Book - Sex and internal secretions (1961) 2

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Young WC. Sex and internal secretions. (1961) 3rd Eda. Williams and Wilkins. Baltimore.
Section A Biologic Basis of Sex Cytologic and Genetic Basis of Sex | Role of Hormones in the Differentiation of Sex
Section B The Hypophysis and the Gonadotrophic Hormones in Relation to Reproduction Morphology of the Hypophysis Related to Its Function | Physiology of the Anterior Hypophysis in Relation to Reproduction
The Mammalian Testis | The Accessory Reproductive Glands of Mammals | The Mammalian Ovary | The Mammalian Female Reproductive Cycle and Its Controlling Mechanisms | Action of Estrogen and Progesterone on the Reproductive Tract of Lower Primates | The Mammary Gland and Lactation | Some Problems of the Metabolism and Mechanism of Action of Steroid Sex Hormones | Nutritional Effects on Endocrine Secretions
Section D Biology of Sperm and Ova, Fertilization, Implantation, the Placenta, and Pregnancy Biology of Spermatozoa | Biology of Eggs and Implantation | Histochemistry and Electron Microscopy of the Placenta | Gestation
Section E Physiology of Reproduction in Submammalian Vertebrates Endocrinology of Reproduction in Cold-blooded Vertebrates | Endocrinology of Reproduction in Birds
Section F Hormonal Regulation of Reproductive Behavior The Hormones and Mating Behavior | Gonadal Hormones and Social Behavior in Infrahuman Vertebrates | Gonadal Hormones and Parental Behavior in Birds and Infrahuman Mammals | Sex Hormones and Other Variables in Human Eroticism | The Ontogenesis of Sexual Behavior in Man | Cultural Determinants of Sexual Behavior
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SECTION A Biologic Basis of Sex

Role of Hormones in the Differentiation of Sex

R. K. Burns, Ph.D., D.Sc. Hon.

Carnegie Institution Of Washington, Department Of Embryology, The Johns Hopkins University, Baltimore, Maryland


Contents

I. The Hormone Theory of Sex Differentiation

The modern era in the study of the pliysiology of sex differentiation is iisually dated from the sohition of the freemartin iiroblem through the simultaneous but entirely independent studies of Lillie (1916, 1917) and Keller and Tandler (1916). The theoretic explanation of the anomaly proposed by these authors was generally accepted and had two far-reaching effects ; it at once provided a simple, functional concept of the nature of embryonic sex differentiation which was readily susceptible of experimental test, and it directly stimulated the pioneer experiments in the field — the attempt to control sex differentiation in chick embryos by grafting gonad tissue to the chorioallantoic membrane (Minoura, 1921) and the application of the technique of parabiosis to the problem in amphibian embryos (Burns, 1924, 1925). However, as is usually the case with epoch-making theories, the concept of hormonal control of embryonic sex differentiation had roots going far back into the past.

The effects of castration in domestic animals and in humans had been familiar since earliest times, and it was long appreciated that in the vertebrates the gonads are necessary for maintaining the structural integrity of the accessory organs of reproduction and for the regulation of their functional acti^vities. It was first clearly demonstrated by Berthold (1849) that this control is exercised through the agency of a substance (of a nature as yet unknown) produced in the gonads and carried throughout the body in the circulating blood (for the historical background of this experiment see Forbes, 1 949 ) . Thus the conception of a blood-borne agent capable of controlling the growth and activities of distant structures, was established long before the name hormone was given to such substances. The theory that the differentiation of the genital structures in the embryo is controlled by a hormone, or hormones, produced by the embryonic gonads was a natural outgrowth of this knowledge. This view was first proposed as an hypothesis by Bouin and Ancel in 1903, suggested by the observation of an unusually rich interstitium in the testes of pig embryos during the period of sex differentiation. No direct evidence in support of the hypothesis was forthcoming, however, until the hormone theory, in virtually its present outlines, was formulated by Lillie and by Keller and Tandler as an explanation of the freemartin.

The freemartin^ had been familiar to breeders of cattle for centuries as a sexually abnormal calf, born as twin to a normal male, and its anatomy had been accurately described by John Hunter in the eighteenth century (for references see Lillie, 1917). The external genitalia and mammary glands are typically female in character and the animal was usually regarded as a female, but in rare cases the clitoris may be greatly enlarged and peniform (Numan, 1843, see Lillie, 1917, Fig. 29; Buyse, 1936). Internally, however, elements of the genital tracts of both sexes are present and frequently well developed, and later investigators were often in disagreement as to the primary sex of the creature. The gonads of the freemartin are rudimentary in form but usually show the histologic structure of an abnormal testis which is almost invariably sterile (for an exception see Hay, 1950, and also Hart, cited by Lillie, 1917, page 417). In many cases, however, the gonads are intersexual, showing varying degrees of agenesis of the ovarian cortex associated with rudimentary tubular structures in the medullary or hilar regions (Chapin, 1917; Willier, 1921). Commonly a well developed male duct system is present, but the development of the female genital tract is variable and in the more modified cases it may be virtually absent.

It is unnecessary to go into the various lines of evidence which were used to establish the fact that the freemartin is zygotically a female (Lillie, 1917, 1923) ; the point has recently been demonstrated cytologically by the Barr method (Moore, Graham and Barr, 1957). It will be useful, however, to review the circumstances which pointed to the cause of the anomaly. A freemartin is always associated at birth with a male twin (which is normal) and never with another female; in addition, the dizygotic origin of the pair was demonstrated by Lillie in many cases. Furthermore, for the birth of a freemartin it is necessary that the placentas of the twins be united, with the presence of vascular anastomoses (Fig. 2.1). In the absence of such connections the female twin is always normal. There is a correlation between the degree of abnormality

  • For a discussion of the origin of this name see Forbes (1946).


Fig. 2.1. Twin calves removed fiom the uterus, showing chorionic fusion and anastomosis between major vessels; male twin, left; freemartin, right. (After F. R. Lillie, J. Exper. Zool., 23, 371-452, 1917).


observed and the extent of the vascular union, and also with the stage of development at which the anastomosis was presumably established. The constancy with which these conditions appear pointed inevitably to the conclusion that the abnormal development of the female twin is caused by the transfusion, from an early stage of development, of a hormone produced by the gonads of the male twin. The invariable dominance of the male member of the pair was explained provisionally on the basis of histologic studies (Lillie and Bascom, 1922; Bascom, 1923) which indicated that the testis is active endocrinologically long before the ovary (rf. Bouin and Ancel, 1903). This conclusion has been supported in recent years by the results of castration in mamnialian embryos (q.i\). Reccnll_\' eNidciice lias come from (|uitc (hlferent sources that in most twins or muUiplc biilhs in cattle, placental anastomoses are established at an early stage. Calves, even when of different sex and with other characteristics indicating dizygotic or polyzygotic origin, possess identical complements of l)lood factors (red cell agglutination types), which can only l)e explained on the basis of an early exchange of blood (Owen, Davis and Morgan, 1946). Since erythrocytes are comparatively short-lived cells, it is indicated in these cases that in-imitive erythroblasts must have been exchanged early in development and colonized the hemopoietic tissues of the recipients. It has been shown also (Anderson, Billingham, Lampson and Medawar, 1951) that diz3^gotic twin calves of dif


II. Methods of Experimental Analysis

The demonstration in the case of the freemartin of the probable nature of the transforming agent and its mode of transmission at once suggested means of attacking the problem of embryonic sex differentiation experimentally. At first grafting methods were mainly employed, using the embryos of birds and amjihibians.

A. Crafting of Gonads or Gonad Tissues in Bird Embryos

Historically, the first experiments were those of JNIinoura (1921) who trans})lanted pieces of adult testis or ovary to the chorioallantois of chick embryos during the period of sex differentiation. Such grafts become vascularized and are then in communication witli the host embryo by way of the umbilical circulation. Various modifications of

ferent sex are, with few exceptions, tolerant to grafts of each other's skin. this is true of skin exchanges in monozygotic twins (as would be exjiected) but is never found in other degrees of rehitionship. As in the preceding case, the ex])lanalion is found in an early transfusion of blood l)etwe(ni the twins. The exceptional cases are doubtless to be explained (as in the freemartin study) on the occasional failure of placental anastomosis to occur. This evidence is cited for its bearing on tlic point of early exchange of blood; the fact that blood cells and other elements are exchanged does not seem to be of significance for the sex hormone theory.


the embryonic genital structures of the host (especially of the Miillerian ducts) were noted and attributed to the influence of the grafted tissues. But later investigators failed to confirm these findings, and it was shown eventually that the anomalies observed by ]\Iinoura were unspecific in character and bore no constant relation to the sex of the grafted tissue (for a review and discussion see AVillier, 1939). Similar modifications were also found after transplantation of various nongonadal tissues, and are apparently induced by changes in the physical environment incidental to operation, such as lowering of the temperature and the humidity (Willier and Yuh, 1928). Evidently the original experiments had not been adequately controlled with respect to such factors.

Obviously this type of experiment does not correspond exactly with conditions in the freemartin. The grafted tissue came from adult gonads, and there was no way of determining the hormone output of such grafts or whether, indeed, they produced hormones at all. Furthermore, sexual differentiation in the host embryos has generally begun before transplantation to the chorioallantois is practicable. This objection was avoided, however, by modifying the exl)eriment. Sexually undifferentiated embryonic gonads were transplanted to the chorioallantoic membrane of a host embryo already well advanced in sex differentiation. In this case changes might be anticipated in the grafts. However, this experiment, as well as transplantation of the gonad-forming region of the blastoderm (Willier, 1927, 1933), also gave negative results. The grafted gonads differentiated to a variable degree, depending on the state of development of the primordia at the time of transplantation, but when sexual differentiation occurred it showed no constant relation to the sex of the host.

These failures to obtain evidence that gonad tissues growing on the chorioallantois influence the differentiation of host structures, or are themselves modified by the hormones of the host, raised serious doubt as to the role of hormones in embryonic sex differentiation in birds ; and this feeling was not entirely removed by the demonstration some years later that the sexual differentiation of the chick can be readily modified by treatment with pure hormone preparations. It was not until embryonic gonads were transplanted directly into the body cavity of another embryo (Bradley, 1941 ; Wolff, 1946) that unmistakable evidence was obtained. The conditions under which this result was achieved — close proximity of the graft to the developing host structures — suggested that the failure to obtain positive results by chorioallantoic grafting was perhaps largely a matter of the quantity or concentration of the hormone. Recently, however, a true "freemartin effect" has been reported in twin chicks of different sex, which developed from an egg with two yolks (Lutz and Lutz-Ostertag, 1958). There was local development of cortex on the left testis of the male twin, and a marked inhibition of the ]Miillerian ducts of the female, effects paralleling those produced by intracoelomic gonad grafts. This is the only recorded case of a natural freemartin in birds.

B. Grafting Experiments in Amphibian Embryos

The principal experimental procedures developed for amphibian embryos are illustrated diagrammatically in Figure 2.2, which shows the different modes of grafting, and the resulting vascular relationships, as compared with the freemartin (Fig. 2.1). The first experiments actually undertook to reproduce as nearly as possible the situation which arises by chance in the freemartin. The method devised was parabiosis— the grafting together of two embryos in the manner of "Siamese twins" (Burns, 1925) so that in later development there is a common circulation (Figs. 2.2A and 2.9). When members of such a pair happen to be genetically of the same sex, normal sexual differentiation would be expected to follow; but in pairs of different sex opportunity for cross-circulation of sex hormones is provided. Circulatory anastomosis is established in such pairs long before the beginning of sex differentiation in the gonads, and so a favorable situation is provided for testing the possibility of hormone action. The results obtained by this method vary greatly, depending on the species under study and on various experimental conditions, as will appear later.



Fig. 2.2. Diagram illustrating different modes of grafting in amphibians in order to bring about vascular continuity between individuals, and association of gonads of different sex. A. Homoplastic twins in salamanders. The body cavities are largely separated and vascular communications between the gonads are remote. For combination of dissimilar species see Fig. 2.9B. B. Anuran twins, showing side-to-side or head-to-tail union; reversal changes appear only under the first condition, when the gonads are in close proximity. (After E. Witschi, in Sex mid Internal Secretions, The Williams & Wilkins Company, 1932). C. Orthotopic transplantation of the gonad primordium by the method illustrated in Fig. 2.4, resulting in two gonads of opposite sex resident in a single individual (Humphrejs method).


The grafting of gonads alone (as opposed to the union of whole organisms) can be carried out in embryonic stages of development or in early larval life. The latter method was tried first. The gonads, attached to a segment of the mesonephric bodies, were removed from young larvae at or soon after the onset of sex differentiation and inserted into the body cavity of older larvae (Burns, 1928) . The development and activity of such grafts depends on the extent to which they become attached and vascularized. When graft and host are of different sex the grafts typically become intersexual, developing the structure of ovotestes; and when a large and well differentiated graft is in close proximity to the gonads of the host the latter may be similarly modified (Fig. 2.3). This method of grafting has the disadvantage, however, that reversal is usually incomplete, and when graft and host gonads show reciprocal modification it is sometimes difficult to determine the primary sex of either.


The method described above was soon greatly improved upon by the development of a technique for transplanting, at an earlier stage, the prospective gonad-forming tissue from one embryo to another (Humphrey 1928a, b). At first such grafts were placed in ectopic locations, but later it was found advantageous to place them in the normal (orthotopic) position in an embryo from which the corresponding gonad primordium had been excised (Fig. 2.4). After such an operation the host embryo bears on one side its own gonad and on the other a gonad which, in approximately half of all cases, has come from an embryo of the other sex (Fig. 2.2C). This method has important advantages over those previously described. A single embryo bearing an orthotopic graft survives better and is more easily reared tlian are parabiotic pairs, and gonads grafted in tlio orthotopic position usually develoj) better than in foreign surroundings. Most important of all, the donor embryo may be reared, thus establishing with certainty the original sex of the grafted gonad. This precise method has yielded unequivocal results which have in general confirmed and extended those obtained by parabiosis.



Fig. 2.3. Transplantation of the salamander gonad in early larval life (Burns, 1928): previously unpublished photographs. A. A large, but somewhat degenerate, grafted ovary lies just anterior to (above) the gonads of the host, which show changes in external form in the vicinity of the graft. B. Cross-section of host's right testis at the level of the white line, showing normal medullary development with well differentiated testis lobules, and peripherally a strongly developed cortex.


Fig. 2.4. Diagrams illustrating Humphrey's orthotopic transplantation method. A. Position of the gonad- and mesonephros-forming area of the embryo (stippled) which is excised and reimplanted in the corresponding position in a host embryo from which the primordium has just been removed. B. Cross-section of host at later stage showing position of the implanted material (between heavy lines) at the left. In the mesodermal layer a part of the lateral mesoderm lies below, the gonad- and mesonephros-forming material above, with the Wolffian duct at the top. Medial to the Wolffian duct lies the mass of primordial germ cells (more densely stippled).


C. Use of Pure Hormones as Sex Differentiating Agents

Early attempts to influence embryonic sex differentiation by the use of crude hormone preparations were almost entirely unsuccessful because of lack of potency, or the toxicity of the extracts. However, the isolation and eventual synthesis of steroid hormones made available a variety of active and nontoxic substances, and the use of pure hormones largely superseded grafting techniques. Direct administration of standard hormone preparations has the great advantage that dosages can be exactly known and regulated; also the timing of treatments is readily controlled and varied.

The first successful experiments using pure hormones were carried out on chick embryos. Similar results were obtained at almost the same time by several groups of investigators (Kozelka and Gallagher, 1934; Wolff and Ginglinger, 1935; Dantchakoff, 1935, 1936; Willier, Gallagher and Koch, 1935, 1937j who introduced the hormones, in oily or in aqueous solution, into the incubating egg. Striking transformations were produced, involving both the structure of the gonads and the accessory organs of sex. In the best cases reversal of the gonads was histologically almost complete.

The effects of crystalline sex hormones have also been investigated in many species of amphibians. Two methods have been utilized. Larvae may be treated individually by repeated injections, or in groups by continuous immersion, the hormone being dissolved in the water in which the larvae are reared. The latter method is particularly convenient for anuran tadpoles. Treatment can be started very early, the concentration is readily varied, and in many cases complete transformations have been obtained with the use of extremely low concentrations.

In mammalian embryos experimental study of sex differentiation was long delayed by the lack of operative techniques adequate for dealing with embryos in utero. The advent of pure hormones made possible the first successful experiments in this field.


In placental forms, in spite of a very high mortality, large doses of crystalline hormones can be administered to the mother during early stages of pregnane}" with pronounced effects on the genital systems of the embryos (for a review of the earlier experiments see Greene, 1942, and for a recent summary Jost, 1955) . About the same time, experiments were begun using the pouch young of a marsupial, the North American opossum (Burns, 1939a, b; Moore, 1941). So undeveloped are young marsupials at birth that virtually the entire course of morphologic sex differentiation takes place postnatally, and the embryos in the pouch are directly accessible for experimentation. Hormones were administered by injection (Burns) or by inunction, the application of an ointment containing the hormone to the skin. Except for minor differences attributable to dosage or other experimental factors, the results were similar, and in general agreement with those obtained in placental mammals by treatment during pregnancy.


D. Sex Differentiation in ihe Absence of Hormones

Although the evidence obtained by grafting techniques and by administration of hormones shows that the differentiation of embryonic genital structures may be profoundly modified or even completely reversed, such evidence is not in itself conclusive with respect to the central problem, the role of hormones in the normal differentiation of sex. The transmissible substances responsible for sex reversal in grafting experiments have not been isolated or identified and it may be argued that experiments with pure hormones show merely that the differentiating embryonic sex primordia are capable of reacting when hormones are introduced experimentally. Such evidence docs not prove, however, that embryonic gonads actually produce such hormones. For this question the crucial test is the capacity of the embryonic genital structure to develop in the absence of gonads or removed from all hormonal influence.

Evidence on this point has been forthcoming in recent years and gives strong supi)ort to tiie hormone theory. Two different experimental approaches have been developed. Early castration of the embryo has now been achieved in both mammals and birds, and improved methods of culturing embryonic organs in vitro have made is possible to observe for a sufficient time the development of sex primordia in complete physiologic isolation.

Since 1947 castration has been successfully performed in amniote embryos by two techniques, surgical castration and irradiation of the gonad region (for summaries see Jost, 1950; Wells, 1950; Raynaud, 1950; Wolff, 1950; Huijbers, 1951). In all cases serious failures of sexual differentiation follow removal or destruction of the gonads. Finally, the cultivation in vitro of individual sex primordia in virtual absence of hormonal influences has yielded results similar in all respects to those of castration. The results of these experiments will be taken up in detail as they relate to the development of particular structures.

III. The Bisexual Organization of the Early Enihryo as the Basis of Sex Reversal

The capacity of vertebrate embryos to undergo a reversal of sex, either spontaneously, as in various developmental anomalies of undetermined etiology ("intersexuality," "hermaphroditism"), or as a result of experiment, is based on the fact that every individual, regardless of genie sex constitution, passes in early development through a sexually undifferentiated or indifferent" phase. During this period virtually all of the embryonic structures necessary for the development of either sex are laid down morphologically and are present for a certain time as discrete 'primordia. The extent to which the primordia of the genetically recessive sex are developed and the length of time during which they are present vary in different groups and species. This fact is of great importance in the experimental transformation of sex. In species in which the structures of the recessive sex are imperfectly represented, or are present for only a brief period in early development, opportunity for sex reversal is correspondingly limited; but in other cases the rudimentary structures of the recessive sex (as for example Miillerian ducts in males or vestigial prostatic glands in females) persist indefinitely and mav even survive in the adults of some species. The existence of a considerable degree of embryonic bisexuality in most groups (see Fig. 2.22) provides a definite morphologic basis for experimental transformation of sex and for the sporadic occurrence of sex anomalies as well. The derivation of the various embryonic primordia which give rise to the male and female genital systems, and their history in the normal differentiation of sex, have been extensively reviewed by AVillier (1939) and will not be taken up again in detail. The main features of normal development will be outlined briefly when dealing with the experimental behavior of individual structures. It must be remembered, however, that the individual parts of the genital system have widely different embryonic origins, and are morphologically and physiologically very dissimilar, and at any particular stage of development may vary greatly in their relative maturity and so in their reactivity to hormones. Many of the basic structures (e.g., the embryonic sex ducts, the urinogenital sinus) are taken over bodily from other systems and only secondarily acquire a sexual status. It cannot be expected, therefore, that all parts of the developing sex complex will be capable at all times of responding harmoniously to experimental conditions which are often of necessity rigid and artificial or improperly timed. The recognition of such differences aids in understanding the variability so frequently encountered in the reactions of sex structures to hormones, and the importance of such experimental factors as the timing of treatment and dosage.

IV. Experimental Reversal of Sex Differentiation in the Gonads

A. Bisexual Organization of the Gonad and the Physiologic Mechanism of Sex Differentiation

The sexually undifferentiated gonads of most amphibians exhibit bisexual organization in a primitive and simple form. In early larval life the gonad, irrespective of its future sex, contains two histologically distinct components in which male and female potentialities are segregated. Internally, there is a hilar or centrally placed mass, the medulla, which has the developmental potentiality of a testis. Surrounding the medulla is a peripheral zone, the cortex, which is specifically female in potency. The topographic relationships of medulla and cortex are illustrated diagrammatically in Figure 2.5, and as they appear histologically in male salamanders in Figure 2.QA and B. Male and female potentialities are evidently pre-established in the medullary and cortical components at an early stage since final differentiation as a testis or an ovary does not involve the transformation of one sex comj^onent into the other but rather the gradual predominance of one element and the recession of the other.


Fig. 2.5. Diagrammatic representation of the male and female components of the sexually undifferentiated amphibian gonad and their roles in sex differentiation: the medulla is stippled, the cortex is plain. The broken arrows indicate the mutually antagonistic or inhibitory actions exerted between the two components in the course of sexual differentiation (Witschi).


