The determinant role ascribed to steroid hormones in sexual differentiation of the reproductive tract of the embryo implies the presence of target cells for sex steroids. An autoradiographic technique adapted for diffusible compounds was employed to characterize and localize cells which concentrate either [3H]oestradiol (E2) or [3H]dihydrotestosterone (DHT) in their nuclei. This paper describes the topographical distribution of cells containing receptor sites for oestrogen or androgen in various tissues of the reproductive tract of chicken embryos from day 6 to 15 of incubation. Receptor sites for oestradiol are present in the mesenchyme of the cloaca and in urodeum and vascular body. In the lower part of the Wolffian duct, only epithelial cells display nuclear labelling. In the Müllerian duct, nuclear receptor sites for [3H]oestradiol are observed not before day 15. Receptor sites for DHT are localized in the mesenchyme of the cloacal region from day 7 to 15. The Wolffian, but not the Müllerian duct contains receptor sites for DHT in the nuclei of epithelial and mesenchymal cells. Cross-competition experiments between [3H]E2 or [3H]DHT and unlabelled DHT or E2 respectively, show that 2 different types of receptor sites exist. The observations indicate: (a) complementary roles for oestrogenic and androgenic hormones in embryonic sexual differentiation; (b) precocity of receptors for sex hormones during embryonic development; (c) importance of mesenchyme in differentiation processes which are sex-steroid dependent.

The prevailing role of steroid hormones in the differentiation of the sexual phenotype during embryonic and fetal life is demonstrated by experiments of castration, grafts of gonads and hormonal treatments (Jost, 1950; Raynaud, 1950; Wolff, 1950). On the basis of the results of these experiments in birds and mammals, two successive phases in the course of differentiation were described: during the first period a neutral phenotype consisting in organs of both sexes is formed in all embryos irrespective to their genetic sex; then, during a second period their sex-specific development is controlled by steroid hormones. The sex steroids either stimulate the differentiation of structures and organs characteristic of the genetic sex of the embryo, or inhibit the development of those pertaining to the opposite sex. Recent studies analyse the role of sex steroids on tissular interactions in two different organs: the mammary rudiment (Kratochwil, 1977) and the urogenital sinus (Cunha & Lung, 1979; Lasnitzki & Mizuno, 1979). A noticeable exception is the Müllerian duct whose normal regression in male embryos is induced by testicular secretions of a non-steroidal nature (Tran & Josso, 1977).

While numerous experimental studies have shown the effects of steroid hormones on anatomical sexual differentiation, little is known about the processes involved at the cellular level before differences are expressed at morphological levels. The present work is an attempt to describe one of the cellular aspects which contributes to the formation of a male or a female phenotype from a sexually undifferentiated one, that is, the binding of sex-steroid molecules to nuclear receptors in certain cells of target organs.

By means of an autoradiographic technique adapted for diffusible compounds, cells concentrating steroid hormones in their nuclei are visualized after injection of radiolabelled hormone to the embryo. The uptake of hormone molecules in the nucleus attests to the presence of nuclear receptors for this type of hormone, provided the specificity and limited binding capacity of the receptor can be assessed. Cells labelled in the nucleus after injection of radiolabelled hormone are considered as target cells for the hormone, that is, cells which receive the message carried by steroids and thereby trigger a chain of events leading to sexual differentiation.

The experimental conditions do not allow one to quantitate the nuclear labelling accurately. Several factors such as the actual thickness of the sections and the endocrine status of each embryo affect the labelling to an extent that cannot be estimated. In addition, mesenchymes have ill-defined boundaries and consequently, do not lend themselves to accurate quantitation. For these reasons the present work is primarily a description of the distribution of target cells for sex steroids. Comparisons and quantitative estimations are made only when justified by specific conditions.

The present study includes the urogenital tract (Müllerian, and Wolffian ducts and ureter), and the other accessory sex organs of the cloacal region. The latter, which corresponds to the urogenital sinus of mammals, contains all the structures which will form the organs necessary for copulation and the laying of eggs. Chicken embryos of 6 – 15 days of incubation were used in order to encompass the period during which sexual differentiation occurs in the organs.

