Cromosomas sexuales y desarrollo gonadal en el modelo anfibio xenopus tropicalis (anura, pipidae)

Resumen

The species X. tropicalis has homomorphic sex chromosomes and it has been assumed for a while that this species has a ZZ/ZW sex chromosome system as this is the situation in other species of the genus Xenopus, including X. tropicalis. The sex of the offspring of sex-reversed animals, together with the manipulation of the sex chromosome constitution using gynogenetic offspring (double haploids, gynogenetic diploids and triploids) reveals that the sex chromosome system in X. tropicalis has, at least, three different sex chromosomes: Y, W and Z, defined by their role in the sexual development of the individual. Thus, the presence of the Y chromosome determines the individual will develop as a male, independently of the other sex chromosome present (W or Z). W type sex chromosomes determine that the sex of the individual will be female always that they are not together with a Y chromosome. Finally, the individuals with two Z chromosomes will be phenotypic males. According to this sex chromosome system, in this species there are three types of males (YW, YZ and ZZ) and two types of females (ZW and WW). This peculiarity defines 6 different types of genetic crosses versus the unique possible in species with just 2 sex chromosomes and affects the sex ratios obtained in each case. Thus, there are 4 possible genetic crosses that produce offspring with 1:1 sex ratio, together with two pairs that produce uncommon sex skewed ratios: males ZZ x females WW give rise to all female descendant while the pair formed by YZ males and ZW females produce offspring with a 3 males:1 female sex ratio. The evolutionary role of these skewed sex ratios in the maintenance of this uncommon sex chromosome system deserves further analysis in natural populations. The sex determining gene in this species is completely unknown, although the sex of triploid animal indicates there is no dosage effect for the sex determining gene since ZZW triploids are female and YWW are male. That is, two Z chromosomes are not enough to trigger male development in presence of one W chromosome, and the same applies to W chromosomes, two copies are not able to avoid the masculinizing effect of the Y chromosome. From the point of view of the evolution of the sex chromosomes, it is of great interest to address the morphological difference between these three sex chromosomes. Morphological differentiation has been analysed in detail using more sensitive cytogenetic techniques (GISH, CGH, chromosome painting). The results obtained in this species reveals no differences between any of its sex chromosomes, but allow obtaining some interesting information about the chromosomes of X. laevis and X. tropicalis. Genomic in situ hybridization (GISH) technique reveals a high amount of repetitive DNA mainly located at the tips of all chromosomes, in the short arms of chromosome pairs 3 and 8 and in the secondary constriction of chromosome 9. Nevertheless, no differences were observed in the sex pair. GISH pattern comparison between X. tropicalis and X. laevis reveals that the accumulation of repetitive sequences at the tip of the chromosomes is a characteristic of both species, although X. laevis shows a smaller amount of these repetitive sequences. Also, the existence of intense signals in the short arms of chromosome pairs 12 and 16 of X. leavis (syntenic of pair number 3 in X. tropicalis), point to the existence of a repetitive DNA sequence present in both species and probably in the ancestry species from which genus Silurana an Xenopus evolved. Chromosome painting, on the other hand, indicates the sex chromosome pair is not conserved between X. tropicalis and X. laevis, as in both species different chromosome pairs harbour the sex determining gene. Sex linked markers are of great interest in species with homomorphic sex chromosomes, as genetic sex cannot be established and the sex of an individual only can be determined when sex-linked morphological differences appears. In this context sex-linked markers have been intensively looked for among SSLPs, sex-specific AFLPs and sex-linked scaffolds. This search provided 26 sex-linked markers that can be used to establish the genetic sex in this species. Genetic mapping of these markers provided detailed information about the location of the sex determining region, on the terminal end of chromosome 7 of X. tropicalis, very close to the telomere. The presence of the sex determining gene on this region does not affect its sex recombination rate in females ZW compared to males ZZ, but a striking increased recombination rate has been observed in YZ males compared to ZW and ZZ individuals. Thus, the genetic distance in this region in YZ males is 2,23 and 2,96 times bigger compared to ZZ and ZW animals, while this differences are reduced to 1,33 bigger when males ZZ are compared to ZW females. Molecular pathways involved in gonadal development in X. tropicalis are also not well known and two approaches have been used to gather new information that allow to put together the pieces of the machinery that run sexual differentiation in this species. Hormonal exposure during larval development has been shown to cause sex-reversal in numerous species. The window of hormonal sensitivity to estrogen and anti-androgen compounds during gonadal development in X. tropicalis has been delimitated in order to correlate this timed sensitivity with cellular changes that have been observed during gonadal development. Estradiol treatment at different stages reveals that complete sex reversal can only be produced when estradiol is added before initiation of gonadal differentiation, after that point, incomplete sex-reversal is frequently observed as ovotestes are observed when initiation of treatment is delayed, until no effect is detected if the estrogen is added after the differentiation of the gonad. Despite this, it is worth to note that estradiol treatment hast to continue even if gonadal has differentiated since in treatments between NF 46 y NF 56 a transitory sex reversal has been observed. Fadrozole, on the other hand did not caused complete sex-reversal with the dose used in this work, although ovarian phenotype can be observed in treated genetic females. The analysis of treated samples showed skewed sex ratios only in treated animals. This can lnly be explained if fadrozole treatment has a selective effect on female mortality. On the other hand, the dose used only produces a delay in female development, with complete sex reversal observed only in 4 individuals. To get a better understanding of gonadal development in X. tropicalis, the expression pattern of several genes orthologs to genes involved in gonadal development in mammals were analysed in this species. The analysis of gene expression of genes involved in testes development (Sox9, Dmrt1 and AMH) revealed male specific expression for sox9 only from NF50 to NF55. From this stage sox9 is also expressed in females. This result contrast with the expression pattern of this gene in other vertebrates that do not show male specific expression pattern in the developing gonad. Amh is the only gene analized with a male specific expression pattern and points to an important role of this gene on the first stages og testis development. Finally, dmrt1 is expressed on both sexes along all the period analysed. This expression pattern contrast with its important role as testis determining factor in other species. Regarding the expression pattern of female specific genes, both foxl2 and cyp19a1 are female specific from NF 50 to NF 66, being expressen in different domains of the developing gonad. These genes are involved in ovarian differentiation in other vertebrate species. Thus this process seems to be highly conserved across evolution, from fish to birds. Mammals are not cyp19a1 dependen as ovarian development is insensible to estrogen exposure. Finally, germ cell markers are expressed during gonadal development in X. laevis. Ddx4 is expressed in low levels in both sexes up to stage NF 56 when expression in females rise up compared to males. This change can be related with germ cell entry into meiosis. On the other hand, Stra8 is present at higher levels in females according to its role in regulating germ cell entry into meiosis. Germ cell marker expression seems to be related with germ cell entry into meiosis.