Development and loss of androgen responsiveness in the embryonic rudiment of the mouse mammary gland

DEVELOPMENTAL

BIOLOGY

61,

358-365 (1977)

Development and Loss of Androgen Responsiveness in the Embryonic Rudiment of the Mouse Mammary Gland KLAUS Znstitut

fiir Molekularbiologie,

Osterreichische

KRATOCHWIL Akademie

Received July 21,1977;

der Wissenschuften,

accepted August

A,5020

Salzburg,

Austria

12,1977

The androgen-responsive phase in the development of the mammary gland was determined by exposing rudiments of various developmental stages to testosterone in vitro. Although testosterone causes destruction of the mammary epithelium in 14-day male fetuses, it failed to prevent formation of mammary buds in explanted 11-day skin. It was found that mammary rudiments become responsive to androgens only late in Day 13 of gestation, and that they are no longer responsive on Day 15 and later. Both acquisition and loss of androgen responsiveness do occur on time in explanted glands, indicating intrinsic developmental changes in the rudiment. The experiments and their results are schematically summarized in Fig. 2. INTRODUCTION

During embryonic development, some organs not only acquire specific shape (morphogenesis) and establish specific biosynthetic pathways (cellular differentiation), but they also become sensitive to, or even dependent on, exogenous regulative signals, such as hormones. Hormone responsiveness is properly regarded as a differentiative function; it requires at least the presence of specific, though mostly undefined, cellular products, the hormone receptors, and its appearance and loss in normal and pathological development could be used as a valid criterium for the differentiative state of a given cell type. In some cases, such changes in hormone responsiveness may allow us to detect developmental processes in the absence of obvious morphological or biochemical changes. We know in only a few cases the precise stage at which an organ becomes susceptible to hormonal regulation, and this seldom coincides with the time of first release of the respective hormone. Whenever an organ becomes hormone-dependent (such as the Wolfian duct on testosterone), it

can afford to do so only when sufficiently high levels of the respective hormone are already present. In contrast, some tissues have been shown to become responsive to a hormone at an early stage, well before the hormone is released. For instance, Tata (1968, 1970) has demonstrated metamorphic competence (i.e., responsiveness to thyroid hormones) in Xenopus larvae after 40-60 hr after fertilization, i.e., about 3 weeks before metamorphosis normally occurs. Acquisition of hormone responsiveness, therefore, need not coincide with the onset of actual hormonal control and must be determined independently of the latter. We have been interested in the development of androgen responsiveness in the embryonic mammary rudiment of the mouse since androgens cause the first known hormone reaction in this organ, several days before the first recorded action of lactogenic hormones (Ceriani 1970,a,b, in the rat gland). Fetal androgens are responsible for the destruction of mammary rudiments in 1Cday male fetuses (Raynaud, 1947a), and in culture it has been shown that they exert their effect directly on the gland, independently of

358 Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0012-1606

KLAUS

KRATOCHWIL

Development

other hormones and irrespective of the genetic sex type of the tissue (Kratochwil, 1971). During the reaction mesenchymal cells first condense around the epithelial bud which then separates from the epidermis and subsequently undergoes partial or complete necrosis (Raynaud and Raynaud, 1953; Schwartz, in preparation). This androgen reaction in male fetuses occurs around 36 hr after visible gonad differentiation and about 3 days after formation of the mammary rudiments from a presumably androgen-insensitive organ, the skin. At this time (in the 1Cday embryo) the gland buds are passing through a conspicuous phase of very low proliferative and virtually no morphogenetic activity, the “resting period’ lasting from the formation of the epithelial bud in 11.5day embryos until the outgrowth of the primary sprout at 16.5 days (Balinsky, 1950). The near coincidence of morphological testis differentiation (and presumed first release of testicular hormones) and of androgen response of the mammary rudiment does not allow us to determine, from observations in vivo, the precise time of acquisition of hormone responsiveness. In organ culture, however, mammary glands can be exposed to testosterone at any time of their development. As we have shown previously (Kratochwil, 19711, explanted mammary rudiments provide a suitable in vitro system for these investigations. Such rudiments respond very reliably to testosterone, not one gland in over 1500 having yet failed to respond to testosterone at concentrations of 1 ti. It is reported here (a) that the first 2 days of mammary gland development are not affected by testosterone as the gland acquires its androgen responsiveness only at around 13.5 days, and (b) that the responsive phase lasts for less than 2 days. MATERIALS

