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Hermaphroditism induced in the female chick by testicular grafts
GENERAL
AND
COMPARATIVE
41, 66-75 (1980)
ENDOCRINOLOGY
Hermaphroditism
Induced
in the Female
Chick
by Testicular
Grafts
R. STOLL, M. RASHEDI, AND R. MARAUD Laboratoire
d’Histologie,
Embtyologie.
U.E.R. 33076
MPdicale Bordeaux,
I, Universite’ France
Bordeaux
II,
146, rue LPo Saignat,
Accepted November 8, 1979 The implantation of one or two testes from 13-day-old chick embryos in the extraembryonic coeloma of 3-day-old female embryos modifies the differentiation of their gonads to various degrees, from developmental inhibition only to hermaphroditism, with one atrophic testis accompanied by an ovary or an ovotestis, and sometimes to total inversion with two atrophic testes. Our previous and present work shows that such an effect is due to the testicular hormone which is responsible for the retrogression of Miillerian ducts and also provokes testicular induction through ovarian cortical inhibition.
The fundamental role played by the testis in the sexual differentiation of external genitalia and gonaducts of higher vertebrates is well documented (review by Stoll and Maraud, 1974), but very little is known at the present time about factors governing the induction of testis differentiation itself. In birds, some aspects of gonadal morphology, particularly the rudimentary state of the fowl’s right gonad, which is derived from the primary sex cords of the germinative epithelium, have allowed for the gonadal inversion following the left ovariectomy in the domestic fowl. This procedure results in a testicular type of development of the right rudimentary gonad (Caridroit and Pezard, 1925; Domm, 1927; Benoit, 1932; Reyss-Brion, 1978). Several authors have studied the influence of embryonic testicular grafts on embryonic female gonads. These gonads became more or less atrophic in certain mammals (Jost, 1947; MacIntyre, 1956; Holyoke, 1956) and in the domestic fowl (Groenendijk-Huijbers, 1973; Thiebold and Reichhart, 1973), but no intersexuality has been observed, except by Turner and Asakawa (1964) who transplanted ovaries and testes next to each other in the kidney capsule of castrated mice and showed that the fetal testis could actually cause the development of an ovotestis. However such
results give little information about factors governing embryonic testicular differentiation. In our present work we have studied the influence of an early graft of one or, more often, two entire embryonic testes on the gonadal differentiation in female chick embryos as well as the posthatching gonadal evolution of these birds. MATERIALS
66 0016-6480/80/050066- 10$01.00/0 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any fom reserved.
AND METHODS
Testes were removed from 13-day-old embryonic chick donors and inserted in 3-day-old chick embryos by using a technique adapted in our laboratory to the grafting of voluminous organs in very young embryos. Through a little aperture in the shell membranes of the egg, one or two testes were inserted in the extraembryonic coeloma of the umbilical cord of the embryo. The donor’s and host’s ages were chosen on the basis of our previous works. We have observed that, at this period, the grafted testes are highly active to cause the retrogression of Milllerian ducts and the atrophy of the ovary (St011 et al., 1975, 1978). By way of control, some embryos were grafted with neutral tissue, a fragment of intestine, and some were not operated on. Donors and hosts belong to the Derco or Harco strains, the latter showing sex-linked feather pigmentation. Embryos of different groups were sacrificed between 8 and 18 days and immediately weighed. In the group of testis-grafted females, we have only taken in account the individuals whose graft was recovered in a well-developed state and adhered to the omphalomesenteric vessels of the umbilicus. Other implantations, caused by displacement of the graft on the yolk sac area, gave poor results on the genital tract of
FEMALE
CHICK
grafts were sacrificed, the size of the implants was greater as the host was older. Their size and structural development were slightly delayed compared to the testes of normal male donors of the same age, probably because of the time needed for the setting up of an adequate irrigation. When the grafted embryos were of the male sex the structure of the implant was the same as in the female host, which excludes an effect of the host’s environment on the testicular implant itself. The female embryos bearing an adequate testicular graft (194 cases) showed a complete lack of the Mtillerian ducts associated with four different types of gonads (Table 1). The type 1 embryo is only represented by gonadal inhibition, which is the minimal response to the action of the grafted testes, the ovary and the right rudiment being reduced in size (116 cases). These embryos are more frequently observed among females grafted with one testis (77%) than with two testes (48%). The three other types consist of a sexual inversion of the female gonads toward the male sex (78 cases) and are more frequent in females grafted with two testes, a condition necessary to obtain types 3 and 4. Type 2 is rep-
the host. Female chickens, grafted with testicular implants during the embryonic life, were also sacrificed 2 and 8 weeks after hatching, but in these cases, the graft was not to be found again. In the course of this research, we have first studied the morphological aspects of the gonadal inversion, using standard anatomical and histological methods. Next, because hermaphroditism results from ovarian atrophy, we have investigated quantitatively this last phenomenon in 13-day-old female embryos whose gonadal differentiation was over. As the dissection and weighing of a modified gonad lack precision, we have measured their external surface area on enlarged photographs (x 18.5) by means of a Kontron-Digiplan planimeter. The gonadal constituents (cortex and medulla, except medullary lacunae) have been measured by the same method, applied to micrographs (X 160) of median transversal sections of the gland. Only the measure of the section area has been made by the same method on the right rudiment. These numerical data were compared with the corresponding values of the control subjects.
RESULTS
The results were similar in the two strains used and consequently they were fused. The embryos with implants were of the same height and weight as control embryos which excludes any retardation of development as the cause of a gonadal inhibition or inversion. When the female embryos with testicular TABLE EFFECTS
OF A TESTICULAR
GRAFT
ON THE
FEMALE
67
HERMAPHRODITISM
1
SEXUAL
CHICK
DIFFERENTIATION
OF GONADS
IN THE
EMBRYO”
Number of embryos obtained in each type” Grafted with one testis
Grafted with two testes
Age in days
Type 1
Type 2
8 9 10 12 13 16 18
2 4 5 9 30 1 8
0
2 2 1 5 2 5
6 17 7 5 17 0 5
6 5 4 2 13 4 6
Total
59
17
57
40
a Type Type Type Type
1: female 2: female 3: female 4: female
with with with with
‘We
two atrophic gonads. a left ovary and a right testis. a left ovotestis and a right testis. two testes.
1
Type 2
Type 3
Type 4
10
STOLL,
FIG. 1. (A) (G x 45). (B) left ovary (0) Gonads of an (D-E) Higher
RASHEDI,
AND
MARAUD
Gonads of a normal female chick embryo, 18 days old. (0) Left ovary; (r) right rudiment. Gonads of an 18-day-old female embryo, grafted at 3 days with an embryonic testis. The is atrophied and the right rudiment transformed into a typical testis (t). (G x 45). (C) l&day-old female embryo, two transformed in testes (t) by the testicular graft. (G x 45). magnification of the left (D) and right (E) testes of the embryo shown in C. (G x 270).
