/ . Embryo!, exp. Morph. Vol. 55, pp. 331-342, 1980
Printed in Great Britain © Company of Biologists Limited 1980
331
Estrogen target cells in gonads of the chicken
embryo during sexual differentiation
BY JEAN-MARIE GASC 1
From the Department of Anatomy, University of North
Carolina, U.S.A.
SUMMARY
Estrogen target cells were searched for in the differentiating gonads of the chicken embryo
in order (1) to establish at the cellular level that steroid hormones can play a physiological role
in gonadal sexual differentiation, and (2) to localize their sites of action. An autoradiographic
technique carried out with frozen sections was employed to demonstrate the presence of binding sites for radiolabelled hormone in the nuclei of the target cells.
Target cells for [3H]estradiol are found similarly in gonads of both male and female embryos
from 5i (stage 27 of H and H) to 7 days of incubation. Estrogen target cells are observed in
the germinal epithelium of the left but not the right gonad, and in the medulla of both the
right and left gonads. In the medulla, numerous cells inside the cords are a target for estradiol.
The germ cells, difficult to identify unmistakably in the experimental conditions, do not seem
to be a target for estrogen hormones. A 100-fold excess of unlabelled estradiol abolishes the
nuclear labelling, which is only slightly reduced after a similar excess of unlabelled dihydrotestosterone. It is concluded that the nuclear binding sites have a limited capacity for steroid
hormones and are specific for estrogen hormones. The lack of clear and consistent nuclear
labelling after [3H]dihydrotestosterone injection confirms the specificity of the [3H]estradiol
nuclear labelling. At day 10 of incubation, only the undifferentiated remnant of the germinal
epithelium in the left testis still displays labelled cells after [3H]estradiol injection.
These observations confirm the determinative role currently ascribed to the estrogen
hormones in the cortical differentiation, but they also emphasize that this role extends to the
medulla of both gonads. In light of this presence of estrogen receptor sites in the medullary
cords as well as in the germinal epithelium, one can assign the estrogen hormones more
specific and diversified roles than currently believed. These roles also appear very precocious
in the process of gonadal differentiation. Finally, the absence of target cells for estrogen
hormones in the germinal epithelium of the right gonad accounts for the lack of cortical
differentiation on the right side.
INTRODUCTION
Following the original works by Dantchakoff (1935), Willier, Gallagher &
Koch (1935) and Wolff & Ginglinger (1935) showing the feminizing effects of
exogenous estrogen hormones on the gonads of male chicken embryos during
sexual differentiation, many studies have been aimed at establishing the mechanisms and physiological significance of this experimental gonadal sex reversal.
Estrogen hormones were proposed to account for the ovarian differentiation in
1
Author's permanent 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.
332
J.-M. GASC
bird embryos (Wolff, 1950). According to this theory, the ovarian differentiation
occurs in the left female gonad as an estrogen-stimulated cellular proliferation
of the cortex, which gives rise to the cortical cords, that is, the fertile part of the
ovary. The cortical proliferation does not occur in the female right gonad,
despite its production of estrogen hormones (Weniger, 1965; Scheib & Haffen,
1968; Woods & Erton, 1978). In male embryos testicular differentiation results,
in the absence of cortical proliferation, from the transformation of the medullary
cords into the testicular cords.
These histological changes, characteristic of sexual differentiation, are observed in gonads from 7 days of incubation onwards. Other sex-dependent
changes in the development of the gonads are described as occurring simultaneously or previously to histological differentiation: asymmetry of germ cell
distribution (Van Limborgh, 1968), asymmetry of gonadal growth rates
(Mittwoch, Laksmi Narayanan, Delhanty & Smith 1971; Gasc, 1978), and
histochemical and biochemical evidences for sex steroid synthesis (Scheib &
Haffen, 1974; Woods & Podczaski, 1974; Woods & Erton, 1978).