AND

METHODS

Mouse embryos were BALBc x C3Hf hybrids, the day of detection of a vaginal plug counting as Day 0 of pregnancy. Be-

of Hormone Responsiveness

359

cause of the normal variation in the developmental stage between litters of the same chronological age, or even within one litter, additional morphological criteria were used for staging the embryos more precisely. This was most difficult in 11-day embryos, in which case the extent of eye pigmentation was taken as an indicator. Thirteen-day embryos were compared by the degree of branching of their submandibular salivary gland; the sublingual gland could be used for lkday embryos as well as the extent of humeral ossification. Embryos that did not fall within close limits of developmental variation were eliminated. The gland rudiments of the entire litter were pooled before explantation, and each experiment was done with glands of one litter only. The explantation procedure for 12-day or older rudiments was as described previously (Kratochwil, 1969). Wherever the sex type of the donor embryo could be determined (12.5-day embryos and older), only the glands from female embryos were used to avoid possible pre-exposure to androgens. In 11-day embryos, where the gland rudiments are just about to be formed, the entire lateral flank was placed, epidermis upward, on thin filter disks (Millipore THWP, 25-pm thick, 0.45pm nominal pore size) supported by stainless steel grids. Each grid carried one filter with either three glands or one llday flank and was placed in a petri dish with a central depression (designed by Grobstein, 1956) containing about 1.5 ml of medium (80% Eagle’s MEM, 10% horse serum, both from Flow Laboratories; 10% embryo extract made from g-day chick embryos, supplemented with 2 r& glutamine; penicillin and streptomycin at 50 units/ml each). Testosterone was first made up as a 0.01 M alcoholic stock solution which then was diluted 1:lOOO with horse serum to make a 10 pM stock solution in serum. Standard testosterone concentration in the medium was 0.1 pM (or 29 rig/ml), about 100 times the minimal

360

DEVELOPMENTALBIOLOGY

required level (Kratochwil, 1971, 1975). The response in vitro. It was previously shown (Kratochwil, 1971) that explanted mammary rudiments respond to androgens with the same processes as observed in 1Cday male fetuses. Only morphological criteria are available to determine whether and when a response does occur. The first recognizable reaction is a conden-

VOLUME 61, 1977

sation of mesenchymal cells around the stalk of the anlage; in a 1Cday gland this occurs between 12 and 16 hr after hormone application. A few hours later the lower part of the gland epithelium is separated from the epidermis (which in the explant usually forms a cyst), and all these processes can very easily be followed in living explants (Fig. 11. In this paper, “full re-

FIG. 1. Stages in the androgen response of 13-day mammary explants as seen: (a, b, c) in living explants (X 100) and (d, e) in histological sections (x 230). Note that the process can well be followed in living explants. (a) A 13-day mammary gland rudiment, explanted with accompanying epidermis, after 44 hr in culture, not exposed to testosterone (control). The glandular bud is connected to the epidermis which has rounded up to form a cyst. A histological section of a comparable explant is seen in Fig. Id. lb) A 13day mammary explant cultured for the same time as in a, but exposed to testosterone for 13 hr. Note mesenchymal condensation beginning around the gland bud. te) A 13-day mammary explant as in a, but exposed to testosterone for 28 hr. The mesenchymal condensation has now shifted to the “stalk” of the gland, and the distal portion of the gland epithelium separates from the epidermal cyst. A histological section of a comparable explant is seen in Fig. le. (d) Histological section of an explanted 13-day mammary gland after 44 hr in culture, not exposed to testosterone (control). (e) Histological section of an explanted 13-day mammary gland after 44 hr in culture, of which 28 hr were with exposure to testosterone. Mesenchymal cells have condensed around the “stalk” of the gland which is about to rupture (further sections show that it is still continuous in this particular explant).

