Keywords

Introduction

Biological species which reproduce sexually require an assembly of two types of haploid gametes, i.e., eggs and sperm. These sex cells have specialized functions and are produced in the female and male gonads, respectively (Capel 2017). Sex determination (SD) is the fundamental developmental process which leads to a binary gonadal fate decision (either ovary or testis) with specific sexual characteristics and therefore becomes critical for the survival of biological species (DeFalco and Capel 2009). Interestingly, fishes are the only class of vertebrates which show a larger plasticity in gonadal development and are represented by both gonochorism (one sex at a time) and hermaphroditism (more than one sex) species (Devlin and Nagahama 2002). The only known exception of unisexual species is Poecilia formosa (Devlin and Nagahama 2002). Gonochoristic teleosts develop as either males or females only and retain such uniform sexual identity throughout their life span. On the other hand, in hermaphrodite fishes (protogynous or protandrous), the developmental pathways leading to the formation of either testis or ovary are plastic and susceptible to the sex reversal signals significantly beyond embryogenesis and early larval stages. In gonochoristic species, such developmental decision toward sex is irreversibly taken long before adulthood is reached (DeFalco and Capel 2009; Devlin and Nagahama 2002). Teleosts exhibit natural hermaphroditism where an individual changes from one sex to the other during adulthood. Some teleosts have gonads containing both mature ovaries and testes (synchronous hermaphroditism). In sequential hermaphroditism, some fishes change sex from male to female (protandrous sex change, e.g., black porgy Acanthopagrus schlegeli), others change from female to male (protogynous sex change, e.g., bluehead wrasse Thalassoma bifasciatum), and a few change sex in both directions for multiple times (bidirectional sex change, e.g., monogamous coral-dwelling gobies Gobiodon and Paragobiodon) (DeFalco and Capel 2009; Devlin and Nagahama 2002). In many cases of sequential hermaphroditism, sex change is controlled by social cues such as the disappearance of the dominant male or female from a group (Capel 2017).

In endothermic vertebrates like birds and mammals, the trigger for the sex-determining pathway is exclusively genetic (known as genetic sex determination GSD), whereas in poikilotherms, in addition to such GSD, an environmental cue, mostly temperature, also acts as the trigger, which is called environmental-/temperature-determined sex (ESD/TSD) (Capel 2017; DeFalco and Capel 2009).

Teleosts are classically categorized as ray-finned bony fishes with homocercal tails which represent almost half of all living vertebrate species (Devlin and Nagahama 2002) and show a wide variety of sex determination (both GSD and ESD mode) mechanisms (Capel 2017; DeFalco and Capel 2009; Devlin and Nagahama 2002). In many fishes, sex determination is genetic (GSD), i.e., males and females have different alleles or even different sets of genes that specify their reproductive fate and morphology (Devlin and Nagahama 2002). Additionally, sex may also be determined by environmental (ESD) variables such as temperature, pH, salinity, and social conditions (Devlin and Nagahama 2002; Baroiller et al. 2009). Thus, fishes are a very interesting group among the vertebrates for the study of sex determination and differentiation.

The mechanisms of sex determination (SD) and differentiation in fishes are highly diverse and plastic which have been extensively reviewed from time to time (DeFalco and Capel 2009). This chapter aims to briefly discuss the environmental, endocrine, and genetic aspects of sex determination (SD) in teleost fishes.

Environmental Mode of Sex Determination (ESD)

ESD system is mediated by temperature, pH, population density, and visual-endocrine cues all together (Capel 2017; DeFalco and Capel 2009; Baroiller et al. 2009; Editorial 2009).

Temperature

Temperature-induced SD or TSD is the major environmental factor in teleosts (Baroiller et al. 2009; Editorial 2009). It was first demonstrated in Atlantic silverside Menidia menidia (Baroiller et al. 2009). However, in most species, both GSD and ESD coexist, and either of the two or both mechanisms may be cooperative to drive SD. Despite having a strong GSD, temperature-induced sex reversal has been reported in the Nile tilapia Oreochromis niloticus and pejerrey Odontesthes bonariensis where high temperature prefers the male progenies (Baroiller et al. 2009). Other reported species include Japanese flounder Paralichthys olivaceus and medaka Oryzias latipes having male heterogametic GSD system or blue tilapia Oreochromis aureus, turbot Scophthalmus maximus, and half-smooth tongue sole Cynoglossus semilaevis having female heterogametic GSD system and finally seabass Dicentrarchus labrax or domestic strains of the zebrafish Danio rerio having a polygenic GSD system (Baroiller et al. 2009; Editorial 2009).