The synthesis and secretion of sex steroids, supposed to be the triggering
agents of ovarian differentiation, are to date the only endocrinological aspects
of sexual differentiation experimentally demonstrated. Since steroids affect their
target cells after binding to the nucleus (Jensen & DeSombre, 1973), we postulated the presence of nuclear receptor sites for estrogen in the gonads of the
chicken embryo at the time when the left female gonad undergoes ovarian
differentiation. This study aims to localize the target cells for estradiol in the
female and male gonads of 5|- to 10-day embryos. In order to assess the specificity of the receptor sites for estrogens, the nuclear binding of an androgen
hormone (dihydrotestosterone or DHT) was also searched for.
Autoradiography after radiolabelled hormone injection, as extensively used
by Stumpf & Sar (1975), was employed to identify and localize the target cells
for sex steroids. Previous studies have shown the presence of sex steroid receptor sites in target organs such as the bursa of Fabricius (Gasc, Sar & Stumpf,
1979), the cloacal region (Gasc, Stumpf & Sar, 1978) and the anterior pituitary
(Gasc et al. 1919a, b) of the chicken embryo. We now report that cells in the
gonads are also target for sex steroid hormones. The results of the present study,
by localizing the cells containing nuclear receptor sites, show which cells can be
sensitive to estrogen hormones and respond to their stimulus during ovarian
differentiation. These observations emphasize the role of estrogen hormones in
the process of gonadal sexual differentiation and reveal novel aspects of their
action in gonadal histogenesis.
MATERIALS AND METHODS
Eggs of White Rock chicken from a commercial poultry farm were incubated horizontally at 38 °C; a small window was opened in the shell above the
Estrogen target cells in gonads of chicken embryo
333
Table 1. Number and sex of embryos used in each age and treatment group
Doses in micrograms (jug) of hormone per embryo (E2: estradiol; DHT: dihydrotestosterone). Stages of development according to Hamburger and Hamilton (1951).
Doses of hormone injected
Age of the embryos
Stage
27
lc?
Stage
29
Of Of
'
'
001 fig 3 H-E 2
19, lc?
1/tg E2 and 001/tg 3 H-E 2
1/tg DHT and 001/xg 3 H-E 2
002 fig 3 H-DHT
003 fig 3 H-DHT
Stage
28
?
Stage
30
Day
7
Day
10
19, 2c?
19, lc?
19, lc?
19, lc?
29,2c?
29,2c?
29,2c?
embryo. After 4 days of incubation the genetic sex was determined by a caryological technique using blood cells to observe mitotic chromosomes (Omura,
1970; Gasc, 1973). At 5|, 6, 7 and 10 days of incubation, the embryos were
intravenously injected with either [2,4,6,7,16,17-3H]-estradiol (3H-E2), specific
activity 141 Ci/m mole, or with [1,2,4,5,6,7,16,17-3H(N)] dihydrotestosterone
(3H-DHT), specific activity 190 Ci/m mole. Embryos received either 0-01 fig
3
H-E2 in 005 or 0-1 ml 5% ethanol isotonic saline solution, or 0-02 /*g (5| to
7-day embryos) or 0-03 /*g (10-day embryos) 3 H-DHT in similar conditions.
Solutions of radiolabelled hormones were injected with a 30-gauge needle into
an extraembryonic vein. In addition some embryos received a 100-fold excess of
unlabelled hormone (E2 or DHT) on the chorioallantoic membrane, 30 min before the radiolabelled hormone injection. Details on the number, sex and age of
the embryos and the hormones they received are shown in Table 1. The stage
of development of 5£- and 7-day embryos was determined at the time of decapitation, according to the table of Hamburger & Hamilton (1951); the 10-day
embryos were staged according to the time of incubation. All embryos were
sacrificed by decapitation one hour after radiolabelled hormone injection. The
whole body (5£ to 7 days) or the dissected gonadal region (10 days) was
mounted on a brass holder, partially embedded in minced fresh rat liver, and
frozen in liquefied propane. The embryos were stored in liquid nitrogen prior to
cutting in a cryostat at - 35 °C (Model W. R., Harris Mfg. Co., North Billerica,
Mass., U.S.A.). Sections, 3/tm thick, were cut and then mounted under safelight on emulsion-precoated slides (NTB3, Kodak) according to the thaw-mount
autoradiography technique described by Stumpf & Sar (1975). After 6-11
months exposure, slides were photographically processed (D19 developer and
Rapid fixer, Kodak), and stained with methyl green-pyronin.
