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/. Embryol. exp. Morph. Vol. 25, 1, pp. 141-153, 1971 Printed in Great Britain In vitro analysis of the hormonal basis for the sexual dimorphism in the embryonic development of the mouse mammary gland By KLAUS KRATOCHWIL 1 From the Institut fiir Krebsforschung, Universitdt Wien SUMMARY Factors underlying the sexual dimorphism in the embryonic development of mouse mammary glands were analysed in vitro and the following results were obtained: 1. Mammary gland rudiments of 13-day male embryos, explanted immediately before the onset of their regression, were perfectly capable of developing into female-type glands in vitro. Even some of the glands of 14-day male embryos, where the regression process had already begun, recovered after explantation and underwent female-type morphogenesis. 2. Combined explantation of 13-day testes with mammary rudiments of female embryos of 12-14 days gestation resulted in male-type regression of the glands. 3. The addition of testosterone to the culture medium caused a similar regression of explanted (female) mammary-gland rudiments. The minimal effective concentration of the hormone was 10~9M, or 000029/*g/ml. 4. Cultured mammary rudiments of 15-day female embryos were no longer responsive to the presence of testis explants. They failed to undergo regression and continued their development in vitro. From these results the following conclusions were drawn: (a) The sexual dimorphism in the embryonic development of mouse mammary glands is caused by their suppression in males and not by their stimulation in female embryos. (b) The androgenic hormones in male foetuses are solely responsible for the regression of the mammary rudiments. They exert their effect directly on the gland without the need for involvement of other endocrine organs. (c) The genetic sex of the gland itself has no influence on its developmental capacities as: (i) glands of male embryos are able to develop in the absence of androgens, and (ii) glands of female embryos undergo typical male-type regression in vitro when exposed to the presence of foetal testes or of testosterone. INTRODUCTION The development of the mammary glands in mouse embryos starts during the 12th day of gestation. Five pairs of rudiments arise as epidermal thickenings in all embryos, the sex of which cannot be determined at that early stage. One day later, morphological differentiation of the gonads makes it possible to distinguish between male and female litter-mates. Mammary-gland development proceeds in an identical way in both sexes for one more day, but at the end of 1 Author's address: Institut fur Krebsforschung, Universitat Wien, A-1095 Vienna, Austria. 142 K. KRATOCHWIL the 14th day a sexual dimorphism becomes apparent. In male embryos a conspicuous condensation of mesenchymal cells is seen around the epithelial bud, the stalk of the Anlage becomes constricted and eventually the gland rudiment is detached from the epidermis (Turner & Gomez, 1933; Raynaud, 1941 b, 1961; Raynaud & Raynaud, 1953a, b; Cowie & Folley, 1961). No nipples are formed in male embryos. The disconnected distal portion of the epithelial rudiment either continues its regression and disappears completely, or it may still undergo some modest development. Considerable variation exists between different strains of mice, and the degree of development was also found to vary with the position of the gland (Richardson & Cloudman, 1947). The fifth pair of glands regresses without prior separation from the epidermis (Raynaud & Raynaud, 19536). There is experimental support for the assumption that this inhibition of mammary-gland development in male embryos is caused by testicular hormones. Raynaud & Frilley (1947, 1949) succeeded in destroying the gonads of 13-day embryos in utero by localized X-irradiation. Mammary-gland development then followed the female-type pattern in all embryos. By contrast, injections of testosterone-propionate into pregnant mothers resulted in an involution of the mammary-gland rudiments even in female embryos (Raynaud, 1947a, 1949; Hoshino, 1965). Finally, the administration of a synthetic steroid with known antiandrogenic effect to pregnant mothers prevented the regression of the mammary glands in male embryos (Elger & Neumann, 1966). So far, all the evidence comes from experiments performed in vivo and the interpretation of the last two experiments is further complicated by the fact that the hormone, or its antagonist, respectively, were given to the mother animal. Possible consequences of castration on other endocrine organs, and the effect of a hormonal imbalance created in the pregnant mother must be considered. From these results it cannot be conclusively decided whether at the stage of the 13-day mammary rudiment, the presence or absence of androgens is the only factor determining the further fate of the gland. In addition, as recently pointed out by