82 



BIOLOGIC BASIS OF SEX 



equivocal results which have in general con- 

 firmed and extended those obtained by para- 

 biosis. 



C. USE OF PURE HORMONES AS SEX 

 DIFFERENTIATING AGENTS 



Early attempts to influence embryonic 

 sex differentiation by the use of crude hor- 

 mone preparations were almost entirely un- 

 successful because of lack of potency, or 

 the toxicity of the extracts. However, the 

 isolation and eventual synthesis of steroid 

 hormones made available a variety of ac- 

 tive and nontoxic substances, and the use of 

 pure hormones largely superseded grafting 

 techniques. Direct administration of stand- 

 ard hormone preparations has the great ad- 

 vantage that dosages can be exactly known 

 and regulated; also the timing of treat- 

 ments is readily controlled and varied. 



The first successful experiments using 

 pure hormones were carried out on chick 

 embryos. Similar results were obtained at 

 almost the same time by several groups of 

 investigators (Kozelka and Gallagher, 1934; 

 Wolff and Ginglinger, 1935; Dantchakoff, 

 1935, 1936; Willier, Gallagher and Koch, 

 1935, 1937j who introduced the hormones, 

 in oily or in aqueous solution, into the in- 

 cubating egg. Striking transformations were 

 produced, involving both the structure of 

 the gonads and the accessory organs of sex. 

 In the best cases reversal of the gonads was 

 histologically almost complete. 



The effects of crystalline sex hormones 

 have also been investigated in many species 

 of amphibians. Two methods have been 

 utilized. Larvae may be treated individually 

 by repeated injections, or in groups by con- 

 tinuous immersion, the hormone being dis- 

 solved in the water in which the larvae are 

 reared. The latter method is particularly 

 convenient for anuran tadpoles. Treatment 

 can be started very early, the concentration 

 is readily varied, and in many cases com- 

 plete transformations have been obtained 

 with the use of extremely low concentra- 

 tions. 



In mammalian embryos experimental 

 study of sex differentiation was long delayed 

 by the lack of operative techniques ade- 

 quate for dealing with embryos in utero. 

 The advent of pure hormones made possible 

 the first successful experiments in this field. 



In i^lacental forms, in spite of a very high 

 mortality, large doses of crystalline hor- 

 mones can be administered to the mother 

 during early stages of pregnane}" with pro- 

 nounced effects on the genital systems of the 

 embryos (for a review of the earlier experi- 

 ments see Greene, 1942, and for a recent 

 summary Jost, 1955) . About the same time, 

 experiments were begun using the pouch 

 young of a marsupial, the North American 

 opossum (Burns, 1939a, b; Moore, 1941). 

 So undeveloped are young marsupials at 

 birth that virtually the entire course of 

 morphologic sex differentiation takes place 

 postnatally, and the embryos in the pouch 

 are directly accessible for experimentation. 

 Hormones were administered by injection 

 (Burns) or by inunction, the application of 

 an ointment containing the hormone to the 

 skin. Except for minor differences attributa- 

 ble to dosage or other experimental factors, 

 the results were similar, and in general 

 agreement with those obtained in placental 

 mammals by treatment during pregnancy. 



D. SEX DIFFERENTIATION IN THE 

 ABSENCE OF HORMONES 



Although the evidence obtained by graft- 

 ing techniques and by administration of 

 hormones shows that the differentiation of 

 embryonic genital structures may be pro- 

 foundly modified or even completely re- 

 versed, such evidence is not in itself con- 

 clusive with respect to the central problem, 

 the role of hormones in the normal differ- 

 entiation of sex. The transmissible sub- 

 stances responsible for sex reversal in graft- 

 ing experiments have not been isolated or 

 identified and it may be argued that experi- 

 ments with pure hormones show merely that 

 the differentiating embryonic sex primordia 

 are capable of reacting when hormones are 

 introduced experimentally. Such evidence 

 docs not prove, however, that embryonic 

 gonads actually produce such hormones. For 

 this question the crucial test is the capacity 

 of the embryonic genital structure to de- 

 velop in the absence of gonads or removed 

 from all hormonal influence. 



Evidence on this point has been forth- 

 coming in recent years and gives strong 

 supi)ort to tiie hormone theory. Two differ- 

 ent experimental approaches have been de- 

 veloped. Early castration of the embryo has 



