Cytogenetic and Genetic Evidence of Male Sexual Inversion by Heat Treatment in the Newt Pleurodeles Poireti

CHROMOSOMA Chromosoma (Bet1) (1984) 90:261-264 Springer-Verlag 1984

Cytogenetic and genetic evidence of male sexual inversion by heat treatment in the newt Pleurodeles poireti

C. Dournon 1, F. Guillet 1, D. Boucher 2, and J.C. Lacroix 2 1 Laboratoire de Biologic Animale; 2Laboratoire de G6n~tique du D~veloppement, Universit~ P. et M. Curie, 9 quai St Bernard 75230 Paris Cedex 05, France

Abstract. Larvae of Pleurodeles poireti were maintained the lengthy reproductive cycle of the amphibians. It should during their development at a high temperature (31 ~ C). be interesting therefore to identify the genotypic sex of an In several species of amphibians, such a treatment is known experimental animal by cytogenetic or other genetic criteria, to change the sex ratio through the inversion of genotypic i.e. through sex chromosomes or sex-linked characters re- females into phenotypic males. Pleurodeles poireti is an ex- spectively. ception. It is the first reported amphibian in which heat In a geographical race of P. poireti, the sex chromo- induces an inversion of genotypic males into functional phe- somes can be distinguished in preparations of lampbrush notypic females. The sexual genotype of standard and ex- chromosomes of the oocytes (Lacroix 1970). Moreover, perimental phenotypic females was determined through he- Ferrier et al. (1980, 1983), have shown that in P. waltlii terochromosomes in lampbrush stage. In the present study, the enzyme peptidase I shows a sex-linked polymorphism. we have utilised another technique for identification of sex- The effects of heat treatment on sexual differentiation ual genotype, applicable to both phenotypic males and fe- of larvae of P. poireti are presented here. We show that, males. It is based on the differential expression of a sex- contrary to the results obtained under similar conditions linked gene, the peptidase 1. in P. waltlii, the sex ratio is biased in favour of females. Demonstration of sexual inversion of genotypic males is based on cytogenetic and genetic analysis.

Introduction Materials and methods The amphibians, urodeles as well as anurans, constitute Heat treatment. The procedure followed was the one that a particularly favourable material for studies on phenotypic was previously developed for P. waltlii (Dournon 1981). functional sexual inversion. Obtaining unisexual offspring The experimental embryos developed at room temperature from animals having undergone gynogenic or androgenic (20~ 3 ~ C) from the egg until the stage immediately pre- treatment demonstrates the phenotypic sexual inversion of ceding hatching (stage 33a of the developmental table of one of the parents. Crosses between normal and sex-re- P. waltlii, GaUien and Durocher 1957). From hatching until versed individuals allow one to define the homogametic the end of metamorphosis, the larvae were placed at a tem- and heterogametic sex for each of the studied species when perature of 31~ 1~ C, and from the end of metamorphosis no sex chromosome can be cytologically detected, as is often until sexual maturity (6-8 months) the animals were main- the case in amphibians (for review see Foo~e 1964). tained at a temperature of 230-27 ~ C and thereafter at room The two species of Pleurodeles, P. waltlii and P. poireti temperature. (urodeles) have often been utilised for such studies. It was Control individuals developed from the egg to the adult thus demonstrated that in P. waltlii, the males are homoga- stage at room temperature. tactic ZZ: crosses between a normal male and a neo-female (genotypic male inversed into phenotypic female by estra- Early identification of sexualphenotype. The sexual pheno- diol benzoate treatment) give an exclusively male progeny type of the gonads was identified on living animals about (Gallien 1951). The homogametic nature of males in P. 3 months after metamorphosis, a stage at which a simple poireti was demonstrated by a similar experimental proce- examination of gonad morphology under a dissecting mi- dure (Lacroix 1970). Conversely, the phenotypic inversion croscope provides an unambiguous diagnosis. This exami- of heterogametic ZW female gonads to functional male ones nation was performed on anaesthetized animals through obtained in P. waltlii by grafts of embryonic gonads pro- a lateral opening of the abdomen. duced females of the sexual genotype WW. Theses females Analysis of sex chromosomes of phenotypic females. In the gave rise to only female offspring when mated with stan- race of P. poireti utilized here, the W chromosome carries dard males (Collenot 1973, 1975). Sexual inversion of geno- a specific morphological differentiation in the lampbrush typic females into males has also been induced in this spe- stage. In the oocyte nuclei, the sexual bivalent (IV) is hetero- cies by heat treatment (Houillon and Dournon 1978 ; Dour- zygotic or heteromorphic (-/+) for this differentiation in non and Houillon 1983). the heterogametic ZW individuals, and homozygotic In these studies, genetic identification of the sex-reversed (-/-) in the homogametic ZZ individuals (Lacroix 1970). individuals was done by an analysis of the sex ratio of Lampbrush chromosomes from oocytes of 11 phenotyp- the progeny. Such a procedure is necessarily long due to ic females from the experimental group, were analysed to 262