Jean & Delost (1969), evidence for a direct action of testicular hormones on the mammary gland rudiments is still missing. Both these questions can be answered by an in vitro system. Embryonic mammary glands of the mouse, grown in organ culture, undergo typical morphogenesis (Kratochwil, 1969), and explanted testes of mouse embryos secrete androgens in vitro (Weniger, Ehrhardt & Fritig, 1967). If the sexual dimorphism in the development of the mammary gland is indeed caused by androgens alone, then rudiments excised from male embryos before the beginning of regression should continue their development after explantation and yield female-type glands. Secondly, the addition of embryonic testes or of testosterone to cultures of mammary glands from female embryos should cause their regression in order to prove a direct effect of the hormones on the gland. The results reported in this paper are able to confirm both of these assumptions. Embryonic mammary gland in vitro 143 MATERIALS AND METHODS Mouse embryos were obtained by mating random bred Swiss (GP) females with C3H males, the detection of a vaginal plug counting as day zero of pregnancy. Dissection and explantation of mammary gland rudiments were performed as previously described in detail (Kratochwil, 1969). Testes of 13-day or 15-day embryos were cut in half before explantation. Both mammary glands and testes were explanted on to the platform of a filter assembly which was suspended on the surface of the liquid medium in an organ culture dish (the method of Grobstein, 1956). The explants—usually two on a filter disc of 3-5 mm diameter—were covered with a plasma clot made up by mixing equal volumes of rooster plasma and 9-day chick embryo extract. Culturing the explants on the translucent filter substrate (Millipore THWP, with a thickness of 25 ft and a pore size of 0-45 /*) allowed continuous observation during their development in vitro. Photographs of living cultures were made with a Zeiss Ultraphot II. The medium consisted of 80 % Eagle's MEM (Grand Island Biological Comp.), 10 % horse serum (Flow Laboratories) and 10 % chick embryo extract, prepared from 9-day embryos. Glutamine was added to a final concentration of 2 mM and 50 units each of penicillin and streptomycin were incorporated in the medium. Each culture dish contained 1 ml of medium. It was changed daily except for the experiments with explanted testes where the medium was replaced only every second day to allow greater accumulation of testicular hormones. When testosterone was used the required amount of a 10"1 M alcoholic solution (28-8 mg/ml) was added to the medium. In all dilution series the concentration of ethanol was kept constant throughout the experiment and only that of testosterone was varied. Control explants were also exposed to the same concentration of ethanol. Normal in vivo development of embryonic mammary rudiments in Swiss xC3H mice. Because of the variation in mammary-gland development between different mouse strains, especially in male foetuses, the normal fate of the glands in our material had to be checked. In newborn females all five pairs are in a considerably advanced state of development, the branched ducts extending very far into the fat pads. In male litter-mates no traces of the third and fifth pair of glands can be found. The other three glands are detached from the epidermis—no nipples are present—and they exhibit very rudimentary development with modest growth and little branching. The best structures are formed by the second pair of glands. RESULTS Two types of experiments were performed. In the first, series A, the developmental capacities of mammary-gland explants from male and female embryos were compared. In series B, gland rudiments of female embryos were cultured 144 K. KRATOCHWIL in the presence of foetal testes or of testosterone. All experiments were done with mammary rudiments at various stages of development. A. In vitro development of mammary-gland rudiments from male and female embryos Explantation of 12-day mammary-gland rudiments. At this early stage the sex of the embryos could not be determined. Mammary-gland rudiments were taken from all embryos of several litters and the glands of individual embryos were kept separate throughout the explantation procedure. With this precaution it would have been possible to detect differences in the development of explants derived from various embryos. However, virtually all glands from all embryos developed (Table 1). Differences in the frequency or rate of development of glands derived from particular embryos were never observed. Table 1. Development of explanted mammary-gland rudiments from male or female embryos Sex of embryo Age of embryo (days) Male 12 13 14 38/40 18/49 Female 65/67 40/42 58/61 Explantation of 13-day rudiments. In this group the sex of the donor embryos was known at the time of explantation. All five gland pairs were used for cultivation and