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Specificity of tissue interaction and origin of mesenchymal cells in the androgen response of the embryonic mammary gland
Cell, Vol. 19. 465-471,
February
1980.
Copyright
0 1980 by MIT
Specificity of Tissue Interaction and Origin of Mesenchymal Cells in the Androgen Response of the Embryonic Mammary Gland Heideiinde Durnberger and Klaus Kratochwii institut fur Molekularbiologie der osterreichischen Akademie der Wissenschaften Biiirothstrasse 11 A-5020 Saizburg, Austria
Summary in the androgen-induced destruction of the mammary rudiments of 14 day male mouse fetuses, the hormone acts directly only on the mesenchyme, which then condenses around the epitheiiai gland buds and-in some unknown way-causes their necrosis. in this paper we report that an organspecific but not species-specific influence of mammary epitheiium on the surrounding mesenchyme is required to allow its response to the hormone. This epitheiiai “signal” has a very short range; its transmission may depend on contact between the two tissues. The requirement for epithelial contact, however, may only exist for those mesenchymai ceils that initiate the reaction at the tissue interface, whereas the hormone must act on ail the ceils that eventually form the mesenchymal condensation. Mesenchyme of the mammary region only is competent to produce this testosterone response. All the mesenchymai ceils required for the reaction are already present at the epitheiiai-surface at least 2 days before the hormone response occurs, and our experiments exclude the participation of any ceils that could have arrived at the mammary bud through migration from more distant sites. introduction The mammary buds of male mouse fetuses are partially or completely destroyed on days 14 and 15 of gestation. The mesenchyme surrounding the epitheiiai gland buds condenses and the “stalk” of the gland stretches and finally ruptures, the gland epithelium thereby becoming separated from the epidermis. Depending on the strain of mice and the position of the gland, most or ail ceils of the gland epitheiium become necrotic, whereas adjacent epidermis remains unaffected (Raynaud and Raynaud, 1953; P. Schwartz, manuscript in preparation). it has been shown that this destruction is the result of a direct action of androgenic hormones on the rudiment (Kratochwil, 1971), and experimental tissue combinations, using the androgen-insensitive Tfm mutant (Lyon and Hawkes, 19701, have allowed the identification of the mesenchyme as the target tissue for the hormone (Kratochwil and Schwartz, 1976; Drews and Drews, 1977). -We therefore conclude that mammary epithelium is affected (and destroyed) by testosterone-activated mesenchymal ceils rather than by the
hormone directly, and that the androgen response of the mammary rudiment thus represents an instance of hormone-induced tissue interaction. in this paper we analyzed further this interaction between mammary mesenchyme and epitheiium. Since our present tentative model of testosterone action in the gland implies possibly target-specific cell-killing, we were interested in two questions. The first concerns the specificity of this tissue interaction: do testosterone-responsive mesenchymal cells “recognize” mammary epitheiium as their target in the androgen reaction? On the other hand, can the presence of mammary epitheiium provoke a similar testosterone response in nonmammary mesenchyme? The second question concerns the origin of the mesenchymal ceils in the characteristic condensation around the epitheiiai bud: is this structure formed solely by the cells lining the epitheiium or does it arise through an accumulation of (specialized?) ceils that migrate from more distant sites to the mammary bud in response to the hormone? This paper demonstrates that the epithelio-mesenchymai interaction in the androgen response of the mammary gland is strictly organ-specific, while lacking species specificity, and that no mesenchymal ceils from distant sites take part in the reaction. Results Epitheliai and Mesenchymai Specificity “Recognition” of Mammary Epithelium In 13 day embryos, mammary and nonmammary (dermai) mesenchyme cannot be distinguished on a morphological basis. Nevertheless, testosterone causes the formation of very conspicuous mesenchymal condensations exclusively around the mammary buds. We therefore investigated whether the presence of mammary epithelium is required in addition to the hormone for the mesenchymai reaction. The degree of specificity of this requirement was tested in experimental tissue combinations by replacing mammary epithelium with other epithelia-either epithelia of other organ rudiments of the mouse (11 day pancreas, 12 day lung, 13 day salivary gland) or mammary epitheiium of other species, rat and rabbit. The two species were selected because their mammary rudiments differ in their responsiveness to testosterone: while the rat gland reacts in a way similar to that of the mouse (Neumann and Eiger, 1966; our own observations), no sexual dimorphism is seen in embryonic rabbit glands, and we have found them to be totally unresponsive even to high levels of testosterone in vitro (our unpublished observations). Experimental associations of 12 day mammary epithelia with 12 day mesenchyme of the mammary region yielded explants responding characteristically to testosterone in more than two thirds of all cases
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(Table 1; see also Kratochwil and Schwartz, 1976). As shown below, this response did not depend on precise association of the epithelium with those mesenchymal cells that had been in contact with the mammary bud before tissue separation. We could therefore consider the entire mesenchyme of the mammary region as competent for the response, and we have checked this by placing 12 day mammary epithelia (usually 2-5) close to the test epithelium. Trypsin-isolated pancreas epithelia grew well on mammary mesenchyme developing characteristic acini. The mesenchymal cells did not show any obvious orientation towards these epithelia as they did with (simultaneously explanted) mammary epithelia. Addition of testosterone had no detectable effect on the mesenchyme lining pancreas epithelium, whereas the characteristic condensations were formed around mammary epithelia (Table 1). Even when pancreatic and mammary epithelia had fused, the reaction of the mesenchyme was strictly limited to the mammary portion of the compound epithelium (Figure 1). Epithelial necrosis was also restricted to mammary cells, even in fused epithelia. The same results were obtained with lung and salivary epithelia. Although these epithelia are known to be more specific in their mesenchymal requirement (Grobstein, 1967), they were well maintained on mammary mesenchyme for the length of the experiments (3-4 days in culture). Testosterone had no effect on the mesenchyme lining these epithelia, while the typical condensations were formed again around co-explanted mammary epithelia (Table 1, Figure 2). Table 1. Epithelial Specificity of the Androgen Response Reaction of Mammary Mesenchyme of 12 Day Mouse Embryos When Combined with Epithelia of Various Sources Mesenchymal (Condensation)
Source
of Epithelium
With Testosterone
Reaction
In Controls a
12 day mouse
mammary
a (148/l
11 day mouse
pancreas
o/12 (O/l 5)
O/5 (O/7)
12 day mouse
lung
o/12 (O/l 7)
O/Q (O/l 6)
13 day mouse
salivary
o/11 (O/l 7)
O/l 1 (O/l 5)
13 day rat mammary
15/21 (29/61)
O/25 (O/47)
15 day rabbit
Q/13 f42/74)
o/11 (O/35)
mammary
The mammary epithelia of 13 day rat embryos proved to be difficult to grow on mouse mesenchyme: of a total of 345 experimental combinations, only 108 glands were present after 3 days in culture. Therefore, only histological sections of explants in good condition were considered. Despite the limited material, it became evident that mouse mammary mesenchyme did form the characteristic testosterone-induced condensations around rat epithelia (Figure 3). The same positive result was obtained with 15 day rabbit mammary epithelia, which, in contrast to rat glands, developed well on mouse mesenchyme (Figure 4, Table 1). Mesenchymal Competence Mesenchymes from various nonmammary sources, as well as mammary mesenchyme from rat and rabbit embryos, were tested for their capacity to respond to testosterone when combined with trypsin-isolated 12 day mouse mammary epithelium. As mentioned, association of these epithelia with 12 day mouse mammary mesenchyme produced a typical response in most explants. It appeared that mesenchyme of the entire mammary region was competent, as condensations were equally observed when the epithelia were deliberately placed at some distance from the (former) position of the gland buds in the mesenchyme. The same combinations were done with deeper layers of “mammary” mesenchyme, which was obtained after removing the uppermost layers of the dermis with the skin. In addition, the mesenchyme was turned upside down before combining it with mammary epithelia. This loose mesenchyme, predominantly constituting the prospective fat pad of the gland, allowed only poor maintenance and development of mammary epithelia; only 76 out of 204 epi-
79)
(O/l
32)
Upper fjgures for each combination represent glands scored in histological sections. The lower figures in parentheses give the number of all cultures observed as living explants. Cultures were maintained for at least 3 days. Testosterone concentration was 0.1 FM. * The histology of the response in these combinations was known from an earlier study (Kratochwil and Schwartz, 1976).
