/ . Embryol. exp. Morph. Vol. 44, pp. 1-13, 1978
Printed in Great Britain © Company of Biologists Limited 1978
\
Growth and sexual differentiation in the gonads of
chick and duck embryos
By JEAN-MARIE GASC 1
From the Institut cTEmbryologie du C.N.R.S. et du College de France
Nogent sur Marne, France
SUMMARY
The sex-dependent morphological asymmetry between gonads of bird embryos is generally
considered to represent the critical point at which the definitive pattern of sexual differentiation is established. A quantitative study of growth in gonads of chick embryos (6- to
11-day-old) and of duck embryos (7- to 11-day-old) has been carried out in order to clarify
in what manner growth of gonads is involved in sexual differentiation. Growth has been
estimated by the determination of total protein and DNA content, and of DNA synthesis
[3H]Tdr incorporation in vitro).
Increase of protein and DNA content follow different growth curves according to the sex
and the laterality of gonads. As early as day 6 (chick) or day 7 (duck) significant differences
of protein content exist between gonads: left > right in both sex embryos, and male > female
on both sides. Differences in DNA content of gonads are large even at the earliest stages. In
the 6-day-old chick embryo both testes contain less DNA than the left ovary and more DNA
than the right ovary. In the 7-day-old duck embryo both testes contain the same amount of
DNA as the left ovary and twice as much DNA as the right ovary. Consequently, the protein/
DNA ratio has a different value according to the sex of the gonad.
In the 6-day-old chick embryo and in the 7-day-old duck embryo absolute values of DNA
synthesis (cpm/gonad) are higher in left gonads than in right gonads, and higher in male
than in female gonads. When calculated as a ratio to the protein content (cpm/protein) DNA
synthesis is lower in the left ovary than in the three other types of gonads, both in chick and
duck embryos. When calculated as a ratio to the DNA content of gonads (cpm/DNA),
DNA synthesis is lower in the left ovary than in the three other gonads in the chick, and
lower in the left ovary and in testes than in the right ovary in the duck.
The results show that growth of gonads is sex-dependent at a very early stage. The meaning
of this sex character is discussed with special reference to the role of steroid hormone
secretion.
INTRODUCTION
One of the earliest detectable differences between male and female bird
embryos is the degree of morphological asymmetry between the gonads. In the
chick, this sex-dependent asymmetry first becomes perceptible in 7-day-old
embryos, when the difference in size between the ovaries in female embryos is
greater than that between the testes in males. It is also at this stage that histological differences between the gonads of male and female chick embryos can
first be clearly recognized (Venzke, 1954; Stahl & Carlon, 1973). This stage
1
Author's address: The University of North Carolina, Department of Anatomy, 111 Swing
Building, Chapel Hill, North Carolina 27514, U.S.A.
2
J.-M. GASC
is therefore considered to represent the critical point at which the definitive
pattern of sexual differentiation is established.
Growth rate differences between male and female gonads underlie their sexdependent asymmetry. Anatomical and histological observations are not
sufficient to compare precisely the differential development of male versus
female, and of right versus left gonads. Only quantitative estimations are able
to reveal the exact pattern of gonadal growth and to show in what manner this
growth is involved in sexual differentiation. The purpose of this work was to
describe gonadal growth quantitatively, in order to emphasize the role of
growth as an early character in sexual differentiation.
Until recently gonadal growth has only been measured as increase in wet
weight (Breneman, 1941; Venzke, 1943; Romanoff, 1967). These results are not
reliable for younger embryos when the gonads are still very small. In a recent
article Teng & Teng (1977) reported protein, RNA and DNA determinations
in ovaries of the chick embryo. The lack of information about male gonads and
the stages of development studied by these authors (days of incubation 8-18)
preclude these results from shedding any light on the sexual differentiation of
the gonads. However, measurements on gonadal volume by Mittwoch,
Narayanan, Delhanty & Smith (1971) provide evidence in favour of the early
sexual differentiation of gonadal growth.
The present investigation attempted to estimate the two main aspects of total
gonadal growth, i.e. cellular proliferation and cellular differentiation, by
examining DNA and protein. Tn rapidly growing organs, such as the embryonic
gonads, cellular proliferation is high and DNA synthesis, which is a measure
of this proliferation, is predominant. However, differentiation involves protein
as well as DNA synthesis. Measurements of the total protein and DNA content,
as well as the rate of DNA synthesis, have therefore been used in order to
estimate gonadal growth during sexual differentiation, from day 6 to day 11 of
incubation for the chick, and from day 7 to day 11 for the duck embryos. Before
day 6 for the chick and day 7 for the duck it is not possible to separate intact
gonads from the mesonephros on which they lie.
