Effects of 17β-estradiol on early gonadal development and expression of genes implicated in sexual differentiation of a South American teleost, Astyanax altiparanae

Highlights

• 17β-estradiol (E2) exposure during early gonadal development affected male secondary characteristics of A. altiparanae;

• E2 treatment did not change gonadal sex ratio, induced testis-ova and altered genes related to sex differentiation;

• Altered expression could be considered a compensatory mechanism, or a mechanism of gonadal resilience;

Abstract

Gonadal sex differentiation in teleost fish shows greater plasticity as compared to other vertebrates, as it can be influenced by a variety of factors such as exogenous sex steroids. Exogenous estrogens, such as 17β-estradiol (E2), can induce feminization when administered during early embryonic development. However, the mechanisms underlying the E2-induced feminization are not fully understood, especially in Neotropical species. Therefore, the aim of this study was to evaluate the effects of E2 administration on the phenotypic sex characteristics, histological assessment of the gonads, and the expression of selected genes in Astyanax altiparanae exposed to dietary E2 prior to gonadal differentiation. At 4 days post-hatch (dph), groups of 30–40 undifferentiated larvae were fed with a diet containing varying amounts of E2 for 28 days, and fish were sampled at 90 dph. Previous studies revealed that ovary formation in A. altiparanae occurred at 58 dph, whereas the first sign of testis formation was found at 73 dph. In relation to the control, E2 exposure increased the proportion of phenotypic females in 120% and 148.4% for 4 and 6 mg E2/Kg, respectively. However, histological analysis revealed that treatments did not affect gonadal sex ratio between males and females, but induced intersex (testis-ova) in the group treated with 6 mg E2/Kg food. Treatment with E2 also altered gonadal transcript levels of a selected number of genes implicated in sexual differentiation. Males overexpressed dmrt1, sox9 and amh following E2 treatment as compared to control. Females showed increased mRNA levels of dmrt1 and sox9, which might be related to the down-regulation of cyp19a1a after E2 exposure. In summary, E2 exposure during early gonadal development affected male secondary characteristics without changing the gonadal sex ratio, and altered expression of genes implicated in sexual differentiation.

Introduction

The most critical stage in vertebrate sex determination is one in which the bipotential gonad anlage will become a testis or ovary. This process involves cell fate and differentiation, and both programs are regulated through cascades and networks of multiple genes (Graham et al., 2003; Herpin et al., 2013; Herpin and Schartl, 2015). Over the past decades, accumulated knowledge has shown that while the genetic machinery triggering the gonadal sex differentiation is diverse among vertebrates, the downstream components seem to be evolutionarily more conserved and appear to converge on the regulation of a few central common effectors (Graham et al., 2003; Herpin et al., 2013; Capel, 2017). In vertebrates, activation of male pathway initiates through up-regulation of Sox9 (Sry-related HMG box 9), followed by Sf1 (steroidogenic factor–1), Fgf9 (Fibroblast growth factor 9) and Dmrt1 (Doublesex and Mab-3 related transcription factor 1). Dmrt1 is not only important for maintaining the male pathway but also in suppressing the female networks. There are two female networks, one involving Foxl2 (Forkhead box transcription factor L2) and Esr1,2 (Estrogen receptor 1 and 2), while the other one comprises Rspo1 (R-spondin 1), Wnt4 (Wnt family member 4)/β-catenin and Fst (Follistatin) (Brennan and Capel, 2004; Yao et al., 2004; Herpin and Schartl, 2015; Biscotti et al., 2018).

Teleost fish exhibits the most diverse mechanisms of sex determination and differentiation among vertebrates (Nagahama, 1994; Devlin and Nagahama, 2002; Kobayashi et al., 2013). Additionally, multiple triggers can regulate the process of sexual differentiation including genetic or environmental factors (hormones, temperature, pH, oxygen, social condition and others) (Kobayashi et al., 2013; Godwin, 2010; Castañeda Cortés et al., 2019). Among these factors, sex steroid hormones are considered the major inducers of gonadal sex differentiation, and are also responsible for the maintenance of the differentiated gonad in fish as reviewed previously (Liu et al., 2017; Li et al., 2019).

