True hermaphroditism in chimaeric mice

Transcription

1 J. Embryol. exp. Morph., Vol. 12, Part 4, pp , December 1964 Printed in Great Britain True hermaphroditism in chimaeric mice by ANDRZEJ K. TARKOWSKI 1 From the Institute of Zoology, University of Warsaw WITH FOUR PLATES INTRODUCTION IN a preliminary communication on the development of mouse chimaerae formed from fused eggs (Tarkowski, 1961) three cases of intersexuality were reported among newborn animals. Sexing was based exclusively on macroscopic examination of the reproductive system. The present paper deals with the microscopic structure of this system in chimaeric hermaphrodites, with special reference to the structure of the gonads. The occurrence of intersexes among animals developed from fused eggs was considered as one of the proofs of their chimaeric constitution (Tarkowski, 1961). Abnormalities similar to those displayed by these chimaerae have been described in the mouse and related rodents in sporadic spontaneous cases of hermaphroditism. However, while the genesis of the latter is unknown, there are strong indications that hermaphroditism among chimaerae develops on the basis of sex-chromosome mosaicism. This type of mosaicism can be expected a priori to occur in a number of individuals developed from two eggs randomly selected. Insight into the problems of abnormal sexual differentiation in rodents may thus be obtained. Such an approach is encouraged by the results of recent karyological studies in man which have furnished a wealth of information regarding the role of sex chromosomes in normal and hermaphroditic sexual differentiation. All spontaneous rodent hermaphrodites so far described were adolescent or adult animals. The data reported in the present work are, on the other hand, derived from early post-natal material. Investigation of the reproductive system at such an early stage, when it still reflects primary structural relationships, can be of value for a better understanding of the way in which these particular sexual disturbances develop. Some general remarks concerning the sexual differentiation of chimaerae together with a hypothetical explanation of the observed sex ratio have been presented elsewhere (Tarkowski, 1963). 1 Author's address: Institute of Zoology, University of Warsaw, Warsaw 64, Poland.

2 736 ANDRZEJ K. TARKOWSKI MATERIALS AND METHODS The material employed in this work comprises five 13-13^ days old embryos and fourteen neonatal animals 0-3 days old. The reproductive system of each embryo was sectioned in situ after the ventral body wall and the viscera had been removed. The reproductive system of each newborn animal was dissected from the body before being sectioned. Sections were cut at 6 or 10 /x. and stained either with Ehrlich haematoxylin and eosin or with azure II-eosin 5 with or without haematoxylin. The animals examined fell into three different classes on the basis of the genotypes of the eggs and the combinations of fusion employed (Tarkowski, 1961). However, no distinction in this respect is made between them in the present paper. RESULTS A. Embryos According to Brambell (1927) sex in the mouse can be distinguished histologically at 11 or 12 days post coitum. Among five 13-13^-day chimaeric embryos only one had well differentiated testes. In the four other embryos sexing was unsuccessful and the gonads either represent ovaries or are still in a undifferentiated state. Since the same difficulty was met in distinguishing the sex of control LAB Grey embryos of the corresponding age it must be assumed that this strain is characterized by a much slower rate of sexual differentiation than the animals investigated by Brambell. Support for such an explanation comes from another observation made by the above author (Brambell, 1927, p. 398), who reported ' Some early prophase stages can be distinguished as early as stage 9(12 days/?.c), but it is not till a day or two later (Stages 12 and 13, 13 and 14 days p.c.) that all the germ cells exhibit them....' In the gonads of chimaeric or control LAB Grey embryos of the age days no meiotic prophase stages whatsoever can be observed and there is no indication that they might be initiated in the following -1 day of development. The 13-13^-day stage was thus too young for distinguishing sex and the present experiment does not provide evidence on the general sex ratio among chimaerae. A complicating factor in distinguishing the sex of chimaerae at an early phase of sexual differentiation is the fact that some embryos are potentially hermaphrodites, and that their gonads would have developed into ovotestes. How differentiation proceeds in such cases, and whether it is delayed, remains unknown. B. Newborn animals Among fourteen newborn animals which died shortly after birth three cases of intersexuality were encountered. All three proved to be true hermaphrodites, i.e. their gonads contained both ovarian and testicular tissue. The general appearance of the reproductive system and the distribution of the ovarian and

3 Hermaphroditism in mice 737 testicular tissue between the two gonads was different in each case. Histological examination of the reproductive system of the remaining eleven newborn animals did not reveal any deviation from normality. Two animals which survived and attained maturity proved to be fertile males (Tarkowski, 1963). Hermaphrodite animals First case {animal no. 31/1) This animal lived for hr. from the time of Caesarian section, which was performed in the evening of the 19th day of gestation. An external view of the reproductive system is shown in Plate 1, Fig. A. The right side of the reproductive system is typically female. The ovary, containing germ cells in pachytene, diplotene and dictyate stages, is accompanied by a normal oviduct and uterus. The left side is abnormal. The gonad, which is an ovotestis, is situated on a thick, bent duct and is drawn down to a point half way between the extreme positions usually occupied by ovary and testis. Since the longitudinal axis of the gonad lies somewhat oblique to the anterioposterior axis of the body, and to the plane of sectioning as well, the terms 'cranial end' and 'caudal end' will be used rather arbitrarily to refer only to the first and last section in the series. Starting from the cranial end the gonad begins as a testis with well differentiated sex cords (Plate 1, Fig. B). However, these sex cords contain both spermatogonia and gonocytes undergoing meiosis (Plate 1, Fig. C). All gonocytes are from the pachytene to dictyate stages. This means that at least some of them follow the female type of meiosis and can be called obcytes. In the sex cords gonocytes steadily increase in number, while spermatogonia are reduced in numbers. Proceeding more caudally ovarian tissue makes its appearance on the latero-mesometrial surface of the gonad. In the neighbouring tissue sex cords steadily decrease in number and become poorly differentiated from the stroma. In relation to the ovarian tissue, the testicular tissue becomes spatially more and more restricted to the opposite side of the gonad. Spermatogonia vanish from the sex cords altogether and all germ cells are undergoing meiosis. Still more caudally, on the medio-mesometrial side of the gonad close to the tissue where the testicular characters are best retained, there appears a crescent of ovarian tissue. This sector is, however, partially separated from the rest of the gonad by loose mesenchymal tissue (Plate 1, Fig. D). At the level of the rete, connecting the gonad with the epidydymis (Plate 1, Fig. D), the last sex cords disappear. The layer of mesenchymal tissue separating the ovarian sector from the rest of the gonad soon disappears also and the whole remaining gonad displays ovarian characteristics. The tunica albuginea is not well developed. The germinal epithelium retains the character of a nearly cuboidal epithelium over most of the surface of the gonad. It is only in the very few places where the adjacent mesenchymal tissue

