/ . Embryol. exp. Morph. Vol. 63, pp. 207-223, 1981
Printed in Great Britain © Company of Biologists Limited 1981
207
Sexual differentiation of the urogenital tract
in the chicken embryo: autoradiographic
localization of sex-steroid target
cells during development
By JEAN-MARIE GASC1 AND WALTER E. STUMPF2
From the Department of Anatomy, The University of
North Carolina at Chapel Hill, U.S.A.
SUMMARY
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 Miillerian 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 Miillerian 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.
INTRODUCTION
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
1
Author's address: Institut d'Embryologie du C.N.R.S. et du College de France,
49bis, avenue de la Belle Gabrielle, 94130-Nogent-sur-Marne, France.
2
Author's address: Department of Anatomy, The University of North Carolina at
Chapel Hill, U.S.A.
208
J-M. GASC AND W. E. STUMPF
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 Miillerian
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 sexsteroid 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 (Miillerian, 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
Target cells for sex steroid in urogenital tract
209
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).
MATERIAL AND METHODS
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 fog
(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/ig (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 jtig (2 females and 2 males at day 7) or 0-03 (2 females and 2 males
at day 10), or 004/^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
I 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 - 3 5 °C
(W.R. model, Harris Manufacturing Co., North Billerica, Massachusetts,
U.S.A.). Sections, 3 //m thick, were mounted on slides coated with photo-
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J-M. GASC AND W. E. STUMPF
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).
RESULTS
Oestrogen target cells
3
Cells concentrating [ H]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.
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).
An identical distribution is observed in male and female embryos, except
for the structures that display an anatomical difference between sexes, such
as the Mtillerian duct and the vascular body on day 15 (Fig. 1C). The vascular
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 Miillerian
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, Miillerian duct; Ud, urodeum; Ur, ureter; VB,
vascular body; WD, Wolffian duct.
Target cells for sex steroid in urogenital tract
(a)
Coel
d and 9 7 day
(b)
WD
VB
dand9 10 day
WD
9 15 day
211
212
J-M. GASC AND W. E. STUMPF
Coel
(a)
WD
6 and 9 7 day
Ur
(b)
WD
dand9 10 day
6 15 day
Target cells for sex steroid in urogenital tract
213
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 at. 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 Mullerian 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).
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
Fig. 2. Distribution of androgen target cells in urogenital tract of the chicken embryo
at 7 (a), 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 wide*
spread 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.
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J-M. GASC AND W. E. STUMPF
Target cells for sex steroid in urogenital tract
215
3
differences exist. [ H]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%.
Mullerian duct. The Miillerian 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.
Wolffion 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. 5 A). 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
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; x 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; x 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; x 1100. (E) When an excess of unlabelled DHT
competes with [3H]DHT for the receptor sites the labelling is abolished. Exposure
time: 9 months; xllOO.
Abbreviations: e, endodermic epithelium; m, mesenchyme.
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J-M. GASC AND W. E. STUMPF
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;
x 1100. (B) in 15-day embryos the vascular body, in mates, 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; xllOO. (C) in 12-day embryos [3H]E2 also is concentrated in
numerous cells of the vascular body. Exposure time: 14 months; xllOO. (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; x 1100.
Target cells for sex steroid in urogenital tract
217
3
nuclear concentration of [ H]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).
DISCUSSION
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
218
J-M. GASC AND W. E. STUMPF
Target cells for sex steroid in urogenital tract
219
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 Miillerian 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 Miillerian 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 Miillerian 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 Miillerian 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 Miillerian 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 Miillerian 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.
Fig. 5. Autoradiograms of the Wolffian and Miillerian 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 Miillerian 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, x 1100; C and D, x 750.
220
J-M. GASC AND W. E. STUMPF
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),butnoandrogen
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 ^differentiation 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 Miillerian 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 &
Target cells for sex steroid in urogenital tract
221
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
3
[ H]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.
8
EMB 63
222
J-M. GASC AND W. E. STUMPF
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.
REFERENCES
BERENS VON RAUTENFELD, D. (1978). Die Kloake als Testorgan fur die Wirkung endogener
und exogener Geschlechtssteroide beim Huhn (Gallus domesticus). ZentBl Vet. Med. A
25, 23-40.
BERENS VON RAUTENFELD, D., BUDRAS, K.-D. & GASSMANN, R. (1976). A morphological
study of antibody transport in the transparent fluid flowing from the lymph folds of the
copulatory organ into the cloacal lumen of the cock (Gallus domesticus). Z. Mikrosk.anat. Forsch. 90, 989-1008.
CUNHA, G. R., CHUNG, L. W. K., SHANNON, J. M. & REESE, B. A. (1980). Stromal-epithelial
interactions in sex differentiation. Biology of Reproduction 22, 19-42.
CUNHA, G. R. & LUNG, B. (1979). The importance of stroma in morphogenesis and functional
activity of urogenital epithelium. In Vitro 15, 50-71.
DREWS, U. & DREWS, U. (1977). Regression of mouse mammary gland anlagen in recombinants of Tfm and wild-type tissues: testosterone acts via the mesenchyme. Cell
10,401-404.
ERICKSON, A. E. & PINCUS, G. (1966). Modification of embryonic development of reproductive and lymphoid organs in the chick. /. Embryol. exp. Morph. 16, 211-229.
