HORMONES IX DIFFERENTIATION OF SEX 



f9 



the embryonic genital structures of the host 

 (especially of the Miillerian ducts) were 

 noted and attributed to the influence of the 

 grafted tissues. But later investigators failed 

 to confirm these findings, and it was shown 

 eventually that the anomalies observed by 

 ]\Iinoura were unspecific in character and 

 bore no constant relation to the sex of the 

 grafted tissue (for a review and discussion 

 see AVillier, 1939). Similar modifications 

 were also found after transplantation of 

 various nongonadal tissues, and are appar- 

 ently induced by changes in the physical 

 environment incidental to operation, such 

 as lowering of the temperature and the hu- 

 midity (Willier and Yuh, 1928). Evidently 

 the original experiments had not been ade- 

 quately controlled with respect to such fac- 

 tors. 



Obviously this type of experiment does 

 not correspond exactly with conditions in 

 the freemartin. The grafted tissue came 

 from adult gonads, and there was no way 

 of determining the hormone output of such 

 grafts or whether, indeed, they produced 

 hormones at all. Furthermore, sexual dif- 

 ferentiation in the host embryos has gen- 

 erally begun before transplantation to the 

 chorioallantois is practicable. This objection 

 was avoided, however, by modifying the ex- 

 l)eriment. Sexually undifferentiated embry- 

 onic gonads were transplanted to the chorio- 

 allantoic membrane of a host embryo 

 already well advanced in sex differentiation. 

 In this case changes might be anticipated 

 in the grafts. However, this experiment, as 

 well as transplantation of the gonad-form- 

 ing region of the blastoderm (Willier, 1927, 

 1933), also gave negative results. The 

 grafted gonads differentiated to a variable 

 degree, depending on the state of develop- 

 ment of the primordia at the time of trans- 

 plantation, but when sexual differentiation 

 occurred it showed no constant relation to 

 the sex of the host. 



These failures to obtain evidence that 

 gonad tissues growing on the chorioallantois 

 influence the differentiation of host struc- 

 tures, or are themselves modified by the 

 hormones of the host, raised serious doubt 

 as to the role of hormones in embryonic sex 

 differentiation in birds ; and this feeling was 

 not entirely removed by the demonstration 



some years later that the sexual differentia- 

 tion of the chick can be readily modified by 

 treatment with pure hormone preparations. 

 It was not until embryonic gonads were 

 transplanted directly into the body cavity 

 of another embryo (Bradley, 1941 ; Wolff, 

 1946) that unmistakable evidence was ob- 

 tained. The conditions under which this re- 

 sult was achieved — close proximity of the 

 graft to the developing host structures — 

 suggested that the failure to obtain positive 

 results by chorioallantoic grafting was per- 

 haps largely a matter of the quantity or 

 concentration of the hormone. Recently, 

 however, a true "freemartin effect" has been 

 reported in twin chicks of different sex, 

 which developed from an egg with two yolks 

 (Lutz and Lutz-Ostertag, 1958). There was 

 local development of cortex on the left 

 testis of the male twin, and a marked inhibi- 

 tion of the ]Miillerian ducts of the female, 

 effects paralleling those produced by in- 

 tracoelomic gonad grafts. This is the only 

 recorded case of a natural freemartin in 

 birds. 



B. GR.\FTIXG EXPERIMENTS IX AMPHIBIAN 

 EMBRYOS 



The principal experimental procedures de- 

 veloped for amphibian embryos are illus- 

 trated diagrammatically in Figure 2.2, 

 which shows the different modes of grafting, 

 and the resulting vascular relationships, as 

 compared with the freemartin (Fig. 2.1). 

 The first experiments actually undertook to 

 reproduce as nearly as possible the situation 

 which arises by chance in the freemartin. 

 The method devised was parabiosis— the 

 grafting together of two embryos in the 

 manner of "Siamese twins" (Burns, 1925) 

 so that in later development there is a 

 common circulation (Figs. 2.2A and 2.9). 

 When members of such a pair happen to be 

 genetically of the same sex, normal sexual 

 differentiation would be expected to follow; 

 but in pairs of different sex opportunity for 

 cross-circulation of sex hormones is pro- 

 vided. Circulatory anastomosis is estab- 

 lished in such pairs long before the begin- 

 ning of sex differentiation in the gonads, 

 and so a favorable situation is provided for 

 testing the possibility of hormone action. 

 The results obtained by this method vary 



