When grown as renal grafts in adult male hosts, the upper (cranial), middle and lower (caudal) portions of fetal mouse and rat Wolffian ducts developed into epididymis, epididymis plus ductus deferens, and seminal vesicle, respectively. In heterotypic tissue recombinants, the epithelia from upper and middle Wolffian ducts were instructively induced to undergo seminal vesicle morphogenesis by neonatal seminal vesicle mesenchyme. Functional cytodifferentiation was examined in these recombinants using antibodies against major androgen-dependent, seminal vesicle-specific secretory proteins. The instructively induced Wolffian duct epithelia synthesized normal amounts of all of the secretory proteins characteristic of mature seminal vesicles, as judged by immunocytochemistry on tissue sections and gel electrophoresis plus immunoblotting of secretions extracted from the recombinants. In heterospecific recombinants composed of rat and mouse tissues, the seminal vesicle proteins induced were specific for the species that had provided the epithelium. This showed that the seminal vesicle epithelium in the recombinants was derived from instructively induced Wolffian duct epithelium and not from epithelial contamination of the mesenchymal inductor. Upper Wolffian duct epithelium, instructively induced to undergo seminal vesicle morphogenesis, did not express epididymis-specific secretory proteins, showing that its normal development had been simultaneously repressed.

Heterotypic tissue recombinants, in which epithelium and mesenchyme from different embryonic organs are combined, have provided a particularly important and versatile means for investigating the essential role of reciprocal epithelial-mesenchymal interactions in glandular organogenesis (reviewed in Cunha, 1976a; Cunha et al. 1980a, 1983a, 1985, 1987; Kedinger et al. 1986a; Haffen et al. 1987). In many heterotypic recombinants, a variety of instructive inductions have been reported in which the mesenchyme reprogrammed the normal developmental fate of the epithelium.

However, most instructive inductions have been characterized purely on a morphological basis (Cunha et al. 1983a, 1985,1987; Kedinger et al. 1986b; Haffen et al. 1987). While the induced change in epithelial differentiation is unambiguous in some inductions, in others the epithelium has responded in a heterogeneous fashion or, while clearly abandoning its normal developmental fate, has developed in a somewhat indeterminate manner (Cunha, 1972a,b,c, 1976b; Matsushita, 1984; Yasugi, 1984; Lacroix et al. 1985).

Several groups have also addressed the crucial question of whether functional cytodifferentiation accompanies instructively induced changes in epithelial morphogenesis. In some cases, this does not seem to have been the case. For instance, while mammary gland epithelium was induced by salivary gland mesenchyme to undergo the pattern of branching morphogenesis characteristic of salivary gland (Kratochwil, 1969), the epithelium retained its simple columnar cytodifferentiation and synthesized the milk protein, α-lactalbumin (Sakakura et al. 1976). Chick epidermis underwent mammary gland morphogenesis with mouse mammary gland mesenchyme but still became keratinized (Propper, 1969). A similar finding was reported in tissue recombinants constructed with salivary gland mesenchyme and palatal epithelium (Tyler & Koch, 1977). The gastric glands that were induced in quail chorioallantoic epithelium by chick proventricular mesenchyme failed to express pepsinogen (Yasugi & Matsushita, 1982; Yasugi, 1984). In contrast, functional cytodifferentiation did appear to accompany morphogenesis in many other cases. Examples include the induction of (i) disaccharidases and other epithelial brush border enzymes by intestinal mesenchymes (Masui, 1982; Ishizuya-Oka & Mizuno, 1984; Lacroix et al. 1984; Matsushita, 1984), (ii) region-specific keratins by integumental mesenchymes (Dhouailly et al. 1978; Sawyer et al. 1984), (iii) tooth markers by tooth mesenchyme (Kollar & Baird, 1970; Slavkin et al. 1984), (iv) lens crystallins by the optic vesicle (Karkinen-Jaaskelainen, 1978) and (v) prostatic proteins and androgen receptors by urogenital sinus mesenchyme (Cunha et al. 1980c, 19836; Neubauer et al. 1983). However, even in these cases where functional cytodifferentiation did appear to have occurred, the expression of marker proteins was often quantitatively lower than for normal tissue or there were differences in ontogeny or response to hormones (Neubauer et al. 1983; Lacroix et al. 1984; Kedinger et al. 1986b). Rarely has a full complement of functionally diagnostic markers been examined following an instructive induction, so that induced changes in epithelial function may only represent partial expression of a new phenotype. Furthermore, the possibility that the presumed induced epithelium had arisen from contamination of the mesenchyme by its homotypic epithelium, rather than from instructive induction, has not always been rigorously excluded. Finally, only a few instructive inductions have been screened for the expression of functional markers diagnostic of the tissue that would normally have developed from the epithelium (Dhouailly et al. 1978; Masui, 1982; Neubauer et al. 1983; Sawyer et al. 1984; Kusakabe et al. 1985). Thus, it has not been possible to exclude a phenotypically mixed developmental response in most heterotypic recombinants.