define the homo- or heterozygotic nature of the sexual biva- Table l. Sex ratio of descendents of standard couples of Pleurodeles lent. The chromosomal preparations were obtained follow- poireti as a function of rearing temperature ing routine procedures from ovarian biopsy of sexually ma- ture animals (Lacroix and Loones 1974). These individuals No. of ~ No. of 9" No. of 9 Total were then appropriately catalogued and maintained to study their progeny. Control 47 (52.8)" 0 (0) 42 (47.2) 89 (20~ 3~ C) Genetic study on the offsprings. The experimental phenotyp- Heat treatment 12 (20.7) 4 (6.9) 42 (72.4) 58 ic females having obtained sexual maturity were bred with (31~ 1 ~ c) standard ZZ males and the sex ratio of the progeny of each of them was analysed. a Percentages are given in parentheses Enzymatic analysis. A sex-linked polymorphism of the enzyme peptidase 1 has been demonstrated by electrophoresis These results clearly show an influence of the high tem- in P. waltIii. This polymorphism depends on a pair of codo- perature on the sexual differentiation of gonads. This influ- minant alleles Pep-lA and Pep-lB. It allows the identifica- ence is shown firstly by the presence of a high percentage tion of the genotypic sex of different individuals (Ferrier of intersexual individuals. As a matter of fact, no case of et al. 1980, 1983). In this study, the method has been spontaneous intersexuality in normal stocks of Pleurodeles adapted to P. poireti. have been reported. This intersexuality thus corresponds Erythrocyte haemolysates were subjected to electro- to a partial modification of the sexual phenotype of the phoresis in horizontal starch gels according to the technique gonads. Secondly, the influence of heat treatment is corrob- of Wright et al. (1976) using Tris-citrate for 16 h at 4 ~ C. orated by the significant deviation of the sex ratio in favour The peptidase was revealed in the presence of valyl-leucine of the female phenotype, suggesting that some genotypic by incubating the lower part of the starch gels in the medi- ZZ males are sex-reversed (heat-induced neo-females). um described by Lewis and Harris (1967) for 1 h at 37 ~ C. Proof of sexual inversion of genotypic ZZ males

Results Cytogenetic analysis. The proposed interpretation was checked by karyological analysis of the lampbrush chromo- Influence of heat treatment on sexual differentiation somes of experimental females once they had reached adult Three lots of animals derived from different couples have stage. This analysis was done on 11 females of the second been utilised. The experimental animals were taken from experimental batch (Table 2). Of these, 5 are heteromorphic all three batches while the controls animals were taken from for the sexual bivalent IV and 6 are homomorphic (Fig. 1). only two of them. Of 110 control animals 89 reached a The 6 homomorphic individuals thus really have a male stage when the sexual phenotype could be identified: 22 ZZ genotype. from the first batch, 67 from the second. Of the 89 individ- Genetic analysis of the progeny. Three of the ZZ females uals, 47 (53%) were males and 42 (47%) females (Table 1). were crossed with standard male and gave rise to descen- Amongst the 125 experimental heat-treated animals, 58 dents. The progeny of two of these females were almost reached the stage of sexual phenotype identification, 15 of completely decimated by a parasitic infection, the third 50 individuals for the first, 20 of 25 for the second, and batch developed normally and gave 66 animals. Raised at 23 of 50 for the third batch. Of these 58 animals, 12 (21%) room temperature, all these individuals were males (Ta- had the male phenotype, 42 (72%) the female phenotype, ble 2). The unisexual nature of this progeny confirms the and 4 (7%) were intersexual (Table 1). In the intersexual sex inversion of males into functional females as well as animals, the gonads were differentiated into testes in their the homogametic nature of males in this species. anterior parts and into ovaries in their posterior parts. The stage of development of the two juxtaposed territories of Electrophoretic pattern of peptidase 1. The procedure for the intersexual gonads was comparable to that of unisexual the detection of peptidase 1, adapted to P. poireti, allowed gonads of other experimental animals of the same age. us to obtain distinct electrophoretic patterns for the two

Table 2. Cytogenetic, enzymatic, and genetic (sex ratio) analyses of 11 experimental females of Pleurodelespoireti

No. of Structure of Sexual Spectrum of Descendents females bivalent IV genotype peptidase i No. of d' No. of ~ Total

1-81 -- / -- ZZ fast 66 0 66 2-81 - / + ZW slow 3-81 - / -- ZZ fast 4-81 - / + ZW slow 5-81 - / - ZZ fast 6-81 -- / + ZW slow 7-81 -- / + ZW slow 8-81 --/- ZZ fast 9-81 -- / - ZZ fast 2 10-81 -- / + ZW slow 11-81 - / - ZZ fast 263

