Neonatal seminal vesicle mesenchyme induces a new morphological and functional phenotype in the epithelia of adult ureter and ductus deferens

Mesenchyme, defined as loose embryonic connective tissue, is critically involved in a myriad of secondary inductions involved in the development of the integu-mental (Kratochwil, 1987; Sawyer, 1983), urinary (Ekblom, 1984; Saxén, 1970), gastrointestinal (Haffen el al. 1987; Kedinger et al. 1986), skeletal (Hall, 1987; Wolpert, 1981) and urogenital systems (Cunha, 1976a; Cunha et al. 1980a, 1983a, 1987). Mesenchyme induces specific patterns of epithelial morphogenesis resulting in the formation of a broad spectrum of epithelial forms such as branched ductal networks, planar epithelial surfaces, simple epithelial ducts and tubules and many highly unique epithelial patterns found in teeth, feathers and sense organs, to name but a few (Bemfield et al. 1973, 1984; Cunha, 1976b; Cunha et al. 1980b, 19836; Higgins et al. 1989a,b; Rawles, 1963). Extensive evidence suggests that during these morphogenetic processes epithelial proliferation is regulated by the mesenchyme, perhaps via paracrine factors (Alescio and Pipemo, 1967; Bigsby and Cunha, 1986; Chung and Cunha, 1983; Cooke et al. 1986; Goldin and Wessells, 1979; Norman et al. 1986; Rutter et al. 1978). Mesenchyme-induced epithelial development culminates in the emergence of specific types of epithelial cytodifferentiation and the expression of tissue-specific macromolecules (Cunha et al. 19806, 19836; Haffen et al. 1982; Higgins et al. 1989a,b; Kedinger et al. 1986; Kollar and Baird, 1970).

The adult counterpart of embryonic mesenchyme is stroma, which constitutes the non-epithelial component of an organ. The predominant cells of stroma are fibroblasts and smooth muscle. The role of epithelial-stromal interactions in adulthood has received considerably less attention than embryonic mesenchymal-epithelial interactions. In part this is because adult epithelial cells are thought to be irreversibly determined and terminally differentiated (Slack, 1985). Nonetheless, various lines of evidence support the idea that adult epithelial cells remain responsive to the inductive influences of stromal cells.

Many examples of developmental plasticity in adulthood concern the induction of regional variation in adult epidermal differentiation by heterotypic dermis (Bernimoulin and Schroeder, 1980; Billingham and Silvers, 1968; Karring et al. 1975; Mackenzie and Hill, 1984; Spearman, 1974). However, these findings are best characterized as connective tissue-induced modulations in epidermal differentiation since such minor changes in epidermal thickness and patterns of keratinization are encompassed within the basic stratified squamous epidermal phenotype. In other experimental models, mesenchymal or stromal cells have also elicited changes in morphology, differentiation or growth of adult epithelial cells (Dudek and Lawrence, 1988; Haslam, 1986; McGrath, 1983; Norman et al. 1986; Sakagami et al. 1984; Sakakura et al. 1979a,b; Sugimura et al. 1986). On the other hand, the profound changes elicited by urogenital sinus mesenchyme in epithelium of the adult urinary bladder provides one of the most striking examples of a mesenchyme-induced alteration in adult epithelial differentiation. In this model, embryonic urogenital sinus mesenchyme elicited prostatic differentiation in epithelial cells of the adult urinary bladder, which entailed the morphogenesis of a branched ductal network, a marked stimulation in epithelial proliferation, the differentiation of a simple columnar secretory epithelium and the expression of several prostate-specific markers (Cunha et al. 19836; Neubauer et al. 1983).

Is such a profound developmental reprogramming as represented by this last example more widely applicable to other adult epithelia? Recently, a closely related mesenchyme derived from the seminal vesicle (SVM) has been reported to be able to induce seminal vesicle (SV) differentiation from embryonic epithelium from the middle and upper Wolffian duct (prospective ductus deferens and epididymis, respectively). Significantly, the induced epithelial cells expressed the full complement of seminal vesicle secretory (SVS) proteins (Higgins et al. 1989a,b). Since the embryonic Wolffian ducts normally give rise to the epithelia of the epididymis, SV, ureter (UR) and ductus deferens (DD), this system provides a suitable model in which morphological and functional aspects of the reprogramming of postnatal epithelia can be studied.

