Expression profiles of amhy and major sex-related genes during gonadal sex differentiation and their relation with genotypic and temperature-dependent sex determination in pejerrey Odontesthes bonariensis.

To shed light on the mechanisms of and interactions of GSD and TSD in pejerrey, we investigated how the transcriptional profiles of amhy and amha are affected by feminizing (17 °C) and masculinizing (29 °C) temperatures during the critical period of sex determination/differentiation and their relation with the expression profiles of AMH receptor type II (amhrII), gonadal aromatase (cyp19a1a), and 11 beta-hydroxysteroid dehydrogenase 2 (hsd11b2). Careful consideration of the results of this study and all information currently available for this species, including similar analyzes for an intermediate, mixed-sex promoting temperature (25 °C), suggests a model for genotypic/temperature-dependent sex determination and gonadal sex differentiation that involves a) cyp19a1a-dependent, developmentally-programmed ovarian development as the default state that becomes self-sustaining in the absence of a potent and timely masculinizing stimulus, b) early, developmentally-programmed amhy expression and high temperature as masculinization signals that antagonize the putative female pathway by suppressing cyp19a1a expression, c) increasing stress response, cortisol, and the synthesis of the masculinizing androgen 11-keto-testosterone via hsd11b2 with increasing temperature that is important for masculinization in both genotypes but particularly so in XX individuals, and d) an endocrine network with positive/negative feedback mechanisms that ensure fidelity of the male/female pathway once started. The proposed model, albeit tentative and non-all inclusive, accounts for the continuum of responses, from all-females at low temperatures to all-males at high temperatures and for the balanced-, genotype-linked sex ratios obtained at intermediate temperatures, and therefore supports the coexistence of TSD and GSD in pejerrey across the range of viable temperatures for this species.

Spatiotemporal Correlations between amh and cyp19a1a Transcript Expression and Apoptosis during Gonadal Sex Differentiation of Pejerrey, Odontesthes bonariensis.

Sex determination in pejerrey is genetically prescribed by the Y chromosome-linked anti-müllerian hormone amhy but is also strongly influenced by water temperature during the critical period of sex determination. Its gonadal differentiation is characterized by a cephalocaudal and left-to-right histological gradient in both sexes that presumably helps prevent discrepant intersex development in different regions of the gonads in response to ambiguous thermal and genetic stimuli, but the relation of this gradient to molecular processes of sex differentiation is unknown. In this study, we investigated the spatiotemporal expression patterns of amh, gonadal aromatase (cyp19a1a), and apoptosis in relation to the histological gradient in ovaries and testes at an intermediate, sexually neutral temperature. The location and timing of expression of amh, cyp19a1a, and apoptosis seemed to be highly coordinated with the time of gonadal sex differentiation and the histological gradient of gonadal sex differentiation. Apoptosis occurred predominantly in the anterior region of the right gonads and is surmised to be a process to delay differentiation in this area compared to the left gonad, possibly as a means to ensure uniform development in both gonads. Aromatase expression early during development was noted even in putative XY males, supporting the notion of primacy of female development in pejerrey gonads. Thus, apoptosis may be particularly important to prevent discrepant gonadal differentiation in XY individuals where genetic pro-male (amhy), pro-female (cyp19a1a), and thermal stimuli may antagonize.

Coexistence of genotypic and temperature-dependent sex determination in pejerrey Odontesthes bonariensis.

In this study, we examined whether a homolog of the master sex-determining gene amhy of Odontesthes hatcheri is present and plays any role in testis determination of pejerrey O. bonariensis, a species otherwise known for its strong temperature-dependent sex determination (TSD). Screening of wild and laboratory-reared pejerrey for amhy revealed a high, although not complete linkage with phenotypic sex. The sex ratio in an amhy+/-/amhy-/- full sibling progeny reared during the thermolabile period of sex determination at an intermediate temperature of 25°C was 68.7% male:31.3% female; all amhy+/- fish developed as males whereas about 2/3 and 1/3 of the amhy-/- were female and male, respectively. Expression analyses revealed that amhy transcription began during embryo stage and decreased by the end of sex determination period. The autosomal amha was present in all individuals regardless of amhy genotype; its expression increased significantly from the end of the same period in the gonads of all amhy+/- but only in part of the amhy-/- animals. After histological gonadal differentiation, all gonads of amhy-/- animals with amha ISH signals were testes and those without it were ovaries. These results suggest that amhy is important for testicular differentiation in pejerrey, at least at intermediate temperatures. Thus, we hypothesize that amhy+/- animals differentiate as males by expression of either amhy alone or amhy and amha together whereas the amhy-/- probably rely solely on amha expression. These findings represent the first clear genomic evidence that genotypic and environmental sex determinants can coexist in species with marked TSD such as the pejerrey. The finding of amhy will make possible to monitor wild pejerrey populations for mismatches between genotypic and phenotypic sex and may prove instrumental for field studies addressing the effects of endocrine disruptors or abnormal temperatures on reproduction and the ecological relevance of TSD for this species.