All determinations were performed on both chick and duck embryos to
enhance the generality of our conclusion, as well as to point out some species
differences.
MATERIALS AND METHODS
Animals. Eggs of both White Leghorn chick and Vekinduck(Anas platyrhyncos)
were incubated at 38 °C in a humidified incubator. The sex of 6- and 7-day-old
chick embryos and of 7- and 8-day-old duck embryos was determined by
examination of the heterochromosomes in blood cells of the extra-embryonic
circulation (Omura, 1970; Gasc, 1973). This technique does not disturb normal
embryonic development. The sex of older embryos was identified by morphological examination of gonads.
Growth and sexual differentiation in avian gonads
3
[*R]Thymidine incorporation. Gonads were separated from the mesonephros
with a microscalpel and placed on a semi-solid culture medium. Contaminating
pieces of mesonephric tubules were completely removed. Groups of gonads
(groups of seven for 6-day-old chick and 7-day-old duck gonads and groups of
five for older gonads) were transferred to a second culture medium (Wolff &
Haffen, 1952) containing [3H]Tdr (10/tCi/ml; 16 Ci/mM) and unlabelled
thymidine (50 x 10~6 mM/ml). It has been previously demonstrated (Gasc &
Liebart, 1972) that this medium is suitable for the incorporation of [3H]Tdr in
gonads. When unlabelled thymidine is not added to the culture medium
labelling is not homogeneously scattered in the whole organ, but an autoradiographic control showed that in our culture conditions [3H]Tdr incorporation is homogeneous even for older gonads (11 -day-old). After a 4 h culture on
this medium, the gonads were washed and homogenized in Tyrode (1 ml for
each group, either 7 or 5). These homogenates were used for protein and DNA
determinations and for radioactivity counting.
Protein determination. 0-1—0-3 ml of gonad homogenate was used for the
protein determinations (Lowry, Rosebrough, Farr & Randall, 1951), with
bovine serum albumin as a standard. Additional protein determinations were
performed on other gonads, immediately after removal from the mesonephros.
Despite the absence of the 4 h culture, protein contents were identical in both
sets of measurements and mean values of protein were calculated from
determinations of both sets.
DNA determination. 0-2-0-4 ml of gonad homogenate was stored at — 18 °C.
In order to reach the threshold of sensitivity for Burton's technique (Burton,
1956), aliquots of several experiments were pooled before being precipitated in
cold PCA (final concentration 0-5 N). After centrifugation the supernatant was
discarded and the sediment was twice hydrolyzed (15 min; 70 °C) in a small
volume (0-2-0-5 ml) of 0-5 N-PCA. Both hydrolyzates were pooled and 0-3 ml
reagent was added to a 0-150 ml measure of these. After an overnight incubation
(30 °C) optical density (600 nm) was measured in 0-5 ml microcuvettes. Samples
of 1-10 jug calf thymus were used as standards. Standard curves proved fully
reliable between 2 and 50 jug, since the variation did not exceed 5 %. Additional
DNA determinations were performed on gonads not cultivated prior to homogenization. Mean values of DNA content in gonads were calculated from these
two sets of values.
Radioactivity counting. 0-5 ml aliquots of homogenate were precipitated with
cold TCA (final concentration 5 %) and filtered on Whatman filter (GFB). Then
niters were washed with 0-5 % TCA and dried. Radioactivity fixed on the niters
(i.e. [3H]Tdr incorporated in DNA) was counted two or three times and mean
values calculated. Values of [3H]Tdr incorporation are listed in tables and
graphs in absolute terms (cpm/gonad), as well as relative to protein (cpm//*g of
protein) and relative to DNA (cpm//tg of DNA). Each cpm/protein ratio was
calculated from cpm and protein determinations performed on the same group
J.-M. GASC
200
100
100
50
I
30
20
10
0-5
10
0-2
3 -
01
7
8
9
10
Age (days)
11
-
7
8
9
10
Age (days)
Fig. 1. Increase of the amount of protein (1 a) and DNA (1 b) in gonads of the chick
embryo between days 6 and 11 of incubation. Semilogarithmic plot. Left ovary, •*•;
right ovary, • ; left testis, # ; right testis, A.
of gonads. A standard deviation (S.D.) value could, therefore, be calculated for
the mean of these ratios of [3H]Tdr incorporation/protein (i.e. rate of DNA
synthesis/protein). Since samples of several groups of gonads were pooled for
each DNA determination, and additional DNA determinations without cpm
measurement were carried out, cpm/DNA values could only be calculated as
ratio of cpm and DNA mean values, without S.D. values.
