Sex Differentiation of Avian Gonads In Vitro Numerous in vivo

AMER. ZOOL.. 15:257-272 (1975).
Sex Differentiation of Avian Gonads In Vitro
KATV HAFFE.N
Unite de Recherches 61 de 1'INSERM, 67200 Strasbourg-Hautepierre, France
SYNOPSIS. The analysis of avian sex differentiation in vitro has been limited to the following
problems: morphological sex differentiation of gonads cultured in vitro; analysis of the
chemical nature of the hormonal secretion; differentiation of germ cells in relation to their
somatic environment. Morphological sex differentiation of avian gonads occurs in vitro.
Differentiated gonads of the chick embryo carry out biosynthesis of sex hormones from
several radioactive precursors. Female gonads in particular synthesize estrogens while male
gonads synthesize testosterone. Some experiments have given evidence of estrogen synthesis by undifferentiated female gonads. Embryonic gonads of quail, like those of chick, are
able to synthesize sex steroids from radioactive precursors. However, in the quail and mainly
in the testes, a delayed appearance and a lower activity of the enzyme system 3/3-HSDHA5-4-isomerase was found. Histoenzymological results corroborate the biochemical ones.
Combination of culture and grafting experiments have shown that male germ cells when
they are forced into female differentiation by early colonization of a female gonad degenerate after entering the premeiotic stage. The reasons for this delayed failure of sex differentiation of "male oocytes" have certainly to be searched for at the level of perturbation in the
mechanisms of meiosis.
INTRODUCTION
Numerous in vivo investigations involving sex hormone administration, castration
experiments, coelomic grafts of gonads, as
well as in vitro culture of gonads and sex
ducts, have shown that the differentiation
of sex characters is under the influence of
the hormonal secretion of the gonads and
that the embryonic gonads produce secretions which have the same effect as steroid
hormones (for reviews see Wolff, \962a,b;
Wolff and Haffen, 1965; Haffen, 1970).
The present paper deals essentially with
some more or less recent contributions of
the organ culture technique to the following problems of gonad differentiation.
The first part, dealing with the spontaneous sex differentiation of the gonads
and their secretory activities, introduces the
second part, which describes the research
work on the chemical nature of the hormonal secretion and the cellular localization of steroid biosynthesis in differentiating gonads. The third part is devoted to the
study of germ cell differentiation as
influenced by their somatic environment.
SEX DIFFERENTIATION AND SECRETORY ACTIVITIES OF GONADS CULTURED IN VITRO
Spontaneous sex differentiation
In order to define their intrinsic autodifferentiating ability, organ culture experiments of embryonic gonads have been carried out by Wolff and Haffen (1952a) in the
duck, Weniger (1961) in the chick, and
Haffen (1964) in the quail.
The 614-day stage is commonly admitted
to represent the sex differentiation stage in
the chick and corresponds to the 8-day
stage in the duck and to the 5V2-day stage in
the quail.
Gonads from these three species of avian
embryos isolated before the stage of sex
differentiation (6 to 7 days in duck, 4 to 5
days in chick, and 5 days in quail) pursue
their development when cultured in vitro.
Genetically male gonads differentiate into
typical testes. Left gonads isolated from
genetically female embryos give rise to an
ovary containing a thick cortex and a more
or less vacuolated medulla, while the right
gonad generally regresses in culture and
257
258
KATY HAFFEN
becomes rudimentary as it does in the normal embryo. Germ cells were present in the
testicular cords and also in the ovarian cortex, where they were seen dividing in duck
and chick embryonic gonads. In ovaries isolated from quail embryos, the oogonia were
in the early stage of meiosis after 6 days of
culture.
Secretory activities of gonads
The secretory activities of embryonic
gonads have been demonstrated by
parabiosis experiments.
Thefeminizing action of female gonads. Pairs
of left, undifferentiated gonads from 6- to
7-day ducklings were set up in parabiosis.
The corresponding right gonads, cultured
singly, were used as controls to determine
the genetic sex. In those experiments in
which different sexes were in parabiosis,
the female gonad differentiated into an
ovary and the male gonad developed into
an ovotestis consisting of testicular medulla
surrounded by an ovarian cortex (Wolff
and Haffen, 1952b). The same results were
obtained by culturing an undifferentiated
left male gonad with a 7- to 10-day right
female gonad. The right female gonad, although regressing, feminized the left male
gonad (Wolff and Haffen, 1952c). Furthermore, a young male germinal epithelium
explanted between the fifth and sixth day
and associated with a female medulla from
between 9 and 13 days incubation differentiated to form an ovarian cortex (Haffen,
1960). Weniger (1961) linked in parabiosis
very young chick gonads of 4 and 5 days
incubation during 4 days. When different
sexes were linked, the female gonad, left
or right, strongly feminized the left genetically male gonad.
These results indicate that the hormonal
secretion from the gonads starts very early.
The masculinizing action of male gonads.
