Figure 1.
amhy gene structure, phylogenetic relationship, and broodstock genotyping.
A: Structure of the amhy gene in O. bonariensis, size of exons, UTRs, and TGF-beta domain, and the respective identity values in relation to O. bonariensis amha. The third intron contains a 0.5 kb insertion in relation to amha. B: Phylogenetic tree (Neighbor-Joining method) for the amino acid sequences of O. bonariensis and O. hatcheri Amhy and Amha and the Amh of other teleosts. Numbers indicate bootstrap values based on 10000 replicates. C: amhy-based sex genotyping in O. bonariensis broodstock using primers that amplify part of the 5′ flanking region and part of the amhy gene (1896 bp); amha gene was used as positive control (2441 bp). The dotted-boxes indicate parents used in the rearing experiment and asterisks indicate disagreement between the amhy-based genotype and phenotypic sex. NC: negative control.
Table 1.
Relationship between phenotypic (gonadal) sex and amhy genotype in wild pejerrey and laboratory-reared broodstock.
Figure 2.
Expression of amhy and amha mRNAs in tissues and embryos.
A: Tissue distribution of amhy and amha mRNAs in juvenile pejerrey (RT-PCR). B: Expression profile of amhy and amha during embryogenesis in amhy+/− and amhy−/− genotypes (RT-PCR). β-actin was used as endogenous control. NC: negative control.
Figure 3.
Quantification of amhy, amha and cyp19a1a mRNAs during sex differentiation.
A to C: Abundance of mRNA transcripts of amhy (A) and amha (B) in amhy+/− genotypes and of amha in amhy−/− genotypes (C) during larval development at 25°C (n = 3 to 6 per time point; qRT-PCR). D: Abundance of amha mRNA transcripts in relation to cyp19a1a in amhy+/− and amhy−/− genotypes at 4 and 6 weeks after hatching (qRT-PCR); arrows indicate two arbitrarily-defined, opposing patterns of gene expression. β-actin was used as endogenous control. Values with different letters are statistically different from one another (One-Way ANOVA with Bonferroni's post-test, p<0.05).
Figure 4.
Spatial expression of amhy and amha mRNAs in differentiating gonads.
Localization amhy and/or amha transcripts by ISH (left panels) and light microscopic histology (right panels) of gonads in 4 and 10 week old larvae reared at 25°C. Transcripts were detected in all amhy+/− genotypes (presumptive amhy and/or amha signals) and in about half of the amhy−/− genotypes (amha signals). At 10 wah, the expression was detected in developing testis but not in developing ovaries. Scale bars indicate 10 µm.