Not only are the two components distinctly segregated in the indifferent gonad, they have separate origins. It has long been recognized that the medulla in both sexes is derived from the mesonephric blastema in the form of a series of cellular strands, the medullary cords or rete cords, which grow into the genital ridge at an early stage. At first similar in appearance in the two sexes, their later differentiation follows very different patterns. In the ovary the cords expand distally, forming a series of saccular cavities, the ovarian sacs. Most of the germ cells are excluded from these sacs and come to lie in a peripheral layer beneath the peritoneal epithelium covering the gonad. This zone becomes the cortex. In prospective testes the cords branch and proliferate rapidly, enveloping and incorporating the majority of the germ cells in a compact central mass, the medulla ( Fig. 2.6 ; for a fuller description see Willier, 1939). Thus the relative proportions of cortex and medulla in the sexes depend on the pattern of differentiation of the medullary cord, which results in a very unequal allocation of the germ cells between the two components. Probably the sex genotype acts primarily by determining the developmental pattern of the medullary cord. The role of the germ cells in the formation of the gonad appears to be a purely passive one since the nongerminal tissues are capable of producing the typical structure of a testis or an ovary in the absence of all germinal elements (for a review of this subject see Burns, 1955b).


FiG. 2.6. Sections showing the histologic appearance of cortex and medulla in early larval stages under various conditions. A. Normal testis of an Amby stoma tigrinum larva; note the thin cortical zone, comparable to a germinal epithelium, which covers most of the surface. B. The cortex of an intersexual testis dissected free from the medullary core, from the punctatum member of an Ambtjstoma tigrinum, 9 ; A. Punctatum, $ pair (c/. Burns, 1935, plate 4). C. Intersexual testis of the male member of a tigrinum-tigrinum pair, showing tl)e relative development of the medullary and cortical components (see Burns, 1930).



The origin of the medullary component of the gonad from the mesonephric blastema and its dominant role in gonad formation has been demonstrated in a striking experiment by Houillon (1956). Formation of the gonad is dependent on the normal development of the mesonephros, and this can be repressed or entirely prevented by blocking the development of the primary nephric duct (pronephric ductj at an early stage. In the absence of the nephric duct the mesonephric blastema is reduced in quantity and delayed in its appearance; typically only a few mesonephric tubules develop and these are poorly differentiated. In consequence of the suppression of the mesonephric blastema, medullary cords are lacking or poorly developed, and the result is a vestigial gonad consisting chiefly of a rudimentary cortex. The essential role of the medullary cords in gonadogenesis is also demonstrated in the gonads of toads (Witschi, 1933) in which the so-called "organ of Bidder" corresponds to an anterior segment of the genital ridge in which medullary cords are absent.

The proportion of cortex to medulla as laid down in embryonic gonads depends primarily on genie constitution, but the stage of development is a factor in the representation of the two elements at any particular time, since during the progress of sexual differentiation one component (corresponding to the genetically determined sex) shows an increasing predominance from stage to stage. Furthermore, the morphologic representation of the two sex components in the indifferent gonad is subject to variation in different groups, species, or races. In some species the recessive component is weakly represented or virtually absent, even in early development, or when present its existence may be of brief duration. In such cases capacity for sex reversal is reduced or lacking. In other species, in which the recessive sex component is well developed, or when it persists over a considerable period of time, capacity for experimental reversal is correspondingly increased.

The alternative behavior of the cortical and medullary components of the gonad in normal differentiation, as well as their behavior under experimental conditions, long ago suggested that the physiologic mechanism of sex differentiation consists essentially of an antagonistic interaction between the two elements, in which the genetically dominant component gradually inhibits its antagonist (Fig. 2.5). This concept of "corticomedullary antagonism" (Witschi, 1932) has been generally accepted as the basic mechanism in the histologic differentiation of the gonad and forms the starting point for the inductor theory of sex differentiation.^ A number of seemingly unrelated experimental procedures which are capable of inducing sex reversal all appear to have a common base of action by influencing or controlling this simple mechanism. For example, sex reversal is in some cases readily induced by external or environmental influences of an unspecific character, which apparently produce their effects by depressing or destroying the dominant gonad component.

Classical experiments of this type are the

^ As originally formulated, this theory postulated simply that each gonad component produces a substance which specifically inhibits the differentiation of the other. These substances, called medullarin and corticin, were considered to be similar in character and to behave like the embryonic inductors of earlier development, being transmitted by diffusion and having strictly localized effects. Subsequently the theory was elaborated to allow for stimulatory as well as inhibitory action, with each inductor system assigned a dual role; accordingly, positive and negative factors were assumed and so designated, e.g., medullarin* and medullarin'. More recently it has been proposed that interactions between the sex inductors are of the nature of an immunologic reaction (Chang and Witschi, 1956), the positive factor appearing first in the role of an antigen which stimulates the other system to produce an antibody, the inhibitory factor (for the development of the inductor theory see Witschi, 1934, 1939, 1942, 1950, 1957). To account for the great taxonomic variability in the action of the sex inductors thej' are assumed to be proteins.



use of extremes of temperature to induce reversal of differentiation in the gonads of anuran larvae (Witschi, 1929; Piquet, 1930; Uchida, 1937 1 . The reversal is due primarily to an unfavorable effect on the dominant component, high temperatures causing cortical degeneration in females and low temperatures inhibiting medullary development in males. Also, simple surgical interventions or even pathologic injury may have the same effect. Castration in certain cases results in complete reversal of sex through the reactivation and renewed development of a recessive gonad component left behind at operation. In adult male toads removal of the testes permits the organs of Bidder to develop into ovaries, which may become fully functional (Ponse, 1924). A comparable case is found in the reversal of sex which takes place in female chicks castrated soon after hatching. Removal of the dominant left ovary is followed by development of the rudimentary right gonad, composed largely or entirely of medullary tissue, into a small testis. Finally, rare cases of partial or complete sex reversal in adult hens, which occur as a result of pathologic destruction of the functional ovary, appear to have the same morphologic basis (Crew, 1923; for a discussion see Domm, 1939 1 .

But although sex differentiation in most vertebrates ends in the complete dominance of one sex component, remarkable deviations from this plan are known in certain groups and species. An extreme is found in the prevalence of hermaphroditism, in varying degree, in many teleost fishes and in cyclostomes (see ch. 17) which may be of the juvenile type and temporary, or may persist in adults. In toads the curious structure known as Bidder's organ is present in adults of both sexes; it represents a local region of the genital ridge in wliich medullary cords are never formed and further differentiation does not occur. Since it corresponds morphologically to the cortical component of the gonad it retains throughout life the potentiality of an ovary. This condition is apparently possible in toads because of the very low level of antagonism in this genus. Stranger still is the situation found in the female of certain insectivores (the mole, Godet, 1949, 1950; the desman, Peyre, 1952, 1955j in which the medulla of the adult ovary is testis-like and developed to a remarkable degree. Except during the reproductive period it greatly exceeds the cortex in bulk. Its cords are tubular in form, resembling testis tubules, and a well developed interstitium indicates an endocrine activity which is reflected in the strong masculinization of the genital tract. The clitoris is large and penis-like, and male accessory glands, absent or rudimentary in the females of most mammals, are well developed. Another species in which the ovarian medulla is highly developed, at least throughout fetal life, is the horse (Cole, Hart, Lyons and Catchpole, 1933). Thus many patterns are found with respect to the persistence of the heterotypic sex component of the ovary and its final fate.

B. Sex Reversal in Amphibian Gonads

1. Constitutional Differences and the Character of the Reversal Process

Modern amphibians, far from being a homogeneous group, are extremely diversified in structure and function and are often highly specialized. Such diversification obviously has had a long evolutionary history. Correspondingly, the processes of sex reversal as evoked experimentally in amphibian gonads, often follow very different histologic and physiologic patterns in different groups, species, or races. These differences must rest ultimately on genetic constitution ; more immediately they are predetermined, in labile fashion at least, in the structural and physiologic organization of the gonad primordium which is in itself a complex system.

The organization of the early gonad may vary greatly (according to species and according to sex) with respect to the cortical and medullary elements as laid down histologically in the primordium — is the heterotyi:)ic sex component of the primordium well represented or is it quantitatively deficient fiom the beginning? The subsequent beliavior of the heterotypic component is also important— does it jiersist and regress slowly over a considerable period of time or is its existence transient? Furthermore. how does it react when the normal balance of the differentiation process is experimentally disturbed — does it respond readily by growth and differentiation or is it relatively inert and refractory? In particular cases the capacity of a gonad for reversal under experimental conditions obviously depends on which of the various situations prevails. Another variable concerns the humoral activity of the gonad, as regards the time of onset and the factors of quantity or rate of production. Species differ widely in this respect and marked sex differences are also found. Presumably all such characteristics exist as predispositions within the gonad primordium, but they are not as a rule irreversibly determined.

In addition the process of reversal, as seen histologically, may be influenced by experimental conditions such as the procedure employed, the stage of development at which reversal is initiated and the duration of the experiment. If conditions are favorable at the beginning of sex differentiation, reversal may take place directly without leaving obvious histologic traces, i.e., an individual of one sex may adopt the developmental pattern of the other virtually from the start. If, on the other hand, transformation is not initiated until sex differentiation is well advanced, various stages of intersexuality will appear in the transforming gonads, until one sex component finally establishes complete dominance and the other disappears. The first situation commonly occurs when larvae are reared in optimal concentrations of hormone dissolved in the aquarium water; exposure to the hormone is continuous from a stage long antedating the appearance of morphologic sex differentiation, and all embryos regardless of sex genotype develop as one sex. However, the same result may occur also in grafting experiments (parabiosis or gonad transplantation) in which heteroplastic combinations of different species assure decisive predominance of one sex from the beginning by virtue of great inequality in size and in rate of development. The second situation is encountered when the conditions of the experiment do not lead to establishment of dominance at an early stage. Reversal sets in late, intersexual stages may be prolonged, and complete transformation mav never occur.


2. Parabiosis and Grajting of the Gonad or the Gonad Primordium

Experiments of this kind involve the interaction of embryonic or larval gonads through the agency of substances of a humoral nature but of unknown chemical constitution. In some species, or under certain experimental conditions, the effects may be limited and highly localized, appearing only when the interacting gonads are in contact or in close proximity. Transport of the humoral agent takes place apparently by diffusion through the intervening tissues (Figs. 2.25 and 2.3 j . In other cases the effects are exerted over great distances and the substances must of necessity be carried in the blood. This does not necessarily mean, however, that different substances are involved in the two cases. As will be shown, hormones in low concentrations may have only local effects and, given a sufficient concentration in the blood stream, there is no reason to suppose that the so-called "inductor substances" could not act at a distance. The mode of transport does not seem to be crucial for the definition of these substances (for discussions see Willier, 1939, page 134, and Burns 1949, 1955b).

In most species of amphibians which have been investigated the male is the dominant sex. In grafting experiments, whether the method is parabiosis or transplantation of embryonic gonads, testes as a rule induce sex reversal in ovaries without being greatly modified themselves. In some cases dominance of the testis is so extreme that no real reversal of the ovary occurs, only an almost complete suppression and sterility. This type of response is seen, for example, in parabiotic pairs of the wood frog (Witschi, 1927) and in the newt Triturus (Witschi and McCurdy, 1929), and is probably correlated with a constitutional inadequacy of the medullary component in the embryonic ovaries of the species in question. In other species, on the other hand, a severe initial repression of the ovary is followed by a delayed reversal, which may have a prolonged course but which eventuall}^ may be quite complete. In parabiotic pairs of certain species of Ambystoma (Fig. 2.7) there is a severe inhibition of the ovary before active transformation is initiated, and when reversal begins it may be confined to local regions of the ovary. Transformation may set in independently at several sites, resulting in localized masses of testicular tissue which are, however, histologically normal (Fig. 2.8). Ultimately all renmants of cortex disai)i)ear and transformation is complete. Such individuals are capable of breeding as males (this depends on the new testis establishing proper connections with the duct system) notwithstanding they have the genotype of the opposite sex (for a discussion see Humphrey, 1942).


Fig. 2.7. An extreme degree of inhibition and reduction in certain regions of an ovary under the dominance of a well developed testis (Humphrey, 1942). A. Level showing sterile medulla above, with degenerate cortical zone below. B. Medulla with rete cord and a single germ cell above, small cortical remnant below. C. Region showing complete atrophy. (From Biological Symposia, Vol. IX, Jacques Cattell Press, Lancaster, Pa.)



Fig. 2.8. Sections Uuoiifiii a tian.sforniing o\;ny in an older case. Tlir cortex is extremely reduced and the medidlary area is well differentiated as a testis. In B, except for the cortical renmant, the histologic picture is that of a normal testis of intermediate development, with well defined lobules (Humphrey, 1942; cf. Fig. 2.7).



To insure invariable predominance of the ovary in sex reversal it is usually necessary to provide a marked advantage in size and rate of development in favor of the female. This can be done experimentally by resorting to heteroplastic combinations (Fig. 2.9). In parabiotic pairs composed of two species of very different size {e.g., Amhystoma tigrinum-Amhy stoma maculatum) and with a corresponding difference in growth rate, when the members are of different sex the larger species is almost invariably dominant (Fig. 2.11; Burns, 1935). When the large partner is a female the ovaries are enormously larger than the testes of the male and are always normal. The testes in some cases undergo reversal almost from the beginning of differentiation, and toward metamorphosis are represented by very small ovaries which contain a few well developed ovocytes. However, in most indi


viduals transformation sets in after considerable differentiation has occurred, the testis cords becoming hollowed out to form ovarial sacs (Fig. 2.11) while the cortex persists and grows rapidly. At metamorphosis males are either completely transformed or the process is far advanced. Complete transformation of this type has also been reported by Witschi (1937) in A. tigrinum-A. jefjersonianum pairs.

A similar result is obtained when single gonad primordia are transplanted heteroplastically by Humphrey's method (Fig. 2.4) . In individuals bearing gonads of different sex, when the ovary is of the larger species it is dominant regardless of whether it belongs to the host organism or was derived from the graft. Histologically the reversal process is the same as in parabiotic pairs (Humphrey, 1935a, b). For fuller discussions of species and racial differences as they affect physiologic sex dominance and the capacity of the gonads to undergo reversal in different species see Witschi (1934, 1957). Humphrey (1942), and Gallien (1955).



Fig. 2.9. Heteroplastic combinations uniting diiierent species of salamander {Amhystoma tigrinum and A. punctatum) which differ greatly in eventual size and rate of growth. A. Ventral view of paired embryos just after operation, showing fusion in the cervical region (punctatum member at left). B. A pair after metamorphosis showing the great difference in size; the larger animal is the tigrimnii member.



The progress of sex reversal, and the mechanism by which the transformation is effected, can be analyzed histologically only when it takes place as a secondary process, after a certain amount of sex differentiation has previously occurred. In this case both histologic components are distinctly represented but the normally recessive component seeks to become dominant; ovaries become testes by regression of the differentiated cortex accompanied by growth and differentiation of the medullary element (Figs. 2.8 and 2.10), and testes are converted into ovaries by the reverse process (Figs. 2.6C and 2.11A-D). The mechanism is flexible, however, and there is much variability, even among individuals in the same experiment, with respect to the stage at which reversal sets in, its progress, and its final outcome. In some cases removal of the dominant gonad after reversal is far advanced may be followed by a second reversal toward the original sex (Humphrey, 1942).

However, transformation is not always a secondary process, set in action only after a certain amount of differentiation has already occurred; as noted above, when the ciuantitative disparity between the interacting gonads is sufficiently marked reversal may proceed from the earliest stages of differentiation. In this case the term "reversal" is less apt since there were no previous histologic steps to be retraced. Transformation is indicated chiefly by the unbalanced sex ratios at the end of the experiment; but in certain cases it is confirmed by characteristic histologic peculiarities (Burns, 1935, Fig. 28; Witschi, 1937, Fig. 39). In the heteroplastic grafting experiments of Humphrey, on the other hand, direct proof is available since in many cases the sex of the grafted gonad, although conforming with that of the host, differs from the sex of the donor animal which is reared to provide a direct control.

Although primary reversal of sex differentiation, as described above, occurs more readily when a marked disparity in size leads to early dominance, it may also occur under conditions which greatly retard developinent and delay the beginning of sex differentiation. This appears to be the ease in the first parabiosis experiments (Burns, 1925) in which the method of joining interfered with feeding and resulted in a severe retardation of growth. Such pairs developed so slowly that sex differentiation was delayed for weeks and in many cases for months. When it eventually took place members of a pair were almost invariably of the same sex (c/. Humphrey, 1932) although there was great variation in the size and stage of differentiation of the gonads. In this experiment the usual physiologic dominance of the male was also disturbed, male-male and female- female pairs appearing in nearly equal numbers.^ The manner in which extremes of temperatures induce sex reversal in tadpoles through a differential inhibitory effect on the medullary or the cortical component of the embryonic gonad has been referred to earlier. The above result may have a similar physiologic basis if, under prolonged repression, the usually dominant male gonad should prove to be more susceptible to unfavorable conditions than the female.



Fig. 2.10. Two views of an ovary of Atnbystoma tigrinuin uudcigoing reversal under the influence of the testes of a male partner. The cortical zone, witli characteristic early ovocytes, is still prominent; however, medullary development is i)roceeding and in the region rei^resonted in B, testis lobules are forming. (From l^ K. Buiiis, J. Exper. Zool., 55, 123-129, 1930; 60,339-387, 1931.)


Fig. 2.11. Stages in the transformation of testis to ovary in the male (punci.ii luii ) imniber of an Ambystorna tigrinum-A. punctatum pair, joined in heteroplastic parabiosis (Burns, 1935). yl to D show, at successive levels in the same gonad, the degeneration of the medulla by vacuolation of the rete canals and lobules, accompanied by persistence and growth of the cortex. E shows a section through one of the dominant and entirely normal tigrinum ovaries; a gross picture of these ovaries is seen in F. (From R. K. Burns, Anat. Rec, 63, 101-129, 1935.)



^ The author's interpretation of the results in this experiment has often been questioned by Witschi. However, all but a small part of this material was subsequently re-examined by Humphrey (1932) whose study confirmed the original conclusions except for minor details in\-olving at the most only eight pairs. In Humphrey's opinion there were certain histologic indications that these



3. Administration of Steroid Hormones

Since sex hormones of adult type became available in pure form their effects on the differentiation of sex have been tested in many species of amphibia. They are readil}' administered in two ways, by injecting directly into the body cavity or, in aqueous solution, by adding them to the water in which the larvae are reared. The results on the whole are striking; in certain species there

pairs were originally heterosexual, although transformation had proceeded to the point where a complete reversal was imminent.


is complete transformation of ovary to testis or of testis to ovary, and in some cases the transformed individuals have been proved functional and capable of breeding. In other si)ecies, however, negative, equivocal, and in many cases paradoxical results have been obtained by the use of the same substances. A hormone that completely transforms all individuals of the opposite sex in one species may have only a weak or impermanent effect in another, or no effect at all in a third. Obviously the gonads of different species differ greatly in their responses to steroid hormones. There is also a correlation with sex. In some species the gonad of one sex undergoes reversal with relative ease whereas that of the other is difficult or impossible to transform, although increased dosages are sometimes effective. In certain species in which the sex chromosome complex is known it is the homogametic sex that is readily reversible (Gallien, 1955). It is also clear that experimental conditions strongly influence the result. The time factor, that is to say the stage of differentiation at which treatment is initiated, is obviously important; in general, the most complete transformations are obtained when the hormone has been present from the beginning of the differentiation process. Dosage is likewise of great importance and the optimal dosage varies greatly from one species to another. A negative response at one dosage may become positive when the dosage is increased; on the other hand, strong "paradoxical effects" (stimulation of the characters of one sex by the hormone of the other) are often encountered with high dosages which are absent at lower levels. At present it is not possible to give consistent explanations for all such contradictory results ; however, better understanding is gained when they are classified into convenient categories.

The effects of male hormones on sex differentiation in frogs. On the whole, the most successful reversals obtained by the use of steroid hormones have been in frogs of the family Ranidae, of which some six species have now been studied with similar results. Both male and female hormones induce reversal of sex in young tadpoles, but male hormones are more effective and far more consistent in their effects. In Rana temporaria treatment with testosterone propionate transforms all genie females into males. The transformation is complete and permanent; moreover, transformed individuals are capable of functioning in their new capacity (Gallien, 1944). A similar transformation has been obtained in Rana sylvatica, a phenomenally low concentration of the hormone (1/500,000,000 parts dissolved in the aquarium water) inducing complete histologic transformation (Mintz, 1948). Comparable results have been reported after use of the male hormone in several other species of this genus (Table 2.1). In the case of Rana catesbiana it was found necessary to "prime" the gonads by simultaneous treatment with gonadotrophin, otherwise they were unresponsive; when so treated, however, complete transformation is obtained. The gonadotrophic substance alone initiates a precocious differentiation of sex but without any tendency to transformation, serving only to precipitate the normal differentiation process. Complete and permanent masculinization of females by testosterone propionate has also been reported recently in a tree frog, Pseudacris (Witschi, Foote and Chang, 1958), and a virtual transformation at the age of metamorphosis in Rhacophonts (Iwasawa, 1958), indicating that other anuran families may resemble the Ranidae in their reactions to the male hormone. In marked contrast, male hormone is without effect on gonad differentiation in the toad (Chang, 1955).

The effects of female hormones in the Ranidae. These are variable and less conclusive. The results frequently depend on dosage and the effective dose may vary greatly in different species. Estradiol benzoate in weak doses has a slightly feminizing action in male tadpoles of Raiia temporaria (Gallien, 1941), but stronger doses produce complete feminization in an "undifferentiated race" (one in which sex differentiation of males occurs relatively late) of the same species, all tadpoles at metamorphosis being females (Gallien, 1940, 1955) . It is noteworthy that in this case both genie constitution (race) and the dosage of the hormone may be factors in the result. In other ranid species estradiol, administered in low dosages during the period of sex differentiation, has a completely feminizing effect ; at meta



TABLE 2.1

The ejects of synthetic male and female sex hormones on the differentiation of the gonads in various species of amphibians. The cases listed are those in which a complete, or near complete, and histologically norma transformation was achieved by the age of metamorphosis. In some species the reversal wa permanent and functional. For details see text.

