Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology
Effects of 17β-estradiol on early gonadal development and expression of genes implicated in sexual differentiation of a South American teleost, Astyanax altiparanae
Graphical abstract
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.
Section snippets
Animals and experimental design
The specimens used in this study were obtained from the Center of Aquaculture of São Paulo State University (CAUNESP), Jaboticabal (São Paulo State, Brazil). All experimental procedures were performed according to the São Paulo State University Animal Care and Use Committee Protocol (CEUA - 9496). Throughout the experiment, water quality parameters were monitored daily. The average monitored conditions were pH 6.6 ± 0.7 (pH meter YSI, model PH100), dissolved oxygen 6.2 ± 0.6 mg/L (oximeter YSI
Early exposure to E2 affected the male secondary sex characteristics of A. altiparanae
Analysis of secondary sex characteristics revealed that E2 exposure affected the phenotypic sex of A. altiparanae when compared to control (Table 2; Supplemental Table 1). In the control group, the phenotypic sex ratio (male:female) was 0.88 (51:58), and after treatment, phenotypic sex ratios changed significantly to 0.56 (45:80) and 0.26 (21:80) in the groups that received 4 mg and 6 mg E2/Kg, respectively (Table 2). Our results showed that treatment with E2, in a concentration-dependent
Discussion
The present study examined the effects of E2 on the phenotypic characteristics, histological assessment of the gonads, and the expression of selected genes in the gonads of a South American fish, A. altiparanae, exposed to dietary E2 for 28 days during the period that precedes the gonadal differentiation. The results showed a concentration-dependent effect of E2 on the phenotypic sex of A. altiparanae. Early exposure to E2 (4 and 6 mg/Kg) significantly affected the establishment of male
Declaration of Competing Interest
The authors declare that they have no competing interests.
Acknowledgements
We would like to thank the members of Reproductive and Molecular Biology group (UNESP - Botucatu) and Laboratory of Histology (UNESP - Jaboticabal) for the collaborative research. The authors are also thankful to Ms. Keila Emilio de Almeida for the technical support in histology, and Dr. Nivaldo Ferreira do Nascimento for technical support during the animal sampling. This work was supported by TWAS/CNPq, Brazil (grant number - 190111/2014-3); and São Paulo Research Foundation (FAPESP), Brazil
References (65)
- et al.
Intersex in teleost fish: are we distinguishing endocrine disruption from natural phenomena?
Gen. Comp. Endocrinol.
(2013) - et al.
Monosex male production in finfish as exemplified by tilapia: Applications, problems, and prospects
- et al.
Effects of GnRH and the dual regulatory actions of GnIH in the pituitary explants and brain slices of Astyanax altiparanae males
Gen. Comp. Endocrinol.
(2019) - et al.
Testes of Astyanax altiparanae: the Sertoli cell functions in a semicystic spermatogenesis
Micron
(2014) - et al.
Characterization of undifferentiated spermatogonia and the spermatogonial niche in the Lambari fish Astyanax altiparanae
Theriogenology
(2017) - et al.
Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences
Aquaculture
(2002) Neuroendocrinology of sexual plasticity in teleost fishes
Front. Neuroendocrinol.
(2010)- et al.
Distribution of GnRH in the brain of the freshwater teleost Astyanax altiparanae
Micron
(2013) - et al.
Ovarian aromatase and estrogens: a pivotal role for gonadal sex differentiation and sex change in fish
Gen. Comp. Endocrinol.
(2010) - et al.
Sex determination: switch and suppress
Curr. Biol.
(2011)
β-Estradiol 17-Valerate Affects Embryonic Development and Sexual Differentiation in Japanese Medaka (Oryzias latipes)
Roles of estrogens in fish sexual plasticity and sex differentiation
Gen. Comp. Endocrinol.
Direct feminization of lumpfish (Cyclopterus lumpus L.) using 17β-oestradiol-enriched Artemia as food
Aquaculture
Masculinization of the European sea bass (Dicentrarchus labrax) by treatment with an androgen or aromatase inhibitor involves different gene expression and has distinct lasting effects on maturation
Gen. Comp. Endocrinol.