Many of the problems associated with the systems mentioned above could be circumvented by using the seminal vesicles (SV) of the male rodent as a model system. As summarized in the accompanying paper (Higgins et al. 1989), the SV of the sexually mature rat have a highly characteristic morphology and produce large amounts of several tissue-specific secretory proteins (Higgins et al. 1976; Ostrowski et al. 1979; Fawell & Higgins, 1986), for which immunological (Fawell et al. 1986, 1987) and nucleotide probes (McDonald et al. 1983; Williams et al. 1983) are available. Another major advantage of SV over many other glandular organs including the prostate is that the SV epithelium (SVE) is not regionally specialized into distinct lobes but is functionally homogeneous, each epithelial cell apparently synthesizing all of the major SV secretory (SVS) proteins (Fawell & Higgins, 1986; Aumuller & Seitz, 1986). The SV of the mouse are very similar to rat SV and share the advantages described above with regard to both morphological and functional markers (Chen et al. 1987; Fawell et al. 1987; Higgins et al. 1989). Of considerable value is the fact that SVS proteins of the rat and mouse are electrophoretically and immunologically distinct (Fawell et al. 1987; Higgins et al. 1989) and thus can be distinguished in interspecies (heterospecific) tissue recombinants as described in the accompanying paper (Higgins et al. 1989). Being rudimentary at birth, the SV are also particularly suitable for the construction of tissue recombinants since the undifferentiated SV mesenchyme (SVM) and SVE of neonatal SV may be readily and cleanly separated. Finally, since the SV are androgen-dependent, the growth and development of SV tissue recombinants may be manipulated via the host’s androgen status.

The SV develop during late fetal life from the lower portion of the paired Wolffian ducts (WD). The upper and middle portions of the WD are the progenitors of the epididymis and ductus deferens, respectively, each of which is structurally and functionally distinct and quite different from the SV. Nevertheless, the epithelia of the upper and middle WD (WDE) are embryologically closely related to SVE, so it seems highly likely that they might be instructively induced to undergo SV morphogenesis by SVM. Whether this is accompanied by SV functional cytodifferentiation could be definitively explored by examining the expression of the tissue-specific SV marker proteins. Furthermore, since rat epididymis also synthesizes tissue-specific androgendependent secretory proteins (Brooks & Higgins, 1980; Brooks & Tiver, 1983; Brooks et al. 1986), for which antibody probes are available, the effect of SVM on the normal functional development of upper WDE (prospective epididymis) can also be checked.

Inductions involving SV and ductus deferens tissue have been described previously (Cunha, 1972a,b,c, 1976b) but none have been analysed functionally. The accompanying paper describes methods for exploring functional SV cytodifferentiation as applied to homotypic SV recombinants (Higgins et al. 1989). Here we use those methods to show that WDE from prospective epididymis and ductus deferens may be instructively induced by neonatal SVM to form fully functional SV tissue.

The sources and details of all materials and methods used in this study will be found in the preceding paper (Higgins et al. 1989) unless specifically mentioned here.

Construction of tissue recombinants

Balb/c mice and Sprague-Dawley rats were used. Seminal vesicles were obtained from neonatal males 0–48 h post partum. Fetal male mice and rats (15th and 16th day of gestation, respectively) were obtained from timed pregnant females (day 0, detection of vaginal plug). Fetal reproductive systems were dissected in Dulbecco’s modified Eagle’s medium under a binocular dissecting microscope. Wolffian ducts were divided into upper (cranial), middle and lower (caudal) regions as indicated in Fig. 1. For some recombinants, the upper and middle portions of the WD were not separated from each other. Epithelia and mesenchymes were separated by microdissection after trypsinization at 4°C and then recombined in vitro in various heterotypic (WD plus SV) and heterospecific (rat plus mouse) combinations as described in the Results section. Tissue recombinants were grafted under the renal capsule of adult male athymie (‘nude’) mice and grown in vivo for 4–5 weeks.

Fig. 1.

Fetal male reproductive tract. Whole-mount preparation of the reproductive tract from a fetal male mouse (15th day gestation) showing testes (T), Mullerian ducts (M), urogenital sinus (U) and WD (W). At this stage, the lower part of the WD shows a characteristic expansion (SV) from which the SV will later develop. On the right, the white lines mark the sites at which the WD was separated from the testis and divided into upper (cranial), middle and lower (caudal) portions (see text). Bar, 500 μm.

Fig. 1.

Fetal male reproductive tract. Whole-mount preparation of the reproductive tract from a fetal male mouse (15th day gestation) showing testes (T), Mullerian ducts (M), urogenital sinus (U) and WD (W). At this stage, the lower part of the WD shows a characteristic expansion (SV) from which the SV will later develop. On the right, the white lines mark the sites at which the WD was separated from the testis and divided into upper (cranial), middle and lower (caudal) portions (see text). Bar, 500 μm.

Analysis of tissue recombinants and WD rudiments

Tissues were fixed with paraformaldehyde, paraffin embedded and sectioned (6 μm) for histological staining with haematoxylin and eosin or Hoechst dye 33258. In a few instances, tissues were embedded in methyl methacrylate and sectioned at 2 μm.

