/. Embryol. exp. Morph. 73, 59-68, 1983
Printed in Great Britain © The Company of Biologists Limited 1983
59
Growth pattern of the sex ducts in foetal mouse
hermaphrodites
By C. YDING ANDERSEN 1 , A. G. BYSKOV AND
J. GRINSTED
From the Finsen Laboratory, Copenhagen
SUMMARY
In this report the morphology of the gonads and the growth of the Wolffian and Mullerian
duct in foetal mouse true hermaphrodites (16 days p.c.) have been studied and compared to
that of normal mice. The ducts from the hermaphrodites were placed in one of five groups
according to the proportion of male and female characteristic of the gonad.
When more than 85 % of the gonadal tissue was masculine, the Wolffian ducts showed the
same percentage of cells in mitosis (mitotic index, MI) as normal males. The MI of the
Wolffian ducts was lower, but constant, if the gonad contained between 0 and 85 % of testicular tissue. The number of Leydig cells in the gonads showed a linear relationship with the
percentage of testicular tissue. Apparently, the MI of the Wolffian duct does not increase with
increasing 'maleness' and with the number of Leydig cells. Four possibilities are put forward
to explain the constant level of MI: (i) The Leydig cells of hermaphrodites may be deficient
in producing testosterone. (2) The Leydig cells may produce testosterone at a normal rate but
the epithelial cells of the Wolffian duct may not respond to increasing levels of testosterone
by increasing their mitotic activity. (3) The presence of female gonadal tissue may directly or
indirectly inhibit the mitotic activity of the epithelial cells of the Wolffian duct. (4) The
epithelial cells of the Wolffian duct may respond to a low threshold level of testosterone, but
maximal response is only triggered by a critical higher hormone level present only in group V.
In hermaphrodites, the Wolffian duct attached to a gonad without testicular tissue and
without Leydig cells, has a MI which is significantly higher than in normal females. It is
suggested that circulating testosterone from the contralateral gonad is responsible for this high
MI.
In the Mullerian duct a mitotic index similar to that of the normal females was only found
when the gonad contained from 0 to 15 % of testicular tissue. If a gonad contained more than
15 % of testicular tissue, the MI of the attached Mullerian duct was much lower equalizing that
of normal males. No influence on the growth of the Mullerian duct could be observed from
the contralateral gonad.
INTRODUCTION
The Mullerian and Wolffian ducts are present in the early embryo of both
sexes. After the sex of the gonads are distinguishable morphologically the ducts
begin to differentiate.
In the male, the Mullerian duct regresses due to the action of the antiMiillerian
hormone (AMH), which is probably secreted by the Sertoli cells (Josso, Picard
1
Author's address: The Finsen Laboratory, The Finsen Institute, Strandboulevarden 49,
2100 Copenhagen 0., DK-Denmark.
60
C. Y. ANDERSEN, A. G. BYSKOV AND J. GRINSTED
& Tran, 1980). The Wolffian duct is stimulated to grow by testosterone produced
by the Leydig cells (Setchell, 1978 for review).
In the female, the processes are quite different. The Miillerian duct grows
autonomically, whereas the Wolffian duct regresses due to a lack of stimulation
from androgens. The Miillerian duct grows since AMH probably is absent in the
female (Pelliniemi & Dym, 1980, for review). It appears that the ducts become
phenotypically female if they are not actively induced to become masculine
(Jost, 1971).
In order to study varying degrees of influence from testicular tissue on the
development and growth of the ducts, we have looked at true hermaphrodites.
These are characterized by having ovarian as well as testicular tissue in their
gonads. Since all degrees of male-femaleness (from 100 % ovarian tissue to
100 % testicular tissue) can be seen, these were suitable to study the interaction
between the masculinity of the gonad and the growth and differentiation of the
Wolffian and Miillerian ducts. The growth pattern of the ducts, evaluated by the
percentage of mitosis, was studied in true hermaphrodites of the foetal mouse
and compared to that of normal male and female foetuses.
MATERIALS AND METHODS
The hermaphrodites used in this study were the same as the ones used by
Whitten, Beamer & Byskov (1979), and were produced by pairing female mice
of different strains wkh Balb/c-WT males.
Gonads with mesonephric tissue attached were dissected from the foetuses on
day 16p. c. The morning when the copulatory plug was found, was defined as day
1 post coitum {p.c). A total of 48 ducts and gonads from 32 foetuses were
studied. In 16 foetuses both gonads and ducts were evaluated, whereas only one
gonad and duct from each of the other foetuses could be used.
For comparison eight normal 16-day-old foetuses, four males and four
females, of Balb/c strain were used.