Adjacent to the cloaca, and in continuity with it, the bursa of Fabricius is also a target organ for steroid hormones (Erickson & Pincus, 1966). The observations on this organ of the immune system are reported in a separate article (Gasc & Stumpf, 1981).

A preliminary note on the distribution of oestrogen target cells in the cloacal region of 12-day chicken embryos has been previously published (Gasc, Stumpf & Sar, 1978).

Eggs of White Rock chickens from a local poultry farm were incubated at 38°C in a humidified incubator. On day 4 of incubation the genetic sex of the embryo was determined by a caryological technique (Omura, 1970; Gasc, 1973). The eggs were resealed with adhesive tape and incubated until injected with radiolabelled hormone on day 6, 7, 10, 12 or 15 of incubation.

Injections were made intravenously in an extraembryonic vessel using 0·05 to 0·1 ml 5% ethanol-isotonic saline solution as a vehicle. Radiolabelled hormones were either [2, 4, 6, 7, 16, 17-3H]oestradiol ([3H]E2), specific activity 141 Ci/mmol, or [1,2,4, 5,6,16,17-3H]dihydrotestosterone([3H]DHT), specific activity 190 Ci/mmol (New-England Nuclear, Boston, Massachusetts, U.S.A.). Competition experiments between labelled and unlabelled steroid hormones were carried out by applying a 100-fold excess of unlabelled hormone on the chorioallantoic membrane, 30 min before the injection of radioactive hormone.

[3H]Oestradiol injections

Animals injected with [3H]E2 received either 0·01 μ g (at day 6: 2 females and 2 males; at day 7: 1 female and 1 male; at day 10: 2 females and 2 males; at day 12: 1 female and 1 male), or 0·02 μg (at dayl2: 2 males; at day 15: 1 female and 1 male). Other animals, in addition to [3H]E2 received also unlabelled oestradiol (1 female and 1 male at day 10; 2 females at day 15), or unlabelled DHT (1 female and 1 male at day 12; 2 females at day 15) under the conditions above described for competition experiments.

[3H]Dihydrotestosterone injections

Animals injected with [3H]DHT received either 0·02 μ g (2 females and 2 males at day 7) or 0·03 (2 females and 2 males at day 10), or 0·04 μ g (1 female and 1 male at day 12; 2 females and 2 males at day 15). Other animals, under the conditions reported above for competition experiments, in addition to [3H]DHT received also unlabelled DHT (2 females and 1 male at day 15), or unlabelled testosterone (1 female and 1 male at day 15), or unlabelled oestradiol (1 female and 1 male at day 12; 1 female and 1 male at day 15) or unlabelled progesterone (1 female and 1 male at day 15).

All animals were sacrificed by decapitation 1 h after radiolabelled hormone injection. The cloacal and gonadal regions were dissected and frozen in liquefied propane on a brass holder with minced rat liver as supporting tissue. Blocks were stored in liquid nitrogen before being cut in a cryostat at − 35°C (W.R. model, Harris Manufacturing Co., North Billerica, Massachusetts, U.S.A.). Sections, 3 μ m thick, were mounted on slides coated with photo graphic emulsion (Kodak NTB3) following the thaw-mount autoradiographic technique (Stumpf & Sar, 1975). After 5 to 14 months exposure slides were developed and fixed (D19 and Rapid fixer by Kodak) and then stained with methyl green-pyronin. The poor stainability of unfixed frozen sections accounts for the lack of contrast in our illustrations.

Schematic drawings were prepared from autoradiograms in order to show the topographical distribution of the target cells for radiolabelled hormones. The anatomical description and terminology are based on Romanoff (1960).

Oestrogen target cells

Cells concentrating [3H]E2 in their nuclei are found in the mesenchyme of the cloacal region at all stages studied from 6 to 15 days of incubation. In contrast, epithelial structures of the cloaca do not contain cells labelled in the nucleus. The pattern of distribution of labelled cells is shown schematically in Fig. 1 on sections located near the junction between the urogenital ducts and the urodeum.