Balb/c mice and Sprague-Dawley rats were obtained from the Cancer Research Laboratory (University of California, Berkeley) and local vendors (Simonsen, Gilroy, CA and Bantin-Kingman, Fremont, CA). Adult athymie mice were obtained from Harlan, Inc. (Indianapolis, IN). Neonatal male rats and mice were used within 24 h of birth (day 0). All animals received water and laboratory chow ad libitum and were housed under standard laboratory conditions.

The preparation and characterization of polyclonal rabbit antibodies (IgG fraction) monospecific for the androgen-dependent proteins (proteins I – V) of rat SV have been described before (Fawell and Higgins, 1986; Fawell et al. 1986,1987). The antibody to mouse SVS (anti-mouse SVS) has been described by Higgins et al. (1989a). Sources and descriptions of all reagents used will be found in Higgins et al. (1989a,b)

SV were excised from 0-day-old neonatal rats and mice. UR and DD were excised from 0-to 60-day-old rats and mice. The epithelium and mesenchyme or stroma of these organs were separated following tryptic digestion and recombined as described earlier (Cunha, 1976c; Cunha et al. 1983b; Sugimura et al. 1986). In homospecific recombinants, the mesenchyme and epithelium were from the same species, either mouse or rat. In heterospecific recombinants, tissues from both rat and mouse were combined, eg., mouse SVM+rat DDE or rat SVM+mouse DDE. After overnight culture, the tissue recombinants were grafted under the renal capsule of adult male hosts anesthetized with Avertin (tertiary amyl alcohol plus tribromoethanol). For homospecific tissue recombinants, syngeneic male hosts were used. For heterospecific recombinants, athymie nude mice were used. Up to six grafts were placed on each kidney. This study is based on the analysis of 279 tissue recombinations.

Hosts were killed 4 weeks after grafting and the tissue recombinants were dissected from the renal capsule. Secretion was recovered from within the cystic recombinants solubilized in SDS and stored as described (Higgins et al. 1989a). The remaining tissue was then fixed by immersion overnight in 4% paraformaldehyde, embedded in paraffin, sectioned at 6 μ m and air-dried onto poly-L-lysine-coated slides (Higgins et al. 1989a). For histologic analysis sections were stained with hematoxylin and eosin. Nuclear staining with Hoechst dye 33258 was as described (Cunha and Vanderslice, 1984).

Anti-mouse SVS and the antibodies against rat SVS proteins IV and V were used in immunocytochemistry. As described earlier (Higgins et al. 1989a) the anti-mouse SVS reacts with mouse but not rat SV, while anti-rat SVS IV or V reacts with rat but not mouse SV. The methodology has been described in detail earlier (Higgins et al. 1989a). Briefly, deparaffinized tissue sections were reacted successively with the primary antisera, biotinylated donkey anti-rabbit IgG, PBS-Tween and the Vectastain peroxidase ABC reagent. The colored reaction product was developed using diaminobenzidine and H₂O₂ as described (Cunha et al. 1989).

Protein samples prepared in PBS – SDS were analyzed by electrophoresis in polyacrylamide (10 – 20% linear gradient) slab gels using a discontinuous buffer system (Laemmli, 1970) with 0.1% SDS throughout as described by Brooks and Higgins (1980). Proteins (20 – 50 μg per lane) resolved by this method (SDS – PAGE) were visualized by staining for 1 h in 0.1 % Coomassie blue in acetic acid:methanol:H₂O (1:3:6 by vol) followed by prolonged destaining in the same solution without the dye.

Mouse SVS proteins were identified immunologically using Western blotting (Burnette, 1981) with anti-mouse SVS. However, for the rat SVS proteins the large number of samples was more conveniently screened with each of the five rat SVS antibodies by immunodot blot procedures rather than five separate Western blots on each occasion (Higgins et al. 1989a).

Immediately after SDS – PAGE, resolved proteins were transferred electrophoretically from the unstained gels to nitrocellulose sheets using a BioRad transblot apparatus containing 0.15 M glycine, 20 HIM Tris (Fawell et al. 1986). Electrophoretic transfer of proteins to the nitrocellulose sheets was then checked by staining the nitrocellulose paper with India ink (Hancock and Tsang, 1983).