FEMALE
CHICK
HERMAPHRODITISM
69
resented by females having a small right In the left ovary of the types 1 and 2 the testis accompanying an atrophic left ovary medulla derived from primary sex cords is (57 cases) (Fig. 1B). Type 3 shows an as- reduced in volume. It is surrounded by the sociation of a right atrophic testis and a left thickened germinal epithelium, which ovotestis with reduced cortex and medulla gives, after 9 days, secondary sex cords in which the reticular part contains a few forming an atrophic ovarian cortex. testicular cords or tubules (11 cases). Type The right gonad of g-day-old embryos of 4 is represented by females possessing two type 1 is made of primary sex cords reduced atrophic testes (10 cases). The left testis, in number, which later give the rudimentary bigger than the right gonad, sometimes gonad. In types 2, 3, and 4 the right testis is presents scattered fragments of epithelium, constituted by the primary sex cords which the remnants of the almost entirely inhibdid not develop their lacunary structure as ited germinal epithelium. This degree of in a normal female embryo. After 9 days, intersexuality, comparable with total inverthese cords progressively become a reticusion, is only recognizable in embryos with a lum of few testicular cords including Sertoli sex-linked feather color (Figs. lC-E). cells and gonocytes. In Table 2 the quantitative data first show On the contrary, some characteristics of that an implant of a neutral tissue inhibits the embryos of types 3 and 4, such as the slightly, but not significantly, the ovary of lack of the left cortex, can be seen only in the 13-day-old host. On the contrary, a tes- older embryos. This fact explains their abticular implant always causes a gonadal at- sence among the young embryos of Table 1. rophy which is statistically significant comUntil 8 weeks after hatching, these repared with a normal female or a female with sults persisted and showed that the sexual an implant of neutral tissue. A correlation inversion was definitive. Seven pullets only exists between the gonadal inhibition and presented reduced but histologically normal hermaphroditism. The more important the gonads (type 1). The seven others were atrophy is, the more marked gonadal inver- hermaphroditic and, fortuitously, of the sion is, the graft of two testes giving the third type. They indeed presented a left best results. So the external surface area of ovotestis, reduced in size, whose ovarian the left gonad decreases from 4.965 mm2 in part possessed growing ovocytes and follicontrol embryos to 2.91 mm2 in embryos of cles (Figs. 2A, B). The hilus of the ovotestis the fourth type in which the left gonad is showed small nodules with few isolated inverted into a testis (Table 2). The cortex seminiferous tubules containing germinal and medulla which constitute the ovary are and Sertoli cells (Figs. 2B, D). almost equally inhibited, but in the female A little testis existed on the right side and of the fourth type the cortex seems more was made of some seminiferous tubules, sensitive to the inhibitory influence of the with germinal and Sertoli cells, inter-tubular testicular implants since the differentiation Leydig cells, and connective tissue. The of a left testis corresponds to the lack of tubules opened in a voluminous “retecortex and the conservation of a small testis” which connected with an epididymis medullary part. whose cells presented stereocilia (Figs. The effects of testicular implants are al- 2A, C). ready visible when the chick is 8 days old so their action is initiated earlier, during the DISCUSSION first stages of gonadal differentiation whose The first signs of normal female gonadal development begins at 6.5 days and is over differentiation generally appear in the 6.5 about 13-14 days, according to Willier day chick embryo (Willier, 1939). At this (1939). time, the bilaterally thickened germinal
-
Right gonad
f
9,721
14,634
6,522
TABLE
2
Type
2
ON THE GONADS
with one testis
GRAFT
Type 1
IN &DAY-OLD
Type
2
Grafted
FEMALE
-55.65%d
90,768 + 4,37ab -38.25%d 218,942 r 13,273* -32.65%d 59,023 -c 6,118"
15
2,21Eb
-64.30%* 26,841
-41.46%d 44,442 4.4196 -73.67%d
-67.46%d 29,291 6,438" -82~G+%~
55,351 9,3d56 -62.34%d 105,775 18,547b
t t
5.89 -95.73%6 8,137 1,488" -95.18%d
20,156
1
+ 0.027* -9o.%%d 7
3
7 0.426
Type 4
1.467 f 0.176" -68.8%*
Type 3
1 1‘+ -c + -84.09%* 13
61,220 5,205" -58.35%d 116,051 10,549"
t SE
-50.69%d
84,128 8,293* -42.76%d 190,306 19,143*
13
f k k
-41.47%6
section (in p mZ) : Mean
77,562 2 13,853" -47.23%d 146,265 + 21,2W -SS.Ol%d 27,031 I? 5.646"
5
Surface area of median transversal
-34.43%6
EMBRYOS
with two testes
CHICK
External surface area of the left gonad (in mm*): Mean * SE 30 17 13 5 3.092 + O.llOb 2.091 f 0.299* 2.325 + 0.169b 2.760 -c 0.176b
Type 1
Grafted
OF A TESTICULAR
D Not statistically significant as determined by Student’s r test, P < 0.30. b Statistically significant as determined by Student’s f test, P < 0.001. ’ Comparison made between mean values of intestine-grafted and control embryos. d Comparison made between mean values of testis-grafted and intestine-grafted embryos. e Surface area of the medullary zone, except lacunae.
168,803
325,118
-
Medulla’
f
k
-
146,993
-
Cortex
-5.02%'
40 4.716 2 0.165"
23
40 4.965 e 0.174
Number
Number Area
Controls
EFFECT
Grafted with intestine
INHIBITORY
> $ z
g "
;
FIG. 2. (A) Gonads of a 2-month-old female pullet grafted with a testis during its embryonic life. It is hermaphroditic and possesses a left ovariotestis (0) and a right testis (t). (G x 23). (B) Detail of the ovariotestis, including a portion of the medullary zone. Its cortical zone is filled with follicles with oocytes (00). The medullary zone contains some seminiferous tubules (St). (G x 170). (C) Higher magnification of the testis (t). The seminiferous tubules are connected to the epididymis (e) through an abundant “rete testis” (rt). (G x 76). (D) A seminiferous tubule of the ovarian medullary zone. Its structure is similar to that of a testicular one. (G x 425).