EMB 55
334
9 10 days
6 10 days
Fig. 1. Schematic distribution of estrogen target cells in the gonads of the chicken
embryo. Target cells for [3H]estradiol, represented by the dots, display the same
pattern of distribution in male and female gonads at day 6; at day 10, only the
epithelium of the left testis contains cells showing nuclear concentration of estradiol.
FIGURE 2
Autoradiograms of gonads of chicken embryos (6 and 6£ days of incubation) injected with [3H]estradiol (3H-E2). When 3H-E2 is injected alone, radioactivity is
concentrated in cell nuclei of the left germinal epithelium (A and B), the left (A and
C) and right (D) medulla, but not the right germinal epithelium (D). When a 100fold excess of unlabelled estradiol is applied to the embryo prior to 3H-E2 injection,
the nuclear concentration of radioactivity is abolished in the left germinal epithelium (F) and in the medulla of both gonads (not shown); in contrast, when a 100fold excess of dihydrotestosterone is similarly applied, the nuclear concentration of
radioactivity is not abolished in the left germinal epithelium (E) and the medulla
(G). No difference is seen between male (A, B, C, D, E, F) and female embryos (G).
Note the absence of nuclear labelling in the layer of cells located between germinal
epithelium and medulla of the left gonad (A) and the retention of radioactivity in the
coelomic fluid (A, B, D, E, F). Abbreviations: GE, germinal epithelium; Md, medulla; Coel., coelomic cavity. Time of exposure: 11 months. Stained with methyl greenpyronin. A, D, E, F, G: x 850; B and C: x 1400.
Estrogen target cells in gonads of chicken embryo
\~*?< /^£'£''-'
335
C(?el
i»
•••«•*•
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336
J.-M. GASC
Fig. 3. Autoradiograms of gonads of 7-day chicken embryos injected with [3H]dihydrotestosterone (3H-DHT). Neither the germinal epithelium (A), nor the medulla,
(B) contains cells displaying a clear concentration of radioactivity in their nuclei.
In the medulla, a weak and inconsistent nuclear concentration is sometimes observed (arrows) but not as clearly as after [3H]estradiol injection (compare with
Fig. 2). Abbreviations: coel., coelomic cavity; GE, germinal epithelium. Stained with
methyl green-pyronin. Exposure time: 10 months, x 850.
RESULTS
Stage-27 to-30-31 embryos
In male and female embryos injected with 3H-E2, from stage 27 onwards, a
nuclear concentration of hormone is observed in both the germinal epithelium
and the medulla. The pattern of distribution of labelled cells is shown in the
schematic drawing (Fig. 1). Labelled cells are present in large number in the
germinal epithelium of the left gonad (Figs. 2 A, B) but absent in the germinal
epithelium of the right gonad (Fig. 2D). The medulla also displays many labelled
cells (Figs. 2 A, C) in similar density in both gonads of the same embryo, and in
both sexes. In the left gonad at stages 29 and 30, a strip of tissue several cell layers
thick, intercalated between the germinal epithelium and the deep medulla,
appears void of labelled cells or contains cells with very low level of nuclear
labelling (Fig. 2 A). The corresponding region in the right gonad forms, with the
adjacent unlabelled germinal epithelium, a thick area of unlabelled cells bordering the deep medulla (Fig. 2D).