KLAUS KRATOCHWIL

Development

sponse” means complete separation of the mammary epithelium from the epidermis, and the response was considered to have started when a mesenchymal condensation was unmistakably diagnosed, which was about 6 hr after the first indication. For this reason the onset of the response in an explant could not be determined with an accuracy better than *6 hr. RESULTS

Exposure of 11-day Skin in Vitro

to Testosterone

of Hormone

Responsiveness

361

onset of the responsive phase. Skin of llday embryos was explanted as before, and the cultures were divided into several groups. These groups were switched from normal medium to testosterone medium at various times after explantation, the interval between two groups being 12 hr. From then on, the explants were kept in testosterone medium for the rest of the culture period. It was assumed that all explants that had been transferred to testosterone medium before they had become responsive to the hormone would react at the same time, irrespective of the time of hormone addition. Only if the hormone was added after the acquisition of responsiveness, would the explants react with a delay, corresponding to the time of hormone addition. As can be seen from Fig. 2a, all groups of explants having received testosterone less than 57 hr after noon on Day 11 responded at the same time as explants that had been cultured in testosterone medium from the beginning. The first group to show some minor delay in its response was the one which was exposed to testosterone after 57 hr in culture (in one out of four experiments, this group still responded without delay with respect to glands cultured in testosterone medium from the time of explantation). All subsequent groups responded with a further delay of about 12 hr relative to each other. Therefore, delayed addition of testosterone had no influence on the time of the response, as long as the hormone was given during the first 2 days in culture. Since the first group with a delayed response was switched to testosterone medium 57 hr after explantation, hormone responsiveness must have been acquired in these explants just before that time.

At midday of Day 11, the mammary rudiments are being formed as individual thickened placodes of the epidermis. The skin of the whole lateral body wall was explanted and cultured in medium containing 0.1 fi testosterone. Although no mammary anlagen could be observed under the dissecting microscope at the time of explantation, visible mammary buds did form on the day after explantation, despite the presence of testosterone in the medium. These buds persisted for 2 more days in vitro, but were then surrounded by a condensation of mesenchymal cells and the epithelial rudiments disappeared completely. This result was observed in seven experiments involving 67 mammary glands. Destruction of the mammary buds in these explants occurred less than half a day later than in male embryos of comparable age in utero (Fig. 2). Such a minor delay is not unusual after explantation. In three experiments, explants of 11-day skin were exposed to higher levels of testosterone (10 or 100 a). Even at these (entirely unphysiological) concentrations, testosterone neither prevented the formation of mammary buds nor caused their precocious destruction. Since these results indicated that the Exposure of Mammary Rudiments of Various Developmental Stages to Testostermammary rudiment acquires its testosterone one responsiveness only some time after its formation, the following experiment In these experiments individual gland was done to determine more precisely the rudiments of 12- to 16-day female mouse