Visual-Endocrine Cue

In bluehead wrasse Thalassoma bifasciatum which shows a protogynous sex change, the behavioral, ecological, and neuroendocrine bases have been well studied (Capel 2017; Baroiller et al. 2009; Todd 2016). Female bluehead wrasses show rapid aggression during courtship leading to the behavioral sex change independent of their gonads. Such male-typical behavior is associated with the neuroendocrine axis which includes the expression of arginine vasotocin, whereas estrogen (E2) implants block such behavioral sex change (Todd 2016). Social interactions and environmental stimuli operate together through both hypothalamus-pituitary-gonadal (HPG) and hypothalamus-pituitary-interrenal (HPI) axes via neuroendocrine factors including kisspeptin (KP), dopamine (DA), gonadotropin-releasing hormone (GnRH), and arginine vasotocin (AVT) and steroids like 17β-estradiol (E2) and testosterone (T). Both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) stimulate the survival, proliferation, or maturation of the germ cells (Capel 2017; Baroiller and D’Cotta 2006; Todd 2016). Corticosteroids produced from adrenal glands act on the gonads to block the aromatase enzyme (encoded by Cyp19a1a) that produces E2 from T in the females. However, cortisol activates the enzymes encoded by Cyp11c1 and Hsd11b2, which convert T to its bio-active form 11-ketotestosterone (11-KT) in the males. Therefore, cortisol seems to be the major player in fishes for sex reversal/determination by regulating the balance between the function of Cyp19a1a and Cyp11c1 or Hsd11b2, thereby fixing the E2:11-KT ratio (Capel 2017; Todd 2016).

Population Density or Size

Sex change in gobiid fish is determined by either behavior or size. For example, in a fixed population of Okinawa rubble Trimma okinawae, larger male fishes remain as male, while the smaller males become females. Intriguingly in the absence of males, the largest female can even change its sex to male (Capel 2017; Todd 2016).

Endocrine Factors

The key regulation of the production of two gonadal steroids, i.e., estrogen (E2) and androgen (T or 11-KT), determines the sexual fate in fishes (Capel 2017; Baroiller and D’Cotta 2006; Todd 2016). In teleosts, E2 and 11-KT are the major steroids that promote ovarian or testicular differentiation and function, respectively. The production of either E2 or 11-KT depends on the bioconversion T via the aromatase enzyme coded by Cyp19a1a gene or the 11β-hydroxylase enzyme coded by Cyp11b gene and 11β-hydroxysteroid dehydrogenase (11β-HSD) enzyme coded by Hsd11b2 gene, respectively (Capel 2017; Todd 2016).

Administrations of non-aromatizable androgens disrupt the female developmental pathway, and supplementations of aromatase inhibitors further suppress ovarian E2 production in female fishes. Interestingly, it is essential to note here that sex reversal is not fully sustained following the withdrawal of hormonal stimuli suggesting that, although sex steroids potentially induce the gonadal transdifferentiation, a robust regulatory genetic network is required further for the switch to shift the sex-specific hormonal production and maintain the gonadal fate (Capel 2017; Baroiller and D’Cotta 2006; Todd 2016).

During gonadal transdifferentiation in sex-changing fishes, a dramatic shift in plasma sex steroids has been reported (Todd 2016). For example, in protogynous sex change, a severe decline in plasma E2 leads to ovarian degeneration followed by a gradual rise in 11-KT production with spermatogenic onset. Alternatively, in protandrous sex change, plasma E2 rises with decline of 11-KT level (Todd 2016). However, in bidirectional sex change, only plasma E2 (but not 11-KT) level follows such sexual directional pattern. Poor circulatory 11-KT in gobiid fishes results into lack of secondary male characteristics in these species and facilitates the rapid switching between sexual phenotypes (Todd 2016). Exogenous stimulation of sex steroids are reported to induce either masculinization or feminization in many teleosts such as Nile tilapia, rainbow trout, and Japanese flounder (Baroiller et al. 2009; Todd 2016).

