Busulfan-mediated germ cell depletion does not alter gonad differentiation in the urodele amphibian Pleurodeles waltl.

It is not known in urodele amphibians whether germ cells (GCs) are indispensable for gonadal differentiation. In order to address this question in the newt Pleurodeles waltl, we first cloned the ortholog of VASA which is known as a GC marker in many species. Male (ZZ) and female (ZW) larvae were then exposed to the alkylating agent busulfan (25 µg/ml for 3 days) just after hatching (stage 36). In the main body of busulfan-treated larvae, PwVASA mRNA expression decreased before gonad differentiation in both sexes (at stage 50). This suggested GC depletion which was confirmed by histology, with a complete absence of GCs observed slightly earlier in females (stage 54) than in males (stage 54 + 60 days). In busulfan-treated ZW larvae, the presence of the typical central cavity and expression of a high level of aromatase mRNA confirmed the ovarian phenotype. In busulfan-treated ZZ larvae, the presence of a medulla surrounded by a thin cortex and a low level of aromatase mRNA confirmed the testis phenotype. At the juvenile stage, efferent ducts and lobules were present in the first testis lobe. Taken together, these data suggest that GC depletion does not alter gonad differentiation in P. waltl.

Sexual development of the urodele amphibian Pleurodeles waltl.

Pleurodeles waltl is a urodele amphibian that displays a ZZ/ZW genetic mode of sex determination involving a putative W-borne dominant determinant. This determining pathway can be environmentally inhibited since heat treated ZW larvae undergo a functional female to male sex reversal. Moreover, both genetic sexes can be reversed by treatment of larvae with steroid hormones suggesting they are the major players in the differentiation process. Indeed we demonstrated that i) aromatase expression and activity increase just before ovarian differentiation, ii) aromatase inhibitors induce a female to male sex reversal, iii) estrogens induce male to female sex reversal whereas the opposite is obtained with non-aromatizable androgens, iv) steroidogenic factor 1 and estrogen receptor alpha both display a female-enriched expression following the increase in aromatase activity. The role of endogenous hormones was investigated in a parabiosis model. Surprisingly, in ZW/ZZ associations, the ZW gonad could not differentiate suggesting that the ZZ parabiont produces an inhibiting factor, prior to ovarian differentiation. The role of AMH in this process is discussed, keeping in mind that Mullerian ducts are maintained in males. The development of antibodies and new molecular tools in the near future should help us to better understand the sexual development of this vertebrate.

Female-enriched and thermosensitive expression of steroidogenic factor-1 during gonadal differentiation in Pleurodeles waltl.

In the urodele amphibian Pleurodeles waltl, sex differentiation is genetically controlled, that is, ZZ male vs ZW female, but may be influenced by temperature, which induces a female-to-male sex reversal. We investigated whether steroidogenic factor 1 (SF-1) could be involved in Pleurodeles sex differentiation or in temperature-dependent sex reversal by cloning a Pleurodeles SF-1 cDNA and examining its developmental expression. The 468-amino-acid deduced protein is highly conserved in comparison with other species. In ZZ and ZW control larvae, SF-1 mRNA is detected at the first stage of the thermosensitive period (TSP) in the gonad-mesonephros-interrenal complex (GMI). By the end of TSP at stage 55, SF-1 is expressed in the gonad (Gd) and in the mesonephros-interrenal (MI) both in ZZ and ZW larvae. During this stage, a transient, ZW-specific increase of SF-1 transcription occurs not only in Gd but also in MI, this increase starting earlier in Gd than in MI. Therefore, in P. waltl, an SF-1 upregulation occurs after the onset of the ovarian-specific increase of aromatase mRNA expression. At the end of metamorphosis, the SF-1 transcription level in Gd and MI is nearly the same in both ZZ and ZW larvae. Besides, after long-term heat treatment leading to sex reversal, SF-1 mRNA upregulation is not observed in ZW larvae, in either Gd or MI. However, SF-1 expression is not decreased after a 48-h heat shock applied at the end of the TSP, suggesting that temperature has no inhibitory effect by itself in long-term heat treatment. Estradiol benzoate treatments show that, at the end of the TSP, SF-1 gene transcription could be controlled by the estrogen level. This is in accordance with the female-enriched SF-1 expression and the decreased SF-1 expression following long-term, sex-reversing heat treatment, which is known to decrease aromatase expression and activity. Thus, it is unlikely that SF-1 is directly involved in Pleurodeles temperature-dependent sex reversal.

Steroids, aromatase and sex differentiation of the newt Pleurodeles waltl.