ACTION OF MALE HORMONE ON FEMALES


SPECIES


INVESTIGATORS


RESULT


COMMENT


RANA TEMPORARIA


GALLIEN 1938, 1944


COMPLETE TRANSFORMATION


PERMANENT AND FUNCTIONAL


RANA SYLVATICA


MINTZ 1948


COMPLETE TRANSFORMATION AT METAMORPHOSIS


DOSAGE 1/500 000 000

IN ACQUARIUM WATER


RANA PIPIENS


FOOTE 1938


COMPLETE TRANSFORMATIONTREATMENT FOR 65 DAYS



RANA CATESBIANA


PUCKETT 1939, 1940


COMPLETE TRANSFORMATION AT METAMORPHOSIS


ADMINISTERED WITH GONADOTROPIN


RANA CLAMITANS


MINTZ, FOOTE 8 WITSCHI 1945


COMPLETE TRANSFORMATIONTREATMENT FOR 95 DAYS


SOME PRODUCED SPERM


RANA AGILIS (DALMATINA)


VANNINI 1941, PADOA 1947


COMPLETE TRANSFORMATION



PSEUDACRIS NIGRITA


WITSCHI, FOOTE 8 CHANG 1958


COMPLETE TRANSFORMATION


EFFECT PERMANENT


RHACOPHORUS SCHLEGELII


IWASAWA 1958


TRANSFORMATION ALMOST COMPLETE


AT THE AGE OF

METAMORPHOSIS


ACTION OF FEMALE HORMONE ON MALES


PLEURODELES WALTLII


GALLIEN 1954


COMPLETE TRANSFORMATION


PERMANENT AND FUNCTIONAL


XENOPUS LAEVIS


GALLIEN 1953


COMPLETE TRANSFORMATION


PERMANENT AND FUNCTIONAL


RANA TEMPORARIA


GALLIEN 1941, 1944


COMPLETE TRANSFORMATION


EFFECT NOT PERMANENT


RANA ESCULENTA


PADOA 1938, 1942


COMPLETE TRANSFORMATION AT METAMORPHOSIS


AT LOW DOSAGES ONLY


RANA SYLVATICA


WITSCHI 1952, 1953


COMPLETE TRANSFORMATION AT METAMORPHOSIS


AT LOW DOSAGES ONLY


RANA CATESBIANA


PUCKETT 1939, 1940


COMPLETE TRANSFORMATION AT METAMORPHOSIS


ADMINISTERED WITH GONADOTROPIN


BUFO AMERICANUS


CHANG 1955


COMPLETE TRANSFORMATION AT METAMORPHOSIS


EFFECT NOT PERMANENT j


morphosis all tadpoles are females (Table 2.1). This result has been reported in R. esculenta (Padoa, 1942), in R. sylvatica (Witschi, 1951, 1952, 1953), and Puckett (1939, 1940) obtained the same effect in R. catesbiana when a gonadotrophin was given simultaneously with the estrogen. However, such transformations, although histologically complete, are not in all cases permanent (Gallien, 1955) ; moreover, the effects of high dosages of the same hormone may be quite different, as wdll be shown. For other species only partial transformations have been found, as in R. clamitans (jMintz, Foote and Witschi, 1945) and in R. pipiens (Foote, 1938). A similar type of incomplete transformation by the female hormone has recently been reported in the tree frog, Pseudacris (Witschi, Foote and Chang, 1958) , again suggesting that in their pattern of response to sex hormones the Hylidae resemble the Ranidae.

Various other anuran species have yielded divergent results. In the primitive frog, Xenopus laevis, complete and functional transformation of males is effected by an aqueous solution of estradiol benzoate (Gallien, 1953), and in the toad {Bufo americanus, Chang, 1955) low doses of estradiol completely transform testes into ovaries although high doses have little effect. Finally, in Discoglossus the effect of estradiol is purely feminizing but the transformation is incomplete, the males exhibiting all degrees of intersexuality without obvious relation to dosage (Gallien, 1955).

Paradoxical ejfects. Thus far emphasis has been placed chiefly on cases in which sex hormones have acted in a sex-specific manner, each type of hormone directly or indirectly promoting the development of structures of the appropriate sex, while inhibiting or behaving in neutral fashion toward those of the other. Reference has been made more than once, however, to the fact that in other cases the effects are just the reverse of theoretical expectation and opposed to the concept of hormones as specific sex-differentiating agents. Anomalous or paradoxical results of this kind have appeared in experiments with both types of hormone, and involve not only the gonads but other sex structures as well.

Such a result was first reported by Padoa (1936) who found that a crystalline form of female hormone (Crystallovar) had a strong masculinizing effect on the sexual differentiation of tadpoles of Bana esculenta, all gonads developing as testes. This unexpected result (the so-called "paradoxical effect") was confirmed by others and has been found to be usually associated with the use of high dosages. In the course of time it was shown in this and in two other species [Rana temporaria, Rana sijlvatica) that the same hormone (estradiol) may have diametrically opposite effects when administered in different dosages. As was emphasized earlier, low doses have a proper feminizing action, producing all female individuals according to theoretical expectation; with high dosages, on the contrary, only males are obtained, and at intermediate levels all individuals become intersexual (Padoa, 1938, 1942; Gallien, 1941, 1955; Witschi, 1952, 1953). Indeed, identical amounts of the same substance may have opposite effects when different solvents are employed. Administered in oil the effect on the gonads is feminizing but in aqueous solution complete masculinization occurs (Gallien, 1941). This also would appear to be a dosage effect since in aqueous solution the rate of uptake is presumably much faster than in oil. There are no histologic indications in the above experiments as to how the paradoxical effect is mediated. But, although such effects are found in various ranid species, they do not occur in Discoglossus regardless of dosage, thus emphasizing the importance of species differences in the phenomenon (Gallien, 1955).

Male hormones also produce paradoxical effects on the gonads and, although the doses employed have generally been high, it again appears that the result often depends on the species tested. The same dose of the same substance may have opposite effects in different species. Testosterone or cthinyl-testostcrone in large doses have strong feminizing effects on the testes of the salamander Pleurodeles, whereas the development of the ovaries is retarded but otherwise unaffected, a typical paradoxical effect (Gallien, 1950, 1955). Ethinyl-testosterone has the same effect in Discoglossus, but in Rana temporaria this hormone has only the expected masculinizing action. Such differences in response may arise from differences in sensitivity on the part of the gonads or gonad components; a dose which is relatively large for one species may not be so in the case of another.

The effects of sex hormones in urodele amphibians. In urodele amphibians the effects of sex hormones on the differentiation of the gonads are perhaps even more variable; however, as opposed to the situation in the Anura, it is the female hormones which are more effective in producing reversal than the male (Gallien, 1955). In only one instance, the newt Pleurodeles, has a functional transformation of sex been achieved (Gallien, 1954). In this species prolonged treatment with estradiol benzoate completely reverses the differentiation of all males, some of which become capable of laying eggs. Varying degrees of transformation have been reported in other urodele genera after shorter periods of treatment, in Amby stoma (Burns, 1938a; Ackart and Leavy, 1939; Foote, 1941) and in Hynobius (Hanaoka 1941a). There was great variation in the timing of treatment and in the dosages employed in these experiments; the incomplete character of the reversal may be due in part to such factors, but the role of species variability must also be great.

On the other hand the male hormone, in marked contrast to its dominating role in the Anura, has but a limited transforming action in Urodeles. Indeed, it frequently, but not always, produces paradoxical effects icf. Burns, 1939c; Foote, 1941; Bruner, 1952). In Pleurodeles, in which the males arc completely transformed by estradiol, the effect of testosterone is limited to a severe inhibition, which affects the gonads of both sexes but is more extreme in males (Gallien, 1955). Medullary development is almost completely suppressed, and after an interval of recovery the vestigial gonads give rise almost exclusively to rudimentary ovaries, a result mentioned pi'e\-i()usly in discussing paradoxical effects. In this case, however, there is clear histologic evidence as to how the effect is mediated. Reversal is caused indirectly by a severe inhibition of mesonephric development. Since the sex cords which give rise to the medulla of the testis are derived from the mesonephric blastema, inhibition of this tissue prevents their formation. In the absence of proper medullary development, the cortical rudiment of the testis eventually becomes active to produce an ovary. This, apparently, is another example of spontaneous differentiation of the heterotypic gonad component when released from domination.

A summary of the effects of steroid hormones in am'phihians. Sex hormones of adult type, such as testosterone and estradiol, have effects which vary greatly in different taxonomic groups of amphibians and also according to experimental conditions, such as dosage, timing, and duration of treatment. In many species their effects are specific to a degree, closely simulating the effects expected of natural hormones. Histologically complete and in some cases functional transformations of the gonads have been produced in a number of species, involving two orders and several families (Table 2.1). Nevertheless, in other species only partial or temporary reversals are obtained, and negative or even paradoxical results have come from use of the same hormones. Constitutional differences between taxonomic units obviously underlie some of the conflicting results. Sex genotype is also involved, because in a particular species reversal may proceed easily in one direction whereas in the other it is difficult or impossible to produce. Following in part Gallien (1955), the results may be tentatively grouped as follows:

a. In the higher anurans of the family Ranidae, and perhaps also in the Hylidae, the male hormone induces complete, and in many species a permanent reversal of sex. The action of female hormones on the contrary is highly variable; transformation may be incomplete or unstable, and with high dosages paradoxical or masculinizing effects often appear. On the other hand, loiv doses of the same hormone have in many cases proper feminizing effects in the same species. In one species (Rana catesbiana) complete reversal of sex in both directions has been obtained.


b. In certain urodeles and lower anurans, female hormones induce a transformation of male gonads which may be complete and stable, as in Pleurodeles and Xenopus, or partial, as in Discoglossus and various species of Ambystoma. The extent to which partial reversals are attributable to particular experimental conditions is uncertain. Male hormones in general are much less effective, and are prone to induce a paradoxical inhibition or a feminization of the testis. These effects have in some cases been shown to be mediated indirectly, through an inhibition of nephrogenesis which suppresses the differentiation of the medullary sex cords. The paradoxical effects of female hormones, on the other hand, are in many cases a matter of high dosages; how such effects are exerted is unknown but the action is probably indirect. This point will be discussed elsewhere in connection with the problem of paradoxical effects on other sex structures.

C. Sex Reversal in Avian Gonads

1. Orgariization of Avian Gonads

In avian as in amphibian gonads a specific morphologic basis for sex reversal exists during early development in the form of medullary and cortical components which have the usual potentialities. In birds, however, the situation is complicated by the peculiar lateral asymmetry which affects in some degree the entire genital system and which is especially pronounced in the female of most species (Fig. 2.12). The summary which follows is based primarily on the chick (for a fuller account see Willier, 1939). In the left embryonic ovary the preponderance of the cortex is great, even in the early stages, whereas in the rudimentary right ovary the cortex is essentially absent, being briefly represented by a transient germinal epithelium which disappears even earlier than that of the testis (Wolff, 1948) . In fact, the right ovary virtually ceases to develop at a stage when only medullary tissue (primary sex cords) has been laid down. In the male the asymmetry is morphologically less marked but it is expressed nevertheless in the better development and longer survival of the germinal epithelium (potential cortex) on the left testis. These



Fig. 2.12. Diagrams sliowing lateral differences in gonad organization in the chick embryo with respect to the representation of cortical and medullary elements; and differences in reaction to sex hormones based on these differences. (After N. T. Spratt, Jr., and B. H. Willier, Tabulae Biologicae, 17, 1-23, 1939). Note the stronger representation of the cortical component in the left gonad in both sexes, and its influence on the responses of gonads to hormones in relation to dosage. For details see text.


structural differences are correlated with different capacities for sex reversal under experimental conditions, as will appear.^

The experimental study of sex differentiation in birds has been limited largely to two domestic species, the chick and the duck. Histologic sex differences first appear in the gonads of these species around the seventh and the ninth days of incubation, respectively, but the future pattern of development is essentially determined much earlier. This is shown by the fact that when the sexually indifferent gonads of chicks are transplanted at the genital ridge stage to the choi'ioallantoic membrane of another embryo, they continue in most cases to develop independently, in accordance with genotype, giving rise to typical testes or ovaries, and in the case of gonads of female constitution to characteristic right or left ovaries as well

° The spontaneous reversal of sex that frequently follows removal of the dominant left ovary in the young female chick is an example, and the basis for this phenomenon lies in the predominantly medullary ciharacter of the rudimentary right ovary as described earlier.


(Willier, 1933, 1939j. The same capacity for self-differentiation has been demonstrated under the more radical conditions of isolation. Histologically undifferentiated gonads of either species when cultured in vitro differentiate into testes, or into right and left ovaries of characteristic structure (Wolff and Haffen, 1952a). In some cases there is injury to the germinal tissue under culture conditions; the gonads may show a reduction in the number of germ cells, or in some cases complete sterility, but otherwise the structure is normal (Fig. 2.13). Duck gonads seem to be more hardy under conditions of culture than those of the chick, and in general show better growth and liistologic differentiation.

2. Effects of Administering Pure Hormones

As noted earlier, sex reversal in the gonads of birds was not demonstrated experimentally until \)uve hormones became available. The first successful experiments were those of Kozelka and Gallagher (1934) ; Wolff and Ginglingcr (1935) ; Willier, Gallagher and Koch (1935, 1937) ; and Dantchakoff (1935, 1936) who introduced steroid hormones into incubating eggs before the beginning of sex differentiation. The results vary in detail but are consistent in the main outlines; they may be stated briefly, following chiefly the reports of Willier, Gallagher and Koch and of Wolff and Ginglinger.


Fig. 2.13. Histologic difYerentiation in the gonads of duck embryos developing in vitro, after isolation just at the beginning of sexual differentiation (Wolff and Haffen, 1952a). A. Normal form and histologic differentiation of the testis in comparison with an ovary developing under the same conditions (B). C and D show, respectively, the structure of these gonads under higher magnification. (From Et. Wolff and K. Haffen, J. Exper. Zool., 119, 381-404, 1952.)



Female hormones (estrone or estriol) do not significantly affect the differentiation of embryonic ovaries but testes are highly transformed. Because of the better development and longer survival of the germinal epithelium the left testis is more amenable to reversal than the right. Relatively low doses convert it into an ovotestis. The distal ends of the medullary cords become hollowed out into tubular structures like the medullary cords of the ovary (Fig. 2.14.4) ; at the same time a zone of cortex develops peripherally, arising as a proliferation of the germinal epithelium. A small, unchanged medullary mass usually persists at the hilus. But with larger doses even this may disappear and the cortex becomes much thicker.


Such cases are practically indistinguishable from ovaries. The right testis, however, is more difficult to transform. In the above experiments it was not greatly modified at lower dosages ; even when the left testis was almost completely transformed into an ovary the right never entirely lost its testicular character. Because of the poor development of the germinal epithelium its capacity to produce cortex is limited. However, Wolff (1948) made a special study of the right gonad in both sexes, assuming that stimulation of the gonad at an earlier stage, before regression of the germinal epithelium can be detected in either sex, might reveal a greater capacity for cortical differentiation. To insure rapid action a water soluble form of the hormone was used. In this way a considerable differentiation of cortex was obtained. The importance of a persistent search for the proper experimental conditions is again demonstrated.

Male hormones, on the other hand, are less effective in transforming the embryonic ovaries of birds. Again lateral differences in reaction are found due to the different liistologic constitution of the right and left primordia, and the results also differ when different forms of the male hormone are employed. After treatment with testosterone the cortex of the left ovary is reduced in thickness and shows degenerative changes; however, it does not entirely disappear. At the same time there is hypertrophy of the medulla and some of its cords acquire the solid structure of testis cords. The result is an ovotestis. Because of its predominantly medullary constitution the rudimentary right ovary is converted superficially into a testis-like gonad. The medullary mass hypertrophies and some of the cords arc transformed into testis cords. In general, larger amounts of hormone are required to transform ovaries than for the conversion of testes (for summaries see Willier, 1939; Wolff, 1950). After hatching there is a tendency for experimentally modified gonads to revert toward the original sex (Wolff, 1938) . Similar conditions of reversal have been produced in the embryonic gonads of ducks by hormone treatment (Lewis, 1946) .



Fig. 2.14. Hi.Ntolojiic m'X rraiisl'ormation in the te.sles uf cluck eiiil)iyu« lrcate(i with female sex hormones. A. Ovotestis produced from a left testis by treatment with a dose of 0.2 mg. of estriol. Note the presence of a thick cortex peripherally, and the reduction of the testicular tissue to a hilar mass of medullary cords. The intervening highly vacuolated tissue is characteristic of ovarian medullary cords. B. Section through the cortex and medullary region of a left tcslis coinplolcl}- transfoinied into an ovary by a dose of 2.0 mg. of estrone. Note the thick cdilrx (aboxc) coxcicd \>y a liciniinal epithelium, and the loss of structure in the medullary ii'gioii Ixlow. (From B. H. W'illior, in Sex and Internal Secretio7is, 2nd ed., The Williams & Wilkins Co., 1939.)




The problem of the jiaradoxical action of hormones presents itself again in the case of a^•ian gonads. Certain male hormones of ui'inary origin (androsterone, dehydro-androstcrone) have a marked feminizing effect, like that produced by t'cnuile hormones. In relatively large doses both substances induce cortical differentiation in testes, especially the left, which may be transformed into an" ovotestis (Willier, 1939; Wolff 1938). Other androgenic substances have like effects, but again it has been shown that they ai-e not jiroduced by low dosages. However, as the concentration of the hormone is raised the degree of intersexiiality and the number of intersexual gonads steadily increase (Wolff, Strudel and Wolff, 1948). Since various accessory sex structures also show paradoxical reactions the problem will appear again.

3. Effects of Grafting Gonads into the Coelomic Cavity

The demonstration that steroid sex hormones are capable of inducing sex transformation in avian gonads led to a reinvestigation of earlier failures to obtain reversal by means of chorioallantoic grafting. Eventually it was shown that the difficulty was largely a matter of the method. When gonad primordia are transplanted directly into the coelomic cavity of a host embryo of different sex, varying degrees of transformation, or even a virtual reversal, are obtained.

The first experiments of this type were only a partial success (Bradley, 1941). Embryonic gonads of the chick and the duck, isolated at 96 to 120 hours of incubation, were inserted into the body cavity of host embryos through a small slit in the somatopleure, using both homoplastic and heteroplastic host-graft coml)inations. In the case of the chick, host embryos were always considerably younger than donors. In all cases the grafts underwent primary sex differentiation in accordance with genotype, and only a small minority showed specific modifications. The results were rather inconclusive because in no case were the changes of a conspicuous character, and there was great variability in the growth and differentiation of the grafts, making it difficult to assess the significance of the modifications. In some cases changes of the same type appeared in the gonads of the host embryo.

The modifications noted by Bradley fall into three main classes. (1) Vacuolation of the medullary cords of testes growing in female hosts (in a few cases) caused them to resemble the hollow medullary cords of ovaries. (2) In some cases ovaries growing in male hosts developed solid medullary cords of male type. (3) Rudimentary right ovaries (always testis-like in character) had a tendency to become enlarged when growing in male hosts. A similar effect was sometimes seen in the right ovaries of hosts bear


ing testis grafts. No ready explanation was available for the inconstant occurrence of these effects or for their quantitative variability, because no clear correlation was found between the degree of modification and the relative proximity of the interacting gonads. Finally, similar changes appeared in a few cases when the host-graft combinations involved the same sex; consequently the specific character of the modifications was left in doubt.

The matter was clarified by the experiments of Wolff (1946) who used a modification of the method with better results. Grafts taken from older embryos (6 to 11 days) were implanted into hosts of about 50 hours of incubation. Under these conditions a striking transformation of gonad differentiation was obtained in the host, and in addition the developing gonaducts [q.v.) were strongly modified. Ovaries grafted into male hosts induced differentiation of cortex on the left testis to such an extent that it sometimes approached the structure of an ovary. The right testis (which was usually more distant from the graft) was less modified but its growth was inhibited. On the contrary, implantation of a testis in the same manner produced no important effect on the differentiation of the ovaries of the host but the development of the Miillerian duct was strongly inhibited indicating that the graft is endocrinologically active. In their histologic character the effects of gonad grafts are similar to those produced by crystalline hormones, differing only, as a rule, in being more localized in relation to the position of the graft. The demonstration in this experiment that, physiologically, the ovary is the dominant gonad in birds is consistent with the earlier observation that relatively larger doses of pure hormones are required for the transformation of ovaries than for testes.

These positive results after so many failures suggested that the ineffectiveness of chorioallantoic grafts in the earlier experiments was possibly a matter of hormone production, failure of the graft to maintain a sufficient level of the hormone in the blood. This view is substantiated by the later experiments of Huijbers (1951) who showed that multiple grafts of well differentiated testes on the chorioallantois have marked effects on the accessory sex structures of the host, similar to those induced by intra-embryonic grafts.

4. Sex Reversal in Vitro

More recently the technique of culture in vitro has been employed by Wolff and his collaborators with great success to study the development of embryonic gonads, and it has been possible to produce typical reversal of sex differentiation in vitro by two methods. Prospective ovary and testis of the chick or duck, isolated at the very beginning of sex differentiation and placed in close contact in the culture dish,^ become firmly fused, facilitating the transmission of humoral influences. As in the case of gonad grafts in the coelomic cavity, the ovary under such conditions proves to be the dominant gonad (Wolff and Haffen, 1952b). It readily induces cortical differentiation on the testis, which becomes an ovotestis, and may even approximate closely the structure of a normal ovary of the same age (Fig. 2.15). The same type of transformation occurs in testes after introduction of estradiol benzoate into the culture medium.