Various authors have shown by grafts and
injections in ovo that androgens have little
or no influence on the sex differentiation of
female gonads. These results have been
confirmed by the parabiosis experiments in
vitro described above. But it is known that a
male hormone is being formed by a young
testis from the time of sex differentiation
(Wolff, 1946). It acts on certain effectors,
such as the Miillerian ducts. A left Mullerian duct from a 7- to 8-day male or female
was explanted and associated between two
testes of 8 to 9 days. The male hormone
formed by the testes caused the duct to
regress over the entire region with which it
was in contact (Wolff et al., 1952; LutzOstertag, 1954).
Diffusion of embryonic sex hormones into the
culture medium. Weniger (1962) showed that
hormones produced by embryonic gonads
diffuse into the culture medium. He substituted the target organ (left testis or Mtillerian ducts) for female or male gonads. On
media into which ovarian hormone had diffused, the testes were feminized. On media
into which testicular hormone had diffused, the Miillerian ducts regressed.
ANALYSIS OF THE CHEMICAL NATURE OF THE
HORMONAL SECRETION
It has been suggested by Wolff and
Ginglinger (1935), Wolff (1950), Willier
(1939, 1942, 1952) that sex hormones produced by the morphologically undifferentiated gonad can be viewed as sex differentiator substances directing transformation
of the indifferent gonad into either an
ovary or a testis. These two working
hypotheses have stimulated research on the
chemical characterization of the hormonal
secretion of embryonic avian gonads at various stages of their development.
Production of steroids by embryonic gonads of the
chick
Steroids have been detected in the blood,
allantoic and amniotic fluids, as well as in
the gonads. Stoll and Maraud (1956) detected trace amounts of 17-ketosteroids in
the amniotic and allantoic fluid of 6'/2-day
chick embryos, while Ozon (1965, 1969)
demonstrated that estrogens are first present in these fluid compartments as well as in
the blood of 10-day embryos. Gallien and
Le Foulgoc (1957) identified estrogens in
the 10-day ovaries. Unfortunately, the
techniques used to detect the hormones in
the embryonic fluids and gonads have
AVIAN GONADS IN VITRO
failed to characterize estrone and estradiol
in the culture media of embryonic female
gonads. Weniger (1966) found estrogenic
activity in these media after extraction by
the biological test of Allen and Doisy. Positive results were obtained with extracts of
media on which female gonads from 7 to 10
days had been cultivated; the results were
negative with male gonads and other organs.
259
Scheib et al., 1974).
Weniger and coworkers overlaid the
labeled precursor on the surface of the
media on which the explanted gonads were
cultured, and extraction was performed on
the culture media including the gonads.
The techniques for identification of steroid
hormones are described in detail in
Weniger (1969, 1970) and in Weniger and
Zeiss (1971).
Biosynthesis of steroid hormones from labeled Estrogen biosynthesis
precursors by cultured avian embryonic gonads
From Na-l-14C acetate. Weniger et al.
(1967), Weniger (1970a), and Akram and
Weniger (1969) have studied estrogen
formation from this precursor by avian
embryonic gonads cultivated in vitro for at
least 24 hr. They have shown labeled estrone and estradiol synthesis by 7- to 9-day
ovaries of chick embryos (Weniger et al.,
1967), and by 12-day ovaries of duck and
pentado embryos (Akram and Weniger,
1969). Sodium acetate-l-14C was also incorporated into estriol and epiestriol by
16-day ovaries of chick embryos (Weniger,
1969). When the precursor was supplied to
4-, 5-, and 6-day undifferentiated gonads,
labeled estrone and estradiol were found in
the culture medium after 24 hr (Weniger
and Zeis, 1971). The radiochemical purity
has been established in the case of estrogens
formed by 6-day gonads (Weniger and Zeis,
1971) and for epiestriol produced by 14- to
16-day ovaries (Weniger, 1970a). Cedard et
al. (1968) and Guichard et al. (1973a) have
also observed a production of estrogens
from Na-l- 14 C acetate1 by their radiochemical purity. Human chorionic gonadotrophin (HCG), which has been shown by
Connell et al. (1966) to stimulate testosterone production by the testis of 2-day
chicks, increased estrogen production in
10- and 18-day ovaries and induced a discrete synthesis of estradiol in 18-day testes
(Haffen etal., 1969; Guichard etal., 1973a)
(Table 1).
This study was performed by two groups
of workers. Cedard and Haffen (1966),
Haffen and Cedard (1968), Haffen et al.
(1969), Guichard etal. (1973a,6), and Scheib
et al. (1974) have developed the quantitative aspect of this biosynthesis as a function
of the stage of development and of the nature of the labeled precursor in chick and
quail in order to investigate the biosynthetic pathways ending in steroid sex hormones. On the other hand, Weniger et al.
(1967), Akram and Weniger (1969), Weniger (1969, 1970), and Weniger and Zeis
(1971) have concentrated their efforts on
the qualitative but early aspect of this biosynthesis in order to check Wolffs hypothesis of estrogens being responsible for
the morphological changes which characterize sex differentiation of female gonads.
These investigations were realized in organ
culture according to the technique devised
by Wolff and Haffen (1952a).