The critical roles of kisspeptin (KP) and isotocin (IT) have also been well documented in sex-changing fishes (Todd 2016). KP stimulates GnRH release, whereas IT, the teleost ortholog of mammalian oxytocin, is associated with social and sex-specific reproductive behaviors. Alterations in the expression of either KP (coded by Kiss2) or its receptor (coded by Kiss1r) are reported in orange-spotted grouper Epinephelus coioides during sex reversal (Todd 2016). In the forebrain of bluehead wrasse, increased expression of IT has been found during sex change (Todd 2016). However, in bluebanded goby, poor IT activity has been observed in the pre-optic area (POA) of males as compared to that of the females (Todd 2016).

The stress response is regulated through the hypothalamic-pituitary-interrenal (HPI) axis and thereby modulates the transitional changes in behavior, metabolism, and growth during sex reversal. The corticotropin-releasing hormone (CRH) and glucocorticoid steroids (GCs) like cortisol respond to the environmental stress and potentially determine the gonadal fate (Capel 2017; Baroiller et al. 2009; Editorial 2009; Todd 2016). Elevated temperature induces gonadal masculinization via increased cortisol levels leading to downregulation of aromatase enzyme (Todd 2016). Furthermore, cortisol also stimulates Hsd11b2 expression, which catalyzes the production of both 11-KT and cortisone, the deactivated metabolite of cortisol (Baroiller and D’Cotta 2006; Todd 2016). A transient surge in serum cortisol has been recorded during both protandrous (cinnamon clownfish) and protogynous (bluebanded goby) sex change. However, long-term cortisol supplementation has shown to promote protogynous sex change in three-spot wrasse (Todd 2016). A schematic illustration of the neuroendocrine crosstalk between the HPG and HPI axes regulating steroidogenesis and sexual behavior/fate in teleosts has been described in Fig. 9.1.

Fig. 9.1
figure 1

A schematic illustration of the neuroendocrine crosstalk between the HPG and HPI axes regulating steroidogenesis and sexual behavior/fate in teleosts

Full size image

Genetic Mode of Sex Determination (GSD)

The GSD mode of sex determination is best understood in mammals and our knowledge has largely emerged from the mouse. After the discovery of the Sry gene in the 1990s, the molecular basis of mammalian sex determination (SD) and differentiation has been investigated in greater details (Capel 2017; DeFalco and Capel 2009; DeFalco 2014). In mice, during embryonic (E) age of 11.5, the SD results from the initial switch of either the Sry-dependent testis differentiation (in XY gonad) or Sry-independent ovary differentiation (in XX gonad). However, recent advancement in this field has indicated that Dmrt1 and Foxl2 are the two key transcription factors that, respectively, maintain the masculinity and femininity intact in adult mammals (DeFalco 2014). For example, the loss of Foxl2 in adult female mice ovaries results in reprogramming of the granulosa and theca cell lineages into Sertoli-like and Leydig-like cell lineages (Matson et al. 2011). Similarly, the conditional ablation of Dmrt1 results into a female reprogramming in adult testes (Matson et al. 2011). Therefore, the evolutionary significance of these two genes is of great interest and now being studied even in non-mammalian vertebrates like fowl, frog, and fishes (Capel 2017; Herpin and Schartl 2015; Bertho 2016).

In fishes, GSD could be monofactorial having a single master SD gene such as Dmy in medaka Oryzias latipes and/or could be polyfactorial involving several genes on multiple chromosomes such as in zebrafish Danio rerio (Capel 2017; Herpin and Schartl 2015). Other than Dmy, Amhy in the Patagonian pejerrey Odontesthes hatcheri, Gsdf in Oryzias luzonensis, Amhr2 in fugu Takifugu rubripes, SdY in rainbow trout Oncorhynchus mykiss, and Sox3 in Oryzias dancena have been identified as candidates for GSD (Herpin and Schartl 2015). Here we discuss some of the key molecules identified till date that regulate GSD in teleosts.