Statistical analysis. Each protein, DNA, cpm and cpm/protein determination
refers to one measurement performed with one homogenate of gonads. The
mean value of determinations and the corresponding standard deviation were
calculated (Lamotte, 1957). The statistical significance between mean values
was evaluated by the Student's t test. The correlation between right and left
gonads was also calculated according to Lamotte (1957).
RESULTS
I. Protein and DNA content in chick gonads
The amount of protein and DNA in chick embryo gonads increases progressively between days 6 and 11 of incubation. Fig. 1 shows a sex difference,
in that growth curves for protein and DNA are parallel for testes but not for
ovaries. In both testes and in the right ovary, the amount of protein (Fig. \a)
increases regularly between days 6 and 11 (9-10 jug of protein per day), but in
Growth and sexual differentiation in avian gonads
10
to
O
• cf* gonads
+ O gonads
e
5
O
3
4
5
I
I
I
6
7
8
9
10
Amount of protein in the left gonad (/(g)
11
12
13
Fig. 2. Scatter diagram of the protein content in gonads of the 6-day-old chick
embryo. Each point represents the mean amount of protein in the left gonad
(abscissa) and in the right gonad (ordinate) of a sample of seven pooled gonads.
the left ovary the amount of protein increases more rapidly than in the other
three gonads, in an almost exponential manner. The protein content of the left
ovary is equal to the one of the right testis in the 6-day-old embryo, but is
4-7 times higher in the 11-day-old embryo (Fig. la).
The DNA growth curves (Fig. 1 b) show a similar pattern, with accelerated
growth of the left ovary.
The protein and DNA contents of 6-day-old embryos are given in Table 1.
In embryos of both sexes, the left gonad contains more protein than the right
(P < 001). Moreover, the left testis contains more protein than the left ovary,
though not significantly so (P > 0-05), and the right testis contains more protein
than the right ovary (P < 0001). This early asymmetry in protein content is
illustrated in the scatter diagram of Fig. 2. The cluster of male points can be
distinguished from that of female points, and the shape of the clusters shows
that the protein content in right and in left gonads is highly correlated
(r = +0-9). Since protein determination in gonads of both sexes was carried
out in the same experiment, the differences between male and female points are
likely to be real. For the same protein content in the left gonad, the right gonad
contains more protein in a male than in a female embryo.
In 6-day-old chick embryo gonads, sex differences in DNA content are
evident (Table 1). Both testes have almost the same DNA content (P > 005),
whereas the left ovary contains twice as much DNA as the right (P < 001).
Protein content in
gonads (/*g)
DNA content in
gonads (fig)
Protein/DNA
7-59 ± 1 59
(15)
0-192±0025
(3)
39-5
Left
Right
Left
o gonads
Right
Left
$ gonads
Right
Left
Right
9-22 ± 1 56
(13)
0-235±0043
(3)
39-3
$ gonads
7-day-oId duck embryo
507 ±1-15 8-67 ±1-77 6-86 ±1-30 9-30 ±1-22 702 ±117 11-41 ±1-66
(15)
(17)
(17)
(16)
(16)
(13)
0091±0010 O136±OO8O 0-124±0-082 0-229±0039 0090±0014 0-254±0086
(3)
(3)
(3)
(3)
(3)
(3)
560
63-8
55-3
40-6
780
44-9
gonads
6-day-old chick embryo
Mean values ± S.D. («), Number of determinations. Each determination of protein was performed with a sample of seven pooled gonads;
each DNA determination with a minimum of 28 pooled gonads.
Table 1. Amount of protein {/ig/gonad) and DNA {(ig/gonad), and the ratio {protein/ DNA) in gonads of
the 6-day-old chick and 7-day-old duck embryos
O
w
k
S"1
Growth and sexual differentiation in avian gonads
30 -
•2 10
z °'5
0-2
9
10
Age (days)
9
10
Age (days)
Fig. 3. Increase of the amount of protein (3 a) and DNA (3 b) in gonads of the duck
embryo from day 7 to day 11 of incubation. Semi-logarithmic plot. Left ovary, *•;
right ovary, • ; left testis, • ; right testis, A.
