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 en- zyme 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.