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.     

Postnatal development of vagina, clitoris and urethral glands of the golden hamster

We have made a histological study of the postnatal development of the clitoris, preputial glands, urethral glands and vagina of the golden hamster. The 'phallic groove' of the clitoris is closed at 10 days of life, then the urethra has a cuboidal stratified, a stratified squamous and a stratified keratinized epithelium. The preputial glands are composed of branched saccular glands. These glands develop, with few changes during their maturation period. Formation of the urethral glands begins at 5 days and the alveoli are fully developed at puberty. The hamster vagina has two origins; the upper part is Müllerian, the caudal part is sinusal. The wall of the Müllerian vagina has a cylindrical epithelium at birth, which becomes 'double epithelium' at puberty and thereafter changes cyclically in connection with the estrous cycle. The sinusal vagina is solid at birth, its lumen being formed in the first 10 days of life and its wall having a cuboidal stratified epithelium. At 15 days it becomes a stratified keratinized epithelium, which will later line the vaginal pouch. At the 5th day, an ectodermic invagination (stratified keratinized epithelium) is observed in the zone of the future introitus. At the time of vaginal opening this zone forms the distal segment.     

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 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.     

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.     

Ultrastructural localization of glucose-6-phosphatase and alkaline phosphatase in the vaginal epithelium of rat

The effect of ovarian hormones on the activities of glucose-6-phosphatase and alkaline phosphatase in the vaginal epithelium was studied in immature and ovariectomized rats, using ultracytochemical techniques. Comparative studies were done on normal rats at the luteal phase and on day 14 of pregnancy. Various vaginal cells show different degrees of response to progesterone and diethylstilbestrol (DES) with regard to glucose-6-phosphatase activity. Intense glucose-6-phosphatase activity was observed in the cisternae of granular endoplasmic reticulum (rER), Golgi saccules and vesicles, and nuclear envelope of both basal cells and stromal cells of progesterone treated rats, whereas in the basal cells and stromal cells of DES-treated and control animals the enzyme was totally lacking. Detectable glucose-6-phosphatase activity was also observed, however, in the rER cisternae and Golgi complex of keratohyalin-secreting squamous intermediate cells of the vaginal epithelium of DES-treated rats. Alkaline phosphatase was also found on the limiting membranes of secretory granules of mucocytes in animals at the luteal phase and during pregnancy. DES and progesterone in the doses used did not affect alkaline phosphatase activity in the rat vagina. Overall, progesterone enhances glucose-6-phosphatase activity in basal cells of the rat vagina prior to completion of mucification. Alkaline phosphatase was found in all cells involved in mucin secretion.     

Androgens and the development of the vagina

Today it is generally held that the vagina develops from sinovaginal bulbs and that the lower third of the definitive vagina is derived from the urogenital sinus. Here we show that the entire vagina arises by downward growth of Wolffian and Müllerian ducts, that the sinovaginal bulbs are in fact the caudal ends of the Wolffian ducts, and that vaginal development is under negative control of androgens. We designed a genetic experiment in which the androgen receptor defect in the Tfm mouse was used to examine the effects of androgens. Vaginal development was studied by 3D reconstruction in androgen-treated female embryos and in complete androgen-insensitive littermates. In androgen-treated females, descent of the genital ducts was inhibited, and a vagina formed in androgen-insensitive Tfm embryos as it does in normal females. By immmunohistochemical localization of the androgen receptor in normal mouse embryos, we demonstrated that the androgen receptor was expressed in Wolffian duct and urogenital sinus-derived structures, and was entirely absent in the Müllerian duct derivatives. We conclude that the Wolffian ducts are instrumental in conveying the negative control by androgens on vaginal development. The results are discussed under evolutionary aspects at the transition from marsupial to eutherian mammals.     

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.     

