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 synthe-
sis 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-HSDH-
A5-4-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 degener-
ate after entering the premeiotic
stage. The reasons for this delayed failure of sex differenti-
ation
of "male oocytes" have certainly to be searched for at the level of perturbation in the
mechanisms of meiosis.
INTRODUCTION
Numerous in vivo investigations involv-
ing 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 secre-
tions 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 follow-
ing problems
of gonad differentiation.
The first part, dealing with
the spon-
taneous
sex differentiation of the gonads
and their secretory
activities, introduces the
second part, which describes
the research
work
on the chemical nature of the hor-
monal secretion and the cellular localiza-
tion
of steroid biosynthesis in differentiat-
ing gonads. The third part
is devoted to the
study
of germ cell differentiation as
influenced by their somatic environment.
SEX DIFFERENTIATION AND SECRETORY AC-
TIVITIES OF GONADS CULTURED IN VITRO
Spontaneous sex differentiation
In order to define their intrinsic autodif-
ferentiating ability, organ culture experi-
ments of embryonic gonads have been car-
ried 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

258KATY HAFFEN
becomes rudimentary as it does in the nor-
mal embryo. Germ cells were present in the
testicular cords and also in the ovarian cor-
tex, where they were seen dividing in duck
and chick embryonic gonads. In ovaries iso-
lated 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.
The feminizing 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, al-
though regressing, feminized the left male
gonad (Wolff and Haffen, 1952c). Further-
more, 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 differen-
tiated 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 genet-
ically 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 Mulle-
rian 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; Lutz-
Ostertag, 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 substi-
tuted the target organ (left testis or Mtille-
rian ducts) for female or male gonads. On
media into which ovarian hormone had dif-
fused, the testes were feminized. On media
into which testicular hormone had dif-
fused, 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 pro-
duced by the morphologically undifferen-
tiated gonad can be viewed as sex differen-
tiator 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 var-
ious 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) de-
tected 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 pres-
ent 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 VITRO259
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. Posi-
tive 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 or-
gans.
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
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 quantita-
tive aspect of this biosynthesis as a function
of the stage of development and of the na-
ture of the labeled precursor in chick and
quail in order to investigate the biosynthet-
ic pathways ending in steroid sex hor-
mones. On the other hand, Weniger et al.
(1967), Akram and Weniger (1969), Weni-
ger (1969, 1970), and Weniger and Zeis
(1971) have concentrated their efforts on
the qualitative but early aspect of this bio-
synthesis in order to check Wolffs hypoth-
esis of estrogens being responsible for
the morphological changes which charac-
terize 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 synthesiz-
ed 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 em-
bryonic extract, does not transform the
precursor into biologically active steroids.
The detailed techniques used for identifica-
tion of steroid hormones are not described
here (for that purpose, see Haffen and
Cedard, 1968; Guichard et al., 1973a;
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 es-
trone 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 in-
corporated 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-14C acetate1 by their radiochem-
ical purity. Human chorionic gonadotro-
phin (HCG), which has been shown by
Connell et al. (1966) to stimulate testos-
terone production by the testis of 2-day
chicks, increased estrogen production in
10- and 18-day ovaries and induced a dis-
crete synthesis of estradiol in 18-day testes
(Haffen etal., 1969; Guichard etal., 1973a)
(Table 1).
From 3H-pregnenolone and 14C-pro-
1 Any other labeled steroid, synthesized from Na-1-
14C acetate, could be detected in the culture media.
