Undifferentiated Gonads of Pejerrey ( Odontesthes bonariensis ) Larvae Exposed to Masculinizing Temperatures Produce 11-ketotestosterone when Incubated with a Tritiated Precursor

Introduction: The involvement of androgens as early mediators of testis differentiation in fish is still a matter of debate [1]. Larval gonadal size limits the possibility to establish whether an early production of 11-oxigenated androgens precedes testicular differentiation. In a previous study we reported the expression of genes involved in androgen synthesis (i.e. cyp11a y la cyp11b) in larval trunks [2] and the production of 11-ketotestosterone was reported in larval trunks by EIA [3]. In this context the aim of the present work was to study the androgen synthesis capacity of gonads and peritoneum of Odontesthes bonariensis in larvae exposed to masculinizing temperatures (29oC) before morphological differentiation of the testis. First we validate the nature of androgens produced by adult testis, liver and spleen and then we studied the androgen production in larval gonads at 29oC. Methods: Pejerrey larvae were raise for 5 weeks at 29oC in order to obtain a whole male population. At 5 that moment, 100 fish were sampled and individually immerse in ice-cold water until movement ceased. Due to technological constrains in taking then apart, the gonads and peritoneum were dissected together under binocular microscope and pooled in ice cold L-15 medium. Adult testis at the onset of spermatogenesis, spleen and liver were also obtained and placed on L-15 medium. Tissue samples were incubated for 18 hs in 500 μL of L-15 medium in the presence of tritium labeled precursors. 17P was used for gonad and peritoneum, and A4 for spleen and liver. The reaction was arrested by ethanol addition to 80% followed by tissue disaggregation using a glass-glass grinder. The supernatant was evaporated under nitrogen until 20% its original volume qws reached and then extracted three times with dichloromethane (DM): methanol (M) (9DM:1M). This fraction was re-suspended and applied to a silica TLC plate, cleaned by a non-polar mobile phase run and then resolved by using benzene acetone (2:1). The resolved radioactive steroids were revealed by autoradiography, the characteristic Rf computed and radioactive zones isolated by scratching the silica followed by extraction using three rounds of 9DM: 1M. The bands extracts corresponding to 11-ketotestosterone (11-KT) and 11β-hydroxyandrostenedione (11β-OHA4) were separately analyzed by HPLC using a RP-C18 column and 0.5 mL fractions were obtained and their radioactivity content determined by scintillation counting. The 11β-OHA4 obtained from kidney extracts was analyzed by co-crystallization to constant specific activity.