Fig. 1

Distribution of oestrogen target cells in the urogenital tract of the chicken embryo at 7 (a), 10 (b) and 15 (c) days of incubation. Cells containing receptor sites for [3H]oestradiol ([3H]E2) are localized in autoradiograms of frozen sections. Schematic drawings of transverse sections near the junction between urogenital ducts and urodeum. Black dots on left side represent cells which concentrate the radiolabelled hormone in their nuclei. The relative intensity of labelling is indicated by the size of the dots, and the density of labelled cells by the density of dots. (a) In 7-day embryos, male or female, cells labelled with [3H]E2 are concentrated in the mesenchyme bordering the cloacal epithelium (detail on Fig. 3 A). (b) In 10-day embryos the condensation of mesenchymal cells forming the vascular body appears on each side of the urodeum in embryos of both sexes, as a large cluster of labelled cells (detail on Fig. 4C). (c) In 15-day embryos cells labelled with [3H]E2 are abundant along the epithelium of all sections of the cloaca. A higher density and intensity is observed at the junction of the urogenital ducts, while the Müllerian duct displays a weaker labelling (detail on Fig. 5D). The embryo represented here is a female and the vascular body is already involuted. In male embryos the vascular body contains a high density of [3H]E2-labelled cells. Abbreviations for Figs 1 & 2: BF, bursa of Fabricius; Cd, coprodeum; Coel, coelomic cavity; M, muscle; MD, Müllerian duct; Ud, urodeum; Ur, ureter; VB, vascular body; WD, Wolffian duct.

Fig. 1

Distribution of oestrogen target cells in the urogenital tract of the chicken embryo at 7 (a), 10 (b) and 15 (c) days of incubation. Cells containing receptor sites for [3H]oestradiol ([3H]E2) are localized in autoradiograms of frozen sections. Schematic drawings of transverse sections near the junction between urogenital ducts and urodeum. Black dots on left side represent cells which concentrate the radiolabelled hormone in their nuclei. The relative intensity of labelling is indicated by the size of the dots, and the density of labelled cells by the density of dots. (a) In 7-day embryos, male or female, cells labelled with [3H]E2 are concentrated in the mesenchyme bordering the cloacal epithelium (detail on Fig. 3 A). (b) In 10-day embryos the condensation of mesenchymal cells forming the vascular body appears on each side of the urodeum in embryos of both sexes, as a large cluster of labelled cells (detail on Fig. 4C). (c) In 15-day embryos cells labelled with [3H]E2 are abundant along the epithelium of all sections of the cloaca. A higher density and intensity is observed at the junction of the urogenital ducts, while the Müllerian duct displays a weaker labelling (detail on Fig. 5D). The embryo represented here is a female and the vascular body is already involuted. In male embryos the vascular body contains a high density of [3H]E2-labelled cells. Abbreviations for Figs 1 & 2: BF, bursa of Fabricius; Cd, coprodeum; Coel, coelomic cavity; M, muscle; MD, Müllerian duct; Ud, urodeum; Ur, ureter; VB, vascular body; WD, Wolffian duct.

Fig. 2

Distribution of androgen target cells in urogenital tract of the chicken embryo at 7 (fl), 10 (b) and 15 (c) days of incubation. Same symbols and abbreviations as Fig. 1. The radio-labelled hormone injected was [3H]dihydrotestosterone ((3H]DHT). (A) In 7-day embryos cells labelled with [3H]DHT are scattered in the mesenchyme of the urodeum, coprodeum and bursa of Fabricius. The epithelium of the Wolffian duct displays some weakly labelled cells. Note the group of intensely labelled cells apart from the urogenital tract. (b) in 10-day embryos labelled cells are abundant in the mesenchyme, specially in the condensation which forms the vascular body (detail on Fig. 4a). (c) in 15-day embryos cells labelled with [3H]-DHT are widespread in the cloacal area to the exclusion of the ureter which always remains free of nuclear labelling. In the vascular body, represented here in a male embryo, the concentration of androgenic hormone is particularly high when compared to other labelled cells of the cloacal region.

Fig. 2

Distribution of androgen target cells in urogenital tract of the chicken embryo at 7 (fl), 10 (b) and 15 (c) days of incubation. Same symbols and abbreviations as Fig. 1. The radio-labelled hormone injected was [3H]dihydrotestosterone ((3H]DHT). (A) In 7-day embryos cells labelled with [3H]DHT are scattered in the mesenchyme of the urodeum, coprodeum and bursa of Fabricius. The epithelium of the Wolffian duct displays some weakly labelled cells. Note the group of intensely labelled cells apart from the urogenital tract. (b) in 10-day embryos labelled cells are abundant in the mesenchyme, specially in the condensation which forms the vascular body (detail on Fig. 4a). (c) in 15-day embryos cells labelled with [3H]-DHT are widespread in the cloacal area to the exclusion of the ureter which always remains free of nuclear labelling. In the vascular body, represented here in a male embryo, the concentration of androgenic hormone is particularly high when compared to other labelled cells of the cloacal region.