Values of protein/DNA ratio bring further evidence of differences between
male and female gonads (Table 1). The testes and the right ovary have the same
value for this ratio (~ 60) whereas the left ovary has a much lower value (39-5).
Thus for the same amount of protein the left ovary has a higher DNA content
than the other gonads.
II. Protein and DNA content in duck gonads
Growth curves (protein, Fig. 3a; DNA, Fig. 36) for the right and left testis
of the duck embryo are parallel, whereas growth curves of the right and left
ovaries are quite distinct. Growth remains fairly steady until near the end of the
period studied, when it slows down rapidly except in the left ovary. The amount
of protein and DNA in the left ovary begins to exceed that in the testes between
days 10 and 11 in the duck as compared to days 7 and 8 in the chick (Fig. 1).
During the period of development that has been studied, growth of the testes
appears similar to that of the left ovary in the duck and the right ovary in the
chick.
Mean values of protein and DNA content in gonads of the 7-day-old duck
embryo are reported in Table 1. The amount of protein is higher in the left
gonad than in the right gonad, and higher in the male gonad than in the female
J.-M. GASC
(a) Chick
(b) Duck
S 1-5
EL 1 0
5
0-5
7
8
9
Days of incubation
10
S
9
10
Days of incubation
Jl
Fig. 4. [3H]Tdr incorporation in gonads of the 6- to 11-day-old chick (a) and 7- to
11-day-oJd duck (b) embryos, in cpm/gonad. Left ovary, *k; right ovary, • ; left
testis, • ; right testis, A.
homologous gonad. The sex difference between left testis and left ovary, and
between right testis and right ovary, is in both cases highly significant (P < 0001).
The DNA content of gonads in the 7-day-old duck embryo also shows sex
differences. Testes and left ovary contain at least twice as much DNA
(~ 0-200fig) as the right ovary (0090jug). This similarity between testes and
the left ovary has been noted previously (Fig. 3 b). The difference between the
right ovary and the right testis is significant (P < 002), as is the difference
between the right ovary and the left ovary (P < 001).
A comparison between chick and duck embryos shows that in both species,
the testes contain the same amount of DNA, but this amount is almost twice as
high in the duck as in the chick. This difference between chick and duck gonads
is also evident from the protein/DNA ratio (Table 1). This ratio is equal in the
testes and in the left ovary of the duck embryo (~ 40), whereas in the chick
embryo the left ovary is the only gonad with a low ratio (39-5).
III. [3H]Tdr incorporation in chick gonads
Between days 6 and 10 of incubation [3H]Tdr incorporation (estimated in
cpm/gonad, i.e. absolute value of incorporation) increases sevenfold in the left
Growth and sexual differentiation in avian gonads
9
3
ovary and two- to threefold in other gonads. [ H]Tdr incorporation is higher
in the left than in the right gonad of both male and female embryos, but this
difference is always greater between ovaries than between testes (Fig. 4 a).
Incorporation is constant in both testes between 7 and 10, but increases in both
ovaries.
In the 6-day-old embryo, absolute values of incorporation in the gonads
depend on their sex and laterality (Table 2). The difference between the right
ovary and the right testis is significant (P < 001), but that between the left
ovary and the left testis is not.
When incorporation is calculated with respect to the protein content in gonads
(i.e. rate of incorporation/protein), values are higher for right than for left
gonads, in embryos of both sexes (Table 2). In the 6-day-old embryo the difference between right and left ovary is not significant (P > 0-1) whereas the
difference between testes is significant (P = 0-05). The differences between
ovaries and testes are not significant.
The lower part of Table 2 gives [3H]Tdr incorporation relative to the DNA
content of the gonads. Values are almost equal in both testes and in the right
ovary, but much lower for the left ovary. Thus the left ovary, which has (in the
6-day-old embryo) the higher DNA content (Table 1), has the lower rate of
incorporation/DNA. Moreover, the three gonads (right ovary and both testes)
which are relatively low in DNA content (see protein/DNA ratios in Table 1)
have the highest [3H]Tdr incorporation rate/DNA. No s.D. value could be
calculated for these ratios and therefore no statistical significance estimated (see
Materials and Methods).