Cedard and coworkers have introduced
the steroid precursor(s) into the medium in
which a number of embryonic gonads were
cultured. One to 3 days later the synthesized radioactive hormones were detected in
the culture media. The authors found that
the transformation products accumulated
in the culture medium rather than in the
explants. They could also verify that the
culture medium itself, whether it contains a
synthetic nutrient mixture or diluted embryonic extract, does not transform the
From 3H-pregnenolone and 14C-proprecursor into biologically active steroids.
The detailed techniques used for identifica- 14 1 Any other labeled steroid, synthesized from Na-1C acetate, could be detected in the culture media.
tion of steroid hormones are not described
cholesterol was found and HCG stimulated its
here (for that purpose, see Haffen and Labeled
production by 10-and 18-day-old testes and by 10-day
Cedard, 1968; Guichard et al., 1973a; ovaries (Haffen et al., 1969).
NO
en
o
TABLE 1. Biosynthesis of testosterone and estrogens by chick embryonic gonads cultured in the presence of radioactive precursors'. (After Guichard et at., 1973a.)
Stage in
days of
ncubation Precursors
Preg-7a 3 H
Prog-4-14C
DHA-4-14C
l¥i
14
10
Na-Acet-l- C
Preg-7a 3 H
Prog-4-MC
DHA-4-'"C
Testo-6,7a 3 H
14
18
a
=
=
0
=
d
=
b
Mean no. ofgonads
Mean amount
per experiment
Specific of precursor
Pairs of
activity per experiment No. of
(mCi/mM)
exp Ovaries'1 testes
(/iCi)
454
58.5
52
41
454
58.5
52
864
41
Na-Acet-l- C
454
Preg-7a 3 H
58.5
Prog-4- l4 C
DHA-4- M C
52
Testo-6,7a 3 H 864
13.6
1.36
0.3
100
12.3
1.1
0.76
1
100
1
1
2
3
2
2
5
1
3
12.3
3
1.1
1
3
1
1
3.75
Testosi erone b
dpm
%
72
72
173
76
76
30 350
26 490
0.23
2.40
190
nul
nul
50
81
50
67
81
67
63
63
88
98
20
26
26
22
20
nul
nul
26
26
22
86 600
22 130
24 000
0.87
2.32
2.31
24
Testes
Ovaries
Estrogens0
%
dpm
Testosterone"
Estrogens0
dpm
%
dpm
%
0.05
0.24
0.39
9200
2680
2930
0.04
0.12
1.17
60 660
29 100
7 260
0.46
3.30
1.0
6 000
2 180
1 460
1720
19540
0.63
0.17
nul
nul
1.0
nul
nul
95 500
4 00
0.05
mil
nul
18 500
26 500
0.16
2.2
3800
0.27
47 000
5.2
1800
0.18
nul
nul
20000
19540
1.55
0.68
36 420
3.8
420
0.03
0.09
1840
8560
1380
11100
18200
0.13
0.07
0.17
1.15
0.20
2 240
nul
nul
nul
nul
249 630
89 280
86 620
2.2
6 200
2 100
5 420
0.01
0.10
0.42
8.5
7.7
Data are expressed in dpm and in percent of the ether soluble fraction corrected to recovery of carrier hormones
After oxidation into A4-androstenedione
Estrone + 17/3-estradiol after methylation
At 7'/2 days of incubation, both left and right ovaries were explanted; at 10 and 18 days, only the left ovary was cultured.
>
H
-<
D
>
z
Tl
W
AVIAN GONADS IN VITRO
gesterone2. Tritiated as well as 14C-labeled
estrogens have been detected in the culture
media of differentiated embryonic gonads
isolated from chick and quail (Guichard et
al., 1973a,b). In chick, no demonstration of
a quantitative difference between male and
female gonads could be made, owing to the
poor yields (Table 1; Fig. 1), nor of an increase with age, while such a difference was
striking in quail (Table 2). In the latter
species, a higher level of synthesis was
found in the female compared with the
male at the same stage. Estrogens were
generally formed in higher amounts from
progesterone than from pregnenolone.
Undifferentiated 15-day chick embryonic'
gonads produced estrogens over 24 hr of
culture from 14C-progesterone (Weniger
and Zeis, 1971).
261
formed from 3 H-pregnenolone and 14Cprogesterone indicated an increasing
synthesis of testosterone by testes as a function of age, together with a better yield
from progesterone. Ovaries synthesized
testosterone at a much lower rate than
testes, the yield remaining constant with
age (Table 1; Fig. 1). The radiochemical
purity could be assessed only in the case of
14
Ctestosterone
formed
from
progesterone by 10- and 18-day testes and
for testosterone derived from 3 Hpregnenolone by 18-day testes (Guichard et
al., 1973a).
In quail, calculation of the yield of formation of testosterone has also shown an increasing synthesis in testes between the
10th and 15th day, with a better yield from
progesterone and a higher production in
From 4-i4C-Dehydroepiandrosterone (DHA). male than in female gonads (Table 2).