362

DEVELOPMENTAL BIOWGY

embryos were cultured in the presence of 0.1 fl testosterone. The response of these glands, with their eventual separation from the epidermis, could be more easily followed in the living explant than in llday skin (Fig. 1). All glands of 12- to 1Cday embryos responded. (In these experiments each group consisted of 20 glands only. In the course of our other investigations, however, a total of 1500 12- to 14-day glands were exposed to testosterone, and all responded to the hormone. The time course of the testosterone reaction was always the same.) Glands of 12- and 13-day embryos responded after different times in vitro, but at the same developmental stage (Fig. 2b). The same held true in comparison with 11-day rudiments. Up to Day 13, the time of the hormone response correlated with the developmental stage of the rudiment and not with the time of hormone exposure. Only glands of 14-day (female) embryos responded later, but these glands had been explanted and exposed to testosterone at a time when the rudiments of male litter-mates were already undergoing the androgen-induced destruction. Most surprisingly, gland rudiments of 15 and Is-day (female) embryos were no longer affected by testosterone in vitro and continued their female-type development (Fig. 2b). It was attempted to determine more precisely at what time between Days 14 and 15 the gland becomes refractory to the hormone. Fourteen-day glands were explanted in hormone-free medium and testosterone was added to different groups at different times between 1200 hr (noon) of Day 14 and 1800 hr of Day 15 (Table 1). It was seen that the degree of the response decreased already late in Day 14. A mesenchymal condensation was observed in all explants switched to testosterone medium at 1800 hr and in most glands transferred to testosterone medium at 2400 hr of Day 14. A full response, however, with separation of the gland epithelium from the epidermis, was no longer observed in

VOLUME 61, 1977

all rudiments. When glands were exposed to testosterone in the early hours of Day 15 (total developmental age), even the mesenchymal condensation was rarely observed, and, from midday of Day 15 onward, the mammary rudiments no longer showed any indication of a testosterone response. Exposure to Testosterone of 12-day Explants That Had Been Precultured in Hormone-Free Medium

This experiment was done to see whether mammary glands lose their testosterone responsiveness also when kept in organ culture. Twenty rudiments of 12day embryos were explanted and cultured in hormone-free medium for 3 days. After that period they were given testosterone medium. None of these glands responded (Fig. 2~). They continued their development in the female-type pattern, giving rise to a well-developed branched duct system connected to the epidermal cyst and even showing formation of a nipple sheath. To check whether the observed loss of testosterone responsiveness was not a culture artifact, 20 1Zday glands were exposed to testosterone after only 2 days in vitro (total developmental age: 14 days). All of them responded. (For other purposes, more than 200 glands were treated in the same way, with the same result.) DISCUSSION

Since the androgen-induced mammary gland destruction in male fetuses takes place about 36-48 hr after morphological differentiation of the testes, the time of this response was considered to reflect the initiation of endosecretory activity of the young testis (Raynaud, 1971). It was therefore not possible to deduce from these observations in uiuo the earliest time of androgen responsiveness in the mammary gland. In this paper it is shown that exposure of very early stages of the mammary rudiment to testosterone did not cause their precocious reaction. The mammary

KLAUS

KRATOCHWIL

Development

In vivo development day of development:

in female

of Hormone

363

Responsiveness

of mammary rudiments

11

16

embryos

in male embryos time of androgen

response

Respo nse in vitro a) 11-day explants

f

b) 12-day to 16-day explants

c) 12-day explants precultured in hormone-free medium

J

FIG. 2. Upper part: Morphology of the mammary gland bud during the “resting period’ in female and male embryos. The androgen response in uivo takes place on Day 14 (condensation of mesenchymal cells around the gland bud and its separation from the epidermis; see Fig. le). Lower part: Schematic representation of the experiments to determine the androgen responsive phase of the mammary rudiment. A thin line symbolizes culture in the absence, and a heavy line represents culture in the presence of 0.1 fl testosterone. The bar at the end of the line indicates the time of the androgen response. The androgensensitive period, as determined by these experiments, is shown by light shading. (a) Response of explanted 11-day skin to testosterone in vitro. Testosterone (t) was either present in the medium from the time of explantation, or it was added to different groups at different times at intervals of 12 hr (at 9 AM and 9 PM,

364

DEVELOPMENTAL BIOLOGY TABLE

1

RESPONSE OF EXPLANTED 14-DAY MAMMARY RUDIMENTS TRANSFERRED TO TESTOSTERONE MEDIUM AT VARIOUS TIMES BETWEEN 1200 HR (NOON) OF DAY 14 AND 1800 HR OF DAY 15’ Time of testosterone addition