71
72
STOLL,
RASHEDI,
epithelium, consisting of epithelial and germ cells, produces the primary or medullary sex cords. These last penetrate the mesenchyme of the gonads and become separated from the epithelium by primary albuginea (Swift, 1915, 1916). In the ovary, the primary sex cords form the medullary part of the gonad and yield a compact zone near the cortex as well as a central reticular zone somewhat about the ninth day (Willier, 1927; Brode, 1928). On the ninth day, the germinal epithelium of the left side develops the secondary or ovigerous sex cords which increase in size essentially by the multiplication of germ cells (Swift, 1915). In 13- to 1Cday embryos these cords also become separated from the epithelium and form the ovarian cortex. The right rudimentary gonad is reduced to the primary sex cords which produce tubules or cords containing some germ cells and bordering lacunae. The gonocytes disappear within 3 weeks after hatching (Brode, 1928). In the normal male, only the primary sex cords develop and are totally isolated from the germinal epithelium at 9 days of age. They fuse and form a reticulum of testicular cords giving seminiferous tubules about hatching time (Swift, 1916; Venzke, 1954). Our results indicate that the grafted testes act specifically on the host’s ovary. Indeed, the graft of the fragment of an ovary from an ll- or 13-day-old embryo onto a female embryo (Rashedi et al., 1977), or of the fragment of a neutral tissue such as intestine does not influence the ovarian differentiation of the host as the testicular graft does. This graft then releases a hormone in the bloodstream of the host. Its effects are already visible in the 8-day-old embryo and persist afterward. This hormone modifies the differentiation of the female gonads and gives hermaphroditism by inhibiting the development of primary and secondary sex cords. The formation of secondary sex cords seems more responsive to the influence of the graft than the medullary ones. Thus, a
AND
MARAUD
strong inhibition results in an entire gonadal inversion because secondary sex cords are totally inhibited and do not develop at all. Meanwhile primary, or medullary cords, being only partially inhibited, give bilaterally a small testis. In a normal female embryo, the ovarian cortex develops and prevents the primary sex cords of the right rudiment and left medullary zone from developing into a testicular structure. We think that gonadal inversion and intermediary states of hermaphroditism, here described, are the consequences of a more or less important inhibitory action of the testicular graft upon cortical structures. Free from cortical inhibition under the influence of the graft the primary sex cords can develop into a testis. Such an interpretation agrees with that of Benoit (1932) who explains in this way how a chick ovariectomy allows the right rudiment to become a testis and how the left gonad forms an ovotestis in the case of an incomplete ovariectomy. We also think that this mechanism is responsible for the testicular differentiation in the male and that the testicular size is regulated by the action of the inhibitory hormone elaborated by the testis itself before it is submitted to hypophyseal control. We can speculate whether the grafted testis acts directly on the gonadal differentiation of the female embryo or through the hypophysis. The answer to the latter hypothesis is negative since gonadal inhibition due to the graft is similar in both normal and hypophysectomized female chick embryos (Rashedi et al., 1975). Hermaphroditic structures developed during the embryonic life persist after hatching and are then submitted to the hypophyseal influence, as shown by the ovarian part with its follicles and growing ovocytes. According to Maraud (1963) the development of an epididymis indicates the secretion of androgens by the right testis. All these points concerning pullets need further investigations. Another fundamental question is relative
FEMALE
CHICK HERMAPHRODITISM
73
to the nature of the testicular hormone se- normally occurs in the male embryos, but creted by the graft and responsible for to an agenesia of the Miillerian tract occurgonadal inhibition and hermaphroditism. ring before this phase, during the undifferOur previous works show that such an ef- entiated state (Gaarenstroom, 1939; Stoll, fect is due to the testicular hormone re- 1950; Stoll and Maraud, 1952; Stall et al., sponsible for Mullerian retrogression. In 1972). Besides, it has been shown that tesfact, the graft of a testis from embryonic tosterone and other androgens stimulate donors of increasing ages, and therefore, of Mullerian ducts of the chick embryo, culdecreasing anti-Mitllerian power (Maraud tured in vitro (Weniger and Zeis, 1973). So et al.. 1966; Stoll et al., 1970) shows that steroidal androgens can be discarded as inthe degrees of retrogression of the Mulleductors of the Mullerian retrogression. rian tract and of the ovarian inhibition of Furthermore, in all these works, no fact the female host embryo are closely proporindicates any influence of androgens on the tional (St011 er al., 1975, 1978). This also gonadal differentiation of the female emappears in this present work where experibryo and peculiarly testosterone is unable mental conditions make out that Miillerian to cause an atrophy of the right gonadal ducts are always missing while gonads are rudiment, as shown by testis implantation strongly inhibited and even sexually in- in the female chick embryo which was preverted. We think that the two effects are viously submitted to ovariectomy due to the same hormone. (Groenendijk-Huijbers, 1973; GroenendijkIn mammals, the study of the phenomeHuijbers and Schaik, 1976). non occurring in freemartin cows has led At the present time, the biochemical Vigier et al. (1977) to consider this possibilstructure of the hormone is unknown in ity among others, on the basis of chronobirds but its molecular weight, higher than logical concordances. 1000, is not the same as that of sterolic anThe problem of the chemical nature of drogens (Weniger et al., 1975). this hormone can be discussed, since sevIn mammals, this substance has been eral authors think that steroidal androgens, concentrated from testis-incubation media which are effectively secreted by the em- and its protein composition has been estabbryonic testes during this period (see lished (review by Josso et al., 1977). Woods and Podczaski, 1974), are inductors of the Mullerian duct retrogression in the REFERENCES male sex. This opinion is founded on the Benoit, J. (1932). L’inversion sexuelle de la Poule dtterminee par l’ablation de l’ovaire gauche. Arch. fact that testosterone or other steroidal an2001. Exp. Gen. 73, l-112. drogens given to the female chick embryo Brode, M. D. (1928). The significance of the asymbefore its sexual differentiation cause the metry of the ovaries of the fowl. J. Morphol. 46, deficiency of the Mullerian tract l-57. (Dantchakoff, 1938; Willier et al., 1935, Caridroit and Pezard (1925). Poussee testiculaire autonome a I’inttrieur des greffes ovariennes et au1937; Willier et al., 1938; Wolff, 1935; Wolff toplastiques chez la Poule domestique. C. R. et al., 1948). The same effect is obtained in Acad. Sci. 180, 2067-2069. the quail (Lutz-Ostertag, 1%5). These hor- Dantchakoff, V. (1938). Rtalisation du sexe a volonte mones also cause “in vitro” the necrosis of par induction hormonale. III: Inversions et deviacultured ducts (Wolff et al., 1952; Scheibtions dans I’histogenese sexuelle chez I’embryon de Poulet train? par l’hormone male. Bull. Biol. Fr. Ptleger, 1953). Be/g. 72, 187-231. In fact, deficiencies of ducts, as observed Domm, L. V. (1927). New experiments on ovariotomy in the female embryos treated by androand the problem of sex inversion in the fowl. J. gens, are not due to their retrogression Exp. Zoo/. 48, 31- 119. during their sexual differentiation, as it Gaarenstroom, J. H. (1939). Action of sex hormones
STOLL,
74
RASHEDI,
on the development of the Miillerian ducts of the chick embryo. J. Exp. ZooI. 82, 31-46. Groenendijk-Huijbers, M. M. (1973). Influence of gonadal hormone administration and gonadal implantation on the compensatory growth of the embryonic chicken right ovary after early sinistral castration. Verb. Anaf. Ges. 67, 193-196. Groenendijk-Huijbers, M. M., and Van Schaik, J. P. J. (1976). Effects of hemi-castration, testis implantation and administration of testosterone propionate on the female embryonic genital tract in various breeds and strains of chickens. Verb. Anat. Ges. 70, 179-182. Holyoke, E. A. (1956). The differentiation of embryonic ovaries and testes grafted together in adult hosts in the rabbit. Anat. Rec. 124, 307. Josso, N., Picard, J. Y., and Dien Tran (1977). The antimullerian hormone. Rec. Progr. Horm. Res. 33, 117- 167. Jost, A. (1947). Recherches sur la differentiation sexuelle de I’embryon de Lapin. Arch. Anat. Microsc. Morphol. Exp. 36, 151-200, 242-315. Lutz-Ostertag, Y. (1965). Action du propionate de testosterone sur les canaux de Muller de l’embryon de Caille (Corurnix cotrrrnixjaponica). C. R. Acad. Sri. 461, 4501-4504. MacIntyre, M. N. (1956). Effect of the testis on ovarian differentiation in heterosexual embryonic Rat gonad transplants. Anaf. Rec. 124, 27-45. Maraud, R. (1963). Recherches experimentales sur les facteurs inducteurs de la formation de l’tpididyme du Coq. Arch. Anaf. Microsc. Morphol. Exp. 52, 84- 127. Maraud, R., Stoll, R., and Coulaud, H. (1966). Action de la greffe testiculaire sur les canaux de Muller de l’embryon de Poulet. C. R. Sot. Biol. 160, 964-966. Rashedi, M., Stoll, R., and Maraud, R. (1975). Action de la greffe testiculaire sur I’ovaire de l’embryon de Poulet ayant subi une decapitation partielle. C. R. Sot. Biol. 169, 1464-1466. Rashedi, M., Stoll, R., and Maraud, R. (1977). Action d’un greffon ovarien sur l’ovaire de l’embryon femelle de Poulet. C. R. Sot. Biol. 171, 585-589. Reyss-Brion, M. (1978). Histochemical changes in the right gonad of the fowl after ovariectomy. Congr. Anat.
