/. Embryol. exp. Morph. Vol. 65, pp. 173-184, 1981 173 Printed in Great Britain Company of Biologists Limited 1981 Sex differentiation in bilaterally allophenic animals produced by cloning of two bipartite male/female chimaeras of Lineus sanguineus By S. SIVARADJAM 1 AND J. B] ERNE 1 From the Department of Biology, University of Reims, Reims, France SUMMARY Two bipartite chimaeras were constructed in Lineus sanguineus by grafting the lateral halves from a phenotypically dark-brown male onto the anatomically complementary halves from a phenotypically light-brown female. Regeneration of a large number of pieces transected from these two bilaterally allogeneic chimaeras produced two clones of bilaterally allophenic nemertines (c?/$ and?/<?). Sex differentiation in the cloned worms started with a transitory stage of gonad developmental autonomy, termed the primary gynandromorphous state; at this stage there were young testes in the originally male lateral halves and juvenile ovaries in the originally female ones, the only abnormality then was that the ovarian development was more advanced than the testicular development relative to those in male and female controls. Then, unilateral sex reversal occurred, with feminization of the testes, i.e. oogenesis took the place of spermatogenesis in the many male gonads located in either the right or the left side of allophenic worms according to the symmetry patterns of the two clones. Finally, when the gonads reached maturity, both sides of allophenic L. sanguineus contained only ovaries with ripe oocytes. The complete feminization of these allophenic worms and the previously observed masculinization of 'heterosexual' chimaeras in L. ruber suggest that a diffusible factor controls gonadal differentiation in worms of the prevailing sex, which is the female sex in L. sanguineus and the male sex in L. ruber. RESUME Deux chimeres bipartites ont ete realisees chez Lineus sanguineus en greffant les moities laterales d'un male de phenotype brun-fonce avec les moities anatomiquement complementaires d'une femelle de phenotype brun-clair. La regeneration des innombrables troncons decoupes sur ces deux chimeres bilateralement allogeniques a produit deux clones de nemertes bilateralement allopheniques (<?/? et $/c?). La differentiation du sexe chez les clonants debute par un stade transitoire d'autonomie du developpement gonadique appele etat gynandromorphe primaire. Cet etat est caracterise par la presence de jeunes testicules dans les moities laterales d'origine male et par celle d'ovaires juveniles dans les moities d'origine femelle. La seule et discrete anomalie identifiable a ce stade est une legere avance du developpement ovarien sur celui des testicules. Par la suite, 1 Author's address: Laboratoire de Biologie Generate, Faculte des Sciences, Universite de Reims, B.P. 347, 51062 Reims, France.
174 S. SIVARADJAM AND J. BIERNE une inversion du sexe intervient unilateralement par feminisation des testicules. En effet, des foyers d'ovocytes viennent se substituer aux spermatogonies, spermatocytes et spermatozo'ides presents dans chacune des nombreuses gonades males localisees soit a droite, soit a gauche, selon que les Vers ressortissent a l'un ou l'autre clone. Finalement, quand les gonades sont mures, seuls des ovaires remplis d'ovocytes prets a etre pondus sont presents dans les deux flancs des L. sanguineus allopheniques. La complete feminisation des Vers allopheniques, aussi bien que la masculinisation des chimeres heterosexuees observee anterieurement chez L. ruber suggerent qu'un facteur diffusible controle la differentiation gonadique chez les Vers du sexe prevalent, c'est-a-dire le sexe femelle chez L. sanguineus et le sexe male chez L. ruber. INTRODUCTION Genotypic factors of sex determination and phenotypic mechanisms of sex differentiation have been examined in several groups of metazoa. So far, vertebrates (especially mammals) and arthropods (especially insects and crustaceans) have been extensively studied, either because of the valuable comparisons that may be drawn with human organogenesis of sex characteristics or because of the experimental accessibility, the abundance, and the ubiquity of the animals. By contrast, few such studies have been made of animals of other phyla, and no data are available concerning the basic events of sexual development and their interrelationships. However, some of the organisms which have attracted the least attention from biologists may contribute to the