Book - Sex and internal secretions (1961) 2

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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be followed by excretion of considerable quantities of estrogen in the urine, which disappears when the male hormone is withdrawn (for the older literature see Burrows 1949, Ch.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

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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.

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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).

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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).

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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

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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

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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

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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.

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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.

Burns. R. K. 1925. The sex of parabiotic twins in Amphibia. J. Exper. ZooL, 42, 31-89.

Burns, R. K. 1928. The transplantation of larval gonads in urodele amphilDians. Anat. Rec, 39, 177-191.

Burns, R. K. 1930. The process of sex transformation in parabiotic Amblystoma. I. Transformation from female to male. J. Exper. Zool., 55, 123-129.

Burns, R. K. 1931. The process of sex transformation in parabiotic Amblystoma. II. Transformation from male to female. J. Exper. Zool., 60, 339-387.

Burns, R. K. 1934. The transplantation of the adult hypophysis into young salamander larvae. Anat. Rec, 58, 415-429.

Burns, R. K. 1935. The process of sex tran.-^formation in parabiotic Amblystoma. III. Con

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version of testis to ovary in heteroplastic pairs of A. tigrinum and A. punctatum. Anat. Rec, 63, 101-129.

Burns, R. K. 1938a. The effects of crystalline sex hormones on sex differentiation in Arnblystoma. I. Estrone. Anat. Rec, 71, 447-467.

Burns, R. K. 1938b. Hormonal control of sex differentiation. Am. Naturahst, 72, 207-227.

Burns, R. K. 1939a. The differentiation of sex in the opossum {Didelphys virginiana) and its modification by the male hormone testosterone propionate. J. Morphol., 65, 79-119.

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