Low quantities of radioactive estrogens
As in the chick, only testosterone prowere detected in the culture media of un- duced by testes could be recrystallized to a
differentiated chick gonads (6 and 7 days of constant isotopic ratio (Guichard et al.,
incubation). This synthesis was found to 1973ft).
increase between 7V&, 10, and 18 days. No
From 4-14C DHA. Radioactive testossignificant synthesis of estrogens was found terone was present in the culture media of
in male gonads at IVi and 10 days, but took 7-day chick embryonic gonads (Fig. 1). Beplace at 18 days (Table 1; Fig. 1) (Haffen tween 7Vi, 10, and 18 days, labeled testosand Cedard, 1968; Guichard et al., 1973a). terone was produced in increasing amounts
Transformation of this precursor into es- by male gonads. The synthesis of this
not be detected in the culture
trogens took place in embryonic quail steroid could
ovaries and testes of 10 and 15 days incuba- media of 7lA- and 10-day ovaries, but was
tion. Singularly, no increase of this estro- found with 18-day ovaries (Table 1; Fig. 1).
gen production could be detected in the The radiochemical purity of testosterone
female, while it was found in the male be- isolated from the culture media could be
tween these two stages (Scheib et al., 1974) assessed (Haffen and Cedard, 1968;
Guichard et al., 1973a).
(Table 2).
In quail, no significant synthesis of testosterone was found with 10- and 15-day
Testosterone biosynthesis
ovaries or with 10-day testes, while it became significant with 15-day testes. DHA
3
l4
From H-pregnenolone and
C-progeswas
found to be a less efficient precursor
terone. Tritiated and 14C-labeled testos- than progesterone (Scheib et al., 1974)
terone was present in the culture media of (Table 2).
ovaries and testes isolated from 7V2-, 10-,
and 18-day chick embryos, and from 10- Conversion of pregnenolone into progesterone2
and 15-day quail embryos. In chick, the
calculation of the yield of testosterone
This transformation was quantitatively
estimated
by determination of the radioac23
H-pregnenolone and 14 C-progesterone were tivity of the tritiated progesterone derived
added simultaneously into the culture medium. Since from 3 H-pregnenolone (Fig. 2). Tritiated
their specific activities were very different, the molar
3
ratio of the precursors was equilibrated by addition of progesterone derived from H-pregnenolone by gonads of both sexes in
non-labeled pregnenolone.
262
p moles
1300 +
KATY HAFFEN
PRECURSORS:
• • PROGEST.
lilTO D.H.A
B
1000- •
500- -
100-
18
10
18
FIG. 1. Production of testosterone (left diagram) and
of estrogens (right diagram) by 6- to 18-day chick
embryonic gonads from 14C-progesterone and DHA.
Data are expressed in /umoles of steroid formed by an
equal number of gonads (20 pairs of gonads at 6 and
7Vt days; 20 pairs of testes and 20 left ovaries at 10 and
18 days). (From Haffen and Cedard, 1968, and from
Guichard et al., 1973a).
chick and by female gonads in quail could
be recrystallized to a constant isotopic ratio
(Guichardetal., I973a,b). The comparative
study between chick and quail revealed that
in both species, the enzymatic activity of the
3/3-HSDH-As-4 isomerase increased with
age, but was always more important in chick
than in quail. This was particularly striking
in the male. This delayed manifestation of
the enzymatic activity in quail testes is
confirmed by histoenzymologic results described later. In the female, a difference
existed between quail and chick, but was far
less striking.
quail (Haffen and Cedard, 1968; Scheib
et al., 1974).
Interpretation and significance of these results
The differences related to the conversion
of pregnenolone into progesterone on one
hand, and into DHA on the other, have
raised the question of the biosynthetic
pathways ending in sexual steroids, and in
particular testosterone.
The generally admitted scheme of
steroid sex hormone biosynthesis displays
two alternative metabolic pathways, the
A4-3 ketosteroid pathway, and the AsFormation of DHA and Ab-androstene-3fi, hydroxysteroid pathway, depending on
whether the 3/3 -HSDH-As-4 isomerase is
17/3-diol
involved before or after conversion of C20
No formation of DHA from 3 H- steroids into C19 steroids. It should be
pregnenolone has been demonstrated so pointed out that A4-androstenedione, a
far in the culture media of explanted chick common intermediate metabolite of both
gonads, while it steadily accumulated in the biosynthetic pathways, could not be demedia of quail gonads (Guichard et al., tected in the culture media while it was
were incubated for 15
I973a,b). On the contrary, As-androstene found when gonads
14
3/3, 17/3-diol was found in the culture media and 30 min with C progesterone as a preof gonads of both sexes in chick as well as in cursor (Galli and Wassermann, 1972,
AVIAN GONADS IN VITRO
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263
1973). The only intermediate metabolite
which could be detected in organ culture
experiments was DHA formed by quail
gonads from 3H-pregnenolone (Guichard
et al., 1973i).