Immunocytochemistry

Paraffin-embedded tissue sections were screened with antibodies against mouse or rat SVS proteins (anti-mouse SVS, anti-rat SVS protein IV or anti-rat SVS protein V) as described (Higgins et al. 1989). Epididymal cytodifferentiation was assessed using polyclonal goat antibodies (IgG fraction) monospecific for rat epididymal sperm-binding glycoproteins B/C and D/E (anti-epididymal protein B/C and anti-epididymal protein D/E, respectively) (Brooks & Higgins, 1980; Brooks & Tiver, 1983; Brooks et al. 1986), generously donated by the late David Brooks, University of Adelaide, South Australia. Each was diluted 1:1000 in phosphate-buffered saline containing 5 % bovine serum albumin before use. The secondary antibody was biotinylated rabbit anti-goat (IgG) from Amersham Corp. (Arlington Heights, IL). Bound antibodies were visualized using an avidin-biotinylated horseradish peroxidase detection system.

Secretory proteins

These were solubilized in sodium dodecylsulphate (SDS) and analysed by polyacrylamide gel electrophoresis in the presence of SDS (SDS–PAGE). Their identities were confirmed immunologically by Western blotting with anti-mouse SVS or by immunodot blotting with antibodies against the individual rat SVS and epididymal proteins as described (Higgins et al. 1989).

Immunocytochemistry of normal epididymal and SV tissue

The specificities of the antibody probes to be used in this study were first checked. Normal rat and mouse epididymis gave identical results in immunocytochemistry with either of the antibodies against epididymal proteins B/C and D/E (Fig. 2A,B). The epithelial cells of most ducts exhibited strong staining of the supranuclear zone, known to contain secretory vesicles and the Golgi complex (Brandes, 1974). Luminal secretion and sperm were also stained. However, a minority of ducts showed background staining or else only the luminal surface of the epithelial cells was stained (not shown). Neither of these antibodies cross-reacted with either rat or mouse SV tissue (Fig. 2C) nor was there any positive staining of rat or mouse epididymal tissue by any of the antibodies against rat or mouse SVS proteins (Fig. 2D).

Fig. 2.

Immunocytochemistry of normal tissues. (A) Mouse caput epididymis with anti-epididymal protein B/C; (B) rat caput epididymis with anti-epididymal protein D/E. In both, note staining of apical regions of epithelial cells, luminal secretion and sperm. (C) Mouse SV with anti-epididymal protein B/C; (D) mouse caput epididymis with anti-mouse SVS. In both, note background staining. Bars, 25 μm.

Fig. 2.

Immunocytochemistry of normal tissues. (A) Mouse caput epididymis with anti-epididymal protein B/C; (B) rat caput epididymis with anti-epididymal protein D/E. In both, note staining of apical regions of epithelial cells, luminal secretion and sperm. (C) Mouse SV with anti-epididymal protein B/C; (D) mouse caput epididymis with anti-mouse SVS. In both, note background staining. Bars, 25 μm.

Development of Wolffian duct rudiments

Fig. 1 shows the reproductive tract from a male mouse on the 15th day of gestation, equivalent in the rat to the 16th day. At this stage, the WD are undifferentiated and their epithelia may be readily separated from the surrounding mesenchymes by treatment with trypsin. The lower (caudal) part of the WD shows a characteristic epithelial expansion marking the future site where the SV rudiment will bud off later in gestation (17th day) (Lung & Cunha, 1981). This expansion was used to delineate the lower WD for the purpose of constructing recombinants. The rest of the WD was divided into upper (cranial) and middle portions as shown in Fig. 1.

The normal developmental fates of these portions of mouse and rat WD were confirmed by growing them as renal grafts in adult male hosts for 4 weeks. The results are summarized in Table 1. Histologically, the grafts of upper WD formed normal epididymis. Sections revealed multiple duct-like structures which contained a weakly eosinophilic secretion and were lined by a columnar epithelium whose apical surface was covered by stereocilia. Functional cytodifferentiation had accompanied morphogenesis. With either anti-epididymal protein B/C or D/E, both rat and mouse upper WD rudiments gave the same pattern of strong immunocytochemical staining as normal epididymal tissue (not shown). In SDS–PAGE, proteins extracted from upper WD grafts (Fig. 3, lane 17) resembled those secreted by normal caput epididymis (Fig. 3, lane 19). Immunodot blots were used to confirm the presence of proteins B/C and D/E in extracts of upper WD grafts just as in normal epididymal secretions (Fig. 4).

Table 1.

Development of Wolffian duct rudiments and tissue recombinants grown as renal grafts in male hosts

Development of Wolffian duct rudiments and tissue recombinants grown as renal grafts in male hosts
Development of Wolffian duct rudiments and tissue recombinants grown as renal grafts in male hosts
Fig. 3.

Electrophoretic analysis of proteins secreted by WD grafts and tissue recombinants. Proteins (20–50 μg per lane) from WD grafts and tissue recombinants were separated by SDS–PAGE and stained with Coomassie blue. The identity of the sample in each of the lanes (numbered alternately) is shown on the Figure. The positions of mouse SVS proteins 1–6 (lane 13) and rat SVS proteins I–V (lane 14) are shown by the scales at the left and right, respectively; for their apparent relative molecular masses see the accompanying paper (Higgins et al. 1989). Note (i) the absence of SVS proteins in lanes 1, 7, 12, 16–20, (ii) the presence of mouse SVS proteins 1–6 in lanes 2, 4, 5, 8, 9, (iii) the presence of rat SVS proteins I–V in lanes 3, 6, 10, 11, 15, and (iv) that the rat serum sample (lane 20) also contained globin, which had the same electrophoretic mobility as mouse SVS protein 6 and rat SVS protein V.

Fig. 3.

