Chapter

ATHERINOPSID AS MODELS FOR THE STUDY OF TEMPERATURE-DEPENDENT SEX DETERMINATION: PHYSIOLOGY OF GONADAL SEX DIFFERENTIATION IN PEJERREY BONARIENSIS

Juan I. Fernandino1, Ricardo S. Hattori2, Carlos A. Strüssmann2 and Gustavo M. Somoza1, 1Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús. (CONICET-UNSAM), Chascomús, Argentina 2Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Tokyo Japan

ABSTRACT

The development of a functional gonad implies consequences not only for the phenotypic sex but also for the reproductive capacity during adulthood. In recent years, Atherinopsid have been extensively studied because they present a variety of and sometimes contrasting mechanisms of sex determination/differentiation. In the present work we review the sex determining mechanisms in different species belonging to this family. As an example, , commonly known as pejerrey, was singled out in order to discuss morphological aspects of the gonadal differentiation process in males and females and its relation to water temperature, the expression profile of some selected genes involved in this process, and the involvement of cortisol in the masculinizing process induced by warm temperature. The well described process of sex determination/differentiation in O. bonariensis and other Atherinopsid species makes this family an emerging model to study the interactions between the environment and genotypic factors that ultimately lead to determination of the gonadal fate.

 Contact Information and Reprint Request: Gustavo Manuel Somoza. Av. Intendente Marino Km. 8.2 (B7130IWA). Chascomús. Provincia de Buenos Aires. Argentina. E-mail: [email protected]. 2 Juan I. Fernandino, Ricardo S. Hattori, Carlos A. Strüssmann et al.