Histol.
Embryol.
Varna.
Scheib-Pfleger, D. (1953). Roles des hormones sexuelles sur le developpement “in vitro” des canaux de Muller de l’embryon de Poulet femelle differencit. C. R. Acad. Sri. 236, 1446-1448. Stoll, R. (1950). Sur la differentiation sexuelle de l’embryon de Poulet. Arch. Anar. Microsc. Morphol. Exp. 39, 415-422. Stoll, R., Faucounau, N., and Maraud, R. (1972). Action des androgenes steroliques sur les canaux de Miiller de l’embryon de Poulet. C. R. Sot. Biol. 166, 858-861.
AND MARAUD Stoll, R., and Maraud, R. (1952). Action des hormones sexuelles sur l’evolution des canaux de Muller de l’embryon de Poulet. Arch. Anat. Microsc. Morphol. Exp. 41, 260-280. Stoll, R., and Maraud, R. (1974). Le r61e de testicule dans la differentiation sexuelle des gonoductes chez l’embryon des Vertdbrts amniotes. Bull. Assoc.
Anal.
58, 695-764.
Stoll, R., Maraud, R., and Coulaud, H. (1970). Sur la secretion de l’inducteur de la regression mullerienne chez l’embryon de Poulet: Chronologie des relations hypophyso-testiculaires. C. R. Sot. Biol. 164, 1013- 101.5. Stoll, R., Rashedi, M., and Maraud, R. (1975). Action de l’hormone testiculaire de regression mullerienne sur la developpement de l’ovaire de l’embryon de Poulet. C. R. Sot. Biol. 169, 927-930. Stoll, R., Rashedi, M., and Maraud, R. (1978). Sur l’hormone de regression mullerienne, agent inducteur du testicule chez l’embryon de Poule. Bull. Assoc. Anat. 176, 13 1- 143. Swift, C. H. (1915). Origin of the definitive sex-cells in the female chick and the relation to the primordial germ cells. Amer. J. Anat. 18, 441-470. Swift, C. H. (1916).0rigin of the sex cords and definitive spermatogonia in the male chick. Amer. J. Anat. 20, 375-410. Thiebold, J. J., and Reichhart, J. H. (1973). Inhibition de developpement ovarien par la greffe de gonades embryonnaires chez le Poulet. C. R. Sot. Eiol. 167, 1958- 1960. Turner, C. D., and Asakawa, H. (1964). Experimental reversal of germ cells in ovaries of fetal mice. Science 143, 1344. Venzke, W. G. (1954). The morphogenesis of the indifferent gonad of chicken embryos. Amer. J. Vet. Res. 15, 300-308. Vigier, B., Prepin, J., Perchellet, J. P., and Jost, A. (1977). Developpement de l’effet free-martin chez le foetus de Veau. Ann. Med. Vet. 121, 521-536. Weniger, J. P., Mack, G., and Holder, F. (1975). L’hormone responsable de la regression des canaux de Mtiller chez l’embryon de Poulet male n’est pas un androgene. C. R. Acad. Sci. 280, 1889- 1891. Weniger, J. P., and Zeis, A. (1973). Action feminisante des androgenes sur le testicule et le canal de Muller d’embryon de Poulet “in vitro.” Arch. Anat. Microsc. Morphol, Exp. 62, 145- 150. Willier, B. H. (1927). The specificity of sex, of organization and of differentiation of embryonic chick gonad as shown by grafting experiments. J. Exp. Zool. 46, 409-467. Willier, B. H. (1939). The embryonic development of sex. Sex Intern. Secretions, 64- 144. Willier, B. H., Gallagher, T. F., and Koch, F. C. (1935). Sex modification in the chick embryo re-
FEMALE
CHICK
HERMAPHRODITISM
suiting from injection of male and female hormones. Proc. Nat. Acad. Sci. USA 21,625-631. Willier, B. H., Gallagher, T. F., and Koch, F. C. (1937). The modification of sex development in the chick embryo by male and female sex hormones. Physiol. Zool. 10, lOl- 122. Willier, B. H., Rawles, M. E., and Koch, F. C. (1938). Biological difference in the action of synthetic male hormones on the differentiation of sex in the chick embryo. Proc. Nat. Acad. Sri. USA 24, 176- 182. Wolff, Et. (1935). Sur la transformation experimentale des femelles genetiques en intersexuees provoquee par I’injection d’hormones males aux
75
embryons de Poulet. C. R. Acad. Sci. 201, 1055-1057. Wolff, Et., Lutz-Ostertag, Y., and Haffen, K. (1952). Sur la regression et la n&rose “in vitro” des canaux de Mtiller de l’embryon de Poulet sous l’action directe des hormones males. C. R. Sot. Biol. 146, 1793- 1795. Wolff, Et., Strudel, G., and Wolff, Em. (1948). L’action des hormones androgbnes sur la differenciation sexuelle des embryons de Poulet. Arch. Anat. Histol. Embryol. 31, 237-310. Woods, J. E., and Podczaski, E. S. (1974). Androgen synthesis in the gonads of the chick embryo. Gen. Comp. Endocrinol. 24, 413-423.