Despite growth and histological changes that affect the gonads between stages
27 and 30-31, the distribution of estradiol-concentrating cells remains the same
during the entire period. No sex- or age-related differences were observed in
either the density of labelled cells or the average intensity of labelling. This
does not exclude possible limited differences that could be measured on larger
Estrogen target cells in gonads of chicken embryo
337
Cod I
1
A
Fig. 4. Autoradiograms of testis of 10-day chicken embryos after [3H]estradiol
(3H-E2) injection. Few cells of the remnant of germinal epithelium in the left testis still
concentrate 3H-E2 in their nuclei (A) and no cell of the testicular cords (B). Abbreviation: coel., coelomic cavity. Stained with methyl green-pyronin. Exposure time:
6 months, x 850.
numbers of embryos of each age and sex using quantitative determinations.
After 6 days (stages 29-30), when the medulla acquires its distinct cordonal
structure, labelled cells are located inside the cords (Fig. 2C). The presence of
target cells for estrogens outside the cords, that is, among the interstitial cells,
cannot be ruled out since the cord boundaries are not clearly marked in our
preparations. However, most of the cells taking up 3H-E2 in their nuclei are
found in structures that can be histologically defined as cords and, consequently,
are likely to be the medullary cords.
Among the staining procedures compatible with the thaw-mount autoradiography methyl green-pyronin gives the best results. However, it does not afford
a clear distinction between, somatic and germ cells. The latter appear large and
somewhat less stained than the former, but the identification is often uncertain.
While some of the largest cells are not labelled, others, that can also be germ
cells, clearly concentrate 3H-E2 in their nuclei. However, in no instance did a
cell unmistakably identified as a germ cell show a clear labelling,
In the embryos treated with unlabelled estradiol prior to the 3H-E2 injection, competition between unlabelled and labelled molecules of estradiol
for the receptor sites prevents the nuclear labelling. Under these conditions cells
in the gonad do not show nuclear concentration of radioactivity (Fig. 2F). In
contrast, prior treatment with unlabelled DHT does not abolish the nuclear
labelling due to the concentration of 3H-E2 (Figs. 2E, G). The intensity of the
labelling is, however, lower than in embryos that received only 3H-E2 injection.
This indicates a limited competition between E2 and DHT during transport or
binding to the nucleus.
Further indication of a limited competition is supplied by results after 3 HDHT injection. The autoradiograms of gonads are, in this case, characterized
338
J.-M. GASC
by the presence of radioactive hormone both in cytoplasm and nuclei, but
display only a weak and usually inconsistent nuclear concentration (Figs. 3 A,
B). In most instances nuclear uptake of 3 H-DHT is masked by a high retention
in the cytoplasm, whose affinity for DHT is then apparently not significantly
different from the nucleus.
10-day Embryos
In male embryos injected with 3H-E2, after 6 months exposure, the left testis
displays labelled cells in the outer layer of cells bordering the coelom. The cells
concentrating 3H-E2 in their nuclei form a thin discontinuous layer on the outer
aspect of the mass of testicular cords (Fig. 4A). This layer of target cells for
estrogen is not observed in the right testis. In both testes, neither cells of the
testicular cords nor interstitial cells appear to be labelled with 3H-E2 (Fig. 4B).
In female gonads no cells display a distinct nuclear uptake of 3H-E2. Neither
cortical cells nor medullary cells appear labelled when compared to the cells in
the epithelium of the left testis. Since these autoradiograms have been exposed
for only 6 months, nuclear concentration of 3H-E2 in other cell types cannot be
excluded and may become visible after longer exposure times, though to a lesser
extent than in the left testis epithelium.
