In fish, genetic and environmental factors may interact to determine gonadal fate (Hattori et al., 2020). Genetic factors establish the genotypic sex at the time of fertilization while environmental factors act later during the critical period of sex determination (CPSD) when the gonad is still bipotential. Strong environmental stimuli transduced as physiological stress (e.g., high temperature, hyper salinity, crowding) can override the genetic predisposition and produce individuals with mismatching genotypic and phenotypic sex. Despite the growing attention that environmental stress on fish sex determination have received in the last decades, their interaction with genetically programmed processes are still elusive. Environmentally induced gonadal sex determination (particularly masculinization) in fish seems to involve a stress reaction and cortisol (Hattori et al., 2020; Strüssmann et al., 2021), epigenesis (e.g., methylation; Piferrer, 2013; Valdivieso et al., 2021), or oxidative stress (Corona-Herrera et al., 2018; Mukai et al., 2022). In the former, which occurs in pejerrey Odontesthes bonariensis and the Japanese medaka Oryzias latipes, exposure to a stressful stimulus like excessive heat causes a rise in circulating cortisol, leading to a blockade of the ovarian differentiation signaling pathway and activation of genes of the testis-developing cascade (Fernandino et al., 2013; Goikoetxea et al., 2017). This occurs because high circulating cortisol levels lead to the activation of the enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD), which breakdowns cortisol and at the same time synthetizes 11-ketotestosterone (11-KT) as a by-product (Fernandino et al., 2013). Thus, the mechanism of cortisol-induced masculinization in the gonads is somehow understood, but the upstream regulation of this process in the brain and pituitary has received little attention so far (Fernandino et al., 2012; Hattori et al., 2009). In a recent study in O. latipes, Castañeda-Cortés et al. (2019) showed that exposure to heat during early development led to the upregulation of the corticotropin-releasing hormone b (crhb) and its receptors crhr1 and crhr2 in the brain, causing masculinization of genotypic females. By knocking out the receptors, it was possible to null the masculinizing effect of heat whereas concomitant administration of the downstream effector of the hypothalamus-pituitary-interrenal (HPI) axis, cortisol, rescued the knockout effect, unquestionably implicating the brain in thermal-induced masculinization via the HPI axis (Castañeda-Cortés et al., 2019).

The pejerrey O. bonariensis (Atheriniformes, Atherinopsidae) is a fish species in which genotypic sex determination (GSD) coexists with a marked temperature-dependent sex determination (TSD) (Yamamoto et al., 2014). In this species, all-male progenies are obtained at high temperatures (male promoting temperatures, MPT) and all-female progenies at low temperatures (female promoting temperatures, FPT). Intermediate temperatures produce varied proportions of males and females (mixed-sex promoting temperatures, MixPT), reflecting the combined effects of TSD and GSD, whose main player is the Y chromosome-linked Anti-Müllerian-hormone gene (amhy) (Yamamoto et al., 2014; Zhang et al., 2018). Earlier studies in this species have pointed out the possibility that gonadal sex determination, and more specifically the effects of temperature, could start in the brain (Miranda et al., 2001, 2003). These studies unveiled the increased abundance of gonadotropin releasing hormone 1 (Gnrh1)-, follicle stimulating hormone (Fsh)-, and luteinizing hormone (Lh)-secreting immunoreactive cells in the brain and pituitary (Miranda et al., 2001, 2003) and the presence of Fsh and Lh receptors in the undifferentiated gonads before the first signs of gonadal differentiation (Shinoda et al., 2010) during the CPSD. Other studies showed that the expression of brain aromatase (cyp19a1b) in the head preceded that of gonadal aromatase (cyp19a1a) in the trunk of undifferentiated larvae (Karube et al., 2007; Strobl-Mazzula et al., 2008) and that the brains of pejerrey larvae during the CPSD at a masculinizing temperature had more abundant cyp19a1b transcripts than at a feminizing temperature (Strobl-Mazzula et al., 2008). It has been also shown that Kisspeptin-encoding gene kiss2 levels increased during the CPSD at MPT conditions (Tovar-Bohórquez et al., 2017). Finally, a stress response and cortisol have been implicated in TSD as well as other forms of ESD of pejerrey (Fernandino et al., 2012, 2013; García-Cruz et al., 2020; Hattori et al., 2009) but the molecular players involved in this process and how they are influenced by the genotype and the environment remain unknown.