Protein samples in PBS – SDS were spotted (2 μ1aliquots containing 2 μ g protein unless otherwise indicated) onto nitrocellulose sheets or strips and air-dried (Higgins et al. 1989a).

Western and immunodot blots were incubated successively at room temperature in PBS containing 5 % BSA, PBS, and 5 % BSA – PBS – Tween containing the primary antibody in heat-sealed plastic bags as described by Higgins et al. (1989a). The blots were then incubated in PBS-Tween, 5% BSA-PBS – Tween, 5% BSA-Tween containing [^l25I)iodo-Protein A (l × 10⁶disintsmin^-1 ml^-1), PBS-Tween. Finally, they were air-dried, mounted on cardboard and autoradiographed at – 70°C with Kodak X-Omat film (Eastman-Kodak, Rochester, NY) and Cronex Lightning Plus intensifying screens (Dupont, Wilmington, DE).

For steroid autoradiography, specimens were incubated for 1h in 10 nM of [³H]dihydrotestosterone (³H-DHT, Amer-sham, specific activity 100 – 150 Ci mmol^-1 ) with or without a 300-fold excess of radioinert DHT (Steraloids, Wilton, NH) at 37°C. After labelling they were placed into nylon mesh bags (Tetko, Elmsford, NY) and washed for 3 to 4h with constant stirring in a 1 liter flask of PBS which was changed at 30min intervals. After washing, the tissues were then embedded in OCT medium (Miles, Elkhart, IN) and frozen in liquid propane as described by Shannon et al. (1982). Frozen sections (4 mm) were cut, thaw-mounted onto emulsion-coated slides (NTB-II emulsion, Eastman Kodak) and exposed autoradiographically. The autoradiograms were processed photographically and stained with hemotoxylin and eosin.

In all cases (19/19), SVM grafted beneath the renal capsule by itself formed masses of fibroblastic cells (Fig. 1) devoid of SVE morphology. SDS – PAGE, immunocytochemistry, Western blot and protein dot blot analysis demonstrated the complete absence of SVS proteins in these SVM grafts (Figs 5 and 7). Deletion of the primary antisera (not illustrated) completely eliminated immunocytochemical staining as described earlier (Fawell and Higgins, 1986; Higgins et al. 1989a).

Homotypic recombinations of UR, DD and SV developed as expected. Namely, transitional urothelium differentiated in URM+URE recombinants (Fig. 2). DDM+DDE tissue recombinants formed simple ductal structures having a star-shaped luminal contour lined by tall columnar epithelial cells with stereocilia (Fig. 3). SVM+SVE recombinants developed the complex morphology characteristic of SV (Fig. 4). Neither URM + URE nor DDM + DDE recombinants expressed SVS proteins as judged by immunocytochemistry (not illustrated), SDS –PAGE or immunoblotting (Figs 5 and 7). As expected SVM+SVE recombinants formed SV tissue which produced the full complement of SVS proteins (Figs 5 –7). When tissue recombinants were constructed with rat SVM+mouse SVE, the 6 major SVS proteins characteristic of the mouse SV were observed (Figs 5 –6), whereas tissue recombinants constructed with mouse SVM+rat SVE expressed the 5 major secretory proteins of the rat SV (Figs 5,7).

Almost all of the SVM+URE tissue recombinants in this study were heterospecific (rat mesenchyme+mouse epithelium or vice versa). In this way, staining with Hoechst dye 33258 (Cunha and Vanderslice, 1984) could be used to verify that any morphological or functional changes that occurred in the epithelium of the tissue recombinants was due to inductive actions of the mesenchyme on the heterospecific epithelium and not due to contaminating homospecific epithelial cells. Tissue recombinants composed of rat SVM+0-to 60-day-old mouse URE (Table 1) formed histologically recognizable SV tissue (Fig. 8A). Such recombinants exhibited intense staining of the apical portion of the epithelial cells with anti-mouse SVS but not with antibodies to rat SVS (Fig. 8B –C). Staining with the Hoechst dye verified that the epithelium was mouse in origin (not illustrated). Reciprocal tissue recombinants composed of mouse SVM+rat (0 to 60 day) URE (Table 1) also differentiated into SV tissue (Fig. 8D). In this case, the epithelium was rat in origin based upon Hoechst dye staining (not illustrated), a finding corroborated by the fact that the induced SVE cells stained immunocytochemically with antibodies to rat, but not mouse, SVS proteins (Figs8E,F). Epithelial age did not influence the outcome of the experiments.