Labelled cells are encountered in the mesenchyme at all levels between the coprodeum and the opening of the proctodeum, including the genital tubercle. They are more numerous in the close vicinity of epithelial structures of the cloaca (Fig. 3 A and B), and also, from day 10 onwards, in the vascular body (Fig. 4C).

Fig. 3

Autoradiograms of the urodeum (middle portion of the cloaca) from chick embryos injected with [3H]oestradiol or [3H]dihydrotestosterone. (A) In 7-day embryos target cells that concentrate 3H-E2 in their nuclei are found exclusively in the mesecnchyme lining closely the endodermic epithelium which appears free of labelling. Exposure time 5 months; x 1100. (B) In 12-day embryos target cells for [3H]E2 are localized in the mesenchyme in close contact with the epithelium. Exposure time: 14 months; × 1700. (C) When an excess of unlabelled oestradiol is injected in addition to [3H]E2 the two types of hormone molcules compete for the same receptor sites, and the nuclear labelling is abolished (12-day embryo). Exposure time: 14 months; × 1700. (D) In 15-day embryos target cells that concentrate [3H]DHT are located primarily in the mesenchyme. Note that unlike [3H]E2-labelled cells, the [3H]DHT-labelled cells are not clustered along the endodermic epithelium. Exposure time: 7 months; × 1100. (E) When an excess of unlabelled DHT competes with [3H]DHT for the receptor sites the labelling is abolished. Exposure time: 9 months; × 1100. Abbreviations: e, endodermic epithelium; m, mesenchyme. .

Fig. 3

Autoradiograms of the urodeum (middle portion of the cloaca) from chick embryos injected with [3H]oestradiol or [3H]dihydrotestosterone. (A) In 7-day embryos target cells that concentrate 3H-E2 in their nuclei are found exclusively in the mesecnchyme lining closely the endodermic epithelium which appears free of labelling. Exposure time 5 months; x 1100. (B) In 12-day embryos target cells for [3H]E2 are localized in the mesenchyme in close contact with the epithelium. Exposure time: 14 months; × 1700. (C) When an excess of unlabelled oestradiol is injected in addition to [3H]E2 the two types of hormone molcules compete for the same receptor sites, and the nuclear labelling is abolished (12-day embryo). Exposure time: 14 months; × 1700. (D) In 15-day embryos target cells that concentrate [3H]DHT are located primarily in the mesenchyme. Note that unlike [3H]E2-labelled cells, the [3H]DHT-labelled cells are not clustered along the endodermic epithelium. Exposure time: 7 months; × 1100. (E) When an excess of unlabelled DHT competes with [3H]DHT for the receptor sites the labelling is abolished. Exposure time: 9 months; × 1100. Abbreviations: e, endodermic epithelium; m, mesenchyme. .

Fig. 4

Autoradiograms of the vascular body, or lymphobulus, of chick embryos injected with either [3H]oestradiol or [3H]dihydrotestosterone. (A) in 10-day embryos, as the vascular body still displays a loose mesenchymal structure, cells that concentrate [3H]DHT in their nuclei are already numerous. Exposure time: 7 months; × 1100. (B) in 15-day embryos the vascular body, in males, appears more compact; more than 50% of the cells are labelled with [3H]DHT. Although this embryo also received a 100-time excess unlabelled oestradiol the nuclear labelling is little affected. In female embryos at the same age, the vascular body is involuted. Exposure time: 7 months; × 1100. (C) in 12-day embryos [3H]E2 also is concentrated in numerous cells of the vascular body. Exposure time: 14 months; × 1100. (D) When a 100-times excess of unlabelled oestradiol competes for the same receptor sites as [3H]E2 the nuclear labelling is abolished. Exposure time: 14 months; × 1100.