Figure 1. 11 Day Pancreas and 12 Day Mammary Epithelium Placed on 12 Day Mammary Mesenchyme Where the Two Epithelia Fused But Remained Distinguishable Due to Different Staining Properties The black line marks the border between the two epithelia. Testosterone caused the formation of a mesenchymal condensation around mammary epithelium only. 3 days in culture with 0.1 pM testosterone.
Cell Interaction 467
in Androgen
Response
of Mammary
Gland
Figure 2. 13 Day Salivary (Left) and 12 Day Mammary Placed on 12 Day Mammary Mesenchyme
Epithelium
Again, the two different epithelia had fused (the black line marking the border). Testosterone caused a mesenchymal condensation only around mammary epithelium. One part of it is about to be pinched off from the rest of the epithelium -a process that occurs in the normal androgen response of mammary glands.
Figure 4. A Combination 12 Day Mouse Mammary 3 Days
of 15 Day Rabbit Mammary Epithelium Mesenchyme. Exposed to Testosterone
A typical mesenchymal condensation rabbit mammary epithelium became gland.
Figure 3. A Combination of 13 Day Rat Mammary Epithelium (with Epidermis) with 12 Day Mouse Mammary Mesenchyme Exposed to Testosterone for 3 Days As in the male mouse gland, the mesenchymal condensation is about to separate the mammary bud from the epidermis. Deformation of the mammary epithelium (lower left) is also very typical for the normal testosterone response.
thelia could be scored after 3 days in vitro. Nevertheless, testosterone-treated cultures formed characteristic mesenchymal condensations around mammary epithelia, whereas no such reaction was seen in control explants (Table 2). Maintenance of epithelial mammary buds was even inferior on dermis of the shoulder region (“nonmammary” dermis), although the development of hair rudiments in regular patterns indicated an otherwise successful epidermal-dermal recombination. Most gland buds (401 of 504) flattened and could no longer be recognized after 2 or 3 days in vitro. No mesenchymal condensations could be observed in living
with for
has formed and a piece of separated from the rest of the
explants exposed to testosterone, yet a very weak indication of a mesenchymal reaction was found in 12 out of 34 glands examined histologically. Since our experiments above had shown that rabbit mammary epithelium can be substituted for mouse tissue, and since the rabbit gland was found to be much more resistant to the procedures used for tissue separation, we used rabbit mammary epithelia as additional “probes” to test the competence of shoulder dermis. Of 117 such combinations, 62 could be scored, 32 of which were exposed to testosterone. Only in two explants was some mesenchymal condensation seen, and this could also be verified in histological sections. These condensations, however, were by far less massive than those formed by mesenchyme of the mammary region associated with rabbit glands. No effect of testosterone could be observed in combinations of mammary epithelia with pancreatic, pulmonary or salivary mesenchyme (Table 2). None of these mesenchymes allowed typical mammary development, but the epithelium was well maintained on pancreas mesenchyme. Mouse mammary epithelia grew very poorly in association with mammary mesenchyme of 13 day rat and of 15 day rabbit embryos, and therefore only a few explants could be scored from a large number of experiments. In combinations involving rat mesenthyme, the addition of testosterone caused the formation of mesenchymal condensations around the epithelium. By contrast, all combinations with rabbit mesenchyme remained entirely unaffected by the hormone.