With respect to the histologic character of the reversal process, the resemblances between the effects of gonad grafts implanted in the body cavity, crystalline hormones injected into the whole organism, and the results of the same procedures applied to isolated gonad primordia in vitro are extremely close. The in vitro studies demonstrate again the autonomous character of the differentiation process, and its flexibility in the presence of extraneous hormones is shown to be independent of the organism as a whole. Hormones in vitro evidently act directly on the gonad mechanism.

D. The Problem uf Sex Reversal In Mammalian Gonads

1. Bisexual Potentialities in the Emhrijoiiic Gonads of Mam^nals

In marked contrast with the striking effects of steroid sex hormones on the differentiation of the gonads of birds and various

" Combinations of gonads in the culture dish must initially be made at random but the other gonad of each donor is cultured separately in order to establish its sex.


species of amphibians has been the failure thus far to ol)tain comparable effects in mammalian embryos with the exception of a single species, the North American opossum, a marsupial. Essentially negative results have been reported for a number of species of placental mammal, in which pregnant females were treated with relatively large dosages of sex hormones during the period of sex differentiation. Experiments of this type were carried out in the rat by Greene, Burrill and Ivy (Greene, 1942), and in the guinea pig (Dantchakoff, 1936, 1937), the mouse (Turner, 1939, 1940; Raynaud, 1942j , the rabbit (Jost, 1947 a) , the hamster (Bruner and Witschi, 1946; White, 1949), and the monkey (Wells and van Wagenen, 1954). With the single exception of the opossum (to be described later) the modifications induced are minor in character and are of three types: (1) a general retardation of growth and development of the gonads, without obvious signs of sex reversal, which occurs in both ovaries and testes and may be produced by either type of sex hormone;'^ (2) a variable degree of hypertrophy of the medullary elements of ovaries after treatment with male hormone, reported in only a few cases (Dantchakoff, 1939; Jost, 1947a; Wells and van Wagenen, 1954) ; and (3) the occasional persistence of localized patches of germinal epithelium on the surface of well differentiated testes, a condition which sometimes appears after treatment with either type of sex hormone. IVIinor changes of this character have not generally been accepted as convincing evidence of sex reversal.

Notwithstanding this array of negative findings, the failure of the embryonic gonads of placental mammals to respond definitely to sex hormones can hardly be attributed to an inherent lack of bisexual potentiality. During the early stages of their development they show, histologically, the same evidences of bisexual structure as the gonads of other vertebrates, although typically the bisexual phase is of relatively brief duration and the recessive sex component is often weakly represented (Fig. 2.16). In the ovary, medullary cords representing the male component are present but tend to become vestigial in most species.^ In the embryonic testis the germinal epi '^ Notable exceptions should bo mentioned in the case of certain species such as the mole (Godet, 1950) and the desman or "water shrew" (Peyre, 1955) in which, as a normal condition, the ovarian medulla is so strongly developed as to resemble a testis, and so active physiologically as to produce strong masculinization of many parts of the genital tract. A somewhat similar development and hypertrophy of the medullary component also occurs in the fetal ovary of the horse (for the literature on this unusual condition see Cole, Hart, Lyons and Catchpole, 1933; Parkes, 1954).


  • This effect is of common occurrence and is

best explained as a depression of the gonadotrophic function of the anterior pituitary, a mechanism whichis well established in adult organisms (Moore and Price, 1932).


Fig. 2.15. Sex transformation in the testis of the duck, isolated in vitro at the beginning of sexual differentiation and cultured in close contact with an embryonic ovary (Wolff and Haffen, 1952b). A. The ovary of such a combination, showing the thick covering germinal epithelium and the vacuolated condition of the medullary region. This young ovary is essentially normal in structure. B. Intersexual condition induced in the testis under the influence of the ovary. The heavy germinal epithelium representing the cortex is as well developed as in a normal ovary ; the meduUaiy region retains largely the compact structure of a testis, but signs of vacuolation are appearing. This gonad is an ovotestis. C and D represent, respectively, the ovary and the completeh' transformed testis in another experiment. The two gonads in this case show almost identical structure, featuring the thick cortex and vacuolated medulla of an ovary. In these experiments the other testis, cultured alone, developed normal testicular structure. (From Et. Wolff and K. Haffen, Arch. x\nat. microscop. et Morphol. exper., 41, 184-207, 1952.)



Fig. 2.16. Diagrams illustrating schematically the main features of gonad differentiation in amniote embryos with reference to the origin of the medullary and cortical components. A. Origin of the primary sex cords (medullary cords) from the germinal epithelium. B. Gonad at the indifferent stage of sexual differentiation ; the well developed primary sex cords i-epresent the male or medullary component, whereas the germinal epithelium represents, potentially, the cortical component. C. Differentiation of a testis consists in the further development of the primary sex cords, and the reduction of the germinal epithelium to a thin, serous membrane, accompanied by development of the tunica albuginea. D. Differentiation of an ovary consists in reduction of the primary sex cords to medullary cords of the ovary, whereas the cortex is formed by continued development of cortical cords from the germinal epithelium.




thclium usually disapi)ears early, and with its involution the potentiality for cortical development is permanently lost. On the other hand, hermaphroditismus verus not infi'ec|iu>ntly occurs as a developmental anomaly iu many mammalian species (including man), indicating the existence of a basic bipotentiality. As an example, an extensive literature dealing with this subject in rodents has recently been summarized by Hollander, Gowen and Stadler (1956) and Kirkman (1958). Also it must be remembered that the classsical example of an embryonic gonad transformed by the action of a sex hormone is found in the freemartin. In some freemartin gonads morphologic transformation may be extreme, although the resulting testis-like structure is histologically abnormal and is almost invariably sterile (Willier, 1921 ).»

2. Bisexual Potentiality in the Embryonic Ovary of the Rat

One of the best known cases illustrating a well marked capacity for bisexual differentiation in a mammalian gonad is provided by the embryonic ovary of the rat. The gonads of rat embryos have been isolated at various stages, both before and during the period of histologic differentiation, and transplanted to various locations in adult hosts of both sexes, normal and castrate, beneath the capsule of the kidney (Buyse, 1935; Mclntyre, 1956), subcutaneously (Moore and Price, 1942), to the omentum (Holyoke, 1949) and into the anterior chamber of the eye (Torrey, 1950). In general, differentiation of the transplanted gonad proceeds without reference to the sex or the hormonal status of the host (certain minor exceptions will be noted later) ; however, there is a great difference in the behavior of testis and ovary after transplantation with respect to their capacity for autonomous differentiation. There is virtual agreement among all investigators that the testis primordium from the beginning of its development possesses a remarkably stable organization, and develops more or less normally in the various foreign environments, even when isolated before the beginning of histologic sex differentiation.^*^ The case of the ovary is entirely different; its organization appears to be extremely labile and it is incapable of fully autonomous development until a relatively late stage of differentiation, after a well formed cortex is present. Before this stage (w^iich according to Torrey is reached about the 17th day of development) ovaries in a high percentage of cases either do not develop at all, or are prone to undergo spontaneous reversal, due apparently to incapacity of the prospective cortical component to develop effectively in abnormal tissue environments. On the other hand, the medullary component of the transplanted ovary suffers no such handicap and frequently assumes the lead in development. The embryonic ovary of the rat thus provides a flexible system for the study of the morphogenetic capabilities of the cortical and medullary components at different stages of development and under different experimental conditions.

^ For an important exception in which a freemartin testis is well supphed with germ cells and essentially normal in appearance see Hay (1950).

" Torrey considers that the self-differentiating capacity of the testis probably dates from the laying down of the early gonadal blastema, i.e., the material of the primary sex cords, which occurs as early as the eleventh day of development.



When transplanted early in development prospective ovaries may give rise (Buyse, 1935) to structures of four types: (1) poorly developed grafts of indeterminate sex, (2) atypical ovaries of retarded development, (3) ovotestes in which both sex components are readily identifiable, and (4) rudimentary testes. As a group, ovaries are adversely affected by transplantation. Some fail entirely to develop the specific structure of gonads (type 1, above) and those that do give rise to ovaries that are greatly retarded (type 2). On the other hand, the medullary component of the prospective ovary resembles the testis in possessing considerable powers of self-differentiation. Thus in many cases the two components develop together, resulting in an ovotestis; in still others the cortical element fails completely to survive and the medulla alone develops, giving rise to a rudimentary testis. The development of types 3 and 4 is favored by the fact that cortical differentiation is almost always severely repressed. This may have the effect of releasing the medullary element from an inhibition normally imposed by the dominant cortex. In all cases, however, the phenomenon of reversal was found to be unrelated to the sex of the host] it seems to occur spontaneously, as it were, in consequence of a disturbance in the normal balance between cortical and medullary systems.

A similar behavior is seen wdien entire reproductive tracts of rat embryos, including the gonads, are transplanted subcutaneously into hosts of various ages, male or female, and into castrate hosts of both sexes (Moore and Price, 1942). Again it was found thai the sex or the hormonal status of the host has no apparent influence on the result.

Testes develop normally except that in castrate hosts there is some hypertrophy of the interstitial tissue, presumably in response to the gonaclotrophin of the host (a phenomenon also reported by Jost, 1948b I. Again many prospective ovaries give rise to gonads in which both cortex and medulla are well differentiated, the hypertrophied medullary cords sometimes approaching the structure of testis tubules. Cells resembling the interstitial cells of the testis are also found around these transformed medullary cords in grafts developing in castrate hosts. On the whole the ovarial cortex is better developed than in the experiments of Buyse, because perhaps the gonads were usually older and better differentiated at the time of transplantation.

Similar forms of develojiment are found when embryonic gonads are transplanted to the omentum of adult hosts (Holyoke, 1949). Testes develop in a virtually normal manner regardless of the sex of the host; this author, however, describes certain effects which appear relatively late, after the testis has acquired its characteristic tubular structure. These are: (1) repression of tubule growth in some cases, with degenerative changes, a condition which was observed only in grafts growing in female hosts; (2) an increase in the amount of interstitium present, similar apparently to that reported by Moore and Price. Transplanted ovaries display the same variability as in the foregoing experiments; cortical development is adversely affected and sometimes fails altogether. In all cases in which cortical structure could be well identified there was also more or less hypertrophy of the medulla, sometimes to the point where the gonads were classified as ovotestes. This latter condition was reported only in male hosts, and this is perhaps the only change that might be interpreted as a reversal of sex conditioned by the sex of the host. On this point the findings differ from those of Buyse, of Moore and Price, and of Torrey, all of whom reported similar changes but without relation to the host's sex.

A further study of the problem was made by Torrey (1950). In his experiments the embryonic gonads were transplanted to the anterior chamber of the eye, using hosts of various ages and of both sexes. He con


firmed the main conclusions of Buyse and of Moore and Price (Holyoke's study was not then available for consideration), namely, that regardless of the stage at which the primordium is isolated, testes are capable of an autonomous and virtually normal development, irrespective of the type of host in which they develop, whereas ovaries vary greatly in the state of differentiation attained, depending on the stage of development at which they are isolated.

Torrey paid particular attention to the importance of developmental age in the fate of grafted ovaries. When transplanted before the appearance of a definite cortical zone (zone of secondary sex cords), prospective ovaries show little capacity for development of cortex; on the contrary (as found by l^revious workers), there is a marked tendency to hypertrophy of the medullary component, leading in some cases to testis formation. This tendency is not influenced by the sex of the host; rather, it seems to be inherent in the state of organization of the primordium at the time of transplantation. In the young ovary the medullary component (primary sex cords) is already in existence; the cortex does not appear as a discrete tissue until much later, and only after a well defined cortex is present is the gonad capable of development as an ovary. The fate of the transplanted ovary appears, then, to be primarily a iiiatter of the selfdifferentiating capacities of the elements already formed and present in the primordium at the time of its isolation. The development of these elements is influenced also by the temperature of the eye chamber, a point not directly involved in the present discussion.

In the foregoing experiments it has been emjihasized that the hormonal environment provided by the host seems to have no important influence on the sexual differentiation of the transplanted gonads (for a partial exception as noted above see Holyoke) . The question thus arises whether the hormones of embryonic gonads might be more effective. An answer to this question was sought by Mclntyre (1956) who transplanted the eml)ryonic testis and ovary together beneath the capsule of the kidney of adult castrate hosts of both sexes. The gonads were ])laccd in close contact in order to determine whether hormones or other diffusible substances might be produced, capable of modifying the differentiation process. As a control procedure, ovaries were transplanted alone, or in association with nongonadal tissues, into noncastrate male hosts.

The results in most respects correspond with those already described. The testis was found to develop normally regardless of the sex of the host or the presence of a contiguous ovary. The behavior of grafted ovaries differed, however, according to whether they were associated with an embryonic testis, or developed alone or with other tissues (control operations). In the jEirst case the ovaries were strongly modified along the lines previously described. Some differentiated poorly or hardly at all, others showed fairly good development of the cortex with some primary follicles, but in the medullary area tubular structures resembling testis tubules were found. Still others had a few well formed follicles in the cortex, but again tubular structures were present in the medulla which sometimes contained ovocytes. The two last mentioned categories would seem to correspond to the "ovotestes," or ovaries with "transformed medullary cords" described by previous writers. In contrast, however, ovaries grafted alone, or with nongonadal tissues, were found to differentiate in an almost normal fashion. It is at this point that the findings depart from those of other investigators. The conclusion was reached that the modifications observed in ovaries associated with embryonic testes are due to a substance produced by the testis, and considered to be of the nature of a medullary inductor.

However, both with respect to the severity of cortical inhibition and the degree of masculinization of medullary structures, these modified ovaries do not appear to differ significantly from those described by previous investigators when ovaries were grafted alone. The significance of the results rests then upon failure to obtain similar changes in the control ovaries. The age and state of differentiation of the control gonads at the time of transplantation is important. They were from donor fetuses of 15 days development, and although older than any used by Buyse they were still within the period during which Torrey found the ovary to be extremely labile in its differentiation. According to Torrey only a small proportion of ovaries aged 15 to 16 days developed as such (5 of 17 cases) whereas at an age of 17 days — after the cortical zone is established — ovaries are obtained almost without exception (10 of 11 cases). Further experiments are needed to clarify this matter.

In all of the foregoing experiments there was general agreement (for a partial exception see Holyoke) that the hormonal environment provided by adult hosts of both sexes, intact or castrate, has no important influence on the sexual differentiation of the transplanted gonads, even in the case of ovaries which are still in a labile state. The reason for this is not clear. Possibly it is a matter of insufficient concentration of the host hormone (compare the case of the chick, p. 99), or it may be that after transplantation a certain interval, perhaps a critical one, elapses before vascularization makes the graft accessible to the hormones of the host. On the other hand, it has been pointed out that in the embryos of placental mamtnals pure hormones thus far have produced no significant changes in the differentiation of the gonads. The cjuestion remains whether there is an essential difference between the sex hormones of adults and the hormones or sex-differentiating substances elaborated by embryonic gonads.

3. Experimental Transjormation of the Testis in the Opossum

Up to now the clearest experimental demonstration of sex reversal in the gonad of any mammal, and the only one to be produced by a steroid hormone, has been obtained in the gonads of young opossums (Didelphis virginiana) . In this species the embryonic testes, if taken in time, are readily transformed into ovotestes or even into "ovaries" of remarkably normal histologic structure by the action of estradiol dipropionate (Burns, 1950, 1955a, 1956b). The hormone is administered at short intervals, beginning at a stage of development corresponding to stage B in Figure 2.16. This is the condition found at birth in litters born at stage 34 of McCrady's series (McCrady, 1938). It is characterized by the presence of well developed primary sex cords which are just in process of separation from the overlying germinal epithelimii. The germinal epitheliimi, however, is still present as a layer of low, columnar cells and at this stage represents, potentially, the female component of the young testis. Normally the germinal epithelium does not survive long after birth. In the course of the first day of postnatal life irreversible changes occur which lead to its rapid involution. It is the presence at stage 34 of a viable germinal epithelium which makes possible the subsequent conversion of the testis into an ovary, since it is this layer which must produce the cortical zone of the transformed gonad by the proliferation of secondary sex cords. In so doing it plays precisely the same role as the corresi)onding layer in the development of the ovary.

Treatment with estradiol dipropionate has been carried out over varying periods up to an age of 30 days postpartum or somewhat longer, after which survival becomes difficult (Burns, 1939b). At the present time 46 male fetuses have been studied histologically, comprising all the surviving males of 13 litters. Without exception, every specimen shows histologic modifications of the type described below, the stage of transformation attained varying only with the length of treatment. The process of transformation consists at first of a gradual inhibition and suppression of testicular differentiation, accompanied by persistence of the germinal epithelium. At the same time the differentiation of the interstitial tissue is severely repressed (compare A and B, Fig. 2.17). Atrophy of the interstitial tissue has also been described by Raynaud (1950) in the testes of mouse embryos treated with estrogen. After an interval (which varies in different experimental litters and is apparently influenced by dosage) the germinal e})it helium again becomes active, producing secondary sex cords which form a cortical zone of varying thickness depending on the length of treatment (Fig. 2.181.

The first essential in obtaining transformation of the testis is the timing of the first treatment, which must not be later than stage 34 if the germinal epithelium is to be preserved. In earlier experiments, in which treatment was begun after stage 35, there were no significant effects on the differentiation of the gonads, even in cases where almost complete transformation of the accessory sex structures had occurred. It is now evident that in these experiments the first application of the hormone came too late to prevent involution of the germinal epithelium, thus precluding development of a cortex. Also of importance is the dosage, which must be kept at a low level to secure a good result. This point is crucial for the survival of germ cells in the developing cortex. High dosages always result in complete sterilitij of the cortical zone, even when this layer is otherwise well developed (Fig. 2.18). With lower doses, however, germ cells are found in limited numbers in the cortex, sometimes sparsely scattered, sometimes in small groups, and not infrequently these cells display the cytologic characters of young ovocytes (Fig. 2.20). Often there is a considerable growth of the cytoplasm and well formed primordial follicles are seen (Fig. 2.20B).




Fig. 2.17. The effects of female hormone on differentiation of the testis in young opossums. A. Normal testis about 10 days after birth. Note the thin, .serous character of the epithehum covering the testis (originally the germinal epithelium), the presence of a distinct tunica albuginea, the prominent testis cords (prospective tubules), and the richly developed interstitium. B. Testis (at somewhat higher magnification) of a young male aged 14 days, modified by the action of the female hormone estradiol dipropionate. Note the greatly reduced condition of the testis cords and interstitial tissue, the thick, spongy character of the tunica albuginea, and especially the survival of the germinal epithelium long after the stage at which it normally undergoes involution.




Fig. 2.18. A. Testis of an opossum aged 20 days, converted into an ovotestis by the action of estradiol dipropionate. Internally the reduced and disorganized medullary region is seen, separated from the external cortical zone by the well defined, fibrous tunic layer. The large, irregular ca\ity in the upper center, lined by a heavy eiuthelium, is the rete testis. B. Detail at higher power of the cortex, showing the structure of the cortical cords (which are sterile) and the highly developed germinal epithelium.




Treatment with relatively low doses of estradiol (of the order of 0.2 to 0.3 fxg. per day) from birth to an age of 20 days produces a remarkable transformation of the testis, which retains hardly any normal features (Burns, 1956b). Rather, it presents the appearance of a somewhat atypical ovary (Fig. 2.19B). The only remnant of testicular structure is a small, central nodule at the junction of the rete canals, and the massive cortical zone is covered externally by a thick germinal epithelium. The cortex of the transformed testis contains germ cells in considerable numbers, including a few large ovocytes. Views of cortical areas in gonads of this group are shown in Figure 2.20.4 and B. In more recent experiments, using still lower doses and a longer period of treatment (thus far the longest experiment has extended to an age of 33 days postpartum ) , the result is even more striking, the structure of the cortex in many cases approximating that of normal ovaries. Always, however, certain remnants of testicular structure ]icrsist in the medullary region (Fig. 2.21, compare A and B). The number of ovocytes in the cortex is enormously



Fig. 2.19. A. The testis of a normal male opossum aged 20 days for comparison with that of another male, B, treated for 20 days with a low dosage of estradiol dipropionate as described in the text. Only a remnant of testis structure survives as a nodular mass in the medullary region, representing straight tubules at the point of junction with the rete canals. Note the well developed cortical zone witli numerous germ cells and a heavy germinal epithelium.


greater than in the preceding experiment. It is not clear whether this is due mainly to the lowered dosage or to what extent it is a result of multiplication of ovogonia present in smaller numbers in the younger gonads (see Fig. 2.195). In any case, dosage in some manner influences the survival and multiplication of the gonia. Although the cortex of the transformed testis is always well developed, there is great variability in the extent to which testicular structures have survived in the medullary zone. Some gonads exhibit well preserved male sex cords and present the picture of a typical ovotestis, whereas in others (Fig. 2.21B} only traces of the male elements remain in the form of degenerate sex cords and patches of fibrous tissue. In these cases transformation is all but complete.

