Genotype-dependent activation of crh-family genes during heat-induced masculinization in pejerrey Odontesthes bonariensis.

AARON TORRES-MARTÍNEZ; DONG HUNG SONG; RICARDO S. HATTORI; GUSTAVO M. SOMOZA; JUAN I. FERNANDINO; YOJI YAMAMOTO; CARLOS A. STRÜSSMANN

Faro

Congreso; Joint Conference of the European Society for Comparative Endocrinology and of the International Society for Fish Endocrinology; 2022

European Society for Comparative Endocrinology e International Society for Fish Endocrinology

In recent years, a connection between stress, stress-related CHR-family genes, and sex determination in fish has emerged. Previous studies from our research group in pejerrey Odontesthes bonariensis, a species with both, genetic and environmental-sex determination, demonstrated the involvement of cortisol in stress (so far thermal and crowding)-induced masculinization during the critical period of sex determination (CPSD: 2-6 weeks after hatching). However, the involvement of the brain via the hypothalamus-pituitary-interrenal axis (HPI) in theregulation of this process in pejerrey has not been investigated. There are also no studies on the possibility of genotypic-specific stress responses of larvae to the masculinizing conditions. Therefore, in this study we analyzed the expression patterns of several members of the CRH family (a group of genes regulating stress and anxiety) including corticotropin-releasing hormone a and b (crha and crhb), urotensin 1 (ust1), urocortins 2 and 3 (ucn2 and ucn3), and corticotropin-releasing binding protein (crhbp) as well as their receptors crhr1 and crhr2 in the heads of XX and XY pejerrey larva during the CPSD at a masculinizing temperature (29°C). Genes found to be upregulated were further analyzed by in situ hybridization (ISH). The deduced amino acid sequences of crhb, uts1, ucn2, and ucn3 exhibited all the structural elements of functional mature peptides, despite ucn2 been nonfunctional in some vertebrates. Notwithstanding our multiple attempts to clone crha, we failed to identify this gene in pejerrey. Quantification by RT-qPCR showed that crhb, ucn2, ucn3 and their receptor crhr2 were upregulated only in the heads of XX larvae but not in XY during heat-induced masculinization. uts1, crhbp, and crhr1, on the other hand, did not show any statistical difference between genotypes and therefore may not be involved in this process. ISH showed that ligands and the receptor of CRH-family genes were expressed in the tuberal hypothalamus, in areas of great neuroendocrine relevance. The fact that all the three overexpressed genes in XX larvae signal through crhr2 (based on affinity studies conducted in other species) and had closespatiotemporal relation during the CPSD suggests that these genes might be operating in a synergic manner to induce the activation of the HPI axis or perhaps the organization of stress-mediated sexual dimorphism in the neural architecture. Overall, upregulation of crhb and urocortins might be necessary to induce a developmental reprograming of the brain of XX larvae during heat-induced masculinization, which is not necessary in XY due that it is already programmed.