AND
COMPARATIVE
41, 66-75 (1980)
ENDOCRINOLOGY
Hermaphroditism
Induced
in the Female
Chick
by Testicular
Grafts
R. STOLL, M. RASHEDI, AND R. MARAUD Laboratoire
d’Histologie,
Embtyologie.
U.E.R. 33076
MPdicale Bordeaux,
I, Universite’ France
Bordeaux
II,
146, rue LPo Saignat,
Accepted November 8, 1979 The implantation of one or two testes from 13-day-old chick embryos in the extraembryonic coeloma of 3-day-old female embryos modifies the differentiation of their gonads to various degrees, from developmental inhibition only to hermaphroditism, with one atrophic testis accompanied by an ovary or an ovotestis, and sometimes to total inversion with two atrophic testes. Our previous and present work shows that such an effect is due to the testicular hormone which is responsible for the retrogression of Miillerian ducts and also provokes testicular induction through ovarian cortical inhibition.
The fundamental role played by the testis in the sexual differentiation of external genitalia and gonaducts of higher vertebrates is well documented (review by Stoll and Maraud, 1974), but very little is known at the present time about factors governing the induction of testis differentiation itself. In birds, some aspects of gonadal morphology, particularly the rudimentary state of the fowl’s right gonad, which is derived from the primary sex cords of the germinative epithelium, have allowed for the gonadal inversion following the left ovariectomy in the domestic fowl. This procedure results in a testicular type of development of the right rudimentary gonad (Caridroit and Pezard, 1925; Domm, 1927; Benoit, 1932; Reyss-Brion, 1978). Several authors have studied the influence of embryonic testicular grafts on embryonic female gonads. These gonads became more or less atrophic in certain mammals (Jost, 1947; MacIntyre, 1956; Holyoke, 1956) and in the domestic fowl (Groenendijk-Huijbers, 1973; Thiebold and Reichhart, 1973), but no intersexuality has been observed, except by Turner and Asakawa (1964) who transplanted ovaries and testes next to each other in the kidney capsule of castrated mice and showed that the fetal testis could actually cause the development of an ovotestis. However such
results give little information about factors governing embryonic testicular differentiation. In our present work we have studied the influence of an early graft of one or, more often, two entire embryonic testes on the gonadal differentiation in female chick embryos as well as the posthatching gonadal evolution of these birds. MATERIALS
66 0016-6480/80/050066- 10$01.00/0 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any fom reserved.
AND METHODS
Testes were removed from 13-day-old embryonic chick donors and inserted in 3-day-old chick embryos by using a technique adapted in our laboratory to the grafting of voluminous organs in very young embryos. Through a little aperture in the shell membranes of the egg, one or two testes were inserted in the extraembryonic coeloma of the umbilical cord of the embryo. The donor’s and host’s ages were chosen on the basis of our previous works. We have observed that, at this period, the grafted testes are highly active to cause the retrogression of Milllerian ducts and the atrophy of the ovary (St011 et al., 1975, 1978). By way of control, some embryos were grafted with neutral tissue, a fragment of intestine, and some were not operated on. Donors and hosts belong to the Derco or Harco strains, the latter showing sex-linked feather pigmentation. Embryos of different groups were sacrificed between 8 and 18 days and immediately weighed. In the group of testis-grafted females, we have only taken in account the individuals whose graft was recovered in a well-developed state and adhered to the omphalomesenteric vessels of the umbilicus. Other implantations, caused by displacement of the graft on the yolk sac area, gave poor results on the genital tract of
FEMALE
CHICK
grafts were sacrificed, the size of the implants was greater as the host was older. Their size and structural development were slightly delayed compared to the testes of normal male donors of the same age, probably because of the time needed for the setting up of an adequate irrigation. When the grafted embryos were of the male sex the structure of the implant was the same as in the female host, which excludes an effect of the host’s environment on the testicular implant itself. The female embryos bearing an adequate testicular graft (194 cases) showed a complete lack of the Mtillerian ducts associated with four different types of gonads (Table 1). The type 1 embryo is only represented by gonadal inhibition, which is the minimal response to the action of the grafted testes, the ovary and the right rudiment being reduced in size (116 cases). These embryos are more frequently observed among females grafted with one testis (77%) than with two testes (48%). The three other types consist of a sexual inversion of the female gonads toward the male sex (78 cases) and are more frequent in females grafted with two testes, a condition necessary to obtain types 3 and 4. Type 2 is rep-
the host. Female chickens, grafted with testicular implants during the embryonic life, were also sacrificed 2 and 8 weeks after hatching, but in these cases, the graft was not to be found again. In the course of this research, we have first studied the morphological aspects of the gonadal inversion, using standard anatomical and histological methods. Next, because hermaphroditism results from ovarian atrophy, we have investigated quantitatively this last phenomenon in 13-day-old female embryos whose gonadal differentiation was over. As the dissection and weighing of a modified gonad lack precision, we have measured their external surface area on enlarged photographs (x 18.5) by means of a Kontron-Digiplan planimeter. The gonadal constituents (cortex and medulla, except medullary lacunae) have been measured by the same method, applied to micrographs (X 160) of median transversal sections of the gland. Only the measure of the section area has been made by the same method on the right rudiment. These numerical data were compared with the corresponding values of the control subjects.
RESULTS
The results were similar in the two strains used and consequently they were fused. The embryos with implants were of the same height and weight as control embryos which excludes any retardation of development as the cause of a gonadal inhibition or inversion. When the female embryos with testicular TABLE EFFECTS
OF A TESTICULAR
GRAFT
ON THE
FEMALE
67
HERMAPHRODITISM
1
SEXUAL
CHICK
DIFFERENTIATION
OF GONADS
IN THE
EMBRYO”
Number of embryos obtained in each type” Grafted with one testis
Grafted with two testes
Age in days
Type 1
Type 2
8 9 10 12 13 16 18
2 4 5 9 30 1 8
0
2 2 1 5 2 5
6 17 7 5 17 0 5
6 5 4 2 13 4 6
Total
59
17
57
40
a Type Type Type Type
1: female 2: female 3: female 4: female
with with with with
‘We
two atrophic gonads. a left ovary and a right testis. a left ovotestis and a right testis. two testes.
1
Type 2
Type 3
Type 4
10
STOLL,
FIG. 1. (A) (G x 45). (B) left ovary (0) Gonads of an (D-E) Higher
RASHEDI,
AND
MARAUD
Gonads of a normal female chick embryo, 18 days old. (0) Left ovary; (r) right rudiment. Gonads of an 18-day-old female embryo, grafted at 3 days with an embryonic testis. The is atrophied and the right rudiment transformed into a typical testis (t). (G x 45). (C) l&day-old female embryo, two transformed in testes (t) by the testicular graft. (G x 45). magnification of the left (D) and right (E) testes of the embryo shown in C. (G x 270).