4 738 ANDRZEJ K. TARKOWSKI elements are tangentially orientated that the epithelium becomes flat and resembles that covering normal testes at a corresponding age. Sex ducts. The ovary is associated with normal oviduct and uterus. The ovotesticular gonad is associated with an epidydymis (Plate 1, Fig. D) an oviduct is lacking. The epidydymis is continuous with the vas deferens, which runs alone at first. The Miillerian duct begins blindly at a point about a third to a half of the length of the Wolffian duct. The size of its lumen is only slightly smaller than that of the right uterus (Plate 1, Fig. E). On the side of the ovotestis there is a seminal vesicle, but its lumen ends blindly and does not join the corresponding vas deferens (Plate 1, Fig. F). Slightly below the level where the two uteri join there appears abruptly on the ovarian side the Wolman duct, whose lumen rapidly enlarges to the size of that of the vagina and acquires a similar cleft-like shape (Plate 1, Fig. G). It runs independently only for 120 /x and merges finally with the vagina. The opposite vas deferens retains a small circular or oval lumen throughout its length (Plate 1, Figs. E, F, G). The most caudal portion of the ducts is partially destroyed and so the final fate of the left vas deferens is unknown. It would seem, however, that it merges with the vagina slightly below the entrance of the right Wolffian duct. Second case (animal no. 61\3) This animal was born at night and found dead the following morning. PLATE 1 Figures A-G refer to the reproductive system of the animal no. 31/1. FIG. A. External view of the whole reproductive system, excised from the body. The right side (left on the picture) is of typically female type. The left side is abnormal. The gonad which is an ovotestis is accompanied by an epidydymis. Sex ducts on this side are represented by both the vas deferens (continuous throughout the whole length) and the uterus which begins blindly somewhere about half of the length of vas deferens. x 15. FIG. B. Testicular part of the ovotestis. Sex cords are well differentiated, x 100. FIG. C. Sex cord from the testicular territory of the ovotestis containing, apart from several gonia (spermatogonia), three gonocytes in meiosis. The gonocyte situated most centrally is in late pachytene, the two others are in diplotene. x 800. FIG. D. Section of the ovotestis at the level of rete. The gonadal tissue is of intermediate type with a preponderance of ovarian characters. On the medio-mesometrial surface of the gonad there is a small crescent of typically ovarian tissue partially separated from the rest of the gland by loose mesenchymal tissue. Attached to the gonad is an epidydymis. x 100. FIG. E. Section through the sex ducts at the level where the Miillerian duct already is accompanying the Wolffian duct on the ovotesticular side. Its lumen is only slightly smaller from that of the uterus on the female side, x 100. FIG. F. Section through the sex ducts at a more caudal level, after the bodies of two uteri have joined together (the disruption is an artifact). A seminal vesicle is present on the ovotesticular side only, x 100. FIG. G. Section through the sex ducts after the two uteri have fused together to form a vagina (middle duct). The small circular duct on the right side of the picture is the Wolffian duct belonging to the ovotesticular side, the big cleft-like one on the left side represents the retained caudal portion of the Wolffian duct from the female side of the reproductive tract. xloo.

5 /. Embryol. exp. Morph. Vol. 12, Pan 4 PLATE 1 A. K. TARKOWSKI {Facing page 738)

6 J. Embryol. exp. Morph. Vol. 12, Part 4 PLATE 2 A. K. TARKOWSKI (Facing page 739) K

7 Hermaphroditism in mice 739 Externally the reproductive system is of normal male type, except that a thin fold of tissue appears along each of the vasa deferentia. Sectioning revealed that both gonads were ovotestes. Right gonad. At the cranial end of the gonad the tissue is of intermediate type with a preponderance of ovarian characters on the medio-mesometrial side. Apart from a very few spermatogonia, the germ cells in this territory are all oocytes. These oocytes, situated cortically, are in pachytene and diplotene stages, while those placed nearer the medulla have already entered the resting phase and are enveloped by follicle cells (Plate 2, Fig. H). On the opposite side of the gonad there are poorly differentiated sex cords, which contain only gonocytes at different stages of meiosis (Plate 2, Fig. I). Gradually the ovarian tissue becomes restricted to the medio-mesometrial surface of the gonad (Plate 2, Fig. J) while in the remaining part the testicular characters become progressively more accentuated (Plate 2, Fig. J). At the level where the blood vessels enter the gonad the sex cords are already well developed and clearly differentiated from the stroma. More caudally, the ovarian tissue becomes clearly separated from the rest of the gonad by a layer of loose mesenchymal tissue (Plate 2, Figs. J, K), and eventually disappears completely. At this level most of the germ cells are spermatogonia. This holds true even for the sex cords lying closest to (but separated by mesenchymal tissue from) the ovarian sector (Plate 2, Fig. K). After the ovarian sector disappears the gonad is typically testicular to the caudal end. In this part the germ cells are exclusively spermatogonia. Left gonad. At the cranial end the gonad is of the ovarian type (Plate 3, Fig. L). Slightly below the rete, structures resembling sex cords make their appearance and the tissue acquires an intermediate character. However, in the medio-mesometrial angle of the gonad the ovarian characters are retained or even accentuated. In the remaining part of the gonad sex cords become more numerous, better developed and more clearly differentiated from the stroma. These characters are most marked in the regions situated most distally from the PLATE 2 Figures H-K refer to the right ovotesticular gonad of the animal no. 61/3. FIG. H. A fragment of the gonadal tissue from the ovarian territory showing oocytes at the beginning of the resting phase. Follicular cells have already begun to envelop the oocytes. x800. FIG. I. A sex cord from the same level of the gonad as the fragment shown on Fig. H, but from the opposite side. All germ cells in the sex cord are undergoing meiosis. x 400. FIG. J. Section through the whole ovotestis at the level where ovarian tissue is already restricted to a small sector situated on the medio-mesometrial surface of the gonad. Loose mesenchymal tissue is wedged in between the two territories, x 100. FIG. K. A fragment of the medio-mesometrial angle of the gonad showing under higher magnification the separation between the ovarian and testicular territories. The sex cords, although they lie close to the ovarian tissue, contain spermatogonia only, x

