Book - Sex and internal secretions (1961) 2
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BIOLOGIC BASIS OF SEX
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).
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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
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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
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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
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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
AcKART, R. J. AND Leavy, S. 1939. Experimental reversal of sex in salamanders by the injection of estrone. Proc. Soo. Exper. Biol. & Med., 42, 720-724.
Ander.son, D., Billingh.am, R. E., L.mvip.son, G. H., AND Medaw.ar, p. B. 1951. The use of skin grafting to distinguish between monozygotic and dizygotic twins in cattle. Heredity, 5, 379-397.
Angevine, D. M. 1938. Pathologic anatomy of hypophysis and adrenals in anencephalv. Arch. Path., 26, 507-518.
=*See Etkin (1936) Etkin and Huth (1939) Gorbman (1950) foi- the pituitary-thyroid relationship; Kaltenbach (1953a and b) for the thyroid hormone; Katsch, Gordon and Charipper (1948) and Weinstein, Schiller and Charipper (1950) for adrenal hormones as they affect certain genital tissues.
Baggett, B., Engel, L. L., Savard, K., and Dorfman, R. I. 1956. The conversion of testosterone3-C" to C"-estradiol-17/3 by human ovarian tissue. J. Biol. Chem., 221, 931-941.
Barr, M. L. 1957. Cytologic tests of chromosomal sex. In Progress in Gynecology, \o\. 3, pp. 131-141. New York: Grune and Stratton.
B.ARR, M. L., AND Grumbach, M. M. 1958. Cytologic tests of chromosomal sex in relation to sexual anomalies in man. Recent Progr. Hormone Res., 14, 225-344.
Ba.scom, K. F. 1923. The interstitial cells of the gonads of cattle, with especial reference to their embryonic development and significance. Am. J. Anat., 31, 223-259.
Berthold, a. a. 1849. Transplantation der Hoden. Arch. Anat. Physiol, wiss. med., 16, 4246.
Blizzard, R. M., and Alberts, M. 1956. Hypopituitarism, hypoadrenalism, and hypogonadism in the newborn infant. J. Pediat. 48, 782792.
Bouin, p., and Ancel, P. 1903. Sur la signification de la glande interstitielle du testicule embryonnaire. Compt. rend. Soc. bioL, 55, 16821684.
Bradley, E. M. 1941. Sex differentiation of chick and duck gonads as studied in homoplastic and heteroplastic host-graft combinations. Anat. Rec, 79, 507-529.
Brewer, D. B. 1957. Congenital absence of the pituitary gland and its consequences. J. Path., 73, 59-i69.
Bronski, M. 1950. Development of embryonic guinea pig Miillerian ducts in anterior eye chamber grafts. Proc. Soc. Exper. Biol. & Med., 75, 426-429.
Bruner, J. A. 1952. Further quantitative studies on the effects of androgens on sex determination in Amblystoma (abst.). Anat. Rec, 113, 564.
Bruner, J. A., and Witschi, E. 1946. Testosterone-induced modifications of sex development in female hamsters. Am. J. Anat., 79, 293-320.
Burns, R. K. 1924. The sex of parabiotic twins in amphibians (abst.). Anat. Rec, 27, 198.
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