Fig. 4

Autoradiograms of the vascular body, or lymphobulus, of chick embryos injected with either [3H]oestradiol or [3H]dihydrotestosterone. (A) in 10-day embryos, as the vascular body still displays a loose mesenchymal structure, cells that concentrate [3H]DHT in their nuclei are already numerous. Exposure time: 7 months; × 1100. (B) in 15-day embryos the vascular body, in males, appears more compact; more than 50% of the cells are labelled with [3H]DHT. Although this embryo also received a 100-time excess unlabelled oestradiol the nuclear labelling is little affected. In female embryos at the same age, the vascular body is involuted. Exposure time: 7 months; × 1100. (C) in 12-day embryos [3H]E2 also is concentrated in numerous cells of the vascular body. Exposure time: 14 months; × 1100. (D) When a 100-times excess of unlabelled oestradiol competes for the same receptor sites as [3H]E2 the nuclear labelling is abolished. Exposure time: 14 months; × 1100.

An identical distribution is observed in male and female embryos, except for the structures that display an anatomical difference between sexes, such as the Mullerian duct and the vascular body on day 15 (Fig. 1C). The vascular body is a dense cluster of cells, laterally located on each side of the urodeum. In a preliminary note this structure was referred to as a ‘cluster of cells’ (Gasc et al. 1978) which corresponds to the vascular body, or Lymphobulbus, described by Berens von Rautenfeld, Budras & Gassman (1976; and personal communication) in the young chick and adult cock. The vascular body is not yet present on day 7, and it disappears in female embryos between days 12 and 15. Throughout its existence, more than 50% of its cells are radioactively labelled after [3H]E2 injection, about 60% at day 12 (Fig. 4C). On day 15 of incubation this structure, then absent in females, is well developed in males and the cells display an intense nuclear labelling.

Mullerian duct

No nuclear concentration of labelled oestradiol could be found in the epithelium of this structure at any stage studied. Stromal cells which surround the Müllerian duct take up [3H]E2 but only in day-15 female embryos (Fig. 1C, and 5C and D); the intensity of the nuclear labelling is lower than in cells of the cloacal mesenchyme or the vascular body.

Wolffian duct

Epithelial cells of the Wolffian duct in 12- and 15-day embryos are labelled with [3H]E2 on a short stretch starting from the merging with the cloaca and ending approximately half way to the dorsal side of the abdominal cavity. Mesenchymal cells along the Wolffian duct are not labelled (Fig. 5B).

Fig. 5

Autoradiograms of the Wolffian and Müllerian ducts of embryos injected with either [3H]oestradiol or [3H]dihydrotestosterone. Cells concentrating [3H]DHT in their nuclei are observed both in the luminal epithelium and the mesenchyme of the Wolffian duct (A) while cells concentrating [3H]E2 are exclusively located in the epithelium (B). Note that the labelling with [3H]DHT in the mesenchyme increases outward the Wolffian duct. In the Müllerian duct no cell concentrating [3H]E2 is observed in 10-day embryos (C) while at day 15 of incubation a weak concentration of [3H]E2 appears in the mesenchyme (D). This nuclear labelling appears much lower than in adjacent structures of the same embryo such as the urodeum or the luminal epithelium of the Wolffian duct. A and C: 15-day embryos; B and D: 12-day embryos. A, C and D: transversal sections; B: longitudinal section. Abbreviations: e, luminal epithelium; m, mesenchyme. Exposure times: A, 9 months; B, 7 months; C, 12 months; D, 14 months. Magnification: A and B, × 1100; C and D, × 750.

Fig. 5

Autoradiograms of the Wolffian and Müllerian ducts of embryos injected with either [3H]oestradiol or [3H]dihydrotestosterone. Cells concentrating [3H]DHT in their nuclei are observed both in the luminal epithelium and the mesenchyme of the Wolffian duct (A) while cells concentrating [3H]E2 are exclusively located in the epithelium (B). Note that the labelling with [3H]DHT in the mesenchyme increases outward the Wolffian duct. In the Müllerian duct no cell concentrating [3H]E2 is observed in 10-day embryos (C) while at day 15 of incubation a weak concentration of [3H]E2 appears in the mesenchyme (D). This nuclear labelling appears much lower than in adjacent structures of the same embryo such as the urodeum or the luminal epithelium of the Wolffian duct. A and C: 15-day embryos; B and D: 12-day embryos. A, C and D: transversal sections; B: longitudinal section. Abbreviations: e, luminal epithelium; m, mesenchyme. Exposure times: A, 9 months; B, 7 months; C, 12 months; D, 14 months. Magnification: A and B, × 1100; C and D, × 750.