8 740 ANDRZEJ K. TARKOWSKI ovarian tissue. Down to this level of the gonad spermatogonia are lacking altogether and all germ cells, irrespective of the type of tissue in which they are situated, are undergoing meiosis. Stages from pachytene to dictyate are displayed by oocytes situated in the ovarian tissue as well as in the sex cords themselves (Plate 3, Figs. M, N). Separate spermatogonia begin to appear in the sex cords on the side of the gonad opposite to the ovarian sector (Plate 3, Figs. M, N). Slightly below the entrance of the blood vessels loose mesenchymal tissue has grown in under the ovarian sector, separating it from the rest of the gonad (Plate 3, Fig. O). The mesenchymal tissue forming this ingrowth resembles, and is continuous with, that underlying the germinal epithelium covering the testicular parts of the gonad. At this level sex cords are already well differentiated. At the level where the ovarian sector disappears spermatogonia become dominant for a number of sections but are always accompanied by gonocytes. In the caudal end of the gonad the tissue is of rather testicular type but sex cords are again less clearly differentiated from the stroma. At the caudal apex germ cells are very scanty, but all of them are undergoing meiosis. In particular parts of the gonad the degree of development of the tunica albuginea varies according to the testicular or ovarian character of the underlying tissue. Sex ducts. In the structure of the somatic part of the reproductive system male characters dominate. The Miillerian ducts begin very far posteriorly in the region of the most caudal portions of the Wolffian ducts. The left duct makes its appearance earlier than the right one and is better developed (Plate 4, Fig. P). They come closer and closer together but remain separated from each other throughout their whole length. Most caudally this separation takes the form of a strand of tissue composed only of the two fused epithelial linings of the ducts (Plate 4, Fig. R). The Miillerian ducts finally approach the sinus urogenitalis but whether they merge with it or end blindly cannot be resolved because this part of the reproductive system is partially destroyed. The Wolffian ducts and seminal vesicles are well developed. However, on the left side the lumen of the vas deferens merges with the lumen of the seminal vesicle, not close to its base (as it does on the right side and as it would in a normal male), but near its blind PLATE 3 Figures L-0 refer to the left ovotesticular gonad of the animal no. 61/3. FIG. L. Section through the cranial end of the gonad where the tissue is of the ovarian type. xloo. FIG. M. Section of a sex cord showing two germ cells in the pachytene stage of meiosis and one spermatogonium. x 800. FIG. N. A fragment of the testicular territory. The sex cord situated most superficially contains a growing oocyte. In one of the adjacent sex cords two spermatogonia are visible, x 400. FIG. O. Section through the gonad at the level where ovarian tissue is restricted to the mediomesometrial surface of the gland and is already clearly separated from the remaining testicular territory by a layer of loose mesenchymal tissue, x 100.

9 J. Embryol. exp. Morph. Vol. 12, Port 4 A. K. TARKOWSKI {Facing page 740)

10 J. Embryo!. exp. Aforph. Vol. 12, Part 4 PLATE 4 A. K. TARKOWSKI {Facing page 741)

11 Hermaphroditism in mice 741 apex (Plate 4, Fig. S). The ejaculating ducts come close to the sinus and presumably (the appropriate sections are lacking) they merge with it. In part of the sinus urogenitalis that is preserved prostatic buds can be seen protruding from its wall (Plate 4, Fig. P). Third case {animal no. 59j2) This animal died on the 3rd day of post-natal life. Externally the reproductive system presents a typical case of lateral hermaphroditism, with an ovary-like gonad and female accessory structures on one side and a testis-like gonad with male accessory structures on the opposite side. The figure and drawing illustrating its external morphology were published previously (Tarkowski, 1961). Due to the prolonged manipulations necessary for making drawings and taking macroscopic pictures the tissue is rather badly preserved, rendering cytological analysis very difficult. The left gonad is a normal ovary. Oocytes situated in the medullary part have already entered the resting phase and are surrounded by follicle cells. The stage of meiosis of the more cortically placed oocytes cannot be clearly ascertained because of profound pycnosis of the nuclei. The sex ducts on this side are of typically female type. The right gonad is an ovotestis. It is mainly comprised of testicular tissue; the ovarian tissue forms a small crescent along the latero-mesometrial surface of the gonad (Plate 4, Fig. T). The bad state of preservation of this tissue led to the overlooking of this small sector during provisional inspection of slides and the gonad was erroneously described in a previous report (Tarkowski, 1963) as a testis. PLATE 4 Figures P, R and S refer to the sex ducts of the animal no. 61/3. FIG. P. Section through the ventral wall of the sinus urogenitalis (the rest of the sinus was destroyed during dissection) with the prostatic buds protruding from it. Two Wolffian ducts (the outer ones) and two Miillerian ducts (in the middle) can be seen below the sinus. The left Miillerian duct (right on the picture) is better developed than the opposite one. xloo. FIG. R. Sex ducts in the more caudal region. The Mullerian ducts, though lying close together, remain separated from each other by the two fused epithelial linings of their walls. On the left side (right on the picture) the vas deferens and ejaculating duct (partially disrupted) are visible. On the opposite side the section is at the level where the vas deferens and ejaculating duct are fused together, x 100. FIG. S. Section through the seminal vesicles. Only the right vesicle (left on the picture) is normal. In the opposite one the middle duct represents the vas deferens just before it merges with the lumen of the seminal vesicle close to its apex, x 100. FIG. T. Section through the ovotestis of animal no. 59/2. The dominating part of the gonad is testicular in character. The ovarian tissue occupies the latero-mesometrial angle of the gonad and is continuous with the neighbouring testicular territory. The ovarian part of the gonad is covered on the outside by a membrane which begins on its surface near the border separating the two territories and merges with the epidydymis. x 100.

