Book - Sex and internal secretions (1961) 2
HORMONES IN DIFFERENTIATION OF SEX
115
B
D
Fig. 2.24. Diagrammatic representation of the effects of relatively large doses of testosterone propionate, administered from birth to an age of 50 days, on the development of the sex ducts in young opossums. A. Sex ducts as they appear in a normal male at 50 days; the vas deferens (Wolffian duct), epididymal tubules and remnants of mesonephric tubules are shown in black; the atrophic Miillerian duct is unshaded and the testis stippled. B. The effects of the male hormone on the male duct system appear throughout; however, large dosages also induce a paradoxical growth and development of the uterine and tubal regions of the Miillerian duct, but the vaginal segment is absent as in the normal male. C. The sex ducts as they appear in a normal female at 50 days — breakdown and disappearance of the Wolffian duct and associated structures, regional development of the Miillerian duct into tubal, uterine, and vaginal segments. The contribution of the urinogenital sinus to the vaginal canal is indicated in stipple. D. The effects of the male hormone in a female subject : note the preservation and great hypertrophy of the male duct system which is not, however, as large or as well differentiated as in the treated male; note also the striking paradoxical effect of a large dosage of androgen on the female genital tract which, at the same dosage level, is far greater than in the treated male (B). These paradoxical effects of androgen on the Miillerian duct dorivativos disappear entirely at lower dosages.
carried out in a number of species of birds and mammals. ^'^
Early castration of avian cml)ryos is followed by persistence and development of Miillerian ducts in both sexes (the chick and the duck, Wolff and Wolff, 1951; Huijbers, 1951). After total castration in males both ducts persist and develop, but partial castration results in regression as usual. In females, in which the right duct normally regresses, both ducts persist and are well developed. ^^ Thus, in the absence of the gonads the Miillerian ducts follow the same pattern of development regardless of sex (Fig. 2.25). It is clear that the testes are
'Mu the rabbit (Jost, 1947b); the mouse (Raynaud and Frilley, 1947) and the rat (Wells, 1946, 1950); in the chick and the duck embryo (Wolff, 1950; Wolff and Wolff, 1951; and Huijbers, 1951).
" In birds involution of the right Miillerian duct of the female is normally conditioned in some manner by the ovaries, and it has been shown further that the presence of either ovary is sufficient (Wolff and Wolff, 1951). The exact nature of the inhibitory factor in this interesting case is not known.
necessary for normal inhibition of the ducts in male embryos, and it was pointed out earlier that a graft of the embryonic testis has the same effect in females (Wolff, 1946). The ovaries, on the contrary, have no positive role in the development of the Miillerian ducts, but actually inhibit the right duct; in their absence both ducts develop without hormonal conditioning.
Among mammalian embryos the case of the rabbit is best known (Jost, 1947b). Castration of the female again has no important consequences; the Miillerian ducts continue to develop and shortly before birth are only slightly smaller than in normal females. In male castrates, on the other hand, the situation is very different; if the operation is performed early enough the Miillerian ducts, instead of regressing, persist and develop, becoming practically indistinguishable from those of castrate females (Table 2.2, Fig. 2.26). Thus, as in birds, castrates of either sex follow identical patterns of development.
Nevertheless, if castration is delayed be
116
BIOLOGIC BASIS OF SEX
MALE
CASTRATE TYPE
FEMALE
Fig. 2.25. Diagrams summarizing the effects of castration on the development of the syrinx (top row) oviducts, and genital tubercle (below) in bird embryos (chick and duck). The left column shows the normal condition in the male : note the complete retrogression of the Mlillerian ducts (broken lines) under the influence of the embryonic testes (black). In the right column, the normal female condition showing the retrogression of the right oviduct. The arrows indicate the inhibitory action normally exerted by the ovaries on the syrinx, right oviduct and genital tubercle. The castrate type, which appears regardless of genetic sex, is seen in the center. In castrates both oviducts persist in complete form and are as well developed as the normal left oviduct ; the syrinx and genital tubercle, however, assume the male form in the absence of the inhibition exerted by the ovaries. Note the extreme asymmetry of the male syrinx and tubercle, as compared with the primitive symmetrical form retained in the normal female. (After Et. Wolff and Em. Wolff, J. Exper. ZooL, 116, 59-97, 1951.)
TABLE 2.2
Effects of castration in male rabbit fetuses studied at the age of 2S days (After A. Jost, Recent Progr. Hormone Res., 8, 379-418, 1953.)
AGE AT CASTRATION
MULLERIAN DUCTS
WOLFFIAN DUCTS AND DERIVATIVES
PROSTATIC GLANDS EXTERNAL GENITALIA
19 DAYS (2 coses)
PERSISTENT
20-21 DAYS (llcoses)
PERSISTENT CAUDAL REMNANTS
(in 3cases)
VENTRAL BUDS PRESENT
22-23 DAYS (Scases)
UTERO-VAGINAL SEGMENTS PERSISTENT
CAUDAL REMNANTS
HYPOSPADIC
23 DAYS (4 cases)
ABSENT- SMALL ABSENT (=NORMAL) SEMINAL VESICLES PRESENT
WELL DEVELOPED
24 DAYS (3 coses)
ABSENT ( = NORMAL)
WELL DEVELOPED
Unilateral castration is followed by normal development.
Castration effects are prevented by Testosterone propionate given at operation.
yond a certain stage (about the 22nd day rather limited period during which involu of gestation in the rabbit; Jost, 1947b, c) tion of the oviducts is determined, as was
the ducts subsequently undergo involution shown also for the chick in the case of male
as usual (Table 2.2). Evidently there is a hormones administered experimentally.
HORMONES IN DIFFERENTIATION OF SEX
A -m B
11^
^'#
ms^sL-^^; ^m&
Fi(j. 2 2b Ihc ctlLit^ ul e i^lialiou uu llif dux uluiniuul uf the sex ducts iii the rabbit. A.
Persistence ot the Mullenan duct (uterine level) above, and the vaginal canal, below, in a
castrate male. B. The same structures seen in a castrate female as compared with the condition in the noimal female shown in C . Note almost complete involution of the male duct
except for remnants (CW) in the castrate male, and compare with the castrate and the
normal female. Castration of the female (S) has little effect on the pattern of development
which follows; on the other hand, castration of the male results in involution of the
Wolffian duct and development of the Mlillerian duct, a reversal of the normal pattern. (From
A. Jost, Arch. Anat. microscop. et Morphol. exper., 36, 271-315, 1947.)
Once conditioning has occurred the effect is irreversible.
In view of the decisive role of the embryonic testis in the development of the Mlillerian ducts, as shown by castration experiments, it is significant to note the condition of the genital tracts in certain "lateral gynandromorphs" of genetic origin in mice (Hollander, Gowen and Stadler, 1956) which have an ovary on one side of the body and a testis on the other. Both gonads are usually small and hypoplastic. Without exception, however, on the side of the testis the Mlillerian duct derivatives are absent entirely and the male duct system is developed. Contralaterally, a female genital tract is always found although it shows great variation in size. An almost identical condition has recently been described in a gynandromorphic hamster (Kirkman, 1958). The manner in which the influence of the testis tends to be limited to its own side of the body in these cases is of special interest, and will be considered later in dealing with the localized character of hormone effects. It is undoubtedly related to the early stage at which the testis begins to exert its effect and probably also to its reduced size in nearly all cases.
The development of the Mullenan ducts
after isolation in vitro. At this point, in the case of mammalian embryos, an obvious question presents itself. Is development of Mlillerian ducts in male castrates in fact a purely autonomous process, due to release from an inhibition normally exerted by the testes or, in the absence of the gonads, does some other humoral factor intervene to assure their development? It is known that in various species of mammals estrogens are present in the placenta and fetal fluids in considerable amounts (for references see Price, 1947; Parkes, 1954), and the possibility arises that development of the ducts after castration may be maintained under the influence of an estrogenic substance of nongonadal or of maternal origin. This question has been answered, in the negative, by experiments designed to test the selfdifferentiating capacity of the ducts under conditions of physiologic isolation. When explanted in vitro (with pieces of the associated mesonephric bodies) the Mlillerian ducts of rat embryos, regardless of the sex of the donor embryo, survive and continue their development in the same manner as in castrate fetuses (Jost and Bergerard, 1949; Jost and Bozic, 1951). In these experiments the ducts were isolated at ages of 15 to 16 days, and 16 to 17 days re
118
BIOLOGIC BASIS OF SEX
spectively. Similar behavior is seen in grafts to the eye chamber of castrate hosts (the guinea pig, Bronski, 1950) and after transplantation of the entire embryonic genital tract into castrate and noncastrate hosts of various ages (the rat, Moore and Price, 1942). Although the experimental environments in all of these cases cannot be considered hormone-free in the strict sense (Jost and Bozic, 1951), the results indicate that the development of the female sex ducts when removed from the influence of the embryonic testis is a matter of autonomous differentiation.
The above experiments show that in rat embryos the fate of the JMlillerian ducts is undetermined up to an age of 16 to 17 days at least (Jost and Bozic), since at this age these ducts in male embryos still retain their capacity for autonomous development when removed from the inhibiting influence of the testis. Experiments on the same species (Price, 1956; Price and Pannabecker, 1956 1 indicate that involution is irreversibly determined at about the age of 17 days. The genital tracts of male fetuses were removed at 17.5 days of gestation and cultured in vitro. At this stage the first signs of involution can be detected in the region of the ostium but the posterior extremities of the ducts are still growing. After explantation the ducts continue to regress regardless of whether the testes are included in the explant or not, whereas those of female fetuses under the same conditions develop normally. It seems that the fate of the Miillerian ducts in the male becomes irreversibly fixed within the brief period of a day by exposure to the testis hormone.
This question has })een investigated in some detail in the chick. It had been shown earlici- that after transplantation to the cliurioalhintois, Miillerian ducts from embryos of either sex differentiate completely if isolated before the beginning of sex differentiation. But if transplanted later than the 10th day of incubation the ducts of male embryos invariably degenerate; beyond this age their involution has been finally determined (Wolff and Ostertag, 1949). Making use of the technique of culture in vitro the analysis has been carried farthei' (Wolff and Lutz-Ostertag, 1952). When isolated
before the appearance of sex differentiation in the gonads, again the Miillerian ducts of both sexes undergo a complete differentiation, as in castrate embryos; but if isolated after the 9th day the ducts of male emIjryos i^romptly undergo involution. However, if the Miillerian ducts of female embryos are cultured in the presence of an embryonic testis, a typical involution takes place (although at the same time the adjacent Wolffian ducts develop normally). Furthermore, when testosterone propionate is added to the medium it has the same effect as the embryonic testis (Wolff, Lutz-Ostertag and Haffen, 1952; for a summary see Wolff, 1953b). These results hardly leave the role of the male hormone in doubt. It follows also that the action of the hormone in vitro must be direct. Evidence has been obtained to show that the process of involution of the ducts is of the nature of an autolysis produced by the action of proteolytic enzymes which are presumabh' activated l)v the male hormone (Wolff, 1953b).
Summary and conclusions. On the basis of present knowledge, the following general statements may be made concerning the role of sex hormones in the differentiation of the Miillerian ducts.
a. Female hormone, in most species, stimulates precocious growth and development of the Miillerian ducts and their derivative structures in embryos of either sex when given in adequate dosages. Administered at an early stage it prevents the normal involution of the ducts in male embryos after which development may continue without further treatment. In certain mammalian species, however (hamster, field mouse, man), negative results have been reported, which may possibly be related to the level of estrogen prevalent during gestation in these species or to dosage.
b. Male hormone, if administered early, has an inliibitory action on the Miillerian ducts in the embryos of birds and amphibians. In some species the primary de\-el()l>nient of the duct is entirely suppressed; in other cases only a partial inhibition occurs. In mammals, the hormone of the embryonic testis ]M-o(lnccs involution of the (hicts in males at tlic beginning of sex dif
HORMONES IN DIFFERENTIATION OF SEX
119
ferentiation, and testis grafts inhibit the Miillerian ducts of females; on the other hand, male hormones of adult type fail to inhibit the ducts, or produce only a limited and localized involution in a few species.
c. The time factor is critical in the hormonal control of the jNIiillerian ducts. To suppress primary differentiation of the ducts in birds and amphibians, male hormone must be administered before or during the formative stage of development. However, the fully formed duct remains sensitive to hormones up to the onset of sex differentiation. Female hormone administered before this stage insures retention and development of the ducts in males, and during the same period they are subject to inhibition by male hormone or by the embryonic testis. Beyond this point the final development or the involution of the ducts has been irreversibly determined and hormones are without effect.
d. In the absence of previous hormonal conditioning, as after early castration or early isolation in vitro, the Miillerian ducts of either sex are capable of an autonomous differentiation, comparable to development in normal females; this development can be prevented, however, and involution induced, by introduction in vitro of an embryonic testis, or (in birds) by the addition of male hormone to the medium.
e. In amniote embryos the testis hormone is the controlling factor in the differentiation of the Miillerian ducts. The involution of the ducts in males is conditioned by the testes, whereas in the absence of gonads there is an almost normal development of the ducts in embryos of either sex. This does not mean, however, that the embryonic Miillerian ducts are insensitive to female hormone; in sufficient concentration it produces hypertrophic de^'elopment in both sexes.
f. The evidence from cultivation in vitro shows that the effects of hormones on the Miillerian ducts are exerted directly and are independent of the organism as a whole.