Cell 468
Origin of the Cells in the Mesenchymal Condensation We have approached this question by studying the testosterone response of glands surrounded by mesenchyme containing mixed populations of androgensensitive (normal) and androgen-insensitive cells. Use was made of the Tfrn mutant (Lyon and Hawkes, 1970), which renders affected cells insensitive to androgenic steroids-presumably by specifying a nonfunctional androgen receptor (Gehring, Tomkins and Ohno, 1971; Attardi and Ohno, 1974). We had found before that mammary rudiments of Xrfm/Y embryos (hemizygous for Tfm) are insensitive even to high levels (10 PM) of testosterone (Kratochwil and Schwartz, 1976). Androgen Response of Experimental XT’“/ YWild-Type Mesenchymal Combinations In one series of experiments, combinations between tissues of Xrfm/Y embryos on one hand, and of normal
embryos on the other, were prepared in such a way that mammary epithelia with very few adhering mesenchymal cells of one type (androgen-insensitive or normal, respectively) were placed on large amounts of mesenchyme of the opposite type, thereby creating a situation in which the mesenchyme at the epitheliomesenchymal interface differed in its sensitivity to testosterone from the more distant mesenchyme (see the schematic presentation in Figure 5). When the few mesenchymal cells at the epithelial interface were of normal genotype (as in Figure 5A), a typical response occurred. In almost all (104 out of 108) of such combinations done on day 13, mesenchymal condensations were formed and the gland buds separated from the epidermis. Histological sections also revealed the disappearance of the basal lamina and epithelial necrosis-both consistent fea-
Table 2. Mesenchymal Specificity of the Androgen Response: Reaction Provoked by the Presence of Mammary Epithelium in Mesenchyme from Various Sources (a) Combinations
with 12 day Mouse
Mammary
Mesenchymal (Condensation)
Epithelium Reaction
With Testosterone
In Controls
12 day mouse mammary superficial layers
’ (140/
B
12 day mouse mammary deeper layers
11/12
Source
of Mesenchyme
179)
(O/132)
W/41)
O/5 (O/35) O/8 (O/53)
12 day mouse
shoulder
0/34b (O/50)
11 day mouse
pancreas
O/l 1
o/12
(O/20)
(O/28)
12 day mouse
lung
O/16 (O/35)
o/10 (O/40)
13 day mouse
salivary
o/10
o/9 (O/27)
(O/26) 13 day rat mammary 15 day rabbit (b) Combinations
mammary
5/7 (22/36)
O/6
o/11 (O/35)
o/2 (O/20)
with 15 Day Rabbit
(O/32)
Mammary
Mesenchymal (Condensation) Source
of Mesenchyme
12 day mouse 13 day mouse
shoulder salivary
Epithelium Reaction
With Testosterone
In Controls
2/10
W32)
o/7 (O/30)
o/9 (O/22)
o/11 (O/28)
See legend for Table 1. a Histology done in Kratochwil and Schwartz (1976). b In 12 of these glands we observed an orientation of the mesenchymal cells differing from that in control sections, but no condensation.
Figure 5. Highly Schematic Presentation of the Situation Created in Our Reciprocal Combinations of Normal, Androgen-Sensitive (Dark Cells) and of Tfm Mutant (Androgen-Insensitive) Tissues Skin carrying the mammary buds was stripped from the underlying mesenchyme so that only few mesenchymal cells adhered to mammary epithelium (as shown in Figure 6). and was placed on mesenthyme of the opposite genotype. In both combinations, the mesenthyme at the epithelial surface differed in its androgen sensitivity from the distant mesenchyme. (The genotype of the epithelium is of no relevance for the response: Kratochwil and Schwartz, 1976). Our experiments have shown that combination A responds to tastosterone, whereas combination S does not.
Cell Interaction 469
in Androgen
Response
of Mammary
Gland
tures of the androgen response in vivo (P. Schwartz, manuscript in preparation). Even when explanted without additional mesenchyme, such rudiments were responsive. The same result (17 positive explants out of 20) was obtained when the combination was done on day 12-that is, 2 days before the androgen response is occurring in vivo. In the reciprocal experiments, where only the few mesenchymal cells at the epithelial surface were androgen-insensitive, all the others being of normal genotype (Figure 5B), no indication of a response was seen in 61 associations involving 13 day tissues, and 35 combinations done with 12 day tissues were equally unresponsive. 12 of these were studied histologically, and in only one of them a small patch of mesenchymal cells was seen with an arrangement reminiscent of a mesenchymal condensation. Sections of mechanically separated glands revealed that small areas of the epithelial surface can be completely denuded of any adhering mesenchymal ceils (Figure 6). In these places, the epithelium presumably can come in direct contact with the normal, androgensensitive mesenchyme when the tissues are combined at explantation. Androgen Response of XT’“/X’ Heterozygous Mammary Rudiments In these experiments, we have taken advantage of the mesenchymal heterogeneity (with respect to androgen sensitivity) that prevails without any experimental manipulation in XTfm/X+ heterozygous female embryos. As the Tfm locus is on the X chromosome, the random occurrence but stable inheritance of X chromosome inactivation (Lyon, 1961) results in the establishment of individual clones that are either fully responsive or entirely unresponsive to testosterone (Drews and Alonso-Lozano, 1974; Ohno, Geller and Kan, 1974a; Ohno, Geller and Young Lai, 1974b). We expected that the effect of testosterone on these clones and their distribution with respect to the epithelium might yield information on how the mesenchymal condensation forms during the androgen response. A total of 468 gland rudiments derived from 59 female 13 day embryos of XT‘“’ carrier mothers were explanted and exposed to testosterone. Approximately half of these embryos (31-that is, 52.5%) yielded glands that all (a total of 244) responded typically to the hormone. These embryos were assumed to be of X+/X’ genotype. The gland rudiments gathered from the other 28 embryos (224 glands) exhibited a weak and atypical response to testosterone. It was found that all the glands from one particular embryo responded in a rather uniform fashion. In living explants, the most frequent observation was the formation of only small mesenchymal condensations which did not surround the entire gland bud, and deformation of the gland epithelia. In a number of glands no mesenchymal
lel Figure 6. A Piece of 13 Day Mouse bination Experiments of Figure 5
Skin As Prepared
It can be seen that the surface of the mammary mesenchymal cells in limited areas.
for the Com-
epithelium
is free of
condensation could be observed in culture. Although the gland epithelia always remained connected to the epidermis, no nipple sheath was seen to develop. Histological examination of 57 such glands revealed that the epithelial buds were surrounded by highly heterogeneous mesenchyme: small patches of densely packed cells interspersed in apparently unaffected loose mesenchyme (Figure 7). Sections of some glands revealed very small mesenchymal condensations not observed in the living state. The mesenchymal condensations were always in contact with mammary epithelium, but their cells showed no tendency to displace the loose mesenchyme from the epithelial surface. At the site of such a mesenchymal condensation the gland epithelium was frequently indented, resulting in the deformation of the epithelial bud seen in living explants. Although the buds never separated from the epidermis as a whole, groups of epithelial cells were detached from the rest of the gland in areas of larger mesenchymal condensations. In these places no epithelial basal lamina could be detected, while it was only discontinuous in other regions. We have found only a few necrotic epithelial cells, and their distribution did not correlate with the position of the mesenchymal condensations. Discussion Our experimental tissue combinations have shown that an androgen response specifically requires both mammary epithelium and mammary mesenchyme. The “recognition” of the target epithelium by testosterone-activated mesenchyme is organ-specific but not species-specific, thereby sharing the characteristics of all known instances of embryonic (morphogenetic) tissue interactions (Grobstein, 1967). Mammary mesenchyme-but not the epithelium-must be derived from a species that shows an androgen response in vivo (mouse and rat), suggesting that the
Cdl 470
Figure 7. A Mammary Rudiment of a Presumed Xrtnr/X+ Heterozygous Female Embryo after 3 Days in Culture with Testosterone A patch of condensed mesenchyme has formed (at the left). whereas the remaining mesenchyme appears unaffected. We assume that the condensation represents a clone expressing the X+. the other mesenchyme being unresponsive due to XTrm activity.
