Epithelial ultrastructure and the distribution of an estradiol sensitive antigen in the vagina of adult mice

Summary The distribution of an antigenic material specific for the cervicovaginal epithelium (CVA) was studied in the vaginal epithelium of the adult mouse with immunofluorescence and immunoferritin techniques. The antigen localization has been correlated to the fine structure of the vaginal epithelium in various states of functional activity. The antigen distribution in adult ovariectomized mice and in ovariectomized mice treated with estradiol was compared with that in normal cycling mice. CVA was found to be associated with the exterior of the cell membrane of vaginal epithelium cells, being part of the glycocalyx.Two cell types, mucous or keratinized, are derived from the germinative cell layer of the vaginal epithelium, depending on the hormonal environment. Mucous cells with morphological features that characterize cells about to cornify have been demonstrated. Fluorescence as well as ferritin particles, indicating the presence of antigen-antibody complexes, were seen within the mucous droplets of the mucous cells. The CVA production is apparently connected with vaginal mucus formation. The CVA distribution in the adult vaginal epithelium is discussed in relation to the distribution demonstrated earlier in the cervicovaginal epithelium of neonatal mice.This investigation was supported by grants from the Norwegian Cancer Society (Landsforeningen mot Kreft) and the Norwegian Research Council.     

Spatial relationship of the genital microflora to the vaginal epithelium of femal rats: transmission electron microscopy

Bacterial associations with vaginal epithelium were examined by transmission electron microscopy in castrate rats before and after estrogen treatment. Bacteria were associated with keratinized epithelium and appeared to be most abundant on exfoliated vaginal cells.     

Role of the epithelial-stromal interaction during the development and expression of ovary-independent vaginal hyperplasia

The tissue interactions involved in the induction and perpetuation of ovary-independent vaginal hyperplasia were studied by growing recombinants prepared with vaginal epithelium and stroma from untreated and neonatally estrogenized mice. As expected, recombinants prepared with untreated tissues developed an atrophied epithelium, while those prepared with estrogenized epithelium and stroma exhibited epithelial hyperplasia in ovariectomized hosts. Recombinants prepared with estrogenized stroma and untreated epithelium and the reciprocal recombination of untreated stroma and estrogenized epithelium also exhibited ovary-independent hyperplasia in many cases. This suggests that the expression of ovary-independent hyperplasia is due to irreversible changes in vaginal epithelium and inductive activities in vaginal stroma. Development of ovary-independent hyperplasia in response to neonatal exposure to estradiol is facilitated when the epithelial-stromal association is maintained and is blocked if this association is disrupted. Finally, Takasugi's (1971, Proc. Japan Acad. 47, 193–198) hypothesis, that the age-dependent loss in sensitivity of the vagina to permanent, irreversible effects of estradiol at 5 days postpartum is due to maturational changes in the epithelium, was confirmed through analysis of the developmental response of heterochronal vaginal recombinants.     

Protein kinases activated by cAMP in the genital tract of spayed mice treated with oestradiol-17beta.

The cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37), has been studied in the vaginal epithelium, vaginal stroma, endometrium, and whole uterus of spayed mice treated with oestradiol-17 beta, and in the vaginal epithelium and uterus of spayed mice. Two protein kinase isoenzymes (PK I and PK II) were found in whole uterus, endometrium, and vaginal stroma. Vaginal epithelium contained only one isoenzyme (PK II). Oestradiol treatment increased PK I relative to PK II in the uterus. The isoenzyme pattern in the vaginal epithelium was unaltered after such treatment. The total protein kinase activity was 70% higher in uterine extracts (cytosol) than in extracts from vaginal epithelium. Oestradiol treatment did not influence the total protein kinase activity in either tissue.     

Lectin histochemistry of mouse vagina during the estrous cycle

Estrous cycle-related histochemical changes in the vaginal epithelium of sexually mature female mice were studied with 30 fluorescein isothiocyanate (FITC)-labeled lectins. On the basis of the staining pattern the lectins were divided into five groups: I, seventeen lectins that reacted with mucinous surface layer of proestrus. This group comprised two subgroups: Ia, seven lectins that reacted exclusively with the mucinous layer, and Ib, ten lectins that reacted with mucinous cells and the underlying squamous epithelium of proestrus; II, two lectins that reacted with squamous epithelium of proestrus only but were unreactive with mucinous cells; III, three lectins that reacted in a phase-specific manner with squamous epithelium; IV, six lectins that showed increased luminal surface reactivity in diestrus and/or metestrus; and V, eleven lectins that were unreactive with vaginal epithelium. These data indicate that the cyclic changes in the morphology of the vaginal epithelium are accompanied by distinct lectin reactivity patterns.     