MSX2 promotes vaginal epithelial differentiation and wolffian duct regression and dampens the vaginal response to diethylstilbestrol

In utero exposure to diethylstilbestrol (DES) leads to patterning defects in the female reproductive tract (FRT) and a propensity to the development of vaginal adenocarcinomas in humans. In the mouse, DES treatment similarly induces a plethora of FRT developmental defects, including stratification of uterine epithelium and presence of glandular tissue in cervix and vagina. Uterine abnormalities are associated with repression of the homeobox gene Msx2, and DES leads to an altered uterine response in Msx2 mutants including a dilated uterine lumen. Here we investigate the role of Msx2 in normal vaginal development and in FRT response to DES. During vaginal development, Msx2 is required for Tgfbeta2 and Tgfbeta3 expression and for proper vaginal epithelial differentiation. Moreover, Msx2 is involved in caudal Wolffian duct regression by promoting apoptosis. Consistently, neonatal DES exposure represses Msx2 expression in the Wolffian duct epithelium and inhibits its apoptosis and subsequent regression. Intriguingly, although DES treatment also represses Msx2 expression in the vaginal epithelium, a much more severe DES-induced vaginal phenotype was observed in Msx2 mutant mice, including a complete failure of Müllerian vaginal epithelial stratification and a severely dilated vaginal lumen, accompanied by loss of p63 and water channel protein expression. These results demonstrate a critical role for Msx2 in counteracting the effect of DES on FRT patterning and suggest that the response to DES may be highly variable depending on the genotype of an individual.     

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.     

Outgrowth in vitro of cervical epithelium separated from the uterovaginal anlage of newborn mice

Summary After gentle trypsinization, the pseudostratified columnar Müllerian epithelium that lines the uterine cervix of newborn mice could be separated from the enclosing stromal tissue. Pure epithelial tubes explanted in vitro and were allowed to grow in a standard medium for 3–4 days forming a confluent colony of rather closely-fitting cells. The cell sheet was studied by a preparatory technique that allows examination of a large number of cells with preserved intercellular spatial orientation. Attempts were made to identify cultured cells according to the morphology of cell types in the cervicovaginal epithelium in vivo.Electron micrographs revealed that, close to the explant, the cultured cell sheet exhibited several features similar to the Müllerian epithelium in vivo. Outside these central areas of the colony was a broad transitional zone consisting of thin platelike cells distinguished by an abundance of microfilaments. At the periphery of the colonies, bulky cells possessing microvilli and a vacuolated cytoplasm tended to overlap adjoining platelike cells. These bulky cells had a morphology resembling that of the superficial cells seen in the upper vagina and common cervical canal of immature and diestrous animals. The epithelial development in the cultures apparently simulated the transformation in vivo from a pseudostratified Müllerian epithelium in the newborn to a stratified epithelium resembling that of the uppermost vagina and common cervical canal of immature animals. Judged by morphological and cytochemical criteria, the Müllerian cells in the outgrowth obviously had many changed features. It thus seems questionable whether the cells grown in vitro are comparable with the corresponding cells in vivo when used for experiments requiring the controlled conditions of the culture environment.Supported by grants from the Norwegian Research Council for Science and the Humanities and from the Norwegian Cancer Society     

Diethylstilbestrol induces vaginal adenosis by disrupting SMAD/RUNX1-mediated cell fate decision in the Müllerian duct epithelium

Women exposed to diethylstilbestrol (DES) in utero frequently develop vaginal adenosis, from which clear cell adenocarcinoma can arise. Despite decades of extensive investigation, the molecular pathogenesis of DES-associated vaginal adenosis remains elusive. Here we report that DES induces vaginal adenosis by inhibiting the BMP4/Activin A-regulated vaginal cell fate decision through a downregulation of RUNX1. BMP4 and Activin A produced by vaginal mesenchyme synergistically activated the expression of ΔNp63, thus deciding vaginal epithelial cell fate in the Müllerian duct epithelial cells (MDECs) via direct binding of SMADs on the highly conserved 5′ sequence of ΔNp63. Therefore, mice in which Smad4 was deleted in MDECs failed to express ΔNp63 in vaginal epithelium and developed adenosis. This SMAD-dependent ΔNp63 activation required RUNX1, a binding partner of SMADs. Conditional deletion of Runx1 in the MDECs induced adenosis in the cranial portion of vagina, which mimicked the effect of developmental DES-exposure. Furthermore, neonatal DES exposure downregulated RUNX1 in the fornix of the vagina, where DES-associated adenosis is frequently found. This observation strongly suggests that the downregulation of RUNX1 is the cause of vaginal adenosis. However, once cell fate was determined, the BMP/Activin-SMAD/RUNX1 signaling pathway became dispensable for the maintenance of ΔNp63 expression in vaginal epithelium. Instead, the activity of the ΔNp63 locus in vaginal epithelium was maintained by a ΔNp63-dependent mechanism. This is the first demonstration of a molecular mechanism through which developmental chemical exposure causes precancerous lesions by altering cell fate.     