Keywords: Atherinopsidae, GSD, TSD, apoptosis, steroids, stress, cortisol

1. MECHANISMS OF SEX DETERMINATION IN ATHERINOPSID FISH

Atherinopsid fish are native from continental and marine waters of the Americas and they are usually referred to as “New World silversides” (Dyer, 2006; Bloom et al., 2012). In the last three decades several species belonging to this group have been intensively investigated with the focus on the sex determination and gonadal differentiation processes. Sex in vertebrates is generally determined by two mechanisms: genetic (GSD) and environmental (ESD) sex determination. In species with GSD, sex is genetically programmed at fertilization and follows the actions of specific gene(s) usually located in the sex chromosomes, whereas in ESD sex is determined by environmental factors (Valenzuela et al., 2003; Ospina-Álvarez and Piferrer, 2008; Mank and Avise, 2009; Angelopoulou et al., 2012). The best known environmental factor involved in ESD is temperature, defined as temperature-dependent sex determination (TSD). In this mechanism the phenotypic sex is driven by temperature during a sensitive period early in development (Devlin and Nagahama, 2002; Grossen et al., 2011; Matsumoto and Crews, 2012). TSD was first discovered in reptiles (see Bull and Vogt, 1979) and later found for the first time in fish by Conover and Kynard (1981) in the menidia (L.), an Atherinopsid fish. Since then it has been demonstrated that temperature can affect in different degrees the sex determination in other Atherinposid species such as Menidia peninsulae (Middaugh and Hemmer, 1987), Odontesthes bonariensis (Strüssmann et al., 1996a), Odontesthes argentinensis and Odontesthes hatcheri (Strüssmann et al., 1996b). TSD has also been demonstrated in many other fish species (Stüssmann and Patiño, 1999; Ospina- Álvarez and Piferrer, 2008; Baroiller et al., 2009a,b; Luckenbach et al., 2009; Siegfried, 2010) and it has been reported as the most important, or at least, the best studied environmental factor influencing sex differentiation in teleosts including tilapia (Devlin and Nagahama, 2002; Baroiller et al., 2009a,b). Although TSD has been described in different fish orders, no relationship has been found with the phylogenetic position, suggesting that such a mechanism has emerged more than once during teleost evolution (Devlin and Nagahama, 2002; Mank et al., 2006). It is now also clear that there are some teleost species in which GSD can be influenced by environmental factors and thus the phenotypic sex is determined as a result of a combination of the two mechanisms (Goto-Kazeto et al., 2006; Baroiller et al., 2009a, b; 2009b; Luckenbach et al., 2009). This is also true for those species in which a sex determining gene was identified as in medaka Oryzias latipes (Matsuda et al., 2002) and rainbow trout Onchorynchus mykiss (Yano et al., 2012), where high temperature has been demonstrated to produce skewed sex ratios (Sato et al., 2005; Hattori et al., 2007; Magerhans et al., 2009; Magerhans and Hörstgen-Schwark, 2010). However, Atherinopsids provide an excellent material to study the interactions of GSD/TSD because members of this family span from a “pure” TSD to a classical GSD model. For example, the brackish water species pejerrey O. bonariensis presents a very strong TSD whereby all-female and all-male populations can be obtained when larvae are raised at Atherinopsid Fishes and Temperature-Dependent Sex Determination 3 low (17º C) and high (29º C) temperatures, respectively (Strüssmann et al., 1996a, 1997). The marine pejerrey O. argentinensis, also shows a clear correlation between sex ratios and temperature: more females are produced at lower (18 and 21º C) than at higher (25º C) temperatures (Strüssmann et al., 1996b). The situation in the Patagonian pejerrey O. hatcheri, in which a clear GSD was described (Hattori et al., 2010; 2012; Koshimizu et al., 2010) is different. In this species, the larvae reared at intermediate temperatures (17-23º C) show sex ratios around 1:1 but low (13-15º C) and high (25º C) temperatures produce female- and male-skewed sex ratios, respectively (Strüssmann et al., 1996b, 1997). This is important because Hattori et al. (2012) described the sex determining gene (amhy) in this species. This indicates, as already said, that the temperature during the period of sex determination can affect sex ratios, even in cases where a clear and defined genetic component exists (Hattori et al., 2010; 2012; Koshimizu et al., 2010). It is important to note that amhy has been found also in O. bonariensis (unpublished results). Although its role in this species is still unknown, it supports the notion that TSD and GSD are the extremes of a continuum (Strüssmann and Patiño, 1999). The objective of the present chapter is to review the physiology of the sex differentiation process in the best studied of these species, the pejerrey O. bonariensis. The pejerrey is a species which inhabits brackish waters in the southern region of the South American continent. The strong sexual thermolability makes of pejerrey a unique and ideal model to study how temperature can influence the feminization and masculinization processes.

2. HISTOLOGICAL FEATURES OF GONADAL SEX DIFFERENTIATION

In differentiated gonochoristic species, i.e., those species in which the gonads differentiate directly into either an ovary or a testis, sex is determined during a critical period of embryonic and/or larval stages that precedes the appearance of sex-specific histological or cellular differences. In pejerrey, this time frame was estimated to be between 1 and 3 weeks after hatching (wah) at 27º C, 2 and 4 wah at 24º C, and 3 and 5 wah at 17 ºC (Strüssmann et al., 1997), which in a size scale ranges from 11 to 18 mm of body length. Thus, the temperature experienced during these early developmental stages determines whether the primordial gonad will follow the male or female pathway. The first signals of morphological sex differentiation start to be detected shortly after this period and appear first in females and then in males (at intermediate temperatures). The primordial ovary is characterized by somatic cell outgrowths, which then will lead to the formation of the ovarian cavity, by the onset of meiosis in the primordial germ cells, and by the formation of blood vessels. On the other hand, the testis differentiation is characterized by the formation of rudiments of the main sperm duct in the medullary region of the gonad and formation of characteristic germ cell cysts in its periphery. The germ cells in the male gonad enter meiosis a few weeks later (Ito et al., 2005). 4 Juan I. Fernandino, Ricardo S. Hattori, Carlos A. Strüssmann et al.

Figure 1. Schematic representation of the gradient of histological sex differentiation in pejerrey gonads. The dotted arrows indicate the wave of differentiation. L: left; R: right.