understanding of sex determination and differentiation because of their unusual properties. This is true of nemertines of the strictly gonochoristic genus Lineus, which can be converted to 'heterosexual' worms by the grafting technique of Bierne (1966, 1967a); this technique is easily carried out between adults of the two sexes (Bierne, 19676, 1968). MATERIALS AND METHODS (a) Production of symmetrical FU (cf/$) and UF ($/< ) graft chimaeras In Lineus species, particularly L. ruber, sagittally cutting two animals of opposite sex and then grafting female lateral halves with anatomically complementary male halves makes it possible to construct two symmetrical 'heterosexual' chimaeras (Bierne, 1970#, b, 1975). We have applied this experimental procedure to a pair of L. sanguineus of both sexes because this species, unlike L. ruber, is endowed with the power of complete regeneration and can reproduce asexually. The two worms were chosen for contrasting pigmentation to emphasize by an external marker the chimaeric status in parabiotic fusions of their lateral halves. Thus, each chimaera formed by grafting the right half of one worm onto the left half of a worm of the opposite sex had different colours on the two sides of the body; this bilaterally chimaeric phenotype was passed on to the newly differentiated body part grown as the posterior end of the composite worm, elongated. The first animal was taken from a clone termed QJ4, obtained by vegetative multiplication of a male L. sanguineus specimen collected on the Brittany coast
Sex differentiation in bilaterally allophenic Lineus 175 of the English Channel. Unlike nemertines of many other clones reared in our laboratory, the C^4 worms have the convenient characteristic of strongly resisting attack by parasites, especially orthonectides and gregarines, which commonly infest nemertines. All QJ4 worms are dark brown when they are adults, i.e. sexually mature. The second animal was from another laboratory clone (called U?l) which we selected for the light-brown pigmentation of the adult worms and for their normal oogenesis. This clone had been produced by asexual reproduction of a female L. sanguineus collected on the Uruguayan shores of the Scuth Atlantic Ocean, because the females from the French coast of the Channel, unlike the males, show abortive gametogenesis (Gontcharoff, 1951; Bierne, 1970a). The special features of the full parabiosis in Lineus resulting from grafting together two halves from adults were : (1) Single-worm status. The half worms grafted together were so closely fused that they formed a single worm, a bilaterally 'heterosexual', allogeneic chimaera. Like a normal nemertine, such a chimaera can be cloned. (2) Morphological and functional normality. By fusion of foreign, symmetrical lateral halves, anatomically normal worms were reconstructed. Multiple examinations of histological sections showed the continuity of all their tissues and structures; their internal content was the faithful image of that of a single functional animal. Externally, too, they looked and acted like normal worms, except that the different skin pigmentation phenotypes of the two halves were retained as the worms grew. (3) No effective immune response. Extensive studies on transplantation in all species of adult Lineus have shown that allografts are accepted for a very long time (Bierne, 1970a, 1972, 1975, 1980; Langlet & Bierne, 1973, 1977); for example, several bilaterally 'heterosexual' chimaeras obtained by grafting together half worms of different sexes from L. ruber in 1967 are still surviving today in our laboratory. The full parabiosis led to two symmetrical chimaeras, called FU and UF (F, France; U, Uruguay): chimaera FU formed from the light-brown female right half (U$l) and the dark-brown male left half (Q?4), and chimaera UF, the reverse, i.e. the mirror image of chimaera FU. (b) Cloning of FU ( /$) and UF ($/ ) chimaeras The original FU and UF graft chimaeras were initially fed well and regularly, so that they elongated in the usual way for nemertines, by growing posteriorly. Then the body, at the level of the gonads and intestine, was cut into many short transverse sections, from which small, anatomically complete chimaeric worms arose by cephalic and caudal regeneration; these small nemertines also elongated in the usual way, by posterior growth. Meanwhile the head part of each graft chimaera was also maintained; these, too, after wound healing and regeneration, grew into large chimaeras.