Since this work is still in progress interpretation must be tentative. It seems that the primary effect of the female hormone is a strong inhibition of the testis, affecting both the primary sex cords and the interstitium. Both influences are apparent at an early stage (Fig. 2.17). Inhibition of testicu


lar differentiation i:)resumably i-)ermits survival of the germinal epithelium, to be followed later by a renewal of activity producing secondary sex cords and the cortex. This course of events may simply be the result of release from an inhibition normally imposed by the differentiating testis. Counterparts are seen, for example, in the spontaneous development of the medullary component in transplanted rat ovaries when cortical differentiation is interfered with, in the development of the rudimentary cortex in Pleurodeles after the medulla has been suppressed by testosterone (p. 94), or in the development of the heterotypic sex component after castration in the toad or the newly hatched chick. In the light of current knowledge regarding the secretory capacity of the embryonic testis (for discu.ssions see Jost, 1953, 1957; Burns, 1955b, and later in this chapter) it is probable that the primary condition for survival of the germinal epithelium is suppression of the interstitial tissue; cortical differentiation is presumably the consequence of escai)e from an inhibition normally exerted by the testis hormone. For this there is no direct evidence; however, in typical ovotestes, with a well developed cortex, the tubular elements may also at times be very well preserved but the interstitium is degenerate. This interpretation does not require positive stimulation by the female






Fig. 2.20. A. View at higher magnification (X 1000) of the cortex of ;i traiisiorme.l te.stis containing gonia, and other germ cells showing the early meiotic prophase stages of young ovocytes. B. Cortex of another transformed testis of the same experimental group showing the formation of primordial follicles (X 1000).



hormone to promote cortical differentiation, but it does not exclude the possibility that this may occur. The germinal epithelium of transformed testes is strongly hypertrophied in comparison with that of normal ovaries of the same age (Figs. 2.18B, 2.195, and 20) , a condition which is seen also in the ovaries



Fig. 2.21. A. The normal ovary of a young female aged 30 days; note the highly developed cortex and the absence of conspicuous structures in the medullary area. B. Transformed testis of a young male treated with estradiol dipropionate for a period of 33 days ; for details see text. Observe the remarkable development of the cortex associated, however, with distinct remains of testicular structure in the medulla.


of females receiving estradiol. Also, precocious growth of a certain number of follicles commonly occurs in the cortex of transformed testes (Figs. 2.195 and 2.20). This effect, however, may be exerted indirectly in response to gonadotrophic stimulation.

It should be noted that in the only other case of an embryonic mammalian gonad transformed by hormone action, that of the freemartin, the reversal involves the conversion of ovary to testis. In the opossum the situation is reversed. In those cases of comjjlete, or near complete, transformation in amphibians, in which the sex chromosome complex has been determined, it appears that it is always the homogametic sex that is readily transformed (Gallien, 1955). This generalization would seem to apply also in birds and in the case of the freemartin. The opossum, however, is certainly an exception; the male in this species is heterogametic (Painter, 1922; Tijo and Puck, 1958; Graham, 1956). In certain fishes at least sex genotype is apparently of no conseciuence .-^ince functional sex reversal proceeds eciually well in either direction (Yamamoto, 1953, 1958).


V. The Role of Hormones in the Development of the Accessory Sex Structures

The heterogeneous character of the various structures comprising the genital complex of the embryo has been previously emphasized as providing a basis for great variability in their behavior under experimental conditions. On the basis of embryonic origin and morphologic relationships the accessory sex structures fall into three principal groups: (1) the embryonic sex ducts and related structures which are taken over from the primitive nephric system; (2) derivatives of the cloaca or the urinogenital sinus, derived at an early stage from the primitive gut; and (3) external organs of sex. Because of the great diversity of the so-called secondary sex characters in vertebrates, and because as a rule they become sexually differentiated only in jiostpubertal life, these structures can be considered only in special cases.

Two distinct stages can be recognized in the development of the accessory sex structures: an early phase which is independent of sex, and wliich follows a \-ii-tuallv identical course in all individuals, and a later phase, the period of sexual differentiation, which is chiefly hormone conditioned. During the first phase the primordial structures necessary for the development of both sexes are laid down and develop in similar or identical fashion up to a certain point, at which stage each embryo possesses, morphologically and for a certain time, the capacity to develop into an individual of either sex (Fig. 2.22A). In this early, indifferent phase of development the sex primordia show little reactivity to hormones, capacity for response evidently requiring a certain degree of maturation in the reacting organs or tissues. The onset of sexual differentiation of the accessory structures follows the appearance of sexual differentiation in the gonads, and this is the phase of development in which control by hormones is predicated.

A. Differentiation of the Embryonic Gonaducts

A complete account of the origin and normal development of the embryonic sex ducts has been given by Willier (1939) and Burns (1955b) . In the embryos of most vertebrates both sex ducts are present and equally developed throughout the sexually undifferentiated period. In many amphibians this primitive condition is retained throughout larval life or indefinitely ; the male, or Wolffian ducts, function as nephric ducts in both sexes and in females they are permanently retained in this capacity. The Milllerian ducts persist throughout life in the males of




Fig. 2.22. Early development and sexual differentiation in the genital tracts of young opossums. A. The lusrxu.il stage of development in a female embryo ±10 days of age, showing the paired gonaducts of both sexes, the sexually indifferent stage of the urinogenital sinus, and the undifferentiated genital tubercle or phallus. B. Male and female at about 35 days, when sexual differentiation is far advanced, showing the structures which develop from the primitive sex ducts, and dimorphic development of the sinus region. The phallus shows chiefly a difference in size, without marked morphologic divergence. (From R. K. Burns, Survey Biol. Progr., 1, 233-266, 1949.)



many species as complete if somewhat rudimentary canals. In amniote embryos, on the other hand, the ducts of the genetically recessive sex are typically transient structures. Sexual differentiation consists in the retention of one sex duct with development of its derivative structures, whereas the other either disappears completely or survives only in a more or less vestigial condition (Fig. 2.22B). Under these circumstances experimental reversal of sex, to be successful, must be properly timed with respect to the state of development of the heterotypic duct. Once regression has been determined it is impossible to preserve the duct.

1. The Mullerian Ducts: the Effects of Female Hormones

The effects of female hormones, whether produced by grafted ovarian tissue or administered in pure form, may be stated generally as follows: in female subjects as a rule they accelerate sexual differentiation, inducing a precocious hypertrophy of the Mullerian ducts which with large doses may become extreme. In males female hormones cause persistence of the Miillerian ducts followed by differentiation in varying degrees depending on timing of treatment, dosage, and the special status of the duct in the species under consideration. There are many deviations from this pattern, however, arising in part from basic group or species differences and in part from the many experimental variables."


  • For reviews and references to a large literature covering amphibians, birds, and mammals see Humphrey, 1942; Wolff, 1938; Willier, 1939; Ravnaud, 1942; Greene, 1942; Moore, 1947; Jost, 1947a, 1948a, 1955; Ponse, 1949; Burns, 1949, 1955b; Stoll, 1950.


In most amphibians the IVIiillerian ducts of both sexes respond readily to sex hormones during larval life. In Triturus {Triton) , after castration, both sex ducts remain indefinitely in a more or less undifferentiated condition, thus providing an ideal basis for sex reversal. Grafting gonads into castrates of either sex readily induces differentiation of the appropriate duct (de Beaumont, 1933). Furthermore, in the males of various species which have undergone complete sex reversal the later development of the Mullerian ducts is always in accordance with the altered sex of the gonad, and the ducts may eventually become completely functional (see e.g., Humphrey, 1942; Ponse, 1949; Gallien, 1955). The reaction of the Miillerian ducts to female hormones (estradiol, estrone) varies in different species and is greatly influenced by the stage at which treatment occurs and by dosage as well. In Ambystoma the ducts show a marked hypertrophy in females and the response in males is almost as great. The backward growth of the incomplete ducts is also accelerated (Burns, 1938a; Foote, 1941). Large doses, paradoxically, may arrest the backward extension of the duct (as do male hormones, q.v.) but the part already laid down becomes greatly hypertrophied (for a summary see Gallien, 1955).


The effects of the female hormone in bird embryos are particularly striking (Wolff, 1938, 1950; AVillier, 1939; Gaarenstroom, 1939; Stoll, 1948). In male embryos both oviducts persist and hypertrophy as does also the right duct of the female, which normally undergoes involution (Figs. 2.23 and 2.12). Once established these effects are permanent, development continuing even after hatching (Wolff, 1938). However, the period of susceptibility to the hormone is limited. Retention and permanent development can be assured only by treatment up to the seventh day of incubation (this is the socalled "stabilization effect" of Wolff) ; later treatment is without effect for the preservation of the ducts, irreversible changes having occurred which determine their regression with finality (Wolff, 1953b). The hormone of the embryonic ovary has the same effects. Ovaries grafted into the body cavity of male embryos cause persistence and development of\he oviducts (Wolff, 1946). The effect of the hormone appears to be a direct one since it occurs independently of any effect on the gonads.

In mammalian embryos the effects are in general similar, but marked species differences have been found. Female hormones cause accelerated development of the Miillerian duct derivatives in females, and with high dosages oviducts, uteri, and vaginal canals all show great hypertrophy. In male embryos the ducts are frequently retained and also differentiate regionally into oviduct, uterine tube, and vaginal canal (Greene, 1942; Burns, 1939b, 1942a and b; Moore, 1941; Raynaud, 1942; Jost, 1947a). Details of structure depend on the state of development of the rudimentary Miillerian ducts in the males of the species in question. The vagina may be defective or absent entirely in males of certain species, as in the opossum, in which the duct is usually incomplete, without a connection to the urinogenital sinus. In other species the effects are slight or lacking entirely (Raynaud, 1950, the field mouse; White, 1949, the hamster; Davis and Potter, 1948, man).



Fig. 2.23. The effects of sex hormones on development of the sex ducts in chick embryos. A. Normal female embryo of 18 days, showing development of the left oviduct with shell gland, and retrogression of the right. B. Genetic male embryo, 18 days, treated with 2.0 mg. estrone. Both oviducts are present and greatly hypertrophied. Compare with C for the normal condition. C. Normal male embryo at 17 days of incubation. Note the paired Wolffian ducts and absence of both oviducts. D. Genetic female embryo at 17 days, after treatment with 1.0 mg. androsterone, showing absence of both oviducts and extreme hypertrophy of the Wolffian ducts. For the normal female anatomy compare with A, and for normal size of male ducts see C. (From B. H. Willier, in Sex and Internal Secretions, 2nd ed., The Williams & Wilkins Co., 1939.)



The effects of male hormones. The effects of male hormones on the ]\liillerian ducts are more variable; strong inhibitory effects are obtained in many species and under proper experimental conditions; but with large doses stimulating or "paradoxical effects" often appear, such as have been described in the case of the gonads. The time of administration of the hormone is important. Both in chick embryos and in larval amphibians treatment with androgens before the appearance of the duct, or during the formative period, may result in total suppression.^^ In amphibians, in which the ]\Iiillerian duct develops slowly, a particularly interesting situation is found. Early administration of testosterone propionate prevents development entirely (Burns, 1939c; Foote, 1941, Amhystoma) or may leave only the ostial rudiment (Gallien, 1955, Pleurodeles) ; however, treatment during the period of formation may result in suppression of the unformed portion of the duct, while the part already laid down persists and with large doses may even be strongly hypertrophied (Mintz, 1947). Here is a striking paradox in which different regions of the same structure (which are, however, developmentally of different age) react in an opposite way to the same treatment.

  • See, for example, for amphibians, Burns, 1939c ; Foote, 1941; Hanaoka, 1941b: for the chick, Willier, 1939; Wolff, 1938, 1950; Gaarenstroom, 1939; Stoll, 1948; Huijbers, 1951. Exceptions must be noted, however, in a few cases: the field mouse (Raynaud, 1950); the hamster (Bruner and Witschi,'l946, White, 1949); man (Davis and Potter, 1948) in which no clear effects were observed, whether because of true species differences or other experimental variables is not clear.


In chick embryos also, early treatment with male hormone may completely suppress development of the Miillerian ducts (Figs. 2.23 and 2.12; see also Gaarenstroom, 1939; Stoll, 1948, 1950) and a similar effect is produced by grafts of the embryonic testis (Wolff, 1946; Huijbers, 1951). Again, however, suppression of the ducts depends on certain rather precise conditions, the dose must be adequate and the hormone must act at the proper stage of development. They can be suppressed completely before the 6th or 7th day of development (Stoll, 1948; Huijbers, 1951) but later treatment is ineffective. Thus (as was found also for the stabilizing effect" of female hormone on the Miillerian ducts of male embryos) there is a limited period of development during which the ducts are susceptible to inhibition by male hormones. In contrast with these clear-cut results, however, a paradoxical hypertrophic effect of certain male hormones (androsterone, dehydro-androsterone and related compounds) has been reported on the ^Miillerian ducts of chick embryos after rather large doses (Willier, 1939; Wolff. 1938; Wolff, Strudel and Wolff, 1948).

In the embryos of mammals effective inhibition of the Miillerian ducts by male hormones has not been found, but suppression of regional parts of the duct sometimes occurs. The ostial portion is suppressed in the hedge-hog (Mombaerts, 1944) and the vaginal segment (the last part to be laid down) is frequently inhibited in female opossums (Burns, 1942a, b) and in mice (Raynaud, 1942). In the mouse and in the rabbit failure of the posterior ends of the ducts to unite to form vagina and corpus uteri has been reported (Raynaud, 1942; .lost, 1947a). In female opossum embryos treated with testosterone propionate the vaginal canals are absent in about half of all cases and arc always absent in males (in which, as noted earlier, the terminal portion of the Miillerian duct is lacking) . However, a paradoxical stimulation of the Miillerian duct and its derivatives also takes place in opossums of both sexes when large doses (25 to 100 ^g. per day) are employed (Fig. 2.24; see also Moore, 1941, 1947; Burns, 1939a. 1955b) an effect which completely disappears when the dose is lowered to ±5 /xg. or less (Burns, 1942a, b).

In contrast with the failure of androgenic hormones to inhibit effectively the Miillerian ducts of mammalian embryos it is known that they are normally inhibited by the hormone of the fetal testis. In castrated male fetuses of the rabbit the ducts persist instead of regressing and develop almost as well as in normal females; conversely, the embryonic testis when grafted into a female fetus inhibits the Miillerian duct in the vicinity of the graft (Jost, 1953, 1955). On the other hand, a crystal of testosterone propionate implanted in the same manner lacks this inhibiting power. Testosterone also fails to inhibit development of the Miillerian ducts in castrate males although in all other respects it fully compensates for the absence of the testis (Jost, 1947b, 1953, 1955). This discrepancy has led to the suggestion (Jost) that in mammals another substance may be required for the inhibition of the Mullerian ducts. In the fetal rat, on the other hand (Price, 1956), neither testosterone nor the presence of the fetal testis influences the differentiation of the Mullerian ducts in genital tracts when isolated at an age of 17.5 days and cultured in vitro. In this case, however (as suggested by Price), it is likely that development of the Miillerian ducts has already been irreversibly determined before the time of explantation. This question will come up again in a discussion of the stage at which irreversible determination occurs in the rat.


The effects of castration on development of the Miillerian ducts. Although the effects of steroid hormones on the development of the Miillerian ducts are on the whole consistent with theory (failure of the ducts of mammalian embryos to be inhibited by male hormone is a notable exception) such results do not constitute evidence that sex hormones are present and active in the normal differentiation of sex. A direct test of this question is provided by castration of the eni])ryo. The effects of castration in amphibian larvae have been previously mentioned (p. 112); after removal of the gonads both sex ducts fail to differentiate further, persisting indefinitely in the condition in which they were at operation. In recent years this difficult operation lias been carried out in a number of species of birds and mammals.


Fig. 2.24. Diagrammatic representation of the effects of relatively large doses of testosterone propionate, administered from birth to an age of 50 days, on the development of the sex ducts in young opossums. A. Sex ducts as they appear in a normal male at 50 days; the vas deferens (Wolffian duct), epididymal tubules and remnants of mesonephric tubules are shown in black; the atrophic Miillerian duct is unshaded and the testis stippled. B. The effects of the male hormone on the male duct system appear throughout; however, large dosages also induce a paradoxical growth and development of the uterine and tubal regions of the Miillerian duct, but the vaginal segment is absent as in the normal male. C. The sex ducts as they appear in a normal female at 50 days — breakdown and disappearance of the Wolffian duct and associated structures, regional development of the Miillerian duct into tubal, uterine, and vaginal segments. The contribution of the urinogenital sinus to the vaginal canal is indicated in stipple. D. The effects of the male hormone in a female subject : note the preservation and great hypertrophy of the male duct system which is not, however, as large or as well differentiated as in the treated male; note also the striking paradoxical effect of a large dosage of androgen on the female genital tract which, at the same dosage level, is far greater than in the treated male (B). These paradoxical effects of androgen on the Miillerian duct dorivativos disappear entirely at lower dosages.



Early castration of avian cml)ryos is followed by persistence and development of Miillerian ducts in both sexes (the chick and the duck, Wolff and Wolff, 1951; Huijbers, 1951). After total castration in males both ducts persist and develop, but partial castration results in regression as usual. In females, in which the right duct normally regresses, both ducts persist and are well developed. ^^ Thus, in the absence of the gonads the Miillerian ducts follow the same pattern of development regardless of sex (Fig. 2.25). It is clear that the testes are necessary for normal inhibition of the ducts in male embryos, and it was pointed out earlier that a graft of the embryonic testis has the same effect in females (Wolff, 1946). The ovaries, on the contrary, have no positive role in the development of the Miillerian ducts, but actually inhibit the right duct; in their absence both ducts develop without hormonal conditioning.


  • In the rabbit (Jost, 1947b); the mouse (Raynaud and Frilley, 1947) and the rat (Wells, 1946, 1950); in the chick and the duck embryo (Wolff, 1950; Wolff and Wolff, 1951; and Huijbers, 1951).
  • In birds involution of the right Mullerian duct of the female is normally conditioned in some

manner by the ovaries, and it has been shown further that the presence of either ovary is sufficient (Wolff and Wolff, 1951). The exact nature of the inhibitory factor in this interesting case is not known.


Among mammalian embryos the case of the rabbit is best known (Jost, 1947b). Castration of the female again has no important consequences; the Miillerian ducts continue to develop and shortly before birth are only slightly smaller than in normal females. In male castrates, on the other hand, the situation is very different; if the operation is performed early enough the Miillerian ducts, instead of regressing, persist and develop, becoming practically indistinguishable from those of castrate females (Table 2.2, Fig. 2.26). Thus, as in birds, castrates of either sex follow identical patterns of development.



Fig. 2.25. Diagrams summarizing the effects of castration on the development of the syrinx (top row) oviducts, and genital tubercle (below) in bird embryos (chick and duck). The left column shows the normal condition in the male : note the complete retrogression of the Mullerian ducts (broken lines) under the influence of the embryonic testes (black). In the right column, the normal female condition showing the retrogression of the right oviduct. The arrows indicate the inhibitory action normally exerted by the ovaries on the syrinx, right oviduct and genital tubercle. The castrate type, which appears regardless of genetic sex, is seen in the center. In castrates both oviducts persist in complete form and are as well developed as the normal left oviduct ; the syrinx and genital tubercle, however, assume the male form in the absence of the inhibition exerted by the ovaries. Note the extreme asymmetry of the male syrinx and tubercle, as compared with the primitive symmetrical form retained in the normal female. (After Et. Wolff and Em. Wolff, J. Exper. ZooL, 116, 59-97, 1951.)

TABLE 2.2

Effects of castration in male rabbit fetuses studied at the age of 2S days (After A. Jost, Recent Progr. Hormone Res., 8, 379-418, 1953.)


AGE AT CASTRATION


MULLERIAN DUCTS


WOLFFIAN DUCTS AND DERIVATIVES


PROSTATIC GLANDS EXTERNAL GENITALIA


19 DAYS (2 coses)


PERSISTENT


20-21 DAYS (llcoses)


PERSISTENT CAUDAL REMNANTS

(in 3cases)


VENTRAL BUDS PRESENT


22-23 DAYS (Scases)


UTERO-VAGINAL SEGMENTS PERSISTENT


CAUDAL REMNANTS


HYPOSPADIC


23 DAYS (4 cases)


ABSENT- SMALL ABSENT (=NORMAL) SEMINAL VESICLES PRESENT


WELL DEVELOPED


24 DAYS (3 coses)


ABSENT ( = NORMAL)


WELL DEVELOPED


Unilateral castration is followed by normal development.

Castration effects are prevented by Testosterone propionate given at operation.


Nevertheless, if castration is delayed beyond a certain stage (about the 22nd day rather limited period during which involu of gestation in the rabbit; Jost, 1947b, c) tion of the oviducts is determined, as was the ducts subsequently undergo involution shown also for the chick in the case of male as usual (Table 2.2). Evidently there is a hormones administered experimentally.


Fig. 2 2b Ihc ctlLit^ ul e i^lialiou uu llif dux uluiniuul uf the sex ducts iii the rabbit. A. Persistence ot the Mullenan duct (uterine level) above, and the vaginal canal, below, in a castrate male. B. The same structures seen in a castrate female as compared with the condition in the noimal female shown in C . Note almost complete involution of the male duct except for remnants (CW) in the castrate male, and compare with the castrate and the normal female. Castration of the female (S) has little effect on the pattern of development which follows; on the other hand, castration of the male results in involution of the Wolffian duct and development of the Mlillerian duct, a reversal of the normal pattern. (From A. Jost, Arch. Anat. microscop. et Morphol. exper., 36, 271-315, 1947.)


Once conditioning has occurred the effect is irreversible.

In view of the decisive role of the embryonic testis in the development of the Mullerian ducts, as shown by castration experiments, it is significant to note the condition of the genital tracts in certain "lateral gynandromorphs" of genetic origin in mice (Hollander, Gowen and Stadler, 1956) which have an ovary on one side of the body and a testis on the other. Both gonads are usually small and hypoplastic. Without exception, however, on the side of the testis the Mullerian duct derivatives are absent entirely and the male duct system is developed. Contralaterally, a female genital tract is always found although it shows great variation in size. An almost identical condition has recently been described in a gynandromorphic hamster (Kirkman, 1958). The manner in which the influence of the testis tends to be limited to its own side of the body in these cases is of special interest, and will be considered later in dealing with the localized character of hormone effects. It is undoubtedly related to the early stage at which the testis begins to exert its effect and probably also to its reduced size in nearly all cases.