FEMALE
CHICK
HERMAPHRODITISM
69
resented by females having a small right In the left ovary of the types 1 and 2 the testis accompanying an atrophic left ovary medulla derived from primary sex cords is (57 cases) (Fig. 1B). Type 3 shows an as- reduced in volume. It is surrounded by the sociation of a right atrophic testis and a left thickened germinal epithelium, which ovotestis with reduced cortex and medulla gives, after 9 days, secondary sex cords in which the reticular part contains a few forming an atrophic ovarian cortex. testicular cords or tubules (11 cases). Type The right gonad of g-day-old embryos of 4 is represented by females possessing two type 1 is made of primary sex cords reduced atrophic testes (10 cases). The left testis, in number, which later give the rudimentary bigger than the right gonad, sometimes gonad. In types 2, 3, and 4 the right testis is presents scattered fragments of epithelium, constituted by the primary sex cords which the remnants of the almost entirely inhibdid not develop their lacunary structure as ited germinal epithelium. This degree of in a normal female embryo. After 9 days, intersexuality, comparable with total inverthese cords progressively become a reticusion, is only recognizable in embryos with a lum of few testicular cords including Sertoli sex-linked feather color (Figs. lC-E). cells and gonocytes. In Table 2 the quantitative data first show On the contrary, some characteristics of that an implant of a neutral tissue inhibits the embryos of types 3 and 4, such as the slightly, but not significantly, the ovary of lack of the left cortex, can be seen only in the 13-day-old host. On the contrary, a tes- older embryos. This fact explains their abticular implant always causes a gonadal at- sence among the young embryos of Table 1. rophy which is statistically significant comUntil 8 weeks after hatching, these repared with a normal female or a female with sults persisted and showed that the sexual an implant of neutral tissue. A correlation inversion was definitive. Seven pullets only exists between the gonadal inhibition and presented reduced but histologically normal hermaphroditism. The more important the gonads (type 1). The seven others were atrophy is, the more marked gonadal inver- hermaphroditic and, fortuitously, of the sion is, the graft of two testes giving the third type. They indeed presented a left best results. So the external surface area of ovotestis, reduced in size, whose ovarian the left gonad decreases from 4.965 mm2 in part possessed growing ovocytes and follicontrol embryos to 2.91 mm2 in embryos of cles (Figs. 2A, B). The hilus of the ovotestis the fourth type in which the left gonad is showed small nodules with few isolated inverted into a testis (Table 2). The cortex seminiferous tubules containing germinal and medulla which constitute the ovary are and Sertoli cells (Figs. 2B, D). almost equally inhibited, but in the female A little testis existed on the right side and of the fourth type the cortex seems more was made of some seminiferous tubules, sensitive to the inhibitory influence of the with germinal and Sertoli cells, inter-tubular testicular implants since the differentiation Leydig cells, and connective tissue. The of a left testis corresponds to the lack of tubules opened in a voluminous “retecortex and the conservation of a small testis” which connected with an epididymis medullary part. whose cells presented stereocilia (Figs. The effects of testicular implants are al- 2A, C). ready visible when the chick is 8 days old so their action is initiated earlier, during the DISCUSSION first stages of gonadal differentiation whose The first signs of normal female gonadal development begins at 6.5 days and is over differentiation generally appear in the 6.5 about 13-14 days, according to Willier day chick embryo (Willier, 1939). At this (1939). time, the bilaterally thickened germinal
-
Right gonad
f
9,721
14,634
6,522
TABLE
2
Type
2
ON THE GONADS
with one testis
GRAFT
Type 1
IN &DAY-OLD
Type
2
Grafted
FEMALE
-55.65%d
90,768 + 4,37ab -38.25%d 218,942 r 13,273* -32.65%d 59,023 -c 6,118"
15
2,21Eb
-64.30%* 26,841
-41.46%d 44,442 4.4196 -73.67%d
-67.46%d 29,291 6,438" -82~G+%~
55,351 9,3d56 -62.34%d 105,775 18,547b
t t
5.89 -95.73%6 8,137 1,488" -95.18%d
20,156
1
+ 0.027* -9o.%%d 7
3
7 0.426
Type 4
1.467 f 0.176" -68.8%*
Type 3
1 1‘+ -c + -84.09%* 13
61,220 5,205" -58.35%d 116,051 10,549"
t SE
-50.69%d
84,128 8,293* -42.76%d 190,306 19,143*
13
f k k
-41.47%6
section (in p mZ) : Mean
77,562 2 13,853" -47.23%d 146,265 + 21,2W -SS.Ol%d 27,031 I? 5.646"
5
Surface area of median transversal
-34.43%6
EMBRYOS
with two testes
CHICK
External surface area of the left gonad (in mm*): Mean * SE 30 17 13 5 3.092 + O.llOb 2.091 f 0.299* 2.325 + 0.169b 2.760 -c 0.176b
Type 1
Grafted
OF A TESTICULAR
D Not statistically significant as determined by Student’s r test, P < 0.30. b Statistically significant as determined by Student’s f test, P < 0.001. ’ Comparison made between mean values of intestine-grafted and control embryos. d Comparison made between mean values of testis-grafted and intestine-grafted embryos. e Surface area of the medullary zone, except lacunae.
168,803
325,118
-
Medulla’
f
k
-
146,993
-
Cortex
-5.02%'
40 4.716 2 0.165"
23
40 4.965 e 0.174
Number
Number Area
Controls
EFFECT
Grafted with intestine
INHIBITORY
> $ z
g "
;
FIG. 2. (A) Gonads of a 2-month-old female pullet grafted with a testis during its embryonic life. It is hermaphroditic and possesses a left ovariotestis (0) and a right testis (t). (G x 23). (B) Detail of the ovariotestis, including a portion of the medullary zone. Its cortical zone is filled with follicles with oocytes (00). The medullary zone contains some seminiferous tubules (St). (G x 170). (C) Higher magnification of the testis (t). The seminiferous tubules are connected to the epididymis (e) through an abundant “rete testis” (rt). (G x 76). (D) A seminiferous tubule of the ovarian medullary zone. Its structure is similar to that of a testicular one. (G x 425).