Electrophoretic analysis of proteins secreted by WD grafts and tissue recombinants. Proteins (20–50 μg per lane) from WD grafts and tissue recombinants were separated by SDS–PAGE and stained with Coomassie blue. The identity of the sample in each of the lanes (numbered alternately) is shown on the Figure. The positions of mouse SVS proteins 1–6 (lane 13) and rat SVS proteins I–V (lane 14) are shown by the scales at the left and right, respectively; for their apparent relative molecular masses see the accompanying paper (Higgins et al. 1989). Note (i) the absence of SVS proteins in lanes 1, 7, 12, 16–20, (ii) the presence of mouse SVS proteins 1–6 in lanes 2, 4, 5, 8, 9, (iii) the presence of rat SVS proteins I–V in lanes 3, 6, 10, 11, 15, and (iv) that the rat serum sample (lane 20) also contained globin, which had the same electrophoretic mobility as mouse SVS protein 6 and rat SVS protein V.

Fig. 4.

Immunodot blotting of epididymal secretory proteins. Protein samples (2 μg in 2 μl) from WD rudiments and tissue recombinants were applied to nitrocellulose paper and probed with anti-epididymal protein B/C (A) or anti-epididymal protein D/E (B) followed by [125I]iodo-Protein A and autoradiography. The layout of the samples is given in the template (solid and open circles indicate positive and negative immunological reactions, respectively). Note the similarity in the patterns of reactivity with the two antibodies and that proteins B/C and D/E are only detected in extracts of epididymis and upper and middle WD rudiments (see text).

Fig. 4.

Immunodot blotting of epididymal secretory proteins. Protein samples (2 μg in 2 μl) from WD rudiments and tissue recombinants were applied to nitrocellulose paper and probed with anti-epididymal protein B/C (A) or anti-epididymal protein D/E (B) followed by [125I]iodo-Protein A and autoradiography. The layout of the samples is given in the template (solid and open circles indicate positive and negative immunological reactions, respectively). Note the similarity in the patterns of reactivity with the two antibodies and that proteins B/C and D/E are only detected in extracts of epididymis and upper and middle WD rudiments (see text).

Both rat and mouse middle WD grafts were heterogeneous in their development (Table 1). Structures characteristic of ductus deferens were routinely observed, i.e. ducts, some star-shaped in cross section, fined by a pseudostratified epithelium adorned with stereocilia, and surrounded by thick layers of smooth muscle. However, in other parts of these same grafts, structures resembling epididymis were found. These areas had all the histological and immunocytochemical characteristics of the epididymal tissue found in upper WD grafts, and epididymal proteins B/C and D/E were detected in the secretion of these middle WD grafts (Fig. 3, lane 16; Fig. 4). This developmental heterogeneity in middle WD rudiments implies that more than the upper third of the fetal WD is developmentally devoted to the formation of the epididymis.

While upper and middle WD grafted into the renal site unequivocally expressed their expected developmental fates, none of the 49 upper WD rudiments and 36 middle WD rudiments (Table 1) showed any signs of SV tissue, whether judged histologically or probed immunocytochemically with the appropriate antibodies against mouse or rat SVS proteins. Similarly, SVS proteins were not detected by SDS–PAGE (Fig. 3, lanes 16,17), Western blotting (Fig. 5, lanes 4,5) or immunodot blotting (Fig. 6). In contrast, lower WD rudiments (Table 1) underwent the expected morphogenesis when grown in adult male hosts for SV 3–4 weeks. Tissue resembling either epididymis or ductus deferens was not detected. Seminal vesicle functional cytodifferentiation was confirmed in these rudiments (Table 1) by the same criteria as for normal SV tissue. All of the tall columnar cells in the highly convoluted simple epithelium were stained strongly in their apical region with antibodies against rat or mouse SVS proteins, as was the luminal secretion. Stromal cells were not stained. Secretion from rat lower WD rudiments had an SDS–PAGE protein profile identical to that of normal rat SVS (Fig. 3, lanes 14 & 15). Immunodot blots confirmed that all five rat SVS proteins were present and in the same proportions as in rat SVS (Fig. 6). Analysis of the proteins secreted by mouse lower WD rudiments by SDS–PAGE revealed the presence of all six mouse SVS proteins (Table 1). Western blots with anti-mouse SVS showed strong signals for SVS proteins 3–6, with much weaker signals for proteins 1 and 2, just as in normal mouse SVS (Fig. 5, lanes 6,7). Epididymal proteins were not detected in either mouse or rat lower WD grafts (Fig. 4).

Fig. 5.

Western blotting of mouse SVS proteins secreted by WD rudiments and tissue recombinants. Secretory protein samples (20–50 fig), resolved by SDS–PAGE and transferred electrophoretically to nitrocellulose paper, were probed with anti-mouse SVS followed by [125I]iodo-Protein A and autoradiography (6h exposure). The identity of the sample in each of the lanes (numbered alternately) is indicated on the Figure, with the positions of mouse SVS proteins 1–6 shown by the scale on the left. Note the strong signals from mouse SVS proteins 3–6 in lanes 6, 9, 10, 12–14 comparable to those for mouse SVS (lane 7).

Fig. 5.