The development of the Mullenan ducts after isolation in vitro. At this point, in the case of mammalian embryos, an obvious question presents itself. Is development of Mlillerian ducts in male castrates in fact a purely autonomous process, due to release from an inhibition normally exerted by the testes or, in the absence of the gonads, does some other humoral factor intervene to assure their development? It is known that in various species of mammals estrogens are present in the placenta and fetal fluids in considerable amounts (for references see Price, 1947; Parkes, 1954), and the possibility arises that development of the ducts after castration may be maintained under the influence of an estrogenic substance of nongonadal or of maternal origin. This question has been answered, in the negative, by experiments designed to test the selfdifferentiating capacity of the ducts under conditions of physiologic isolation. When explanted in vitro (with pieces of the associated mesonephric bodies) the Mlillerian ducts of rat embryos, regardless of the sex of the donor embryo, survive and continue their development in the same manner as in castrate fetuses (Jost and Bergerard, 1949; Jost and Bozic, 1951). In these experiments the ducts were isolated at ages of 15 to 16 days, and 16 to 17 days respectively. Similar behavior is seen in grafts to the eye chamber of castrate hosts (the guinea pig, Bronski, 1950) and after transplantation of the entire embryonic genital tract into castrate and noncastrate hosts of various ages (the rat, Moore and Price, 1942). Although the experimental environments in all of these cases cannot be considered hormone-free in the strict sense (Jost and Bozic, 1951), the results indicate that the development of the female sex ducts when removed from the influence of the embryonic testis is a matter of autonomous differentiation.

The above experiments show that in rat embryos the fate of the JMlillerian ducts is undetermined up to an age of 16 to 17 days at least (Jost and Bozic), since at this age these ducts in male embryos still retain their capacity for autonomous development when removed from the inhibiting influence of the testis. Experiments on the same species (Price, 1956; Price and Pannabecker, 1956 1 indicate that involution is irreversibly determined at about the age of 17 days. The genital tracts of male fetuses were removed at 17.5 days of gestation and cultured in vitro. At this stage the first signs of involution can be detected in the region of the ostium but the posterior extremities of the ducts are still growing. After explantation the ducts continue to regress regardless of whether the testes are included in the explant or not, whereas those of female fetuses under the same conditions develop normally. It seems that the fate of the Miillerian ducts in the male becomes irreversibly fixed within the brief period of a day by exposure to the testis hormone.


This question has })een investigated in some detail in the chick. It had been shown earlier that after transplantation to the cliurioalhintois, Mullerian ducts from embryos of either sex differentiate completely if isolated before the beginning of sex differentiation. But if transplanted later than the 10th day of incubation the ducts of male embryos invariably degenerate; beyond this age their involution has been finally determined (Wolff and Ostertag, 1949). Making use of the technique of culture in vitro the analysis has been carried farthei' (Wolff and Lutz-Ostertag, 1952). When isolated before the appearance of sex differentiation in the gonads, again the Mullerian ducts of both sexes undergo a complete differentiation, as in castrate embryos; but if isolated after the 9th day the ducts of male embryos promptly undergo involution. However, if the Miillerian ducts of female embryos are cultured in the presence of an embryonic testis, a typical involution takes place (although at the same time the adjacent Wolffian ducts develop normally). Furthermore, when testosterone propionate is added to the medium it has the same effect as the embryonic testis (Wolff, Lutz-Ostertag and Haffen, 1952; for a summary see Wolff, 1953b). These results hardly leave the role of the male hormone in doubt. It follows also that the action of the hormone in vitro must be direct. Evidence has been obtained to show that the process of involution of the ducts is of the nature of an autolysis produced by the action of proteolytic enzymes which are presumabh' activated l)v the male hormone (Wolff, 1953b).

Summary and conclusions.

On the basis of present knowledge, the following general statements may be made concerning the role of sex hormones in the differentiation of the Miillerian ducts.

a. Female hormone, in most species, stimulates precocious growth and development of the Mullerian ducts and their derivative structures in embryos of either sex when given in adequate dosages. Administered at an early stage it prevents the normal involution of the ducts in male embryos after which development may continue without further treatment. In certain mammalian species, however (hamster, field mouse, man), negative results have been reported, which may possibly be related to the level of estrogen prevalent during gestation in these species or to dosage.

b. Male hormone, if administered early, has an inliibitory action on the Miillerian ducts in the embryos of birds and amphibians. In some species the primary de\-el()l>nient of the duct is entirely suppressed; in other cases only a partial inhibition occurs. In mammals, the hormone of the embryonic testis ]M-o(lnccs involution of the (hicts in males at tlic beginning of sex differentiation, and testis grafts inhibit the Miillerian ducts of females; on the other hand, male hormones of adult type fail to inhibit the ducts, or produce only a limited and localized involution in a few species.

c. The time factor is critical in the hormonal control of the jNIiillerian ducts. To suppress primary differentiation of the ducts in birds and amphibians, male hormone must be administered before or during the formative stage of development. However, the fully formed duct remains sensitive to hormones up to the onset of sex differentiation. Female hormone administered before this stage insures retention and development of the ducts in males, and during the same period they are subject to inhibition by male hormone or by the embryonic testis. Beyond this point the final development or the involution of the ducts has been irreversibly determined and hormones are without effect.

d. In the absence of previous hormonal conditioning, as after early castration or early isolation in vitro, the Miillerian ducts of either sex are capable of an autonomous differentiation, comparable to development in normal females; this development can be prevented, however, and involution induced, by introduction in vitro of an embryonic testis, or (in birds) by the addition of male hormone to the medium.

e. In amniote embryos the testis hormone is the controlling factor in the differentiation of the Miillerian ducts. The involution of the ducts in males is conditioned by the testes, whereas in the absence of gonads there is an almost normal development of the ducts in embryos of either sex. This does not mean, however, that the embryonic Miillerian ducts are insensitive to female hormone; in sufficient concentration it produces hypertrophic de^'elopment in both sexes.

f. The evidence from cultivation in vitro shows that the effects of hormones on the Miillerian ducts are exerted directly and are independent of the organism as a whole.

3. The Male Duct System

The male sex duct, or Wolffian duct, develops first as the duct of the pronephros (primary nephric duct) and subsequently serves as the mesonephric duct in both sexes. As the mesonephros is replaced by the metanephros in amniote embryos, the Wolffian duct loses its excretory function and its fate in the two sexes is different. In the female it disappears entirely or survives only as vestiges, but in males it acquires a new status as the male sex duct. At the same time a certain number of mesonephric tubules, which connect the duct with the rete canals of the testis, become a part of the epididymis, and the seminal vesicle develops as a diverticulum of the duct near its junction with the urinogenital sinus. During its early history as the duct of the mesonephros the Wolffian duct is unresponsive to sex hormones, but with the onset of sex differentiation it becomes responsive to the male hormone.

The role of male hormone in the differentiation of the Wolffian ducts. In the female of amphibians experimental transformation of ovaries into testes is followed by hypertro])hy and masculinization of the AVolffian ducts (Humphrey, 1942), and transplantation of testes into larval castrates of either sex has the same effects (de Beaumont, 1933). Administration of male hormones produces marked hypertrophy of the Wolffian ducts in larval amphibians of either sex [e.g., Burns, 1939c; Foote, 1941; for summary see Gallien, 1955). A similar response occurs in the embryos of birds and has also been reported in many species of mammals (Figs. 2.23, 2.24). ^^ This is the case in female embryos as well as in males. Development of epididymal tubules (from the epoophoron) also takes place in females (Fig. 2.24D) which also commonly develop seminal vesicles (Greene, 1942; Raynaud, 1942; Wells and van Wagenen, 1954 L With higher dosages all these structures undergo extreme hypertrophy.

Female hormones, on the contrary, appear to have little influence on the development of the Wolffian ducts, although in a few cases a jiartial involution of the ducts has

^" This effect has been widely reported : see Willier (1939), Wolff (1938, 1950) for the chick; Greene (1942) for the rat; Raynaud (1942), Turner (1940) for the mouse; Jost (1947a) for the rabbit; Godet (1949) for the mole; Burns (1939a and b, 1945a); and Moore (1941) for the opossum.


been reported (Raynaud, 1942; Greene, 1942). It should be noted that the latter observations are in mammalian embryos (mice and rats) in which the hormone of the testis (see below) is essential to insure retention of the ducts. Partial involution in males under the influence of a female hormone is possibly only a result of interference with the normal activity of the testis. On the other hand, a paradoxical action of female hormone, causing partial retention and hypertrophy of the male duct, has been occasionally reported (e.g., Greene, 1942; Moore, 1941). Although the method of administration in these cases does not permit accurate estimation of the dosage it was evidently rather large. With these exceptions, the effects of sex hormones on the male duct system are consistent with theory. The effects of castration on the development of the male sex duct are striking and agree with the results of hormone administration. They show that in all species the presence of the embryonic testis is necessary to induce sexual differentiation of the duct, and to insure its retention in mammalian embryos. In amphibian larvae (Triturus, syn. Triton) the Wolffian ducts persist after castration in their capacity as nephric ducts, but remain in a sexually undifferentiated condition (de Beaumont, 1933). In bird embryos also there is no significant alteration of the Wolffian ducts after castration. It is in mammals that the ducts become dependent on the testis and its hormone for survival as well as for sexual differentiation. In rabbit embryos castrated before the 22nd day of gestation (Jost, 1947b) the ducts in both sexes completely regress, following the female pattern of development (Fig. 2.26) ; involution in castrates is prevented, however, and normal development is maintained by prompt administration of male hormone. A more variable atresia of the ducts also occurs in fetal rats after castration (Wells and Fralick, 1951). In mice Raynaud reports a difference in reaction in the two sexes. After castration the Wolffian ducts of males undergo a complete involution but they may be partially retained in females. It is suggested that in this species the ovaries may play a positive role in the involution of the duct in females (Raynaud, 1950).


The development of the Woffian ducts in vitro. Further evidence that retention and sexual differentiation of the Wolffian ducts and associated structures (epididymal tubules and seminal vesicles) are dependent on the testis and its hormone is provided by the behavior of the male duct system after isolation, using the technique of organ culture. Development in isolation provides a parallel to development in the castrate fetus, with exclusion, however, of possible influence by hormones of maternal or placental origin or from some extragonadal source in the fetus.

When the mesonephric bodies of rat embryos, including long segments of the gonaducts, are removed at 15 to 16 days of gestation and cultured without the gonads, the Wolffian ducts of both males and females degenerate, but at the same time the Miillerian ducts survive and develop normally. The degeneration of the Wolffian ducts cannot, therefore, be due to unfavorable conditions in the medium (Jost and Bergerard, 1949) . The same result was obtained using slightly older fetuses of ±16.5 days (Jost and Bozic, 1951). However, the most complete study of this question is that of Price and Pannabecker (Price, 1956; Price and Pannabecker, 1956) who explanted male genital tracts of 17.5-day rat fetuses under various conditions designed to test the role of the embryonic testis and the male hormone. When both testes are included with the explant, development of the male duct system proceeds normally up to an age of 21.5 days (approaching term for the normal fetus I and the seminal vesicles develop as usual. Normal development ensues also when only one testis is left with the implant. But if one testis is removed and the lateral halves of the genital tract are spread widely apart on the surface of the medium, development is normal only on the side where the testis is present ; on the other side serious defects appear; the duct is thin and weakly developed, and the seminal vesicles are small or even lacking. Finally, if both testes are removed the Wolffian ducts regress completely. However, the addition of male hormone to such a prci^aration fully compensates for the al)scncc of the testes and development of the male duct system is again normal.



Fig. 2.27. The effects of castration on the development of the prostatic glands in the rabbit. A. The sinus region in a male fetus, aged about 27 days, castrated before the 20th day of gestation; above is the canal of the urinogenital sinus, below it the dark, bilobed structure represents the vaginal cord as it unites with the wall of the sinus. No sign of prostatic buds is seen. B. The sinus region in a young male of the same age castrated at about 21 days (20 days, 20 hours). Two large prostatic buds are seen ventral to the vaginal cord which were present at the time of castration. No further development has occurred. C. The sinus region in a male fetus of 28 days, castrated at the age of 23 days. Castration at this age is followed by essentially normal development. (From A. Jost, Arch. Anat. microscop. et Morphol. exper., 36, 271-315, 1947.)



Fig. 2 2S Hi-told.i In k lu ( - be twcin noimal h i i i i I , i i \ternal

genitalia {B) m i.ihlut htu^i^ aitcM -e\ual diffeientiation Mk uiinoj-nut il uu itu^ i-^ larger in the fem<ile, and is onh p.uth >uiioundcd b^ the pieputial fold, wliith maik'- off the glans clitoridis from the surrounding tissues. In the male the urethral cleft is narrower and completely enclosed within the preputial fold. The paired erectile bodies are seen above the urethral cleft. Castration at an early stage alwaj^s results in genitalia of female type (A) regardless of the sex of the castrated embrvo. (From A. Jost, Arch. Anat. microscop. et Morphol. exper., 36, 271-315, 1947.)


Altogether, the evidence clearly indicates that the male hormone is the essential determining factor in the survival and sexual differentiation of the male sex ducts and seminal vesicles. Notwithstanding the minor exceptions noted above, the female hormone evidently has little role. The reason for the insensitivity of the Wolffian ducts to the female hormone may possibly be found in their long phylogenetic history as nephric ducts in both sexes, in which capacity they must be retained in some groups beyond the period of sex differentiation or even permanently.


B. Derivatives of the Cloaca and Urinogenital Sinus

Sexual dimorphism of the amphibian cloaca chiefly takes the form of special cloacal glands which in males become highly developed at the breeding season, causing the prominent swelling of the cloacal region so conspicuous in males. In the females of various species they may be absent, present in a rudimentary state, or in some cases differently specialized (Noble, 1931). For their development and maintenance these glands depend almost entirely on the testis. After experimental transformation of sex, the subsequent differentiation of the cloacal glands


TABLE 2.3

Effects of hormones on derivatives of the urogenital sinus and the external genitalia in mammalian embryos

ACTION OF MALE HORMONE ON FEMALES


SUBJECT OF EXPERIMENT


FORM OF SINUS


VAGINAL DEVELOPMENT


SINUS EPITHELIUM


PROSTATE FORM OF GENITALIA


OPOSSUM


MALE TYPE


SUPPRESSED IN 50% OF CASES


HYPERTROPHIC* BUT NOT CORNIFIED


H 1 G H LY DEVELOPED


MALE TYPE


RAT


MALE TYPE


SINUS PORTION SUPPRESSED



HIGHLY DEVELOPED


MALE TYPE


MOUSE


MALE TYPE


SINUS PORTION SUPPRESSED



WELL DEVELOPED


MALE TYPE


RABBIT


MALE TYPE


SUPPRESSED



WELL DEVELOPED


MALE TYPE


MONKEY


MASCULINIZED


SINUS PORTION SUPPRESSED


STRATIFIED SQUAMOUS


LARGE OR VARIABLE


MASCULINIZEDPENIS-LIKE


ACTION OF FEMALE HORMONE ON MALES


OPOSSUM


FEMALE TYPE


SINUS PORTION

HYPERTROPHIC


VAGINAL TYPEHIGHLY 60RNIFIED


COMPLETELY SUPPRESSED


FEMALE TYPE


RAT


FEMALE TYPE


SINUS PORTION

WELL DEVELOPED



SUPPRESSED


FEMALE TYPE


MOUSE


FEMALE TYPE


VAGINAL CORD

WELL DEVELOPED


STRATIFIED SQUAMOUSMETAPLASTIC


SUPPRESSED


FEMALE TYPE


» r/7/5 effect appears only with large dosages


corresponds to the altered sex of the gonad. Castration of mature males results in retrogression of the glands and after early castration the cloaca remains sexually undifferentiated in both sexes (de Beaumont, 1933). However, testis tissue grafted into castrates (de Beaumont) , or treatment with male hormone {e.g., Burns, 1939c), readily induces development of male cloacal glands in individuals of either sex (for a review see Humphrey, 1942).

In the development of mammalian eml)ryos the urinogenital sinus is separated at an early stage from the cloacal region of the hind-gut by formation of the perineal septum. In its primitive condition the sinus is a short canal, extending from the neck of the bladder to the exterior, with a meatus at the base of the genital tubercle. The paired gonaducts open into it near the neck of the bladder (Fig. 2.22). Sexual differentiation in females chiefly involves anatomic and histologic changes associated with development of the vagina, to which the urinogenital sinus makes an important contribution; at the same time the male sex ducts regress and largely disappear (Fig. 2.22B). In placental mammals fusion of the posterior ends of the Miillerian ducts as they approach the urinogenital sinus gives rise to the unpaired, median vagina, but in marsu


pials the ducts remain separate and paired lateral vaginal canals are formed (Fig. 2.22B). In male embryos the main features of sinus differentiation are the involution of the ]\Iiillerian ducts with absence of vaginal development, and the differentiation of elaborate prostatic glands. Sex hormones show a high degree of specificity in their effects on the sinus structures, inducing development of typically male or female forms. Results are available for a number of species belonging to several orders of mammals,^ and are in agreement except for minor details (for some representative species see Table 2.3).

The histologic aspects of the differentiation of the sinus are well illustrated in young opossums. The effects of male hormone (testosterone propionate) in male and female pouch young are compared in Figure 2.29. The effect of the hormone in males is

^\See Greene (1942) for the rat: Raynaud (1942), Turner (1940) for the mouse; Jost (i947a) for the rabbit; Godet (1950) for the mole; Wells and van Wagenen (1954) for the monkey; Burns (1939a, b) and Moore (1941) for the opossum. Simimaries for these and other species are to be found in Colloques Intcrnationaux : La Differencialion Sexuelle choz les Vertebre.*, Masson et Cie., Paris, 1951. As an exception, the effects in the hamster are rather slight (Bruner and Witschi, 1946; White, 1949).




Fig. 2.29. The effects of testosterone propionate on the development of the urinogenital sinus and prostatic glands in young opossums. A. Extreme hypertrophy of the sinus and prostate in a male aged 50 days, treated from birth ; compare with the condition in a normal male of the same age (C). The effect of the same dose of hormone in a littermate female is shown in B. Female opossums normally never develop prostatic rudiments, as shown in D and E, representing cross-sections through the urinogenital sinus somewhat below (D) and at the point of junction {E) of the lateral vaginal canals (c/. Fig. 2.225). Note the great difference in the volume of prostatic tissue in the treated male (A) as compared with the treated female (B), although dosage and other conditions were the same.


merely to exaggerate the normal processes of development. With large doses there is a moderate hyperplasia of the sinus epithelium in males and a tremendous hypertrophy of the prostatic glands. But in females a striking deA'elopment of prostatic glands also occurs (although normally the female possesses no prostatic rudiments) together with a change in form, resulting in a sinus that is typically male. Quantitatively, these effects are proportional to dosage, but with the same dosage an interesting sex difference is constantly observed with respect to the magnitude of the response. The prostate (Fig. 2.29.4, B) is invariably more strongly developed in male subjects than in females. This difference in size apparently depends on an inherent difference in growth capacity in homologous tissues of different sex genotype when exposed to the same intensity of stimulation (Burns, 1942b, 1956a). This effect appears regularly in the case of many other sex structures of the


opossum as will be seen. The induction of prostatic glands and a male form of sinus is of regular occurrence in female mammalian embrvos exposed to male hormones (Table 2.3).'

Two special points concerning prostatic differentiation in young opossums are of interest. Brief treatment of female embryos with androgen, just at the time when the prostatic buds are appearing in males, is sufficient to induce buds which are then capable of continued differentiation after the hormone is withdrawn (Moore, 1945 ». This is an unusually clear case of permanent conditioning of a sex structure by brief exposure to a hormone at a critical stage in development. Also of interest is the fact that by gradually reducing the dosage of male hormone a level is reached, at approximately 5 /xg. per day, which induces prostatic buds in young females which are identical in size and appearance with those of normal males of the same age (Burns, 1942a) . Without attempting to allow for the constitutional sex factor mentioned above, this amount of androgen would appear to be roughly equivalent to the hormonal activity of the embryonic testes at this period.

Female hormone has opposite effects on the urinogenital sinus and its derivatives in young opossums. Estradiol dipropionate completely suppresses prostatic differentiation in males and transforms the sinus epithelium into a stratified squamous epithelium of vaginal type (Fig. 2.30) ; in fact the histologic picture is one of intense proliferation and cornification like that of the adult vagina at estrus. Moreover, a single dose of estrogen administered during the 15th day of pouch life, shortly before the prostatic buds would normally appear, results in complete suppression of the prostate, an effect which is also permanent (Burns, 1942a, b, c) . Thus, there is a relatively short period during which induction and continued development of ])rostatic glands in females, or their permanent suppression in males, is wholly conditioned by the presence of the appropriate hormone. Quantitatively as well as qualitatively the reactivity of the embryonic sinus epithelium to estradiol is remarkable. Again a sex difference, as measured by growth and proliferation is seen, this time in favor of the female. Transformation to a typical vaginal epithelium in the estrous phase can be induced in very young male embryos, long before the time of appearance of prostatic buds (Fig. 2.31; Burns, 1942c). It is hardly surprising that an epithelium of this type permanently loses all capacity to produce prostatic tissue.

The effects of castration on the development of the urinogenital sinus and its derivatives in mammalian embryos follow the pattern previously described for the sex ducts. The male form of sinus is incapable of developing in the absence of the testes, whereas morphogenesis of the female form is not significantly affected by castration



Fig. 2.30. The effects of estradiol dipropionate on the development of the urinogenital sinus and prostate in young opossums. A. The normal sinus of a young male aged 30 days, showing the condition of the prostatic buds, for comparison with a normal female of the same age (fi). Note the bilobed form of the sinus canal in the male as compared with the typical pentangular form in the female. C. The effect of the female hormone in a male littermate of the same age, treated from the time of birth. Complete suppression of the prostatic glands has occurred, the form of the sinus canal is typically female, and the sinus epithelium has been transformed into a thick stratified squamous epithelium, like that of the adult vagina, in a state of pronounced keratinization and desquamation. The effect of an identical dose in a female subject is similar but much more intense.



Fig. 2.31. The effects of a stronger dosage of estradiol dipropionate on the urinogenital sinus at a much earher stage. A. Young male treated from birth to an age of about 12 days, for comparison with the normal male sinus (inset, B) of the same age. Compare the character of the sinus epithelium with that in the older specimen shown in Figure 2.30C This condition of the sinus epithelium is induced at an age which precedes by se^■eral days the normal appearance of the prostatic buds, which never develop.