Foxl2/Foxl3

Foxl2 is a member of the large family of Forkhead Box (Fox) domain transcription factors (Bertho 2016). Since their discovery, some Fox genes like Foxc1, Foxl2, Foxl3, and various FoxOs have been reported to regulate the ovarian function and Foxj2, Foxp3, or Foxo1 for spermatogenesis, respectively (Bertho 2016). The Foxl2 gene is a highly conserved transcriptional factor expressed in the somatic cells of ovary and is essential for ovarian development and maintenance in mammals and fishes (Uhlenhaut et al. 2009; Bertho 2016; Nishimura and Tanaka 2016; Bhat et al. 2016a, b). In female gonads, Foxl2 is considered to suppress Dmrt1 or its orthologs and upregulates the expression of female programming genes including Cyp19a1a, Rspo1, and Wnt4/β-catenin, thereby ensuring the production of E2 (Bertho 2016; Nishimura and Tanaka 2016). In teleosts, two paralogs of Foxl2, Foxl2a, and Foxl2b are present originated from teleost-specific genome duplication (Bertho 2016). However, recent phylogenetic analyses revealed that Foxl2b is found in tetrapods, including reptiles, birds, and marsupials (Bertho 2016; Nishimura and Tanaka 2016). Therefore, Foxl2a and Foxl2b are currently renamed as Foxl2 and Foxl3, respectively (Nishimura and Tanaka 2016). In salmonids and seabass, Foxl2 displays a clear sexually dimorphic expression pattern in the differentiating and adult gonads with elevated expression in ovaries as compared to testes (Nishimura and Tanaka 2016). During adulthood, Foxl2 mainly present in follicular ovarian cells, i.e., granulosa cells and theca cells, surrounding the oocytes (Bertho 2016).

In medaka Oryzias latipes, Foxl2 is also restricted to ovarian tissues only (Bertho 2016; Nishimura and Tanaka 2016). Throughout the transition of germline stem cells to oocytes, the FOXL2 protein is first present in the germline stem cells and thereafter maintained during meiosis until oogenesis is completed (Bertho 2016). During oocyte formation, no FOXL2 protein is detected in the ovarian cord/follicular cells. However, FOXL2 protein is localized in the surrounding cells progressively with maturation of the oocyte (Bertho 2016). Unlike mammals, FOXL2 protein is localized in the nuclei of all granulosa cells only not in theca cells in medaka (Bertho 2016).

On the other hand, Foxl3 is expressed in germ cells and acts as a major determinant of sexual fate decision in medaka Oryzias latipes (Nishimura et al. 2015). In the undifferentiated gonads, the expansion of germ cells is reported by stem cell-like self-renewal proliferation denoted as type I division. By the hatching stage of the female embryos, a subset of germ cells undergoing type I division initiates a type II cystic division leading to the meiotic onset. Foxl3 transcript and FOXL3 protein are initially detected in type I germ cells of both male and female embryos. Thereafter, FOXL3 is only detected in a subset of mitotically active type I germ cells in female embryos, but not in mitotically quiescent germ cells in males (Nishimura et al. 2015). Both Foxl3/FOXL3 are expressed in type II germ cells but gradually start disappearing with meiotic progression and are completely lost in oocytes (Nishimura and Tanaka 2016). However, by 10 days post-hatching, such expression pattern of Foxl3/FOXL3 becomes undetectable in male fishes (Nishimura and Tanaka 2016).