failure of the rabbit gland to respond to testosterone is due to its lack of mesenchymal competence. We have found no evidence for migration of specialized mesenchymal cells to the mammary bud in response to the hormone. Obviously, all the mesenchymal cells required for the reaction on day 14 are already situated at the epithelial surface 2 days before (experiment of Figure 5A), and no mesenchymal cells from slightly more distant sites become attracted to the epithelial bud (experiment of Figure 58). Finally, if the mesenchymal condensations arose through migration of responding cells to the mammary bud, the X+-expressing clones would have had to displace the nonresponsive mesenchyme from the epithelial surface in XTfm/X’ heterozygous glands. The actual distribution of the densely packed clones in these glands indicated that the condensation process around the mammary bud is not caused by the epithelium attracting the mesenchymal cells, but rather by increased adhesiveness between the mesenchymal cells themselves: as seen in Figure 7, the condensed mesenthyme (presumably an X+ phenoclone) is in contact with the epithelium but does not spread over its surface. Since the reaction of the mesenchyme could only be initiated at the epithelial surface, we assume that
these mesenchymal cells require two “signals”-testosterone and close proximity to mammary epithelium. As shown, the influence of mammary epithelium is organ-specific, and apparently has a very short range, which again is typical for organogenetic tissue interactions (Grobstein, 1967). At this stage, we do not yet know whether the mesenchymal cells must be exposed to this epithelial influence at the time of hormone action or even before. As the mesenchymal condensation in vivo eventually reaches a considerable thickness, we consider it improbable that all of its cells had contact with the epithelium. While the need for epithelial contact may therefore exist only for the mesenchymal cells initiating the reaction, the hormone must be acting on all cells of the condensation, as shown by the inability of androgen-insensitive clones to take part in the reaction of adjacent X+ phenoclones in XT’m/X+ heterozygous glands. While we have found that all XT’“/X’ heterozygous glands responded to testosterone, but never to the full extent of normal rudiments, Ohno et al. (1974a) reported that the mammary glands of Xrfm/X+ males (sex-reversed by the dominant autosomal Sxr allele) were affected either fully or not at all. Most interestingly, in many animals all the glands were affected in the same way. This result could have been explained if the destruction of the mammary rudiments was mediated by a mobile cell population, not adhering to clonal borders, the overall composition of which would decide the fate of all mammary glands of an individual. Our experiments directed at this question have clearly ruled out this possibility. We think that the reason for the apparent discrepancy in our results lies merely in the criteria used for the androgen response. Ohno et al. (1974a) have scored the presence or absence of nipples in newborn animals. Nipple formation can be observed in explanted mammary rudiments (Kratochwil, 1969) but it was conspicuously absent in the testosterone-treated XTfm/X’ glands of this study. By the criteria of Ohno et al. (1974a), therefore, our “incompletely responding” glands would all have been scored as pure “male.” The large number of “female” glands found by the previous authors can be explained by the much higher proportion of XTfmexpressing (nonresponsive) clones in their material: in addition to the Tfm mutation, their X chromosome also carried an extreme mutant allele (0”“) of Cattanach’s “controlling element” site that causes preferential activity (approximately 80%) of this X in (Oh”)/(O’) heterozygotes (Cattanach, 1970; Ohno et al., 1973; Drews et al., 1974). Despite the fact that the testosterone-induced tissue interaction in the embryonic mammary rudiment eventually involves target-specific cell killing, its characteristics-as studied in this paper-are those of typical morphogenetic tissue interactions. We have found no resemblance to cytotoxic processes that require participation of blood-borne or other mobile cells.
27:
Interaction
Experimental
in Androgen
Response
of Mammary
Gland
Procedure0
Drews. U.. Blecher, S. R.. Owen, 0. A. and Ohno. S. (1974). Genetically directed preferential X-activation seen in mice. Cell 7, 3-6.
Animals Normal mouse embryos were obtained by mating BALB/c females with C3Hf males, the day of plug detection designated as day 0 of pregnancy. Androgen-sensitive mammary as well as all nonmammary mouse tissues were derived from such embryos. Androgen-insensitive embryos were from Xr’“/X’ carrier mothers, the Xr’“’ chromosome being maintained in our mouse colony derived from three pairs donated by M. F. Lyon. Embryos with testes can be either X+/Y (normal) or Xrm/Y (androgen-insensitive). The genotype was determined for each embryo by exposing at least three test mammary glands to the hormone. Their female littermates are either X+/X+ or Xr‘“‘/X+ heterozygotes. and no other X-linked genes allow ready distinction between these two genotypes in early 13 day embryos. The testosterone response of the mammary glands from such embryos, however, clearly fell into two categories, and therefore we assume that they could be classified by this criterion (see Results). Rats and rabbits were from local dealers and were mated in our laboratory. Explantation Procedures All mammary tissues were explanted on day 12 or 13. well before the gland enters its testosterone-responsive phase on day 14 (Kratochwil. 1977). Details of the culture procedure are given in previous papers (Kratochwil. 1969. 1971). Clean epithelio-mesenchymal separation was achieved after incubation in a 3% trypsin-pancreatin solution (Grobstein, 1953). When mammary buds with few adhering mesenchymal cells were needed, the skin of the embryos was stripped from the underlying mesenchyme (the result is seen in Figure 6). Glands and experimental combinations were cultured at the medium-gas interface on a filter or agar film spanning a hole in a stainless steel grid. In both situations, the development of the explants could be monitored in transmitted light. The culture medium was 60% Eagle’s MEM. 10% horse serum (both from Flow laboratories, Irvine. Scotland), 10% embryo extract from 9 day chick embryos. Glutamine (final concentration 2 mM) and Penicillin-Streptomycin (each 50 U/ ml) were added. Testosterone was applied at 0.1 AM concentration, approximately 100 times the minimal effective concentration. Histological Procedures Cultures were fixed in 3% glutaraldehyde (in Millonig’s buffer). postfixed with 2% 0~0, and embedded in Epon. Semithin (I Am) sections were stained with toluidine blue. Acknowledgments This work was supported by e contract with the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact. Received
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Kratochwil, K. and Schwartz, P. (1976). Tissue interaction in endrogen response of embryonic mammary rudiment of mouse: identification of target tissue for testosterone. Proc. Nat. Acad. Sci. USA 73, 4041-4044.