Functional cytodifferentiation of the induced URE cells was also analyzed by SDS –PAGE, Western blot and protein dot blot using antibodies specific to rat or mouse SVS proteins (Figs 7, 9 –10). Based upon the analysis of secretory proteins extracted from 43 individual tissue recombinants the SVS proteins detected were found to correspond to the species of the epithelium utilized. Thus, in rat SVM+mouse URE recombinants, the proteins characteristic of mouse SVS were detected (Figs 9,10). Likewise, in mouse SVM+rat URE recombinants, the five major rat SVS proteins were expressed. For all SVM + URE tissue recombinants analyzed by SDS –PAGE (n=11) in which neonatal epithelium was used, the entire spectrum of SVS proteins was detected by SDS –PAGE, Western blot and dot blot methods. However, when adult rat URE was utilized, rat SVS protein I was often present in exceedingly low levels as judged by SDS –PAGE (Fig. 9), even though its presence could be verified by more sensitive dot blot methods (Fig. 7).

One feature unique to SVE (not shared with URE) is the expression of androgen receptors (AR). Neither epithelial nor stromal cells of the UR exhibit nuclear concentration of ³H-DHT (Fig. 11 A). By contrast, nuclear androgen binding (indicative of AR) is a prominent feature in both the epithelium and stroma of the SV (Fig. 1 IB). When URE was induced by SVM to differentiate into SV tissue, the induced epithelium expressed nuclear ³H-DHT binding sites (Fig. 11C). This nuclear binding was abolished by coincubation with a 300-fold excess of radioinert DHT (Fig. HD).

DDE from 0-to 60-day old rats or mice (Table 1) was also induced by SVM to differentiate into SV tissue in both rat SVM+mouse DDE and mouse SVM+rat DDE recombinants (Fig. 12A,D). The species origin of the induced SVE was verified as above with Hoechst dye staining and was appropriate for the types of tissue recombinants constructed (not illustrated). The inductive effect of SVM on DDE was further corroborated by SDS –PAGE, Western blotting and through use of species-specific antibodies on tissue sections, which showed that the appropriate SVS proteins were expressed in these tissue recombinants (Figs 9,10,12B,-C,E,F). Thus, rat SVM+mouse DDE recombinants were stained with antibodies to mouse (but not rat) SVS (Fig. 12B,C), whereas mouse SVM+rat DDE recombinants were stained with antibodies to rat (but not mouse) SVS (Fig. 12E,F). Once again these immunocytochemical findings were verified by SDS–PAGE, Western blot or protein dot blot analysis of the secreted proteins (Figs 7, 9, 10). Tissue recombinants constructed with rat SVM+mouse DDE expressed the 6 major mouse SVS proteins, while the reciprocal tissue recombinants (mouse SVM+rat DDE) expressed the 5 major rat SVS proteins (Figs 7, 9, 10). The full spectrum of SVS proteins was observed in all tissue recombinants (n =23) analyzed irrespective of the initial age of the epithelium.

Mesenchyme-induced changes in epithelial differentiation described herein were examined from several standpoints: gross morphological organization, epithelial cytodifferentiation, and the expression of AR and tissue-specific secretory proteins. In SVM+URE recombinants, epithelial cytodifferentiation was radically changed from the transitional phenotype characteristic of a urothelium, which lacked AR (Cunha et al. 1980c), to a simple columnar secretory epithelium (Brandes, 1974; Price and Williams-Ashman, 1961), which expressed both AR and SVS proteins. It is likely that the expression of AR preceded, and is a prerequisite for, the production of SVS proteins. In the normal course of SV development, epithelial AR appear on 2 to 3 days postpartum (Cooke, 1988; Shima et al. 1990), whereas SVS protein synthesis begins after day 10 (Fawell and Higgins, 1986). This striking change in epithelial cytodifferentiation in SVM+URE recombinants is directly comparable to the induction of prostatic differentiation in epithelium of the urinary bladder (Cunha et al. 19806, 19836; Neubauer et al. 1983). Here, too, a stratified AR⁻ urothelium differentiated into an AR⁺ simple columnar secretory epithelium. Such profound changes in cytodifferentiation are accompanied by major reprogramming of epithelial biochemistry and functional differentiation. The induction of SV differentiation from adult DDE involves the conversion of a pseudostratified epithelium, which has prominent stereocilia, to a simple columnar epithelium lacking stereocilia. Again these changes in epithelial cytodifferentiation resulted in a complete change in functional differentiation of the epithelial cells, and the expression of SVS proteins. However, in this case the DDE already possessed AR prior to the construction of the tissue recombinants (Cooke, 1988) and presumably continued to express AR during the induction and differentiation of SV tissue.