Fig. 1. Phase-contrast micrograph of heterochromosomes of Pleurodeles poireti in the lampbrush stage. The W chromosome carries a sequence of loops characterizing its differential segments (bracket); this sequence is absent in the Z chromosome. The bivalent IV from an oocyte of a standard ZW female (lower) is heterozygotic (+/-) for this sequence. The bivalent IV from an oocyte of the ZZ neo-female 1-81 (upper), is homozygotic (-/-). Bar represents 30 gm sexes. Furthermore, these patterns are different from those Discussion obtained for P. watdii (Ferrier et al. 1980, 1983). For the controls, the analysis was done on a sample of 10 animals In all the amphibians previously studied, the anurans Rana of each sex. In the males, the enzyme is located in a fast temporaria and R. sylvatica (Witschi 1914, 1929), R. tempor- moving band, whereas in the females the band containing aria and Bufo vulgaris (Piquet 1930), R..japonica (Yoshikura the enzyme moves more slowly (Fig. 2). These results show 1959), R. catesbeiana (HSii et al. 1971) and urodeles Hyno- that there is a correlation between the enzyme expression bius retardatus (Uchida 1937), Pleurodeles waltlii (Houillon and the sex of the individuals; however, they do not distin- and Dournon 1978), an unusual high temperature orients guish whether the correlation is due to the sexual genotype the sexual differentiation of the gonads towards testes. or to the phenotype, as the two are the same in these control Pleurodeles poireti is thus an exception: it is the first am- animals. phibian in which heat treatment has been found to orient Of the 20 experimental animals examined by this proce- the gonads of genot.ypic males towards an ovarian differen- dure, 8 were phenotypic males and 12 were phenotypic fe- tiation. This result is all the more surprising because the males, including the 11 utilised for cytogenetic analysis. For sexual inversion is obtained under conditions comparable the 8 males, the electrophoretic pattern was identical to to those utilised for P. waltlii. Actually, the two species, that of standard males. Of 12 females, 5 showed a spectrum besides being homogametic for the mate sex, are closely identical to that of standard females (a slower moving band) allied giving rise to hybrids fertile up to the third generation whereas 7 females showed a spectrum comparable to that (Lacroix 1968). of standard males (a faster moving band). There is a perfect In our experiments, we did not obtain sexual inversion correlation between the karyological and electrophoretic of all the genotypic males. It is probable that application analyses for the 11 females studied by the two techniques of a temperature slightly higher than 31 ~ C during the heat- (Table 2). The heat-induced neo-females thus have an elec- sensitive period, which remains yet to be determined pre- trophoretic pattern for peptidase 1 of the male type, which cisely for P. poireti, could ameliorate the efficiency of sexual indicates that the expression of this enzyme depends on inversion as has been observed for P. wahlii (Dournon, the genotypic sex. These observations also suggest that pep- in preparation). However, it should be noted that all the tidase 1 gene is sex-linked in P. poireti as in P. waltlii. heat-induced neo-females present complete and regular 264