176 S. SIVARADJAM AND J. BIERNE Fig. 1. Dorsal view of an allophenic nemertine from the UF ('Uruguay/France') clone. The background paper is marked off in millimetres. Fig. 2. Ventral view of an allophenic nemertine from the UF clone. The background paper is marked off in millimetres. Subsequently both types of chimaeric worms (those regenerated from the cephalic ends and those from the many sections) were in turn transected. In this way, we produced two clones, FU and UF, of chimaeric nemertines, which retained the bipartite phenotype of the original allogeneic chimaeras (Figs. 1, 2 and 6). We emphasize that the phenotypically bipartite nature of the worms became evident only after the sections had completely regenerated, i.e. a developmental process occurs before the dual phenotype becomes evident, as in chimaeric mice obtained by fusing embryos. We use the term 'allophenic' (coined by Mintz (1970) for mice produced from chimaeric embryos) for cloned nemertines produced from chimaeric adults. (c) Special rearing conditions The allophenic nemertines were reared at constant temperature (12 C± 1 ) in darkness to eliminate factors, such as photoperiod and thermoperiod, that act on the biological rhythms. Each worm was isolated in a small, labelled glass bottle, both for easy observation of its growth and to prevent any interaction with other partners. Because feeding is an important variable factor in nature,
Sex differentiation in bilaterally allophenic Lineus 177 all the allophenic nemertines were given the same food, calf liver once a week. Thus the rearing conditions excluded the influence of external factors. (d) Sexual differentiation in cloned allophenic newer lines \t might be objected that only the interactions between grafted tissues from adults were studied. But we wish to emphasize how allophenic worms regenerated from transected pieces of graft chimaeras (in which sexual differentiation has already taken place), can serve as appropriate material for research into sexual development. The procedure was as follows. We amputated all of the gonads of 32 allophenic nemertines by transection behind the mouth, thus placing the worms in a state of complete sexual undifferentiation (Fig. 3, section 6). In this situation, which can be compared with sexual undifferentiation in embryo parabiosis, it is also possible to investigate the cellular and hormonal interactions that come into play during sexual differentiation. Fig. 3 also summarizes all the experimental manipulations diagrammatically, together with the numbers of each type of specimen examined. Gonadogenesis was studied under two different conditions: (1) in the permanent presence of the brain, which can produce a gonad-inhibiting hormone (Bierne, 1964, 1966, 1970a, b; Bierne & Rue, 1979) (Fig. 3, sections 1, 2, 4 (top) and 6); and (2) temporarily without the influence of the brain (Fig. 3, sections 3, 4 (bottom) and 5). We observed the development of allophenic nemertines of the two clones in both situations. For the histological studies, the worms at each anatomical state of sexual development (as defined below) were fixed in Bouin's fluid, embedded in paraffin, sectioned at 7 jam, and routinely stained with haematoxylin and eosin. RESULTS We report here studies of 279 allophenic worms during the first sexual cycle which followed the experimental manipulations (Fig. 3). Whether or not the chimaeric animals were permanently under the influence of the brain, the same sequence of events of sex differentiation occurred. The only differences observed were a more rapid formation and earlier development of the gonads in the temporarily brainless pieces than in the corresponding parts with brains. Although the brain influenced the timing of the sexual differentiation, it did not control the sex phenotypes. The gonads of all the allophenic worms eventually passed through three different anatomical stages, corresponding to three states of sex differentiation. (a) Primary gynandromorphous state The first anatomical state of the developing allophenic nemertines, called the primary gynandromorphous state by reference to the autonomous development