Differentiation of the müllerian (paramesonephric) duct epithelium in the rat

Differentiation of the Müllerian duct epithelium was studied in 15- to 21-day female rat foetuses. The proximal segment of the Müllerian duct is formed by the 15th day; it runs parallel to the Wolffian duct and the two are wrapped in a common basement membrane. On the 16th day the genital ducts are clearly separate; the Müllerian duct has a slit-like lumen and is lined with simple columnar epithelium. Throughout the whole of the given period the epithelium retains a relatively indifferent appearance. Characteristic findings from the 18th day include the apical migration of centrioles and the formation of solitary cilia.     

Pre-natal innervation of the human female genital tract

In the human fetus of 14 weeks, ganglia on either sides of the Müllerian uterovaginal canal contained two types of cells. In the 16th week, axons invaded the basal zone of the stratified squamous epithelium at the sides of the upper vagina. In the 20th week, vesicular nuclei typified the large neurons in the midportion of the cervico-vaginal ganglion. During the 22nd week, capsulated ganglia invaded the wall of the upper vagina forming three concentrically disposed strata. Non-capsulated clusters invaded its lamina propria. At the 24th week, axons were shaded after reaching the superficial zone of the stratified vaginal epithelium. In the 28th week, satellites surrounded the mature neurons and sheath cells enveloped the axons. Ganglia invaded the splitted muscle layer of the upper vagina at 30 weeks. Intraepithelial fibres invaded the whole thickness of the endometrium, the columnar epithelium of the cervix and uterine tube at 40 weeks. Nerve cells were detected among the basal epithelial cells of the lower vagina and its subepithelial plexus.     

Epithelial-stromal tissue interaction in paramesonephric (Müllerian) epithelial differentiation.

During organogenesis, the middle to caudal portion of Müllerian epithelium differentiates into uterine and vaginal epithelia in females. Functional differentiation of uterine and vaginal epithelia occurs in adulthood, and is regulated by 17beta-estradiol (E(2)) and progesterone. In this report, the roles of mesenchyme/stroma in differentiation of uterine and vaginal epithelia were studied in tissue recombination experiments. At birth, Müllerian epithelium was negative for uterine and vaginal epithelial markers. Tissue recombinant experiments showed that uterine and vaginal gene expression patterns were induced in neonatal Müllerian epithelium by the respective mesenchymes. Differentiated adult uterine and vaginal epithelia did not change their original gene expression in response to heterotypic mesenchymal induction. In the adult vagina, E(2) induced expression of involucrin, a CCAAT/enhancer-binding protein beta and cytokeratin 1 via estrogen receptor alpha (ERalpha). Tissue recombination experiments with wild-type and ERalpha knockout mice demonstrated that epithelial gene expression is regulated by E(2) via epithelial-stromal tissue interactions. Uterine/vaginal heterotypic tissue recombinations demonstrated that functional differentiation of uterine and vaginal epithelia required organ-specific stromal factors. In contrast, stromal signals regulating epithelial proliferation appeared to be nonspecific in the uterus and vagina.     

Embryonic cholinesterase activity during morphogenesis of the mouse genital tract

Summary In the genital tract of male and female mouse embryos cholinesterase activity is described that is independent from innervation. The enzyme activity is localized in the mesenchyme at the junction of Wolffian and Müllerian ducts with the urogenital sinus. During male development prostate buds and vesicular glands grow out into the cholinesterase-active mesenchyme. During female development the active mesenchyme participates in the downgrowth of the vaginal anlage. Ultrastructurally the cholinesterase activity is localized in the perinuclear cisterna and in smooth endoplasmic reticulum of the mesenchymal cells. The enzyme activity disappears with definitive differentiation of the tissue. The embryonic cholinesterase is a component of a primitive muscarinic system. Its relation to the morphogenetic action of testosterone and its possible general functions are discussed.     