It seems that organ culture favors the
accumulation of end products (testosterone
and estrogens) rather than products of intermediate metabolism. One should anyway keep in mind that the lack of detection
of a metabolite does not necessarily mean
that it does not play a role in the biosynthetic chain. Thus, the identification of the
biosynthetic pathways leading to formation
of testosterone is based on comparison of
the data obtained from the 3H/14C ratio in
progesterone and that obtained in testosterone produced in double labeling experiments (3H pregnenolone + 14C progesterone)2. A higher 3H/14C ratio in progesterone than in testosterone indicates a preferential passage through the A4-3ketosteroid pathway, while on the contrary
a higher 3H/14C ratio in testosterone than in
progesterone reflects the prevalence of the
As-hydroxysteroid pathway and a certain
deficiency of the 3/3-HSDH-As-4 isomerase
at the level of the conversion of pregnenolone into progesterone. Figure 3 gives the
comparison of the different ratio values determined for chick and quail. In chick, a
near equal 3H/14C ratio was found in progesterone and in testosterone, while in quail,
the 3H/14C ratio was higher in testosterone
than in progesterone. This indicates that in
chick, conversion into testosterone can take
place through both pathways, while in
quail, the As-hydroxysteroid pathway predominates strikingly, thus confirming the .
enzymatic deficiency of the As-3j8-HSDHA5-4 isomerase at the level of the conversion
of pregnenolone into progesterone.
Finally, quail testis synthesises at the end
of the incubation period noticeable
amounts of estrogens from 14C-DHA. This
could be explained by the fact that it displays signs of intersexuality after the 10th
day of incubation (Scheib, 1971a). In chick,
male gonads would produce estrogens
from 14C-DHA(Haffen, and Cedard, 1968)
and 14C-Na acetate (Akram and Weniger,
1967) only when experimentally feminized.
264
KATY HAFFEN
Chick
30
Quail
n
20
10-
0J
10 10
18 15
10 10
18 15
OVARIES
TESTES
FIG. 2. Conversion of pregnenolone into progesterone (see footnote 2) by ovaries and testes of chick
(10-and 18-day embryos) and of quail (10- and 15-day
embryos). Data are expressed as the percentage of the
3
H/ 14 C ratio in progesterone compared with the ratio
in the ether soluble fraction. (From Guichard et al.,
by these authors fit together as far as the
localization of the enzymatic activity in the
ovary is concerned but differ for the testis.
Furthermore, they differ as well in the time
of its appearance.
First appearance of enzymatic activity
In the chick embryo, As-3/3-HSDH has
been reported to be present initially in the
genital ridge of 2Vz days (Woods and
Weeks, 1969), in the undifferentiated
gonads of 6V2 days (Scheib and Haffen,
1968), in the differentiated gonads of 8
days (Narbaitz and Kolodny, 1964; Chieffi
et al., 1964) and of 9 days (Boucek et al.,
1966).
Cellular localization of enzymatic activity
Woods and Weeks (1969) observed the
As-3/3-HSDH from 2V2 days onward in both
the germinal epithelium and mesenchymal
cells of the underlying gonadal blastema. In
the ovary, the enzymatic activity is concentrated in the medullary cords and weaker in
the cortex.
Narbaitz and Kolodny (1964), Chieffi et
Cellular localization of steroid synthesis
al., (1964), and Scheib and Haffen (1968,
1969) have shown that no reaction took
Steroid producing cells show some re- place in the ovarian cortex during emmarkable cytological and cytochemical fea- bryonic development. The As-3/3-HSDH is
tures. The problem of localization and dif- confined to those interstitial cells derived
ferentiation of steroidogenic cells has been from the medullary cords (Fig. 4A-C).
studied by various authors both by electron Furthermore, the reaction is at every stage
microscopic and histochemical techniques.
These researches do not deal with in vitro
CHICK
QUAIL
cultures. We shall thus mention only one of
them, namely the histoenzymologic study,
^ H Progesteron
which has provided precious data as to the
L J Testosteron*
cellular localization of steroid biosynthesis.
As seen in the preceding section, the A53/3-HSDH is a key enzyme in steroid biosynthesis. The histochemical method of Levy
et al. (1959) tested on adult mammals has
been applied to the avian embryonic
gonads in order to check the stage at which
the enzymatic reaction appears and to establish its localization at the cellular level as
10
15
described by various authors (Chieffi et al.,
FIG. 3. 3H/14C ratio in progesterone and testosterone
1964; Narbaitzand Kolodny, 1964; Scheib produced by chick (IVi to 18 days) and quail emand Haffen, 1968, 1969; Woods and bryonic testes (10 and 15 days). (From Guichard et al.,
Weeks, 1969). Indeed, the results obtained 1973a,i.)
u
AVIAN GONADS IN VITRO
FIG. 4. As-3/3-HSDH activity in ovaries and testes of
chick and quail embryos. In the ovaries (A-C) the enzymatic reaction is confined to cell clusters of the
medulla (m). The cortex (co) shows no reaction. In the
testes (D-F) the enzymatic reaction is present in some
cells of the cords and in the interstitial cells of the
stroma. A, Quail embryonic ovary of 12 days. B, Chick
embryonic ovary of 12 days. C, Chick embryonic ovary
of 8!4 days. D, Quail embryonic testis of 14J4 days.
