Estrogen-induced epithelial proliferation and cornification are uncoupled in sinus vaginal epithelium associated with uterine stroma.

Epithelium from the urogenital sinus-derived portion of the newborn mouse vagina when grown in association with uterine mesenchyme forms a "vaginal" stratified squamous non-cornified epithelium. However, the epithelium of these tissue recombinants composed of sinus vaginal epithelium plus uterine mesenchyme does not undergo the fluctuations in cytodifferentiation normally seen in vaginal epithelium during the estrous cycle (e.g., cornification and mucification). In this report we show that sinus vaginal epithelium in association with uterine mesenchyme proliferated in response to estradiol but failed to cornify in response to diethylstilbestrol (DES), even though both the epithelium and the stroma had estrogen receptors. However, if sinus vaginal epithelium that had been grown in combination with uterine mesenchyme was re-isolated from the tissue recombinant and recombined with fresh vaginal mesenchyme, the epithelium cornified in response to DES. These results indicate that the proliferative and the cytodifferentiation response to estrogen could be uncoupled and that sinus vaginal epithelium required vaginal stroma to cornify in response to DES.

Does sinus vaginal epithelium persist in the adult mouse vagina?

It generally is held that the murine vagina develops from the urogenital sinus and the lower portion of the Müllerian ducts and that both endodermally derived sinus epithelium and mesodermally derived Müllerian epithelium contribute to the adult vagina. We tested Müllerian and urogenital sinus-derived vaginal epithelia for their ability to differentiate in response to various hormonal conditions and compared these responses to those of the in situ vagina. Tissue recombinants were prepared with 0-day Müllerian-derived vaginal epithelium and vaginal mesenchyme. Similarly, tissue recombinants containing urogenital sinus-derived vaginal epithelium were prepared with either 0-day sinus vaginal epithelium or 16-day fetal urogenital sinus epithelium and vaginal mesenchyme. Müllerian- or sinus-derived vaginal mesenchyme was used to construct the tissue recombinants; however, the source of the mesenchyme had no influence on the results. Tissue recombinants were grafted to the renal capsule of female mice, allowed to develop for 1 month, and exposed to various hormonal treatments. In diethylstilbestrol-treated hosts, all tissue recombinants regardless of the source of the epithelium were lined by a cornified epithelium. In contrast, only in tissue recombinants containing mesodermally derived Müllerian vaginal epithelium did the epithelium mucify in response to progesterone plus estrogen or become atrophic in ovariectomized hosts. These are the same epithelial modifications seen in the hosts' vagina. Tissue recombinants containing endodermally derived urogenital sinus epithelium or sinus vaginal epithelium and grafts of intact urogenital sinus maintained a stratified squamous noncornified epithelium in both ovariectomized and progesterone plus estrogen-treated hosts. Furthermore, in tissue recombinants containing Müllerian vaginal epithelium and vaginal mesenchyme, estrogen induced a slightly higher epithelial labeling index than in tissue recombinants containing urogenital sinus epithelium or sinus vaginal epithelium. The epithelial labeling index was the same regardless of the source of the vaginal mesenchyme. These results indicate that Müllerian-derived and sinus-derived vaginal epithelia are not equivalent and suggest that Müllerian vaginal epithelium displaces sinus vaginal epithelium during postnatal development. The replacement may result in part from a slight but consistently higher proliferation rate in Müllerian versus sinus vaginal epithelium.

Normal and abnormal development of the male urogenital tract. Role of androgens, mesenchymal-epithelial interactions, and growth factors.

Androgen-dependent male urogenital development occurs via mesenchymal-epithelial interactions in which mesenchyme induces epithelial morphogenesis, regulates epithelial proliferation, and evokes expression of tissue-specific secretory proteins. Mesenchymal-epithelial interactions continue to be important into adulthood. For example, mesenchyme of the urogenital sinus (UGM) and seminal vesicle (SVM) induce dramatic morphologic and functional changes in various adult epithelia. Since adult epithelial cells are unquestionably responsive to mesenchymes that can elicit expression of alternative morphologic and functional phenotypes, established carcinomas might also be influenced by their connective tissue environment. In this regard, Dunning prostatic tumor has been induced by UGM or SVM to differentiate into tall columnar secretory epithelial cells. This change in cytodifferentiation is associated with a reduction in growth rate and loss of tumorigenesis. The role of soluble growth factors in the mechanism of mesenchymal-epithelial interactions is discussed.

Insulin improves survival but does not maintain function of cultured chick wing bud apical ectodermal ridge.

Previously we demonstrated that high levels of insulin (5 micrograms/ml) permit the survival of isolated chick apical ectodermal ridge in culture (Boutin and Fallon, Dev. Biol., 104:111-116, 1984). Here we address whether lower levels of insulin or insulin-like growth factors (IGFs) can also improve the survival of cultured apical ectodermal ridge and whether ridge function is maintained along with ridge survival. Neither IGF I nor IGF II (100 ng/ml) decreased ridge cell death; however, cell death was significantly decreased with 50 ng/ml insulin. No further improvement was obtained in the presence of both IGF I and insulin. These data suggest that insulin improved the survival of the isolated apical ectodermal ridge by binding its own receptor. To test for the maintenance of function, stage 20 ridges were cultured for 0, 6, 12, 18, or 24 hr with or without insulin (5 micrograms/ml or 5 ng/ml) and used to make recombinant limbs. Isolated ridges cultured for 12 hr or more produced fewer outgrowths and these were rarely distally complete. The medium in which the ridges had been cultured did not influence ridge activity, despite the major differences in cell survival. Recombinants made with ridges cultured with limb mesoderm for 18 hr did not yield outgrowths as often as those with freshly isolated ridges, but most of the limbs that did form were distally complete. These results suggest that the decline in function of cultured, isolated apical ectodermal ridge was not due merely to ridge cell death but rather, at least in part, to its separation from limb mesoderm.

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.

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 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.

Cell death in cultured dorsal and ventral chick wing bud epithelia.

We have examined the fate of cultured stage 20 and 25 dorsal and ventral wing bud epithelia and have found evidence that the requirements of apical ectodermal ridge and nonridge limb ectoderms for in vitro survival are different. As previously reported for the apical ectodermal ridge (Boutin and Fallon, '84), dorsal and ventral ectoderms were extensively necrotic after 12 hours of culture in serum-containing medium. The survival of dorsal and ventral limb epithelia at 18 hours was not improved by a collagen substratum, 10% Nuserum, epidermal growth factor, nerve growth factor, multiplication-stimulating activity, insulin, or insulin, transferrin, and selenium. This is in contrast to our observations on the ridge which remains vital for at least 24 hours in insulin or insulin, transferrin, and selenium.

An analysis of the fate of the chick wing bud apical ectodermal ridge in culture.

Stages 20 and 25 chick apical ectodermal ridge have been cultured in nutrient medium containing fetal bovine serum and the tissues have been examined for dying cells at 0, 6, 12, 18, and 24 hr. By 12 hr, an average of 43% of the cells were dying. By 24 hr, stage 20 ridge had lost its integrity and stage 25 ridge contained an average of 50% dying cells. These results are in agreement with the observations of R. L. Searls and E. Zwilling (1964, Dev. Biol. 9, 38-55) on isolated stage 20 ridge. In subsequent experiments, ridge ectoderm was cultured in serum-containing medium to which insulin (5 micrograms/ml), transferrin (5 micrograms/ml), and selenium (5 ng/ml) or insulin (5 micrograms/ml) had been added. Under these conditions the ectoderms remained viable even after 24 hr in vitro.