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Copyright
0 1980 by MIT
Specificity of Tissue Interaction and Origin of Mesenchymal Cells in the Androgen Response of the Embryonic Mammary Gland Heideiinde Durnberger and Klaus Kratochwii institut fur Molekularbiologie der osterreichischen Akademie der Wissenschaften Biiirothstrasse 11 A-5020 Saizburg, Austria
Summary in the androgen-induced destruction of the mammary rudiments of 14 day male mouse fetuses, the hormone acts directly only on the mesenchyme, which then condenses around the epitheiiai gland buds and-in some unknown way-causes their necrosis. in this paper we report that an organspecific but not species-specific influence of mammary epitheiium on the surrounding mesenchyme is required to allow its response to the hormone. This epitheiiai “signal” has a very short range; its transmission may depend on contact between the two tissues. The requirement for epithelial contact, however, may only exist for those mesenchymai ceils that initiate the reaction at the tissue interface, whereas the hormone must act on ail the ceils that eventually form the mesenchymal condensation. Mesenchyme of the mammary region only is competent to produce this testosterone response. All the mesenchymai ceils required for the reaction are already present at the epitheiiai-surface at least 2 days before the hormone response occurs, and our experiments exclude the participation of any ceils that could have arrived at the mammary bud through migration from more distant sites. introduction The mammary buds of male mouse fetuses are partially or completely destroyed on days 14 and 15 of gestation. The mesenchyme surrounding the epitheiiai gland buds condenses and the “stalk” of the gland stretches and finally ruptures, the gland epithelium thereby becoming separated from the epidermis. Depending on the strain of mice and the position of the gland, most or ail ceils of the gland epitheiium become necrotic, whereas adjacent epidermis remains unaffected (Raynaud and Raynaud, 1953; P. Schwartz, manuscript in preparation). it has been shown that this destruction is the result of a direct action of androgenic hormones on the rudiment (Kratochwil, 1971), and experimental tissue combinations, using the androgen-insensitive Tfm mutant (Lyon and Hawkes, 19701, have allowed the identification of the mesenchyme as the target tissue for the hormone (Kratochwil and Schwartz, 1976; Drews and Drews, 1977). -We therefore conclude that mammary epithelium is affected (and destroyed) by testosterone-activated mesenchymal ceils rather than by the
hormone directly, and that the androgen response of the mammary rudiment thus represents an instance of hormone-induced tissue interaction. in this paper we analyzed further this interaction between mammary mesenchyme and epitheiium. Since our present tentative model of testosterone action in the gland implies possibly target-specific cell-killing, we were interested in two questions. The first concerns the specificity of this tissue interaction: do testosterone-responsive mesenchymal cells “recognize” mammary epitheiium as their target in the androgen reaction? On the other hand, can the presence of mammary epitheiium provoke a similar testosterone response in nonmammary mesenchyme? The second question concerns the origin of the mesenchymal ceils in the characteristic condensation around the epitheiiai bud: is this structure formed solely by the cells lining the epitheiium or does it arise through an accumulation of (specialized?) ceils that migrate from more distant sites to the mammary bud in response to the hormone? This paper demonstrates that the epithelio-mesenchymai interaction in the androgen response of the mammary gland is strictly organ-specific, while lacking species specificity, and that no mesenchymal ceils from distant sites take part in the reaction. Results Epitheliai and Mesenchymai Specificity “Recognition” of Mammary Epithelium In 13 day embryos, mammary and nonmammary (dermai) mesenchyme cannot be distinguished on a morphological basis. Nevertheless, testosterone causes the formation of very conspicuous mesenchymal condensations exclusively around the mammary buds. We therefore investigated whether the presence of mammary epithelium is required in addition to the hormone for the mesenchymai reaction. The degree of specificity of this requirement was tested in experimental tissue combinations by replacing mammary epithelium with other epithelia-either epithelia of other organ rudiments of the mouse (11 day pancreas, 12 day lung, 13 day salivary gland) or mammary epitheiium of other species, rat and rabbit. The two species were selected because their mammary rudiments differ in their responsiveness to testosterone: while the rat gland reacts in a way similar to that of the mouse (Neumann and Eiger, 1966; our own observations), no sexual dimorphism is seen in embryonic rabbit glands, and we have found them to be totally unresponsive even to high levels of testosterone in vitro (our unpublished observations). Experimental associations of 12 day mammary epithelia with 12 day mesenchyme of the mammary region yielded explants responding characteristically to testosterone in more than two thirds of all cases
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(Table 1; see also Kratochwil and Schwartz, 1976). As shown below, this response did not depend on precise association of the epithelium with those mesenchymal cells that had been in contact with the mammary bud before tissue separation. We could therefore consider the entire mesenchyme of the mammary region as competent for the response, and we have checked this by placing 12 day mammary epithelia (usually 2-5) close to the test epithelium. Trypsin-isolated pancreas epithelia grew well on mammary mesenchyme developing characteristic acini. The mesenchymal cells did not show any obvious orientation towards these epithelia as they did with (simultaneously explanted) mammary epithelia. Addition of testosterone had no detectable effect on the mesenchyme lining pancreas epithelium, whereas the characteristic condensations were formed around mammary epithelia (Table 1). Even when pancreatic and mammary epithelia had fused, the reaction of the mesenchyme was strictly limited to the mammary portion of the compound epithelium (Figure 1). Epithelial necrosis was also restricted to mammary cells, even in fused epithelia. The same results were obtained with lung and salivary epithelia. Although these epithelia are known to be more specific in their mesenchymal requirement (Grobstein, 1967), they were well maintained on mammary mesenchyme for the length of the experiments (3-4 days in culture). Testosterone had no effect on the mesenchyme lining these epithelia, while the typical condensations were formed again around co-explanted mammary epithelia (Table 1, Figure 2). Table 1. Epithelial Specificity of the Androgen Response Reaction of Mammary Mesenchyme of 12 Day Mouse Embryos When Combined with Epithelia of Various Sources Mesenchymal (Condensation)