165
First appearance of anzymatic activity in the interstitial
cells of the stroma. F, Chick embryonic testes of 18
days showing the well-developed interstitial tissue in
which a strong enzymatic activity is present. E, Chick
embryonic testis of 10 days showing enzymatic activity
in cell clusters located inside the cords (arrows). Magnification for/*, B, D,E;X\ 10; forC, F: x 450. (From
Scheiband Haffen, 1968, 1969.)
266
KATY HAFFEN
similar in the right gonad which is deprived
of cortex, thus confirming the fact that in
the embryonic ovary, the medulla is the site
of hormonal secretion, first demonstrated
by Wolff and Wolff (1951) (see also Wolff
and Haffen, 1965).
In the testis, Chieffi et al. (1964) have mentioned that As-3j8-HSDH appears only in a
diffuse fashion from the 8th day in the sexual cords where it remains localized
throughout embryonic development. According to Narbaitz and Kolodny (1964),
the testis would be completely deprived of
specific enzymatic activity.
Scheib and Haffen (1968, 1969) gave the
following description of the localization of
the As-3/3-HSDH. A weak activity is visible
in the cord cells at 614 days in the chick
embryo. The activity becomes more widespread between 7 and 8 days and remains
throughout embryonic development. After
the eighth day one can further distinguish
two types of cell clusters in which the As3/3-HSDH reaction is very intense; they are
located at different levels in some cords and
also in the stroma (Fig. 4E-F). Both are still
found at later stages. In the stroma the cell
clusters enlarge and their number increases
during development; they represent the interstitial cells. The fact that some cells in the
cords and the interstitial cells in the stroma
have similar properties—cholesterides
(Scheib, 1959) and As-3/3-HSDH— argues
for Benoit's (1923) theory concerning the
origin of the interstitial cells from the somatic cells of the cords. The specialization
of these cells seems to occur earlier in the
chick (8!4 days) than in the quail {Wh
days). In the latter, only some cells react
more intensively at 13 xh days, whereas at
14'/2 days some large clusters are found
(Fig. AD). This delayed appearance of the
histochemical reaction in interstitial cells in
quail confirms the biochemical results,
which have shown that the enzymatic activity becomes significant only at the end of
the incubation period.
DIFFERENTIATION OF GERM CELLS AS INFLUENCED
BY THEIR SOMATIC ENVIRONMENT
Germ cells undergo ultrastructural, histochemical, and physiological changes
(migratory properties) during embryonic
sexual organogenesis. This important
question has been extensively reviewed by
Dubois and Croisille (1970) so that this
paragraph will not deal with it. Our interest
here is the fate of germ cells which have
been introduced at an early stage into
gonads of the opposite sex.
Indeed, experimental intersexuality
studies (Wolff and Haffen, 1961) which followed those of Wolff and Ginglinger
(1935), have pointed out that germinal
epithelium from a male gonad converted
into an ovarian cortex by estradiol or by a
grafted embryonic ovary does not display
the same evolutive potentialities as normal
ovarian cortex: indeed, it disappears
shortly after hatching as a consequence of
the degeneration of the oocytes it contains.
Association of an ovarian medulla with
the cortical part of an intersexed male
gonad does not promote the normal evolution of the oocytes (Haffen, 1969a).
The question arose then as to whether
the somatic cells or the germ cells themselves impair female differentiation in an
experimentally induced male cortex.
The study of Haffen (1969a), which was
carried out by a combination of culture and
grafting experiments, confirmed the second hypothesis.
Evolution of germ cells after early experimental
introduction into a gonad of the oppostie sex
Haffen (1960), using the technique described by Wolff and Simon (1955) and
Simon (1960), formed chimaeric embryos
at the 9 to 15 somite stage: the anterior part
(fertile and agonadic) of a blastoderm A
was associated with the posterior part
(sterile and gonadic) of blastoderm B. A
chimeric embryo (AB) was thus obtained.
The reverse association led to the formation of a chimeric embryo (BA). In 50% of
the cases, the A and B embryos were of the
opposite sex, while in 25% of the cases, the
male gonocytes from the anterior germinal
crescent colonized the female gonadic region in the posterior part, the inverse situation being found in 25% of the cases. The
chimeric embryos, (AB) and (BA), developed for 48 hr in the culture medium
AVIAN GONADS IN VITRO
and reached a stage corresponding to that
of 3- to 3 J4-day embryos. The left genital
ridges colonized by the gonocytes were isolated and grafted for 10 days into the
coelom of a 3-day embryo. After this first
passage, the explant was introduced into a
second host for 10 more days, then into a
third and a fourth host. At each passage, a
piece of the explant was examined histologically. Under these conditions, the age of
the explant could be estimated as follows:
13 days after grafting I
23 days after grafting II
33 days after grafting III
43 days after grafting IV
When the chimeric embryos were
formed, the sex of neither embryo was
known. To insure this, it was necessary that
one gonad develop from each of both
chimeric embryos.