The germ layer origin of mouse vaginal epithelium restricts its responsiveness to mesenchymal inductors: uterine induction

The epithelium of the mammalian vagina arises from two distinct germ layers, endoderm from the urogenital sinus and mesoderm from the lower fused Müllerian ducts. While previously it has been reported that neonatal vaginal epithelium can be induced to differentiate as uterus, which normally develops from the middle portion of the Müllerian ducts, it has not been determined whether this ability is shared by both mesoderm- and endoderm-derived vaginal epithelia. To test if germ layer origin influences the ability of vaginal epithelium to undergo uterine differentiation, we have isolated sinus-derived and Müllerian-derived vaginal epithelia from newborn mice, combined them with uterine mesenchyme, and grown them for 4 weeks in female mice. Mesoderm-derived Müllerian vaginal epithelium in combination with uterine mesenchyme formed the simple columnar epithelium typical of uterus. Similar results were obtained with neonatal cervical epithelium, another mesodermal Müllerian duct derivative. On the other hand, sinus vaginal epithelium combined with uterine mesenchyme formed small cysts lined by a stratified squamous vaginal-like epithelium. This epithelium never showed evidence of cycling between the cornified and mucified states as is typically seen in vaginal epithelium combined with vaginal stroma. These results indicate that the ability of epithelium to form uterus is limited to mesoderm-derived epithelia and suggest that endoderm-derived sinus vaginal epithelium cannot undergo the typical differentiative modifications in response to the hormonal fluctuations of the estrous cycle when associated with uterine stroma.     

The role of fibroblast growth factors on the differentiation of vaginal epithelium of neonatal mice

The uterus and upper 3/5 of the vagina originate from the Müllerian duct; however, these organs show quite distinct characteristics in morphology and function. To investigate factors controlling vaginal epithelial cell differentiation from a single layer of pseudostratified epithelium to a multi-layered stratified epithelium with keratin, we focused on fibroblast growth factors (Fgfs). Transformation related protein 63 (Trp63) expression, a marker of stratified epithelium, increased in the Müllerian vaginal epithelial cells from days 0 to 5, and keratin 14 (Krt14) was expressed from day 5, suggesting that Trp63-negative vaginal epithelial cells can differentiate into Trp63-positive cells after birth. Fgf7 and Fgf10 were localized in the vaginal stroma but their receptor, Fgf receptor 2IIIb (Fgfr2IIIb), was localized in the vaginal epithelium. Both Fgf9 and its receptor, Fgfr2IIIc, were localized in the vaginal epithelium. Vaginae cultured with FGF10 or anti-FGF9 antibody showed stratified epithelium with an intense Krt14 expression; however, an inhibitor of phosphorylation of mitogen-activated protein kinase 1/3 (MAPK1/3) canceled the effect of FGF10 and anti-FGF9 antibody. Thus, Fgf10 stimulates the differentiation of pseudostratified epithelial cells into stratified cells via MAPK1/3 pathway, and Fgf9 inhibits this differentiation in the neonatal mouse vagina.     

Regulation of the vitamin D receptor and cornifin beta expression in vaginal epithelium of the rats through vitamin D3

The aim of the present study was to determine the respective role of 1,25-dihydroxyvitamin D3 on vaginal epithelium and 1,25-dihydroxyvitamin D3 receptor expression in ovariectomized rats and vitamin D3 treated rats. Bilateral ovariectomies were performed in 20 mature, non-pregnant Wistar female rats. All the animals were divided into 2 groups consisting of 10 rats each. Group I served as control. In group II, animals were injected intramuscularly with vitamin D3 (50, 00 IU/kg). Two weeks after the injections, vaginas of animals in group I and group II were removed removed and processed for immunohistochemistry. Epithelial differentiation, 1,25-dihydroxyvitamin D3 receptor and cornifin beta expression were investigated in vaginal epithelium of control group (ovariectomized) and vitamin D3 treated rats. Vaginal epithelial cells from vitamin D3 treated animals changed into highly- stratified keratinizing layers. 1,25-dihydroxyvitamin D3 receptor and cornifin beta as a marker of squamous differentiation were present in ovariectomized rats treated with 1,25-dihydroxyvitamin D3. In contrast, cornifin beta and 1,25-dihydroxyvitamin D3 receptor were absent in all layers of vaginal epithelium in control group. We demonstrated for the first time that 1,25-dihydroxyvitamin D3 induced proliferation of vaginal epithelium consistent with the cornifin beta expression and 1,25-dihydroxyvitamin D3 up-regulated 1,25-dihydroxyvitamin D3 receptor expression in vaginal epithelium.     