(Table 2.2; Jost, 1947b; Raynaud and Frilley, 1947; Wells, 1950). In castrate males prostatic differentiation is prevented and development of a vagina (correlated with persistence of the Miillerian ducts in male castrates) results in a sinus of female type (Fig. 2.27 A). Male and female castrates are morphologically very similar, and both closely resemble the normal female. Again it is clear that the embryonic testis is the essential factor in male development, whereas the female pattern is independent of hormonal conditioning and also of sex constitution, since in the absence of the gonads it develops spontaneously in castrates of either sex.

The factor of time is again of paramount importance and sharply limits the effectiveness of castration. This holds for the development of other accessory structures (Table 2.2) but is particularly clear in the case of the prostate which will serve to illustrate. In male rabbit embryos castrated on or after the £3rd day of gestation there is only a


slight effect on prostatic development, which continues in a practically normal manner; but if the operation is performed a day earlier there is a distinct reduction in size. In fetuses castrated from the 20th to the 21st day only small ventral buds are found which are already formed at the time of operation, and after castration earlier than 20 days prostatic buds are absent altogether (Fig. 2.27; Jost, 1947b, c). The period from 20 to 21 days, then, is critical for the appearance of the prostatic buds and their further differentiation for which the embryonic testis is essential. However, absence of the testis is fully compensated for by male hormone ; in castrates receiving androgen the development of all male parts proceeds normally. A similar result has been obtained in the fetal rat (Wells, Cavanaugh and Maxwell, 1954). Late castration has little effect, but castration on day 18 results only in buds which do not undergo branching. Earlier castration has not proved feasible in this species.


Fig. 2.32. The effects of sex hormones on the sex type of tlic copulalory stniciur(-s in young opossums. A. The appearance of the phallus, or genital luljerclc, ui ;i normal male (left) and female opossum aged 20 days. Sex is difficult to distinguish by form alone but the phallus is somewhat larger in the male. Sex is readily distinguished at this age, however, by


C. External Genital Structures

Copulatory organs homologous with those of higher vertebrates are not found in amphibians. They are developed to an extent, however, in certain birds and reptiles and become highly specialized in mammals. The copulatory organ in amniote embryos develops from a simple primordium, the genital tubercle, which is common to both sexes. It becomes specially developed as the penis in the male but in females it persists in a more or less rudimentary form, known in mammals as the clitoris. The genital tubercle of birds arises as a small, conical protuberance just within the cloacal orifice. In chickens it is not highly developed, although larger in the male than in the female, but in the males of ducks, geese, and certain other birds it becomes considerably larger and more modified, constituting a penis (Fig. 2.25). In the embryos of mammals (except the Monotremes) the genital tubercle is external in position, arising as an eminence near the ventral rim of the urinogenital meatus.

The developing copulatory organs of birds and mammals react readily to sex hormones and are extremely sensitive to castration. In birds the clearest experimental results have been obtained in duck embryos, because of the more pronounced sexual dimorphism in this species. Treatment with female hormone (estradiol benzoatej before the 12th day of incubation completely arrests development of the penis in males, and the rudimentary clitoris of the female may be even smaller than normal (Wolff, Em., 1950 ) ; beyond this age, however, the hormone is no longer effective, the form of the prospective penis having been finally determined. The effects of the male hormone are less precise and it is not essential for normal development (see the effects of castration below) . Testosterone proprionate produces great hypertrophy but the structure is not entirely normal, the characteristic spiral form of the penis being imperfectly developed (Wolff, Em., 1950). This abnormality is perhaps a result of overdosage as the dosages used were undoubtedly very large. Although in the chick the dimorphism of the genital tubercle is less pronounced than in the duck, it reacts in the same way ; male hormones stimulate and female hormones inhibit growth and morphologic differentiation (Reinbold, 1951).

In mammalian embryos the sex type of the developing genital tubercle, or phallus, is easily controlled by sex hormones (Table 2.3) ; in fact, this structure is unusually susceptible to modification and may be completely transformed. Typical are the results in the rat (Greene, 1942), the mouse (Raynaud, 1942; Kerkhof, 1952), the hamster (Bruner and Witschi, 1946) and in pouch young of the opossum (Burns, 1939a, b; Moore, 1941). The rat and the mouse are similar in their behavior. Male hormone does not affect the development of the penis in males except to produce hypertrophy, but in females the genital tubercle is greatly enlarged and assumes the character of a penis.^' Female hormone has opposite effects; females differentiate normally but in males the tubercle fails to enlarge and a hypospadic condition frequently appears.

In young opossums the form of the copulatory structures is completely controlled in accordance with the type of hormone given, with results which are identical in the two sexes except for a difference in size (Fig. 2.32). The basis for the transformation of the phallus has been analyzed histologically (Burns, 1945b). The various histologic con ^' Strangely enough this is not tyi^ically the case in freemartins. The chtoris as a rule is not greatly modified (Lillie, 1917). There are, however, some striking exceptions (Buyse, 1936; Numan, 1843, illustrated in Lillie, 1917, Fig. 29).


the scrotal sac in males and the presence of the pouch folds and mammary rudiments in females. B. The typical form produced by male hormone (left) and female hormone in specimens treated from birth to an age of 20 days; the normal condition at this age is shown above. This striking difference in form is produced without regard to the sex of the subjects, which in this case are both male (note the scrotal sacs). C. The result of administering male hormone (testosterone propionate) from birth to an age of 50 days, in a female subject (left; note the pouch) and a male littermate. Observe the identity in form but distinctly greater size of the penis in the male. D. Comparison of the effects of estradiol dipropionate, given from birth to an age of 30 days, in a female subject (left) and a male littermate. In both C and D it is shown that the hormones produce genitalia of typical male or female form, regardless of the sex of the subject.


stituents of the organ respond to the appropriate hormone in a highly specific manner. The erectile bodies, the development of which largely determines the form and the size of the penis, are strongly stimulated by male hormone and almost entirely inhibited by female hormone (Fig. 2.33j. Qualitatively, these responses are independent of sex constitution, but with identical dosages marked differences in size, such as were


noted previously in the case of the prostate, the sinus epithelium and derivatives of the sex ducts (Figs. 2.29 and 2.24) , are again observed in the two sexes. Female hormone, in addition to inhibiting the erectile tissue, induces an extreme hyperplasia of the vulvar and periurethral connective tissues (Fig. 2.335). It is this response which produces the gross swelling of the vulvar region, so conspicuous in estrogen treated embryos of


Fig. 2.33. The effects of male and female hormone^ on the differentiation of the histologic constituents of the phallus in young opossums. A. The effects of androgen in a male, aged 30 days, treated from birth onward. There is great hypertrophy of the erectile bodies but otherwise structiue is normal; at the top, the paired corpora cavernosa are imited at the mid-line ; below, the urethral canal, with the bulbo-urethral glands on either side ; laterally, the large bulbs of the corpora spongiosa, with their muscular investments. B. The effects of estrogen in another male littermate. The erectile bodies are almost completely suppressed and there is an enoimous hyperplasia of the periurethral connective tissue. The urethral canal (urinogenital sinus) is greatly enlarged, as in a female, and the sinus epithelium is transformed into stratified scjuamous epithelium like that of the fully developed vaginal canals. The sex of the subject makes no difference in the character of these responses.


both sexes (Fig. 2.32). With increasing dosages all these effects are accentuated.

The phallic structures of mammalian embryos react to castration according to the pattern already established for the sex ducts and the prostate (Jost, 1947b; Raynaud and Frilley, 1947). In both sexes castration is followed by development of external genitalia of female type (Fig. 2.28; Table 2.2) ; the male type of differentiation is dependent on the testis whereas the female form is capable of developing without hormonal conditioning, in a somatic or asexual manner.

At this point it will be useful to recapitulate for mammalian embryos the effects of castration, or of early isolation, on the development of the genital system as a wdiole. It has been shown that in the absence of the gonads, or of any hormonal conditioning, the embryonic sex primordia collectively follow the female pattern of development. In all castrates, regardless of sex, the external genitalia and the derivatives of the urinogenital sinus are of female type, the INIiillerian ducts persist and continue to develop in a virtually normal fashion, whereas the Wolffian ducts undergo involution. Thus castrates of either sex toward term have female genital systems which are anatomically complete and almost as well developed as in normal females.

It is noteworthy that the pattern of development observed in castrate fetuses corresponds closely with a condition in human subjects known clinically as gonadal dysgenesis. Individuals presenting this anomaly either lack gonads entirely or show evidences of gonadal atresia at an early stage of development. Regardless of chromosomal sex as established by the Barr test (Barr, 1957) they possess external genitalia of female type and female genital tracts which, however, are of infantile proportions. Recent evidence indicates that some individuals of this type may lack the Y-chromosome, being of XO constitution (Ford, Jones, Polani, de Almeida and Briggs, 1959; chapter by Gowen) .

In bird embryos the effects of castration on the genital tubercle are similar except that the sex relation observed in mammals is reversed ; in this group the male form of the organ corresponds to the asexual condition, which develops without hormonal conditioning in castrates of both sexes (Fig. 2.25; Wolff and Wolff, 1951). This is not an exceptional finding; it corresponds with the behavior of various other avian sex characters, such as the syrinx {q.v.) , the spurs, and the sex plumage in species such as domestic fowl. The transposed relationship seen here is in line with the dominant role played by the grafted ovary and the greater potency shown by the female hormone in producing sex reversal in the gonads of the chick.

The developmental behavior of the genital tubercle after isolation in vitro has been studied in the duck, with results which correspond with those of castration. Isolated at 7 to 9 clays of incubation, before the beginning of sex differentiation in the gonads, primordia of the genital tubercle always assume the male form as in castrates, regardless of the sex of the donor. By the 10th day, however, the sex type has become fixed, and when isolated after this stage differentiation always follows the sex genotype (Wolff and Wolff, 1952b).

D. Differentiation Of Other Types Of Sex Character

Two further examples will be considered as illustrations of the role of hormones in the development of sex characters of quite different type, the mammary glands, and the syrinx of birds. The mammary glands of field mice have been extensively studied by Raynaud (for a summary see Raynaud, 1950). The rudiments of the glands first appear as bud-like ingrowths of the epidermal epithelium which penetrate the underlying mesoderm but retain a connection with the epidermis by a constricted neck (Fig. 2.34.4, B). This phase of development follows the same course in both sexes. Toward the 16th day of gestation differences appear in males which coincide with the beginning of masculinization of the female genital tract; the mammary buds lose their connection with the epidermis and remain as isolated epithelial nodules in the mesenchyme (Fig. 2.34C) . In females, on the contrary, the buds retain their attachment to the epidermis, and as development continues a circular fold appears surrounding the mammary rudiment, which leads to elevation of the nipple.


130


BIOLOGIC BASIS OF SEX



SS^M




•K^« ;'^ vVi^^#-p^ r-^i -^^-"i^^^^f^^ 7,


^^^^s^^^^i^^ss ^^^^^^^^^^^^




Fig. 2.34. The normal development of the mammary rudiments in embryos of the field mouse, and the effects of sex hormones (for a summary see Raynaud, 1950). A. Early appearance of the mammary thickenings in the female (left) and the male (right). B. Later stage, showing growth of the mammary primordia and penetration into the mesenchymal layer. C. Stage of sexual differentiation: in females the mammary rudiment remains attached to the epidermis and the nipple later develops at this point; in males the rudiment becomes detached from the epidermis and persists as a small epithelial nodule in the underlying mesenchyme. For the effects of hormones and of castration on this pattern see text. D. Nipple development in a male embryo induced by treatment of the mother with estradiol dipropionate. For details see text. (After A. Ravnaud, Arch. Anat. micro.scop. et Morphol. cxper., 39, 518-569, 1950.)


Treatment with sex hormones during the latter half of gestation shows that the processes described above are readily controlled or reversed experimentally. In female fetuses of mothers injected with male hormone, development of the mammary gland follows the male pattern; the mammary buds separate from the epidermis and persist only as nodular rudiments, the nipple fold does not appear and nipples fail to develop. The female pattern of differentiation is converted completely to the male type. The use of female hormones, on the other hand, leads to a somew^hat paradoxical result ; there is an inhibition of the mammary buds rather similar to that exerted by androgen, but nipple development on the contrary is strongly stimulated (Fig. 2.34D). The dosages were large, however, and the effects of female hormones under more physiologic conditions have not as yet been determined. With respect to development of the nipple similar results have been reported in the laboratory mouse (Greene, 1942); male hormone completely inhibits the nijv ples in females whereas female hormone induces typical development in males.

Of particular interest are the effects of castration on mammary development in mice. When the embryonic gonads are destroyed by irradiation the mammary glands continue to develop in both sexes according to the normal female pattern (Raynaud, 1950). This pattern obviously does not depend on the ovary but represents the asexual or anhormonal type of development. Its appearance in castrate males indicates that the regression of the mammary glands in the male is normally determined by the testis. This is in agreement with the results of administering male hormone, as described above. Once more the predominant role of the male hormone in mammalian sex differentiation is demonstrated.

An entirely different type of sex character, and one which exhibits sexual dimorphism in a striking way, is the syrinx of birds. This organ has received special study in the duck (Wolff, Em., 1950) . The syrinx makes it appearance as a vesicular dilatation at the junction of the trachea and bronchial tubes, and at first it is small and symmetrical in form in both sexes. This is the permanent


condition of the syrinx in the female but in males a pronounced asymmetry soon appears, involving an enlargement of the left side of the vesicle with corresponding modifications of the cartilaginous rings. By the 10th day of incubation the asymmetry is extreme (Fig. 2.25). The appearance of dimorphism follows closely the beginning of sex differentiation in the gonads.

Experimental studies have shown that the dimorphism of the avian syrinx is conditioned by the ovary, or by the female hormone (Wolff, Em., 1950). Estradiol benzoate, introduced into incubating eggs, inhibits the development of the male syrinx and the female form appears. However, if large doses are used, or if treatment is too long delayed, a paradoxical result appears, consisting in the development of atypical and intermediate forms (Lewis and Domm, 1948). ]Male hormone (testosterone propionate) in moderate dosages has little effect on the syrinx (a slight enlargement may occur) but with large doses a paradoxical tendency is again found; the male syrinx is inhibited and may actually be reduced in size, resembling somewhat the female form.

Castration again reveals the dominant role of the female hormone in birds. In castrates of both sexes the form of the syrinx is male, both in size and in its asymmetry (Fig. 2.25) ; absence of the ovary is critical but the presence or absence of the testis is of no consequence in the sexual differentiation of this structure. In its response to castration the syrinx thus behaves like the genital tubercle.

The differentiation of the syrinx has also been studied in vitro (Wolff and Wolff, 1952a; Wolff, 1953a) with similar results. When explanted before the onset of sex differentiation, the result is the same as after castration; in an anhormonal environment the male form develops without regard to the sex of the donor. When explanted after the beginning of sex differentiation, however, development proceeds always in accordance wuth genotype. At this stage the form of the female syrinx has already been irreversibly determined. The syrinx developing in vitro responds directly to sex hormones introduced experimentally. Addition of estradiol benzoate to the culture medium has the expected result; regardless of sex constitution, only the female type of syrinx develops. But male hormone (testosterone proprionate) under the same conditions produces, paradoxically, organs of female or of intermediate form. The dosages employed, however, were extremely large (5 mg. and 40 mg. per cc. solvent) ^^ and in the light of the effects of large doses of androgen on the gonads and other structures the anomalous result is not surprising.

VI. The Pituitary and the Differentiation of Sex

It does not appear that the anterior lobe of the pituitary is concerned in the primary differentiation of sex, i.e., in the morphogenesis of the gonads themselves. Early hypophysectomy does not interfere with their histologic differentiation, up to the stage at least where they are fully characterized as ovaries or testes; neither is the process of differentiation appreciably delayed. Later, however, deficiencies of a secondary order may appear in the genital tracts of hypophysectomized animals; the development of various accessory sex structures may be considerably retarded but without any essential change in character. This effect is explainable as the result of diminished secretory activity on the part of the gonads through lack of adequate gonadotrophic stimulation. The question of when the functional interrelation between the gonads and the anterior pituitary is established has been reviewed by Willier (1952, 1955) with special reference to the chick, and .lost (1953, 1955) has dealt with this problem in mammalian embryos and fetuses. In each case gonadotrophic activity is evident shortly after the period of gonad differentiation and covers the period when the sex ducts and other accessory structures are differentiating. Some of the evidence on which these statements are based will be briefly reviewed.

In amj)hibian embryos or early larvae,

^* Under the conditions of culture the exphints develop in close contact with droplets of the hormone solution, and may actually be exposed to extremely high concentrations. Culture with the oil solvent alone, however, shows that the solvent is not the disturbing factor.


hypophysectomized long before the time of sex differentiation, the development of ovaries and testes proceeds normally and without appreciable delay until toward the end of the larval period (Smith, 1932, p. 752; Chang and Witschi, 1955; Chang, 1955). It is also well known that during larval life the gonads are capable of responding readily to gonadotrophic substances by rapid growth and precocious maturation of the germinal elements (e.g., Burns and Buyse, 1931; Burns, 1934). During this period pituitary stimulation merely accelerates the normal processes of development and maturation. It has been shown previously that in many amphibian species administration of steroid sex hormones during the larval period induces transformation of the gonads; however, an interesting case is known in which sex hormones appear to be without effect unless a gonadotrophin is also administered (Puckett, 1939, 1940). The tadpoles of a so-called "undifferentiated race" of Rana catesbiana all have gonads w^iich structurally resemble young ovaries until late in larval life, when differentiation of the males occurs rather abruptly. The administration of gonadotrophin alone to the undifferentiated tadpoles initiates sex differentiation precociously, the two sexes appearing in the usual 50:50 ratio. The administration of sex hormones of either type to tadpoles during the indifferent period is without effect; the gonads are apparently incapable of responding at this stage of development. However, when the sex hormone and gonadotrophin are administered concurrently a striking response occurs; not only is sex differentiation precipitated, as when gonadotrophin was administered alone, but a complete transformation of sex takes place, resulting in all males or all females, according to the type of sex hormone employed. The gonadotrophin is evidently necessary to initiate sexual diff(>rentiation but the type of differentiation which follows is determined by the type of sex hormone. In chick embryos "hypophysectomy" before the onset of sex differentiation has been accomplished in two ways, by partial decapitation, in which the forebrain area is remov(>d surgically after 33 to 38 hours of incubation (Futio, 1940), and bv irradiation of the hypophyseal region (Wolff and Stoll, 1937) . After excision of the forebrain, histologic differentiation of the gonads proceeds normally. Later, however, the interstitial tissue of the testis fails to develop or is deficient in quantity, and the cortex of the left ovary remains rather thin through failure of secondary sex cords to develop in the usual numbers. The development of the gonaducts, on the other hand, is normal in both sexes. Wolff and Stoll reported that, after destruction of the hypophysis by irradiation, differentiation of the gonads continued in a normal manner to the end of incubation and again the gonaducts were found to develop normally. Such embryos, moreover, undergo sex reversal in the usual manner when treated with sex hormones (Wolff, 1937). The available experimental evidence indicates, then, that the hypophysis has no appreciable part in the primary differentiation of sex, a conclusion which is supported by van Deth, van Limborgh and van Faassen (1956).

In mammalian embryos and fetuses, hypophysectomy has been carried out by procedures similar to those described for the chick, namely, partial or total decapitation and irradiation with x-rays. The former method was used on embryos of the rat (Wells, 1947, 1950) and the rabbit (Jost, 1947d, 1950, 1951a), and the latter on the embryos of the mouse (Raynaud and Frilley, 1947; for a summary see Raynaud 1950) . In the case of the rabbit and the rat, the operation was not performed early enough to affect the primary differentiation of the gonads; in the mouse, however, irradiation on the 12th day of gestation, just at the beginning of differentiation, was without effect except for a certain reduction in the number of germ cells when the hypophysis was entirely destroyed. The results differed sharply, however, with reference to the condition of the genital tracts in hypophysectomized male rabbit fetuses, as opposed to those of the rat and the mouse. In the two latter species no significant changes in the development of the accessory genital structures were observed. It may be that in these species the entire process of sexual differentiation is independent of pituitary function, although the possibility is


perhaps not excluded that an extraneous gonadotrophin, of maternal or placental origin, may be substituted. In the rabbit, on the other hand, definite defects were found in the development of certain accessory genital structures, resembling those which follow embryonic castration but somewhat less severe. However, if gonadotrophin is administered following decapitation these deficiencies do not appear; they may therefore be ascribed to lack of gonadotrophic activity (Jost, 1951a, 1953). The defects observed vary in severit}^ according to position ; the anterior regions of the gonaducts and the epididymides, which are near the testes, develop normally, whereas distant structures such as the prostatic glands and external genitalia may be almost as severely affected as in castrate fetuses. This observation indicates that the testis is acting in an intermediary capacity. In the absence of the pituitary its humoral activity is diminished to a level adequate for normal differentiation of nearby structures but insufficient to maintain the development of more distant parts. The point has been previously established that after decapitation there is a decrease in the amount of interstitial tissue.

The problem of the time of onset of gonadotrophic function in its relation to gonad secretion and the dift'erentiation of the genital tract has been studied in the rabbit by Jost (1951a). By examining the genital tracts of decapitated male fetuses at short intervals to determine when the first signs of abnormal development appear, and by varying also the age at which decapitation was performed, he was able to define rather closely the beginning of gonadotrophic function and the period during which it is critical for normal development of the genital structures. Following decapitation on the 19th day of gestation no marked defects in the genital structures appear until about the 22nd day, after which abnormalities become more and more pronounced until the 24th day. If decapitation is delayed until the 24th day no important anomalies are subsequently found. It should be recalled that the latter date coincides with the stage after which castration likewise has no effect on development. The interval from the 22nd to the 24th clay of development thus falls within the period during which testis activity is most essential for normal morphogenesis (p. 125).

