J. Embryol exp. Morph. Vol. 67, pp. 81-87, 1982
Printed in Great Britain © Company of Biologists Limited 1982
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Influence of a temporary embryonic
testis graft on the regression of Miillerian ducts
in female chick embryo
By R. MARAUD 1 , M. RASHEDP AND R. STOLL1
From the Laboratoire d'Histologie et d'Embryologie,
Universite Bordeaux II, France
SUMMARY
Whole testes of 13-day-old chick embryos grafted according to an original method into
the extraembryonic coelom of 3-day-old recipients were next removed up to day 6.
The temporary presence of a testis allowed the Miillerian ducts to retrogress, from day 8,
in a high percentage of female hosts. It can be concluded that the graft secreted into the
blood of the embryo enough 'antiMullerian hormone' to provoke regression of ducts at
least 2 days after its removal and that the reaction of ducts depends on the appearance of
their sensitivity to this hormone.
INTRODUCTION
The grafting of an embryonic testis in female chick embryos provokes the
regression of Miillerian ducts (M.D.) (Wolff, 1947) but graft activity, as
measured by percentage of regression obtained in implanted embryos, varies
according to the donor strain and age (Maraud, Stoll & Coulaud, 1966, 1970),
the maximum effect being obtained with graft from 13-day-old male embryos in
the strain used here (Stoll, Rashedi & Maraud, 1975).
Graft activity is as strong in the intraembryonic coelom (Maraud et ah 1966),
implanted according to Dossel's method (1954), as implanted in the extraembryonic coelom (Stoll, Rashedi & Maraud, 1978). This supports the idea of
an hormonal effect, but the grafted testis does not act through androgenic
steroids because hormones of this type cannot duplicate the action of the graft.
Indeed steroids administered to an embryo before its sexual differentiation
cause an agenesia of M.D., i.e. an interruption of their development, whereas
a graft causes their regression after development (Stoll, 1948, 1950; Stoll,
Faucounau & Maraud, 1972). The testis acts by means of a hormone of
molecular weight greater than 1000 daltons (Weniger, Mack & Holder, 1975),
perhaps analogous to 'antiMullerian hormone' of mammals (Josso, Tran &
1
Authors' address: Laboratoire d'Histologie et d'Embryologie, Universite Bordeaux II,
146, rue Leo Saignat, 33076 Bordeaux Cedex, France.
82
R. MARAUD, M. RASHEDI AND R. STOLL
E.C
E.E.C.
v.v.
Fig. 1(A). Schematic view of a 3-day-old chick embryo (Hamburger-Hamilton
stage 18). The arrows show the two ways used to implant the graft under the chorion
near the umbilical region. (B) Schematic cross section of the same embryo showing
final position of the graft in relation to embryonic membranes and vessels. Symbols:
Am: Amnion; Ch.: chorion; E.C: embryonic coelom; E.E.C: Extraembryonic
coelom; G.: graft; V.V.: vitelline vessels. (Modified after Mathias Duval, 1889).
Picard, 1977) since chick embryonic testis provokes the 'in vitro' regression of
mouse M.D. (Weniger, 1965).
On the other hand, the chronology of duct regression was the same in
a grafted female as that in a normal male (Maraud et ah 1970) extending
between the 8th and the 13th day of embryonic life (Willier, 1939). The question
arises whether the regression begins because of the onset of the secretory
activity in the graft or because of the appearance of the duct sensitivity.
This later point could be ascertained with a method allowing both precocious
grafting of large organs and removal of the graft before the duct regression.
However methods described by various authors permitted either grafting of
small fragments in young embryos (Hamburger, 1938; Dossel, 1954) or grafting
of large fragments in older embryos (Willier, 1924; Groenendijk-Huijbers,
1966) and none of them permitted removal of the graft at an early stage. We
have devised a new, simple method which allows grafting of large fragments
into young embryos and also removal of the graft at various stages of embryonic
Regression of Mullerian ducts in chick embryos
83
Table 1. Influence on the Mullerian ducts (M.D.) of female chick embryos of
a grafted embryonic testis left temporarily (X), compared with a testicular graft
left definitively (XX) or with a graft of neutral tissue (XXX)
?1 embryos
Type of grafting
Embryonic testis
at day 3 (X)
Embryonic testis
at day 4 (X)
Embryonic testis
at day 3 (XX)
Neutral tissue (XXX)
Further manipulation
No. M. D.
Reg. M.D
6
6
12
0
1
13
9
2
2
15
19
0
Graft removed
at day 4
at day 5
at day 6
Graft removed
at day 6
—
No. M.D.: Normal Mullerian ducts; Reg. M.D.: Regressed Mullerian ducts.
life. Thus it was possible to analyse separately a testis graft as inductor and M.D.
as receptors.
MATERIAL AND METHODS
Three-day-old embryos from Derco strain (Red-Cornish $ x White Rock $)
(Hamburger & Hamilton, stage 18, 1951) were grafted with an entire testis from
a 13-day-old male donor according to the following method.
At 3 days the embryo partly lies on its left side, the embryonic membranes
are separated into amnios and chorion and the vitelline membrane has disappeared from the upper part of the egg. The large extraembryonic coelom
situated between chorion and yolk sac is readily accessible and the numerous
vitelline and allantoic vessels will give the graft good vascularization.