Source
of Epithelium
With Testosterone
Reaction
In Controls a
12 day mouse
mammary
a (148/l
11 day mouse
pancreas
o/12 (O/l 5)
O/5 (O/7)
12 day mouse
lung
o/12 (O/l 7)
O/Q (O/l 6)
13 day mouse
salivary
o/11 (O/l 7)
O/l 1 (O/l 5)
13 day rat mammary
15/21 (29/61)
O/25 (O/47)
15 day rabbit
Q/13 f42/74)
o/11 (O/35)
mammary
The mammary epithelia of 13 day rat embryos proved to be difficult to grow on mouse mesenchyme: of a total of 345 experimental combinations, only 108 glands were present after 3 days in culture. Therefore, only histological sections of explants in good condition were considered. Despite the limited material, it became evident that mouse mammary mesenchyme did form the characteristic testosterone-induced condensations around rat epithelia (Figure 3). The same positive result was obtained with 15 day rabbit mammary epithelia, which, in contrast to rat glands, developed well on mouse mesenchyme (Figure 4, Table 1). Mesenchymal Competence Mesenchymes from various nonmammary sources, as well as mammary mesenchyme from rat and rabbit embryos, were tested for their capacity to respond to testosterone when combined with trypsin-isolated 12 day mouse mammary epithelium. As mentioned, association of these epithelia with 12 day mouse mammary mesenchyme produced a typical response in most explants. It appeared that mesenchyme of the entire mammary region was competent, as condensations were equally observed when the epithelia were deliberately placed at some distance from the (former) position of the gland buds in the mesenchyme. The same combinations were done with deeper layers of “mammary” mesenchyme, which was obtained after removing the uppermost layers of the dermis with the skin. In addition, the mesenchyme was turned upside down before combining it with mammary epithelia. This loose mesenchyme, predominantly constituting the prospective fat pad of the gland, allowed only poor maintenance and development of mammary epithelia; only 76 out of 204 epi-
79)
(O/l
32)
Upper fjgures for each combination represent glands scored in histological sections. The lower figures in parentheses give the number of all cultures observed as living explants. Cultures were maintained for at least 3 days. Testosterone concentration was 0.1 FM. * The histology of the response in these combinations was known from an earlier study (Kratochwil and Schwartz, 1976).
Figure 1. 11 Day Pancreas and 12 Day Mammary Epithelium Placed on 12 Day Mammary Mesenchyme Where the Two Epithelia Fused But Remained Distinguishable Due to Different Staining Properties The black line marks the border between the two epithelia. Testosterone caused the formation of a mesenchymal condensation around mammary epithelium only. 3 days in culture with 0.1 pM testosterone.
Cell Interaction 467
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Response
of Mammary
Gland
Figure 2. 13 Day Salivary (Left) and 12 Day Mammary Placed on 12 Day Mammary Mesenchyme
Epithelium
Again, the two different epithelia had fused (the black line marking the border). Testosterone caused a mesenchymal condensation only around mammary epithelium. One part of it is about to be pinched off from the rest of the epithelium -a process that occurs in the normal androgen response of mammary glands.
Figure 4. A Combination 12 Day Mouse Mammary 3 Days
of 15 Day Rabbit Mammary Epithelium Mesenchyme. Exposed to Testosterone
A typical mesenchymal condensation rabbit mammary epithelium became gland.
Figure 3. A Combination of 13 Day Rat Mammary Epithelium (with Epidermis) with 12 Day Mouse Mammary Mesenchyme Exposed to Testosterone for 3 Days As in the male mouse gland, the mesenchymal condensation is about to separate the mammary bud from the epidermis. Deformation of the mammary epithelium (lower left) is also very typical for the normal testosterone response.
thelia could be scored after 3 days in vitro. Nevertheless, testosterone-treated cultures formed characteristic mesenchymal condensations around mammary epithelia, whereas no such reaction was seen in control explants (Table 2). Maintenance of epithelial mammary buds was even inferior on dermis of the shoulder region (“nonmammary” dermis), although the development of hair rudiments in regular patterns indicated an otherwise successful epidermal-dermal recombination. Most gland buds (401 of 504) flattened and could no longer be recognized after 2 or 3 days in vitro. No mesenchymal condensations could be observed in living
with for
has formed and a piece of separated from the rest of the
explants exposed to testosterone, yet a very weak indication of a mesenchymal reaction was found in 12 out of 34 glands examined histologically. Since our experiments above had shown that rabbit mammary epithelium can be substituted for mouse tissue, and since the rabbit gland was found to be much more resistant to the procedures used for tissue separation, we used rabbit mammary epithelia as additional “probes” to test the competence of shoulder dermis. Of 117 such combinations, 62 could be scored, 32 of which were exposed to testosterone. Only in two explants was some mesenchymal condensation seen, and this could also be verified in histological sections. These condensations, however, were by far less massive than those formed by mesenchyme of the mammary region associated with rabbit glands. No effect of testosterone could be observed in combinations of mammary epithelia with pancreatic, pulmonary or salivary mesenchyme (Table 2). None of these mesenchymes allowed typical mammary development, but the epithelium was well maintained on pancreas mesenchyme. Mouse mammary epithelia grew very poorly in association with mammary mesenchyme of 13 day rat and of 15 day rabbit embryos, and therefore only a few explants could be scored from a large number of experiments. In combinations involving rat mesenthyme, the addition of testosterone caused the formation of mesenchymal condensations around the epithelium. By contrast, all combinations with rabbit mesenchyme remained entirely unaffected by the hormone.