The tissue is fixed in Zenker's solution, processed for paraffin embedding,
serially sectioned at 6/im and stained with PAS and haematoxylin.
Based on the histological preparations both gonads of each hermaphrodite
were categorized according to the relative proportions of male and female
gonadal tissue. The male part contained testicular cords with nonmeiotic germ
cells and the female part lacked testicular cords and had germ cells in meiosis.
The male-femaleness of the gonads was quantified by point counting (Gundersen & 0sterby, 1980; 1981) of every 10th section starting with the section in
which 10 points could be counted. The point density of the grid was a priori
selected to give a mean score of 80 per section, which should provide a sufficient
precision (Gundersen & 0sterby, 1980). Points over ovarian and testicular tissue
were counted separately. All point counts of testicular and ovarian tissue of each
gonad were added to give a relative evaluation of the size of the gonad,
Sex ducts in foetal mouse hermaphrodites
61
i.e. total point score, as well as a measurement of the degree of maleness. The
degree of maleness was calculated as the percentage of points over the testes
tissue per total point score. Depending on the percentage of testicular tissue
within the gonads they were placed in one of the following five groups:
Group I:
0-14 % male
Group II:
15-39% —
Group III:
40-60% —
Group IV:
61-85% —
Group V:
86-100% —
In each specimen the Wolffian and the Mullerian duct were identified. At least
250 nuclei were counted in the epithelia of the ducts, using every second section,
and the number of mitoses were recorded, from which the mitotic index, i.e. the
percentage of cells in mitosis (MI) was calculated. However, in three specimens
the epithelium of one or the other duct was degenerated to such an extent that
only 175 nuclei could be counted. The mean value ±S.E. of the MI for the
Wolffian and Mullerian ducts were calculated for each of the five groups as well
as for the normal foetuses; these values were plotted against the proportion of
male tissue (Fig. 1). The MI for the ducts of the different groups was compared
and the differences were evaluated statistically using analysis of varians.
Before any statistical treatment the data were transformed using x = Arcsin
-/p"(p = the observed MI). According to Davies (1971) this is an appropriate
transformation of the data before statistical analysis.
The number of Leydig cells were counted in every 10th section on the same
section which had been used for point counting. The Leydig cells were distinguished from other somatic cells by their PAS-positive-reacting cytoplasm and
their spherical nuclei. The total number of Leydig cells per total point score of
the gonad was calculated and the mean value ±S.E. of each group was plotted
against the percentage of male tissue in that group. Linearity was examined by
linear regression analysis.
Further analyses were carried out to determine whether the contralateral
gonad influences growth of the ducts on the other side of the foetuses. Influence
from the testicular tissue was tested by comparing the MI of the ducts of a normal
female with the MI from the ducts of a hermaphrodite, in which one gonad
belonged to group I (i.e. 0-15 % male tissue) and the contralateral gonad contained more testicular tissue (group II-V, i.e. 15-100 % male tissue). Influence
of the ovarian tissue was tested by comparing the MI from the ducts of a normal
male foetus with the MI from the ducts of a hermaphrodite, in which one gonad
belonged to group V (i.e. 85-100% male tissue) and the contralateral gonad
contained less ovarian tissue (group I-IV, i.e. 0-85 % male tissue).
RESULTS
The MI of Wolffian and Mullerian ducts of all foetuses are shown in Table 1.
The mitotic index of the Mullerian and Wolffian ducts as a function of the
EMB73
62
C. Y. ANDERSEN, A. G. BYSKOV AND J. GRINSTED
Table 1. Mitotic indices of Wolffian (W) and Miillerian (M) ducts of 16-day p.c.
mouse hermaphrodites and normal mouse foetuses
Normal
female
W-M
Number
Mean
Normal
male
W-M
Group I
W-M
Group II Group III Group IV Group V
W-M
W-M
W-M
W-M
2-1-6-7
3-6-7-5
2-7-8-4
2-7-7-9
7-0-6-0
5-6-8-7
5-9-8-3
6-1-8-6
2-5-9-8
6-9-4-4
5-8-2-9
6-4-1-4
7-4-3-1
7-0-2-9
5-7-4-5
5-7-2-4
6-9-1-7
6-1-3-4
7-0-3-5
6-8-4-6
5-2-1-5
5-9-5-1
6-1-3-2
5-2-3-0
5-6-3-1
5-3-2-2
6-2-1-7
4-8-1-9
5-2-2-4
5-0-2-4
5-8-3-5
6-2-5-4
5-4-1-9
6-6-1-4
5-7-2-3
5-4-2-4
7-3-2-4
8-6-1-8
8-7-2-8
7-5-3-5
7-2-2-0
5-2-2-3
8-9-1-4
8-1-1-5
7-4-2-0
5-2-3-3
6-9-2-8
6-8-2-9
7-8-1-8
8-5-1-7
8-0-2-2
8-2-1-7
7-5-2-0
7-9-2-1
7-7-2-0
7-5-2-1
4
5
7
9
12
15
4
2-8-7-6
5-7-8-3
6-4-3-1
6-1-3-6
5-7-2-5
7-5-2-2
7-6-2-1
percentage of testicular tissue in the gonads is shown as a mean ±S.E. in Fig. 1.
