Characterization and expression profile of the ovarian cytochrome P‐450 aromatase (cyp19A1) gene during thermolabile sex determination in pejerrey, Odontesthes bonariensis

Abstract

Cytochrome P450 aromatase (cyp19) is an enzyme that catalyzes the conversion of androgens to estrogens and may play a role in temperature-dependent sex determination (TSD) of reptiles, amphibians, and fishes. In this study, the ovarian P450 aromatase form (cyp19A1) of pejerrey Odontesthes bonariensis, a teleost with marked TSD, was cloned and its expression profile evaluated during gonadal differentiation at feminizing (17 degrees C, 100% females), mixed-sex producing (24 and 25 degrees C, 73.3 and 26.7% females, respectively), and masculinizing (29 degrees C, 0% females) temperatures. The deduced cyp19A1 amino acid sequence shared high identity (>77.8%) with that from other teleosts but had low identity (<61.8%) with brain forms (cyp19A2), including that of pejerrey itself. The tissue distribution analysis of cyp19A1 mRNA in adult fish revealed high expression in the ovary. Semi-quantitative reverse transcription polymerase chain reaction analysis of the bodies of larvae revealed that cyp19A1 expression increased before the appearance of the first histological signs of ovarian differentiation at the feminizing temperature but remained low at the masculinizing temperature. The expression levels at mixed-sex producing temperatures were bimodal rather than intermediate, showing low and high modal values similar to those at the feminizing and masculinizing temperatures, respectively. The population percentages of high and low expression levels at intermediate temperatures were proportional to the percentage of females and males, respectively, and high levels were first observed at about the time of sex differentiation of females. These results suggest that cyp19A1 is involved in the process of ovarian formation and possibly also in the TSD of pejerrey.

Dimorphic Expression of<i> dmrt1</i> and <i>cyp19a1 </i>(Ovarian Aromatase) during Early Gonadal Development in Pejerrey, <i>Odontesthes bonariensis</i>

“…During the same period, cyp19a1 expression remained low at MPT. A previous study using semi-quantitative PCR noted a correlation between the proportions of larvae with increased cyp19a1 expression during the gonadal sex differentiation period and the percentages of females and suggested that cyp19a1 was related to ovarian differentiation in pejerrey (Karube et al, 2007). The results of this study provide confirmation to those findings and are in agreement with results obtained in other teleosts (Kitano et al, 1999;D'Cotta et al, 2001) and reptiles (Jeyasuria and Place, 1997;Gabriel et al, 2001;Murdock and Wibbels, 2003b;Ramsey and Crews, 2007;Rhen et al, 2007) with TSD.…”

“…In teleosts with TSD temperature also appears to regulate gonadal aromatase (cyp19a1) expression (Kitano et al, 1999;D'Cotta et al, 2001;Uchida et al, 2004). In the pejerrey, cyp19a1 showed a sexually dimorphic expression pattern during the first stages of ovarian differentiation and was proposed as a female molecular marker (Karube et al, 2007). However, to the best of our knowledge, the interplay and expression balance between dmrt1 and cyp19a1 at individual levels have not been analyzed so far in fish or any other species with TSD.…”

“…The primer sets were designed using the Primer Express Software (Ver. 2.0, Applied Biosystems) according to published sequences of dmrt1 : 5 -ATC CAG CAC ACC ACA CGA CAT-3 and 5 -TGA TGG GCT GGA GGA CGA T-3 for amplification of a 79-bp fragment; (NCBI GenBank accession AY319416; Fernandino et al, 2006); cyp19a1 : 5 -GCG AGC TGT CTG GCT GAG AA-3 and 5 -AGG AGC AGC AGC ATG AAG AAG A-3 for a 100-bp fragment (EF030342; Karube et al, 2007); and ␤ -actin : 5 -CTC TGG TCG TAC CAC TGG TAT CG-3 and 5 -GCA GAG CGT AGC CTT CAT AGA TG-3 for a 83-bp fragment (EF044319). Relative quantification of gene expression by real time PCR (RQ real time PCR) was performed in 20 l reaction volumes containing 2 !…”

Environmental Effects on Fish Sex Determination and Differentiation

“…2 ) [D'Cotta et al, 2001b;Baroiller et al, 2008]. Elevated cyp19a1 levels have also been found in the pejerrey O. bonariensis fry reared at female promoting temperatures (FPT) of 17 ° C after 6 wph, preceding histological differentiation [Karube et al, 2007;Fernandino et al, 2008b]. Similarly high expression levels of cyp19a1 were found in larvae of Japanese flounder reared at feminizing low temperatures of 18 ° C, but down-regulation occurred in offspring reared at the elevated male promoting temperatures of 27 ° C [Kitano et al, 1999].…”

The effect of rearing temperature in larval development of pejerrey, Odontesthes bonariensis: morphological indicators of development

“…In pejerrey, the critical or temperature-sensitive period occurs from the first to the fifth week after hatching, depending on temperature. Pejerrey larvae reared from hatching at 13-19ºC produced 100% females, at 29ºC 100% males, and mixed sex proportions at intermediate temperatures, 24-25ºC (Strüssmann et al, 1996b(Strüssmann et al, , 1997Karube et al, 2007;Fernandino et al, 2008a). Sex differentiation is also accelerated by rearing temperature; the ovarian differentiation occurs before at 24ºC than at 17ºC, whereas testis development takes place before at 29ºC than at 24ºC (Ito et al, 2005;Fernandino et al, 2008b).…”