Cell 468
Origin of the Cells in the Mesenchymal Condensation We have approached this question by studying the testosterone response of glands surrounded by mesenchyme containing mixed populations of androgensensitive (normal) and androgen-insensitive cells. Use was made of the Tfrn mutant (Lyon and Hawkes, 1970), which renders affected cells insensitive to androgenic steroids-presumably by specifying a nonfunctional androgen receptor (Gehring, Tomkins and Ohno, 1971; Attardi and Ohno, 1974). We had found before that mammary rudiments of Xrfm/Y embryos (hemizygous for Tfm) are insensitive even to high levels (10 PM) of testosterone (Kratochwil and Schwartz, 1976). Androgen Response of Experimental XT’“/ YWild-Type Mesenchymal Combinations In one series of experiments, combinations between tissues of Xrfm/Y embryos on one hand, and of normal
embryos on the other, were prepared in such a way that mammary epithelia with very few adhering mesenchymal cells of one type (androgen-insensitive or normal, respectively) were placed on large amounts of mesenchyme of the opposite type, thereby creating a situation in which the mesenchyme at the epitheliomesenchymal interface differed in its sensitivity to testosterone from the more distant mesenchyme (see the schematic presentation in Figure 5). When the few mesenchymal cells at the epithelial interface were of normal genotype (as in Figure 5A), a typical response occurred. In almost all (104 out of 108) of such combinations done on day 13, mesenchymal condensations were formed and the gland buds separated from the epidermis. Histological sections also revealed the disappearance of the basal lamina and epithelial necrosis-both consistent fea-
Table 2. Mesenchymal Specificity of the Androgen Response: Reaction Provoked by the Presence of Mammary Epithelium in Mesenchyme from Various Sources (a) Combinations
with 12 day Mouse
Mammary
Mesenchymal (Condensation)
Epithelium Reaction
With Testosterone
In Controls
12 day mouse mammary superficial layers
’ (140/
B
12 day mouse mammary deeper layers
11/12
Source
of Mesenchyme
179)
(O/132)
W/41)
O/5 (O/35) O/8 (O/53)
12 day mouse
shoulder
0/34b (O/50)
11 day mouse
pancreas
O/l 1
o/12
(O/20)
(O/28)
12 day mouse
lung
O/16 (O/35)
o/10 (O/40)
13 day mouse
salivary
o/10
o/9 (O/27)
(O/26) 13 day rat mammary 15 day rabbit (b) Combinations
mammary
5/7 (22/36)
O/6
o/11 (O/35)
o/2 (O/20)
with 15 Day Rabbit
(O/32)
Mammary
Mesenchymal (Condensation) Source
of Mesenchyme
12 day mouse 13 day mouse
shoulder salivary
Epithelium Reaction
With Testosterone
In Controls
2/10
W32)
o/7 (O/30)
o/9 (O/22)
o/11 (O/28)
See legend for Table 1. a Histology done in Kratochwil and Schwartz (1976). b In 12 of these glands we observed an orientation of the mesenchymal cells differing from that in control sections, but no condensation.
Figure 5. Highly Schematic Presentation of the Situation Created in Our Reciprocal Combinations of Normal, Androgen-Sensitive (Dark Cells) and of Tfm Mutant (Androgen-Insensitive) Tissues Skin carrying the mammary buds was stripped from the underlying mesenchyme so that only few mesenchymal cells adhered to mammary epithelium (as shown in Figure 6). and was placed on mesenthyme of the opposite genotype. In both combinations, the mesenthyme at the epithelial surface differed in its androgen sensitivity from the distant mesenchyme. (The genotype of the epithelium is of no relevance for the response: Kratochwil and Schwartz, 1976). Our experiments have shown that combination A responds to tastosterone, whereas combination S does not.
Cell Interaction 469
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Response
of Mammary
Gland
tures of the androgen response in vivo (P. Schwartz, manuscript in preparation). Even when explanted without additional mesenchyme, such rudiments were responsive. The same result (17 positive explants out of 20) was obtained when the combination was done on day 12-that is, 2 days before the androgen response is occurring in vivo. In the reciprocal experiments, where only the few mesenchymal cells at the epithelial surface were androgen-insensitive, all the others being of normal genotype (Figure 5B), no indication of a response was seen in 61 associations involving 13 day tissues, and 35 combinations done with 12 day tissues were equally unresponsive. 12 of these were studied histologically, and in only one of them a small patch of mesenchymal cells was seen with an arrangement reminiscent of a mesenchymal condensation. Sections of mechanically separated glands revealed that small areas of the epithelial surface can be completely denuded of any adhering mesenchymal ceils (Figure 6). In these places, the epithelium presumably can come in direct contact with the normal, androgensensitive mesenchyme when the tissues are combined at explantation. Androgen Response of XT’“/X’ Heterozygous Mammary Rudiments In these experiments, we have taken advantage of the mesenchymal heterogeneity (with respect to androgen sensitivity) that prevails without any experimental manipulation in XTfm/X+ heterozygous female embryos. As the Tfm locus is on the X chromosome, the random occurrence but stable inheritance of X chromosome inactivation (Lyon, 1961) results in the establishment of individual clones that are either fully responsive or entirely unresponsive to testosterone (Drews and Alonso-Lozano, 1974; Ohno, Geller and Kan, 1974a; Ohno, Geller and Young Lai, 1974b). We expected that the effect of testosterone on these clones and their distribution with respect to the epithelium might yield information on how the mesenchymal condensation forms during the androgen response. A total of 468 gland rudiments derived from 59 female 13 day embryos of XT‘“’ carrier mothers were explanted and exposed to testosterone. Approximately half of these embryos (31-that is, 52.5%) yielded glands that all (a total of 244) responded typically to the hormone. These embryos were assumed to be of X+/X’ genotype. The gland rudiments gathered from the other 28 embryos (224 glands) exhibited a weak and atypical response to testosterone. It was found that all the glands from one particular embryo responded in a rather uniform fashion. In living explants, the most frequent observation was the formation of only small mesenchymal condensations which did not surround the entire gland bud, and deformation of the gland epithelia. In a number of glands no mesenchymal
lel Figure 6. A Piece of 13 Day Mouse bination Experiments of Figure 5
Skin As Prepared
It can be seen that the surface of the mammary mesenchymal cells in limited areas.
for the Com-
epithelium
is free of
condensation could be observed in culture. Although the gland epithelia always remained connected to the epidermis, no nipple sheath was seen to develop. Histological examination of 57 such glands revealed that the epithelial buds were surrounded by highly heterogeneous mesenchyme: small patches of densely packed cells interspersed in apparently unaffected loose mesenchyme (Figure 7). Sections of some glands revealed very small mesenchymal condensations not observed in the living state. The mesenchymal condensations were always in contact with mammary epithelium, but their cells showed no tendency to displace the loose mesenchyme from the epithelial surface. At the site of such a mesenchymal condensation the gland epithelium was frequently indented, resulting in the deformation of the epithelial bud seen in living explants. Although the buds never separated from the epidermis as a whole, groups of epithelial cells were detached from the rest of the gland in areas of larger mesenchymal condensations. In these places no epithelial basal lamina could be detected, while it was only discontinuous in other regions. We have found only a few necrotic epithelial cells, and their distribution did not correlate with the position of the mesenchymal condensations. Discussion Our experimental tissue combinations have shown that an androgen response specifically requires both mammary epithelium and mammary mesenchyme. The “recognition” of the target epithelium by testosterone-activated mesenchyme is organ-specific but not species-specific, thereby sharing the characteristics of all known instances of embryonic (morphogenetic) tissue interactions (Grobstein, 1967). Mammary mesenchyme-but not the epithelium-must be derived from a species that shows an androgen response in vivo (mouse and rat), suggesting that the
Cdl 470
Figure 7. A Mammary Rudiment of a Presumed Xrtnr/X+ Heterozygous Female Embryo after 3 Days in Culture with Testosterone A patch of condensed mesenchyme has formed (at the left). whereas the remaining mesenchyme appears unaffected. We assume that the condensation represents a clone expressing the X+. the other mesenchyme being unresponsive due to XTrm activity.