The gonads varied from normal ovaries (group I) to normal testes (group V)
(Fig. 2,3,4) and were grouped according to the percentage of testicular tissue
in the gonad. The number of observations in each group is also listed in Fig. 1.
In the Wolffian duct the mitotic index was of the same magnitude in group I,
II, III and IV (P = 0-25). The mitotic index in group V is significantly higher than
in group IV (P = 0-0001). In the Miillerian duct the mitotic index decreases from
group I to a nearly constant low level in group II, III, IV and V (Fig. 1). No linear
relationship is found between the MI of the Mullerian duct and the percentage
of male tissue in the gonad (correlation coefficient 0-67). A linear relationship
exists between the mean value of the relative number of Leydig cells per gonad
and the percentage of male tissue found in the gonad (correlation
coefficient = 0-983) (Fig. 5). The mean of the relative number of Leydig cells is
almost zero in group I and approaches 600 in group V.
By comparing the MI of the ducts from normal male and female foetuses with
the hermaphrodites, it was determined whether a gonad influenced the MI of the
ducts at the other side of the foetus.
The ovarian tissue at one side of a foetus does not seem to influence the growth
of the contralateral ducts with an attached gonad of group V, since the MI of the
Mullerian and Wolffian ducts does not differ significantly from the MI of a
normal male foetus (Table 1).
Sex ducts in foetal mouse hermaphrodites
63
Mitotic index
10
Hermaphrodites
Normal
6
T
•
Normal
I
4 •
I
2 -
Female
15
60
III
% Male tissue
85
40
IV
100
Male
V
Fig. 1. The mitotic index (mean±s.E.) in the Miillerian duct (—O—) and the
Wolffian duct (—#—) as a function of male tissue in the gonad of hermaphrodites. In the hermaphrodites the number of duct systems included in each group
was, group I: 5, group II: 7, group III: 9, group IV: 12, group V: 15. The mitotic
indices of the sex ducts from normal male and females are also shown. The normal
male and females included four duct systems each.
In contrast, the testicular tissue seems to influence the growth of the
contralateral Wolffian duct belonging to group I. The MI of the Wolffian
duct from group I is significantly higher than the MI of normal females (P
= 0-02).
The MI of ducts from foetuses where both sides could be counted is shown in
Table 2. The relative few data make statistical analysis difficult.
However, in foetus number 11 and 16 the two gonads belonging to group I are
both determined to be 100 % pure ovaries. The higher MI for the Wolffian duct,
compared to that of normal female foetuses, support the suggestion of a stimulation from testosterone produced by the other gonad of the foetus containing
testicular tissue.
There is no significant difference between the MI of the Miillerian duct from
group I and normal female (P = 0-46) Table 1. This indicates that the testicular
tissue has no influence on the growth of the Miillerian duct on the other side of
the foetus.
64
C. Y. ANDERSEN, A. G. BYSKOV AND J. GRINSTED
• J •
Sex ducts in foetal mouse hermaphrodites
65
Table 2. Mitotic indices on the hermaphrodites, where the Wolffian (W) and the
Mullerian (M) ducts on both sides of the foetus are counted.
No.
Group I
W-M
1
2
3
70-6-0
Group II
W-M
Group III
W-M
Group IV
W-M
Group V
W-M
5-3-2-2
6-2-1-7
5-2-2-3
6-1-3-4
6-9-4-4
7-0-3-5
6-8-4-6
4
8-9-1-4
8-1-1-5
5
5-2-1-5
6
7
8
9
10
11
12
13
5-9-5-1
6-1-3-2
5-6-8-7
7-4-3-1
5-7-2-4
14
15
16
6-1-8-6
4-8-1-9
5-2-2-4
5-0-2-4
5-8-3-5
6-2-5-4
5-4-1-9
6-6-1-4
5-7-2-3
6-9-2-8
6-8-2-9
7-8-1-8
8-5-1-7
8-0-2-2
8-2-1-7
7-0-2-9
DISCUSSION
This study shows that the growth of the Wolffian duct, expressed as the percentage of cells in mitosis, is not correlated to the number of Leydig cells in the
ipsilateral gonad. Since the growth of the Wolffian duct is dependent on testosterone secreted by the Leydig cells, it could be expected that an increase in the
Fig. 2. A. Gonad with the sex ducts (Mullerian (M), Wolffian (W)) from a 16-day
p.c. mouse hermaphrodite (group I). (x90). B. Mullerian duct with mitosis,
(x 350). C. Wolffian duct degenerating without mitosis, (x 350).
