Influence of a temporary embryonic testis graft on the regression of Müllerian ducts in female chick embryo

The grafting of an embryonic testis in female chick embryos provokes the regression of Müllerian 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 al. 1966), implanted according to Dossel’s method (1954), as implanted in the extra-embryonic 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 ‘antiMüllerian hormone’ of mammals (Josso, Tran & 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 al. 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 life. Thus it was possible to analyse separately a testis graft as inductor and M.D. as receptors.

Three-day-old embryos from Derco strain (Red-Cornish ♂× 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. 1A). 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 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 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 testis-grafted 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.

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.

As previously described, the regression of Müllerian 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 ‘antiMüllerian 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 ‘antiMüllerian 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.