3. The Male Duct System
The male sex duct, or Wolffian duct, develops first as the duct of the pronephros (primary nephric duct) and subsequently
serves as the mesonephric duct in both sexes. As the mesonephros is replaced by the metanephros in amniote embryos, the Wolffian duct loses its excretory function and its fate in the two sexes is different. In the female it disappears entirely or survives only as vestiges, but in males it acquires a new status as the male sex duct. At the same time a certain number of mesonephric tubules, which connect the duct with the rete canals of the testis, become a part of the epididymis, and the seminal vesicle develops as a diverticulum of the duct near its junction with the urinogenital sinus. During its early history as the duct of the mesonephros the Wolffian duct is unresponsive to sex hormones, but with the onset of sex differentiation it becomes responsive to the male hormone.
The role of male hormone in the differentiation of the Wolffian ducts. In the female of amphibians experimental transformation of ovaries into testes is followed by hypertro])hy and masculinization of the AVolffian ducts (Humphrey, 1942), and transplantation of testes into larval castrates of either sex has the same effects (de Beaumont, 1933). Administration of male hormones produces marked hypertrophy of the Wolffian ducts in larval amphibians of either sex [e.g., Burns, 1939c; Foote, 1941; for summary see Gallien, 1955). A similar response occurs in the embryos of birds and has also been reported in many species of mammals (Figs. 2.23, 2.24). ^^ This is the case in female embryos as well as in males. Development of epididymal tubules (from the epoophoron) also takes place in females (Fig. 2.24D) which also commonly develop seminal vesicles (Greene, 1942; Raynaud, 1942; Wells and van Wagenen, 1954 L With higher dosages all these structures undergo extreme hypertrophy.
Female hormones, on the contrary, appear to have little influence on the development of the Wolffian ducts, although in a few cases a jiartial involution of the ducts has
^" This effect has been widely reported : see Willier (1939), Wolff (1938, 1950) for the chick; Greene (1942) for the rat; Raynaud (1942), Turner (1940) for the mouse; Jost (1947a) for the rabbit; Godet (1949) for the mole; Burns (1939a and b, 1945a); and Moore (1941) for the opossum.
120
BIOLOGIC BASIS OF SEX
been reported (Raynaud, 1942; Greene, 1942). It should be noted that the latter observations are in mammalian embryos (mice and rats) in which the hormone of the testis (see below) is essential to insure retention of the ducts. Partial involution in males under the influence of a female hormone is possibly only a result of interference with the normal activity of the testis. On the other hand, a paradoxical action of female hormone, causing partial retention and hypertrophy of the male duct, has been occasionally reported (e.g., Greene, 1942; Moore, 1941). Although the method of administration in these cases does not permit accurate estimation of the dosage it was evidently rather large. With these exceptions, the effects of sex hormones on the male duct system are consistent with theory. The effects of castration on the development of the male sex duct are striking and agree with the results of hormone administration. They show that in all species the presence of the embryonic testis is necessary to induce sexual differentiation of the duct, and to insure its retention in mammalian embryos. In amphibian larvae (Triturus, syn. Triton) the Wolffian ducts persist after castration in their capacity as nephric ducts, but remain in a sexually undifferentiated condition (de Beaumont, 1933). In bird embryos also there is no significant alteration of the Wolffian ducts after castration. It is in mammals that the ducts become dependent on the testis and its hormone for survival as well as for sexual differentiation. In rabbit embryos castrated before the 22nd day of gestation (Jost, 1947b) the ducts in both sexes completely regress, following the female pattern of development (Fig. 2.26) ; involution in castrates is prevented, however, and normal development is maintained by prompt administration of male hormone. A more variable atresia of the ducts also occurs in fetal rats after castration (Wells and Fralick, 1951). In mice Raynaud reports a difference in reaction in the two sexes. After castration the Wolffian ducts of males undergo a complete involution but they may be partially retained in females. It is suggested that in this species the ovaries may play a positive role in the involution of the duct in females (Raynaud, 1950).
The development of the Woffian ducts in vitro. Further evidence that retention and sexual differentiation of the Wolffian ducts and associated structures (epididymal tubules and seminal vesicles) are dependent on the testis and its hormone is provided by the behavior of the male duct system after isolation, using the technique of organ culture. Development in isolation provides a parallel to development in the castrate fetus, with exclusion, however, of possible influence by hormones of maternal or placental origin or from some extragonadal source in the fetus.
When the mesonephric bodies of rat embryos, including long segments of the gonaducts, are removed at 15 to 16 days of gestation and cultured without the gonads, the Wolffian ducts of both males and females degenerate, but at the same time the Miillerian ducts survive and develop normally. The degeneration of the Wolffian ducts cannot, therefore, be due to unfavorable conditions in the medium (Jost and Bergerard, 1949) . The same result was obtained using slightly older fetuses of ±16.5 days (Jost and Bozic, 1951). However, the most complete study of this question is that of Price and Pannabecker (Price, 1956; Price and Pannabecker, 1956) who explanted male genital tracts of 17.5-day rat fetuses under various conditions designed to test the role of the embryonic testis and the male hormone. When both testes are included with the explant, development of the male duct system proceeds normally up to an age of 21.5 days (approaching term for the normal fetus I and the seminal vesicles develop as usual. Normal development ensues also when only one testis is left with the implant. But if one testis is removed and the lateral halves of the genital tract are spread widely apart on the surface of the medium, development is normal only on the side where the testis is present ; on the other side serious defects appear; the duct is thin and weakly developed, and the seminal vesicles are small or even lacking. Finally, if both testes are removed the Wolffian ducts regress completely. However, the addition of male hormone to such a prci^aration fully compensates for the al)scncc of the testes and development of the male duct system is again normal.
HORMONES IN DIFFERENTIATION OF SEX
121
Fig. 2.27. The effects of castration on the development of the prostatic glands in the rabbit. A. The sinus region in a male fetus, aged about 27 days, castrated before the 20th day
of gestation; above is the canal of the urinogenital sinus, below it the dark, bilobed structure represents the vaginal cord as it unites with the wall of the sinus. No sign of prostatic
buds is seen. B. The sinus region in a young male of the same age castrated at about 21
days (20 days, 20 hours). Two large prostatic buds are seen ventral to the vaginal cord
which were present at the time of castration. No further development has occurred. C. The
sinus region in a male fetus of 28 days, castrated at the age of 23 days. Castration at this
age is followed by essentially normal development. (From A. Jost, Arch. Anat. microscop. et
Morphol. exper., 36, 271-315, 1947.)
Fig. 2 2S Hi-told.i In k lu ( - be twcin noimal h i i i i I , i i \ternal
genitalia {B) m i.ihlut htu^i^ aitcM -e\ual diffeientiation Mk uiinoj-nut il uu itu^ i-^ larger in the fem<ile, and is onh p.uth >uiioundcd b^ the pieputial fold, ■wliith maik'- off the glans clitoridis from the surrounding tissues. In the male the urethral cleft is narrower and completely enclosed within the preputial fold. The paired erectile bodies are seen above the urethral cleft. Castration at an early stage alwaj^s results in genitalia of female type (A) regardless of the sex of the castrated embrvo. (From A. Jost, Arch. Anat. microscop. et Morphol. exper., 36, 271-315, 1947.)
Altogether, the evidence clearly indicates that the male hormone is the essential determining factor in the survival and sexual differentiation of the male sex ducts and seminal vesicles. Notwithstanding the minor exceptions noted above, the female hormone evidently has little role. The reason for the insensitivity of the Wolffian ducts to the female hormone may possibly be found in their long phylogenetic history as nephric ducts in both sexes, in which capacity they must be retained in some groups beyond the period of sex differentiation or even permanently.
B. DERIVATIVES OF THE CLOACA AND URINOGENITAL SINUS
Sexual dimorphism of the amphibian cloaca chiefly takes the form of special cloacal glands which in males become highly developed at the breeding season, causing the prominent swelling of the cloacal region so conspicuous in males. In the females of various species they may be absent, present in a rudimentary state, or in some cases differently specialized (Noble, 1931). For their development and maintenance these glands depend almost entirely on the testis. After experimental transformation of sex, the subsequent differentiation of the cloacal glands
122
BIOLOGIC BASIS OF SEX
TABLE 2.3
Effects of hormones on derivatives of the urogenital sinus and the external genitalia in mammalian embryos
ACTION OF MALE HORMONE ON FEMALES
SUBJECT OF EXPERIMENT
FORM OF SINUS
VAGINAL DEVELOPMENT
SINUS EPITHELIUM
PROSTATE FORM OF GENITALIA
OPOSSUM
MALE TYPE
SUPPRESSED IN 50%
OF CASES
HYPERTROPHIC*
BUT NOT CORNIFIED
H 1 G H LY
DEVELOPED
MALE TYPE
RAT
MALE TYPE
SINUS PORTION SUPPRESSED
HIGHLY
DEVELOPED
MALE TYPE
MOUSE
MALE TYPE
SINUS PORTION SUPPRESSED
WELL
DEVELOPED
MALE TYPE
RABBIT
MALE TYPE
SUPPRESSED
WELL
DEVELOPED
MALE TYPE
MONKEY
MASCULINIZED
SINUS PORTION SUPPRESSED
STRATIFIED SQUAMOUS
LARGE OR
VARIABLE
MASCULINIZEDPENIS-LIKE
ACTION OF FEMALE HORMONE ON MALES
OPOSSUM
FEMALE TYPE
SINUS PORTION
HYPERTROPHIC
VAGINAL TYPEHIGHLY 60RNIFIED
COMPLETELY
SUPPRESSED
FEMALE TYPE
RAT
FEMALE TYPE
SINUS PORTION
WELL DEVELOPED
SUPPRESSED
FEMALE TYPE
MOUSE
FEMALE TYPE
VAGINAL CORD
WELL DEVELOPED
STRATIFIED SQUAMOUSMETAPLASTIC
SUPPRESSED
FEMALE TYPE
» r/7/5 effect appears only with large dosages
corresponds to the altered sex of the gonad. Castration of mature males results in retrogression of the glands and after early castration the cloaca remains sexually undifferentiated in both sexes (de Beaumont, 1933). However, testis tissue grafted into castrates (de Beaumont) , or treatment with male hormone {e.g., Burns, 1939c), readily induces development of male cloacal glands in individuals of either sex (for a review see Humphrey, 1942).
In the development of mammalian eml)ryos the urinogenital sinus is separated at an early stage from the cloacal region of the hind-gut by formation of the perineal septum. In its primitive condition the sinus is a short canal, extending from the neck of the bladder to the exterior, with a meatus at the base of the genital tubercle. The paired gonaducts open into it near the neck of the bladder (Fig. 2.22). Sexual differentiation in females chiefly involves anatomic and histologic changes associated with development of the vagina, to which the urinogenital sinus makes an important contribution; at the same time the male sex ducts regress and largely disappear (Fig. 2.22B). In placental mammals fusion of the posterior ends of the Miillerian ducts as they approach the urinogenital sinus gives rise to the unpaired, median vagina, but in marsu
pials the ducts remain separate and paired
lateral vaginal canals are formed (Fig.
2.22B). In male embryos the main features
of sinus differentiation are the involution of
the ]\Iiillerian ducts with absence of vaginal
development, and the differentiation of elaborate prostatic glands. Sex hormones show a
high degree of specificity in their effects on
the sinus structures, inducing development
of typically male or female forms. Results
are available for a number of species belonging to several orders of mammals,^ and
are in agreement except for minor details
(for some representative species see Table
2.3).
The histologic aspects of the differentiation of the sinus are well illustrated in young opossums. The effects of male hormone (testosterone propionate) in male and female pouch young are compared in Figure 2.29. The effect of the hormone in males is
^\See Greene (1942) for the rat: Raynaud (1942), Turner (1940) for the mouse; Jost (i947a) for the rabbit; Godet (1950) for the mole; Wells and van Wagenen (1954) for the monkey; Burns (1939a, b) and Moore (1941) for the opossum. Simimaries for these and other species are to be found in Colloques Intcrnationaux : La Differencialion Sexuelle choz les Vertebre.*, Masson et Cie., Paris, 1951. As an exception, the effects in the hamster are rather slight (Bruner and Witschi, 1946; White, 1949).
HORMONES IN DIFFERENTIATION OF SEX
123
m^M
^\'»^'^
- ^rHi.
Fig. 2.29. The effects of testosterone propionate on the development of the urinogenital sinus and prostatic glands in young opossums. A. Extreme hypertrophy of the sinus and prostate in a male aged 50 days, treated from birth ; compare with the condition in a normal male of the same age (C). The effect of the same dose of hormone in a littermate female is shown in B. Female opossums normally never develop prostatic rudiments, as shown in D and E, representing cross-sections through the urinogenital sinus somewhat below (D) and at the point of junction {E) of the lateral vaginal canals (c/. Fig. 2.225). Note the great difference in the volume of prostatic tissue in the treated male (A) as compared with the treated female (B), although dosage and other conditions were the same.
merely to exaggerate the normal processes of development. With large doses there is a moderate hyperplasia of the sinus epithelium in males and a tremendous hypertrophy of the prostatic glands. But in females a striking deA'elopment of prostatic glands also occurs (although normally the female possesses no prostatic rudiments) together with a change in form, resulting in a sinus that is typically male. Quantitatively, these effects are proportional to dosage, but with the same dosage an interesting sex difference is constantly observed with respect to the magnitude of the response. The prostate (Fig. 2.29.4, B) is invariably more strongly developed in male subjects than in females. This difference in size apparently depends on an inherent difference in growth capacity in homologous tissues of different sex genotype when exposed to the same intensity of stimulation (Burns, 1942b, 1956a). This effect appears regularly in the case of many other sex structures of the
opossum as will be seen. The induction of prostatic glands and a male form of sinus is of regular occurrence in female mammalian embrvos exposed to male hormones (Table 2.3).'