When a 100-fold excess of unlabelled oestradiol competes with [3H]E2 for the binding sites the nuclear labelling is abolished (Fig. 3C and 4D). In contrast, it remains unchanged after a competition by a 100-fold excess of dihydrotestosterone.

Androgen target cells

Nuclear concentration of [3H]DHT is observed in mesenchymal cells of the cloacal region from the coprodeum to the opening of the cloaca, including the genital tubercle. The schematic drawings of Fig. 2 show the pattern of distribution of labelled cells on a cross section near the merging of the urogenital ducts in the cloaca in 7-, 10- and 15-day embryos. Similar distribution are observed in male and female embryos, except at day 15 when anatomical differences exist. [3H]DHT-labelled cells (Fig. 3D) do not appear to line the cloacal epithelium so closely as those labelled with [3H]E2 (Fig. 3B) but appear more scattered throughout the mesenchyme of this region.

The vascular body at day 10 (Fig. 4A) and 12 in both sexes, and at day 15 in males (Fig. 4B) contains a high proportion of cells that concentrate [3H]DHT in their nuclei. At day 12 the percentage of labelled cells amounts to about 70%.

Müllerian duct

The Müllerian duct does not display any nuclear concentration of [3H]DHT at any stage between day 7 and 15, either at the level of the mesonephros or the cloaca. Nor is any high density of labelling observed in the cytoplasm or intercellular matrix.

Wolffian duct

The junction between the Wolffian duct and the urodeum is marked by a high density of labelled cells in the mesenchyme (Fig. 2B and 2C). Numerous labelled cells are also found in the mesenchyme along the Wolffian duct, especially in the outer layers (Fig. 5 A). The labelling is not continuous and uniform along the periphery and the length of the Wolffian duct. On some portions it is weak and sparse while on some others it is more intense. In addition to mesenchymal cells, epithelial cells concentrate [3H]DHT in their nuclei (Fig. 5A). The high concentration of radioactivity sometimes found in the lumen of the Wolffian duct masks in certain instances the labelling in nuclei of the epithelium. In contrast to the [3H]E2 labelling of only a short portion of the Wolffian duct epithelium, the [3H]DHT labelling extends from the urodeum up to the dorsal side of the abdominal cavity. However, in the same animals the Wolffian duct does not display [3H]DHT-labelled cells along its pathway through the mesonephros. Nor are there any cells with nuclear concentration in the mesonephros itself. Unlike the Wolffian duct, the ureter is devoid of cells concentrating [3H]DHT at any level and along any portion of its pathway.

In addition to mesenchyme, cells of the cloacal epithelium display a weak nuclear concentration of [3H]DHT. As this labelling is low, in particular lower than in the neighbouring mesenchymal cells, it is often difficult to distinguish from the background labelling. Though weak and not so conspicuous as in other tissues, the nuclear labelling of the epithelial cells appears consistently in 12- and 15-day embryos. Some large blood vessels in the caudal region are surrounded by labelled mesenchymal cells, bordering the endothelium. These labelled cells are restricted to the very close vicinity of the vessel wall with which they seem to fuse.

In 7-day embryos a group of mesenchymal cells laterally located near the attachment of the limb to the pelvian cartilage shown an unusually high intensity of nuclear labelling after [3H]DHT injection (Fig. 2 A). This cluster of cells never exceeds 20 – 30 cells on each section, and each cell appears very intensely labelled compared to the other mesenchymal cells of the cloacal region. This group of cells does not seem to be adjacent or related to any epithelial structure. By its location these cells do not correspond to the future vascular body, unless they migrate toward the urodeum between day 7 and 10. The absence of [3H]E2 uptake in these particular cells at day 7 also suggests they are not precursors of the vascular body which at day 10 contains cells labelled both after [3H]E2 and [3H]DHT injection.