Western blotting of mouse SVS proteins secreted by WD rudiments and tissue recombinants. Secretory protein samples (20–50 fig), resolved by SDS–PAGE and transferred electrophoretically to nitrocellulose paper, were probed with anti-mouse SVS followed by [125I]iodo-Protein A and autoradiography (6h exposure). The identity of the sample in each of the lanes (numbered alternately) is indicated on the Figure, with the positions of mouse SVS proteins 1–6 shown by the scale on the left. Note the strong signals from mouse SVS proteins 3–6 in lanes 6, 9, 10, 12–14 comparable to those for mouse SVS (lane 7).

Fig. 6.

Immunodot blotting of rat SVS proteins secreted by WD rudiments and tissue recombinants. Secretory protein samples (2 μg in 2 μl except where indicated) from WD rudiments or tissue recombinants were applied to nitrocellulose paper and probed with anti-rat SVS protein I (A), anti-rat SVS protein II (B), anti-rat SVS protein III (C), anti-rat SVS protein IV (D), or anti-rat SVS protein V (E) followed by [125I]iodo-Protein A and autoradiography. The layout of the samples is given in the template (solid and open circles indicate positive and negative immunological reactions, respectively). Note the similarity in the patterns of reactivity with the five antibodies.

Fig. 6.

Immunodot blotting of rat SVS proteins secreted by WD rudiments and tissue recombinants. Secretory protein samples (2 μg in 2 μl except where indicated) from WD rudiments or tissue recombinants were applied to nitrocellulose paper and probed with anti-rat SVS protein I (A), anti-rat SVS protein II (B), anti-rat SVS protein III (C), anti-rat SVS protein IV (D), or anti-rat SVS protein V (E) followed by [125I]iodo-Protein A and autoradiography. The layout of the samples is given in the template (solid and open circles indicate positive and negative immunological reactions, respectively). Note the similarity in the patterns of reactivity with the five antibodies.

Homotypic WD recombinants

Before seeing whether the prospective developmental fates of WDE could be influenced by growth in association with neonatal SVM, we examined the development of homotypic recombinants constructed by recombining mesenchyme and epithelium from the same regions of the WD. However, only homotypic recombinants of lower WD mesenchyme (WDM) + lower WDE underwent successful morphogenesis and functional cytodifferentiation to produce SV tissue (Table 1). As with lower WD rudiments, all the major SVS proteins characteristic of normal SV were found in the secretions recovered from these homotypic lower WD recombinants (Table 1; Fig. 6). In contrast, in homotypic recombinants of upper and middle WD (i.e. upper WDM + upper WDE and middle WDM + middle WDE) grown in vivo for up to 7 weeks, WDE was not detected (Table 1). Neither SV nor epididymal proteins were detected in the extracts of these recombinants (Fig. 3, lanes 7, 12; Fig. 4; Fig. 5, lanes 8, 11; Fig. 6).

Heterotypic WD recombinants

Despite this lack of success with homotypic upper and middle WD recombinants, the developmental responses of WDE to neonatal SVM were examined. However, in such heterotypic recombinants any SV tissue that may develop could have arisen from traces of SVE remaining associated with the SVM after enzymic dissociation. In fact specimens of SVM grafted in the absence of SVE rarely showed any signs of epithelial contamination (Higgins et al. 1989) and did not contain SVS proteins (Fig. 3, lane 1; Fig. 6). To circumvent the problem entirely, rat/mouse heterospecific recombinants were used. Under these conditions, instructive reprogramming of the WDE can be readily distinguished from SVE contamination of the SVM as described in detail in the preceding paper (Higgins et al. 1989).

Permissive induction of lower WDE by neonatal SVM

Since the lower WD is the fetal progenitor of the SV (Price & Williams-Ashman, 1961; Price & Ortiz, 1965; Lung & Cunha, 1981), it was not surprising that mouse lower WDE underwent SV morphogenesis and functional cytodifferentiation in association with rat SVM (Table 1). Mouse, but not rat, SVS proteins were produced (Fig. 3, lane 2; Fig. 5, lane 14). In the reciprocal recombinant, mouse SVM + rat lower WDE, SV morphogenesis also occurred, but in this case rat SVS proteins and not those of mouse SVS were produced (Fig. 3, lane 3). These results exclude the possibility that the SVM had been contaminated with SVE, but affirm that SVM had permissively supported the normal development of the lower WDE.

