Temporal and Spatial expression of amh, cyp19a1 and cyp19a2 in XX and XY genotypes of Patagonian pejerrey during sex differentiation

HATTORI, R.S.; JUAN IGNACIO FERNANDINO; OURA, M.; TAKAGI, K.; STÜSSMANN, C.A.; MANUEL SOMOZA, GUSTAVO; YOKATA, M; WATANABA, S

Calgary

Simposio; 6th International Symposium on Fish Endocrinology; 2008

The process of sex determination in atherinopsids seems to be under strong influence of environmental factors, especially of water temperature, as reported for Menidia menidia, Odontesthes bonariensis, O. argentinensis and O. hatcheri. The molecular characterization of this process is greatly limited by the difficulty in discerning the genotypic sex of embryos/juveniles prior to morphological sex differentiation. Our group has recently developed a strain of Patagonian pejerrey (O. hatcheri) with strong genotypic sex determination (GSD, XX/XY system) and a SNP sex-linked marker, allowing the investigation of genes/processes during very early stages of larvae development. In the present study, we analyzed of sex differentiation genes, in particular the anti-müllerian hormone (amh), gonadal aromatase (cyp19a1) and brain aromatase (cyp19a2) in genotypic males and females of this strain in order to clarify their roles during sex differentiation as well as for estimating the critical period of sex determination in this specie. Fertilized eggs from a single cross were incubated and hatched at 21ºC and the larvae were reared at the same temperature until 12 weeks. Gene expression analysis were performed by in situ hybridization (ISH), RT-PCR, and Real Time PCR in eggs, gonads and heads of larvae of known genotypes (XX and XY) that were sampled at various developmental stages. The caudal fin of each individual was used for sex genotyping using the SNP sex-linked marker. RT-PCR and Real Time PCR revealed the presence of amh transcripts in fertilized eggs from 2 days after fertilization (daf) with increasing expression levels reaching a peak at 10 weeks after hatching (wah). Sexually dimorphic expression was detected in the gonad of 1wah larvae and transcripts were localized in somatic cells of the primordial testis. The strongest ISH signals were concentrated in somatic cells in the medullar area of the gonad, suggesting a possible role in efferent duct formation. In the case of cyp19a1, transcripts first appeared in 1wah larvae of both sexes but increased only in females thereafter.