71
72
STOLL,
RASHEDI,
epithelium, consisting of epithelial and germ cells, produces the primary or medullary sex cords. These last penetrate the mesenchyme of the gonads and become separated from the epithelium by primary albuginea (Swift, 1915, 1916). In the ovary, the primary sex cords form the medullary part of the gonad and yield a compact zone near the cortex as well as a central reticular zone somewhat about the ninth day (Willier, 1927; Brode, 1928). On the ninth day, the germinal epithelium of the left side develops the secondary or ovigerous sex cords which increase in size essentially by the multiplication of germ cells (Swift, 1915). In 13- to 1Cday embryos these cords also become separated from the epithelium and form the ovarian cortex. The right rudimentary gonad is reduced to the primary sex cords which produce tubules or cords containing some germ cells and bordering lacunae. The gonocytes disappear within 3 weeks after hatching (Brode, 1928). In the normal male, only the primary sex cords develop and are totally isolated from the germinal epithelium at 9 days of age. They fuse and form a reticulum of testicular cords giving seminiferous tubules about hatching time (Swift, 1916; Venzke, 1954). Our results indicate that the grafted testes act specifically on the host’s ovary. Indeed, the graft of the fragment of an ovary from an ll- or 13-day-old embryo onto a female embryo (Rashedi et al., 1977), or of the fragment of a neutral tissue such as intestine does not influence the ovarian differentiation of the host as the testicular graft does. This graft then releases a hormone in the bloodstream of the host. Its effects are already visible in the 8-day-old embryo and persist afterward. This hormone modifies the differentiation of the female gonads and gives hermaphroditism by inhibiting the development of primary and secondary sex cords. The formation of secondary sex cords seems more responsive to the influence of the graft than the medullary ones. Thus, a
AND
MARAUD
strong inhibition results in an entire gonadal inversion because secondary sex cords are totally inhibited and do not develop at all. Meanwhile primary, or medullary cords, being only partially inhibited, give bilaterally a small testis. In a normal female embryo, the ovarian cortex develops and prevents the primary sex cords of the right rudiment and left medullary zone from developing into a testicular structure. We think that gonadal inversion and intermediary states of hermaphroditism, here described, are the consequences of a more or less important inhibitory action of the testicular graft upon cortical structures. Free from cortical inhibition under the influence of the graft the primary sex cords can develop into a testis. Such an interpretation agrees with that of Benoit (1932) who explains in this way how a chick ovariectomy allows the right rudiment to become a testis and how the left gonad forms an ovotestis in the case of an incomplete ovariectomy. We also think that this mechanism is responsible for the testicular differentiation in the male and that the testicular size is regulated by the action of the inhibitory hormone elaborated by the testis itself before it is submitted to hypophyseal control. We can speculate whether the grafted testis acts directly on the gonadal differentiation of the female embryo or through the hypophysis. The answer to the latter hypothesis is negative since gonadal inhibition due to the graft is similar in both normal and hypophysectomized female chick embryos (Rashedi et al., 1975). Hermaphroditic structures developed during the embryonic life persist after hatching and are then submitted to the hypophyseal influence, as shown by the ovarian part with its follicles and growing ovocytes. According to Maraud (1963) the development of an epididymis indicates the secretion of androgens by the right testis. All these points concerning pullets need further investigations. Another fundamental question is relative
FEMALE
CHICK HERMAPHRODITISM
73
to the nature of the testicular hormone se- normally occurs in the male embryos, but creted by the graft and responsible for to an agenesia of the Miillerian tract occurgonadal inhibition and hermaphroditism. ring before this phase, during the undifferOur previous works show that such an ef- entiated state (Gaarenstroom, 1939; Stoll, fect is due to the testicular hormone re- 1950; Stoll and Maraud, 1952; Stall et al., sponsible for Mullerian retrogression. In 1972). Besides, it has been shown that tesfact, the graft of a testis from embryonic tosterone and other androgens stimulate donors of increasing ages, and therefore, of Mullerian ducts of the chick embryo, culdecreasing anti-Mitllerian power (Maraud tured in vitro (Weniger and Zeis, 1973). So et al.. 1966; Stoll et al., 1970) shows that steroidal androgens can be discarded as inthe degrees of retrogression of the Mulleductors of the Mullerian retrogression. rian tract and of the ovarian inhibition of Furthermore, in all these works, no fact the female host embryo are closely proporindicates any influence of androgens on the tional (St011 er al., 1975, 1978). This also gonadal differentiation of the female emappears in this present work where experibryo and peculiarly testosterone is unable mental conditions make out that Miillerian to cause an atrophy of the right gonadal ducts are always missing while gonads are rudiment, as shown by testis implantation strongly inhibited and even sexually in- in the female chick embryo which was preverted. We think that the two effects are viously submitted to ovariectomy due to the same hormone. (Groenendijk-Huijbers, 1973; GroenendijkIn mammals, the study of the phenomeHuijbers and Schaik, 1976). non occurring in freemartin cows has led At the present time, the biochemical Vigier et al. (1977) to consider this possibilstructure of the hormone is unknown in ity among others, on the basis of chronobirds but its molecular weight, higher than logical concordances. 1000, is not the same as that of sterolic anThe problem of the chemical nature of drogens (Weniger et al., 1975). this hormone can be discussed, since sevIn mammals, this substance has been eral authors think that steroidal androgens, concentrated from testis-incubation media which are effectively secreted by the em- and its protein composition has been estabbryonic testes during this period (see lished (review by Josso et al., 1977). Woods and Podczaski, 1974), are inductors of the Mullerian duct retrogression in the REFERENCES male sex. This opinion is founded on the Benoit, J. (1932). L’inversion sexuelle de la Poule dtterminee par l’ablation de l’ovaire gauche. Arch. fact that testosterone or other steroidal an2001. Exp. Gen. 73, l-112. drogens given to the female chick embryo Brode, M. D. (1928). The significance of the asymbefore its sexual differentiation cause the metry of the ovaries of the fowl. J. Morphol. 46, deficiency of the Mullerian tract l-57. (Dantchakoff, 1938; Willier et al., 1935, Caridroit and Pezard (1925). Poussee testiculaire autonome a I’inttrieur des greffes ovariennes et au1937; Willier et al., 1938; Wolff, 1935; Wolff toplastiques chez la Poule domestique. C. R. et al., 1948). The same effect is obtained in Acad. Sci. 180, 2067-2069. the quail (Lutz-Ostertag, 1%5). These hor- Dantchakoff, V. (1938). Rtalisation du sexe a volonte mones also cause “in vitro” the necrosis of par induction hormonale. III: Inversions et deviacultured ducts (Wolff et al., 1952; Scheibtions dans I’histogenese sexuelle chez I’embryon de Poulet train? par l’hormone male. Bull. Biol. Fr. Ptleger, 1953). Be/g. 72, 187-231. In fact, deficiencies of ducts, as observed Domm, L. V. (1927). New experiments on ovariotomy in the female embryos treated by androand the problem of sex inversion in the fowl. J. gens, are not due to their retrogression Exp. Zoo/. 48, 31- 119. during their sexual differentiation, as it Gaarenstroom, J. H. (1939). Action of sex hormones
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on the development of the Miillerian ducts of the chick embryo. J. Exp. ZooI. 82, 31-46. Groenendijk-Huijbers, M. M. (1973). Influence of gonadal hormone administration and gonadal implantation on the compensatory growth of the embryonic chicken right ovary after early sinistral castration. Verb. Anaf. Ges. 67, 193-196. Groenendijk-Huijbers, M. M., and Van Schaik, J. P. J. (1976). Effects of hemi-castration, testis implantation and administration of testosterone propionate on the female embryonic genital tract in various breeds and strains of chickens. Verb. Anat. Ges. 70, 179-182. Holyoke, E. A. (1956). The differentiation of embryonic ovaries and testes grafted together in adult hosts in the rabbit. Anat. Rec. 124, 307. Josso, N., Picard, J. Y., and Dien Tran (1977). The antimullerian hormone. Rec. Progr. Horm. Res. 33, 117- 167. Jost, A. (1947). Recherches sur la differentiation sexuelle de I’embryon de Lapin. Arch. Anat. Microsc. Morphol. Exp. 36, 151-200, 242-315. Lutz-Ostertag, Y. (1965). Action du propionate de testosterone sur les canaux de Muller de l’embryon de Caille (Corurnix cotrrrnixjaponica). C. R. Acad. Sri. 461, 4501-4504. MacIntyre, M. N. (1956). Effect of the testis on ovarian differentiation in heterosexual embryonic Rat gonad transplants. Anaf. Rec. 124, 27-45. Maraud, R. (1963). Recherches experimentales sur les facteurs inducteurs de la formation de l’tpididyme du Coq. Arch. Anaf. Microsc. Morphol. Exp. 52, 84- 127. Maraud, R., Stoll, R., and Coulaud, H. (1966). Action de la greffe testiculaire sur les canaux de Muller de l’embryon de Poulet. C. R. Sot. Biol. 160, 964-966. Rashedi, M., Stoll, R., and Maraud, R. (1975). Action de la greffe testiculaire sur I’ovaire de l’embryon de Poulet ayant subi une decapitation partielle. C. R. Sot. Biol. 169, 1464-1466. Rashedi, M., Stoll, R., and Maraud, R. (1977). Action d’un greffon ovarien sur l’ovaire de l’embryon femelle de Poulet. C. R. Sot. Biol. 171, 585-589. Reyss-Brion, M. (1978). Histochemical changes in the right gonad of the fowl after ovariectomy. Congr. Anat.