Instructive reprogramming of upper and middle WDE by neonatal SVM

After 4 weeks of growth as renal grafts, 45% of SVM + upper WDE recombinants and 27 % of SVM + middle WDE recombinants had failed to grow, being recovered as small masses of undifferentiated fibromuscular tissue resembling SVM controls (Table 1). There was no evidence of either SV or epididymal secretory proteins. However, the other 55 % of SVM + upper WDE recombinants and 73 % of SVM + middle WDE recombinants had undergone considerable growth (Table 1) and were recovered engorged with a thick opaque secretion. The tissue of these recombinants showed all the histological features of normal SV (Fig. 7A,B; Fig. 8A). The highly convoluted simple epithelium was made up of tall columnar cells possessing basally located nuclei. The glandular lumen was filled with a strongly eosinophilic secretion. The proportion of SVM + WDE recombinants that showed SV morphogenesis in this study (Table 1) is similar to the success rate for instructive inductions in other systems (Cunha, 1972a,b,c;Sakakura et al. 1976; region of the epithelial cells, as well as the luminal Cunha & Lung, 1978; Fisher & Sawyer, 1979; Yasugi, 1979, 1984). In each tissue recombinant, the apical secretion, gave strong immunocytochemical staining with SV antibodies just as in mature SV. In recombinants constructed with mouse epithelium, i.e. rat SVM + mouse upper WDE or rat SVM + mouse middle WDE, the epithelium and secretion were stained strongly with anti-mouse SVS (Fig, 7D; Fig. 8C) but not with either anti-rat SVS protein IV or anti-rat SVS protein V (Fig. 7E; Fig. 8D). In contrast, where rat epithelium had been used, i.e. mouse SVM + rat upper WDE or mouse SVM + rat middle WDE, positive reactions were obtained with both antirat SVS protein IV and anti-rat SVS protein V (Fig. 9A,B) but not with anti-mouse SVS (Fig. 9C). This strongly indicates that the normal developmental programmes of the upper WDE and middle WDE had been redirected towards SVE under the influence of the neonatal SVM.

Fig. 7.

Instructive induction of SV functional cytodifferentiation in heterotypic recombinants composed of rat SVM and mouse upper WDE. (A,B) Haematoxylin and eosin; note SV morphology. (C) Hoechst dye 33258; note presence of intranuclear fluorescent foci in epithelium (arrowheads) and their absence from stroma (arrows) indicating mouse and rat cells, respectively. (D) Anti-mouse SVS; note epithelial staining. (E) Anti-rat SVS protein V; note background staining (F) Anti-epididymai protein B/C; note background staining. Paraffin-embedded tissue was used for A and D–F, while methyl methacrylate was used for B and C. Bars, 100 μm (A), 25 μm (B–F).

Fig. 7.

Instructive induction of SV functional cytodifferentiation in heterotypic recombinants composed of rat SVM and mouse upper WDE. (A,B) Haematoxylin and eosin; note SV morphology. (C) Hoechst dye 33258; note presence of intranuclear fluorescent foci in epithelium (arrowheads) and their absence from stroma (arrows) indicating mouse and rat cells, respectively. (D) Anti-mouse SVS; note epithelial staining. (E) Anti-rat SVS protein V; note background staining (F) Anti-epididymai protein B/C; note background staining. Paraffin-embedded tissue was used for A and D–F, while methyl methacrylate was used for B and C. Bars, 100 μm (A), 25 μm (B–F).

Fig. 8.

Instructive induction of SV functional cytodifferentiation in heterotypic recombinants composed of rat SVM and mouse middle WDE. (A) Haematoxylin and eosin; note SV morphology. (B) Hoechst dye 33258; note presence of intranuclear fluorescent foci in epithelium (arrowheads) and their absence from stroma (arrows). (C) Anti-mouse SVS; note epithelial staining. (D) Anti-rat SVS protein V; note background staining (arrows show erythrocytes). (E) Anti-epididymal protein B/C, note background staining. Bars, 100 μm (A), 25 μm (B –E).

Fig. 8.

Instructive induction of SV functional cytodifferentiation in heterotypic recombinants composed of rat SVM and mouse middle WDE. (A) Haematoxylin and eosin; note SV morphology. (B) Hoechst dye 33258; note presence of intranuclear fluorescent foci in epithelium (arrowheads) and their absence from stroma (arrows). (C) Anti-mouse SVS; note epithelial staining. (D) Anti-rat SVS protein V; note background staining (arrows show erythrocytes). (E) Anti-epididymal protein B/C, note background staining. Bars, 100 μm (A), 25 μm (B –E).

Fig. 9.

Instructive induction of SV functional cytodifferentiation in heterotypic recombinants composed of mouse SVM and rat upper WDE. (A,B) Anti-rat SVS protein IV and anti-rat SVS protein V, respectively; note epithelial staining. (C) Antimouse SVS; note background staining. (D) Anti-epididymal protein B/C; note background staining. Bars 25 μm.

Fig. 9.

Instructive induction of SV functional cytodifferentiation in heterotypic recombinants composed of mouse SVM and rat upper WDE. (A,B) Anti-rat SVS protein IV and anti-rat SVS protein V, respectively; note epithelial staining. (C) Antimouse SVS; note background staining. (D) Anti-epididymal protein B/C; note background staining. Bars 25 μm.

This conclusion was confirmed by analyses of the secretions recovered from the tissue recombinants. All five rat SVS proteins were present in the case of mouse SVM + rat upper WDE and mouse SVM + rat middle WDE recombinants, and in about the same proportions as in normal rat SVS (Fig. 3, lanes 6,10,11; Fig. 6). In contrast, secretions from rat SVM + mouse upper WDE and rat SVM + mouse middle WDE contained all six mouse SVS proteins as in mouse SVS (Fig. 3, lanes 4,5,8,9; Fig. 5, lanes 9,10,12,13). In none of these recombinants was there any evidence for expression of SVS proteins typical of the species that had provided the SVM, thus excluding the possibility that the SVM had been contaminated by its own epithelium.

The species origins of the epithelium and stroma in each recombinant were confirmed by the characteristic nuclear staining pattern with Hoechst dye 33258 (Cunha & Vanderslice, 1984; Higgins et al. 1989). In rat SVM + mouse upper WDE and rat SVM + mouse middle WDE recombinants, the epithelial nuclei showed fluorescent foci usually seen in murine nuclei, while the stromal nuclei were more uniformly stained as in rat tissues (Fig. 7C; Fig. 8B). The converse was seen with mouse SVM + rat upper WDE and mouse SVM + rat middle WDE recombinants (not shown).