After injection of 3 H-DHT to 10-day embryos, observations made on gonads
of both sexes are similar to what is described at 7 days (see above). In the
medulla, i.e. testicular cords in males and lacunary tissue in females, though no
clear nuclear labelling can be shown, certain cells seem slightly to concentrate
3
H-DHT in their nuclei. Nothing similar is seen in the germinal epithelium of the
left ovary or the epithelium of the other gonads, or in the interstitial cells of the
testes.
DISCUSSION
3
Cells concentrating [ H]estradiol in their nuclei are observed in the gonads at
all ages studied, between 5\ (stage 27 of H and H) and 10 days of incubation
(Fig. 1). The presence of receptor sites for estradiol can be inferred from this
observation. No conclusion can be drawn about physico-chemical properties
of the receptors, except that their binding capacity for estrogens is limited, as
shown by the suppression of the nuclear labelling when unlabelled E2 is offered
in competition with 3H-E2.
The lack of 3H-E2 labelling after competition by a 100-fold excess of unlabelled estradiol, as compared to the limited decrease after the same dose of
unlabelled DHT, indicates receptor specificity for estrogen hormones. The
results obtained after 3 H-DHT injection are consistent with the specificity
of the receptors for estrogen, since a distinct concentration of 3 H-DHT in the
nuclei of estrogen target cells cannot be ascertained.
The presence of target cells for estrogen hormones in the left germinal epithelium, in male as well as in female embryos, is in agreement with experimental
studies showing that estrogens can induce an ovarian type of differentiation in
Estrogen target cells in gonads of chicken embryo
339
male embryos. Our observations, therefore, confirm the direct involvement of
estrogens in the cortical proliferation, that is, the main histological characteristic
of ovarian differentiation. The absence of target cells for estradiol in the germinal
epithelium of the right gonad is related to a most intriguing aspect of sexual differentiation in birds: the lack of cortical differentiation in the right female
gonad. The germinal epithelium of the right gonad receives fewer germ cells
than the left gonad (Van Limborgh, 1968; Dubois & Cuminge, 1978), is thinner
(Stahl & Carlon, 1973), and fails to differentiate into a fertile cortex. The actual
cause-effect relationships between the different possible factors leading to a
rudimentary gonad remain unclear, but the absence of receptor sites is undoubtedly a major factor.
Target cells for estrogens are also found in the medulla, in both gonads of all
embryos either male or female. Although the limits of the medullary cords are not
always distinguishable in our preparations, many cells that can be certainly
localized inside the cords appear to contain nuclear receptor sites. Estradiol
is synthesized by the medullary component of the gonad (Wolff & Haffen, 1952;
Mintz & Wolff, 1954; Woods & Erton, 1978; and see also review in Scheib &
Haffen, 1974) and the cells with steroidogenic activity are seen first inside the
medullary cords (Narbaitz & Adler, 1966; Scheib & Haffen, 1968). Thus, it is
possible that the same cells which produce estrogenic hormones contain specific
receptors in their nucleus. Whether this be true or not, the presence of nuclear
receptor sites in the medullary cells suggests that estradiol probably plays a
physiological role in the development and differentiation of the medulla. This
role may be more important in female gonads, where estrogen synthesis is presumably higher than in male gonads.
Consequently, estrogen hormones appear to be important not only in the
cortical differentiation of the left ovary, as shown by experimental studies, but
also in the differentiation of the medulla of the female gonads. On the basis of the
same experimental studies, an inhibiting role on the differentiation of the
testicular cords can be ascribed to the estrogen hormones. An alternative to this
inhibiting effect is a differentiating effect on the lacunary tissue, the steroidproducing tissue in female gonads.
Whatever the effects of estrogen hormones are in the cortex and in the
medulla, the presence of estrogen receptors does not seem to be a cause of
sexual differentiation since target cells display similar patterns of distribution
in embryos of both sexes. The intensity of the labelling appears also to be similar
in. males and in females. The estrogen receptors are, therefore, more likely a
sexually neutral factor in the process of gonadal differentiation rather than the
origin of the sex differences that appear during this period. The factor(s) responsible for these differences must be searched for at other levels of the process,
such as local differences in estrogen concentration between male and female embryos.
