum granulosum, and a stratum corneum. By electron microscopy, the principal early modifications consisted of a marked increase in ribosomes, desmosomes, and 70 A cytoplasmic filaments, the latter being aggregated into approximately 700 A fibrils. The subsequent establishment of a keratin layer was preceded by the appearance of keratohyaline granules and the disappearance of mitochondria and endoplasmic reticulum in cells immediately above the stratum spinosum and by the development of a transitional cell layer in which there was progressive aggregation of cytoplasmic filaments and disappearance of nuclei, keratohyaline granules, and free ribosomes. In the upper stratum granulosum, transitional cell layer, and stratum corneum there were distinctive modifications in desmosomal structure (composite and modified desmosomes). The morphological and physiological significance of these observations is discussed.

Ultrastructure of spontaneous vaginal keratinization in hanging-drop organ culture (Balb/cCrgl MICE)

The vaginal epithelium of castrate 8.5 week old mice undergoes spontaneous keratinization in the absence of estrogen when placed in hanging-drop organ cultures using chemically defined medium 199. The early stages of in vivo and in vitro keratinization have been shown to be similar and are characterized by marked increases in cytoplasmic ribosomes and filaments which correspond to known biochemical changes induced by estrogen in cells of the female genital tract. Similarly, there are early changes in nuclear chromatin and nucleoli in vitro indicative of active messenger-RNA and ribosomal-RNA synthesis. In the later stages of in vitro keratinization, however, there is, by electron microscopy, a failure of keratohyaline granule formation and incomplete filament aggregation. Thus, estrogen induced in vivo and spontaneous in vitro keratinization are not truly comparable. The changes occurring in the organ culture system used have, however, been shown to be reproducible and, at the light microscopic level, comparable to those previously described in other in vitro studies. The culture vessel used in the present study should offer certain advantages over other organ culture systems.

Pre-trauma oestrogen requirements for induction of decidualization in spayed, progesterone-treated rats.

ABSTRACT The formation of deciduomata was investigated in ovariectomized rats treated with various combinations of oestrogen and progesterone before traumatization. The hormonal treatment was kept constant for all groups of animals during the period between traumatization and autopsy (4 mg of progesterone and 0.2 μg of oestradiol benzoate daily). Uterine weight and carbonic anhydrase activity were measured 96 hours after trauma and compared with those of intact controls receiving traumatization on day 4 of pseudopregnancy. When a single dose of oestrogen was given on the day before traumatization, induction of decidualization was successful under a wide variety of experimental conditions (presence or absence of oestrogen »priming« before the beginning of the progesterone treatment; progesterone treatment of varying duration). On the contrary, treatment with divided doses of oestrogen, given for 3 days before trauma, allowed extensive decidualization only in rats »primed« with oestrogen, and traumatized on the fourth day of a progesterone treatment started 24 hours after vaginal keratinization. It appears therefore that only the »oestrogen surge« hypothesis of Shelesnyak and his collaborators can account for those conditions in which implantation occurs at variable intervals of time after the last oestrus (lactation, hypophyseal autotransplantation, administration of a tranquilizer and early ovariectomy followed by hormonal treatment). On the other hand, both the hypothesis of Shelesnyak and that proposed by Yochim & DeFeo (1963) (i. e. continuous secretion of small amounts of oestrogen during the first three days of pregnancy and pseudopregnancy) could account for the rapid waxing and waning of endometrial sensitivity to deciduoma-inducing stimuli observed in normal pregnancy and pseudo-pregnancy.