INVESTIGADORES
SOMOZA Gustavo Manuel
congresos y reuniones científicas
Título:
Undifferentiated gonads of pejerrey (Odontesthes bonariensis) larvae exposed to masculinizing temperatures produce 11-ketotestosterone when incubated with a tritiated precursor.
Autor/es:
MARTÍN BLASCO; GUSTAVO M. SOMOZA; DENISE VIZZIANO-CANTONNET
Lugar:
Cochin
Reunión:
Congreso; 9th International Symposium on Reproductive Physiology of Fish.; 2011
Resumen:
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, 236:21982206.
[2] BLASCO, M., FERNANDINO,
J.I., GUILGUR, L.G., STRÜSSMANN, C.A., SOMOZA, G.M., VIZZIANOCANTONNET, 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.