Establishment and characterization of clonal cell lines from the vagina of p53-deficient young mice

Clonal cell lines have been established from vagina of prepubertal female p53(-/-) mice. Because the mouse vagina has a dual origin (the cranial three-fifths derived from the Müllerian duct and the caudal two-fifths derived from the urogenital sinus), both parts were separately subjected to cloning. Sixteen epithelial and two fibroblastic cell lines were established from the cranial three-fifths (Müllerian vagina group), and four epithelial and three fibroblastic cell lines were established from the caudal two-fifths (sinus vagina group). They were maintained in Dulbecco's modified Eagle medium and Ham's nutrient mixture F-12 containing 10% fetal calf serum and 17 beta-estradiol at 10(-8) M. Two cell lines (one epithelial and one fibroblastic) were examined using soft agar assay, but no colonies were formed. The doubling time of the cell lines was approximately 24 h, and all of them divided more than 200 times without crisis, suggesting that they were immortalized. All epithelial cell lines expressed cytokeratin 8. However, the epithelial cell lines expressed cytokeratin 14 and cytokeratin 10 when exposed to medium containing different concentrations of Ca(2+). Fibroblastic cell lines expressed vimentin. All epithelial and fibroblastic cell lines expressed estrogen receptor-alpha protein. This is the first successful establishment of clonal cell lines from the normal mouse vagina, and these lines may provide good models in vitro of the vagina for the study of the mechanism of estrogen action.     

Histological localization of hydrolases in the epididymis of the dog]

Localization and activity of five hydrolases (alkaline phosphatase, adenosine triphosphatase, acid phosphatase, nonspecific esterase and leucylamino-peptidase) were evaluated histochemically in the epididymides of mature dogs. In the ductuli efferentes, cilia and apical parts of the epithelial cells displayed high activity of alkaline phosphatase and adenosine triphosphatase. Strong activity of acid phosphatase, nonspecific esterase and leucylamino-peptidase was present in the basal and supranuclear zones of the epithelium of the ductuli efferentes. Stereocilia of all three segments of the ductus epididymidis showed a high activity of alkaline phosphatase. Positive adenosine triphosphatase reaction was confined to the stereocilia of the initial segment. A complex pattern of acid phosphatase activity was observed in the middle segment. The subdivision of the middle segment in four subsegments was therefore suggested. In the epithelium of the initial segment only a few nonspecific esterase-positive cells were seen. The infranuclear and basal areas of the epithelium in the middle segment and the supranuclear zone of the terminal segment displayed distinct nonspecific esterase activity. The possible contribution of the hydrolases to the function of the epididymis is discussed.     

Immunohistochemical localisation of androgen receptor during sex-specific morphogenesis in the fetal mouse

The distribution of androgen receptors (ARs) in paraffin serial sections of day 17 and day 18 male and female mouse embryos was investigated. In the cranial section of the genital tract AR expression was restricted to Wolffian structures while Müllerian ducts and surrounding mesenchyme were AR negative. In the fusion zone with the urogenital sinus the epithelial components of the vaginal bud were clearly distinguished by differential AR expression, which was faint in the Wolffian ducts, totally missing in the Müllerian ducts, and intense in the sinus ridges with the most intense expression in the morphogenetically active mesenchyme, indicating a new mechanism of negative control of vagina formation via androgens. Expression of ARs outside the genital tract was observed: (1) in loose interstitial mesenchyme extending into the retroperitoneal space up to the coeliac artery, indicating androgen effects during ascent of the kidneys and descent of intraperitoneal organs, (2) in the trigone of the bladder indicating androgen involvement in the development of the vesico-ureteral junction, and (3) in loose mesenchyme between striated muscle fibres and around pelvic skeletal elements, indicating mediation of androgen effects on the musculoskeletal system via loose mesenchyme.