Estrogens are produced by the conversion of androgens through cytochrome P450 aromatase, which is encoded by cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a - gonadal type) and cyp19a1b (cytochrome P450, family 19, subfamily A, polypeptide 1b - brain type) genes in most of teleost fish species (reviewed by Le Page et al., 2010; Li et al., 2019). The expression of cyp19a1a and the production of endogenous estrogens occur specifically in the female gonads during the critical period of molecular sexual differentiation (reviewed by Nagahama, 2002; Li et al., 2019). In this context, exposure to 17β-estradiol (E2) before the critical time window of gonadal differentiation has been shown to induce ovarian differentiation in genetic males of different families of fish including Salmonidae, Cichlidae, Anguillidae, Sparidae, Belontiidae, Poecilidae, Cyprinidae and Characidae (Pandian and Sheela, 1995; Piferrer, 2001; Chang et al., 1994; Bem et al., 2012; Díaz and Piferrer, 2017). Conversely, administration of aromatase inhibitors prior to sexual differentiation has been reported to induce masculinization of genotypic female fish, as reported in rainbow trout (Oncorhynchus mykiss) (Guiguen et al., 1999), tilapia (Oreochromis niloticus) (Guiguen et al., 1999), chinook salmon (Oncorhynchus tshawytscha) (Piferrer, 1994), protogynous orange-spotted grouper (Epinephelus coioides) (Tsai et al., 2011) and European sea bass (Dicentrarchus labrax) (Navarro-Martín et al., 2009). Altogether these data indicate that gonadal aromatase and E2 can act as inducers of ovarian differentiation in some teleost species (reviewed by Nagahama, 2002; Li et al., 2019). Treatments with E2 or aromatase inhibitors have been widely used for sex control in aquaculture, in particular for establishing monosex cultures when a species has a pronounced sexual dimorphism in weight and size or growth rate (Piferrer, 2001; Beardmore et al., 2001). However, the mechanisms underlying the E2-induced feminization in fish are not fully understood nor systematically compared, especially for Neotropical species (Fernandino and Hattori, 2018). For example, studies with South American pejerrey (Odontesthes bonariensis) have shown that E2 treatment during early gonadal development decreased the expression of genes related to testicular differentiation, such as amh (anti-Müllerian hormone) and dmrt1 (doublesex and Mab-3 related transcription factor 1) (Fernandino et al., 2008a, Fernandino et al., 2008b). Simultaneously, E2 increased the transcript abundance of cyp19a1a, which is associated with ovarian differentiation (Fernandino et al., 2008a).

Astyanax altiparanae is a South American teleost fish, popularly known as lambari, native to the Upper Paraná basin in Brazil (Garutti and Britsky, 2000). A. altiparanae easily reproduce, have good survival rates for both larvae and juveniles, and display rapid growth rate and larger body size for females (Porto-Foresti et al., 2005). Therefore, the species is of substantial commercial importance (Porto-Foresti et al., 2005) and scientific relevance as a Neotropical experimental model (Gomes et al., 2013; Costa et al., 2014; Adolfi et al., 2015; De Paiva Camargo et al., 2017; Branco et al., 2019). Previous studies have induced meiotic gynogenesis in A. altiparanae and the sex ratio of gynogenetic progenies suggests a XX/XY chromosome system for this species (Do Nascimento et al., 2019). However, sex determining gene is still unknown and no genetic makers are available to genotype individuals regarding the sex.

Considering that A. altiparanae females are more economically attractive than males due to their faster growth rate and larger size (Porto-Foresti et al., 2005), studies to induce feminization in this species are of great interest. Nevertheless, this topic remains poorly investigated in A. altiparanae and the molecular mechanisms underlying the E2-induced feminization are unknown. Therefore, the aim of the present study was to evaluate the effects of dietary E2 on phenotypic sex characteristics, gonadal histology, and gonadal gene expression in A. altiparanae exposed to dietary E2 prior to gonadal differentiation.