comparison was made only between litter-mates. No difference in the behaviour of glands from male or female donors was found, the glands of male embryos developed with the same frequency and at the same rate (Table 1). Outgrowth of the glandular ducts occurred in both groups on the 4th day in vitro after the obligatory resting phase of the gland (Balinsky, 1950; Kratochwil, 1969). Even when at the time of explantation the donor embryos were fairly advanced in their development, as judged by morphological criteria, the rudiments from male embryos were still able to form typical mammary glands in vitro. Explantation of 14-day rudiments. At the time of their explantation the mammary-gland rudiments of 14-day female embryos did not differ significantly from those of 13-day embryos except for a very slight increase in size (Fig. 1). In male 14-day embryos, however, the glands had already started to undergo visible regression: the epithelial knob was smaller than in female litter-mates, connected to the epidermis only by a thin stalk and was surrounded by densely packed mesenchymal cells (Fig. 2). This process appeared to be most advanced in the second and third pair of glands, the third pair (second thoracic gland) Embryonic mammary gland in vitro 145 occasionally became hardly distinguishable under the dissecting microscope from the neighbouring hair rudiments. Almost all of the glands from female donor embryos developed in vitro (Table 1). Outgrowth of the primary sprouts occurred after a 'resting period' of 3 days. In explanted male mammary glands the mesenchymal condensation persisted for about 2 days in culture and the epithelial rudiments regressed further. In the majority of the cases the gland epithelium was no longer detectable after 3 days in vitro. Although epidermal cornification and the formation of hair rudiments proceeded normally, glandular outgrowth failed to occur in these explants. In about one-third of the cultures (Table 1), however, the epithelial rudiments, though reduced in size, were still present after 3 days in vitro. At that time the mesenchymal condensation around the gland had disappeared—probably due to outgrowth of the fibroblasts on the substrate. In these cases the epithelia were found to recover. They started growing again and finally gave rise to glandular structures identical to those formed by explants of female rudiments. The only difference was a delay of about 2 days in the development of these male glands as compared with cultures of mammary glands from female littermates. Table 2. Development of explanted mammary-gland rudiments from 14-day male embryos Developing explants Pair of glands 1 (cervical) 2 (1st thoracic) 3 (2nd thoracic) 4 (1st inguinal) 5 (2nd inguinal) Total 7/11 3/11 2/5 6/11 0/11 18/49 Individual pairs of glands of 14-day male embryos were found to differ in their capacity to develop in vitro. The cervical (first) and the first inguinal (fourth) pair of glands appeared to be least affected by the regression process at the time of their explantation: more than half of them were able to give rise to a mammary gland in vitro. The results obtained with the thoracic rudiments (second and third pair) were inferior and not a single gland was formed by eleven explants of the fifth (second inguinal) pair (Table 2). B. Exposure of mammary-gland rudiments from female embryos to the influence of testicular hormones These experiments were designed to reveal whether testicular androgens act directly on the mammary gland. The rudiments were exposed to the hormones by combined explantation with 13-day testes or by the addition of testosterone 10 EMB 25 146 K. KRATOCHWIL Embryonic mammary gland in vitro 147 to the culture medium. Although the previous experiments (section A) had demonstrated no difference in the in vitro development of rudiments of male or female embryos up to 13 days of age, the following experiments were done with glands from female donors only, in order to avoid the possibility that the glands could have been exposed to testicular androgens before their explantation. The experiments were done with mammary rudiments at different stages of development between day 12 and day 15 of gestation. Rudiments at the end of the gland's resting phase (16-day embryos) could no longer be tested as it has previously been shown that they do not develop in vitro even in the absence of androgens (Kratochwil, 1969). Mammary-gland rudiments of 12- to 14-day embryos combined with 13-day testes. In all experiments the testis explants formed typical and well-developed testis cords in vitro (Figs. 3-5). The mammary rudiments cultured with these testes began to show the first morphological signs of regression as early as 18 h Figs. 1, 2. Histological sections of the first inguinal mammary-gland rudiment (4th pair of glands) of 14-day embryos, at a time when the sexual dimorphism first becomes apparent. Fig. 1. Gland rudiment of a female