178 S. SIVARADJAM AND J. BIERNE 50 30 UF + 20 FU 38 38 23UF+ 15 FU "3~UF+T5~FU" U 26 95 16UF+ 10 FU 62 UF + 33 FU 32 20 UF + 12 FU
differentiation in bilaterally allophenic Lineus 179 of sex characteristics in gynandromorphs, corresponded to the first histologically determined stage of sexual differentiation. In this case, the allophenic worms clearly exhibited the duality of their sexual constitution, having two types of gonads: ovaries in the side of female origin and testes in the side of male origin. The phenotype of the genital organs proved to be dependent on the origin of the territories concerned in their organogenesis (Fig. 4). Gametogenesis in the male and female halves of these 'gynandromorphic' worms, as observed in histological sections, was analysed in terms of the threestage pattern of gametogenesis seen in L. ruber (Bierne, 1970a): stage I, a 'latent period'; stage II, a period of spermatogenesis (in the male) or auxocytosis (in the female); and stage III, a period of spermiogenesis (in the male) or vitellogenesis (in the female). The stage of sexual development in the allophenic L. sanguineus of the present study was clearly different in the male and female halves of a given individual, with that of the female half generally clearly more advanced than that of the male half. When the half of male origin was in stage I (the latent period), the testis contained only gonia, whose number increased gradually. However, at this same time the oocytes, on the female side, were in a period of auxocytosis (stage II) with abimodalsize distribution because many of them were'arrested'in their growth; only one to six oocytes increased much in size. Thus, the gynandromorphous state at this time was characterized by more advanced development of the ovaries than of the testes. Examination of other samples extended the same pattern a step further. When the testes had reached the spermatogenesis period (stage II), during which male gametogenesis had begun, all categories of male germ cells (gonia, primary and secondary spermatocytes, spermatids and spermatozoa) were seen, arranged in successive crescents. At the same time, the oocytes were in a period of vitellogenesis (stage III), with vitelline enrichment of their cytoplasm and a considerable increase of their size, so that they filled the ovarian cavities, situated in the connective packaging round the intestine. (b) Unilateral sex reversal The second anatomical state of the developing allophenic worms, the state of unilateral sex reversal with a feminizing effect, was the middle event of sexual organogenesis. In the female part, gonadal development ran its normal course; Fig. 3. Diagram showing the gonadal differentiation in the various types and the number of allophenic worms studied: 1, in unoperated animals; 2, in animals regenerated from large anterior parts of sexually undeveloped allophenic worms; 3, in animals regenerated from small posterior ends of sexually undeveloped allophenic worms; 4, in animals regenerated from bisected sexually undeveloped allophenic worms; 5, in animals regenerated from small transected pieces of sexually undeveloped allophenic worms; 6, in animals regenerated from heads of allophenic worms. The total number of each type of specimen is indicated in the first line, and its composition (FU or UF) in the second line.
180 S. SIVARADJAM AND J. BIERNE Fig. 4. Transverse section of an allophenic worm from the FU clone, showing a testis on the left side and an ovary on the right (primary gynandromorphous state). Scale bar, 50 /tm. the ovaries underwent no modifications other than those inherent in the gradual maturation of gametes. In the male part, in both clones, however, the testes gradually became typical ovotestes as centres of oocytes appeared among the spermatogenic cells. Thus, genetic sex and gonadal sex began to be dissociated from each other in the parts of male origin, with female gametes developing in the testes. (c) Secondary feminized state The third and last anatomical state of the allophenic nemertines which was recorded, termed the secondary feminized state, corresponded to the final stage of sexual differentiation. The allophenic nemertines were then