The response of female urogenital tract epithelia to mesenchymal inductors is restricted by the germ layer origin of the epithelium: prostatic inductions

The epithelium of the mammalian vagina arises from two distinct germ layers, endoderm from the urogenital sinus and mesoderm from the Müllerian ducts. While neonatal vaginal epithelium can be induced to form prostate which is normally an endodermal derivative, it has not been determined whether this ability to form prostate is shared by both mesoderm- and endoderm-derived vaginal epithelia. To test the competence of vaginal epithelia we have isolated sinus-derived and Müllerian-derived vaginal epithelia from newborn mice, combined them with rat urogenital sinus mesenchyme, and grown the tissue recombinants for 4 weeks in male athymic nude mice. Endoderm-derived sinus vaginal epithelium was induced to form prostatic tissue which expressed prostate-specific secretory proteins in 21 of 23 tissue recombinants. Müllerian-derived vaginal epithelium formed small ducts and cysts lined by a simple epithelium. These latter tissue recombinants lacked any evidence of prostatic secretory proteins. Similarly, endoderm-derived urethral epithelium was induced to form prostate (17 of 17 cases), while mesoderm-derived uterine epithelium was not (0 of 13 cases). Therefore, the ability to form prostatic epithelium was limited to endodermal derivatives of the urogenital tract.     

Visualization of the Langerhans cells in the human vaginal epithelium by the L-dopa histofluorescence method

In this study we used the technique of L-DOPA histofluorescence for visualizing Langerhans cells in the human vaginal epithelium. Biopsy specimens were incubated with L-DOPA and sectioned by cryostat. The sections were exposed to formaldehyde vapour; during this passage, chemical conversion of L-DOPA into a strongly fluorescent compound occurred. Langerhans cells were clearly visualized in the vaginal epithelium; cell bodies and dendritic processes fluoresced sharply. The method is rapid and specific: it represents an useful tool for demonstrating Langerhans cells in the stratified squamous epithelium of vagina.     

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.     

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.     

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.     

Histological studies on the vaginal plate and vaginal epithelium in androgenized rats

Summary The present work is a histological study on the epithelium of the vaginal plate and vagina proper of intact rats, 5–45 days old, and of rats which were injected with 1.5 mg testosterone propionate at the age of 5 days (androgenized) and studied at different ages from 6–45 days. Moreover, the vaginal epithelium in adult androgenized rats was studied.A perforation of the vaginal plate and an external orifice of the vagina was seen in intact rats about 35 days old. The perforation resulted from cornification in scattered, later confluencing, regions in the epithelium of the plate.The androgenized rats developed a precocious vaginal opening at 12 days of age. Also in these animals the perforation of the plate resulted from a cornification in the center of the strongly thickened plate. After a lumen had developed the plate region presented a transitional epithelium. This was transformed into a stratified squamous epithelium at about 35–45 days, that is when the first oestrus occurred in intact controls. In contrast to the strong reaction to testosterone in the epithelium of the vaginal plate no effects could be seen in the vaginal epithelium proper, with the exception of some leucocytic infiltration.The adult androgenized rats had a cornified vaginal epithelium which in two cases showed some leucocytic infiltration. Acknowledgement. This investigation was supported by grants from the Swedish Medical Research Council (no 14X-571-02 and no Y 479) and from the Swedish Cancer Society (no 64164).     

Reactions of the vaginal plate and the vaginal epithelium to oestradiol monopropionate given once to 5 or 26-day old rats

Summary Five-day old rats were given a single injection of varying amounts of oestradiol monopropionate (OeM) in propylene glycol (0.5, 5, 30 and 150 g) and 26-day old rats were given a single injection of 5 or 150 g OeM. The rats were sacrificed at varying times after the injection and the changes in the vaginal plate and the vaginal epithelium proper were studied. As indicated by the cornification of the epithelium the reaction to OeM is stronger in the vaginal plate than in the vaginal epithelium proper. The response of the vaginal epithelium to 5 or 150 g OeM is most extensive in the rats injected at 26 days of age. With 150 g OeM a cornification of the vaginal epithelium was obtained in rats injected when 26 days old but not in those injected when 5 days old. The reactions in the vaginal plate were compared with those found previously after injections of testosterone propionate. Acknowledgement. This investigation was supported by grants from the Swedish Medical Research Council (12 X-579-03, K67-14X-571-03) and from the Swedish Cancer Society (67:46).     