Two special points concerning prostatic differentiation in young opossums are of interest. Brief treatment of female embryos with androgen, just at the time when the prostatic buds are appearing in males, is sufficient to induce buds which are then capable of continued differentiation after the hormone is withdrawn (Moore, 1945 ». This is an unusually clear case of permanent conditioning of a sex structure by brief exposure to a hormone at a critical stage in development. Also of interest is the fact that by gradually reducing the dosage of male hormone a level is reached, at approximately 5 /xg. per day, which induces prostatic buds in young females which are identical in size and appearance with those of normal males of the same age (Burns, 1942a) . With
124
BIOLOGIC BASIS OF SEX
out attempting to allow for the constitutional sex factor mentioned above, this amount of androgen would appear to be roughly equivalent to the hormonal activity of the embryonic testes at this period.
Female hormone has opposite effects on the urinogenital sinus and its derivatives in young opossums. Estradiol dipropionate completely suppresses prostatic differentiation in males and transforms the sinus epithelium into a stratified squamous epithelium of vaginal type (Fig. 2.30) ; in fact the histologic picture is one of intense proliferation and cornification like that of the adult vagina at estrus. Moreover, a single dose of estrogen administered during the 15th day of pouch life, shortly before the prostatic buds would normally appear, results in complete suppression of the prostate, an effect which is also permanent (Burns, 1942a, b, c) . Thus, there is a relatively short period during which induction and continued development of ])rostatic glands in females, or their
permanent suppression in males, is wholly conditioned by the presence of the appropriate hormone. Quantitatively as well as qualitatively the reactivity of the embryonic sinus epithelium to estradiol is remarkable. Again a sex difference, as measured by growth and proliferation is seen, this time in favor of the female. Transformation to a typical vaginal epithelium in the estrous phase can be induced in very young male embryos, long before the time of appearance of prostatic buds (Fig. 2.31; Burns, 1942c). It is hardly surprising that an epithelium of this type permanently loses all capacity to produce prostatic tissue.
The effects of castration on the development of the urinogenital sinus and its derivatives in mammalian embryos follow the pattern previously described for the sex ducts. The male form of sinus is incapable of developing in the absence of the testes, whereas morphogenesis of the female form is not significantly affected by castration
B
Fig. 2.30. The effects of estradiol dipropionate on the development of the urinogenital sinus and prostate in young opossums. A. The normal sinus of a young male aged 30 days, showing the condition of the prostatic buds, for comparison with a normal female of the same age (fi). Note the bilobed form of the sinus canal in the male as compared with the typical pentangular form in the female. C. The effect of the female hormone in a male littermate of the same age, treated from the time of birth. Complete suppression of the prostatic glands has occurred, the form of the sinus canal is typically female, and the sinus epithelium has been transformed into a thick stratified squamous epithelium, like that of the adult vagina, in a state of pronounced keratinization and desquamation. The effect of an identical dose in a female subject is similar but much more intense.
HORMONES IN DIFFERENTIATION OF SEX
125
Fig. 2.31. The effects of a stronger dosage of estradiol dipropionate on the urinogenital sinus at a much earher stage. A. Young male treated from birth to an age of about 12 days, for comparison with the normal male sinus (inset, B) of the same age. Compare the character of the sinus epithelium with that in the older specimen shown in Figure 2.30C This condition of the sinus epithelium is induced at an age which precedes by se^■eral days the normal appearance of the prostatic buds, which never develop.
(Table 2.2; Jost, 1947b; Raynaud and Frilley, 1947; Wells, 1950). In castrate males prostatic differentiation is prevented and development of a vagina (correlated with persistence of the Miillerian ducts in male castrates) results in a sinus of female type (Fig. 2.27 A). Male and female castrates are morphologically very similar, and both closely resemble the normal female. Again it is clear that the embryonic testis is the essential factor in male development, whereas the female pattern is independent of hormonal conditioning and also of sex constitution, since in the absence of the gonads it develops spontaneously in castrates of either sex.
The factor of time is again of paramount importance and sharply limits the effectiveness of castration. This holds for the development of other accessory structures (Table 2.2) but is particularly clear in the case of the prostate which will serve to illustrate. In male rabbit embryos castrated on or after the £3rd day of gestation there is only a
slight effect on prostatic development, which continues in a practically normal manner; but if the operation is performed a day earlier there is a distinct reduction in size. In fetuses castrated from the 20th to the 21st day only small ventral buds are found which are already formed at the time of operation, and after castration earlier than 20 days prostatic buds are absent altogether (Fig. 2.27; Jost, 1947b, c). The period from 20 to 21 days, then, is critical for the appearance of the prostatic buds and their further differentiation for which the embryonic testis is essential. However, absence of the testis is fully compensated for by male hormone ; in castrates receiving androgen the development of all male parts proceeds normally. A similar result has been obtained in the fetal rat (Wells, Cavanaugh and Maxwell, 1954). Late castration has little effect, but castration on day 18 results only in buds which do not undergo branching. Earlier castration has not proved feasible in this species.
126
BIOLOGIC BASIS OF SEX
Fig. 2.32. The effects of sex hormones on the sex type of tlic copulalory stniciur(-s in
young opossums. A. The appearance of the phallus, or genital luljerclc, ui ;i normal male
(left) and female opossum aged 20 days. Sex is difficult to distinguish by form alone but the
phallus is somewhat larger in the male. Sex is readily distinguished at this age, however, by
HORMONES IN DIFFERENTIATION OF SEX
127
C. EXTERNAL GENITAL STRUCTURES
Copulatory organs homologous with those of higher vertebrates are not found in amphibians. They are developed to an extent, however, in certain birds and reptiles and become highly specialized in mammals. The copulatory organ in amniote embryos develops from a simple primordium, the genital tubercle, which is common to both sexes. It becomes specially developed as the penis in the male but in females it persists in a more or less rudimentary form, known in mammals as the clitoris. The genital tubercle of birds arises as a small, conical protuberance just within the cloacal orifice. In chickens it is not highly developed, although larger in the male than in the female, but in the males of ducks, geese, and certain other birds it becomes considerably larger and more modified, constituting a penis (Fig. 2.25). In the embryos of mammals (except the Monotremes) the genital tubercle is external in position, arising as an eminence near the ventral rim of the urinogenital meatus.
The developing copulatory organs of birds and mammals react readily to sex hormones and are extremely sensitive to castration. In birds the clearest experimental results have been obtained in duck embryos, because of the more pronounced sexual dimorphism in this species. Treatment with female hormone (estradiol benzoatej before the 12th day of incubation completely arrests development of the penis in males, and the rudimentary clitoris of the female may be even smaller than normal (Wolff, Em., 1950 ) ; beyond this age, however, the hormone is no longer effective, the form of the prospective penis having been finally determined. The effects of the male hormone are less precise and it is not essential for normal development (see the effects of castration below) . Testosterone proprionate produces great hypertrophy but
the structure is not entirely normal, the characteristic spiral form of the penis being imperfectly developed (Wolff, Em., 1950). This abnormality is perhaps a result of overdosage as the dosages used were undoubtedly very large. Although in the chick the dimorphism of the genital tubercle is less pronounced than in the duck, it reacts in the same way ; male hormones stimulate and female hormones inhibit growth and morphologic differentiation (Reinbold, 1951).
In mammalian embryos the sex type of the developing genital tubercle, or phallus, is easily controlled by sex hormones (Table 2.3) ; in fact, this structure is unusually susceptible to modification and may be completely transformed. Typical are the results in the rat (Greene, 1942), the mouse (Raynaud, 1942; Kerkhof, 1952), the hamster (Bruner and Witschi, 1946) and in pouch young of the opossum (Burns, 1939a, b; Moore, 1941). The rat and the mouse are similar in their behavior. Male hormone does not affect the development of the penis in males except to produce hypertrophy, but in females the genital tubercle is greatly enlarged and assumes the character of a penis.^' Female hormone has opposite effects; females differentiate normally but in males the tubercle fails to enlarge and a hypospadic condition frequently appears.
In young opossums the form of the copulatory structures is completely controlled in accordance with the type of hormone given, with results which are identical in the two sexes except for a difference in size (Fig. 2.32). The basis for the transformation of the phallus has been analyzed histologically (Burns, 1945b). The various histologic con ^' Strangely enough this is not tyi^ically the case in freemartins. The chtoris as a rule is not greatly modified (Lillie, 1917). There are, however, some striking exceptions (Buyse, 1936; Numan, 1843, illustrated in Lillie, 1917, Fig. 29).
the scrotal sac in males and the presence of the pouch folds and mammary rudiments in females. B. The typical form produced by male hormone (left) and female hormone in specimens treated from birth to an age of 20 days; the normal condition at this age is shown above. This striking difference in form is produced without regard to the sex of the subjects, which in this case are both male (note the scrotal sacs). C. The result of administering male hormone (testosterone propionate) from birth to an age of 50 days, in a female subject (left; note the pouch) and a male littermate. Observe the identity in form but distinctly greater size of the penis in the male. D. Comparison of the effects of estradiol dipropionate, given from birth to an age of 30 days, in a female subject (left) and a male littermate. In both C and D it is shown that the hormones produce genitalia of typical male or female form, regardless of the sex of the subject.
128
BIOLOGIC BASIS OF SEX
stituents of the organ respond to the appropriate hormone in a highly specific manner. The erectile bodies, the development of which largely determines the form and the size of the penis, are strongly stimulated by male hormone and almost entirely inhibited by female hormone (Fig. 2.33j. Qualitatively, these responses are independent of sex constitution, but with identical dosages marked differences in size, such as were
noted previously in the case of the prostate, the sinus epithelium and derivatives of the sex ducts (Figs. 2.29 and 2.24) , are again observed in the two sexes. Female hormone, in addition to inhibiting the erectile tissue, induces an extreme hyperplasia of the vulvar and periurethral connective tissues (Fig. 2.335). It is this response which produces the gross swelling of the vulvar region, so conspicuous in estrogen treated embryos of
.,*m^
Fig. 2.33. The effects of male and female hormone^ on the differentiation of the histologic
constituents of the phallus in young opossums. A. The effects of androgen in a male, aged 30
days, treated from birth onward. There is great hypertrophy of the erectile bodies but
otherwise structiue is normal; at the top, the paired corpora cavernosa are imited at the
mid-line ; below, the urethral canal, with the bulbo-urethral glands on either side ; laterally,
the large bulbs of the corpora spongiosa, with their muscular investments. B. The effects of
estrogen in another male littermate. The erectile bodies are almost completely suppressed
and there is an enoimous hyperplasia of the periurethral connective tissue. The urethral
canal (urinogenital sinus) is greatly enlarged, as in a female, and the sinus epithelium is
transformed into stratified scjuamous epithelium like that of the fully developed vaginal
canals. The sex of the subject makes no difference in the character of these responses.
HORMONES IN DIFFERENTIATION OF SEX
129
both sexes (Fig. 2.32). With increasing dosages all these effects are accentuated.
The phallic structures of mammalian embryos react to castration according to the pattern already established for the sex ducts and the prostate (Jost, 1947b; Raynaud and Frilley, 1947). In both sexes castration is followed by development of external genitalia of female type (Fig. 2.28; Table 2.2) ; the male type of differentiation is dependent on the testis whereas the female form is capable of developing without hormonal conditioning, in a somatic or asexual manner.
At this point it will be useful to recapitulate for mammalian embryos the effects of castration, or of early isolation, on the development of the genital system as a wdiole. It has been shown that in the absence of the gonads, or of any hormonal conditioning, the embryonic sex primordia collectively follow the female pattern of development. In all castrates, regardless of sex, the external genitalia and the derivatives of the urinogenital sinus are of female type, the INIiillerian ducts persist and continue to develop in a virtually normal fashion, whereas the Wolffian ducts undergo involution. Thus castrates of either sex toward term have female genital systems which are anatomically complete and almost as well developed as in normal females.
It is noteworthy that the pattern of development observed in castrate fetuses corresponds closely with a condition in human subjects known clinically as gonadal dysgenesis. Individuals presenting this anomaly either lack gonads entirely or show evidences of gonadal atresia at an early stage of development. Regardless of chromosomal sex as established by the Barr test (Barr, 1957) they possess external genitalia of female type and female genital tracts which, however, are of infantile proportions. Recent evidence indicates that some individuals of this type may lack the Y-chromosome, being of XO constitution (Ford, Jones, Polani, de Almeida and Briggs, 1959; chapter by Gowen) .
In bird embryos the effects of castration on the genital tubercle are similar except that the sex relation observed in mammals is reversed ; in this group the male form of the
organ corresponds to the asexual condition, which develops without hormonal conditioning in castrates of both sexes (Fig. 2.25; Wolff and Wolff, 1951). This is not an exceptional finding; it corresponds with the behavior of various other avian sex characters, such as the syrinx {q.v.) , the spurs, and the sex plumage in species such as domestic fowl. The transposed relationship seen here is in line with the dominant role played by the grafted ovary and the greater potency shown by the female hormone in producing sex reversal in the gonads of the chick.
The developmental behavior of the genital tubercle after isolation in vitro has been studied in the duck, with results which correspond with those of castration. Isolated at 7 to 9 clays of incubation, before the beginning of sex differentiation in the gonads, primordia of the genital tubercle always assume the male form as in castrates, regardless of the sex of the donor. By the 10th day, however, the sex type has become fixed, and when isolated after this stage differentiation always follows the sex genotype (Wolff and Wolff, 1952b).
D. DIFFERENTIATION OF OTHER TYPES OF SEX CHARACTER
Two further examples will be considered as illustrations of the role of hormones in the development of sex characters of quite different type, the mammary glands, and the syrinx of birds. The mammary glands of field mice have been extensively studied by Raynaud (for a summary see Raynaud, 1950). The rudiments of the glands first appear as bud-like ingrowths of the epidermal epithelium which penetrate the underlying mesoderm but retain a connection with the epidermis by a constricted neck (Fig. 2.34.4, B). This phase of development follows the same course in both sexes. Toward the 16th day of gestation differences appear in males which coincide with the beginning of masculinization of the female genital tract; the mammary buds lose their connection with the epidermis and remain as isolated epithelial nodules in the mesenchyme (Fig. 2.34C) . In females, on the contrary, the buds retain their attachment to the epidermis, and as development continues a circular fold appears surrounding the mammary rudiment, which leads to elevation of the nipple.