GASC, J.-M. (1973). Sur les resultats d'une technique d'identification precoce du sexe
genotypique des embryons d'oiseaux. C. r. hebd. Seanc. Acad. Sci., Paris 111, 19251928.
GASC, J.-M. (1980). Estrogen target cells in gonads of the chicken embryo during sexual
differentiation. /. Embryol. exp. Morph. 55, 331-342.
GASC, J.-M., SAR, M. & STUMPF, W. E. (1980). Immunocharacteristics of oestrogen and
androgen target cells in the anterior pituitary of chick embryo demonstrated by a combined
method of autoradiography and immunohistochemistry. J. Endocrinol. 86, 245-250.
GASC, J.-M. & STUMPF, W. E. (1981). The bursa of Fabricius of the chicken embryo: localization and ontogenic evolution of sex-steroid target cells. / . Embryol. exp. Morph. 63,
225-231.
GASC, J.-M., STUMPF, W. E. & SAR, M. (1978). Estrogen target sites in the cloacal region
of female and male chick embryos. Cell and Tissue Res. 193, 457-462.
GASC, J.-M. & THIBIER, M. (1979). Plasma testosterone concentration in control and
testosterone treated chick embryo. Experientia 35, 1411-1412.
JOST, A. (1950). Sur le controle hormonal de la differenciation sexuelle du lapin. Archs
Anat. micr. Morph. exp. 39, 577-607.
KRATOCHWIL, K. (1977). Development and loss of androgen responsiveness in the embryonic
rudiment of the mouse mammary gland. Devi Biol. 61, 358-365.
KRATOCHWIL, K. & SCHWARTZ, P. (1976). Tissue interaction in androgen response of
embryonic mammary rudiment of mouse. Identification of target tissue for testosterone.
Proc. natn. Acad. Sci., U.S.A. 73, 4041-4044.
LASNITZKI, I. & MIZUNO, T. (1979). Role of the mesenchyme in the induction of the rat
prostatic gland by androgen in organ culture. /. Endocrin. 82, 171-178.
LE DOUARIN, N. M., MICHEL, G. & BAULIEU, E.-E. (1980). Studies of testosterone-induced
involution of the bursa of Fabricius. Devi Biol. 75, 288-302.
MARAUD, R., CAMBAR, J., VERGNAUD, O. &STOLL, R. (1980). Etude au microscope electronique
de 1'evolution du glomerule des corpuscules de Malpighi mesonephretiques au cours de
la differenciation epididymaire chez le poulet. Biologie cellulaire 38, 61-64.
OMURA, T. (1970). Prediction of sex of embryonal chick from chromosomal examination
of extraembryonal blood. Caryologia 23, 155-158.
RAHIL, K. S. & NARBAITZ, R. (1972). Differentiation of male chick oviducts under hormonal
stimulation. Gen. comp. Endocrin. 18, 315-318.
Target cells for sex steroid in urogenital tract
223
A. (1950). Recherches experimentales sur le developpement de l'appareil genital
et le fonctionnement des glandes endocrines des foetus de souris et de mulot. Archs
Anat. micr. Morph. exp. 39, 518-576.
REINBOLD, R. (1951). Le rudiment de tubercule genital du poulet: developpement embryonnaire et sensibilite aux hormones sexuelles. Bull. biol. Fr. Belg. 85, 347-367.
ROMANOFF, A. L. (1960). The Avian Embryo. New York: The Macmillan Company.
SCHEIB-PFLEGER, D. (1953). Role des hormones sexuelles sur le developpement in vitro des
canaux de Miiller de l'embryon de poulet femelle differencie. C. r. hebd. Seanc. Acad.
Sci., Paris 236, 1446-1448.
STUMPF, W. E., NARBAITZ, R. & SAR, M. (1980). Estrogen receptors in the fetal mouse.
J. Ster. Biochem. 12, 55-64.
STUMPF, W. E. & SAR, M. (1975). Autoradiographic techniques for localizing steroid
hormones. In Methods in Enzymology, Hormone Action, Part A. Steroid Hormones (ed.
B. W. O'Malley & J. G. Hardman), pp. 135-156. New York: Academic Press.
TENG, C. S. & TENG, C. T. (1976). Studies on sex organ development. Oestrogen-receptor
translocation in the developing chick mullerian duct. Biochem. J. 154, 1-9.
TRAN, D. & Josso, N. (1977). Relationship between avian and mammalian antimiillerian
hormones. Biol. of Reprod. 16, 267-273.
WOLFF, EM. (1950). La differenciation sexuelle normale et le conditionnement hormonal
des caracteres sexuels somatiques precoces, tubercule genital et syrinx, chez l'embryon
de canard. Bull. biol. Fr. Belg. 84, 119-193.
WOLFF, ET. (1950). Le role des hormones embryonnaires dans la differenciation sexuelles
des oiseaux. Archs Anat. micr. Morph. exp. 39, 426-450.
WOLFF, ET. & WOLFF, EM. (1951). The effects of castration on bird embryos. /. exp. Zool.
116, 59-98.
WOODS, J. E., SIMPSON, R. M. & MOORE, P. L. (1975). Plasma testosterone levels in the
chick embryo. Gen. comp. Endocrin. 27, 543-547.
RAYNAUD,
{Received 13 August 1980, revised 1 December 1980)
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