Ovary colonized by female germ cells. After
23 days, the cortex was filled with oocytes at
the leptotene stage of premeiosis. After 33
days, the primary follicles contained oocytes at the diplotene stage. After 43 days,
the primary follicles of the ovarian cortex
continued to increase (Fig. 5A-C).
Ovary colonized by male germ cells. A pro-
gressive degeneration of the ovarian cortex
took place (Fig. 5D-E). This degeneration
could be compared with that described for
the cortex of intersexed male gonads. The
first evidence of it was already seen after the
second passage (23 days); in a great number
of "male oocytes," the chromatin was markedly retracted. After 33 days, the degeneration of the "male oocytes" was even more
striking: the nucleus became light and
looked empty, the chromatin was dispersed
and accumulated against the nuclear membrane, which was underlined by a dark line,
or tended to disappear. After 43 days, the
cortical region was represented only by a
few areas of degenerated cells. Follicular
cells did not surround the oocytes in the
course of degeneration: they were grouped
in clusters on the side of the medulla.
It should be pointed out that in some
cases, Haffen (1969a) has observed the existence of some rare primary follicles in the
cortex which was, besides this feature,
mainly occupied by germs cells in the
course of degeneration. Haffen attributed
267
their formation to the presence of female
gonocytes which were found in the posterior part of the chimeric embryo. This was
the most plausible explanation since the
avian germinal crescent has a statistical
value. It may happen that in the course of
the caudocephalic migration of the germinal material, some gonocytes remain
definitely delayed (Fargeix and Theilleux,
1967; Dubois, 1967). In embryos after the
10-somite stage, dispersion of the primary
gonocytes of the germinal crescent could
have begun. It should be pointed out that
the coexistence of such normal follicles and
of degenerating "male oocytes" in the same
ovarian cortex has provided for an additional and particularly convincing argument for this demonstration.
Testes colonized by male or female germ cells.
Testes were grafted from chimeric embryos into three or four successive hosts;
some of them were certainly colonized by
female germ cells and the others, by male
germ cells. No differences could be observed in the evolution of the two categories
of germ cells. Both looked like spermatogonia until 43 days. It should be recalled that in normal development,
gametogenesis is delayed in the male as
compared with the female. The experimental conditions did not allow the meiosis
stage to be reached.
These experiments have shown that
somatic tissues in an ovary can impose a
begining of female sex differentiation on
genetically male germ cells, but that they do
not ensure their subsequent evolution in
the female direction. Indeed, "male oocytes" cannot cross the threshold of the
premeiotic prophase and thus degenerate.
The reasons for this delayed failure of sex
differentiation of "male oocytes" have certainly to be searched for at the level of perturbation in the mechanism of meiosis. An
ultrastructural study of cortical differentiation in chick ovaries and in intersexual
gonads has been made by Narbaitz (1971).
He came to the same conclusion as Haffen
(1969a), i.e., that the primary incompetency responsible for cortical degeneration
lies in the germ cells and not in the somatic
(prefollicular) cells.
The problem of evolution of female
268
KATY HAFFEN
*#--<ii
.;*&£*
N1*^3S
FIG. 5. Grafting experiments of ovaries colonized by
female and by male germ cells into successive embryonic hosts. Differentiation of female germ cells
(/f-C). Degeneration of male germ cells (D-F).A, Graft
II (23 days) oocytes in meiotic prophase; leptotene
structure of the nuclei. B, Graft III (33 days); primary
follicles; diplotene structure of the nuclei. C, Graft IV
(43 days); growing follicles. D, Graft II (23 days);
"male oocytes" in meiotic prophase. Several nuclei
show retracted chromatin (r.ch.). E, Graft III (33
days); numerous degenerating "male cocytes" (D.o.).
Others still show a retracted chromatin (r.ch.). F, Graft
IV (43 days); the ovarian cortex contains only degenerated "male cocytes". Magnification for A, D: x 1125;
forB, C: x 450; forC, F: x 540. (From Haffen, 1969a.)
germ cells in a testis could not be resolved
experimentally by Haffen (1969a); indeed,
as already shown by the experiments of Benoit (1932) (see in Taber, 1964), ablation of
the left ovary shortly after hatching induced the transformation of the right
rudimentary gonad into a testis and the
evolution into spermatozoa of those gono-
AVIAN GONADS IN VITRO
cytes which were still present at the time of
ovariectomy
ovariectomy.
