ABSTRACT
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.
Subject(s)
Homeodomain Proteins/genetics , Pleurodeles/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Sex Differentiation , Transcription Factors/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA Primers , Female , Homeodomain Proteins/chemistry , Male , Molecular Sequence Data , Pleurodeles/physiology , RNA, Messenger/genetics , Receptors, Cytoplasmic and Nuclear/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Steroidogenic Factor 1 , Transcription Factors/chemistryABSTRACT
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.