not noticeably different from the normal females. In many individuals, feminization went so far that both ovaries reached the same size and the same stage of development; no difference could then be perceived and the worms could be mistaken for females from the U$l clone (Fig. 5). The worms had no testes, but only many large ovaries, which contained submature or mature oocytes filling threequarters of the body volume. The sex of the gonads of the genetically male halves had been completely and spectacularly reversed. In this third state the dissociation of genetic sex from phenotypic sex in the genetically male halves of the allophenic worms was complete. Although during the first two states of sexual differentiation the cloned animals
Sex differentiation in bilaterally allophenic Lineus 181 Fig. 5. Transverse section of a feminized allophenic worm from the FU clone. The left gonad, initially a testis, was spectacularly reversed into a bulky ovary. No difference can be observed from the details of a section of a female control. Scale bar, 120 /*m. were asymmetrical both internally, in their gonadal constitution, and externally, in their dual pigmentation, during the last state the gonadal difference disappeared totally: FU and UF worms had identical female gonads (Fig. 5). The chimaeric difference in pigmentation, however, persisted (Fig. 6). It is unfortunate that the sex-specific cutaneous glands present in L. ruber and L. viridis are missing in L. sanguineus, so that they cannot be used to elucidate the differentiation of secondary sexual characteristics, in the latter species. DISCUSSION Tn a study of Lineus ruber chimaeras derived by grafting together lateral halves of opposite sexes, Bierne (1970 a) found that in the early stage of gonadal differentiation during posterior regeneration, the testes developed earlier and faster than the ovaries; later, the female halves showed clear features of masculinization; and in the final stage, all the gonads of the 'heterosexual' chimaeric worms had become testes engaged in intense spermatogenetic activity. The opposite effects were found in the present study of allophenic male/female L. sanguineus, the ovaries developed earlier and faster than the testes and in the final stage the worms possessed only ovaries with ripe oocytes.
182 S. SIVARADJAM AND J. BIERNE Fig. 6. Dorsal view of a feminized allophenic worm from the FU clone, showing persistence of the difference in pigmentation between the two sides of the body. The background paper is marked off in millimetres. In both cases (chimaeric L. ruber and allophenic L. sanguineus) sex differentiation was autonomous at the beginning of gonadaljievelopment, ovaries developed in the parts regenerated from female halves, and testes developed in the part regenerated from male halves. At a slightly more advanced stage, the growth of either the testes (in chimaeric L. ruber) or the ovaries (in allophenic L. sanguineus) was stimulated. Might the subsequent masculinization and feminization, respectively, result from an advance of gonadal differentiation in the dominant sex? Tn vertebrates and other organisms, including plants, the more rapid organogenesis of gonads in the prevailing sex suggested to Mittwoch (1973) the differential-growth theory. But this theory, based on a postulated faster rate of mitosis in the cells of the gonads of the dominant sex, accounts only for endogenous gonadal processes. It does not explain how the dominant sex component can, from a distance, reverse the gonadal sex of the dominated component in male/female composite worms. An exogenous mechanism must be put forward. From this point of view, two kinds of phenomenon could account for the sex reversal of gonads and germ cells in bipartite chimaeric L. ruber and allophenic L. sanguineus: either a migration of genetically determinate and sexually com-