Dmrt1

Doublesex- and mab-3-related transcription factor 1 (Dmrt1) is a critical inducer of testicular morphogenesis mostly conserved in metazoan organisms (Herpin and Schartl 2011 Zafer et al. 2019). It suppresses the transcription of female programming genes like Cyp19a1a, Rspo1, Figla, Gdf9, and Wnt4/β-catenin and further promotes the expression of male-specific genes like Gsdf, Cyp11c1, Sox9/3, Amh, etc. in the testes (Herpin and Schartl 2011; Dar et al. 2020). Male-restricted expression pattern of Dmrt1 has been reported in African catfish Clarias gariepinus, rare minnow Gobiocypris rarus, Nile tilapia Oreochromis niloticus, medaka Oryzias latipes, olive flounder Paralichthys olivaceus, lake sturgeon Acipenser fulvescens, zebrafish Danio rerio, Atlantic cod Gadus morhua, pejerrey Odontesthes bonariensis, rainbow trout Oncorhynchus mykiss, shovelnose sturgeon Scaphirhynchus platorynchus, rohu Labeo rohita, and southern catfish Silurus meridionalis (Herpin and Schartl 2011; Sahoo et al. 2019). In gonochoristic annual breeders like Clarias gariepinus, Oncorhynchus mykiss, and Silurus meridionalis, Dmrt1 has the key role during testicular recrudescence and spermatogenic onset (Herpin and Schartl 2011). Furthermore, in hermaphrodite teleosts like protogynous; black porgy Acanthopagrus schlegeli, gilthead seabream Sparus aurata, protandrous; grouper Epinephelus coioides, wrasse Halichoeres tenuispinis, rice field eel Monopterus albus the expression dynamics of Dmrt1s are shown to be consistent with the testicular development (Herpin and Schartl 2011). Additionally, in pejerrey Odontesthes bonariensis, which shows a strong TSD system, the developmental expression pattern of Dmrt1 coincides perfectly with the rearing temperature (up at male-determining temperatures and down at female-determining temperatures) (Herpin and Schartl 2011). However, in Gadus morhua and Danio rerio, Dmrt1 expressions are detected in the ovarian germ cells too (Herpin and Schartl 2011). Fish Dmrts (Dmrt2, Dmrt3, Dmrt4, Dmrt5) show conserved expression during embryonic development in undifferentiated gonads (Herpin and Schartl 2011). Both male and female gonadal expressions have been reported for Dmrt2 in medaka and Dmrt3 or Dmrt5 in zebrafish (Herpin and Schartl 2011). Male-specific gonadal expression has been observed for Dmrt3/4 in medaka (Herpin and Schartl 2011) and Dmrt4 in olive flounder (Herpin and Schartl 2011). On the contrary, Dmrt4 expression is restricted to ovary in Nile tilapia (Herpin and Schartl 2011).

Dmy

Medaka Oryzias latipes employs an XX/XY SD system in which the Y chromosome bears the master SD gene, the duplicated copy of Dmrt1a on the Y chromosome Dmy/Dmrt1bY (DM domain gene on the Y chromosome/doublesex- and mab-3-related transcription factor 1b on the Y chromosome) (Nanda et al. 2002; Matsuda et al. 2002). It is the only functional gene in the whole Y-specific region of the medaka sex chromosome (Kikuchi and Hamaguchi 2013). Mutations affecting this gene result in male-to-female sex reversal (Kikuchi and Hamaguchi 2013). Furthermore, Dmy transgene-induced testis development in genetic females (XX) specifically indicates that Dmy is not only necessary but also sufficient for triggering male developmental programming (Herpin and Schartl 2011). In XY medaka males, Dmy-driven primordial germ cell proliferation and the determination of pre-Sertoli cells are the primary events together leading to the male-specific primordial germ cell mitotic arrest (Herpin and Schartl 2011). Dmy is also reported to downregulate the hedgehog pathway in differentiating testes by suppressing its receptor Pitch-2 and upregulating its antagonist Hhip in medaka (Herpin and Schartl 2011). Dmy is a transcription factor, and its transcription is suppressed by P element like DNA transposon named Izanagi within its own promoter (Herpin and Schartl 2011). During testicular differentiation in XY gonads, an 11-nucleotide protein-binding motif located in the 3″-UTR of Dmy mediates unique gonad-specific mRNA stability (Herpin and Schartl 2011). Interestingly, such motif is conserved in the 3-UTRs of a wide range of Dmrt1 orthologous genes from Drosophila to mice, suggesting that different taxa may employ an evolutionary conserved RNA regulatory mechanism for this gene (Herpin and Schartl 2015; Kikuchi and Hamaguchi 2013; Herpin and Schartl 2011). In medaka Oryzias latipes, the insertion of transposable element Rex1 into the promoter of Dmy gene results in the binding of transcription factor Sox5, which downregulates Dmy expression. XX mutants for Sox5 thereby show a complete female-to-male sex reversal (Schartl et al. 2018).