In the light of these results the anterior hypophysis was studied cytologically for direct evidences of secretory activity, using the MacManus periodic acid-Schiff (PAS) test (Jost and Gonse, 1953; Jost and Tavernier, 1956). PAS-positive cells are first seen in small numbers, and faintly stained, on the 19th day of development. Thereafter they increase in numbers and in staining reaction, reaching a maximal development during the 22nd and 23rd days; on the 24th day these cells abruptly decrease in number and stainability and almost disappear. The peak of gonadotrophic activity, as indicated by the cytologic evidence, falls again during the 2-day period when the secretory activity of the testis is at its height, as judged by the conseciuences of castration, a remarkable example of endocrine correlation (for a fuller account see Jost, 1953, 1955).

It was once widely believed that in human anencephalic monsters the pituitary is absent or vestigial in character ; more recent studies have revealed, however, that although difficult to identify grossly, anterior lobe tissue can usually be demonstrated by careful histologic examination {e.g., Angevine, 1938). Nevertheless, cases are known in which apparently no anterior lobe tissue is present, and such cases are pertinent to the present discussion. Barr and Grumbacli (1958, and personal communication of Dr. Grumbach) have described such a case in a newborn male infant, in which no malformation of the genital system was evident except that the testes were somewhat smaller than usual. They were not otherwise abnormal, however, and interstitial tissue was present. In a similar case, also a male, reported by Blizzard and Alberts (1956), the external genitalia were small but normal in structure. The testes also were small and undescended, lying in a pelvic position, the tubules were somewhat atrophic and no interstitial tissue was present. No other abnormalities were noted. It is perhaps significant in this case that absence of the interstitial cells is correlated with underdevelopment of the external genitalia and


failure of the testes to descend. In two cases of congenital absence of the hypophysis, a male and a female (Brewer, 1957), development of the gonads and genital system was apparently normal.

Cases in which complete absence of the pituitary has been demonstrated are unfortunately few but of great value since they represent in humans the closest approach to hypophysectomy in experimental animals. The consequences of the deficiency and the conclusions to be drawn in the two cases are similar; the primary differentiation of the gonads is evidently independent of the pituitary but secondary defects may appear later, both in the gonads and in the genital tract. The testes may be underdeveloped, the tubules may show secondary atrophy or degenerative changes, and the interstitial tissue may be reduced or lacking, but in some cases it appears to be well develoj^ed. There is need for a careful correlation of the status of the interstitial tissue in such cases with the presence or absence of defects of the accessory organs. The consequences to the gonad of absence of the pituitary may hinge on whether a secondary source of gonadotrophin is available to the fetus (Jost, 1953). This is a matter which may be expected to vary in different groups or species. As yet too few species have been studied to clarify the point.

VII. Group Differences in the Relations of Hormones to Sex Differentiation

The extensive experimental data reviewed in the foregoing pages show clearly that the embryonic gonads produce sex specific substances which must be regarded as hormones and which act as physiologic agents in the differentiation of sex, controlling not only the development of the various accessory sex structures but in many cases the differentiation of the gonads themselves. That the substances are hormones in the usual sense is shown by the fact that they regulate the development of distant structures in such fashion that they can only be distributed by way of the circulating blood. This, however, does not preclude a sharply localized action under jiroper circumstances. That they are ('hib<)iat('(l in the gonads is demonstrated bv


HORMONES IX DIFFERENTIATION OF SEX


135


many types of grafting experiments, and above all by the results of embryonic castration. At the same time, steroid hormones of the adult type are also capable in many cases of reproducing closely the effects of the embryonic hormones, thus suggesting a basic similarity. This is not to say, however, that in all groups and species the role of hormones in the differentiation of sex is the same, either with respect to their effects on individual structures or their part in the differentiation process as a whole. Along with ontogenetic processes in general, hormonal relationships have evolved differently in the different vertebrate groups.

Amphibians. In amphibians both sex hormones seem to have active and essentially coordinate roles in sexual differentiation. With respect to gonad differentiation, grafted gonads of opposite sex induce transformation of both testes and ovaries by acting selectively on the appropriate gonad components. In many cases the interacting gonads are reciprocally modified, both becoming strongly intersexual ; when this reciprocal effect does not occur it is apparently due to the decisive predominance of one member. Moreover, the gonad components in many cases react in a similar or identical manner to both natural and synthetic hormones. Male hormones induce precocious differentiation of the male duct system and the cloacal glands in individuals of either sex and when administered early may also completely suppress the development of the jMiillerian ducts; conversely, female hormones stimulate differentiation of the ]\Iiillerian ducts but are without effect on such male structures as Wolffian ducts and cloacal glands. But if such evidence demonstrates beyond question that the larval sex structures are capable of reacting specifically to hormones of the proper type, the role of hormones in the normal differentiation of sex is more directly demonstrated by the effects of larval castration. In castrates of either sex the gonaducts and other sex accessories remain indefinitely in an undifferentiated or slightly differentiated condition. The sexually neutral type in amphibians thus tends to be morphologically intermediate between the sexes; however, either sex type may be readily obtained


from the castrate type by transplanting an ovary or a testis (p. 112). The positive role of both hormones in sex differentiation is apparent.

Birds. In bird embryos also steroid sex hormones stimulate precocious growth and differentiation of the appropriate sex primordia, and in general have inhibitory effects on structures of the other sex. There is a high degree of specificity in the interaction between hormone and end organ, and from the results of hormone administration alone it might be inferred that the two hormones have coordinate roles in sex differentiation. However, the results of castration show clearly that the roles of the two hormones are different and unequal. The Miillerian ducts persist and continue to develop in a similar manner in castrates of both sexes (p. 115). The male hormone is evidently the decisive factor in their differentiation since the presence of the testes causes involution of the ducts in males whereas the ovaries are not essential for their development in females. On the other hand the sextype of the genital tubercle and the syrinx is conditioned by the ovaries. Both structures are normally developed in castrate males, for which male hormone is evidently not essential, whereas in castrate females also they closely approach the male condition in both form and size. It is the inhibitory action of the ovary, therefore, that determines the dimorphism of the syrinx and the genital tubercle. ^^ These conclusions are confirmed by the fact that when isolated and cultured in vitro the Miillerian ducts, and the genital tubercle and syrinx, behave exactly as in castrate embryos; however, addition of male hormone to the culture medium causes involution of the Miillerian ducts, and female hormone prevents male differentiation of the tubercle and syrinx.

Significant differences thus appear in the reactions of the accessory sex structures in birds as compared with amphibians. In the

'^This i.s true also of such striking sex characters as phunage type and spurs in adult fowl, whereas the head furnishings are under the control of the male hormone. There is, however, much ■\-ariation in the relationships of secondary sex characters and gonad hormones in birds (Domm, 1939).


136


BIOLOGIC BASIS OF SEX


latter positive stimulation by the proper hormone is necessary to induce final sexual differentiation. In birds, the role of the sex hormones appears to be primarily inhibitory; in the absence of gonads Miillerian ducts develop normally in castrates of either sex and genital tubercle and syrinx spontaneously assume the male form. No positive stimulus is necessary, only release from the inhibitory influence of the opposing gonad. Thus, the castrate type in birds is not undifferentiated or intermediate in type, but is rather a mosaic in which certain characters are typically male, others typically female. Again, however, both hormones are essential for the realization of normal sex differentiation but with the female hormone having the major role with respect to the number of structures controlled.

Mammals. In mammals still another pattern appears in the relation of the accessory sex structures to the gonads and their hormones. Administration of pure steroid hormones demonstrates again that most of the genital primordia, regardless of sex constitution, are capable of reacting to the appropriate hormone, and if excessive dosages are avoided the responses are in most cases specific. To summarize, administration of male hormone does not significantly affect the development of male embryos except to accelerate the rate of differentiation; in females, on the other hand, it induces differentiation of male structures whereas female primordia are inhibited or fail to respond. In like manner, female hormones have a feminizing action on male embryos.

Again, it might be assumed from the results of hormone administration that the two hormones have comparable roles in normal differentiation. In reality, what is demonstrated is the capacities of the sex primordia to respond to hormones experimentally introduced; the true role of hormones in normal differentiation is disclosed only when the embryonic genital tract is required to develop in the absence of the gonads or other hormonal influences. Castration of mammalian embryos reveals that normal differentiation depends chiefly if not exclusively on the male hormone. Castrated embryos, regardless of genie sex, develo):) female cliaracters (Miillerian derivatives,


female sinus form and external genitalia, mammary glands) which are almost as well differentiated in castrates as in normal females (Figs. 2.26-2.28, and Table 2.2). Any possible influence of a maternal hormone in this result appears to be excluded (at least for the sex ducts and their derivatives) by studies of development in vitro. Culture of the isolated gonaducts results in persistence and development of the ]\Iiillerian ducts and involution of the Wolffian ducts, regardless of the sex of the donor embryo (pp. 117, 121) providing always that isolation is carried out before irreversible determination has occurred.

In mammalian embryos, then, the testes and the male hormone are all imjiortant for the normal differentiation of sex. Moreover, the role of the male hormone is a dual one; its presence is essential to insure retention and development of male parts and at the same time to prevent the differentiation of female structures, which are capable of developing autonomously, regardless of the presence or absence of female hormone. The latter apparently has no essential role in primary sex differentiation. At this point it should be recalled that such a conclusion had in fact been forecast much earlier on the basis of castration of the newborn rat (Wiesner, 1934, 1935; see Burns, 1938b). At birth morphogenesis of the genital structures of young rats is far advanced and profound modifications after castration are not to be expected ; however, it was found that in castrate males a marked atrophy promptly appeared in such sex accessories as the seminal vesicles and external genitalia, suggesting that a hormonal influence had been removed, whereas in castrate females development proceeded more or less normally until the approach of puberty. These results were confirmed and extended by LaVelle (1951) in the ncnvboi'n hamster. Wiesner (1934, 1935) proposed that sex differentiation might be explained on the basis of one hormone, the male, the presence or absence of which would account for the two types of development, an hypothesis now confirmed ill a striking way by the results of castration during embryonic and fetal development.

The pre-ciiiiiiciit role of the male hormone


HORMONES IN DIFFERENTIATION OF SEX


137


in mammalian sex differentiation stands in contrast with the situation in birds, in which the female hormone has the major role. The parallel with the well known difference in the sex-chromosome complex has been noted but this provides no immediate explanation and may be merely coincidence. On the other hand, another explanation may be found in the special physiologic conditions incidental to the evolution of intra-uterine development in mammals. A situation in which the female hormone has an active role in sex differentiation might present a serious difficulty with male embryos constantly exposed during development to the influence of the mother's hormones (the presence of considerable amounts of maternal estrogen during pregnancy is an established fact in many species (Price, 1947; Parkes, 1954). Elimination of the role of the female hormone coincident with the evolution of viviparity would then be advantageous. The problem of female development has apparently been met by a change in the status of the female sex primordia which, as amply shown by the results of castration and cultivation in vitro, do not require positive stimulation but develop autonomously unless inhibited l)y the male hormone.

VIII. The Organization of the Sex

Priniordiuni and Its Role in the

Differentiation of Sex

The role of hormones as specific conditioners of sexual differentiation is, however, only one aspect of the problem. Of far greater complexity, and fundamental to the selective character of the differentiation process, are the special attributes of the individual sex primordia which predetermine their reactions to the presence, or absence, of a particular hormone. Each primordium possesses a complex organization, not only as to sex type and morphologic character, but also with respect to such detailed physiologic properties as the timing of receptivity, the thresholds at which responses occur, and definite capacities for growth. It is obvious that specificity of hormone action does not exist independently of specificity of response. This organization of the primordium derives ultimately from the genotype of the


species, operating through the same processes of ontogeny that prescribe the special characteristics of other embryonic parts and systems; it is intrinsic as opposed to the conditioning activities of hormones and other modifying agencies which, with respect to the primordium, are external and secondary. Thus, sex primordia may be expected to show variations in behavior toward hormones which will be peculiar to and integrated with the patterns of development characteristic of particular groups or species.

A. CONSTITUTION AND THE MORPHOLOGIC REPRESENTATION OF SEX PRIMORDIA

It has been pointed out that even in the bisexual or undifferentiated period of development many variations are found among different species in the extent to which the structures of the recessive sex are represented morphologically, i.e., are laid down in the form of discrete primordia. The absence or the deficient representation in one sex of certain heterotypic primordia may be normal for particular species, whose pattern of development thus places special limitations on sex reversal. In some amphibians reversal is difficult or impossible to induce experimentally because of the weak or transient representation of the recessive sex component in the gonad; there is no real transformation, merely a severely inhibited, or vestigial gonad. Certain accessory structures of the recessive sex may also tend to be abortive or imperfectly developed. This is the case for the Miillerian ducts in the males of various species. In young male opossums, for example, the iNIiillerian duct rarely completes its development to the point of union with the urinogenital sinus, or the connection if formed is quickly lost, so that the terminal segment of the duct is lacking. Consequently, it has never been possible to induce vaginal development in male opossums by treatment with female hormones, although the uterus and Fallopian tube are present and highly developed. Moreover, the tendency which leads to absence of this region of the duct in male embryos appears to be present also, but more weakly expressed, in the female. Although the terminal segment


138


BIOLOGIC BASIS OF SEX


of the duct is always present, it is susceptible to inhibition by male hormone while the tubo-uterine portion is never so affected (Burns, 1942b). Thus, specific morphologic defects which appear in experimental results can often be directly related to developmental peculiarities of the species in question and in final analysis are an expression of constitutional factors.

However, constitutional differences which control the morphologic representation of sex primordia do not as a rule involve simply the presence or the absence of a part, but more commonly have a quantitative expression, affecting the extent to which the structures are developed or the length of the period during which they are present and capable of responding. An example is found in the gonads of birds, in which there are marked lateral differences between right and left sides (p. 95). In consequence, the effects of hormones on the right and the left gonads may be different, not qualitatively but in degree. The left ovary, with its strongly developed cortical component (Fig. 2.12), is only moderately affected by doses of male hormone which almost completely transform the rudimentary right gonad (Willier, 1939). The morphologic differences between right and left testes are less marked, but consistently the germinal epithelium is better developed and tends to survive longer on the left side than on the right. Consequently, female hormone readily transforms a left testis into an ovotestis, and with stronger dosages into an almost normal ovary, but the right testis is but slightly affected except when the dosage is very large. It appears also from studies of the effects of graduated dosages that threshold differences for the two sides may be involved, thus a physiologic as well as a morphologic factor is introduced. It is not held that experimental failures or anomalies are always directly traceable to specific morphologic deficiencies; however, the frequency with which such correlations appear indicates the importance of underlying structural variations in modifying the responses of sex primordia under experimental conditions.


B. CONSTITUTIONAL FACTORS AND PHYSIOLOGIC

DIFFERENCES IN THE ORGANIZATION

OF SEX PRIMORDIA

In the foregoing cases obvious morphologic differences provide, at least in part, a basis for observed differences in the experimental behavior of sex primordia. It is unlikely that morphologic differences of this order exist without an underlying physiologic differentiation. On the other hand, under experimental conditions, physiologic differences often become apparent which have no visible morphologic expression, as in the inhibition of the vaginal canals of female opossums cited above. Certain accessory sex structures in birds exhibit lateral differences in sensitivity to hormones which are evidently a reflection of the general tendency to asymmetrical development in this group. In normal females only the left Miillerian duct develops into a functional oviduct ; the right, although originally well developed, regresses at an early stage. After castration, however, both ducts develop equally (Fig. 2.25) as is also the case when the ducts are isolated in vitro. Involution of the right oviduct, then, is conditioned in some way by the ovaries and it has been shown that either the right or left ovary alone is effective (Wolff and Wolff, 1951). Evidently the ovaries exert an inhibitory action on the right oviduct which is not effective on the left. Presumably a threshold difference is involved.^*'

Other examples may be cited. The syrinx and the genital tubercle remain small, symmetrical, and essentially undifferentiated in females, but in males they become large and highly asymmetrical (Fig. 2.25). Unlike the paired structures previously dealt with, these organs are single and median in position and the asymmetry of the male form is due to unequal development of the lateral halves of the organs. It is well established in the case of eacii that the female hormone

""It seems unlikely tliat the inhibitoiy factor in this case is the female hormone since the introduction of estrogenic hormones into incubating eggs causes persistence of the right oviduct. However, the concentration or dosage may be a factor in this curious effect.


HORMONES IN DIFFERENTIATION OF SEX


139


inhibits the male type of development (pp. 127, 131) which, on the other hand, develops spontaneously in both sexes after castration (Fig. 2.25) or after isolation in vitro, without hormonal conditioning. A difference in susceptibility to inhibition by the female hormone apparently masks the inherent difference in growth potential and the primary symmetry of the female structure is preserved.

The marked asymmetries of the genital system in birds thus appear to rest on lateral differences involving such physiologic characteristics as growth potentials and reaction thresholds. These in turn are apparently correlated with a more extensive asymmetry involving the whole organism. Lateral growth differentials are established in the blastoderm of chick embryos as early as the head-process stage. In testing the organ- forming potencies of regional pieces of the blastoderm it was found (Willier and Rawles, 1935; Rawles, 1936) that when corresponding pieces of the same size from right and left halves of the blastoderm are transplanted to the chorioallantoic membrane they consistently show marked differences in capacity for growth and self-differentiation, which are manifested both in the size attained by the graft (growth capacity) and in the quality of the histologic differentiation. Regardless of the particular tissues or organs dealt with, grafts from the left half of the blastoderm are consistently larger and better differentiated than those from the corresponding pieces of the right half. Lillie (1931) also postulated critical differences in growth rate and threshold to sex hormones on the two sides of the body in explaining the occurrence of "gynandromorphic" plumage in adult fowls and its distribution. He pointed out that the sharply defined difference in plumage on the two sides of the body is usually accompanied in gynandromorphs by gross bodily asymmetry (hemihypertrophy) favoring the left side. It would seem that lateral differences in the morphologic and physiologic properties of the sex primordia of birds are not peculiarities of sexual differentiation ; rather they are an aspect of the general pattern of


somatic organization in this group. In the normal differentiation of sex as well as in experimental studies these differences are exploited by hormones whose effects serve merely to exaggerate or to obliterate tendencies inherent in the organization of the individual primordia.

C. INFLUENCE OF SEX GENOTYPE ON THE REACTIONS OF SEX PRIMORDIA

Another example of the way in which constitutional factors operate to modify or set limits to hormone action is seen in the influence of the sex genotype on the responses of sex primordia to hormones, as illustrated especially in young opossums (Burns, 1942b, 1956a). In comparing the effects of identical doses of the same hormone (whether male or female) in embryos of different sex, it was found in the case of many structures that the amount of growth induced was influenced by the sex of the individual. Under identical experimental conditions the effect of a male hormone on the growth of a particular male structure was always greater in male embryos than on the homologous structure in females, and vice versa. This result is well illustrated by the reactions of the genital tubercle or phallus in male and female littermates which received identical doses of testosterone propionate. The transformed phallus of the female cannot be distinguished anatomically or histologically from the male organ, except for a constant and considerable difference in size (Fig. 2.32). Such an effect was cited earlier in the case of the prostate (Fig. 2.29) and it occurs for various other male structures such as the vas deferens I Wolffian duct) and the epididymis (Fig. 2.24). After treatment with female hormone corresponding differences are observed in the response of female structures. The Miillerian ducts of male embryos hypertrophy and undergo a typical differentiation into oviduct and uterus; nevertheless, in size these organs do not approach those produced by the same dosage in females (Fig. 2.24). The same is true in the case of the hyperplastic reaction of the sinus epithelium and for other structures. Differences in size can be 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 certain 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


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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).



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


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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 reversing 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



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.



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).

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Pages where the terms "Historic" (textbooks, papers, people, recommendations) appear on this site, and sections within pages where this disclaimer appears, indicate that the content and scientific understanding are specific to the time of publication. This means that while some scientific descriptions are still accurate, the terminology and interpretation of the developmental mechanisms reflect the understanding at the time of original publication and those of the preceding periods, these terms, interpretations and recommendations may not reflect our current scientific understanding.     (More? Embryology History | Historic Embryology Papers)
Young WC. Sex and internal secretions. (1961) 3rd Eda. Williams and Wilkins. Baltimore.
Section A Biologic Basis of Sex Cytologic and Genetic Basis of Sex | Role of Hormones in the Differentiation of Sex
Section B The Hypophysis and the Gonadotrophic Hormones in Relation to Reproduction Morphology of the Hypophysis Related to Its Function | Physiology of the Anterior Hypophysis in Relation to Reproduction
The Mammalian Testis | The Accessory Reproductive Glands of Mammals | The Mammalian Ovary | The Mammalian Female Reproductive Cycle and Its Controlling Mechanisms | Action of Estrogen and Progesterone on the Reproductive Tract of Lower Primates | The Mammary Gland and Lactation | Some Problems of the Metabolism and Mechanism of Action of Steroid Sex Hormones | Nutritional Effects on Endocrine Secretions
Section D Biology of Sperm and Ova, Fertilization, Implantation, the Placenta, and Pregnancy Biology of Spermatozoa | Biology of Eggs and Implantation | Histochemistry and Electron Microscopy of the Placenta | Gestation
Section E Physiology of Reproduction in Submammalian Vertebrates Endocrinology of Reproduction in Cold-blooded Vertebrates | Endocrinology of Reproduction in Birds
Section F Hormonal Regulation of Reproductive Behavior The Hormones and Mating Behavior | Gonadal Hormones and Social Behavior in Infrahuman Vertebrates | Gonadal Hormones and Parental Behavior in Birds and Infrahuman Mammals | Sex Hormones and Other Variables in Human Eroticism | The Ontogenesis of Sexual Behavior in Man | Cultural Determinants of Sexual Behavior


Reference: Young WC. Sex and internal secretions. (1961) 3rd Eda. Williams and Wilkins. Baltimore.


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