Dournon C (1981) Action d'une temp6rature d'61evage de 30~ sur la morphogen6se, l'inversion fonctionnelle du ph~notype sexuel et la prolif6ration des cellules germinales chez l'Amphi- bien Urod61e Pleurodeles waltlii Michah.. Thesis Doc Etat Sci Nat Univ Pet M Curie, 204 p Dournon C, Houillon Ch (]983) D6terminisme g6n6tique du sexe: d+monstration fi partir d'animaux fi ph6notype sexuel invers~ sous Faction de la temp6rature chez l'Amphibien Urod61e, Pleurodeles waltlii Michah. CR Acad Sci S6r III, 296:779-782 Ferrier V, Jaylet A, Cayrol C, Gasser F, Buisan JJ (1980) Etude 61ectrophor&ique des peptidases ~rythrocytaires chez Pleuro- deles waltlii (Amphibien Urod61e): mise en 6vidence d'une liai- son avec le sexe. CR Acad Sci S6r D, 290:571-574 Ferrier V, Gasser F, Jaylet A, Cayrol C (1983) A genetic study of various enzyme polymorphisms in Pleurodeles waltlii (uro- dele amphibian). II-peptidases: demonstration of sex linkage. Biochem Genet 21 : 535-549 Foote CL (]964) Intersexuality in amphibians. In: Armstrong CN, Marshall AJ (ed) Intersexuality in vertebrates including man. Academic Press, London, New-York, pp 233-272 Gallien L (1951) Sur la descendance unisexu6e d'une femelle de Pleurodeles waltlii Michah. ayant subi pendant sa phase lar- vaire Faction gynog6ne du benzoate d'oestradiol. CR Acad Sci 233 : 828-830 Gallien L, Durocher M (]957) Table chronologique du d~veloppe- Fig. 2. Electrophoretic patterns of peptidase 1 in five experimental ment chez Pleurodeles waltlii Michah. Bull Biol Fr Belg and two control animals. Fast moving bands are from a control 91:97-114 ZZ male (a), female 3-81 (c), and female 5-81 (e). Slow moving Houillon Ch, Dournon C (1978) Inversion du ph6notype sexuel bands are from a control ZW female (b), female 4-81 (d), female femelle sous Faction d'une temp6rature ~lev6e chez l'Amphibien 6-81 (f), and female 7-81 (g). Note the presence of two types of Urod+le, Pleurodeles waltlii Michah. CR Acad Sci S6r D patterns in experimental females 286:]475-1478 t-Isfi CY, Yfi NW, Liang HM (1971) Induction of sex reversal Mfillerian ducts as in standard females. Treatment of the in female tadpoles of Rana catesbeiana by temperature treatment. Endocrinol Jpn ] 8:243-25] larvae of P. poireti with steroid hormones (estradiol ben- Lacroix JC (]968) Variations exp6rimentales ou spontan6es de la zoate) can result in 100% sexual inversion of gonads in morphologie et de l'organisation des chromosomes en 6couvil- genotypic males (Lacroix 1970). However, under these con- lon dans le genre Pleurodeles (Amphibien, Urod61e). Ann Em- ditions Mfillerian ducts often display abnormalities. bryol Morphol t :205-248 The differentiation of sex chromosomes in the lamp- Lacroix JC (/970) Mise en 6vidence sur les chromosomes en 6cou- brush stage allows determination of the genotypic sex. This villon de Pleurodeles poireti Gervais, Amphibien Urod61e, procedure is unequivocal but is applicable only to pheno- d'une structure li6e au sexe, identifiant le bivalent sexuel et typic females with oocytes in vitellogenesis. Demonstration marquant le chromosme W. CR Acad Sci S6r D 271 : 102-104 Lacroix JC, Loones MT (]974) S~lection par les chromosomes of a sex-linked genetic marker permits us to extend the en 6couvillon de lign~es vectrices de mutations chez l'Amphi- analysis of sexual genotype to individuals of either sexual bien Urod~le Pleurodeles poireti. Chromosoma 48 : 297-326 phenotype. This procedure should also be applicable to lar- Lewis WHP, Harris H (1967) Human red cell peptidases. Nature vae prior to metamorphosis, as has been shown for P. waltlii 215:351-355 (Ferrier et al. 1983). Piquet J (/930) D6termination du sexe chez les Batraciens en fonc- To conclude, we have obtained a functional feminiza- tion de la temp6rature. Rev Suisse Zool 37:173-281 tion of genotypic males by increasing the temperature dur- Uchida T (/937) Studies of the sexuality of amphibia. III. Sex ing development of embryos and larvae in the urodela transformation in Hynobius retardatus by the function of high Pleurodeles poireti. This observation is confirmed by cyto- temperature. J Fac Sci, Hokka'ido Imp Univ, Ser 6 (Zool) genetic and genetic analysis. Demonstration of a sex-linked 6: 59-70 Witschi E (1914) Experimentetle Untersuchungen fiber die Ent- gene of a peptidase extends the analysis of sexual genotype wicklungsgeschichte der Keimdrfisen von Rana temporaria. to adults of both sexual phenotypes. Arch Mikrosk Anat Entwicklungsmech 85:9-] 13 Witschi E (1929) Studies on sex differentiation and sex determina- Acknowledgements. This work was supported by grants from the tion in amphibians. II. Sex reversal in female tadpoles of Rana Centre National de la Recherche Scientifique (A.T.P. "Biologic sylvatica following the application of high temperature. J Exp Mol6culaire du g6ne" 1982-]983) and the Minist6re de l'Education Nationale (Direction de la Recherche A R U 1983). Zool 52:267-291 Wright DA, Huang CP, Chuoke BD (1976) Meiotic origin of trip- loidy in the frog detected by genetic analysis of enzyme polymorphisms. Genetics 84:319-332 References Yoshikura M (1959) The action of the pituitary in sex differentia- Collenot A (]973) Obtention, par la m&hode des greffes de gonades tion and sex reversal in amphibians. II. Effects of high tempera- embryonnaires, d'une femelle fi descendance unisexu~e femelle, ture on the gonads of hypophysectomized frog larvae. Kuma- chez le triton Pleurodeles waltlii Michah. Experientia moto J Sci Ser B Sec 2,4:69-101 29 : 885-887 Collenot A (1975) Unisexual female offsprings in the salamander Pleurodeles waltlii Michah. In: R Reinboth (ed) Intersexuality Received May 8, 1984 in the animal kingdom. Springer Verlag, pp 31 ]-3]7 Accepted by W. Hennig