130
BIOLOGIC BASIS OF SEX
SS^M
•K^« ;'^ vVi^^#-p^ r-^i -^^-"i^^^^f^^ 7,
^^^^s^^^^i^^ss ^^^^^^^^^^^^
Fig. 2.34. The normal development of the mammary rudiments in embryos of the field mouse, and the effects of sex hormones (for a summary see Raynaud, 1950). A. Early appearance of the mammary thickenings in the female (left) and the male (right). B. Later stage, showing growth of the mammary primordia and penetration into the mesenchymal layer. C. Stage of sexual differentiation: in females the mammary rudiment remains attached to the epidermis and the nipple later develops at this point; in males the rudiment becomes detached from the epidermis and persists as a small epithelial nodule in the underlying mesenchyme. For the effects of hormones and of castration on this pattern see text. D. Nipple development in a male embryo induced by treatment of the mother with estradiol dipropionate. For details see text. (After A. Ravnaud, Arch. Anat. micro.scop. et Morphol. cxper., 39, 518-569, 1950.)
HORMONES IN DIFFERENTIATION OF SEX
131
Treatment with sex hormones during the latter half of gestation shows that the processes described above are readily controlled or reversed experimentally. In female fetuses of mothers injected with male hormone, development of the mammary gland follows the male pattern; the mammary buds separate from the epidermis and persist only as nodular rudiments, the nipple fold does not appear and nipples fail to develop. The female pattern of differentiation is converted completely to the male type. The use of female hormones, on the other hand, leads to a somew^hat paradoxical result ; there is an inhibition of the mammary buds rather similar to that exerted by androgen, but nipple development on the contrary is strongly stimulated (Fig. 2.34D). The dosages were large, however, and the effects of female hormones under more physiologic conditions have not as yet been determined. With respect to development of the nipple similar results have been reported in the laboratory mouse (Greene, 1942); male hormone completely inhibits the nijv ples in females whereas female hormone induces typical development in males.
Of particular interest are the effects of castration on mammary development in mice. When the embryonic gonads are destroyed by irradiation the mammary glands continue to develop in both sexes according to the normal female pattern (Raynaud, 1950). This pattern obviously does not depend on the ovary but represents the asexual or anhormonal type of development. Its appearance in castrate males indicates that the regression of the mammary glands in the male is normally determined by the testis. This is in agreement with the results of administering male hormone, as described above. Once more the predominant role of the male hormone in mammalian sex differentiation is demonstrated.
An entirely different type of sex character, and one which exhibits sexual dimorphism in a striking way, is the syrinx of birds. This organ has received special study in the duck (Wolff, Em., 1950) . The syrinx makes it appearance as a vesicular dilatation at the junction of the trachea and bronchial tubes, and at first it is small and symmetrical in form in both sexes. This is the permanent
condition of the syrinx in the female but in males a pronounced asymmetry soon appears, involving an enlargement of the left side of the vesicle with corresponding modifications of the cartilaginous rings. By the 10th day of incubation the asymmetry is extreme (Fig. 2.25). The appearance of dimorphism follows closely the beginning of sex differentiation in the gonads.
Experimental studies have shown that the dimorphism of the avian syrinx is conditioned by the ovary, or by the female hormone (Wolff, Em., 1950). Estradiol benzoate, introduced into incubating eggs, inhibits the development of the male syrinx and the female form appears. However, if large doses are used, or if treatment is too long delayed, a paradoxical result appears, consisting in the development of atypical and intermediate forms (Lewis and Domm, 1948). ]Male hormone (testosterone propionate) in moderate dosages has little effect on the syrinx (a slight enlargement may occur) but with large doses a paradoxical tendency is again found; the male syrinx is inhibited and may actually be reduced in size, resembling somewhat the female form.
Castration again reveals the dominant role of the female hormone in birds. In castrates of both sexes the form of the syrinx is male, both in size and in its asymmetry (Fig. 2.25) ; absence of the ovary is critical but the presence or absence of the testis is of no consequence in the sexual differentiation of this structure. In its response to castration the syrinx thus behaves like the genital tubercle.
The differentiation of the syrinx has also been studied in vitro (Wolff and Wolff, 1952a; Wolff, 1953a) with similar results. When explanted before the onset of sex differentiation, the result is the same as after castration; in an anhormonal environment the male form develops without regard to the sex of the donor. When explanted after the beginning of sex differentiation, however, development proceeds always in accordance wuth genotype. At this stage the form of the female syrinx has already been irreversibly determined. The syrinx developing in vitro responds directly to sex hormones introduced experimentally. Addition of estradiol benzoate to the culture medium
132
BIOLOGIC BASIS OF SEX
has the expected result; regardless of sex constitution, only the female type of syrinx develops. But male hormone (testosterone proprionate) under the same conditions produces, paradoxically, organs of female or of intermediate form. The dosages employed, however, were extremely large (5 mg. and 40 mg. per cc. solvent) ^^ and in the light of the effects of large doses of androgen on the gonads and other structures the anomalous result is not surprising.
VI. The Pituitary and the Diiferentiation
of Sex
It does not appear that the anterior lobe of the pituitary is concerned in the primary differentiation of sex, i.e., in the morphogenesis of the gonads themselves. Early hypophysectomy does not interfere with their histologic differentiation, up to the stage at least where they are fully characterized as ovaries or testes; neither is the process of differentiation appreciably delayed. Later, however, deficiencies of a secondary order may appear in the genital tracts of hypophysectomized animals; the development of various accessory sex structures may be considerably retarded but without any essential change in character. This effect is explainable as the result of diminished secretory activity on the part of the gonads through lack of adequate gonadotrophic stimulation. The question of when the functional interrelation between the gonads and the anterior pituitary is established has been reviewed by Willier (1952, 1955) with special reference to the chick, and .lost (1953, 1955) has dealt with this problem in mammalian embryos and fetuses. In each case gonadotrophic activity is evident shortly after the period of gonad differentiation and covers the period when the sex ducts and other accessory structures are differentiating. Some of the evidence on which these statements are based will be briefly reviewed.
In amj)hibian embryos or early larvae,
^* Under the conditions of culture the exphints develop in close contact with droplets of the hormone solution, and may actually be exposed to extremely high concentrations. Culture with the oil solvent alone, however, shows that the solvent is not the disturbing factor.
hypophysectomized long before the time of sex differentiation, the development of ovaries and testes proceeds normally and without appreciable delay until toward the end of the larval period (Smith, 1932, p. 752; Chang and Witschi, 1955; Chang, 1955). It is also well known that during larval life the gonads are capable of responding readily to gonadotrophic substances by rapid growth and precocious maturation of the germinal elements (e.g., Burns and Buyse, 1931; Burns, 1934). During this period pituitary stimulation merely accelerates the normal processes of development and maturation. It has been shown previously that in many amphibian species administration of steroid sex hormones during the larval period induces transformation of the gonads; however, an interesting case is known in which sex hormones appear to be without effect unless a gonadotrophin is also administered (Puckett, 1939, 1940). The tadpoles of a so-called "undifferentiated race" of Rana catesbiana all have gonads w^iich structurally resemble young ovaries until late in larval life, when differentiation of the males occurs rather abruptly. The administration of gonadotrophin alone to the undifferentiated tadpoles initiates sex differentiation precociously, the two sexes appearing in the usual 50:50 ratio. The administration of sex hormones of either type to tadpoles during the indifferent period is without effect; the gonads are apparently incapable of responding at this stage of development. However, when the sex hormone and gonadotrophin are administered concurrently a striking response occurs; not only is sex differentiation precipitated, as when gonadotrophin was administered alone, but a complete transformation of sex takes place, resulting in all males or all females, according to the type of sex hormone employed. The gonadotrophin is evidently necessary to initiate sexual diff(>rentiation but the type of differentiation which follows is determined by the type of sex hormone. In chick embryos "hypophysectomy" before the onset of sex differentiation has been accomplished in two ways, by partial decapitation, in which the forebrain area is remov(>d surgically after 33 to 38 hours of incubation (Futio, 1940), and bv irradiation
HORMONES IX DIFFERENTIATION OF SEX
133
of the hypophyseal region (Wolff and Stoll, 1937) . After excision of the forebrain, histologic differentiation of the gonads proceeds normally. Later, however, the interstitial tissue of the testis fails to develop or is deficient in quantity, and the cortex of the left ovary remains rather thin through failure of secondary sex cords to develop in the usual numbers. The development of the gonaducts, on the other hand, is normal in both sexes. Wolff and Stoll reported that, after destruction of the hypophysis by irradiation, differentiation of the gonads continued in a normal manner to the end of incubation and again the gonaducts were found to develop normally. Such embryos, moreover, undergo sex reversal in the usual manner when treated with sex hormones (Wolff, 1937). The available experimental evidence indicates, then, that the hypophysis has no appreciable part in the primary differentiation of sex, a conclusion which is supported by van Deth, van Limborgh and van Faassen (1956).
In mammalian embryos and fetuses, hypophysectomy has been carried out by procedures similar to those described for the chick, namely, partial or total decapitation and irradiation with x-rays. The former method was used on embryos of the rat (Wells, 1947, 1950) and the rabbit (Jost, 1947d, 1950, 1951a), and the latter on the embryos of the mouse (Raynaud and Frilley, 1947; for a summary see Raynaud 1950) . In the case of the rabbit and the rat, the operation was not performed early enough to affect the primary differentiation of the gonads; in the mouse, however, irradiation on the 12th day of gestation, just at the beginning of differentiation, was without effect except for a certain reduction in the number of germ cells when the hypophysis was entirely destroyed. The results differed sharply, however, with reference to the condition of the genital tracts in hypophysectomized male rabbit fetuses, as opposed to those of the rat and the mouse. In the two latter species no significant changes in the development of the accessory genital structures were observed. It may be that in these species the entire process of sexual differentiation is independent of pituitary function, although the possibility is
perhaps not excluded that an extraneous gonadotrophin, of maternal or placental origin, may be substituted. In the rabbit, on the other hand, definite defects were found in the development of certain accessory genital structures, resembling those which follow embryonic castration but somewhat less severe. However, if gonadotrophin is administered following decapitation these deficiencies do not appear; they may therefore be ascribed to lack of gonadotrophic activity (Jost, 1951a, 1953). The defects observed vary in severit}^ according to position ; the anterior regions of the gonaducts and the epididymides, which are near the testes, develop normally, whereas distant structures such as the prostatic glands and external genitalia may be almost as severely affected as in castrate fetuses. This observation indicates that the testis is acting in an intermediary capacity. In the absence of the pituitary its humoral activity is diminished to a level adequate for normal differentiation of nearby structures but insufficient to maintain the development of more distant parts. The point has been previously established that after decapitation there is a decrease in the amount of interstitial tissue.
The problem of the time of onset of gonadotrophic function in its relation to gonad secretion and the dift'erentiation of the genital tract has been studied in the rabbit by Jost (1951a). By examining the genital tracts of decapitated male fetuses at short intervals to determine when the first signs of abnormal development appear, and by varying also the age at which decapitation was performed, he was able to define rather closely the beginning of gonadotrophic function and the period during which it is critical for normal development of the genital structures. Following decapitation on the 19th day of gestation no marked defects in the genital structures appear until about the 22nd day, after which abnormalities become more and more pronounced until the 24th day. If decapitation is delayed until the 24th day no important anomalies are subsequently found. It should be recalled that the latter date coincides with the stage after which castration likewise has no effect on development. The interval from the 22nd to the 24th clay of development thus falls within the period during which testis activity is most essential for normal morphogenesis (p. 125).
In the light of these results the anterior hypophysis was studied cytologically for direct evidences of secretory activity, using the MacManus periodic acid-Schiff (PAS) test (Jost and Gonse, 1953; Jost and Tavernier, 1956). PAS-positive cells are first seen in small numbers, and faintly stained, on the 19th day of development. Thereafter they increase in numbers and in staining reaction, reaching a maximal development during the 22nd and 23rd days; on the 24th day these cells abruptly decrease in number and stainability and almost disappear. The peak of gonadotrophic activity, as indicated by the cytologic evidence, falls again during the 2-day period when the secretory activity of the testis is at its height, as judged by the conseciuences of castration, a remarkable example of endocrine correlation (for a fuller account see Jost, 1953, 1955).
It was once widely believed that in human anencephalic monsters the pituitary is absent or vestigial in character ; more recent studies have revealed, however, that although difficult to identify grossly, anterior lobe tissue can usually be demonstrated by careful histologic examination {e.g., Angevine, 1938). Nevertheless, cases are known in which apparently no anterior lobe tissue is present, and such cases are pertinent to the present discussion. Barr and Grumbacli (1958, and personal communication of Dr. Grumbach) have described such a case in a newborn male infant, in which no malformation of the genital system was evident except that the testes were somewhat smaller than usual. They were not otherwise abnormal, however, and interstitial tissue was present. In a similar case, also a male, reported by Blizzard and Alberts (1956), the external genitalia were small but normal in structure. The testes also were small and undescended, lying in a pelvic position, the tubules were somewhat atrophic and no interstitial tissue was present. No other abnormalities were noted. It is perhaps significant in this case that absence of the interstitial cells is correlated with underdevelopment of the external genitalia and
failure of the testes to descend. In two cases of congenital absence of the hypophysis, a male and a female (Brewer, 1957), development of the gonads and genital system was apparently normal.