Gsdf

Like Oryzias latipes, Oryzias curvinotus too retains the Dmy, as its SD gene (Herpin and Schartl 2015). However, in Oryzias luzonensis, although Dmy is lost, the SD mode follows a simple Mendelian trait (Kikuchi and Hamaguchi 2013). In 2012, an advanced genetic mapping has identified Gsdf (gonadal soma-derived growth factor) as a strong candidate for the master SD gene in this species (Myosho et al. 2012). Gsdf encodes a secretory protein belonging to the transforming growth factor-β (TGF-β) superfamily. Gsdf is present on both chromosomes (X and Y) in Oryzias luzonensis (Myosho et al. 2012). Transgenic experiments have demonstrated that the allele of this gene residing on the Y chromosome (GsdfY) was sufficient to induce female-to-male sex reversal in XX Oryzias luzonensis (Myosho et al. 2012). Interspecific transgenic experiment between Oryzias latipes and Oryzias luzonensis indicated that GsdfY of Oryzias luzonensis is capable to produce a male phenotype in XX Oryzias latipes in the absence of Dmy (Schartl et al. 2018). The spatial and temporal expression pattern of Gsdf is closely correlated to that of Dmy in Oryzias latipes (Kikuchi and Hamaguchi 2013), and the expression patterns of the further downstream genes in gonadal differentiation, such as Sox9a2, Dmrt1, and Foxl2, are comparable between Oryzias latipes and Oryzias luzonensis (Kikuchi and Hamaguchi 2013). In Oryzias latipes, Gsdf is the target of Dmy and both of them get co-localized in pre-Sertoli cells. Knocking down of Dmy resulted in male-to-female sex reversal induced by suppressing Gsdf or Sox9a and upregulating R-spondin1 or Rspo1 (Chakraborty et al. 2016). Complete female-to-male sex reversal is observed in Oryzias sakaizumii but not in Oryzias latipes by Gsdf supplementation (Kikuchi and Hamaguchi 2013).

Gsdf is also found to be the SD gene in the sablefish Anoplopoma fimbria (Kikuchi and Hamaguchi 2013). GSDF protein has been found as a somatic factor controlling the proliferation of primordial germ cells and spermatogonia in rainbow trout (Kikuchi and Hamaguchi 2013), and its expression in gonads is also observed in medaka Oryzias latipes and zebrafish Danio rerio (Kikuchi and Hamaguchi 2013).

In XY tilapia Oreochromis niloticus, Gsdf action is reported to suppress the E2 production probably via the inhibition of ovarian-differentiating genes (Jiang et al. 2016). The upregulation of Gsdf mRNA was found to be comparable with Dmrt1 in the somatic cells surrounding germ cells (Jiang et al. 2016). In vitro co-transfection of Dmrt1 and Sf1 also activates Gsdf in a dose-dependent manner in this fish (Jiang et al. 2016).

Sox9

The Sry-related HMG box (Sox) gene(s) encode a variety of transcription factors and have been implicated in male SD across the vertebrates (Capel 2017). In mammals, Sox9 is the only known direct target of the SRY transcription factor and in the testis is expressed exclusively in Sertoli cells (Capel 2017, DeFalco and Capel 2009; Bhat et al. 2016a, b). In Nile tilapia Oreochromis niloticus, by 25 days of post-hatching, Sox9 mRNA level is shown to be higher in XY gonads, whereas such expression becomes undetectable in XX gonads (Siegfried 2010). Two paralogous forms of Sox9, namely, Sox9a and Sox9b, are reported in medaka Oryzias latipes and zebrafish Danio rerio. However, in their developing gonads, no such sexual dimorphic expression pattern is observed (Herpin and Schartl 2015; Kikuchi and Hamaguchi 2013).

Sox3

In Indian ricefish Oryzias dancena, Sox3 gene is present in the XY sex chromosome (Takehana et al. 2014). The male-specific region of the Y chromosome bears a cis-regulatory DNA segment that induces the expression of the Y-chromosomal Sox3. Targeted deletion of the Y-chromosomal Sox3 results in developing females, whereas XX fish transgenic for that regulatory segment matures as males confirming the role of Sox3 as a master SD gene in Oryzias dancena. Sox3 has been shown further to initiate the testicular differentiation by upregulating expression of Gsdf (Takehana et al. 2014). However, the back clone of Sox3 from Oryzias dancena fails to induce testicular development in Oryzias latipes (Herpin and Schartl 2015).