In fish, the stress response is a complex process mediated by neuropeptides, neurotransmitters, and receptors. One group of genes key in the stress response belongs to the so-called Crh (corticotropin releasing hormone) family, formed by the corticotropin-releasing hormone (crh), urotensin 1 (uts1) (urocortin 1 in mammals), urocortin 2 (unc2), and urocortin 3 (ucn3), neuropeptides that operate through two different G protein-coupled transmembrane receptors: corticotropin-releasing hormone receptor 1 (crhr1) and corticotropin-releasing hormone receptor 2 (crhr2). The corticotropin-releasing hormone-binding protein (encoded by the crhbp gene) also plays an important role in the stress response by sequestering/releasing some of these ligands. Crh and Uts1 bind to Crhr1 and to Crhr2, which in turn provides binding sites for Ucn2 and Ucn3. Thus, Crhr2 is a promiscuous receptor capable of mediating the transduction of multiple signals (Lovejoy et al., 2014; Inda et al., 2017). In addition to Crh family members, arginine vasotocin (Avt) is another neuropeptide that participates in the stress response (Baker et al., 1996; Gesto et al., 2014; Martos-Sitcha et al., 2019). Many of these peptides have central effects, regulating stress and anxiety in different regions of the brain, while others such as pomc (proopiomelanocortin) encoding adrenocorticotropic hormone (Acth), expressed in the pituitary are necessary to activate HPI axis and trigger the hormonal (sympathetic) stress response leading to the synthesis and release of cortisol. Finally, the glucocorticoid receptors 1 and 2 (GR1 and GR2, respectively) (gr1 and gr2) and a mineralocorticoid receptor (MR) encoded by the nuclear receptor subfamily 3 group C member 2 (nr3c2) are the final targets of cortisol, mediating the physiological actions of this steroid and playing multiple roles in feedback mechanisms and developmental programming (Faught and Vijayan, 2018; Gjerstad et al., 2018; Nesan and Vijayan, 2013).

Based on the above evidence for pejerrey and on similar studies in other species such as the European seabass Dicentrarchus labrax and the Senegalese sole Solea senegalensis (; Moles et al., 2007; Guzmán et al., 2009), it is hypothesized that the brain plays a crucial role in the transduction of environmental signals during the sex determination and differentiation in pejerrey. The pejerrey has a different master sex determining gene (amhy vs. dmy/dmrt1Y) and a much more marked TSD than medaka which is operational at environmentally relevant temperatures, and therefore provides an excellent model to corroborate and extend these findings. In this study, we took advantage of the amhy gene as a marker of genotypic sex to further explore the involvement of the central nervous system in the sex determination of this species and the possibility of a genotype-based dimorphism in thermal stress response during the CPSD. First, we analyzed the expression profile of the Crh family genes (crh, uts1, ucn2, ucn3), their receptors crhr1 and crhr2, the carrier protein crhbp, the neuropeptide avt and the expression of associated genes such as pomc, gr1 and gr2 as well as nr3c2 as the targets of cortisol signaling during the CPSD at masculinizing and feminizing temperatures in relation to genotype. We also determined the localization of the mRNAs of selected genes in the brain and analyzed the whole-body cortisol titers in XX and XY larvae during the CPSD to establish a functional link between the HPI axis and the developing gonads. Finally, we compared the temporal expression of the stress-related genes in the brain with that of brain aromatase (cyp19a1b) and of molecular markers of sex differentiation in the gonads, including amhy, amha, and cyp19a1a, to establish the hierarchical relationship between the brain and the developing gonads during the CPSD.