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Scheib-Pfleger, D. (1953). Roles des hormones sexuelles sur le developpement “in vitro” des canaux de Muller de l’embryon de Poulet femelle differencit. C. R. Acad. Sri. 236, 1446-1448. Stoll, R. (1950). Sur la differentiation sexuelle de l’embryon de Poulet. Arch. Anar. Microsc. Morphol. Exp. 39, 415-422. Stoll, R., Faucounau, N., and Maraud, R. (1972). Action des androgenes steroliques sur les canaux de Miiller de l’embryon de Poulet. C. R. Sot. Biol. 166, 858-861.
AND MARAUD Stoll, R., and Maraud, R. (1952). Action des hormones sexuelles sur l’evolution des canaux de Muller de l’embryon de Poulet. Arch. Anat. Microsc. Morphol. Exp. 41, 260-280. Stoll, R., and Maraud, R. (1974). Le r61e de testicule dans la differentiation sexuelle des gonoductes chez l’embryon des Vertdbrts amniotes. Bull. Assoc.
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Stoll, R., Maraud, R., and Coulaud, H. (1970). Sur la secretion de l’inducteur de la regression mullerienne chez l’embryon de Poulet: Chronologie des relations hypophyso-testiculaires. C. R. Sot. Biol. 164, 1013- 101.5. Stoll, R., Rashedi, M., and Maraud, R. (1975). Action de l’hormone testiculaire de regression mullerienne sur la developpement de l’ovaire de l’embryon de Poulet. C. R. Sot. Biol. 169, 927-930. Stoll, R., Rashedi, M., and Maraud, R. (1978). Sur l’hormone de regression mullerienne, agent inducteur du testicule chez l’embryon de Poule. Bull. Assoc. Anat. 176, 13 1- 143. Swift, C. H. (1915). Origin of the definitive sex-cells in the female chick and the relation to the primordial germ cells. Amer. J. Anat. 18, 441-470. Swift, C. H. (1916).0rigin of the sex cords and definitive spermatogonia in the male chick. Amer. J. Anat. 20, 375-410. Thiebold, J. J., and Reichhart, J. H. (1973). Inhibition de developpement ovarien par la greffe de gonades embryonnaires chez le Poulet. C. R. Sot. Eiol. 167, 1958- 1960. Turner, C. D., and Asakawa, H. (1964). Experimental reversal of germ cells in ovaries of fetal mice. Science 143, 1344. Venzke, W. G. (1954). The morphogenesis of the indifferent gonad of chicken embryos. Amer. J. Vet. Res. 15, 300-308. Vigier, B., Prepin, J., Perchellet, J. P., and Jost, A. (1977). Developpement de l’effet free-martin chez le foetus de Veau. Ann. Med. Vet. 121, 521-536. Weniger, J. P., Mack, G., and Holder, F. (1975). L’hormone responsable de la regression des canaux de Mtiller chez l’embryon de Poulet male n’est pas un androgene. C. R. Acad. Sci. 280, 1889- 1891. Weniger, J. P., and Zeis, A. (1973). Action feminisante des androgenes sur le testicule et le canal de Muller d’embryon de Poulet “in vitro.” Arch. Anat. Microsc. Morphol, Exp. 62, 145- 150. Willier, B. H. (1927). The specificity of sex, of organization and of differentiation of embryonic chick gonad as shown by grafting experiments. J. Exp. Zool. 46, 409-467. Willier, B. H. (1939). The embryonic development of sex. Sex Intern. Secretions, 64- 144. Willier, B. H., Gallagher, T. F., and Koch, F. C. (1935). Sex modification in the chick embryo re-
FEMALE
CHICK
HERMAPHRODITISM
suiting from injection of male and female hormones. Proc. Nat. Acad. Sci. USA 21,625-631. Willier, B. H., Gallagher, T. F., and Koch, F. C. (1937). The modification of sex development in the chick embryo by male and female sex hormones. Physiol. Zool. 10, lOl- 122. Willier, B. H., Rawles, M. E., and Koch, F. C. (1938). Biological difference in the action of synthetic male hormones on the differentiation of sex in the chick embryo. Proc. Nat. Acad. Sri. USA 24, 176- 182. Wolff, Et. (1935). Sur la transformation experimentale des femelles genetiques en intersexuees provoquee par I’injection d’hormones males aux
75
embryons de Poulet. C. R. Acad. Sci. 201, 1055-1057. Wolff, Et., Lutz-Ostertag, Y., and Haffen, K. (1952). Sur la regression et la n&rose “in vitro” des canaux de Mtiller de l’embryon de Poulet sous l’action directe des hormones males. C. R. Sot. Biol. 146, 1793- 1795. Wolff, Et., Strudel, G., and Wolff, Em. (1948). L’action des hormones androgbnes sur la differenciation sexuelle des embryons de Poulet. Arch. Anat. Histol. Embryol. 31, 237-310. Woods, J. E., and Podczaski, E. S. (1974). Androgen synthesis in the gonads of the chick embryo. Gen. Comp. Endocrinol. 24, 413-423.