Cases in which complete absence of the pituitary has been demonstrated are unfortunately few but of great value since they represent in humans the closest approach to hypophysectomy in experimental animals. The consequences of the deficiency and the conclusions to be drawn in the two cases are similar; the primary differentiation of the gonads is evidently independent of the pituitary but secondary defects may appear later, both in the gonads and in the genital tract. The testes may be underdeveloped, the tubules may show secondary atrophy or degenerative changes, and the interstitial tissue may be reduced or lacking, but in some cases it appears to be well develoj^ed. There is need for a careful correlation of the status of the interstitial tissue in such cases with the presence or absence of defects of the accessory organs. The consequences to the gonad of absence of the pituitary may hinge on whether a secondary source of gonadotrophin is available to the fetus (Jost, 1953). This is a matter which may be expected to vary in different groups or species. As yet too few species have been studied to clarify the point.
VII. Group Differences in the Relations of Hormones to Sex Differentiation
The extensive experimental data reviewed in the foregoing pages show clearly that the embryonic gonads produce sex specific substances which must be regarded as hormones and which act as physiologic agents in the differentiation of sex, controlling not only the development of the various accessory sex structures but in many cases the differentiation of the gonads themselves. That the substances are hormones in the usual sense is shown by the fact that they regulate the development of distant structures in such fashion that they can only be distributed by way of the circulating blood. This, however, does not preclude a sharply localized action under jiroper circumstances. That they are ('hib<)iat('(l in the gonads is demonstrated bv
HORMONES IX DIFFERENTIATION OF SEX
135
many types of grafting experiments, and above all by the results of embryonic castration. At the same time, steroid hormones of the adult type are also capable in many cases of reproducing closely the effects of the embryonic hormones, thus suggesting a basic similarity. This is not to say, however, that in all groups and species the role of hormones in the differentiation of sex is the same, either with respect to their effects on individual structures or their part in the differentiation process as a whole. Along with ontogenetic processes in general, hormonal relationships have evolved differently in the different vertebrate groups.
Amphibians. In amphibians both sex hormones seem to have active and essentially coordinate roles in sexual differentiation. With respect to gonad differentiation, grafted gonads of opposite sex induce transformation of both testes and ovaries by acting selectively on the appropriate gonad components. In many cases the interacting gonads are reciprocally modified, both becoming strongly intersexual ; when this reciprocal effect does not occur it is apparently due to the decisive predominance of one member. Moreover, the gonad components in many cases react in a similar or identical manner to both natural and synthetic hormones. Male hormones induce precocious differentiation of the male duct system and the cloacal glands in individuals of either sex and when administered early may also completely suppress the development of the jMiillerian ducts; conversely, female hormones stimulate differentiation of the ]\Iiillerian ducts but are without effect on such male structures as Wolffian ducts and cloacal glands. But if such evidence demonstrates beyond question that the larval sex structures are capable of reacting specifically to hormones of the proper type, the role of hormones in the normal differentiation of sex is more directly demonstrated by the effects of larval castration. In castrates of either sex the gonaducts and other sex accessories remain indefinitely in an undifferentiated or slightly differentiated condition. The sexually neutral type in amphibians thus tends to be morphologically intermediate between the sexes; however, either sex type may be readily obtained
from the castrate type by transplanting an ovary or a testis (p. 112). The positive role of both hormones in sex differentiation is apparent.
Birds. In bird embryos also steroid sex hormones stimulate precocious growth and differentiation of the appropriate sex primordia, and in general have inhibitory effects on structures of the other sex. There is a high degree of specificity in the interaction between hormone and end organ, and from the results of hormone administration alone it might be inferred that the two hormones have coordinate roles in sex differentiation. However, the results of castration show clearly that the roles of the two hormones are different and unequal. The Miillerian ducts persist and continue to develop in a similar manner in castrates of both sexes (p. 115). The male hormone is evidently the decisive factor in their differentiation since the presence of the testes causes involution of the ducts in males whereas the ovaries are not essential for their development in females. On the other hand the sextype of the genital tubercle and the syrinx is conditioned by the ovaries. Both structures are normally developed in castrate males, for which male hormone is evidently not essential, whereas in castrate females also they closely approach the male condition in both form and size. It is the inhibitory action of the ovary, therefore, that determines the dimorphism of the syrinx and the genital tubercle. ^^ These conclusions are confirmed by the fact that when isolated and cultured in vitro the Miillerian ducts, and the genital tubercle and syrinx, behave exactly as in castrate embryos; however, addition of male hormone to the culture medium causes involution of the Miillerian ducts, and female hormone prevents male differentiation of the tubercle and syrinx.
Significant differences thus appear in the reactions of the accessory sex structures in birds as compared with amphibians. In the
'^This i.s true also of such striking sex characters as phunage type and spurs in adult fowl, whereas the head furnishings are under the control of the male hormone. There is, however, much ■\-ariation in the relationships of secondary sex characters and gonad hormones in birds (Domm, 1939).
136
BIOLOGIC BASIS OF SEX
latter positive stimulation by the proper hormone is necessary to induce final sexual differentiation. In birds, the role of the sex hormones appears to be primarily inhibitory; in the absence of gonads Miillerian ducts develop normally in castrates of either sex and genital tubercle and syrinx spontaneously assume the male form. No positive stimulus is necessary, only release from the inhibitory influence of the opposing gonad. Thus, the castrate type in birds is not undifferentiated or intermediate in type, but is rather a mosaic in which certain characters are typically male, others typically female. Again, however, both hormones are essential for the realization of normal sex differentiation but with the female hormone having the major role with respect to the number of structures controlled.
Mammals. In mammals still another pattern appears in the relation of the accessory sex structures to the gonads and their hormones. Administration of pure steroid hormones demonstrates again that most of the genital primordia, regardless of sex constitution, are capable of reacting to the appropriate hormone, and if excessive dosages are avoided the responses are in most cases specific. To summarize, administration of male hormone does not significantly affect the development of male embryos except to accelerate the rate of differentiation; in females, on the other hand, it induces differentiation of male structures whereas female primordia are inhibited or fail to respond. In like manner, female hormones have a feminizing action on male embryos.
Again, it might be assumed from the results of hormone administration that the two hormones have comparable roles in normal differentiation. In reality, what is demonstrated is the capacities of the sex primordia to respond to hormones experimentally introduced; the true role of hormones in normal differentiation is disclosed only when the embryonic genital tract is required to develop in the absence of the gonads or other hormonal influences. Castration of mammalian embryos reveals that normal differentiation depends chiefly if not exclusively on the male hormone. Castrated embryos, regardless of genie sex, develo):) female cliaracters (Miillerian derivatives,
female sinus form and external genitalia, mammary glands) which are almost as well differentiated in castrates as in normal females (Figs. 2.26-2.28, and Table 2.2). Any possible influence of a maternal hormone in this result appears to be excluded (at least for the sex ducts and their derivatives) by studies of development in vitro. Culture of the isolated gonaducts results in persistence and development of the ]\Iiillerian ducts and involution of the Wolffian ducts, regardless of the sex of the donor embryo (pp. 117, 121) providing always that isolation is carried out before irreversible determination has occurred.
In mammalian embryos, then, the testes and the male hormone are all imjiortant for the normal differentiation of sex. Moreover, the role of the male hormone is a dual one; its presence is essential to insure retention and development of male parts and at the same time to prevent the differentiation of female structures, which are capable of developing autonomously, regardless of the presence or absence of female hormone. The latter apparently has no essential role in primary sex differentiation. At this point it should be recalled that such a conclusion had in fact been forecast much earlier on the basis of castration of the newborn rat (Wiesner, 1934, 1935; see Burns, 1938b). At birth morphogenesis of the genital structures of young rats is far advanced and profound modifications after castration are not to be expected ; however, it was found that in castrate males a marked atrophy promptly appeared in such sex accessories as the seminal vesicles and external genitalia, suggesting that a hormonal influence had been removed, whereas in castrate females development proceeded more or less normally until the approach of puberty. These results were confirmed and extended by LaVelle (1951) in the ncnvboi'n hamster. Wiesner (1934, 1935) proposed that sex differentiation might be explained on the basis of one hormone, the male, the presence or absence of which would account for the two types of development, an hypothesis now confirmed ill a striking way by the results of castration during embryonic and fetal development.
The pre-ciiiiiiciit role of the male hormone
HORMONES IN DIFFERENTIATION OF SEX
137
in mammalian sex differentiation stands in contrast with the situation in birds, in which the female hormone has the major role. The parallel with the well known difference in the sex-chromosome complex has been noted but this provides no immediate explanation and may be merely coincidence. On the other hand, another explanation may be found in the special physiologic conditions incidental to the evolution of intra-uterine development in mammals. A situation in which the female hormone has an active role in sex differentiation might present a serious difficulty with male embryos constantly exposed during development to the influence of the mother's hormones (the presence of considerable amounts of maternal estrogen during pregnancy is an established fact in many species (Price, 1947; Parkes, 1954). Elimination of the role of the female hormone coincident with the evolution of viviparity would then be advantageous. The problem of female development has apparently been met by a change in the status of the female sex primordia which, as amply shown by the results of castration and cultivation in vitro, do not require positive stimulation but develop autonomously unless inhibited l)y the male hormone.
VIII. The Organization of the Sex
Priniordiuni and Its Role in the
Differentiation of Sex
The role of hormones as specific conditioners of sexual differentiation is, however, only one aspect of the problem. Of far greater complexity, and fundamental to the selective character of the differentiation process, are the special attributes of the individual sex primordia which predetermine their reactions to the presence, or absence, of a particular hormone. Each primordium possesses a complex organization, not only as to sex type and morphologic character, but also with respect to such detailed physiologic properties as the timing of receptivity, the thresholds at which responses occur, and definite capacities for growth. It is obvious that specificity of hormone action does not exist independently of specificity of response. This organization of the primordium derives ultimately from the genotype of the
species, operating through the same processes of ontogeny that prescribe the special characteristics of other embryonic parts and systems; it is intrinsic as opposed to the conditioning activities of hormones and other modifying agencies which, with respect to the primordium, are external and secondary. Thus, sex primordia may be expected to show variations in behavior toward hormones which will be peculiar to and integrated with the patterns of development characteristic of particular groups or species.
A. CONSTITUTION AND THE MORPHOLOGIC REPRESENTATION OF SEX PRIMORDIA
It has been pointed out that even in the bisexual or undifferentiated period of development many variations are found among different species in the extent to which the structures of the recessive sex are represented morphologically, i.e., are laid down in the form of discrete primordia. The absence or the deficient representation in one sex of certain heterotypic primordia may be normal for particular species, whose pattern of development thus places special limitations on sex reversal. In some amphibians reversal is difficult or impossible to induce experimentally because of the weak or transient representation of the recessive sex component in the gonad; there is no real transformation, merely a severely inhibited, or vestigial gonad. Certain accessory structures of the recessive sex may also tend to be abortive or imperfectly developed. This is the case for the Miillerian ducts in the males of various species. In young male opossums, for example, the iNIiillerian duct rarely completes its development to the point of union with the urinogenital sinus, or the connection if formed is quickly lost, so that the terminal segment of the duct is lacking. Consequently, it has never been possible to induce vaginal development in male opossums by treatment with female hormones, although the uterus and Fallopian tube are present and highly developed. Moreover, the tendency which leads to absence of this region of the duct in male embryos appears to be present also, but more weakly expressed, in the female. Although the terminal segment
138
BIOLOGIC BASIS OF SEX
of the duct is always present, it is susceptible to inhibition by male hormone while the tubo-uterine portion is never so affected (Burns, 1942b). Thus, specific morphologic defects which appear in experimental results can often be directly related to developmental peculiarities of the species in question and in final analysis are an expression of constitutional factors.
However, constitutional differences which control the morphologic representation of sex primordia do not as a rule involve simply the presence or the absence of a part, but more commonly have a quantitative expression, affecting the extent to which the structures are developed or the length of the period during which they are present and capable of responding. An example is found in the gonads of birds, in which there are marked lateral differences between right and left sides (p. 95). In consequence, the effects of hormones on the right and the left gonads may be different, not qualitatively but in degree. The left ovary, with its strongly developed cortical component (Fig. 2.12), is only moderately affected by doses of male hormone which almost completely transform the rudimentary right gonad (Willier, 1939). The morphologic differences between right and left testes are less marked, but consistently the germinal epithelium is better developed and tends to survive longer on the left side than on the right. Consequently, female hormone readily transforms a left testis into an ovotestis, and with stronger dosages into an almost normal ovary, but the right testis is but slightly affected except when the dosage is very large. It appears also from studies of the effects of graduated dosages that threshold differences for the two sides may be involved, thus a physiologic as well as a morphologic factor is introduced. It is not held that experimental failures or anomalies are always directly traceable to specific morphologic deficiencies; however, the frequency with which such correlations appear indicates the importance of underlying structural variations in modifying the responses of sex primordia under experimental conditions.
B. CONSTITUTIONAL FACTORS AND PHYSIOLOGIC
DIFFERENCES IN THE ORGANIZATION
OF SEX PRIMORDIA
In the foregoing cases obvious morphologic differences provide, at least in part, a basis for observed differences in the experimental behavior of sex primordia. It is unlikely that morphologic differences of this order exist without an underlying physiologic differentiation. On the other hand, under experimental conditions, physiologic differences often become apparent which have no visible morphologic expression, as in the inhibition of the vaginal canals of female opossums cited above. Certain accessory sex structures in birds exhibit lateral differences in sensitivity to hormones which are evidently a reflection of the general tendency to asymmetrical development in this group. In normal females only the left Miillerian duct develops into a functional oviduct ; the right, although originally well developed, regresses at an early stage. After castration, however, both ducts develop equally (Fig. 2.25) as is also the case when the ducts are isolated in vitro. Involution of the right oviduct, then, is conditioned in some way by the ovaries and it has been shown that either the right or left ovary alone is effective (Wolff and Wolff, 1951). Evidently the ovaries exert an inhibitory action on the right oviduct which is not effective on the left. Presumably a threshold difference is involved.^*'
Other examples may be cited. The syrinx and the genital tubercle remain small, symmetrical, and essentially undifferentiated in females, but in males they become large and highly asymmetrical (Fig. 2.25). Unlike the paired structures previously dealt with, these organs are single and median in position and the asymmetry of the male form is due to unequal development of the lateral halves of the organs. It is well established in the case of eacii that the female hormone
""It seems unlikely tliat the inhibitoiy factor in this case is the female hormone since the introduction of estrogenic hormones into incubating eggs causes persistence of the right oviduct. However, the concentration or dosage may be a factor in this curious effect.