An aperture was made through the shell of the egg at a point on the middle
of its greatest circumfeience. Pipetting a small amount of albumen makes the
embryo slightly lowered when it comes under this aperture because of the
rotation of the yolk sac. The chorion was opened by tearing with fine watchmaker's forceps either near the head, or near the caudal end of the embryo
carefully avoiding any injury of amnion or yolk sac (Fig. I A). The graft
marked with a few grains of sterile carbon was nudged into the incision and
pushed against the vessels of the future umbilical region (Fig. 1 A, B). The eggshell aperture was then closed with cellulose tape. The graft became vascularized within approximately 24 h. Its differentiation was excellent and it was
easy to recover when embryos were sacrificed, or before, being generally
inserted on extraembryonic membranes near the umbilicus.
The graft was removed from some of the embryos (200), in the same way, at
day 4, 5 or 6. After tearing the chorion and gently pushing the allantois which
84
R. MARAUD, M. RASHEDI AND R. STOLL
M
Fig. 2. (a). Microscopic view of the genital tract of a testis-grafted 7-day-old chick
embryo. Both right and left Miillerian ducts are present, (arrows) G: gonad;
M: mesonephros. (b). Macroscopic view of a normal 14-day female chick embryo.
Both right rudimentary and left Miillerian ducts are present, (arrows), (c) Macroscopic view of a 14-day female chick embryo, grafted at day 3 with a testis and
graft removed at day 6. Miillerian ducts are regressed. Only persists a very small,
spherical remnant (arrow) lying before the left Wolffian duct.
developed for the time of grafting, the implant was carefully excised, to avoid
haemorrhage as far as possible. Since its characteristic shape is retained, it
is possible to ensure that it is removed entirely. We only operated on embryos
where graft was well vascularized by embryonic vessels of the host. Other
embryos where graft was observed free in the coelom and of a ivory-like aspect
Regression of Midlerian ducts in chick embryos
85
were rejected. Incubation was continued until day 7 (10 subjects) or 14 (81
subjects) when embryos were sacrificed. These stages were chosen, in the first
case because M.D. are normally present, and in the second because it is
just above the period of normal regression when any graft-induced duct
regression is easy to recognize macroscopically in female embryos.
Some other embryos were grafted at 4 days and the graft removed at 6 days
(61 subjects).
Two other batches of embryos were used as controls. Some of the testisgrafted ones were sacrificed at day 14 when only those presenting a well vascularized graft were considered (37 embryos). Other embryos (40) were grafted
under the same conditions with a piece of intestine and also sacrificed at 14 days.
Genital tracts of female embryos were observed macroscopically fixed in
Bouin's fluid and treated by conventional histology methods.
RESULTS
The grafting of a 13-day-old whole testis or of a piece of intestine, as control,
was followed by some post-operative death (nearly 20 %). The ablation of the
graft also caused a high percentage of death (nearly 60 %) particularly from
haemorrhage when the testis was implanted near or upon a large vessel of the
host.
The results observed in surviving 14-day-old female embryos from different
batches are reported in Table 1.
The major result is that the regression of M.D. occurred after the removal
of the graft.
The influence of a temporary graft on M.D. was related to the length of time
it was present in the embryo. Female embryos whose testicular graft remained
in place for 1 day (day 3 to day 4) had normal M.D. One case of regression
occurred with a 2-day implantation (day 3 to day 5) and about 50 % of cases
showed regression after a 3-day implantation (day 3 to day 6). With a later
2-day implantation (day 4 to day 6) regression of ducts was observed in 2 out
of 11 females.
The chronology of this regression can be considered as normal, i.e. between
8 and 12 days, because all embryos examined at 7 days possessed M.D. (Fig. 2a)
which proves that regression did not begin, while regression was complete in
14-day-old embryos (Fig. 2 b, c) as in normal male embryos or in testis grafted
female embryos.
Control female embryos maintained under testis graft influence for the whole
duration of the experiment showed a total regression of ducts in more than
90 % of cases.
Control female embryos bearing a graft of neutral tissue (intestine) did not
show any modification of their M.D. which were morphologically normal at
day 14. This proves that the manipulations in themselves had no effect.
86
R. MARAUD, M. RASHEDI AND R. STOLL
DISCUSSION AND CONCLUSION
As previously described, the regression of Miillerian ducts of the chick
embryo begins around day 8 of embryonic life, both in a normal male (Willier,
1939) and in a testis-grafted female (Maraud et al. 1970). Results here observed
showed that the chronology of the regression was the same in graft-removed
embryos, occurring after day 7 and ending before day 14.
When the testis graft was removed from female hosts at day 6, i.e. 2 or 3 days
after grafting and at least 2 days before the beginning of regression, there was
a total regression of ducts in many female recipients. It can be considered that
the graft secreted 'antiMiillerian hormone' which was stored in the embryo
during the 2 or 3 days it was present and provoked destruction of ducts at least
2 days later.
The question is raised as to whether the influence of 1 or 2 days' implantation
(day 3 to day 4 or 5) was weaker than 3 days' implantation because a lower
quantity of hormone was secreted or because the hormone was inactivated
during the 3 or 4 days which elapse before the regression begins. The other type
of 2 days' implantation (day 4 to day 6) which permits the graft to release its
hormone later in development gave a similar result, which showed that the
quantity of hormone secreted in only 2 days was a threshold one since it
provoked the M.D. regression in 18 % of cases.
We conclude that the reaction of ducts beginning near the 8th day under the
influence of the graft is not the result of the appearance of 'antiMullerian
hormone' in embryonic blood, because it is present before the beginning of the
regression in sufficient level, but of the appearance of sensitivity of ducts to
this testicular hormone.
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(Received 28 April 1981, revised 6 August 1981)