In the newt Pleurodeles waltl, genetic sex determination obeys female heterogamety (female ZW, male ZZ). In this species as in most of non-mammalian vertebrates, steroid hormones play a key role in sexual differentiation of gonads. In that context, male to female sex reversal can be obtained by treatment of ZZ larvae with estradiol. Male to female sex reversal has also been observed following treatment of ZZ larvae with testosterone, a phenomenon that was called the "paradoxical effect". Female to male sex reversal occurs when ZW larvae are reared at 32 degrees C during a thermosensitive period (TSP) that takes place from stage 42 to stage 54 of development. Since steroids play an important part in sex differentiation, we focussed our studies on the estrogen-producing enzyme aromatase during normal sex differentiation as well as in experimentally induced sex reversal situations. Our results based on treatment with non-aromatizable androgens, aromatase activity measurements and aromatase expression studies demonstrate that aromatase (i) is differentially active in ZZ and ZW larvae, (ii) is involved in the paradoxical effect and (iii) might be a target of temperature. Thus, the gene encoding aromatase might be one of the master genes in the process leading to the differentiation of the gonad in Pleurodeles waltl.

Effect of cadmium on gonadogenesis and metamorphosis in Pleurodeles waltl (urodele amphibian).

In the amphibian Pleurodeles waltl, steroid hormones play a key role in sex differentiation. Since cadmium has been reported to block receptors of sex steroid hormones, we analyzed the effects of this heavy metal on Pleurodeles larvae gonadogenesis. At stage 42, larvae die in the presence of 10.9 microM Cd in the rearing tap water, with TL(50) of 46.3 h, but the concentration of 5.5 microM is tolerated for more than 60 days. When used at 5.5 microM cadmium accumulation measured by atomic absorption spectrophotometry (AAS) in total homogenates of larvae at stage 54 (after 77 days of exposure to the heavy metal) reached 58.1 microg/g of dry weight. At stage 54, we did not detect inhibitory effects on gonadogenesis in larvae reared in the presence of 5.5 microM Cd since stage 42. When the exposure to 5.5 microM Cd was lengthened after stage 54, metamorphosis was delayed and could not be completed. When larvae were exposed to 10.9 microM Cd from stage 54, metamorphosis did not occur and gonad development was stopped. Our study demonstrates a lack of a direct effect of cadmium on sex determination-differentiation but a strong inhibitory effect on metamorphosis, which impairs further gonadal development.

Differential expression of P450 aromatase during gonadal sex differentiation and sex reversal of the newt Pleurodeles waltl.

A better understanding of vertebrate sexual differentiation could be provided by a study of models in which genetic sex determination (GSD) of gonads can be reversed by temperature. In the newt Pleurodeles waltl, a P450 aromatase cDNA was isolated from adult gonads, and the nucleotide or deduced amino acid sequences showed a high level of identity with various vertebrate species. In adults, aromatase expression was found in gonads and brain. In developing gonads, the expression was found to fit with the thermo-sensitive period (TSP) and was detected in both ZZ and ZW larvae, as well as in ZW submitted during the whole TSP to a masculinizing temperature. In the latter individuals, in situ hybridization and semi quantitative RT-PCR showed that, at the end of TSP, aromatase expression was at the same level than in normal ZZ larvae and was significantly lower than in normal ZW ones. Furthermore, temperature-induced down regulation did not occur when heating was performed at the end of TSP. Our results confirm the importance of aromatase regulation in female versus male differentiation and demonstrate that a down regulation of aromatase expression is involved in the process of sex reversal.

Effects of androgens on sex differentiation of the urodele Pleurodeles waltl.

In nonmammalian vertebrates, steroids have been hypothesized to induce somatic sex differentiation, since manipulations of the steroidal environment of gonads have led to various degrees of sex reversal. Whereas the critical role of estrogens in ovarian differentiation is well documented, studies on androgens have produced a perplexing variety of results depending upon species variations and nature of androgens used. In this way, testosterone induces masculinization of females in some species but provokes paradoxical feminization of males in many other species such as the urodelan Pleurodeles waltl. In reptiles this phenomenon could be interpreted by conversion of exogenous testosterone to estradiol by aromatase. Treatments of Pleurodeles larvae with nonaromatizable androgens bring support to this hypothesis and suggest a role of androgens in sex differentiation. Dihydrotestosterone (DHT) could not induce the paradoxical feminization of ZZ larvae. In addition, DHT as well as 11beta-hydroxy-androstenedione could drive a functional male differentiation of ZW larvae. Moreover, other 5alpha reduced androgens also induced sex reversal of female larvae. Yet, the 5alpha reductase inhibitor CGP 53133 and antiandrogens such as flutamide or cyproterone acetate did not exert any effect on male sex differentiation of ZZ larvae. Though the precise role of androgens is still unknown, especially for 11-oxygenated androgens, our results suggest an implication in male sex differentiation. In this way, testosterone could play a pivotal role in being metabolized either into other androgens during testis differentiation or into estradiol during ovarian differentiation.