IV. \?H]Tdr incorporation in duck gonads.
[ H]Tdr incorporation in duck gonads increases between days 7 and 11 of
incubation (Fig. 4b). In the testes the incorporation curves are parallel and
achieve a level in the 9-day-old gonad intermediate between the right and left
ovary curves. The incorporation level of the testes in the duck embryo plateaus
later and at a higher level than in the chick embryo. The same species difference has
already been observed for the protein and DNA content of gonads (Fig. 1 and 3).
Absolute values of [3H]Tdr incorporation (cpm/gonad) in gonads of the
7-day-old duck embryo show the same differences between sexes as in gonads
of the 6-day-old chick embryo (Table 2). Incorporation is higher in testis than
in ovary (P< 0-01). The rate of [3H]Tdr incorporation/protein in the left ovary
of the 7-day-old duck embryo, as in 6-day-old chick gonads, is the lowest of the
four gonads (Table 2).
Large differences of [3H]Tdr incorporation/DNA rates appear between the
right ovary and the three other gonads in the 7-day-old duck embryo. Estimated
in cpm//tg of DNA, this is twice as high in the right ovary as in the left ovary
or the testes. The statistical significance of this difference could not be estimated
since these rates are ratios of means (see Materials and Methods). Yet the
3
Cpm//*g of DNA
Cpm//*g of protein
Cpm/gonad
Right
363 ±74
(10)
68-7±14-l
(10)
3980
450 ±121
(10)
611 ±8-9
(10)
2345
? gonads
Left
c
482 + 59
(10)
77-7 ±15-3
(10)
3890
Right
gonads
519±71
(10)
64-6 ±10-3
(10)
3820
Left
6-day-old chick embryo
A
Left
644+79
(8)
59-8 + 91
(8)
2530
Right
431 ±75
(8)
62-9 ±15-2
(8)
4790
605 ±105
(8)
69-5 ±11-5
(8)
2530
Right
c? gonads
7-day-old duck embryo
$gonads
485 + 63
(8)
53-3 + 9-7
(8)
2120
Left
[3H]Tdr incorporation
Incorporation is calculated per gonad (cpm/gonad), or as a ratio to the amount of protein (cpm//*g of protein) or DNA (cpm/^g of
DNA). Mean values ± S.D. except for cpm/DNA. This latter rate is a ratio of mean values of cpm/gonad (1st line of the table) and of
DNA/gonad (2nd line of table 1) without S.D. value. («), number of determinations. Each determination was performed with a sample
of seven pooled gonads.
Table 2. [3H]Tdr incorporation (4 h culture) in gonads of 6-day-old chick and 7-day-old duck embryos
O
•M. GAS
Growth and sexual differentiation in avian gonads
11
importance of the difference between the left and the right ovary, when compared to the equal values of this rate in both testes, suggests that this difference
is actual.
The difference between chick and duck embryos for the rate of incorporation/
DNA is conspicuous in Table 2: in the chick, the left ovary is the only gonad
whose rate of incorporation/DNA is relatively low, and in the duck the right
ovary is the only gonad whose rate is relatively high. This species difference
supports our previous observation about the chick gonads: that is, gonads with
the highest DNA content, which also have a low value for the protein/DNA
ratio, have the lowest rate of [3H]Tdr incorporation/DNA.
DISCUSSION
The data presented have implications concerning sexual differentiation in
gonads of bird embryos. Before discussing these implications, three observations
from the results should be pointed out.
(1) A difference has been shown between the linear growth pattern for
protein in testes of the chick embryo, and the accelerated growth pattern of the
left ovary (Fig. 1 a). This difference brings a clear mathematical expression to the
morphological observation of differences of growth between ovaries and testes.
(2) Figs. 1 and 2 show the continuous growth (protein and DNA) of the
right ovary. Therefore the regression of this gonad has not started before day 11
of incubation, and growth is still significant at this stage.
(3) In the 6-day-old chick and the 7-day-old duck embryos, the left ovary has
a lower rate of DNA synthesis than the other three gonads (Table 2). This seems
paradoxical, since the left ovary will grow more than the other gonads during
the following days. Tt is necessary, however, to consider the synthesis rates in
relation to the amount of protein and DNA of the gonads; therefore, the
absolute values of growth can be temporarily larger in the gonad with the smallest
synthesis rates. Furthermore, rates in the following days strikingly decrease in
all gonads except in the left ovary (unpublished results). Thus, the left ovary has
low and slowly decreasing synthesis rates; the other gonads have higher
synthesis rates, decreasing more rapidly.