When unlabelled DHT or testosterone in excess is applied to the embryo with [3H]DHT, the nuclear labelling is abolished in all cells of the cloacal region (Fig. 3E). On the contrary, a 100-fold excess of oestradiol or progesterone does not modify the distribution of labelling but slightly diminishes the intensity of [3H]DHT labelling (Fig. 4B).

The uptake and concentration of radioactivity after injection of either [3H]E2 or [3H]DHT attests to the presence of binding sites for these hormones in certain cell nuclei. The competition exerted by a 100-fold excess of the same unlabelled hormone and the weak or lack of competition by a different unlabelled hormone in large excess suggest that the nuclear binding sites have a limited capacity and a specificity for only one type of hormone, either androgen or oestrogen. Presumably, cells concentrating one or the other hormone in their nuclei are the ones whose activity is directly affected by this type of steroid hormone. They are considered target cells for androgen or oestrogen.

Oestrogen target cells

The presence of oestrogen target cells in the cloacal region of the chick embryo is in agreement with the works of Wolff & Wolff (1951) and Reinbold (1951) who showed the inhibitory effects of oestradiol on the genital tubercle and other structures surrounding the opening of the cloaca by means of hormonal treatments and castration. The present data outline the exact location of the target tissues, and indicate that this differentiating role extends to the whole cloacal region. While the presence of oestrogen target cells at day 12 and 15 of incubation is consistent with the established chronology of sexual differentiation (Reinbold, 1951; Wolff & Wolff, 1951), the localization of target cells as early as day 6 or 7 suggests a much more precocious role of steroids on the sexual phenotype.

In a preliminary note the absence of target cells for oestradiol in the lower part of the Müllerian duct of 12-day embryos was reported (Gasc et al. 1978). The present observations confirm this result but also show the appearance of target cells in the stroma surrounding the Müllerian duct of 15-day female embryos. Though consistently observed this nuclear labelling is much weaker than in other mesenchymal cells of the urodeum on the same section. The reason for this difference in intensity is not clear since the Müllerian duct is a target organ for oestrogen (Scheib-Pfleger, 1953; Rahil & Narbaitz, 1972) which is reported to contain oestrogen receptors from day 8 of incubation onwards (Teng & Teng, 1976).

A high level of occupancy of receptor sites by endogenous unlabelled hormone may account for the weak labelling in stromal cells of the Müllerian duct. Another explanation is that nuclear receptors are present in small number until day 12 and increase strongly between day 12 and 15. This is consistent with the five-fold increase in endogenous nuclear-binding sites for oestradiol observed in the Müllerian duct between day 12 and 15 (Teng & Teng, 1976). An absence or low level of oestrogen-binding sites until day 15 is in agreement with the normal development of the Müllerian duct which is apparently similar in female control embryos and female castrated embryos (Wolff, 1950; Wolff & Wolff, 1951), at least until the last days of incubation.

Androgen target cells

Androgen target cells are also present in large number in the cloacal region, following a definite topographical distribution.

According to Wolff (1950), and Reinbold (1951) testosterone stimulates the growth and development of this region during the last days of incubation. Since testosterone in plasma (Woods, Simpson & Moore, 1975; Gasc & Thibier, 1979), and androgen-binding sites in target tissues are present before day 10 of incubation, one may expect an effect of androgens on the differentiation of the cloacal region to occur earlier in the development.

Of the three sets of urogenital ducts only the Wolffian duct display target cells for androgens. Cells in the epithelium and the mesenchyme concentrate [3H]DHT in their nuclei. This is probably related to the transformation of the embryonic urinary duct into a genital duct, i.e. the ducts deferens. During the same period of development the mesonephros also undergoes a transformation from a urinary function to a reproductive function (epididymis), but no androgen target cells are observed in this organ, not even in the Wolffian derivatives. The absence of androgen-binding sites is consistent with the identical development of the mesonephros in male, female and testosterone-treated embryos from day 7 to 18 of incubation (Croisille, Gasc & Gumpel-Pinot, unpublished results). Our observation confirms that the redifferentiation of the mesonephros is not sex-steroid dependent during embryonic life (Maraud, Cambar, Vergnaud & Stoll, 1980). It is only in the young chicken that testosterone achieves the functional differentiation of the epididymis. As concerns the presence of oestrogen target cells in the lower part of the Wolffian duct, it cannot be related to any known effect of the hormone.