embryo. Fig. 2. Gland rudiment of a male litter-mate showing the beginning of the regression process. The epithelial Anlage, especially its stalk, is surrounded by densely packed mesenchymal cells. The stalk later ruptures, the lower portion of the gland thus becoming separated from the epidermis. Figs. 3-7. Explan ted mammary-gland rudiments exposed to the presence of foetal testes or of testosterone, epid., epidermis; hair, hair rudiments; m.ep., mammary-gland epithelium; mes., condensation of mesenchymal cells. Fig. 3. Early stage of mammary-gland regression in a 14-day rudiment after 40 h in combined culture with foetal testes. The mesenchyme immediately adjacent to the epithelial bud (m.ep.) becomes condensed as seen by the darker zone surrounding the gland rudiment. Living explant. Fig. 4. More advanced stage of regression of a 14-day mammary-gland rudiment, after 3 days in combined culture with foetal testes. The distal part of the gland epithelium has become separated from the epidermis, the area of the former stalk of the Anlage is occupied by a streak of condensed mesenchymal cells. Living explant. Fig. 5. Explant of a 14-day mammary rudiment after 8 days in vitro in the presence of foetal testes. The gland epithelium was detached from the epidermis, its distal portion still undergoing some development. Note the formation of hair rudiments in the epidermal cyst and the good development of testis cords in the testis explant. Living explant. Fig. 6. Two control glands, developed from 14-day mammary rudiments explan ted without testes. After 7 days in vitro typical glandular structures have been formed which remained connected to the epidermal cyst (see upper explant). Living explant, same magnification as Fig. 5. Fig. 7. Explant of the first inguinal mammary-gland rudiment of a 13-day female embryo after 42 h in vitro, the culture medium containing 10~ 6 M testosterone (0-29 /tg/ml). Typical condensation of mesenchymal cells has occurred and the gland epithelium has become separated from the epidermis. The response of the gland is the same as in combined culture with foetal testes—cf. Fig. 4. Living explant. 148 K. KRATOCHWIL after explantation. Typical condensation of mesenchymal cells occurred around the gland epithelium, particularly around the stalk of the Anlage (Fig. 3). In almost all explants the mammary rudiment became detached from the epidermis (Fig. 4). Regression of the gland epithelium proceeded during the second day in vitro, and between 48 and 60 h in culture the mammary rudiments completely disappeared in 57 of 65 explants. Although these cultures were maintained for at least 7 days, no glandular outgrowth was observed. By contrast, the survival and development of epidermal cysts and the formation of hair rudiments in these explants were not affected by the presence of testes. In a few explants the effect appeared to be less severe. Although these glands became detached from the epidermis, their regression remained incomplete, sparing a small distal portion of the epithelial rudiment which subsequently underwent some modest development (Fig. 5). No obvious difference in the response of 12-day, 13-day, and 14-day mammary explants was found (Table 3) except for the observation that 14-day gland rudiments were able to persist for a longer period in culture than the younger stages. The position of the gland had no influence on its response under these experimental conditions. In those experiments where the five individual pairs of glands of 13-day embryos were cultured in separate groups all explants regressed completely. It was observed that in some, but not all, explants of the fifth pair, the glandular epithelium became detached from the epidermis. Although this process is characteristic of the first four glands it does not represent the normal mode of regression in the fifth pair. Table 3. Development of mammary-gland rudiments from female embryos after combined explantation with 13-day testes Age of mammary gland at time of explantation (days) 12* 13 14 15 15 (with 15-day testis) Developing explants With testis Without (control) 0/20 0/22 0/23 16/20 15/20 20/20 19/23 22/23 7/10 — * Sex of 12-day embryos could not be determined. Mammary-gland regression also occurred when the testes were explanted in a transfilter position. In the control series without testes the mammary rudiments persisted and glandular outgrowth occurred in all but five explants between the 3rd and 5th day in culture, depending on the age of the gland at the time of its explantation (Fig. 6). Five of sixty-six control epithelia were lost due to spreading of the filter substrate. Embryonic mammary gland in vitro 149 Mammary-gland rudiments of 15-day embryos combined with testes. In contrast to the results obtained with the earlier stages of the gland, the development of mammary explants from 15-day embryos was not affected by the presence of testis explants (Table 3). Even the