12 742 ANDRZEJ K. TARKOWSKI On the outside the ovarian sector is covered by a membrane which at one end, close to the border between testicular and ovarian parts, merges with the tunica albuginea, and on the other with the epidydymis (Plate 4, Fig. T). A cleft-like space is thus formed over the surface of the ovarian sector. It appears that, where the membrane begins, the germinal epithelium covering the testicular part sinks down under the capsule and that it is continuous with the epithelium covering the ovarian sector. The origin and developmental history of the membrane are not clear. In the sex cords of the testicular region a few germ cells have been observed which appear to be in late diplotene or dictyate stages of a female-type meiosis. Bad preservation of the tissue makes it impossible either to give cytological details or to estimate their frequency. In the ovarian portion the nuclei of most oocytes, especially of those situated more cortically, are not suitable for estimating the stage of meiosis because of pycnosis. Undoubtedly, however, many must have entered the resting phase since they are surrounded by follicle cells. The ovotestis is associated with a normally developed epidydymis, which, in turn, is followed by a vas deferens. A seminal vesicle is present on this side only. It is unfortunate that the whole of the genital apparatus below the junction of the sex ducts underwent destruction, thus making it impossible to describe the relationships between these ducts in the most caudal region. DISCUSSION Though no data are available which allow us to calculate the overall incidence of spontaneous hermaphroditism in the mouse, the phenomenon is certainly a rare one. True hermaphrodites, i.e. animals possessing both ovarian and testicular tissue in their gonads, were described by Danforth (1927, 1932), Blotevogel (1932), Fekete (1937), Fekete & Newman (1944), Hooker & Strong (1944), Klein (1955), Green (1956) and finally Hollander, Gowen & Stadler (1956). The last paper is exceptional since it reports as many as twenty-five animals, all belonging to the Bagg albino (BALB/GW) strain. In this particular case the incidence of the phenomenon was calculated to be 0-05 per cent, in the stock colony and 0 5 per cent, in the experimental group. The explanation of this unusually high incidence is not known. True hermaphrodites have also been reported in other rodents, such as the rat (Burrill, Green & Ive, 1941; Greep, 1942; Arey & Green, 1957; Bradbury & Bunge, 1958; Iglesias, 1958; Maibenco et al. t 1963),field mouse (Asdell, Hamilton & Hummel, 1941), ground squirrel (Wells, 1937), guinea-pig (Jaffe & Papanicolaou, 1927), syrian hamster (Kirkman, 1958) and common vole (Frank, 1960). There are also numerous reports of hermaphroditism in other mammals, including man. The reproductive systems of each of the three hermaphroditic mice described in the present paper, although displaying some peculiarities, do not differ

13 Hermaphmditism in mice 743 substantially from those developed spontaneously. The structure of the somatic part of the reproductive system of these animals can be accounted for by our present knowledge of inductive interactions during sexual differentiation. The present material does not, however, contribute much new information to the understanding of the development of the somatic sexual characters and this problem will not be discussed. The background of true hermaphroditism It is widely believed that the cases of true hermaphroditism in mammals cannot be explained simply on the ground of inductive disturbances during gonadogenesis but must have a more profound background of a karyological nature. One expression of this belief is the adoption by many authors of the term 'gynandromorph' to describe hermaphroditic animals, by inference postulating these individuals to be chromosomal mosaics. Plausible as it is this assumption has remained hypothetical until now, since in no case was a spontaneously occurring rodent hermaphrodite investigated for its chromosomal constitution. Recently, evidence in favour of such a background of true hermaphroditism in mammals has come from chromosomal studies on sexually abnormal human beings (Gartler et ah, 1962). It should be pointed out, however, that, apart from their case, in all human hermaphrodites chromosomal analysis has been carried out on somatic tissues only. Karyological inspection of the somatic tissues of several human hermaphrodites has not revealed mosaicism, they being either wholly female or wholly male (see Polani, 1962, and Overzier, 1963, for references). On the other hand, some cases of mosaicism (see Matthey, 1963, for references) were found in individuals which, though displaying sexual disorders, could not be considered as being true hermaphrodites. Knowledge of the karyotype of somatic tissues only of hermaphrodites could, however, be misleading. As long as the gonadal tissue itself is not investigated karyologically there is no direct proof of the gynandromorphic character of true hermaphroditism. In this situation the findings reported by Gartler et ah (1962) have a special importance. A child examined by the above authors proved at laparotomy to have an ovary on the left side and an ovotestis on the right side. Biopsies taken from the gonads revealed exclusively XX cells in the ovary but both XX and XY types in the ovotestis. What is more important is that in the ovotestis the testicular part showed a preponderance of XY cells, and the ovarian part a preponderance of XX cells. Somatic tissues, like skin and peripheral blood, also displayed the mosaic constitution. This is the only case in which a clear-cut correlation was revealed between the type of gonadal tissue and the sex chromosome constitution of its cells. The three hermaphroditic animals described in the present study, though not different from spontaneous cases in the morphology of the reproductive system, are exceptional because of their origin from fused eggs. Assuming that the primary sex ratio is 1:1 and that XX and XY eggs are fused

14 744 ANDRZEJ K. TARKOWSKI at random, theoretically 50 per cent, of chimaerae produced should be sex chimaerae, i.e. mosaics regarding the complement of sex chromosomes. The actual proportions may deviate from the theoretically expected if the primary sex ratio is not equal. That such a possibility exists was shown by Sundell (1962) who found the sex ratio at the blastocyst stage of the golden hamster to be 180:100 as compared with 106:100 at birth. But since the differential mortality of male embryos, which must be responsible for reducing the sex ratio at birth to 1:1, might well operate among chimaeric embryos as well, the ratio of females: female/males: males among newborn chimaerae should approach the expected figures 25:50:25. If the hypothesis of sex-chromosome mosaicism as a background of hermaphroditism is true, then such cases should be represented among chimaeric animals. In fact, three hermaphrodites were encountered in the post-natal material which consisted of sixteen animals (Tarkowski, 1963). Keeping in mind the extremely low incidence of spontaneous mouse hermaphrodites, this proportion is highly significant. However, this incidence is much below the level theoretically anticipated upon the assumption that sex-chromosome mosaicism must irrevocably lead to hermaphroditism. To account for this unexpectedly low incidence of hermaphrodites among chimaerae (3/16) and for the excess of males (11/16), it was suggested (Tarkowski, 1963) that some sex chimaerae may develop into males despite their mosaic constitution. This hypothesis still needs verification by karyological studies. However, the three cases encountered in this study must have arisen because of sex-chromosome mosaicism. Though the gonadal cells were not studied for their chromosome complements and, therefore, direct evidence is lacking, the probability of a background other than sex-chromosome mosaicism seems to be extremely low. Two of the animals in question, namely nos. 59/2 and 61/3 developed from eggs differing in the factor for pigmentation (pink-eyed dilution versus agouti) and were proved to contain a mixed population of cells in the outer layer of the retina (Tarkowski, 1964). The hermaphroditic condition in these animals, some of whose organs are certainly mosaic, forms a solid basis for claiming that mosaicism in such cases is in fact of XX/XY type. The above considerations show that the production of chimaerae by fusion of eggs may present a fruitful approach to an understanding of the background and mechanism of spontaneous hermaphroditism. It should be made clear, however, that the case of the 'chimaeric hermaphrodite' with its mosaicism of the XX/XY type is not a copy of all spontaneously arising hermaphrodites as far as the type of their sex-chromosome mosaicism is concerned. Chromosomal mosaicism must originate from mitotic errors from the first cleavage division onwards. The earlier such a mitotic irregularity takes place the more profound the effect it will have on the whole organism. The starting point for the development of a sex-chromosome mosaic displaying a hermaphroditic condition must be an XY egg. Non-disjunction of the Y chromosome could lead to an XO/XYY

