Undifferentiated gonads of pejerrey (Odontesthes bonariensis) larvae exposed to masculinizing temperatures produce 11-ketotestosterone when incubated with a tritiated precursor.

MARTÍN BLASCO; GUSTAVO M. SOMOZA; DENISE VIZZIANO-CANTONNET

Cochin

Congreso; 9th International Symposium on Reproductive Physiology of Fish.; 2011

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-KT and 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. Results: The results of the incubation of the 5 larval gonads and peritoneum from male promoting temperature (29ºC) showed that these tissues were able to metabolize 17P to 11-KT (Figure 1). Furthermore, the analysis of the zone corresponding to 11β-OHA4 (fractions 19-21) resulted in no radioactivity after resolution by RP-HPLC. In the adult testis the major androgen observed in the presence of 17P was 11β-OHA4.  On the other hand the incubation of spleen with A4 resulted in 11β-OHA4 whereas liver did not show production of neither 11-KT nor 11β-OHA4 (data not shown). Discussion and Conclusions: In fish, 11-oxygenated androgens are considered the biologically active androgens, but their roles in early gonadal development are not fully understood. Here it was observed that undifferentiated gonads (and/or nearby tissues) of larvae exposed to masculinizing temperatures were able to produce 11-KT from 17P. This data, together with previous information, suggests that 11KT can act as early mediator of testis differentiation. Furthermore, it was observed a clear difference in the 11-oxygenated androgen profile produced by mature male fish gonads, were 11β-OHA4 dominates, indicating that steroidogenic pathways are different in non-differentiated compared to adult fish. Furthermore, as adult spleen is able to produce 11β-OHA4 by using A4, other tissues would be cooperating in the production of 11-oxygenated androgens in fish during early sex differentiation. 11-KT would be involved in the gonadal differentiation process driven by temperature in pejerrey.   Reference List [1] VIZZIANO, D., RANDUINEAU, G., BARON, D., CAUTY, C., GUIGUEN, Y. 2007. Characterization of early molecular sex differentiation in rainbow trout, Oncorhynchus mykiss. Dev Dyn, 236:2198–2206. [2] BLASCO, M., FERNANDINO, J.I., GUILGUR, L.G., STRÜSSMANN, C.A., SOMOZA, G.M., VIZZIANO–CANTONNET, D. 2010. Molecular characterization of cyp11a1 and cyp11b1 and their gene expression profile in pejerrey (Odontesthes bonariensis) during early gonadal development. Comp Biochem Physiol A 156: 110-118. [3] HATTORI, R.S., FERNANDINO, J.I., KISHII, A., KIMURA, H., KINNO, T., OURA, M., SOMOZA, G.M., YOKOTA, M., STRÜSSMANN, C.A., WATANABE, S. 2009. Cortisol-Induced Masculinization: Does thermal stress affect gonadal fate in pejerrey, a teleost fish with temperature-dependent sex determination? PLOS ONE 4(8): e6548.