269
the steroid nature of the hormonal secretion produced by avian embryonic gonads
and suggest the existence of specific enzymes playing a role in the series of transDISCUSSION
formation reactions which lead to estrogen
production through androgens from a
The experiments using organ culture of common precursor.
sex organs of birds set up by Wolff and
It should be noted that Weniger (1970)
Haffen (1952a) led to the following conclu- never isolated labeled testosterone from
sions:
culture media of 10- to 19-day embryonic
The gonads develop outside the or- testes, whatever the labeled precursor was.
ganism into testes or ovaries according to These results are inconsistent with those of
their genetic sex. Male or female develop- Cedard and Haffen (1968), Guichard et al.
ment of a gonad primordium is an example (I973a,b), Scheib et al., (1974), and Galli
of autodifferentiation. The gonads possess and Wassermann (1972, 1973) as well as
the factors necessary for their differentia- with those of Woods and Podczaki (1973).
tion at an early stage of their development. These latter authors have recently demonNatural and synthetic hormones act di- strated by use of an immunohistochemical
rectly on the target organ (testis, Mullerian technique that androgens are first syntheduct) without intervention of any other sized by the sexually undifferentiated gogland in the embryo. The similitude of the nads of both presumptive sexes of the chick
effects of embryonic and adult hormones embryo on day 3'/4 of incubation.
has led Wolff and Ginglinger (1935) and
Histoenzymology has provided imporWillier (1939) to postulate that sex differentant
data as to the cellular localization of the
tiation is realized through an hormonal
steroidogenic
function in embryonic
mechanism and that the same hormones act
gonads.
This
technique
revealed a A5-3/3during the whole of development, from
HSDH
activity
in
the
medullary
cords beembryonic organogenesis to the adult
fore
appearance
of
the
ultrastructural
stage.
characteristics of secretory cells. It furthNew techniques such as radiochro- ermore demonstrated that in the ovary, the
matography, histoenzymology, and elec- enzymatic localization is limited to the sectron microscopy have provided clues as to retory cells in the medulla, which have been
the steroid nature of the gonadal secretion characterized by electron microscopy
and to the cellular support which ensures (Narbaitz and Adler, 1966; Scheib, 1970a).
this function.
The observations of Scheib and Haffen
Embryonic avian gonads cultured in (1968) on chick testis have emphasized that
vitro can utilize various radioactive precur- from the 8th day there are present in the
sors added to the culture medium and con- cords certain cells showing the same Asvert them into steroid sex hormones. Es- 3/3-HSDH activity as the interstitial cells of
trogens are essentially synthesized in the stroma. These results confirm the hisfemale gonads and testosterone in male tological observations of Benoit (1923) who
gonads. Estrogen and testosterone synthe- described such cells in the cords from the
sis from progesterone had been confirmed 12th day onward. They provide arguments
by incubation of gonads in buffered saline in favor of the theory of the epithelial ori(Galli and Wassermann, 1972, 1973). Es- gin of the interstitial cells. By electron mitrogen synthesis in culture media from croscopy Narbaitz and Adler (1966) have
Na-acetate by 6-day-old gonads (Weniger observed the presence of interstitial cells in
and Zeis, 1971) has provided a worthy ar- the testicular stroma from the 9th day ongument to the theory of Wolff, according ward and found some cells with an analoto whom estrogens would be responsible for gous structure in the 16-day cords.
the characteristic morphological changes
Scheib (19706) reports that from the 8th
upon female gonad differentiation.
day there are present in the testicular cords
These results as a whole are in favor of somatic cells with the same ultrastructural
270
KATY HAFFEN
characteristics as interstitial cells. According to this author, the interstitial tissue in
the testes results from the first limited and
then massive differentiation of cells which
were initially included in the cords and
which colonize the interstitial spaces.
The last problem evoked in the present
paper deals with the evolution of male
germ cells, in the chick, when they are
forced into female differentiation by early
colonization of a female gonad. These cells
degenerate after entering the premeiotic
stage. New research should thus elucidate
the causes of this disorder. In the quail, this
disorder seems to be less pronounced. Indeed, application of diethylstilbestrol to
quail embryos induces a feminization of the
gonads which persists partially after hatching until the adult stage. Germ cells are
converted into oocytes, start their growth
and become surrounded by follicular cells,
thus giving rise to large sized follicles (Haffen, 19696). On the other hand, the disorder in meiosis does not seem to exist upon
spermatogenesis of female germ cells in the
right chick gonad, which converts itself into
a testis when left ovariectomy is performed
after hatching (Benoit, 1932).
Reynaud (1969, 1971) has realized interspecific transfers of germ cells (from
turkey to chick) as well as intraspecific
transfers (from one strain to another), by
injecting these cells into the blood stream of
previously sterilized chick embryos. These
experiments demonstrated the existence of
complete gametogenesis of the primordial
germ cells injected. However, they do not
provide clues as to the eventual differentiation of female germ cells in a male soma,
since the injected cells represent a population of heterosexual cells from several germinal crescents.
It should be recalled that in amphibians
and fishes, cytodifferentiation of germ cells
can be completely inverted (see Gallien,
1967). Furthermore in placental mammals,
where experimental sex inversion is impossible, the pioneer work of Mystowska and
Tarkowski (1968, 1970) has shown that
female germ cells which evolve in a
chromosomic chimera do not behave like
spermatogonia; they enter premeiosis as
they would do in an ovary, but are unable to
carry on this differentiation in a male environment, and degenerate.
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