Amhy/Amha

In the Patagonian pejerrey Odontesthes hatcheri, SD is directed by a Y copy of the anti-Müllerian hormone (Amhy) gene that codes for TGF-β superfamily protein (Hattori et al. 2012). Despite having a strong TSD, another pejerrey Odontesthes bonariensis also expressed Amhy at early stages of male-promoting temperatures followed by the expression of the autosomal Amha, showing the coexistence of both GSD and TSD mode in this species (Yamamoto et al. 2014). In the Nile tilapia Oreochromis niloticus, two tandem copies of anti-Müllerian hormone (Amh) gene have been identified Amhy and AmhΔY (a truncated Amh gene lacking the TGF-β domain due to 233-bp deleted region in exon VII and a 5-bp insertion in exon VI). However, knocking down of only Amhy (but not AmhΔY) demonstrated a male-to-female sex reversal, whereas the overexpression of only Amhy in XX fish resulted in female-to-male sex reversal (Li et al. 2015). Very recently, a male-specific duplicate copy of AmhbY (Y chromosome-specific anti-Müllerian hormone paralog b) showing differential expression pattern from its autosomal paralog Amha has been reported as a master regulator SD gene in northern pike Esox lucius (Pan et al. 2019).

Amhr2

The tiger pufferfish Takifugu rubripes employs a XX/XY SD system. A missense single-nucleotide polymorphism (SNP) in the kinase domain of the anti-Müllerian hormone receptor type II (Amhr2) has been found to be associated with the sexual phenotype in this fish (Kamiya et al. 2012). Two Amhr2 subtypes are present that differ by one amino acid (H384D) in the kinase domain. The 384His mutation causes lower activity to the receptor and is encoded on the X chromosome. Females are homozygous for the mutant, whereas the males keep one allele of the wild-type receptor on their Y chromosome. Therefore, a quantitative difference in Amh signaling becomes critical for the male development. Similarly hotei mutation observed in Amhr2 of medaka Oryzias latipes with a compromised Amh signaling shows a male-to-female sex reversal (Capel 2017, Kikuchi and Hamaguchi 2013). In Nile tilapia Oreochromis niloticus, knockout of Amhr2 results in complete (100%) male-to-female sex reversal, but in contrast, with Amhy, only 60% of such effect has been observed (Li et al. 2015). Knockdown of both Amhy and Amhr2 in XY tilapia shows upregulation of the aromatase Cyp19a1a gene as well as higher E2 levels; however, no such rise in Cyp19a1a expression has been observed in Amhy/Amhr2 overexpressed XX fish (Li et al. 2015).

SdY

Rainbow trout Oncorhynchus mykiss belongs to salmonid family and native to tributaries of the Pacific Ocean in Asia and North America. SD in this fish is strictly genetic, with an XX/XY system controlled by a single SD locus. In 2012, Yano and colleagues have identified a male-specific gene, SdY, expressed in the somatic cells surrounding germ cells (Yano et al. 2012). This gene encodes a novel protein that displays sequence homology with the carboxy-terminal domain of interferon regulatory factor 9 (Irf9). IRF9 is a transcription regulatory factor that mediates signaling by type I interferon in mammals. Microinjection of the SdY into eggs resulted in female-to-male sex reversal in XX fish, whereas the targeted inactivation of SdY induced ovarian differentiation in F1 XY fishes (Yano et al. 2012).

Table 9.1 describes the major master regulator gene(s) of SD in teleosts.