Sex differentiation in bilaterally allophenic Lineus 183 petent cells from the dominant halves into the dominated halves, with lysis and/or inhibition of the equivalent resident cells; or else a sexualizing action of a substance emanating from the halves of the prevailing sex upon sexually competent cells of both genetic sexes. Neither of these hypotheses is confirmed or rejected by our current findings. However, we favour the idea of the existence of a diffusible sexualizing factor because of suggestive results of extensive works on sex differentiation in animals of other phyla. Among many speculations about the nature of this putative sexualizing substance, the hormonal theory and the sex-antigen theory (see for review the recent paper by McCarrey & Abott, in Advances in Genetics, 1979) seem most promising. Furthermore, to be consistent with our previous and present data, the sexualizing factor should be androgeneic in L. ruber but gynogeneic in L. sanguineus, perhaps because the male is heterogametic in the former species and the female in the latter. We are grateful to Dr Dei Cas for supplying the L. sanguineus female specimen cloned in U$l, to Miss Laquerriere for technical assistance, and to Mrs Ch. Derangere for typing the manuscript. This work was supported by a grant (Al no. 33666) from the Centre National de la Recherche Scientifique. REFERENCES BIERNE, J. (1964). Maturation sexuelle anticipee par decapitation de la femelle chez rheteronemerte Lineus ruber Miiller. C. r. hebd. Seanc. Acad. Sci.., Paris 259, 4841-4843. BIERNE, J. (1966). Localisation dans les ganglions cerebroi'des du centre regulateur de la maturation sexuelle chez la femelle de Lineus ruber Miiller (Heteronemertes). C. r. hebd. Seanc. Acad. Sci., Paris 262, 1572-1575. BIERNE, J. (1967a). Regeneration posterieure des chimeres interspecifiques chez les Lineidae (Heteronemertes). Bull. Soc. Zool. Fr. 92, 351-359. BIERNE, J. (19676). Sur le controle endocrinien de la differenciation du sexe chez la Nemerte Lineus ruber Miiller. La masculinisation des ovaires des chimeres heterosexuees. C. r. hebd. Seanc. Acad. Sci., Paris 265, 450-477. BIERNE, J. (1968). Facteur androgene et differenciation du sexe chez la Nemerte Lineus ruber Miiller. L'effet 'free-martin' dans la parabiose heterosexuee. C. r. hebd. Seanc. Acad. Sci., Paris 261, 1646-1648. BIERNE, J. (1970a). Recherches sur la differenciation sexuelle au cours de l'ontogenese de la regeneration chez le Nemertien Lineus ruber Miiller. Ann. Sci. Nat. Zool. 12, 181-288. BIERNE, J. (19706). Aspects experimentaux de la differenciation sexuelle chez Lineus ruber (Heteronemertes). Bull. Soc. Zool. Fr. 95, 529-543. BIERNE, J. (1972). Greffe et regeneration chez les Nemertiens: aspects immunologiques. In Vetude Phylogenique et Ontogenique de la Reponse immunitaire et son Apport a la Theorie Immunologique (ed. P. Liacoupoulous et P. Panigel), pp. 47-53. Paris: INSERM. BIERNE, J. (1975). Sex differentiation in regenerating 6/2 Nemertine chimeras. In Intersexuality in the Animal Kingdom (ed. R. Reinboth), pp. 30-40. Heidelberg: Springer- Verlag. BIERNE, J. (1980). Viable animals obtained by grafting pieces from several nemertean adults. Transplantation 29, 74-76. BIERNE, J. & RUE, G. (1979). Endocrine control of reproduction in two rhynchocoelan worms. Int. J. Invertebr. Reprod. 1, 109-120. GONTCHAROFF, M. (1951). Biologie de la regeneration et de la reproduction chez quelques Lineidae de France. Ann. Sc. nat. Zool. 13, 149-235.
184 S. SIVARADJAM AND J. BIERNE LANGLET, C. & BIERNE, J. (1973). Recherches sur l'immunite de greffe chez les Nemertiens du genre Linens. Evolution de transplants homospecifiques et heterospefiques. C. r. hebd. Seanc. Acad. Sci., Paris 276, 2485-2488. LANGLET, C. & BIERNE, J. (1977). The immune response to xenografts in Nemertines of the genus Lineus. In Developmental Immunobiology (ed. J. B. Salmon & J. D. Horton), pp. 17-26. Amsterdam: Elsevier North-Holland. MCCARREY, J. R. & ABBOTT, U. K. (1979). Mechanisms of genetic sex determination, gonadal sex differentiation, and germ-cell development in animals. In Advances in Genetics (ed. E. W. Caspari), pp. 217-290. New York: Academic Press. MINTZ, B. (1970). Gene expression in allophenic mice. In Control Mechanisms in the Expression of Cellular Phenotype (ed. M. A. Padykula), pp. 15-41. New York: Academic Press. MITTWOCH, V. (1973). Genetics of Sex Differentiation. New York: Academic Press. {Received 9 February 1981, revised 30 April 1981)