15 Hermaphroditism in mice 745 mosaic; if this is followed by 'lagging' of one or both chromatids an XO/XY mosaic or an XO/XO individual will ensue. The picture may become even more complicated if one of the gametes is already abnormal, due to non-disjunction during meiosis, or if the mitotic error takes place more than once during cleavage. It is hard to imagine a mechanism of the 'mitotic error' type which in nature could lead to the origin of a XX/XY mosaic, i.e. the type most resembling that displayed by sex chimaerae. For the origin of their XX/XY human mosaic Gartler et ah (1962) postulate, on the ground of additional serological studies, a different and rather exceptional mechanism, namely double fertilization of the egg with two haploid sets of chromosomes. Braden (1957) suggested such a mechanism, precisely speaking, double fertilization following 'immediate cleavage', to account for the origin of the hermaphroditic mice from the BALB albino strain described by Hollander et al. (1956). Braden remarks that among all inbred strains investigated by him only strain A yielded 'immediate cleavage' eggs; it is interesting to note that this strain is related to BALB/GW strain from which all hermaphrodites described by Hollander et al. (1956) were derived. It has been shown both in man and mouse that the Y chromosome causes the development of the male phenotype. Human beings of the constitution XXY, XXXY, XXXXY and XXYY are phenotypically males and all exhibit variations of the Klinefelter syndrome (see Ford, 1961). In the mouse, XXY individuals are also sterile males (see Russell, 1962). On the other hand, individuals with an XO constitution are viable and phenotypically female. In man they are usually sterile, but one exception has been recorded (Bahner et ah, 1960). In the mouse, fertile XO females have been described recently by a number of workers (see Russell, 1962). It can, therefore, be inferred that an essential for the hermaphroditic condition is a type of sex-chromosome mosaicism which in the simplest form (two components) would be either X n O/X n Y n or X n X n /X n Y n. In other words the mosaicism would be characterized by the presence of a Y chromosome or chromosomes in one type of cell and its absence in the other. Each particular mosaic of this general type may show variations in the resulting phenotype, but the general way in which they express themselves in the development and structure of the genital system should be similar. The study of hermaphroditism in general, and the role of sex-chromosome mosaicism in its development in particular, should profit from any type of experimental procedure which can yield sex-chromosome mosaics conforming to the formulae presented above. At the moment the only method which can routinely yield such mosaics is the fusion of eggs. General tendencies in the development of various types of true hermaphroditism On the grounds of the distribution of ovarian and testicular tissue between the two gonads of hermaphrodites four types can be distinguished. These are: (1) ovary-testis, (2) ovotestis-testis, (3) ovotestis-ovary, (4) ovotestis-ovotestis. Apart from the case of Fekete & Newman (1944), which belongs to the second

16 746 ANDRZEJ K. TARKOWSKI group, all spontaneous mouse hermaphrodites fall into the first group. The three cases described in the present work are, so far, the only examples of classes 3 and 4. The numerical distribution of mouse hermaphrodites among these four consecutive groups is 33:1:2:1. The total number of hermaphrodites described in other species of rodents amounts to fourteen (distribution 7:1:1:5). The corresponding figures for man, calculated from the data compiled by Overzier (1963), are: 43:11:38:33 (only 125 'unquestionable' cases were selected from 171 tabulated). Comparable figures compiled by Asdell (1942) for pig are: 17:9:7:15. Thus the comparison of figures for these species does not reveal any common tendencies in the development of hermaphroditism. Though the data available for the mouse would suggest that in this species there is a tendency for a lateral type of hermaphroditism (first group: ovary-testis) to develop, in my opinion the cases described in the literature do not give an accurate estimate of the actual incidence of the four combinations of gonads among hermaphrodites. First, it seems very likely that in routine dissections not aimed at detecting hermaphrodites, only the most extreme abnormalities involving the whole reproductive system such as lateral hermaphroditism are detected, while minor deviations escape notice. Case 61/3 from the present paper may serve as an example. Though the reproductive system of this animal is nearly completely of male type in that only the most posterior portions of the Miillerian ducts are retained, both gonads are ovotestes. As far as could be anticipated from the morphology of this system just after birth, its ultimate structure at maturity would not have deviated much from the normal male type. Secondly, the gonads were not always sectioned and the possibility cannot be excluded that some of them were ovotestes and not 'pure' ovaries or testes as claimed. Animal no. 59/2 from the present work (externally a typical lateral hermaphrodite, with a small ovarian territory in the apparent testis) proves the necessity of serial sectioning of the gonads even in those cases where their external appearance does not show any deviation from normality. Thirdly, on theoretical grounds to be discussed below, there are some indications that several hermaphroditic rodents presented in the literature as examples of the lateral type of hermaphroditism may have, in fact, possessed ovotestes rather than ovaries. Extensive experimental investigations regarding inductive interactions in the development of the reproductive system (see Jost, 1960), show that the ahormonal sex in mammals is feminine and that the embryonic testes are necessary for retention of the Wolffian ducts and for the development of male accessory sex glands. It is accepted that the testes elaborate a substance, presumably identical to the androgenic hormone of the adult, which diffuses through the tissue of the corresponding side of the reproductive system. On the other hand it is not quite clear what factors are involved in the involution of the Miillerian ducts in males. The character of the sex ducts and accessory structures in both spontaneous and experimental chimaeric hermaphrodites, though showing a great diversity,