In none of the tissue sections from these SVM + WDE recombinants (Fig. 7F, Fig. 8E, Fig. 9D), or in samples of the secretion obtained from them (Fig. 4), was there any evidence for the synthesis of epididymal proteins B/C or D/E. Admittedly, sensitive tissue labelling techniques have not been applied to search for extremely low levels of expression, but at least there was no overt sign of heterogeneous gene expression in the epithelium based on immunocytochemistry, SDS –PAGE and immunoblotting techniques. Thus, while instructively inducing SV development in WDE, the neonatal SVM had simultaneously repressed the usual developmental fate of those WD regions that normally serve as epididymal progenitors. Finally, histology and immunocytochemistry demonstrated that the newly induced functional cytodifferentiation was homogenously expressed throughout the entire recombinant.

While the SVM faithfully induced the expression of the complete range of major SVS proteins in heterotypic WDE, a SV pattern of smooth muscle development and differentiation occurred in the SVM itself (not shown). Since this was not evident in SVM controls (Higgins et al. 1989), it presumably resulted from inductive effects of the epithelium on the mesenchyme.

In the heterotypic tissue recombinants described in this paper, a battery of antibody probes has been used to show that SV morphogenesis was accompanied by normal SV functional cytodifferentiation. All of the major SV-specific secretory proteins were produced in the same relative proportions and amounts as in normal SV. Furthermore, by using heterospecific rat/mouse recombinants, the sources of the epithelial and stromal tissues present in each recombinant have been verified. In this way, any possibility that the induced SVE was derived from epithelial contamination of SVM has been unequivocally discounted.

Seminal vesicle development is clearly dependent on a specific mesenchymal-epithelial interaction in which SVM induces and specifies a characteristic morphogenetic process that leads to a functional cytodifferentiation unique to SVE and which involves the expression of gene products, notably SVS proteins, that are likewise unique to SVE. Thus SVE fails to develop unless it is associated with its homotypic mesenchyme (Cunha, 1972a; Higgins et al. 1989) and SVM can induce SVE differentiation in the epithelia from the upper and middle regions of the WD (this study). The newly induced phenotype was unequivocally that of SVE, encompassing the full complement of major SVS proteins. Moreover, in SVM and upper WDE tissue recombinants, the presumptive epididymal epithelium did not express epididymal secretory proteins, demonstrating that the SVM has the ability to repress completely one genetic programme (epididymis) while simultaneously activating an alternative (SV) programme.

The responsiveness of an epithelium to the reprogramming activities of heterotypic inductors is certainly not completely unrestricted in fetal life. Previous work stresses the importance of the germ layer (ecto-, endo- and mesoderm) origin of the epithelium as a determinant of developmental response. There are numerous examples of ectoderm being induced to express new phenotypes within the repertoire of ectoderm. Chick embryo scale and corneal epidermis have been induced to form feathers (Coulombre & Coulombre, 1971) and preputial gland epithelium has been induced to become a hair-bearing epidermis (Cunha, 19726). However, it has not yet been possible to induce epidermis to form gut or genital tract structures. Likewise, endodermal epithelia can be induced by heterotypic mesenchymes to change radically from one endodermal differentiation pattern to another within the repertoire of gut endoderm (Cunha et al. 1983a; Haffen et al. 1987; Kedinger et al. 1986a,b; Yasugi, 1979, 1984). Thus, while ectodermal and endodermal epithelia may exhibit periods in which their developmental fates can be instructively induced to change in response to certain heterotypic mesenchymes, possible developmental endpoints appear to be restricted. The same is probably true for the epithelia of the mesodermally derived WD.

While prospective epididymal and ductus deferens epithelia (upper and middle WDE) were induced by SVM to form SVE, the outcome is radically different when ectodermally or endodermally derived epithelia are associated with SVM. Thus, epidermis formed glandular structures, clearly not SV in character (Cunha, 19726), while salivary gland epithelium continued its normal development (Cunha, 1972c). The endodermally derived epithelium of the urogenital sinus (prospective prostatic epithelium) was permissively induced by SVM to express prostatic differentiation (Cunha, 1972a). Finally, the epithelium of the endodermally derived urinary bladder is induced to undergo prostatic development by SVM (Donjacour & Cunha, 1988). In considering all of the above examples, it seems that epithelia from each germ layer (or subdivision thereof) may have a specific set of developmental options (repertoire) which may be elicited by appropriate mesenchymal inductors.