Response of explants Mesenchymal condensation

Separation of gland epithelium from epidermis

Day 14

1200 hr 1800 hr 2400 hr

12112 20/20 7111

12/12 7120 2/11

Day 15

600 hr 1200 hr 1800 hr

5/20 l/22 O/6

l/20 o/22 O/6

loss of hormone

responsive-

0 Note the gradual ness during this time.

rudiment, therefore, is not responsive to testosterone from its beginning on Day 11 of gestation. From the results obtained with delayed addition of testosterone to 11-day explants (summarized in Fig. 2a), we conclude that the mammary gland acquires its characteristic androgen responsiveness late in Day 13, i.e., about 2 days after its formation. This interpretation is supported by the time course of the androgen response of 12- and 13-day explants, which react after a shorter culture period but at the same developmental stage (Fig. 2b). Rather surprisingly, the androgen-responsive phase of the mammary rudiment is rather short, lasting for only about 36 hr. The end of the responsive phase could

VOLUME 61, 1977

not be determined precisely due to a gradual loss of responsiveness in the night between Days 14 to 15. This early loss of androgen responsiveness apparently is important for the survival of mammary glands in female embryos: With their high sensitivity to testosterone (0.5-l I%), they would otherwise succumb to testosterone produced by the ovaries in later fetal and postnatal life (Price, 1969). Although earlier experiments have shown that the androgen acts directly only on the mesenthyme while epithelial destruction is caused indirectly by testosterone-activated mesenchymal cells (Kratochwil and Schwartz, 1976; Drews and Drews, 1977), we do not know which tissue determines the short responsive phase of the entire rudiment. This phase could reflect a similarly short hormone-responsive period of the mesenchyme, or a short period of susceptibility of the gland epithelium to mesenchymal “attack” or both. Mammary development in the “resting phase” is not only affected by testosterone but also by estrogens (Raynaud, 1947b). Estradiol also acts directly on the gland rudiment and, at a concentration of 0.1 IN, is capable of arresting further mammary development (our own unpublished results). Due to the characteristics of the estradiol response (failure of glandular outgrowth at 16.5 days) we were as yet unable to determine the onset of the sensitive phase. Its end, however, quite inter-

respectively). Four experiments of this type were done, with a total of between 16 and 28 glands in each group. Note that the time of the response in the first four groups is independent of the time of testosterone addition, the fifth group is the first to show some delay. Subsequent groups respond with a delay corresponding to the time of testosterone addition. From this result it is concluded that the explants become responsive before 9 PM of Day 13. (b) Explanted 12- and 13&y glands respond at the same developmental stage as do 11-day explants and glands in uiuo, although they were exposed to testosterone for shorter periods. This suggests that these glands had been explanted and exposed to testosterone before they had become responsive to the hormone. In contrast, glands of ll-day (female) embryos, explanted at a time when the androgen response was already taking place in male littermates, respond with a delay due to delayed exposure to the hormone. Glands of 15- and 16-day (female) embryos no longer respond to testosterone and continue their development, indicating a loss of androgen responsiveness between Days 14 and 15. (c) Explanted 12-day glands were cultured in the absence of testosterone for 2 or 3 days, respectively, and then were exposed to the hormone. When the hormone was added atier 2 days, a typical response was observed. In contrast, when the addition of testosterone was delayed for one more day, no response occurred. Like 15-day glands, these explants were no longer responsive.