HORMONES IN DIFFERENTIATION OF SEX
139
inhibits the male type of development (pp. 127, 131) which, on the other hand, develops spontaneously in both sexes after castration (Fig. 2.25) or after isolation in vitro, without hormonal conditioning. A difference in susceptibility to inhibition by the female hormone apparently masks the inherent difference in growth potential and the primary symmetry of the female structure is preserved.
The marked asymmetries of the genital system in birds thus appear to rest on lateral differences involving such physiologic characteristics as growth potentials and reaction thresholds. These in turn are apparently correlated with a more extensive asymmetry involving the whole organism. Lateral growth differentials are established in the blastoderm of chick embryos as early as the head-process stage. In testing the organ- forming potencies of regional pieces of the blastoderm it was found (Willier and Rawles, 1935; Rawles, 1936) that when corresponding pieces of the same size from right and left halves of the blastoderm are transplanted to the chorioallantoic membrane they consistently show marked differences in capacity for growth and self-differentiation, which are manifested both in the size attained by the graft (growth capacity) and in the quality of the histologic differentiation. Regardless of the particular tissues or organs dealt with, grafts from the left half of the blastoderm are consistently larger and better differentiated than those from the corresponding pieces of the right half. Lillie (1931) also postulated critical differences in growth rate and threshold to sex hormones on the two sides of the body in explaining the occurrence of "gynandromorphic" plumage in adult fowls and its distribution. He pointed out that the sharply defined difference in plumage on the two sides of the body is usually accompanied in gynandromorphs by gross bodily asymmetry (hemihypertrophy) favoring the left side. It would seem that lateral differences in the morphologic and physiologic properties of the sex primordia of birds are not peculiarities of sexual differentiation ; rather they are an aspect of the general pattern of
somatic organization in this group. In the normal differentiation of sex as well as in experimental studies these differences are exploited by hormones whose effects serve merely to exaggerate or to obliterate tendencies inherent in the organization of the individual primordia.
C. INFLUENCE OF SEX GENOTYPE ON THE REACTIONS OF SEX PRIMORDIA
Another example of the way in which constitutional factors operate to modify or set limits to hormone action is seen in the influence of the sex genotype on the responses of sex primordia to hormones, as illustrated especially in young opossums (Burns, 1942b, 1956a). In comparing the effects of identical doses of the same hormone (whether male or female) in embryos of different sex, it was found in the case of many structures that the amount of growth induced was influenced by the sex of the individual. Under identical experimental conditions the effect of a male hormone on the growth of a particular male structure was always greater in male embryos than on the homologous structure in females, and vice versa. This result is well illustrated by the reactions of the genital tubercle or phallus in male and female littermates which received identical doses of testosterone propionate. The transformed phallus of the female cannot be distinguished anatomically or histologically from the male organ, except for a constant and considerable difference in size (Fig. 2.32). Such an effect was cited earlier in the case of the prostate (Fig. 2.29) and it occurs for various other male structures such as the vas deferens I Wolffian duct) and the epididymis (Fig. 2.24). After treatment with female hormone corresponding differences are observed in the response of female structures. The Miillerian ducts of male embryos hypertrophy and undergo a typical differentiation into oviduct and uterus; nevertheless, in size these organs do not approach those produced by the same dosage in females (Fig. 2.24). The same is true in the case of the hyperplastic reaction of the sinus epithelium and for other structures. Differences in size can be
140
BIOLOGIC BASIS OF SEX
detected at an early stage and increase throughout the period of treatment.
To account for the constancy of these differences it seems necessary to assume that sex constitution in some way conditions the reactivity of the primordia, placing certain limitations on rate of growth. When sex constitution and type of hormone administered are the same there is, in effect, a summation of the two factors, but when they are different a conflict occurs. It might seem more simple to suppose that the embryonic gonads and their hormones are involved in this result, rather than to assume a differential reactivity on the part of the sex primordia; hormones of the same type would in one instance reinforce each other whereas in the other case unlike hormones oppose each other. However, this simple hypothesis cannot be sustained. Although the presence of a hormone from the embryonic testis may be safely accepted, there is as yet no evidence in mammals that the ovary produces significant amounts of hormone at this period, thus no supplementary factor can be assumed in the case of females receiving female hormone. In the male, moreover, histologic studies reveal that the size differences observed are much too great to be accounted for by the secretory activity of the embryonic testis ; for example, the difference in size between the male and the female prostate after treatment with male hormone (Fig. 2.29) is many times greater than the volume of the normal prostate, which may be taken as the measure of normal testis activity. The conclusive argument against participation of embryonic hormones in this phenomenon has come, however, from examination of the embryonic testes of experimental animals (Burns, 1956a). There is a great reduction in the size of the testis (and the ovary as well) and histologic study shows complete suppression of the interstitial tissue, the intertubular spaces being filled with a dense, nonstaining connective tissue of mucoid type. In contrast, the normal testis of the same age has a rich interstitium which is well developed as early as the 10th day of pouch life (Fig. 2.17.4 ». Evidence to be summarized later points strongly to the embryonic interstitial tissue as the source of the testis hormone, and in
the absence of this tissue it does not seem that the testis can be a factor in the result.
IX. The Time Factor in the Responses
of Sex Primordia: Receptivity
and "Critical Periods"
Of special importance is the factor of developmental age as it relates to the appearance of receptivity and the timing of determinative changes in sex primordia. This becomes apparent when the reactivity of a primordium to sex hormones, or its capacity for independent differentiation after isolation, is tested at successive stages of development. Typical studies of the second type are the experimental analyses of the appearance of sex-specific organization in the genital ridge of chick embryos (Willier, 1933) and in the differentiating gonads of the rat (Torrey, 1950; see pp. 103, 104). Such studies show that the organization of embryonic gonads with respect to sex type and capacity for self-differentiation is acquired gradually, leading step by step to changes which are stable and irreversible. Such transitions coincide in some cases with distinct morphologic events. In Willier's study of chick gonads fixation of sex-type, with capacity for autonomous differentiation, coincides with the appearance of a distinct germinal epithelium on the genital ridge. In rat gonads (Torrey) the sexes differ greatly in this respect; differentiation of prospective testes becomes an autonomous process from the first laying down of the medullary blastema, whereas the ovary has but little capacity for self-differentiation until much later, after the appearance of a distinct cortical zone.
The fact that at certain stages of development changes of an irreversible nature can be demonstrated has led to the recognition of so-called "critical periods," during which rather abrupt transitions occur from a state of lability to one of complete autonomy. Thereafter, hormones or other extraneous factors no longer have decisive effects. Such stages have been demonstrated for various types of sex primordia and are often narrowly limited in time. In chick embryos continued development of the Miillerian ducts, or their involution, depends normally on the ty{)e of gonad present, but at a cer
HORMONES IX DIFFERENTIATION OF SEX
141
tain stage their fate can be permanently conditioned by hormones administered experimentally (Wolff, 1938; Stoll, 1948). In male embryos involution of the ducts is prevented by administration of female hormone at the proper stage (p. 112), and once "stabilized" in this manner their subsequent development is assured without further treatment. Male hormones administered before this stage induce involution of the ducts in the living embryo or in vitro, but beyond this point have no effect.
The genital tubercle of the female duck shows a critical stage in relation to the embryonic ovaries during the 9th day of incubation (Wolff and Wolff, 1952b). When isolated in vitro before this stage the tubercle always develops the male form, which is also the condition found in castrated embryos. Isolation after the 9th day, however, results always in a structure of female type. About the 9th day of development, then, its future character becomes fixed after which differentiation proceeds without further need of hormonal conditioning. Similar results were obtained in the case of the syrinx. If isolated before the stage of final determination the sex type of both j^-imordia can be readily controlled in vitro by addition of hormones to the medium.
The Wolffian ducts of mammalian embryos behave similarly. In this instance the male hormone is necessary at a certain stage to insure retention of the ducts. Castration of male rabbit embryos before the 22nd day is followed by involution but later castration has little effect; changes of an irreversible nature have occurred which insure continuation of development regardless of hormonal conditioning. A critical period of brief duration also exists for the prostate glands of young opossums, involving the response to both types of sex hormone. Estrogens permanently suppress prostate development in males if a single dose is administered just before the stage when the buds should appear. Male hormones, on the other hand, induce prostatic glands in females at this stage which thereafter continue to develop without further treatment. The effects of castration on the prostate are like those described for the Wolffian ducts. In rabbit embryos the critical period falls
from the 22nd to the 23rd day of gestation after which the operation has but slight effect (Table 2.2).
It appears from much evidence of this kind that sex primordia typically pass through developmental phases which are crucial with respect to the origin, the survival or the future mode of differentiation of the structure in question. At such stages, and for brief periods, formative or suppressive, trophic or involutionary, responses are readily induced by hormones. However, the physiologic status of the primordium itself prescribes the specific quality of the response and the timing as well.
X. Specificity of Hormone Action
and the Significance of
Paradoxical Effects
Perhaps the objection most frequently urged against steroid hormones as specific agents in sexual differentiation is the common occurrence of paradoxical effects, in which a hormone of one type stimulates the differentiation of structures of the other sex, sometimes in a striking manner. Such responses have been encountered in all major groups thus far investigated, and practically every type of sex character may be involved. The frequency with which this phenomenon is associated with high dosages has been noted, with emphasis on the fact that in low concentrations the effects are usually sex specific. Some apparent exceptions to this general rule may, indeed, be due to difficulty in defining a low dose in particular cases in view of the efficiency of extremely low concentrations in certain species (e.g., Mintz, 1948). Specificity of action obviously implies that male hormones stimulate development of male characters in embryos of either sex, whereas female primordia are inhibited or give no positive response; in like manner, female hormones should induce differentiation of female primordia while inhibiting the development of male structures. Convincing examples of specific action in this sense are found in the complete and even functional transformations obtained in various amphibian species with low concentrations of hormones (Table 2.1) ; in the maintenance of normal differentiation after castration by treatment
142
BIOLOGIC BASIS OF SEX
with a crystalline hormone; and in the manner in which many accessory sex structures or their primordia respond to the approiH'iate hormone, both in vivo and in vitro. It should be emphasized further that paradoxical effects seldom appear to the exclusion of normal responses, but usually as accompanying phenomena. For example, large doses of testosterone propionate produce strong hypertrophy of the entire male genital system in opossum embryos as expected; however, they also cause hyper trophy of the Miillerian duct derivatives, a response that disappears at low dosages although the effect on male structures remains. In the interpretation of paradoxical effects the problem of direct vs. indirect action arises. It may be argued tentatively that in sufficient concentration hormones of either type stimulate the primordia of the other sex by direct action ; both sets of primordia are capable of responding but at different thresholds, those of heterotypic structures being very high. Such a situation would permit selective action at ordinary or "physiologic" levels and at the same time account for the appearance of paradoxical effects at higher concentrations. As a matter of fact, the dosages which elicit paradoxical responses are as a rule so far above physiologic levels as to have doubtful significance for normal differentiation. Evidence favoring the thesis of direct action has been cited and in one case at least a typical paradoxical effect has been produced in vitro. Large doses of testosterone propionate have a strong feminizing (i.e., inhibitory) action on the syrinx of the duck in vitro (Wolff and Wolff, 1952a). However, the presumption that the paradoxical action must have been exerted directly does not establish its nature. The high concentration of male hormone obviously has an adverse effect, resembling the inhibitory effect of the female hormone, but the inhibition is possibly of a general nature rather than specific. First attempts to culture the syrinx on a simple synthetic medium also resulted in atypical differentiation (Wolff, Haffen and Wolff, 1953) due apparently to nutritive deficiencies. High concentrations of hormones ?>? vitro mav onlv create general
conditions unfavorable for normal growth and differentiation.
On the other hand, paradoxical effects are certainly in some cases not mediated directly but are of secondary origin. The feminization of the testes that occurs in certain amphibians after treatment with male hormones (p. 94; for a summary see Gallien, 1955) is an example. An early disturbance of mesonephric development interferes subsequently with differentiation of the medullary sex cords, thus preventing testicular development. After metamorphosis, when the hormone is withdrawn, a certain recovery occurs and development is resumed, but in the virtual absence of the medullary component only the cortical rudiment develops. It should be noted, however, that during the hormone phase of this experiment there is inhibition of the cortex as well as suppression of the medulla, and it is only after the male hormone is withdrawn that development of the cortex is resumed. Although the paradoxical effect on the medulla, is pronounced it is indirect, and it does not occur alone but in conjunction with a partial atresia of the cortex. Thus the picture is more complicated than first appears.