The inductions described in this paper represent the first examples where complete functional reprogramming has occurred in adult epithelial cells. In other examples of tissue interactions in which epithelial cytodifferentiation is minimally altered, changes in functional cytodifferentiation did not take place, e.g. tissue recombinants composed of salivary gland mesenchyme+embryonic mammary epithelium (Sak-akura et al. 1976). In this case, the embryonic mammary epithelium formed branched ductal networks resembling salivary gland, but continued to produce α-lactalbumin, a major constituent of milk. In other cases, a mixed response has been obtained, e.g. in interactions between salivary gland mesenchyme and embryonic pituitary epithelium where the outcome (production of ACTH or α-amylase) is usually mixed and dependent upon the age of the epithelium (Kusakabe et al. 1985).

The experimental data in this paper show that URE and DDE from 1-to 60-day old rats and mice can be induced by neonatal SVM to undergo SV differentiation and to express the full complement of the secretory proteins characteristic of the SV. There are two possible interpretations of these findings which cannot be distinguished at present. The induction of fully functional SVE from adult URE and DDE may indicate that determined fully differentiated adult epithelial cells first dedifferentiated and then were reprogrammed to express the SV phenotype. Alternatively, these adult epithelia may contain undetermined and undifferentiated stem cells that were the source of the induced SV tissue. Based upon histological, ultrastructural and immunocytochemical observations such uncommitted embryonic-like cells would be present in adult epithelia in exceedingly small numbers so that SV differentiation would likely to be induced focally by SVM in scattered sites throughout the adult epithelia. This is not supported by preliminary time course studies with SVM+URE recombinants grown for 6, 9 and 12 days in culture which have shown that the conversion of URE (originally organized as a simple tubular structure) into the complex folded and branched SV mucosa occurred globally throughout the tissue recombinant (Cunha and Young, unpublished). Thus, the alternate mechanism, dedifferentiation and subsequent reprogramming, is favored. Since adult epithelial cells are clearly capable of expressing alternative phenotypes when associated with inductive mesenchymes, this suggests that the stability of adult epithelial differentiation in intact glandular organs must be due to ongoing influences of the adult stroma.

Differentiated adult epithelial cells in situ express qualitatively distinct histotypes and faithfully maintain their characteristic histodifferentiation even in rapidly renewing tissues. These features describe the stability of the differentiated state (Ursprung, 1968). How is this stability of the differentiated state maintained in adult epithelial cells? For some epithelia, e.g. from mammary gland and fiver, functional differentiation can be maintained by various extracellular materials without the need for living stromal cells (Blum et al. 1987; Bissel and Barcellos-Hoff, 1987; Reid and Jefferson, 1984). For other epithelia, adult stromal cells may be involved in maintaining adult epithelial differentiation as experimental recombination of adult epithelium with heterotypic stromas can lead to profound changes in adult epithelial cytodifferentiation and function (Cunha et al. 1985). The studies reported herein and many others (Bemimoulin and Schroeder, 1980; Billingham and Silvers, 1966, 1968; Briggaman, 1982; Karring et al. 1975; Mackenzie and Hill, 1984; Spearman, 1974; Cunha, 1975, 19766; Cooke et al. 1987; Cunha, 19766; Cunha et al. 19806, 19836; Neubauer et al. 1983; Daniel and DeOme, 1965; Daniel et al. 1965; Hoshino, 1967; Hoshino, 1978; Dudek and Lawrence, 1988; Norman et al. 1986; Sakagami et al. 1984) demonstrate that adult (or at least postnatal) epithelia can be induced to undergo changes in cytodifferentiation and in some cases biochemical function. While all of these studies emphasize the responsiveness of postnatal epithelial cells to inductive mesenchyme, they do not prove that adult stromal cells themselves have inductive properties. However, adult mammary stroma (the fat pad) has been shown to induce mammary development in both adult and fetal mammary epithelial cells (Daniel and DeOme, 1965; Daniel et al. 1965; Sakakura et al. 1979b), and adult vaginal stroma can induce neonatal uterine epithelium to express vaginal differentiation (Cunha, 1976b). Adult dermal cells possess region-specific inductive activities (Billingham and Silvers, 1966, 1968; Briggaman, 1982; Bemimoulin and Schroeder, 1980; Karring et al. 1975; Mackenzie and Hill, 1984; Spearman, 1974). Moreover, epidermal appendages such as feather and hair are induced to form and grow by cells of the adult dermal papillae (Ibrahim and Wright, 1977; Jahoda et al. 1984; Lillie and Wang, 1943; Oliver, 1968; Wang, 1943). More recently it has been shown that neonatal uterine and vaginal mesenchymes, following culture for 1 to 2 months, retain the ability to instructively and permissi-vely induce responsive epithelia (Cooke et al. 1987). Thus, adult stromal cells do seem capable of functioning either as permissive or instructive inductors.