Fig. 3. A. Gonad with the sex ducts (Mullerian (M), Wolffian (W)) from a 16-day
p.c. mouse hermaphrodite (group V). (x 90). B. A degenerating Mullerian duct,
(x 350). C. The Wolffian duct growing well with mitoses, (x 350).
Fig. 4. A. Gonad with the sex ducts (Mullerian (M), Wolffian (W)) from a 16-day
p.c. mouse hermaphrodite (group IV) with testicular cords and germ cells in meiosis
(arrows), (x 90). B. Mullerian duct without mitoses, (x 350). C. Wolffian duct with
mitoses. (x350).
66
C. Y. A N D E R S E N , A. G. BYSKOV AND J.
GRINSTED
Relative number of leydig cells per gonad
800 • •
700
600
500
400
300 -
I
200
100
40
15
60
85
100
% Male tissue
I
II
III
IV
V
Fig. 5. The relative number of Leydig cells per gonad (mean±s.E.) in each group as
a function of the percentage of male tissue in the hermaphrodite gonad. (Correlations coefficient: 0-983).
0
number of Leydig cells would result in an increase in MI of the attached Wolffian
duct. However, in these foetal mouse hermaphrodites the MI of the ducts is the
same in group I to group IV although the relative number of Leydig cells gradually increases from 0 in a 'pure' female gonad of group I to about 600 in an 84 %
male gonad of group IV. The MI of the Wolffian duct in the normal female
mouse foetus is significantly lower. To explain this constant level of MI in group
I to IV some possibilities are discussed.
One explanation may be that the Leydig cells of true hermaphrodites are
deficient in producing testosterone, so that the number of Leydig cells is not
proportional to the testosterone secretion.
Another explanation is that the Leydig cells produce normal amounts of testosterone but that the target of the testosterone, the epithelial cells of the Wolffian duct, respond differently to testosterone, due to their genetic constitution.
True hermaphrodites are in fact chromosome mosaics, with a mixture of XX and
XY cells (Lyon, 1969; Whitten, 1975; Polani, 1981).
It is also a possibility that the female tissue inhibits the mitotic activity of the
epithelial cells of the Wolffian duct, e.g. by aromatizing testosterone or by
Sex ducts in foetal mouse hermaphrodites
67
producing substances which affect the growth of the Wolffian duct. However,
such an interaction is likely to be proportional to the amount of female tissue,
i.e. the effect should gradually disappear from group I to group IV.
Perhaps the most plausible explanation for the constant level of MI of the
Wolffian duct is that the cells respond to a low threshold level of testosterone,
but that the maximal response is only triggered by a critical higher hormone
level, which is only present in group V.
The relative few data on foetuses where enough cells of the ducts could be
counted on both sides, make it difficult to say to what an extent a gonad affects
the growth of the ducts on the other side of the foetus. However, it seems that
testosterone not only influences the growth of the attached Wolffian duct but
also stimulates the contralateral duct to grow. The statistical significance
between the MI of normal females and group I hermaphrodites seems to show
this. It is further supported by the higher MI of the Wolffian duct from the
two 100% pure ovaries, which have gonads on the other side containing
testicular tissue.
The mitotic index of the Miillerian duct shows that suppression of growth is
obtained when the attached gonad contains more than 15 % of testicular tissue.
It appears that more than 15 % of testicular tissue is needed to produce
antiMiillerian hormone (AMH) in concentrations which result in full inhibition
of growth in the ipsilateral Miillerian duct.
The influence of AMH from one gonad was not expressed in an effect on the
contralateral gonad. The MI of the Miillerian ducts of group I is the same as in
normal females showing that the testicular tissue on the contralateral side does
not influence their growth.
The authors wish to thank Mrs. Lene Ahrenst, Mrs. Ingelise Green and Mrs. Dorrit Hansen
for excellent technical assistance. This work was supported by The Danish Natural Science
Research Council (No. 11-0026), The Danish Medical Research Council (No. 12-2350 and No.
512-2130) and by Nordic Insulin Foundation.
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{Accepted 1 August 1982)