On the other hand, the correlation between the appearance of paradoxical effects and the use of high dosages suggests other possibilities as to the manner in which such effects are mediated. It is a familiar fact in endocrine physiology that prolonged treatment with sex hormones disturbs the normal endocrine balances and may influence the activity of other glands. It is possible that certain paradoxical effects may originate in this way. As yet there is no direct evidence of this in embryonic organisms but it is well known that under abnormal or pathologic conditions both the gonads and the adrenal glands of adult animals are capable of producing the hormones of the other sex. This is the case for certain tumors of the gonads and adrenal cortex, and it is characteristic of the adrenal hyperplasias which produce the adrenogenital syndrome in fetal and postnatal life. It is also well established that under abnormal physiologic conditions ovaries may produce considerable amounts of androgen (cf. Hill, 1937a, b; Deanesly, 1938; for further discussion see Ponse, 1948). A striking case of adrenal disturbance induced by a sex hormone appears in frog tadpoles completely inasculinized by large doses of estradiol. Histologically, the change takes the form of a massive hyperplasia of the adrenal cortical or interrenal tissue (Padoa, 1938, 1942; Witschi, 1953; Segal, 1953) which may attain 10 times the normal volume. In this remarkable case, however, the masculinization of the ovaries is not caused by the adrenal hyperplasia, because in hypophysectomized tadpoles the hyperplasia does not occur but the paradoxical masculinizing effect still persists. Nevertheless, when excessive doses of sex hormones can induce glandular disturbances of this order, the possibility remains that they may be only secondarily involved in the appearance of paradoxical effects.-^
Perhaps the simplest explanation of the paradoxical effects of high dosages lies, however, in the possibility that, when present in excess, a hormone may be transformed in the organism into one of opposite type. In this event two hormones are in fact acting simultaneously and the specificity of the administered substance is not in question. This possibility was first suggested by findings in the adults of several mammalian species, including man. Treatment with large amounts of testosterone may be followed by excretion of considerable quantities of estrogen in the urine, which disappears when the male hormone is withdrawn (for the older literature see Burrows 1949, Ch. VI). This may occur in normal males, in castrates or in eunuchoid types. In female subjects the estrogen thus produced is sufficient to stimulate female characters or functions, e.g., a marked hyperplasia of the vaginal epithelium appears. Without the knowledge that estrogen is being produced this would be regarded as a typical paradoxical effect. Conversion of testosterone to estrone or estradiol also
-^ For fuller discussions of various forms of paradoxical effects and their interpretations see Gallien (1944, 1950, 1955), Wolff (1947), Ponse (1948), Padoa (1950), Jost (1948a) and Burns (1949, 1955b).
takes place in ovariectomized and adrenalectomized women (West, Damast, Sarro and Pearson, 1956). It is evident that neither gonads nor adrenals are necessary for such conversions, which may even occur in vitro (Baggett, Engel, Savard and Dorfman, 1956; Wotiz, Davis, Lemon and Gut, 1956). Finally, it has been established that the injected male hormone is the actual source of the estrogen by the use of testosterone labeled with C'^ (Baggett, Engel, Savard and Dorfman, 1956; Wotiz, Davis, Lemon and Gut, 1956; Heard, Jellinek and O'Donnell, 1955; for a recent review of this subject see Dorfman, 1957). Although it may be technically difficult to demonstrate such conversions in embryonic organisms, there are no grounds for supposing that they cannot occur.
XI. Time of Origin and the Source of Gonad Hormones
Evidence that the embryonic gonads begin to produce their hormones early in the course of sexual differentiation comes from many sources. In the more strongly modified freemartins, conditions indicate that the hormone of the male twin must have been active at an early stage. The ovaries of the female are severely inhibited with almost complete suppression of cortical differentiation (Willier, 1921). Lillie (1917) suggested that in such cases the first action of the male hormone might be to produce, in effect, a "castration" of the female twin by suppression of the ovarial cortex. It now appears from the results of actual castration experiments that this point is probably not significant as there is nothing to indicate that the ovary is active endocrinologically at such an early stage (Bascom, 1923) .
In amphibians, either after parabiosis or transplantation of the gonad priraordium, changes in the gonads can be detected very early in relation to the onset of sex differentiation, and in some circumstances reversal occurs by direct differentiation as a gonad of opposite sex. The gonads involved are as a rule widely separated and the hormonal nature of the transforming agent in these cases is beyond question. Similar indications are found in birds. Embrv
144
BIOLOGIC BASIS OF SEX
onic ovaries grafted into the coelomic cavity induce cortical differentiation in the testes of male hosts at a very early stage, and during the same period testis grafts inhibit the difTerentiation of the IMiillerian ducts. A similar effect is observed when histologically undifferentiated gonads of duck embryos are cultured in vitro in close contact (Wolff and Haffen, 1952b). When ovaries and testes are thus associated the latter exhibit typical reversal changes from the very beginning of sexual differentiation.
In mammals the activity of the testis hormone in the early stages of sex differen
tiation is revealed by the promptness with
which castration effects appear. In the absence of the testes, changes in certain accessory sex structures are evident as soon
as sexual differentiation can be observed.
In the case of the prostate the hormone is
actually necessary for the appearance of
the primary buds. Conversely, implantation
of an embryonic testis into a female rabbit
embryo inhibits the Miillerian ducts and
initiates development of male accessory
structures (Fig. 2.35; for a more detailed
summary see Willier, 1955).
The source of the hormones produced by
Ostium of Mullerian Duct
fted Testl
Fig. 2.35. Localized effects of an embryonic testis grafted to the broad ligament of a female rabbit embryo (Jost, 1947b). The inhibitory effect of the grafted testis has caused a great reduction in the size of the host ovary and has suppressed the Mullerian duct (unshaded) in the vicinity of the graft. These structures are normal on the opposite side. Conversely, the testis has induced complete retention of the Wolffian duct and epididymis (stippled) on the side of the graft and a partial retention on the other side. The influence of the graft is strongest in its immediate vicinity and beyond a certain distance disappears, indicating that the hormone spreads locally by diffusion rather than through the circulation. The results also suggest that the stimulatory action on the male structures is stronger than the inhibitory effect on the Miillerian duct, since it extends further. Such a situation probably results from threshold differences in reactivity of the two end-organs to the testis hormone.
HORMONES IN DIFFERENTIATION OF SEX
145
the embryonic gonads is a matter which can be discussed with some assurance only in the case of the mammahan testis. In the testes of adult mammals the interstitial tissue has long been recognized as the source of the male hormone and, as was pointed out in the beginning, the marked development of this tissue in the testes of pig embryos led to the first suggestion that a hormone might be involved in sexual differentiation (Bouin and Ancel, 1903). In connection with the freemartin studies, an examination was made of the gonads of normal calf embryos and fetuses (Bascom, 1923) which pointed to the well developed interstitial cells as the probable source of the male hormone. This provided a plausible explanation of the invariable dominance of the male twin, because in fetal ovaries no indication of internal secretion could be found until relatively late in gestation ; in the testis, on the contrary, interstitial tissue was seen in increasing amounts from practically the beginning of sex differentiation.
It is unnecessary to multiply cases in which the presence of interstitial tissue in the embryonic testis coincides with indications of hormone activity. On the other hand, instances in which a reduction in testis activity (as evidenced by the condition of the sex accessories) can be correlated with the status of the interstitial tissue are pertinent. Decapitation of rabbit embryos (Jost, 1951a) is followed by definite retardation in the development of certain male accessory structures, although the growth of the embryo as a whole is normal. Examination of the testes in these specimens showed a reduction in size and in the number of interstitial cells; whether there was also cytologic abnormality has not been ascertained. The defects of the sex accessories in the decapitated fetuses resembled those which appear after incomplete or unilateral castration. Structures near the defective testes (epididymides, vasa deferentia) were virtually normal but more distant structures (sinus derivatives, external genitalia) showed failures of development comparable to those produced by complete castration. Inadequacy of the testes to maintain normal development in these cases is appar
ently due to a quantitative deficiency of the
interstitial tissue and the male hormone.
A similar reduction of the interstitial tissue occurs in decapitated rat fetuses (Wells, 1950) and in this instance cytologic changes in the interstitial cells were also seen. In this species, however, no clear effects were observed on the accessory sex organs, as is also the case in fetal mice hypophysectomized by irradiation (Raynaud and Frilley, 1947; Raynaud, 1950). Negative findings in these cases may be attributable to species differences as to the stage at which the interstitial tissue becomes active ; however, it is more probable that the different result is due simply to the longer period of observation in the rabbit, allowing more time for the deficiencies to appear.- Also pertinent in this connection is the behavior of the interstitial tissue of the embryonic rat testis transplanted between the lobules of the seminal vesicle of a castrate adult; the interstitium of the grafted testis undergoes a considerably hypertrophy and the epithelium of the host's seminal vesicle gives a corresponding response (Jost, 1951b). But when the host is also hypophysectomized such grafts are deficient in interstitial cells and there is little or no response by the seminal vesicle (Jost and Colonge, 1949). The correlation between the state of development of the interstitial cells and the evidences of hormonal activity in these cases is direct and striking (for a recent review of this subject see Jost, 1957).
XII. A Comparison of the Effects of
Emhryonic and Adult Hormones
in Sex Differentiation
A problem has long existed as to whether the hormones or hormone-like substances produced by the embryonic gonads are essentially similar in character to adult sex hormones. When the effects of the two types of hormone on the development of embryonic sex primordia are studied under comparable conditions the resemblances are in
"It should be noted that treatment with gonadotrophins prevents the reduction in the interstitial tissue after decapitation, and may even produce hypertrophy of the interstitial cells (Wells, 1950, Jost, 1951a). In one instance also a graft of the fetal hypophysis had the same effect (Jost).
146
BIOLOGIC BASIS OF SEX
many cases extremely close; moreover, some of the apparent discrepancies have since been found to be due not to fundamental differences in the two types of hormone but to differences in experimental conditions as regards such factors as timing and dosage. The most important objections to steroid hormones of adult type as controllers of sex differentiation have been noted previously in various connections. However, a brief recapitulation is in order: they are (1) the frequent occurrence of paradoxical effects; (2) the failure of male hormones to inhibit effectively the Miillerian ducts of mammalian embryos; and (3) their failure in nearly all cases to have significant effects on the differentiation of mammalian gonads. The first objection has been dealt with (p. 141) and will not be disscussed further. In the other cases the failure is not absolute ; moreover, it is confined to a single group, the mammals, and is not of general application. To an extent, group or species differences may be involved. In the hedgehog, for example, the funnel region of the oviduct is inhibited by male hormone (Mombaerts, 1944), and in female opossums the vaginal portion is suppressed on one or both sides in about 50 per cent of all individuals (p. 114). Furthermore, in mouse and rabbit embryos male hormone prevents the union of the posterior ends of the Miillerian ducts to form the vaginal canal (Raynaud, 1942) and corpus uteri (Jost, 1947a). These partial effects in themselves require explanation. Failure of steroid hormones to reproduce more fully the effects of the embryonic hormone may lie, at least in part, in experimental conditions other than the type of hormone. On the other hand it is possible, as suggested by Jost (1953, 1955), that in mammals a special substance is required for the inhibition of the Miillerian ducts other than the ordinary testis hormone.
The failure of steroid hormones to modify the gonads of placental mammals (even when the accessory sex structures are profoundly transformed) is in marked contrast with the striking results obtained in many lower vertebrates and in the opossum. It is also at variance with the strong modifications usually found in freemartin gonads, and this has often been cited as proof that
different types of hormone are involved. However, the freemartin is still almost unique among mammals as an example of gonad transformation induced by another embryonic gonad, and may yet prove to be a special case of a type peculiar to the bovine family.^^ The possibility must be considered that in placental mammals gonad differentiation (as opposed to the differentiation of the accessory sex structures) has come to be under direct genotypic control; nevertheless, the demonstration, after many earlier failures, of a thoroughgoing transformation of the testis in the opossum suggests that certain essential experimental conditions have perhaps not been fully realized. In any case, it may be a difficult matter to determine whether the refractoriness of mammalian gonads to steroid hormones is indeed due to a fundamental difference in the character of the hormones themselves or to a change in the status of the embryonic gonads affecting their reactivity to hormones.
In many other situations it appears that embryonic and adult sex hormones are interchangeable without observable differences in the results. Testosterone propionate or methyl-testosterone, administered to castrated rabbit embryos at the time of operation, prevent the usual castration changes in all male structures, in this respect fully replacing the embryonic testis (Jost, 1947b, 1950, 1953), although they do not inhibit the Miillerian ducts. A similar effect of tes " There is, in fact, a notable scarcity of freemartins in a strict sense in other groups in which, on the grounds of placental fusion, the phenomenon might be expected to occur at least occasionally. For the literature on scattered cases interpreted as freemartins, see Willier (1939) and Witsclii (1939); and for a case (the marmoset, Wislocki) in which no freemartin effect was found although the essential conditions seemed to be present, see Witschi (1939). It is possible that the piesence of the hormone is not the only factor to be considered ; lack of reactivity on the part of the gonad may be the principal factor and one which may vary in different groups, correlated perhaps with the presence of maternal or placental estrogens during pregnancy. There is still a surprising lack of information for many groups; for example, the freemartin condition in sheep (at least as regards sterility) may occur more frecjuently than lias been supi)oyed (sec Stormont, Weir and Lane, 1953).
HORMONES IN DIFFERENTIATION OF SEX
147
tosterone propionate in maintaining the male accessory glands has been shown in castrated rat embryos (Wells, 1950; Wells and Fralick, 1951 ) . In opossum and other mammalian embryos synthetic androgens readily induce in females prostatic glands which are histologically indistinguishable from those of normal males although the latter are known to be conditioned in certain species by the hormone of the embryonic testes. Also, with proper timing, synthetic androgens induce involution of chick Miillerian ducts, either in vivo or in vitro, in a manner not histologically distinguishable from the effects of the embryonic testis (p. 114). Examples of this kind could be multiplied. Furthermore, the female hormone estradiol controls the sex type of various avian sex primordia, even when administered in vitro, closely simulating the normal action of the embryonic ovary. Wlien it is added to the culture medium, testes are transformed into ovotestes in the same fashion as when cultured in association with an embryonic ovary (Wolff and Haffen, 1952b) , and it produces a typical female syrinx or genital tubercle in vitro regardless of the sex of the donor embryo (Wolff and Wolff, 1952a; Wolff, 1953a).