If our interpretation of the results points to amajor role of estrogen hormones in
340
J.-M. GASC
the gonadal sexual differentiation of the chicken embryo, it does not demonstrate
that these hormones are the sole triggering and prevailing agent in the naturally
occurring development of the embryonic gonads. The rather high doses of
estrogen hormones injected to obtain the ovarian differentiation in a genetically
male embryo and the fact that the genetic sex always prevails after hatching
(Wolff, 1936; Erickson&Pincus, 1966; Narbaitz &DeRobertis, 1970; Narbaitz,
1971), whatever the hormonal treatment applied to the chick, favors a scheme of
differentiation involving other factors.
The presence of estrogen target cells in the cortical remnant of the left testis at
10 days is consistent with experimental studies showing the ability of this epithelium to respond to an estradiol treatment until 11 days of incubation (Hampe,
1950). Since the autoradiograms have been in exposure for only 6 months, it is
not possible to conclude that a loss of the receptors has occurred in the other
tissues which contained estrogen target cells at day 7. However, in the left testis
a diminution of the binding affinity in the medulla relative to the cortical remnant is probable. In the female gonads a subtotal saturation of the receptor
sites by estrogen hormones is another possibility to explain the lack of labelling
after 3H-E2 injection. Investigations currently carried out will show if longer
exposure time reveals a weak nuclear labelling in the medullary tissue of male or
female gonads.
The observations of 3 H-DHT labelling in ovaries and testes at 7 and 10 days
are not conclusive. Although in our experimental conditions the nuclear concentration of DHT does not appear to be clear and consistent, the presence of
nuclear receptor sites for androgen is not excluded in certain cells of the testicular cords and of medulla in both female gonads. The presence of androgens in
the blood (Woods, Simpson & Moore, 1975; Gasc & Thibier, 1979) and in the
gonads at 10 days (Woods & Podczaski, 1974; Guichard etal. 1977) is another
clue for a possible role of androgens in the sexual differentiation of the gonads.
Although the first proliferation of sex cords is not a sexual characteristic,
the asymmetry of estrogen target cell distribution may have direct implications
in this controversial question of the gonad histogenesis (Stahl & Carlon, 1973;
Gasc, 1976). Indeed, an identical cellular proliferation in the germinal epithelium
of the right and left gonad, as classically described between 5-| and 6\ days,
is difficult to correlate with the absence of target cells for estradiol in one gonad
and their presence in the other. Directly related to the first proliferation of sex
cords is the presence in the left gonad of a zone devoid of target cells located
between the germinal epithelium and the deep medulla, both containing a large
number of target cells. This zone displays a variable thickness and corresponds
by its location to the previously described ' zone intermediate' (Stahl & Carlon,
1973) and to the 'zone sous-corticale' (Gasc, 1976). A discussion of the nature
and function of this zone is not relevant to this study, but our observations again
indicate the presence between cortex and medulla of a zone displaying specific
properties.
Estrogen target cells in gonads of chicken embryo
341
Finally, two important questions remain: are germ cells target cells for estrogen? at what stage of development do the estrogen receptors appear first in
the gonads? In both instances the techniques available at present failed to give
clear answers. Technical improvements will be required to clarify these two
questions, critical for our understanding of the gonadal differentiation in the
chicken embryo.
The author is greatly indebted to Drs W. E. Stumpf and M. Sar (University of North
Carolina at Chapel Hill) for their constant support and advice during the course of this work
and to Dr R. Narbaitz for his valuable criticism of the manuscript.
Supported by a Fellowship from the Delegation Generate a la Recherche Scientifique et
Technique (Paris, France) to J.-M. Gasc and a PHS grant NS 09914 to W. E. Stumpf.
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NARBAITZ,
(Received 3 August 1979, revised 10 September 1979)