The mechanism whereby SVM reprogrammed the WDE is not clear. However, since SV development is normally androgen-dependent (Price & Williams-Ashman, 1961; Lung & Cunha, 1981; Williams-Ashman, 1983), the instructive inductions by SVM reported here are probably also androgen-dependent. Hence adult male hosts were used but the effect of altering the host’s androgen status has not been examined here as has been done for some other inductions (Cunha, 1973, 19766). At least initially, the presumed effects of androgens on the epithelium must be elicited indirectly via the mesenchyme, since autoradiographic studies (Shannon & Cunha, 1983; Cooke, 1988) have shown that only mesenchymal (and not epithelial) cells of the male accessory organ rudiments exhibit nuclear uptake of radiolabelled androgen during mid-fetal stages, when their development commences under the influence of fetal testicular androgens. Only in late fetal (epididymis and ductus deferens) or early neonatal life (SV and prostate) does the epithelium express androgen receptors (Cooke, 1988; Shannon & Cunha, 1983). Furthermore, during the instructive reprogramming of adult bladder epithelium by urogenital sinus mesenchyme to form prostate, the bladder epithelium is initially devoid of androgen receptors (Cunha et al. 1980c). More conclusively, the bladder epithelium from androgeninsensitive Tfm mice, which lack functional androgen receptors (Gehring et al. 1971; Attardi & Ohno, 1974; Shannon & Cunha, 1984) and have undetectable levels of mRNA for the androgen receptor (Lubahn et al. 1988), have also been induced to form prostate by wildtype urogenital sinus mesenchyme (Cunha & Lung, 1978; Cunha et al. 1980b; Shannon & Cunha, 1984; Sugimura et al. 1986).

Using a variety of developmental systems, much effort has been devoted by others to identifying the nature of the inductive signals between mesenchyme and epithelium. Primary induction in early amphibian embryogenesis, for example the formation of mesoderm and the subsequent neuralization of ectoderm, does not require cell contact, or even living inducer cells, and so probably involves diffusible factors. Some of these have been identified as known growth factors, such as transforming growth factor-β and basic fibroblast growth factor (Kimelman & Kirschner, 1987; Kimelman et al. 1988). Later inductions, illustrated by kidney tubulogenesis (Lehtonen et al. 1975) may require physical contact between living cells. Finally the mesenchyme may control epithelial morphogenesis by the deposition and modification of the extracellular matrix (Grobstein, 1967; Bernfield et al. 1984; Perris et al. 1988).

Regardless of the exact nature of the inductive signals from the mesenchyme, these signals are highly conserved between species, since successful instructive inductions have been reported in chick/quail, mouse/chick, rat/human and rabbit/mouse tissue recombinants in a diverse set of experimental systems (Propper, 1969; Coulombre & Coulombre, 1971; Marin & Dameron, 1972; Sawyer et al. 1972; Sawyer, 1975; Hata & Slavkin, 1978; Kollar & Fisher, 1980; Masui, 1982; Cunha et al. 1983c; Yasugi, 1984; Lacroix et al. 1984, 1985). These findings, in conjunction with our own on rat/mouse SV recombinants, stress the universality of developmental mechanisms in these vertebrates and imply that the mediators of these cell-cell interactions must be highly conserved. As mentioned above, some of these may be protein growth factors encoded by genes whose nucleotide sequences show considerable conservation between species.

While the mesenchyme induces epithelial development, the epithelium is thought to have reciprocal inductive effects on the mesenchyme as emphasized by studies on the developing limb (Stark & Searls, 1974; Summerbell, 1974; Solursh et al. 1981) and kidney (Saxen et al. 1980; Ekblom, 1984). For internal organs, relatively little is known about the effects of the epithelium on mesenchymal development. In the lung (Taderera, 1967), gut (Lacroix et al. 1985) and uterus (Cunha et al. 1989), the epithelium seems to be particularly important in establishing the differentiation and spatial organization of the mesenchymally derived smooth muscle. Control grafts of neonatal SVM (Higgins et al. 1989; this study) and fetal WDM (S. J. Higgins, P. Young & G. R. Cunha, unpublished data) showed no evidence of smooth muscle differentiation. Clearly then, the characteristic spatial arrangement and abundance of smooth muscle that distinguishes epididymis, ductus deferens and SV requires the presence of, and presumably the active induction by, WDE.

The completeness and fidelity of the instructive reprogramming of the WDE by SVM, and the ease and precision with which SV functional cytodifferentiation can be assessed, mean that the SV system offers many advantages over others for the detailed study of epithelial-mesenchymal interactions in the future.

     
  • SDS

    sodium dodecylsulphate

  •  
  • SDS-PAGE

    polyacrylamide gel electrophoresis in the presence of SDS

  •  
  • SV

    seminal vesicle(s)

  •  
  • SVE

    seminal vesicle epithelium

  •  
  • SVM

    seminal vesicle mesenchyme

  •  
  • SVS

    seminal vesicle secretion

  •  
  • WD

    Wolffian duct(s)

  •  
  • WDE

    Wolffian duct epithelium

  •  
  • WDM

    Wolffian duct mesenchyme.

We thank Joel Brody and Craig Yonemura for skilled technical assistance, John Warrallo and Simona Ikeda for photography, Paula Duncan for typing the manuscript, and Steve Zippen and Scott Monroe (Center for Reproductive Endocrinology, UCSF) for the [125I]iodo-Protein A. The work reported in this paper was undertaken during the tenure (by S.J.H.) of an American Cancer Society-Eleonor Roosevelt-International Cancer Fellowship awarded by the International Union Against Cancer. Financial support was also provided by the Medical Research Council, UK (Project Grant G83/046/58CA) and the Yorkshire Cancer Research Campaign, UK (to S.J.H.), by NIH grants HD 21919 and HD 17491 (to G.R.C.) and by NIH grant HD 11979 which supports the Center for Reproductive Endocrinology.

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