17 Hermaphroditism in mice can be understood in the light of these experimental findings. With the few exceptions discussed below the sex ducts in hermaphrodites correspond to the sex of the adjoining gonad; in the presence of ovotestis both ducts can be retained. In the latter case the degree of development of Wolffian and Mullerian duct derivatives differs from case to case, probably depending in each instance on the strength of hormonal activity of the corresponding gonad at the time of sexual differentiation. The conformity of gonad with accompanying ducts steadily decreases in the caudal direction. This seems logical since in the caudal region the sexual apparatus comes under the simultaneous influence of both gonads. Asdell (1942) and Hooker & Strong (1944) were the first to reveal such a general regularity in the development of the reproductive system of spontaneous rodent hermaphrodites. However, some of the reported cases of hermaphrodite rodents do not conform to this general scheme, usually by lacking Wolffian duct derivatives on the side of an ovotestis (Fekete & Newman, 1944) or testis (Blotevogel, 1932) or the presence of Wolffian duct derivatives on the side of an ovary (Burrill et al, 1941; Greep, 1942; Hollander et al, 1956; Bradbury & Bunge, 1958). It is hard to reconcile these deviations, especially the development of Wolffian duct derivatives in the absence of testicular tissue, with what is known from experimental studies on sex differentiation. The presence of the male ducts would be understandable if the gonads were actually ovotestes and not ovaries as claimed. That this might be the case, at least in some of the animals described by Hollander et al (1956), seems more than likely. The authors themselves draw attention to the ovotesticular character of the gonad illustrated in their Plate 2, fig. 11, in the description of this Plate, though not in the text. In my opinion the morphology of the sex ducts in such 'non-comforming' hermaphrodites suggests that the gonads must have been involved in masculimzing hormonal activity at the time of sexual differentiation and that the testicular tissue must have been overlooked or have retrogressed by the time of autopsy. In the case of an ovotestis it can sometimes be difficult to infer the primary character of a given gonad from its adult structure. A secondary obliteration of the primary structure could easily have arisen in the ovotestes of animals nos. 31/1 and 61/3 described in this paper. In these gonads many of the sex cords, even those within the typical testicular territory, contain oocytes in meiosis. In addition, some of the sex cords are not well developed and might, on further development, have come to resemble polyovular follicles. With the growth of oocytes the primary testicular character of such a tissue could become steadily obliterated. Such changes take place, however, after the basic pattern of the rest of the reproductive system has been laid down. Another factor which can complicate the analysis of differentiation, and of the structure of the sex ducts in hermaphrodites, is the cellular composition of these structures. The sex ducts can be composed of either or of both types of cells contributing to the mosaic individual. Moreover, the share of the two types of 1A1

18 748 ANDRZEJ K. TARKOWSKI cells in the genital system may differ on each side or may change along the length of the tract. It is more than likely, for instance, that if the gonad proves to be an ovotestis, the accompanying duct or ducts will also display the mosaic constitution, at least anteriorly. Price & Pannebaker (1959), in their studies on differentiation in vitro of the reproductive system of the rat, have shown that the Wolffian ducts of the male and female differ in their responsiveness to testicular hormone, the ducts of the female being less responsive. Thus, the genetic sex of the cells of the somatic part of the reproductive system may also be of some importance for sexual differentiation, and this could account for the diversity of structure displayed by the ducts found with the ovotestes of hermaphrodites (cf. case 31/1 and 61/3 described in this paper). The ultimate structure of each half of the reproductive system would then depend primarily on the character of the adjoining gonad but also on the composition of the 'target' tissue. To estimate the role of these two factors in the sexual differentiation of hermaphrodites, it would be necessary to know in each case the contribution made by cells of each sex to the ducts, and the character and strength of the hormonal stimulus from the gonad. Since the strength of such a stimulus can be inferred only from the effect which it exerts on the target organ, we are faced with a vicious circle. It seems that this particular problem can be approached only indirectly, by experiments on normal sexual differentiation. The above considerations seem to favour the suggestion that the frequencies of the four types of hermaphroditism in the mouse, and generally in rodents as derived from the literature, may present a false picture of this phenomenon. In my opinion the incidence of the lateral type of hermaphroditism is certainly exaggerated at the expense of other types. If chimaerae obtained through fusion of eggs of different genetic sex represent a good model of spontaneous sex-chromosome mosaics the development of the lateral type of hermaphroditism must be considered an exception rather than a rule. Studies on the distribution of pigment forming cells in chimaerae developed from eggs differing genetically for pigmentation (Tarkowski, 1964) revealed no lateral chimaerism. On the contrary, it seems that the cells descending from both the contributing eggs tend to intermingle throughout the body from an early stage of development This would imply that a gonad of mixed composition is much more likely than a 'pure' ovary or testis. It has been suggested (Tarkowski, 1963) that a gonad of mixed composition may develop into an ovotestis or into a normal testis. If this is true, then the proportion of lateral hermaphrodites and hermaphrodites with ovotestis and testis might be expected to increase secondarily at the expense of those with two ovotestes, or with ovary and ovotestis. Further, some of the sex-chromosome mosaics, notwithstanding the mixed composition of their gonads, would develop into phenotypically normal males, thus concealing the true incidence of sexchromosome mosaicism. The sex ratio among chimaerae (two females, three hermaphrodites, eleven males) favours such a possibility.

19 Hermaphroditism in mice 749 Inductive interactions in the differentiation ofovotestes Somatic differentiation The ovarian and testicular tissues of ovotestes can be continuous with each other or, alternatively, the two territories can be partially separated from each other by a layer of loose mesenchymal tissue. The ingrowth of loose mesenchymal tissue which runs underneath the ovarian tissue finally merges with the tunica albuginea. This continuity implies that the layer in question originates from the primary downgrowth of mesenchyme which in normal development is, according to Brambell (1927), the first indication of the differentiation of the indifferent gonad into a testis. During normal development of the testis this mesenchymal tissue insinuates itself between the germinal epithelium and the epithelial nucleus (the term 'epithelial nucleus' is adopted from Brambell, 1927) to form the tunica albuginea. The most prominent immigration of mesenchymal cells can be seen just along the median surface of the gonad, i.e. in the region which strictly corresponds to the position of the layer separating the ovarian and testicular tissues in ovotestes as mentioned above. It seems logical, therefore, to postulate that it is only when the primary boundary between genetically female and male cells happens to run close to the plane along which this 'stream' of mesenchymal cells normally grows in that the two territories become separated from each other. In this particular place mesenchymal cells must have been 'attracted' by the genetically male tissue to begin ingrowing but not 'allowed' by the genetically female tissue to intervene between it and the germinal epithelium. In consequence the ingrowth of mesenchymal cells passes between the two territories and subsequently, after encircling the ovarian tissue from inside, finds its way under the germinal epithelium covering the testicular tissue. When the primary boundary separating XX from XY territories does not coincide with the plane of normal ingrowth of mesenchymal cells, the layer separating the two kinds of tissue does not develop and the gonadal tissue displays a continuous change from one type to the other. Outside the layer of mesenchymal tissue no strict boundary between testicular and ovarian tissue can be drawn. What is the genetic sex of the cells in tissues of an intermediate nature? The continuous change from ovarian to testicular characters may be a reflection of the changing composition of the tissue by cells of each genetic sex. On the other hand, there is a possibility of secondary obliteration of the primary relationships due to morphogenetic interactions during differentiation of gonadal tissue. We are forced to consider such a possibility by the results of experiments carried out by Holyoke (1956), Macintyre (1956), Beber (1957), Holyoke & Beber (1958) and Macintyre et al (I960), who found that in mammals the embryonic testes inhibit differentiation of ovarian tissue and can exert some masculinizing effect on it. No influence in the opposite direction was observed. Such interactions may take place in the ovotestes of chimaeric and spontaneously developed hermaphrodites thus obliterating the