Table 9.1 Master regulator genes of SD in teleosts
Full size table

Germ Cell Number

Beyond the several environmental cues and genes, the germ cell numbers seem to be a key determinant of sex in fishes. In fact, fishes are the only class of vertebrates where germ cell number plays a key role in SD (Capel 2017; DeFalco and Capel 2009; Baroiller and D’Cotta 2006; Todd 2016). In teleosts, the germline stem cells express Nanos2 and support gametogenesis in both the sexes. Therefore, unlike mammals, germ cells in fishes show high sexual plasticity even in the matured testes and ovaries (Nishimura and Tanaka 2016). In zebrafish, goldfish, medaka, and rainbow trout, germ cells obtained from mature testes and/or ovaries can colonize in larval gonads and can differentiate into either sperm or eggs according to the sex of gonadal somatic cells (Nishimura and Tanaka 2016). In medaka Oryzias latipes, transplantation of genetically male (XY) somatic cells is sufficient to induce spermatogenesis in female (XX) fishes (Nishimura and Tanaka 2016). Emerging evidences from zebrafish Danio rerio (Uchida et al. 2004; Slanchev et al. 2005 Siegfried and Nüsslein-Volhard 2008) and medaka Oryzias latipes (Kurokawa et al. 2007) suggest that germ cell number may drive SD in these species. In zebrafish, if germ cells are depleted during the first day of development, all fishes develop as sterile males (Capel 2017). However, if germ cells are depleted in later stage, as occurs in Fanconi anemia mutation (Fancl), adult females undergo sex reversal to phenotypic males and can further become fertile if some of the germline stem cells persist and populate the testis (Rodriguez-Mari et al. 2010). On the other hand, in medaka, germ cell-depleted fishes develop with male phenotype; by contrast, when the number of germ cells is amplified (e.g., in the hotei mutant, the Amhr2 gene results into a compromised Amh signaling on the mitotic self-renewing germ cells leading to massive germ cell proliferation), fishes show a male-to-female sex reversal (Nakamura et al. 2012). Similarly, loss of germ cells induced by high temperatures in fugu Takifugu rubripes induced sex reversal of females into males (Baroiller and D’Cotta 2006). However, the depletion of germ cells fails to affect the sexual fate of gonadal somatic cells in goldfish Carassius auratus (Goto et al. 2012) or loach Misgurnus anguillicaudatus (Fujimoto et al. 2010).

Conclusion

Various laboratories across the globe for the past five decades have substantially contributed toward our present knowledge regarding the environmental, endocrine, and genetic control on SD mechanisms in fishes. Briefly at the molecular level, in females, the transcription factors like Foxl2 and Sf1 (controlled by gonadotropins like FSH via cAMP pathway) upregulate the expression of Cyp19a1a to produce the aromatase enzyme for the bioconversion of E2 from T. This estrogenic environment further maintains an auto-regulatory feed-forward loop for supporting the ovarian function via promoting female-specific gene expression while suppressing male programming genes. In males, Dmrt1 directly inhibits Cyp19a1a promoter activity, while Dax1 negatively modulates the expression of Cyp19a1a through its suppression of Sf1 and Foxl2. Therefore, such inhibition of aromatase action promotes the male-specific genes for testicular differentiation and function. Furthermore, the environmental stress induces cortisol production, which regulates the balance between the turnover rate of T to either E2 or 11-KT (Capel 2017; Baroiller and D’Cotta 2006; Todd 2016). In summary, a complex antagonistic crosstalk between neuroendocrine signaling and genetic regulatory network promotes either an estrogenic or an androgenic milieu to determine the sexual fate in teleosts. Figure 9.2 schematically represents such antagonistic regulation toward gonadal development and function in fishes.

Fig. 9.2
figure 2

The antagonistic regulatory pathways controlling the differential steroidogenesis and genetic networks determining the sexual fate in teleost fishes

Full size image

Future Direction

Emerging data in the last decade further indicate that unlike mammals, SD in fishes is not brought by a simple linear cascade, but a complex network of multiple regulatory pathways is involved with apparently diversified upstream inducers and relatively conserved downstream effectors (Herpin and Schartl 2015; Kikuchi and Hamaguchi 2013; Herpin et al. 2013). However, in-depth comparative analyses in diverse non-model species are necessary for the better understanding of such regulatory networks. The advancement in next-generation high-throughput sequencing technologies and omics approach may potentially reveal the gonadal transcriptome, miRNome, or methylome for future discovery/identification of other novel putative factor(s) and establish their interaction(s) with known sex-determining loci (Qian et al. 2014; Pan et al. 2016).