Conversely, an example of an embryonic hormone substituting for an adult sex hormone is seen in the effect of a graft of the embryonic testis on the epithelium of the seminal vesicle in an adult castrate rat (Jost, 1948b, 1953; Jost and Colonge, 19491. Within a few days the vesicle epithelium in the vicinity of the graft is completely restored. Although the interstitial tissue of the grafted testis is somewhat hypertrophied under the influence of the host's hypophysis, it is improbable that a radical change in the character of the testicular secretion could be induced so quickly. That the hormone of the graft must be attributed to interstitial cells which cytologically are like those of the adult testis is also significant. It should be noted further that both estrogens and androgens (which can be detected by standard methods of assaying adult sex hormones) have been extracted from chick embryos in the latter half of the incubation period (Leroy, 1948) and from fetal mammalian gonads as well {e.g.. Cole, Hart, Ly
ons and Catchpole, 1933). If special embryonic hormones are necessary for the
control of sex differentiation, it would seem
that hormones of adult type are also being
produced during the same period.
Many minor inconsistencies can be pointed out in comparing the effects of the two types of hormone, but experimental conditions are usually too dissimilar to justify such detailed comparisons. It is noteworthy that the most "normal" results from the use of steroid hormones have been obtained under conditions which most closely approach the ideal, as when larval amphibians absorb the hormone continuously but in low concentration from the surrounding water. In many such experiments involving many species (Table 2.1) all individuals develop in accordance with, the type of liormone used, and without obvious histologic abnormalities. Under similar conditions, however, if the concentration is increased, all gonads become intersexual and very strong doses may actually produce effects exactly opposite to those obtained at very low levels (p. 94).
It is nevertheless too simple to suppose that all difficulties may be avoided simply by empirically arriving at the proper dose. What constitutes the optimal dose is not easy to determine from one species to another for, regardless of absolute concentration, the hormone level in the internal environment of the experimental organism may be greatly affected by such factors as the rates of absorption, utilization, and inactivation. These are factors which vary widely with different hormone preparations, different methods of administration, and also no doubt from one organism to another. In the second place, it is difficult or impossible to adjust the dosage accurately and flexibly from stage to stage, to correspond with the changing conditions of development and the state of the reacting structures; however, a continuing equilibrium is doubtless more nearly approached when doses are relatively low and the hormone enters continuously through the gills or by infusion from a graft. In comparison with normal development experimental conditions must always be arbitrary and inflexible; a dose which permits a normal re^^jionsc
148
BIOLOGIC BASIS OF SEX
in the case of one structure may be quite discordant for others, resulting in serious disharmonies or even in paradoxical reactions. Nevertheless, much can be learned analytically of the processes involved in sex differentiation without requiring perfectly integrated results.
XIII. Embryonic Hormones and Inductor Substances
According to generally accepted theory, the normal differentiation of the gonads is the result of an antagonistic interaction between the cortical and medullary components, in which one element (as prescribed by sex genotype) gradually becomes predominant while the other retrogresses. It has been previously emphasized that in experimental sex transformation no new principles or processes are involved ; the normal mechanism is simply set in reverse. The transformation process as it appears at various stages presents essentially the same histologic picture, whether the impulse to reversal comes from a developing gonad of opposite sex or from an administered hormone. It has been shown further that steroid hormones are capable in many cases of redirecting the differentiation process from its inception, leaving but slight histologic traces of reversal. Thus the processes of sex differentiation in the gonads are amenable to control by hormones at least over a considerable range of the developmental period.
The cjuestion then arises as to the manner in which the antagonistic interactions between cortex and medulla are mediated in normal development, and the nature and relationships of the physiologic agents involved. On this subject differences of opinion have long existed. The well known theory of Witschi^ postulates special inductor substances elaborated by the cortical and medullary tissues. Because the special sphere of the inductor substances is presumed to be the regulation of gonad differentiation, their field of action is thus topographically restricted; when at later stages the gonads begin to exercise control over the developing accessory sex structures, frequently over considerable distances, the action of embryonic hormones is presumed. However, since steroid hormones also may influence, or
even completely control, the mechanism of gonad differentiation, it is important to know whether such control is exerted secondarily, i.e., by regulating the inductor systems, or whether hormones are capable of playing the role of inductors. It must be remembered that the inductor substances have not as yet been isolated or directly identified; their existence and their character are postulated from the nature of the effects attributed to them. Consequently, this problem can only be approached indirectly by comparing the effects of sex hormones under as many conditions as possible with those ascribed to the inductor substances.
Although the activities of the inductor substances are ordinarily confined to the gonads, it is held that under favorable conditions their influence may extend somewhat further, but only within a limited range. This view was originally based on observations in certain parabiosis experiments (Witschi, 1932) and involves the manner in which inductor substances are supposedly transported. In parabiotic pairs of frogs, so closely united that the gonads lie within in a common body cavity, gonads of different sex do not influence each other significantly except when they are in contact or in very close -proximity. The action of the inductor substances, that is to say the intensity of their effects, seems to be roughly proportional to the distance between the interacting gonads (Fig. 2.2B). This observation suggested that the inductor substance is transmitted only by diffusion through the tissues, the concentration declining steadily with distance from the point of origin. Failure to be effective at greater distances presumably indicates that the agent is not distributed through the blood in the manner of a hormone. This, however, may mean only that in early stages of development the humoral substances, whatever their nature, are not produced in sufficient quantity to reach or maintain an adequate level in the bloodstream. In parabiotic salamanders, on the other hand, typical sex reversal also occurs, although the interacting gonads as a rule are widely separated (Fig. 2.2.4). In this case the inducing agent must be bloodborne; nevertheless, the changes in the re
HORMONES IN DIFFERENTIATION OF SEX
149
versing gonads occur at the same time and are of the same histologic character as those attributed to inductor action. Evidently the effects of gonads acting from a distance through the agency of blood-borne hormones are not distinguishable from those attributed to inductor substances when the interacting gonads are in close juxtaposition.
Furthermore, in other experimental situations where hormones are almost certainly involved, effects of a strongly localized character can be observed under proper conditions. When sexually differentiated gonads are grafted into the coelomic cavity of chick embryos (Wolff, 1946) ovaries have a transforming effect on the testes of male hosts which varies according to their relative proximity; and at the same stage of development testis grafts modify the adjacent gonaducts. A similar response appears when well differentiated larval gonads of Amhystoma are transplanted into the body cavity of another larva (Fig. 2.3) and in this case the effects are reciprocal. Also, after unilateral castration of male rabbit embryos (Jost, 1953), the sex accessories on the two sides of the body may show distinct differences in reaction. On the unoperated side normal differentiation of the sex ducts occurs, but on the operated side the Miillerian ducts are not completely inhibited and the Wolffian ducts are only partially preserved (c/. also Price and Pannabecker, 1956). It would seem that the remaining testis produces enough hormone to insure normal development of nearby structures, but at this early stage its output is insufficient to maintain proper development of structures at greater distances. A comparable result was obtained when an embryonic testis was implanted in a female embryo in close proximity to one ovary of the host (Jost, 1947b, 19531. The ovary on the side of the grafted testis was inhibited and atrophic whereas the other was normal (Fig. 2.35), and duct development followed different patterns on the two sides. On the side of the testis graft the Wolffian duct and epididymis persisted and developed but the IMiillerian duct was suppressed in the vicinity of the graft. On the side of the normal ovary these relationships were reversed.
Evidently the pattern of development is determined by proximity to the grafted testis. The same situation as regards the differentiation of the sex ducts and accessory structures is often encountered in so-called "lateral gynandromorphs" which occur sporadically in many mammals. In such cases, where embryonic hormones are clearly involved, the localized aspect of their action often resembles closely the postulated effects of inductor substances. It is interesting to compare the results of the experiments cited above with conditions found in a type of lateral gynandromorphism of doubtful etiology which occurs in a certain genetic strain of mice (Hollander, Gowen and Stadler, 1956). These gynandromorphs have an ovary on one side and a testis on the other, but without relation to laterality. Typically both gonads are small and underdeveloped, with the testes as a rule more severely affected. It is the condition of the accessory sex structures in these cases that is of special interest. On the side of the testis, development of the gonaducts without exception follows the male pattern (24 cases), a vas deferens and epididymis are present, and Miillerian duct derivatives are lacking (Fig. 2.36). This condition corresponds exactly to the role of the testis as the conditioner of male duct development and the inhibitor of the Miillerian duct, as revealed by the results of castration and culture in vitro. The development of the seminal vesicles is variable and the external genitalia, although usually underdeveloped, are nearly always of male type. These conditions in turn can be correlated with the size of the testis and the factor of distance from a gonad which is probably subnormal in its secretory activity. On the side of the ovary the opposite picture prevails; the Miillerian derivatives are always present, although variable in size, whereas the male accessories are either imperfectly developed or in most cases altogether lacking. A similar condition has recently been reported in a gynandromorphic hamster (Kirkman, 1958) . It may be noted that lateral differences of this kind are not infrequently met with in certain human intersexes (Jost, 1958; Wilkins, 1950). Evidence from other types of experiment
150
BIOLOGIC BASIS OF SEX
Fig. 2.36. Composite drawing illustrating the condition of the genital systems in a group
of "gynandromorphic" mice, which have an ovary on one side of the body and a testis on
the other, after Hollander, Gowen and Stadler (1956). On the side of the testis (which as a
rule was partially or entirely descended and is shown as dissected out) a complete male duct
system with seminal vesicle is present; the female genital tract is absent on this side. On the
side of the ovary a complete female genital tract is found, although it varied greatly in size
in different cases. On this side the male duct system was usually absent, but appeared in
whole or in part in about one third of all cases. Compare with conditions induced by a unilateral testis graft shown in Figure 2.35. Abbreviations: Bl., bladder; Ep., epididymis; O,
ovar}^; Ovd., oviduct; R, rectum; S.V., seminal vesicle; T, testis; U, uterus; V.D., vas
deferens.
bears on this point. When the pituitary is absent the normal secretory activity of the embryonic testis may be materially reduced. In male rabbit fetuses deprived of their hyi)ophyses by decapitation, although the testes are present, an apparent decrease in endocrine activity has local effects which reseml)le those of castration (Jost, 1951a, 1953). In their lowered state of activity, the influence of the testes on the accessory sex structures is graduated according to distance. Structures near the gonads, such as the vas deferens and epididymis, are normally developed but the more distant sinus derivatives and external genitalia are of female (i.e., castrate) type. Here again a level of activity adequate to maintain normal development of nearby structures is ineffective at greater distances, and the result is not compatible with the view that the hormone is distributed only thi'ough the blood stream.
Approaching the ciuestion from yet another direction, a clear demonstration of local action by a hormone appears in an experiment cited previously, in which an embryonic testis is engrafted between the lobules of the seminal vesicle of a castrate host; there is complete cytologic recovery of the atrophic epithelium in lobules contiguous with the graft, but the effect diminishes rapidly with distance and soon disappears. Greenwood and Blyth (1935) have also described a sharply circumscribed effect on the feathers of capons. A very small dose of female hormone injected subcutaneously changes the pigmentation of growing feathers at the site of injection, but beyond a very short distance it has no effect. On the other hand, after local implantation of hormone pellets, both localized and more distant effects may be registered at the same time, indicating that both modes of distribution are simultaneously effective [cf.
HORMONES IN DIFFERENTIATION OF SEX
151
Robson, 1951; Grayhack, 1958). A survey of a considerable literature on the local action of sex hormones (Speert, 1948) indicates that, with due consideration for such factors as vascularity and method of application, dual action in the above sense is chiefly a matter of dosage. Larger doses may have strong local effects accompanied, however, by a definite "systemic" action on distant structures. With small doses only local effects appear. Finally, it should be emphasized that localized activity has been demonstrated under suitable conditions in the case of many other endocrine glands and their hormones.^^
Thus numerous parallels and resemblances may be adduced with respect to the behavior of the hypothetical inductor substances and the local effects of sex hormones. On the basis of their histologic effects on gonad differentiation, their mode of distribution and range of action, and the period during which they operate, it seems that no clear or final distinctions can be drawn. Although theoretically the possibility cannot be excluded that hormones act indirectly on gonad differentiation by controlling the existing inductor systems, there are obvious advantages in postulating a single humoral agency; theory is simplified and hypothetic substances are replaced by known entities (for further discussions of this problem see Wolff, 1947; Jost, 1948a, 1953, 1955; Ponse, 1949; Burns, 1949, 1955b; Witschi, 1950, 1957).
XIV. References
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Dantchakoff, V. 1939. Les bases biologiques du "freemartinisme" et de "I'intersexualite hormonale" sont-elles les memes? Compt. rend. Soc. bioL, 130, 1473-1476.
Davis, M. E., and Potter, E. L. 1948. The response of the human fetal reproductive system to the administration of diethylstilbestrol and testosterone propionate during early pregnancy. Endocrinology, 42, 370-378.
Deanesly, R. 1938. The androgenic activity of ovarian grafts in castrated male rats. Proc. Roy. Soc, London, ser. B, 126, 122-135.
DE Be.'^umont, J. 1933. La differenciation sexuelle dans I'appareil in-o-genital du Triton et .son determinisme. Roux, Arch. Entwicklungsmech. Organ., 129, 120-178.
DoMM, L. 1939. Modifications in sex and secondary sexual characters in birds. In Sex and Internal Secretions, 2nd ed., E. Allen, C. H. Danforth and E. A. Doisy, Eds., Ch. 5. Baltimore: The WiUiams & Wilkins Company.
DoRFMAN. R.I. 1957. Biochemistry of the steroid hormones. Ann. Rev. Biochem., 26, 523-560
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