Labeled cholesterol was found and HCG stimulated its
production by 10-and 18-day-old testes and by 10-day
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
incubation Precursors
l¥i
10
18
Preg-7a3H
Prog-4-14C
DHA-4-14C
Na-Acet-l-14C
Preg-7a3H
Prog-4-MC
DHA-4-'"C
Testo-6,7a3H
Na-Acet-l-14C
Preg-7a3H
Prog-4-l4C
DHA-4-MC
Testo-6,7a3H
Mean amount
Specific of
activity per
(mCi/mM)
454
58.5
52
41
454
58.5
52
864
41
454
58.5
52
864
precursor
experiment
(/iCi)
13.6
1.36
0.3
100
12.3
1.1
0.76
1
100
12.3
1.1
1
3.75
No. of
exp
1
1
2
3
2
2
5
1
3
3
3
1
1
Mean no.ofgonads
per experiment
Ovaries'1
72
72
173
50
81
81
63
63
20
26
26
22
24
Pairs of
testes
76
76
190
50
67
67
88
98
20
26
26
22
Testosi
dpm
30 350
26 490
nul
mil
18 500
26 500
nul
nul
86 600
22 130
24 000
eroneb
%
0.23
2.40
nul
nul
0.16
2.2
nul
nul
0.87
2.32
2.31
Ovaries
Estrogens0
dpm
9200
2680
2930
1720
19540
3800
20000
19540
1840
8560
1380
11100
18200
%
0.04
0.12
1.17
0.63
0.17
0.27
1.55
0.68
0.13
0.07
0.17
1.15
0.20
Testes
Testosterone"
dpm
60 660
29 100
7 260
nul
95 500
47 000
36 420
nul
249 630
89 280
86 620
%
0.46
3.30
1.0
nul
1.0
5.2
3.8
nul
2.2
8.5
7.7
Estrogens0
dpm
6 000
2 180
1 460
nul
4 00
1800
420
2 240
nul
6 200
2 100
5 420
%
0.05
0.24
0.39
nul
0.05
0.18
0.03
0.09
nul
0.01
0.10
0.42
>
H
-<
DC
>
Tl
W
z
a = Data are expressed in dpm and in percent of the ether soluble fraction corrected to recovery of carrier hormones
b = After oxidation into A4-androstenedione
0 = Estrone + 17/3-estradiol after methylation
d = At 7'/2 days of incubation, both left and right ovaries were explanted; at 10 and 18 days, only the left ovary was cultured.

AVIAN GONADS IN VITRO261
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 in-
crease 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).
From 4-i4C-Dehydroepiandrosterone (DHA).
Low quantities of radioactive estrogens
were detected in the culture media of un-
differentiated chick gonads
(6 and 7 days of
incubation). This synthesis was found to
increase between
7V&, 10, and 18 days. No
significant synthesis of estrogens was found
in male gonads at IVi and 10 days, but took
place at 18 days (Table 1; Fig. 1) (Haffen
and Cedard, 1968; Guichard et al., 1973a).
Transformation of this precursor into es-
trogens took place in embryonic quail
ovaries and testes of
10 and 15 days incuba-
tion. Singularly, no increase of this estro-
gen production could be detected in the
female, while it was found in the male be-
tween these two stages (Scheib et al., 1974)
(Table 2).
Testosterone biosynthesis
From 3H-pregnenolone and l4C-proges-
terone. Tritiated and 14C-labeled testos-
terone was present in the culture media of
ovaries and testes isolated from 7V2-, 10-,
and 18-day chick embryos, and from 10-
and 15-day quail embryos. In chick, the
calculation of the yield of testosterone
2 3H-pregnenolone and 14C-progesterone were
added simultaneously into the culture medium. Since
their specific activities were very different, the molar
ratio of the precursors was equilibrated by addition of
non-labeled pregnenolone.
formed from 3H-pregnenolone and 14C-
progesterone indicated an increasing
synthesis of testosterone by testes as a func-
tion 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
testosterone formed from 14C-
progesterone by 10- and 18-day testes and
for testosterone derived from 3H-
pregnenolone by 18-day testes (Guichard et
al., 1973a).
In quail, calculation of the yield of forma-
tion of testosterone has also shown an in-
creasing synthesis in testes between the
10th and 15th day, with a better yield from
progesterone and a higher production in
male than in female gonads (Table 2).
As in the chick, only testosterone pro-
duced by testes could be recrystallized to a
constant isotopic ratio (Guichard et al.,
1973ft).
From 4-14C DHA. Radioactive testos-
terone was present in the culture media of
7-day chick embryonic gonads (Fig.
1). Be-
tween 7Vi, 10, and 18 days, labeled testos-
terone
was produced in increasing amounts
by male gonads. The synthesis of this
steroid could not be detected in the culture
media of 7lA- and 10-day ovaries, but was
found with 18-day ovaries (Table 1; Fig. 1).
The radiochemical purity of testosterone
isolated from the culture media could be
assessed (Haffen and Cedard, 1968;
Guichard et al., 1973a).
In quail, no significant synthesis of
testos-
terone was found with 10- and 15-day
ovaries or with 10-day testes, while it be-
came significant with 15-day testes. DHA
was found to be a less efficient precursor
than progesterone (Scheib et al., 1974)
(Table 2).
Conversion of pregnenolone into progesterone2
This transformation was quantitatively
estimated by determination of the radioac-
tivity of the tritiated progesterone derived
from 3H-pregnenolone (Fig. 2). Tritiated
progesterone derived from 3H-preg-
nenolone by gonads of both sexes in