failure of the rabbit gland to respond to testosterone is due to its lack of mesenchymal competence. We have found no evidence for migration of specialized mesenchymal cells to the mammary bud in response to the hormone. Obviously, all the mesenchymal cells required for the reaction on day 14 are already situated at the epithelial surface 2 days before (experiment of Figure 5A), and no mesenchymal cells from slightly more distant sites become attracted to the epithelial bud (experiment of Figure 58). Finally, if the mesenchymal condensations arose through migration of responding cells to the mammary bud, the X+-expressing clones would have had to displace the nonresponsive mesenchyme from the epithelial surface in XTfm/X’ heterozygous glands. The actual distribution of the densely packed clones in these glands indicated that the condensation process around the mammary bud is not caused by the epithelium attracting the mesenchymal cells, but rather by increased adhesiveness between the mesenchymal cells themselves: as seen in Figure 7, the condensed mesenthyme (presumably an X+ phenoclone) is in contact with the epithelium but does not spread over its surface. Since the reaction of the mesenchyme could only be initiated at the epithelial surface, we assume that
these mesenchymal cells require two “signals”-testosterone and close proximity to mammary epithelium. As shown, the influence of mammary epithelium is organ-specific, and apparently has a very short range, which again is typical for organogenetic tissue interactions (Grobstein, 1967). At this stage, we do not yet know whether the mesenchymal cells must be exposed to this epithelial influence at the time of hormone action or even before. As the mesenchymal condensation in vivo eventually reaches a considerable thickness, we consider it improbable that all of its cells had contact with the epithelium. While the need for epithelial contact may therefore exist only for the mesenchymal cells initiating the reaction, the hormone must be acting on all cells of the condensation, as shown by the inability of androgen-insensitive clones to take part in the reaction of adjacent X+ phenoclones in XT’m/X+ heterozygous glands. While we have found that all XT’“/X’ heterozygous glands responded to testosterone, but never to the full extent of normal rudiments, Ohno et al. (1974a) reported that the mammary glands of Xrfm/X+ males (sex-reversed by the dominant autosomal Sxr allele) were affected either fully or not at all. Most interestingly, in many animals all the glands were affected in the same way. This result could have been explained if the destruction of the mammary rudiments was mediated by a mobile cell population, not adhering to clonal borders, the overall composition of which would decide the fate of all mammary glands of an individual. Our experiments directed at this question have clearly ruled out this possibility. We think that the reason for the apparent discrepancy in our results lies merely in the criteria used for the androgen response. Ohno et al. (1974a) have scored the presence or absence of nipples in newborn animals. Nipple formation can be observed in explanted mammary rudiments (Kratochwil, 1969) but it was conspicuously absent in the testosterone-treated XTfm/X’ glands of this study. By the criteria of Ohno et al. (1974a), therefore, our “incompletely responding” glands would all have been scored as pure “male.” The large number of “female” glands found by the previous authors can be explained by the much higher proportion of XTfmexpressing (nonresponsive) clones in their material: in addition to the Tfm mutation, their X chromosome also carried an extreme mutant allele (0”“) of Cattanach’s “controlling element” site that causes preferential activity (approximately 80%) of this X in (Oh”)/(O’) heterozygotes (Cattanach, 1970; Ohno et al., 1973; Drews et al., 1974). Despite the fact that the testosterone-induced tissue interaction in the embryonic mammary rudiment eventually involves target-specific cell killing, its characteristics-as studied in this paper-are those of typical morphogenetic tissue interactions. We have found no resemblance to cytotoxic processes that require participation of blood-borne or other mobile cells.
27:
Interaction
Experimental
in Androgen
Response
of Mammary
Gland
Procedure0
Drews. U.. Blecher, S. R.. Owen, 0. A. and Ohno. S. (1974). Genetically directed preferential X-activation seen in mice. Cell 7, 3-6.
Animals Normal mouse embryos were obtained by mating BALB/c females with C3Hf males, the day of plug detection designated as day 0 of pregnancy. Androgen-sensitive mammary as well as all nonmammary mouse tissues were derived from such embryos. Androgen-insensitive embryos were from Xr’“/X’ carrier mothers, the Xr’“’ chromosome being maintained in our mouse colony derived from three pairs donated by M. F. Lyon. Embryos with testes can be either X+/Y (normal) or Xrm/Y (androgen-insensitive). The genotype was determined for each embryo by exposing at least three test mammary glands to the hormone. Their female littermates are either X+/X+ or Xr‘“‘/X+ heterozygotes. and no other X-linked genes allow ready distinction between these two genotypes in early 13 day embryos. The testosterone response of the mammary glands from such embryos, however, clearly fell into two categories, and therefore we assume that they could be classified by this criterion (see Results). Rats and rabbits were from local dealers and were mated in our laboratory. Explantation Procedures All mammary tissues were explanted on day 12 or 13. well before the gland enters its testosterone-responsive phase on day 14 (Kratochwil. 1977). Details of the culture procedure are given in previous papers (Kratochwil. 1969. 1971). Clean epithelio-mesenchymal separation was achieved after incubation in a 3% trypsin-pancreatin solution (Grobstein, 1953). When mammary buds with few adhering mesenchymal cells were needed, the skin of the embryos was stripped from the underlying mesenchyme (the result is seen in Figure 6). Glands and experimental combinations were cultured at the medium-gas interface on a filter or agar film spanning a hole in a stainless steel grid. In both situations, the development of the explants could be monitored in transmitted light. The culture medium was 60% Eagle’s MEM. 10% horse serum (both from Flow laboratories, Irvine. Scotland), 10% embryo extract from 9 day chick embryos. Glutamine (final concentration 2 mM) and Penicillin-Streptomycin (each 50 U/ ml) were added. Testosterone was applied at 0.1 AM concentration, approximately 100 times the minimal effective concentration. Histological Procedures Cultures were fixed in 3% glutaraldehyde (in Millonig’s buffer). postfixed with 2% 0~0, and embedded in Epon. Semithin (I Am) sections were stained with toluidine blue. Acknowledgments This work was supported by e contract with the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact. Received
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