Microgravity and hypergravity effects on fertilization of the salamander Pleurodeles waltl (urodele amphibian).

Effects of microgravity (microG) on fertilization were studied in the urodele amphibian Pleurodeles waltl on board the MIR space station. Genetic and cytomorphologic analyses ruled out parthenogenesis or gynogenesis and proved that fertilization did occur in microG. Actual fertilization was demonstrated by the analysis of the distribution of peptidase-1 genes, a polymorphic sex-linked enzyme, in progenies obtained in microG. Further evidence of fertilization was provided by the presence of spermatozoa in the perivitelline space and in the fertilization layer of the microG eggs and by the presence of a female pronucleus and male pronuclei in the egg cytoplasm. Experiments in microG and in 1.4G, 2G, and 3G hypergravity showed for the first time that, compared to eggs in 1G, several characteristics of the fertilization process including the cortical reaction and the microvillus transformations were altered depending on the gravitational force applied to the eggs. Microvillus elevation, the most evident feature, was reduced on microG-eggs and amplified on eggs submitted to 2G and 3G. No lethal consequences of these alterations on the early development of microG-eggs were observed.

Sex reversal by aromatase inhibitor treatment in the newt Pleurodeles waltl.

Pleurodeles waltl is a newt with a ZZ male-ZW female sex determination mechanism, and a temperature-sensitive gonadal sex differentiation. Raising larvae at 32 degrees C from stage 42 to stage 54 (thermosensitive period) drives genetic females to differentiate into functional males. Estrogens are intimately linked with temperature action in this species, as well as in other vertebrates with temperature-dependent sex determination. We report here the masculinization of female ZW larvae and one WW larva by aromatase inhibitor treatment. Larvae were treated from stage 52 (before the onset of histological differentiation of the gonads) to stage 56 (metamorphosis), with the non-steroidal inhibitors fadrozole or miconazole. Miconazole proved to be very toxic, but not fadrozole. Fadrozole at a concentration of 300 micrograms/l in the rearing water resulted in complete sex reversal of 9 out of 30 ZW larvae and 1 out 9 WW larvae. Only one individual (ZW) was intersex, all the remainder being typical females. Gonadal aromatase activity was measured in several individuals at different developmental stages during treatment. The activity was low in all individuals at the beginning of the treatment, but varied strongly and was well correlated with gonadal structure at the end of the treatment. Despite these differences in individual responses to treatment inhibiting aromatase, results confirm the important role of estrogens in ovary differentiation in Pleurodeles waltl.

Aromatase activity in larval gonads of Pleurodeles waltl (Urodele Amphibia) during normal sex differentiation and during sex reversal by thermal treatment effect.

Estrogens are involved in gonadal differentiation in birds and in reptiles with temperature-dependent sex determination, suggesting a key role for aromatase, the enzyme complex converting androgens to estrogens. Pleurodeles waltl is an amphibian with ZZ/ZW genotypic sex determination but gonadal differentiation is thermosensitive. The sexual phenotype is in conformity with the sexual genotype at ambient temperature (20 +/- 2 degrees), but ZW animals are sex-reversed in functional males when larvae are reared at 32 degrees from stage 42 to stage 54 (thermosensitive period). Histological sexual differentiation of gonads begins at stage 53. Aromatase activity was measured either in the gonadmesonephric complexes (stages 47 to 52) or in the gonads (stages 53 to 56) of ZZ and ZW larvae reared at ambient temperature or at 32 degrees and of ZW larvae shifted at stages 53, 55, or 56 from ambient temperature to 32 degrees for 48 hr. At ambient temperature, aromatase activity was detected, at low levels, in gonad-mesonephric complexes of both ZZ and ZW larvae at stages 47 and 50. At stage 52, it was significantly higher in ZW individuals than in ZZ ones. Then it remained low in gonads of ZZ males, whereas it markedly increased in gonads of ZW females. At 32 degrees, the gonad-mesonephric complexes (stage 52) and the gonads (stages 53 to 56) of ZW larvae had low aromatase activity similar to that in ZZ individuals. The exposure to 32 degrees for 48 hr of ZW individuals significantly decreased gonadal aromatase activity during the thermosensitive period (shift at stage 53) but not after the thermosensitive period (shifts at stages 55 and 56).(ABSTRACT TRUNCATED AT 250 WORDS)