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.     

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.     

Epithelial-mesenchymal interactions in uterus and vagina alter the expression of the cell surface proteoglycan, syndecan

The cell surface proteoglycan, syndecan, exhibits molecular and histological dimorphism in the mouse uterus and vagina. In the mature vagina, syndecan is localized at the surfaces of the basal and intermediate cells of the stratified epithelium and has a modal molecular mass of ca. 92 kDa. The uterus expresses a larger form of syndecan (ca. 110 kDa) which is detected at the basolateral surfaces of the simple columnar epithelial cells. We have investigated whether epithelial-mesenchymal interactions influence the expression of syndecan in these organs by analyzing tissue recombinants composed of mouse epithelium and rat mesenchyme or vice versa with monoclonal antibody 281-2, which recognizes mouse syndecan. In tissue recombinants composed of newborn mouse uterine epithelium and rat vaginal stroma, the uterine epithelium was induced to form a stratified vaginal epithelium which expressed syndecan in same the pattern and mass typical of vaginal epithelium. Likewise, rat uterine stroma induced newborn mouse vaginal epithelium to undergo uterine development, and this epithelium exhibited a uterine pattern of syndecan expression. Although stromal cells normally express little syndecan in most adult organs, analysis of recombinants composed of mouse stroma and rat epithelium revealed that both uterine and vaginal mouse stromata synthesized syndecan that was larger (ca. 170-190 kDa) than the epithelial syndecans. A quantitative increase in the amount of stromal syndecan was evident when stroma was grown in association with epithelium in comparison to stroma grown by itself. These data suggest that epithelial-mesenchymal interactions influence the amount, localization, and mass of both epithelial and stromal syndecan.     

The permeability of nonhuman primate vaginal epithelium: a freeze-fracture and tracer-perfusion study

The lining of the vaginal mucosa in primates is a keratinized stratified squamous epithelium. As in other structurally similar epithelia, one function of the vaginal epithelium is to provide a barrier between the external environment and the underlying tissues. The vaginal lining is aglandular and the source of true vaginal fluid has been suggested to be the intercellular channels of the epithelium. On the other hand, other structurally similar epithelia have been shown to have a barrier to the movement of water-soluble molecules through these channels. In the present study, we have examined the permeability of rhesus monkey vaginal epithelium to lanthanum and horseradish peroxidase. Both tracer molecules penetrated the intercellular channels in the lower layers of the epithelium, but were excluded from the channels at and above the granular layer. Neither tracer penetrated significantly between cells at the free surface of the epithelium and usually did not penetrate between cells in the upper layers to any degree from the cut edges of the biopsy. These results are consistent with tracer studies in other structurally similar epithelia and strongly suggest that the upper layers of vaginal epithelium present a barrier to the movement of water-soluble molecules through the intercellular channel system. Freeze-fracture analysis of the epithelium revealed gap junctions and desmosomes between cells in the lower layers, but the former disappear in the upper layers. Unlike other keratinizing epithelia that have been described, random intramembranous particles do not disappear from the plasma membranes of the fully differentiated cells. Fracture planes through the upper layers reveal particle-free lamellae in the intercellular spaces, supporting the idea that intercellular lipids may be one of the components that limits the permeability of the intercellular spaces in this epithelium.     

Studies on the differentiation pattern and hormonal sensitivity of an antigenic material specific for the cervicovaginal epithelium in fetal and neonatal mice.

By use of an indirect mixed haemagglutination method for tissue sections, the Müllerian cervicovaginal epithelium of fetal and neonatal mice has been shown to contain a material (CVA) with antigenic properties specific for this epithelium. The method is highly sensitive and permits a semiquantitative estimation of CVA in the epithelium. The studies showed that the cervicovaginal epithelium undergoes a multiphasic differentiation pattern. An estradiol injection 1 day prior to killing the animals strongly increased the amount of demonstrable CVA. The quantitative response to estradiol varied with the age of the animals. Notably, estradiol given as early as the day of birth stimulated CVA accumulation in the vaginal epithelium. Differences between the cervical epithelium and the vaginal epithelium regarding the response to estradiol are described.