The chemical mediators of stromal effects upon adult epithelial growth and differentiation have not yet been defined even though growth-promoting activities have been described in conditioned medium from mammary fibroblasts (Enami et al. 1983; Howard et al. 1976; Kawamura et al. 1986) and adult comeal fibroblasts (Chan and Haschke, 1983). Based upon data reported herein and by others, mesenchymal effects on epithelial cells are not species specific (Cunha et al. 1983c; Fukamachi et al. 1986; Haffen et al. 1983; Kedinger et al. 1981; Kollar and Fisher, 1980; Lacroix et al. 1984), which suggests that the mediators of these cell – cell interactions are highly conserved in higher vertebrates. Previously characterized growth factors such as EGF, TGF α, TGF β, FGF and KGF are likely candidates. Other evidence suggests that extracellular matrix is certainly involved in maintenance of epithelial differentiation (Bissell and Barcellos-Hoff, 1987; Blum et al. 1987; Reid and Jefferson, 1984; Michalopoulos and Pitot, 1975; Chen and Bissell, 1989).

These striking mesenchyme-induced changes in adult epithelial differentiation and function have important implications for understanding the etiology of abnormal cellular proliferation including carcinogenesis. In humans, benign prostatic hyperplasia and prostatic adenocarcinoma are common proliferative lesions of the genital tract (Coffey et al. 1987; McNeal, 1983). Benign prostatic hyperplasia is thought to be due to a reawakening of inductive activity of prostatic stroma cells (McNeal, 1978), an idea that has received a certain degree of experimental support (Cunha et al. 1987). Likewise, mesenchymal-epithelial interactions have been implicated in the genesis and modulation of carcinomas (Cooper and Pinkus, 1977; DeCosse et al. 1973, 1975; Fujii et al. 1982; Hodges et al. WIT, Mackenzie et al. 1979). Of particular interest is the recent report that SVM can modify ductal morphogenesis and elicit secretory cytodifferentiation in carcinoma cells of the Dunning prostatic adenocarcinoma (Hayashi et al. 1990). Similarly, digestive tract mesenchymes from fetal rats can elicit glandular morphogenesis and differentiation in human colon carcinoma cells (Fukamachi et al. 1986, 1987). Since most carcinomas arise postnatally, ongoing epithelial-stromal interactions in adulthood may play an important role in the differentiation, proliferation and malignant properties of emerging carcinomas.

This work was supported by NIH grants: HD 21919, DK 32157 and HD 17491.

 
• SVM=

  seminal vesicle mesenchyme

 
• URE=

  ureter epithelium

 
• DDE=

  ductus deferens epithelium

 
• SVS=

  seminal vesicle secretion

 
• AR=

  androgen receptors

 
• SV=seminal

  vesicle

 
• UR=

  ureter

 
• DD=ductus

  deferens

 
• IgG=

  immunoglobulin

 
• PBS=

  phosphate-buffered saline

 
• SDS=

  sodium dodecylsulfate

 
• DHT=

  dihydrotestosterone.