KLAUS KRATOCHWIL

Development

estingly coincides with the end of the androgen-responsive phase, i.e., between Days 14 and 15 (about 36 hr before the estradiol-sensitive process actually takes place). Finally, the present experiments have shown that mammary rudiments acquire their androgen responsiveness in culture (experiments represented in Fig. 2a) and also lose it again in vitro (Fig. 2~) at the same time as do glands in uiuo. Therefore, both acquisition and loss of androgen responsiveness reflect developmental processes totally intrinsic to the mammary rudiment. This indicates that important developmental changes are taking place during an otherwise entirely inconspicuous phase of the glands development, the “resting period,” which was shown before to reflect itself as an entirely autonomous developmental program of the rudiment (Kratochwil, 1969). This work was supported by Contract 33883 from the National Cancer Institute.

NOl-CB-

REFERENCES BALINSKY, B. I. (1950). On the developmental processes in mammary glands and other epidermal structures. Trans. Roy. Sot. Edinburgh 62, l-31. CERIANI, R. L. (1970a). Fetal mammary gland differentiation in vitro in response to hormones. I. Morphological findings. Deuelop. Biol. 21, 506529. CERIANI, R. L. (1970b). Fetal mammary gland differentiation in vitro in response to hormones. II. Biochemical findings. Deuelop. Biol. 21, 530-546. DREWS, U., and DREWS, U. (1977). Regression of mouse mammary anlagen in recombinants of Tfm and wild-type tissues: Testosterone acts via the mesenchyme. Cell 10, 401-404. GROBSTEIN, C. (1956). Trans-filter induction of tubules in mouse metanephrogenic mesenchyme. Exp. Cell Res. 10, 424-440. KRATOCHWIL, K. (1969). Organ specificity in mesenchymal induction demonstrated in the embry-

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onic development of the mammary gland of the mouse. Develop. Biol. 20, 46-71. KRATOCHWIL, K. (1971). In vitro analysis of the hormonal basis for the sexual dimorphism in the embryonic development of the mouse mammary gland. J. Embryol. Exp. Morphol. 25, 141-153. KRATOCHWIL, K. (1975). Experimental analysis of the prenatal development of the mammary gland. In “Milk and Lactation” (N. Kretchmer, ed.), Modern Problems in Pediatrics Series, Vol. 15, pp. l-15. Karger, Basel. KRATOCHWIL, K., and SCHWARTZ, P. (1976). Tissue interaction in androgen response of embryonic mammary rudiment of mouse: Identification of target tissue for testosterone. Proc. Nut. Acad. Sci. USA 73, 4041-4044. PRICE, D. (1969). Sexual differentiation of the reproduction ducts of fetal guinea pigs: An example of hormonal influences on genetic expression. In “Environmental Influences on Genetic Expression,” (N. Kretchmer and D. Walcher, eds.), Fogarty International Center Proceedings, Vol. 2, pp. 31-45. U. S. Government Printing Office, Washington, D. C. RAYNAUD, A. (1947a). Effet des injections d’hormones sexuelles a la souris gravide sur le developpement des ebauches de la glande mammaire des embryons. I. Action des substances androgenes. Ann. Endocrinol. (Paris) 8, 248-253. RAYNAUD, A. (194713). Effet des injections d’hormones sexuelles a la souris gravide sur le developpement des ebauches de la glande mammaire des embryons. II. Action de fortes doses de substances oestrogenes. Ann. Endocrinol. (Paris) 8, 318-329. RAYNAUD, A. (1971). Foetal development of the mammary gland and hormonal effects on its morphogenesis. In “Lactation,” (I. R. Falconer, ed.), Proceedings, International Symposium, Nottingham, 1970, pp. 3-29. Butterworths, London. RAYNAUD, A., and RAYNAUD, J. (1953). Les principales &apes de la separation d’avec l’epiderme, des Bbauches mammaires des foetus males de Souris; recherches sur les processus de la rupture de la tige du bourgeon mammaire. C. R, Sot. Biol. 147, 1872-1876. TATA, J. R. (1968). Early metamorphic competence of Xenopus larvae. Deuelop. Biol. 18, 415-440. TATA. J. R. (1970). Simultaneous acquisition of metamorphic response and hormone binding in Xenopus larvae. Nature (London) 227, 686-689.