Among the target tissues, the vascular body offers a particular instance of a dual presence of receptor sites in the same cells. In day-12 embryos receptor sites for one or the other of the two types of sex steroid are present in more than 50% of the cells. Since the two types of receptors are different, as shown by cross-competition experiments, one can conclude a dual presence of receptors in the same cells. The fact that the two types of receptors mediate opposite effects, ‘stimulation’ for androgen and ‘inhibition’ for oestrogen, adds to the peculiarity of this structure (Berens von Rautenfeld, 1978).

Sex-steroid target cells in mesechyme

Mesenchyme and mesenchyme-derived tissues would seem to be the main targets for steroid hormones by their preferential uptake of tritiated oestradiol and DHT. Mesenchymes are probably the site where the information carried by steroids is first received before being conveyed to the epithelium. Not only in the chicken embryo but also in the 16-day mouse fetus oestrogen target cells are found exclusively in the mesenchyme of the Müllerian duct and urogenital sinus (Stumpf, Narbaitz & Star, 1980) with a pattern of distribution that displays similarities to the chicken embryo. The importance of certain mesenchymes as target tissue for sex steroids is further substantiated by experiments which aim at testing the sensitivity to testosterone of the two tissue components of a primordium. In the mammary rudiment (Kratochwil & Schwartz, 1976; Kratochwil, 1977; Drews & Drews, 1977), the urogenital sinus (Cunha & Lung, 1979; Cunha et al. 1980), and the prostatic bud (Lasnitzki & Mizuno, 1979) the mesenchyme is the target tissue sensitive to treatment with steroid hormones, while the epithelium remains unchanged in its differentiating capacities. In contrast, it is the epithelium of the bursa of Fabricius which mediates the involution of this immune organ after androgen treatment (Le Douarin, Michel & Baulieu, 1980).

Although a uniform pattern of steroid effects on embryonic tissues cannot be proposed on the basis of our observations, there is increasing evidence for ascribing a key role to mesenchymes in the differentiation processes controlled by steroid hormones. It is a specific contribution of autoradiography to discriminate between two apparently identical mesenchymes on the basis of their affinity for one type of steroid hormone. By means of the technique employed, this property of certain mesenchymes is visualized and thereby more more accessible to investigation.

Precocity of sex-steroid target cells

Very early in the development receptor sites for oestrogens and androgens are observed in target cells. For the youngest embryos we have studied, day 6 for [3H]E2 and day 7 for [3H]DHT, labelled cells are present in the mesenchyme of the cloacal region. Other examples of the precocity of the receptors in embryonic tissues are reported elsewhere (Gasc, 1980; Gasc & Stumpf, 1981). As no competition experiments were carried out with young embryos, the hormonal specificity of the receptors is not directly established. However, the specificity at later stages on the one hand, and the difference in distribution of [3H]E2 and [3H]DHT-labelled cells on the other hand, strongly suggest that receptors for oestrogen and androgen are distinct from each other throughout the embryonic life.

The presence of receptors detected by autoradiography is not by itself unequivocal evidence that steroid hormones actually modify the activity of the cell. However, steroid hormones are present in the embryo at a sufficient concentration known to exert differentiating effects in target organs. Such is the case, for instance, for the embryonic gonads of the chick embryo (Gasc, 1980), and also the mammary rudiment of the mouse fetus which responds to testosterone as soon as this hormone is produced in measurable amounts by the gonads (Kratochwil, 1977). The complimentarity of the results in birds and mammals lends support to admit that receptors are active at day 6 or 7 of incubation in the chick embryo.

Finally, it is noteworthy that in no instance the distribution of [3H]E2- and [3H]DHT-labelled cells appears different in male and female in early stages of development. Unless unsuspected quantitative differences exist, the presence of sex-steroid hormone receptors represents a sexually neutral character that confirms at the cellular level, the classical theory of a neutral phenotypical sex.

The authors are indebted to Dr M. Sar (The University of North Carolina at Chapel Hill) for his help and stimulative advice during the course of this work. Supported by a fellowship from the D.G.R.S.T. (Paris, France) to J.-M. Gasc, and a PHS grant NS 09914 to W. E. Stumpf.

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