20 750 ANDRZEJ K. TARKOWSKI primary boundary between the territories of XX and XY constitution. In this situation it can be postulated that only the territories showing the highest morphological resemblance to testicular and ovarian tissue are composed of populations of cells of predominantly XY and XX type respectively. Since ovarian tissue does not influence the differentiation of testicular tissue the zone displaying intermediate characters can be considered as being composed of either a mixed population of cells or of genetically female cells which were under the influence of the testicular part. The ability of XY tissue to influence the differentiation of tissues of XX constitution in the testicular direction, or at least to inhibit its development in the ovarian direction, should not be overemphasized. In the ovotestes examined, at the level where the ovarian and testicular territories are separated from each other by the layer of loose mesenchymal tissue, this hypothetical morphogenetic stimulus must have been completely ineffective. The presence, in the remaining part of the gonad of the tissue of intermediate type, may indicate, though it need not (see above), that a morphogenetic stimulus elaborated by XY tissue has been in action. Assuming that this is so, the fact that the ovarian tissue can differentiate normally, even in close proximity to the testicular tissue and when the two territories are continuous with each other, means that the hypothetical stimulus must be weak and spatially restricted to the neighbourhood of the site of its elaboration. In the light of these findings it seems that a gonad of mixed composition could develop into a normal testis (as was suggested previously) only in those cases where both kinds of cells are completely mixed up and the genetically female eels do not form a compact territory. Behaviour of germ cells Theoretically, in the gonads of a chimaeric individual which is a sex-chromosome mosaic, germ cells should be of two types XX and XY, each group being derived from one contributing egg. Primordial germ cells originate in the mouse extra-embryonally and afterwards migrate to their definite locations in the germinal ridges (Chiquoine, 1954; Mintz & Russell, 1957; Mintz, 1957, 1959). This has several implications. In the case of lateral hermaphroditism both ovary and testis should contain two types of germ cells. There is no reason whatever to postulate a differential immigration of XX germ cells to the future ovary and of XY germ cells to the future testis. When an ovotestis is formed, it can also be expected to contain two types of germ cell, but the distribution of these two types need not coincide with the genetic sex of the somatic cells in the ovotestis. If the primordial germ cells of one type failed for some unknown reasons to reach the germinal ridges then both kinds of gonadal tissue of a hermaphrodite would contain a single type of germ cell. These considerations are valid, also, for spontaneously developed sexchromosome mosaics in which the 'mitotic error' took place early in

21 Hermaphroditism in mice 75 1 embryogenesis (cleavage) and affected not only somatic cells but also predecessors of the future primordial germ cells. If the 'mitotic error' takes place later in development it might, perhaps, in occasional cases, influence the differentiation of the gonad without affecting the karyotype of the primordial germ cells themselves. When the germ line finally deviates from the somatic line in the development of the mouse is not known. Most probably this takes place quite early and long before the 8th day when the primordial germ cells can first be identified by their high alkaline phosphatase content (Chiquoine, 1954; Mintz & Russell, 1957; Mintz, 1957, 1959). From the late embryonic period onwards the germ cells in the gonads of chimaeric hermephrodites are represented by two distinct types oocytes and spermatogonia. In mature animals, cells representing later stages of spermatogenesis would presumably have been present as well. These two types do not necessarily represent XX and XY germ cells respectively. Experimentally induced complete sex reversal in some fishes (see Gowen, 1961), amphibians (see Burns, 1961) and birds (Miller, 1938) followed by formation of the gametes typical for the acquired sex, as well as the early meiotic prophase stages seen in the gonocytes of the transformed testes of the opossum (Burns, 1956,1961), show clearly that gametogenesis follows the somatic sex of the gonadal tissue and not the genetic sex of the germ cells themselves. It follows that the meiosis-inducing stimuli are elaborated by the ovarian and testicular tissues themselves, that the stimulus inducing meiosis of the female type must be different in nature from that inducing meiosis of the male type, and that the germ cells of male genetic sex are potentially able to start meiosis at the time normally characteristic of females. In the absence of direct information it seems fair to assume that the properties of germ cells and the mechanisms regulating meiotic processes are the same in placental mammals as in the other classes of vertebrates. True mammalian hermaphrodites would provide unequivocal answers to these questions if it were known that the whole population of germ cells in such animals is of one genetic sex only. However, since it is equally likely that cells of both genetic sexes are represented, such material can furnish only indirect information, although this is all that is available at present. In the spontaneously developed rodent hermaphrodites so far described, the authors have noted the coincidence between the character of the germ cells and the somatic sex of the gonadal tissue in which they are situated. The only exception to this rule so far, observed in rodents, consists in finding oocytes in the seminiferous tubules of the ovotestes of the rat (Bradbury & Bunge, 1958). In the newborn chimaeric hermaphrodites an ovary or testis has germ cells that are, in fact, consistent with the somatic sex of the gonadal tissue, whereas in the ovotesticular gonad there is no such strict conformity. While in the ovaries and the ovarian territory of ovotestes all germ cells have entered into meiosis and there are no undifferentiated gonia, in the testicular territory of ovotestes germ cells are represented both by spermatogonia and oocytes.

22 752 ANDRZEJ K. TARKOWSKT The distribution of oocytes and spermatogonia in the testicular tissue reveals a tendency for spermatogonia to be relatively more numerous in those regions which are far away from the ovarian tissue, while in the testicular tissue lying close to the ovarian tissue all germ cells are undergoing meiosis of the female type. It is only at those levels where the two territories are separated from each other by a layer of loose messenchymal tissue that the sex cords situated close to the ovarian tissue may contain spermatogonia instead of oocytes. In the testicular region of the gonad, opposite to the ovarian region, germ cells may be represented either exclusively by spermatogonia (right ovotestis of the animal no. 61/3, ovotestis of the animal no. 59/2) or, both kinds of cells may be present together (left ovotestis of animal no. 61/3, ovotestis of animal no. 31/1). The hermaphroditic animals described in this report died at the age of meiotic prophase in normal female development which considerably precedes the normal time for male meiosis. In normal development of the female mouse the meiotic prophase starts in the ovaries shortly before parturition, and during the few days after birth all oocytes reach the dictyate stage (Borum, 1961; Slizinski, 1961). Therefore, initiation of meiosis of the female type by the germ cells populating ovotestes of the newborn chimaeric hermaphrodites can be attributed only to the action of the 'female' meiosis-inducing stimulus. Only the germ cells which have not entered into meiosis, by this stage of development, can become the male germ cells. The behaviour of germ cells in the ovotestes examined, and especially the presence of oocytes in the testicular tissue, becomes understandable if it is assumed that the meiotic stimulus is elaborated by the ovarian tissue, that it specifically determines the course of meiosis of germ cells, irrespective of their genetic sex, and that it is able to diffuse through the gonadal (testicular) tissue and to act at some distance from the place of its origin. The only difficulty met by this hypothesis is that it is not possible to draw in the testicular territory a clear-cut borderline separating spermatogonia from oocytes. Quite often some of the germ cells situated at the same level of the gonad, or even side by side in the same sex cords, enter into meiosis while the others do not. This differing behaviour of neighbouring germ cells could be explained tentatively on the ground that in the testicular tissue the diffusion of the meiotic stimulus elaborated by the ovarian tissue is very erratic and that the proper threshold is not maintained over the period when all germ cells become ready to react. The fact that in the typically testicular tissue of ovotestes there are both oocytes and spermatogonia, while in the ovarian tissue there are neither undifferentiated gonia nor cells undergoing meiotic divisions, only oocytes being present, can be explained only on the ground that the initiation and course of meiosis in the mouse is governed completely by the kind of meiotic stimulus and does not depend on the genetic sex of the germ cells. Theoretically, the only method which should permit of distinguishing XX and XY germ cells in the gonads of neonatal mouse chimaeric hermaphrodites is