Protein and DNA contents in gonads of the 6-day-old chick and of the
7-day-old duck embryo show sex differences (Table 1 and 2). Protein and DNA
growth curves are also different in male and in female gonads (Figs. 1 and 3).
Consequently the total growth of gonads depends on their sex as early as day 6
in the chick and day 7 in the duck embryos; this dependence was studied until
day 11. The growth pattern differences show that, early in development, growth
mechanisms are controlled by the genetic sex of the embryo.
Among these mechanisms one of the most important is DNA synthesis.
Table 2 shows that DNA synthesis levels in gonads are dependent on the genetic
sex. Sex differences of DNA synthesis levels between gonads are evident both
12
J.-M. GASC
for absolute values and for rates. Gonads relatively rich in DNA (the left ovary
of the chick, and the left ovary and both testes of the duck) have the lowest
DNA synthesis rate. This observation suggests an effect of the genetic sex on
mechanisms of gonadal growth more precocious than we report in this paper.
This assumption is supported by the work of Mittwoch et al. (1971) on the
growth of chick embryo gonads. These authors showed that, as early as day 5
of incubation, the volume of the left gonad is greater than the volume of the
right, in both sexes; while the testis of the 5-, 6- and 7-day-old embryo has
a volume greater than the corresponding ovary in an embryo of the same age.
This difference we observed in measuring protein content in the chick gonads
from day 6 in the chick and day 7 in the duck.
According to Mittwoch (1971), growth is responsible for sexual differentiation in gonads: a gonad with high growth differentiates into an ovary and
a gonad with low growth differentiates into a testis. Our results do not support
this hypothesis. Indeed, although the left ovary of the 6-day-old chick contains
more DNA than the testes, it contains less protein and its DNA synthesis rates
are lower. Furthermore, in the duck embryo the testes have higher DNA
synthesis and content values than the left ovary, and this persists until after
day 9 (Fig. 4b) or 10 (Fig. 3a, b). At this time other sexual characters are well
fixed. Contrary to the hypothesis of Mittwoch (1971), it does not seem possible
to assert that growth has the predominant role in sexual differentiation of gonads.
Rather we consider gonadal growth as one character among others.
Two of these other sexual characters have to be mentioned in this discussion.
The first is the degree of asymmetry in gonocyte distribution between the two
gonads of an embryo. According to the exhaustive study by van Limborgh
(1968#, b; 1970), this asymmetry appears to be sex-dependent in gonads of
young chick (4-day-old) and duck (5-day-old) embryos. The second is hormonal
secretion. According to Scheib & Haffen (1974), steroid hormone secretion
differs in male and female in 6- or 7-day-old embryo. Woods & Podczaski (1974)
have reported a difference in androgen secretion between male and female
gonads on day 5£ of incubation. These results do not come from accurate
measurements but from estimations, and the differences between these values
cannot be accepted without reservation until they become large enough to
overcome the lack of precision, that is, in the 6- or 7-day-old embryo. In a more
recent paper Woods, Simpson & Moore (1975) have reported a difference in
plasma concentration of testosterone between male and female in the 6^-day-old
embryo.
Therefore, steroid secretion exists in the early embryo but there is no clear
evidence of a sex difference before day 6 of incubation (see reviews in Scheib &
Haffen, 1974; and in Weniger, 1974). Thus, the sexual differentiation of hormonal
secretion occurs no earlier than the differentiation of other sex-dependent
characters, such as asymmetry of gonocyte distribution or growth. Hormone
secretion may, therefore, not be the predominant and determinative factor of
Growth and sexual differentiation in avian gonads
13
the early sexual differentiation of gonads. Further, the role of the steroid
receptors must be considered in this new pattern of relationships, not merely
the direct effect of hormones on tissues.
We are very grateful to Professor Et. Wolff who initiated this work on gonadal growth and
provided us valuable advice and encouragement in the course of this study. The results were
submitted on the 21st of January 1976 to the Faculty of Sciences of the Pierre and Marie
Curie University (Paris, France) as a part of a doctoral thesis (C.N.R.S. A.O. 12042).
BRENEMAN, W.
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BURTON,
{Received 11 January 1977, revised 9 September 1977)
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