Sex differentiation and hormonal sex reversal in the bagrid catfish Pseudobagrus fulvidraco (Richardson)
Aquaculture
The role of Amh signaling in teleost fish – multiple functions not restricted to the gonads
Gen. Comp. Endocrinol.
Endocrine sex control strategies for the feminization of teleost fish
Aquaculture
The comparative effectiveness of the natural and a synthetic estrogen for the direct feminization of Chinook salmon (Oncorhynchus tashawytscha)
Aquaculture
Methodology for estradiol treatment in marine larval and juvenile fish: uptake and clearance in summer flounder
Aquaculture
Steroid hormone enrichment of Artemia nauplii
Aquaculture
Dmrt1 directly regulates the transcription of the testis-biased Sox9b gene in Nile tilapia (Oreochromis niloticus)
Gene
Molecular cloning and expression analysis of dmrt1 and sox9 during gonad development and male reproductive cycle in the lambari fish, Astyanax altiparanae
Reprod. Biol. Endocrinol.
Increase of cortisol levels after temperature stress activates dmrt1a causing female-to-male sex reversal and reduced germ cell number in medaka
Mol. Reprod. Dev.
Intersex, hermaphroditism, and gonadal plasticity in vertebrates: evolution of the Müllerian duct and Amh/Amhr2 Signaling
Ann. Rev. Animal Biosci.
A comparative view on sex differentiation and gametogenesis genes in lungfish and coelacanths
Genome. Biol. Evol.
Sex hormone concentrations and gonad histology in brown trout (Salmo trutta) exposed to 17β-estradiol and bisphenol a
Ecotoxicology
One tissue, two fates: molecular genetic events that underlie testis versus ovary development
Nat. Rev. Genet.
Vertebrate sex determination: evolutionary plasticity of a fundamental switch
Nat. Rev. Genet.
The central nervous system acts as a transducer of stress-induced masculinization through corticotropin-releasing hormone B
Development.
Estradiol-17β associated with the sex reversal in protandrous black porgy, Acanthopagrus schlegeli
J. Exp. Zool.
Effectiveness of estradiol valerate on sex reversion in Astyanax altiparanae (Characiformes, Characidae)
Braz. Arch. Biol. Technol.
Estrogen exposure overrides the masculinizing effect of elevated temperature by a downregulation of the key genes implicated in sexual differentiation in a fish with mixed genetic and environmental sex determination
BMC Genomics
Cited by (20)
Exposure to estrone disrupts the endocrine system of western mosquitofish (Gambusia affinis)
2023, Aquatic ToxicologyDevelopmental toxicity and transcriptome analysis of equine estrogens in developing medaka (Oryzias latipes) using nanosecond pulsed electric field incorporation
2023, Comparative Biochemistry and Physiology Part - C: Toxicology and PharmacologyEstrogen pollution of the European aquatic environment: A critical review
2023, Water ResearchCitation Excerpt :Increased concentrations of VTG in fish tissues and serum, and disturbances in VTG synthesis have been found in many studies, which indicates the high sensitivity of fish to the presence of estrogens in the environment (Filby et al., 2010; Hill and Janz, 2003; Jobling et al., 2003; Jones et al., 2000; Schäfers et al., 2007; Song et al., 2020; Vajda et al., 2008). One of the commonly reported negative effects of estrogens on fish is the feminization of many male species, which may lead to changes in primary and secondary sexual characteristics, and thus a reduction in the number of sperm cells produced or morphological changes (Dang and Kienzler, 2019; Filby et al., 2010; Jackson and Klerks, 2020; Karki et al., 2021; Martinez-Bengochea et al., 2020; Sumpter, 1995). There have also been many cases where the presence of estrogens in the environment caused the dominance of female individuals (Hill and Janz, 2003; Jackson and Klerks, 2020; Karki et al., 2021; Teta et al., 2018; Vajda et al., 2008) or the intensification of intersex (Hicks et al., 2017; Jobling et al., 2006) among many fish populations.
- 1
Both authors contributed equally to this manuscript.