larger and more advanced testes of 15-day foetuses could not cause their regression. Although all testes were found to grow as well as in the experiments just described, there was no observable difference in the frequency and rate of mammary-gland development between experimental and control groups. The relatively low percentage of rudiments developing in both groups was due to a greater spreading tendency of the older epithelium. It was not observed that any of the morphological signs of androgeninduced regression was associated with the loss of these explants. The effect of testosterone on explanted 13-day mammary rudiments. Testosterone was incorporated in the medium at concentrations ranging from 10~5 M (2-9 /tg/ml) to 10"11 M, each group consisting of twenty explants. The alcohol concentration in the medium was 0-01 % for the experiments with 10~5-10~7 M testosterone (including controls), and 0-0001 % for the experiments with < 10~7 M testosterone. The response of mammary-gland explants to testosterone was the same as observed in the presence of testes. Condensation of mesenchymal cells around the gland epithelia was seen 30 h after explantation; all glands became separated from the epidermis and, with very few exceptions again, eventually disappeared completely. The formation of epidermal cysts with hair rudiments proceeded normally. The lowest concentration of testosterone still causing regression of all twenty mammary-gland explants was 10~9 M (0-00029 /tg/ml). At the next lower concentrations (10~10 and 1 0 - U M ) testosterone was completely ineffective: all glands developed as in the controls. DISCUSSION For the study of the effect of hormones on organ cultures a completely synthetic medium would be desirable. The use of a semi-defined culture medium in these experiments does not allow an answer to the question whether the embryonic development of the mouse mammary gland is completely independent of hormones. Lasfargues & Murray (1959) reported the formation of mammary buds from explanted skin in medium 199 but they did not comment on the morphology of the gland. Mitoses were not observed in these cultures and the connective tissue showed areas of necrosis. Despite the reservations due to the use of serum and of embryo extract in our medium the experiments allow some conclusions to be drawn concerning the role of hormones in the sexual dimorphism of the mammary gland. From the survival and development in vitro of explanted mammary rudiments from male embryos it appears that the gland—irrespective of its genetic sex type—follows the female-type pattern of development in the absence of androgenic hormones. 150 K. KRATOCHWIL This result fully agrees with the female-type development of mammary rudiments in male foetuses after X-ray destruction of their testes (Raynaud & Frilley, 1947, 1949) and it lends further support to the assumption that this female-type development of the mammary gland does not require a particular hormonal environment, present only in female embryos, but is the one which is expressed in the absence of any sex steroids (Raynaud, 1961). Furthermore, in the experiments with simultaneously explanted testes or with testosterone in the medium, the hormonal 'background' due to the natural medium ingredients was identical in the experimental and control groups. It was shown in these experiments that the regression of the gland was caused solely by the addition of a testis or of testosterone. This strongly suggests that the same mechanism prevails in vivo and that the only cause of the sexual dimorphism in mammarygland development is the presence of androgenic hormones in male foetuses. Continued presence of androgens seems to be required at least during the initial phase of mammary-gland regression, as revealed by the in vitro development of some gland rudiments taken from 14-day male foetuses. Withdrawal of the hormone(s)—by explanation—allowed recovery and subsequent female-type development even of glands already showing the characteristic mesenchymal condensation. It appears, therefore, that the process of tissue destruction in the male mammary gland of the mouse follows rules different from those governing the regression of Miillerian ducts in the mouse (Brewer, 1962), rat (Price & Pannabecker, 1956), and chick embryo (Hamilton & Teng, 1965). In the Miillerian ducts an apparently irreversible pattern of cell death is imposed by the testis during a rather brief period early in development. No survival or even recovery in vitro was observed when explantation occurred after that event. The regression of explanted (female) glands in the presence of foetal testes or of testosterone demonstrates that androgens act directly on the mammary gland without necessary involvement of other endocrine organs. This result supports the assumption of Raynaud & Raynaud (1953 a) that the destruction of mammary rudiments in female foetuses, whose mothers had received