23 Hermaphroditism in mice 753 based on cytological differences displayed by them during meiosis. Male germ cells in meiosis possess a characteristic structure, the so-called' sex vesicle' which appears in zygotene, is prominent through pachyteneand disintegrates before diakinesis (see Geyer-Duszynska, 1963, for details and references). During pachytene the XY bivalent is enclosed in the sex vesicle. No such structure has been found in the course of female meiosis. Since the formation of the sex vesicle must be associated with the intimate peculiarities of the XY bivalent only, it may be expected that the vesicle will be formed in genetically male germ cells irrespective of the kind of meiotic stimulus to which the cells have been submitted. Search for sex vesicles in the meiotic germ cells in the ovotestes of animals nos. 31/1 and 61/3, as well as in the ovary of animal no. 31/1, was unsuccessful. Most gonocytes in these gonads are at the stage from late pachytene onwards and, therefore, are not suitable for this purpose. Apart from a few doubtful cases no sex vesicle could be identified in the pachytene nuclei. This cytological approach did not furnish any conclusive evidence either for or against the theory of a mixed population of germ cells in the gonads of chimaeric hermaphrodites. The character of the population of germ cells in fertile chimaerae can be assessed by breeding with the parental strains. So far, the only chimaeric male which has been tested in this way did not furnish any useful information (Tarkowski, 1963). In this particular case the analysis of crosses was rendered difficult because of the combination of eggs used for fusion (one component was a hybrid hererozygous for pigmentation factors). Knowledge of the fate of primordial germ cells originating from both contributing eggs and their distribution in the gonads of sexually normal chimaeric individuals would be important per se, but it should also provide useful indirect information regarding hermaphrodites. SUMMARY 1. Histological examination of the reproductive systems of fourteen newborn chimaerae developed from fused eggs revealed eleven sexually normal animals and three true hermaphrodites. The genital apparatus of the hermaphrodites is described, the structure of the gonads being given special attention. 2. Of the three hermaphrodites, one had one ovary with female sex ducts and an ovotestis with complete male ducts and incomplete female ones. The second had two ovotestes and a genital apparatus of nearly typically male type; the remnants of Miillerian ducts being retained only caudally. The third had one ovary with female sex ducts and one ovotestis with male sex ducts. 3. The occurrence of true hermaphrodites strongly suggests that spontaneous true hermphroditism also develops from sex-chromosome mosaicism. 4. The available literature on spontaneous hermaphroditism in rodents is critically reviewed. It is postulated that the frequencies of the four types of true hermaphroditism described (ovary-testis, ovary-ovotestis, testis-ovotestis,

24 754 ANDRZEJ K. TARKOWSKI ovotestis-ovotestis) in mice and other rodents do not adequately reflect their actual incidence in nature in that the incidence of the lateral type of hermaphroditism is exaggerated. 5. It is suggested that if the testicular tissue does, in fact, elaborate a morphogenetic stimulus influencing the differentiation of XX gonadal tissue, such a stimulus must be rather weak and act only near the site of its elaboration. 6. The genetic sex of germ cells in chimaeric hermaphrodites remains unknown. It is postulated that the presence in the hermaphrodite gonads of oocytes and male germ cells reflects only the secondary differentiation of germ cells, which need not reflect their genetic sex. While the ovarian tissue of the ovotestes examined contains only oocytes, in the testicular tissue both oocytes and spermatogonia are present. Initiation of meiosis of the female type in the testicular territory is attributed to the action of a meiotic stimulus elaborated by the ovarian tissue and diffusing through the testicular part. RESUME Hermaphrodisme vrai chez des souris-chimeres 1. L'examen histologique des organes de reproduction chez quatorze nouveaunes chimeres issus d'oeufs fusionnes a revele, a cote de onze animaux sexuellement normaux, trois veritables hermaphrodites. L'appareil genital de ceux-ci fait l'objet d'une description ou l'attention est specialement dirigee sur la structure des gonades. 2. Des trois hermaphrodites, l'un avait un ovaire avec voies genitales femelles, et un ovotestis avec voies males completes et voies femelles incompletes. Le deuxieme avait deux ovotestis et un appareil genital presque typiquement male, des restes de canaux de Miiller ne s'etant maintenus que dans la region caudale. Le troisieme avait un ovaire avec des voies genitales femelles, et un ovotestis avec des voies genitales males. 3. L'existence de ces veritables hermaphrodites suggere fortement que l'hermaphrodisme vrai spontane est du a un mosaicisme heterochromosomial. 4. Les donnees de la litterature concernant l'hermaphrodisme spontane chez les rongeurs sont soumises a une revision critique. Elle conduit a penser que les frequences des quatre types d'hermaphrodisme vrai (ovaire-testicule, ovaireovotestis, testicule-ovotestis, ovotestis-ovotestis) decrits chez la Souris et d'autres rongeurs ne refletent pas adequatement leur incidence effective dans la nature, en ce sens que l'incidence du type lateral d'hermaphrodisme est exageree. 5. II est suggere que si le tissu testiculaire exerce effectivement un stimulus morphogenetique influencant la differentiation du tissu gonadique de constitution XX, un tel stimulus doit etre plutot faible et agir seulement a proximite du siege de son elaboration. 6. Le sexe genetique des cellules germinales dans les chimeres hermaphrodites demeure inconnu. On peut soutenir que la presence, dans les gonades

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