injections of testosterone propionate, was caused by a direct action of the hormone on the gland. The sensitivity to testosterone of the 13-day mammary rudiment is several orders of magnitude greater than in the adult gland where it has been found that a concentration of 10"4 M is required to stop all epithelial DNA synthesis (Turkington & Topper, 1967). This unusually high sensitivity of the embryonic rudiment (to 10~9 M testosterone) is an additional argument for the assumption that in normal development also androgens act directly on the mammary rudiment and not via the possible action of the pituitary gland, as was considered by Jean & Delost (1969). However, this response of the mammary gland to androgens is limited to a relatively short time in its development: explanted 15-day rudiments were no longer affected by the presence of testes. Hoshino (1965) applied testosterone propionate in vivo on Embryonic mammary gland in vitro 151 different days of pregnancy and obtained most extensive inhibition of mammary development when the injection was done on day 12 of gestation. Testosterone given on day 15 produced much less effect and none at all on day 16. In view of our in vitro findings it appears that Hoshino's results indeed reflect a changing sensitivity of the mammary-gland rudiments and not alterations in the steroid metabolism of the mother and/or foetus. The experiments done in vivo as well as ours in vitro show that the sex type of the embryo has no influence on the response of the gland. This indifference is one more reason for caution in comparing mammary-gland regression with the involution of the Miillerian ducts. Exposure of the Miillerian ducts from female mouse (Brewer, 1962) and rat (Price, 1957; Price & Pannabecker, 1959) embryos to the presence of testes or of steroidal androgens not only failed to cause their regression but was even found to stimulate their growth in vitro. Controversial results were reported for chick Miillerian ducts exposed to androgens: regression (Wolff, Lutz-Ostertag & Haffen, 1952) and stimulation (Hamilton & Teng, 1965). The fact that mammary-gland regression is directly and solely caused by testicular androgens, without complication by other hormones or by the sex type of the donor embryo, appears to offer experimental advantages not found in other systems. The reliable reproduction of this regression in vitro—with all the morphological features observed in the embryo—could make it a valuable system for the study of hormone-initiated tissue destruction. ZUSAMMENFASSUNG In-vitro Analyse der hormonellen Grundlage des Sexualdimorphismus in der Embryonalentwicklung der Milchdruse der Maus Die Ursachen fur den bei Mausen gefundenen Sexualdimorphismus in der Embryonalentwicklung der Milchdruse wurden mit Hilfe der Organkultur untersucht. Dabei wurden folgende Ergebnisse erzielt: 1. Milchdriisenanlagen, die 13-tagigen mannlichen Embryonen und somit noch vor dem Einsetzen ihrer Regression (13 1/2 Tage) entnommen wurden, entwickelten sich in vitro wie Driisen weiblicher Tiere ohne Anzeichen einer Regression. Selbst ein Drittel aller Driisenanlagen von 14-tagigen mannlichen Embryonen war noch in der Lage, sich in Kultur zu entwickeln, obwohl sie zum Zeitpunkt ihrer Explantation bereits deutlich in Regression begriffen waren. 2. Wurden Milchdriisenanlagen 12- bis 14-tagiger weiblicher Embryonen zusammen mit embryonalem Hoden explantiert, so zeigten alle Driisen dieselben Regressionserscheinungen wie sie in mannlichen Embryonen beobachtet werden. 3. Testosteron im Kulturmedium verursachte ebenfalls Regression explantierter Milchdriisenanlagen weiblicher Embryonen. Die niedrigste noch wirksame Konzentration des Hormons war 10~9 M, oder 0,00029/*g/ml. 4. Milchdriisenanlagen von 15-tagigen weiblichen Embryonen reagierten nicht mehr auf die Anwesenheit gleichzeitig explantierter Hoden. Diese Driisen entwickelten sich in vitro ohne Anzeichen einer Regression. Aus diesen Ergebnissen wurden folgende Schliisse gezogen: (a) Der Sexualdimorphismus der Milchdruse kommt allein dadurch zustande, daB die Entwicklung des Organs in mannlichen Embryonen unterdriickt wird. Die unterschiedliche Entwicklung wird nicht etwa durch eine Wachstumsstimulation in weiblichen Embryonen bedingt. 152 K. KRATOCHWIL (b) Die Regression der Milchdriisen wird durch die Anwesenheit androgener Hormone in mannlichen Embryonen hervorgerufen. Diese Hormone iiben ihre Wirkung unmittelbar auf die Druse aus, ohne Beteiligung anderer endocriner Organe. (c) Das genetische Geschlecht der Druse hat keinen EinfluB auf ihre Entwicklungsmoglichkeiten, da: (i) Driisen mannlicher Embryonen sich bei Abwesenheit androgener Hormone normal entwickeln konnen, und (ii) Driisen aus weiblichen Embryonen auf die Gegenwart von Androgenen mit der fur mannliche Foeten charakteristischen Regression reagieren. REFERENCES BALINSKY, B. I. (1950). 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