An intriguing aspect of the gonadal differentiation in this species is the presence of a cephalo-caudal, left-to-right gradient of differentiation (Strüssmann and Ito, 2005). Regardless of sex, the left-anterior area is the region from where the onset of gonad differentiation begins toward the posterior part of the gonad. On the opposite side, the right gonad begins to differentiate only when 10-30% of the left side is already differentiated (Figure 1). It is also important to stress that blood vessels are formed prior to sex differentiation (Ito et al., 2005), suggesting that extra-gonadal factors may be carried by the bloodstream into the left-anterior region of the gonad to trigger the sex differentiation process. This mechanism is not well understood but one explanation is based on the rarity of intersex gonads not only in wild but especially in laboratory-reared larvae that were subjected to shifts between low-feminizing and high-masculinizing temperatures, which would be expected given the high thermal plasticity of sex in this species. According to this idea, this gradient might be important to prevent the occurrence of gonadal sex ambiguity and therefore the appearance of intersex animals with ovarian and testicular tissue in the same lobe or with a testis in one lobe and an ovary in the other (Strüssmann and Ito, 2005). Another feature of sex differentiation in pejerrey gonads is the occurrence of programmed cell death (apoptosis) linked to testicular differentiation. Thus, a preliminary analysis of apoptosis by the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay revealed that larvae reared at female-producing temperatures (FPT) had none to very few apoptotic cells whereas those from the male-producing temperatures (MPT) had widespread apoptosis in the anterior region of the right lobe between 4 or 5 and 10 wah. In larvae from mixed-sex-producing temperatures (MixPT), both patterns were observed and the proportion of animals with intense apoptosis correlated well with that of animals which differentiated as males (Strüssmann et al., 2008). Nevertheless the role of apoptosis Atherinopsid Fishes and Temperature-Dependent Sex Determination 5 during testis differentiation in pejerrey is still not clear. In the undifferentiated gonochorist zebrafish, Danio rerio, programmed cell death seems to have a clear role in the removal of oocyte-like cells in genotypic males and therefore in testis formation (Uchida et al., 2002; 2004). On the other hand, in medaka, increased apoptosis during differentiation has been shown to produce gonadal sex inversion from female to male (Kurokawa et al., 2007) and is associated with the regulation of the number of germ cells necessary for testicular development (Tanaka et al., 2008).The detection of apoptosis in the lobe with delayed differentiation (right side) and its occurrence mainly in somatic cells point out to a different mechanism or role in pejerrey testis differentiation. For example, as with the histological gradient of sex differentiation, the incidence of apoptosis in the anterior part of the right lobe could be also part of a mechanism to avoid gonadal ambiguity in larvae experiencing temperature changes during and immediately after the critical period of sex determination.

3. GENES INVOLVED IN GONADAL DIFFERENTIATION

Since the ´90s and with the identification of the major sex determining gene SRY in mammals (Sinclair et al., 1990) the interest in the study of the molecular processes involved in gonadogenesis increased rapidly not only in mammals but also in other vertebrates. Interestingly, although the sex determining mechanisms or genes might differ in different species, the downstream molecular pathways involved in the gonadal fate seem to be highly conserved across vertebrates (Barske and Capel, 2008). These genes include for example cyp19a1a or gonadal aromatase, dmrt1 and amh. This section presents an overview of the knowledge gathered so far on these genes in relation to the gonadal sex differentiation in pejerrey.

3.1. Gonadal Aromatase (cyp19a1a)

One of the most studied genes on the gonadal network is the cytochrome P450 aromatase enzyme, involved in the conversion of androgens into estrogens. This enzyme determines the balance between these two groups of steroids and it is essential for gonadal function in vertebrates (Carreau et al., 2002). Aromatase is also important at early stages of development; its expression and/or high activity have been associated with the process of ovarian differentiation in birds (Yoshida et al., 1996; Yang et al., 2008), reptiles (Gabriel et al., 2001; Pieau and Dorizzi, 2004) and also in teleost fish species (Guiguen et al., 2010). It is also important to highlight that there are two different genes that express aromatase enzymes in teleosts, the gonadal form or cyp19a1a, and the referred to as brain aromatase or cyp19a1b (Kishida and Callard, 2001; Jeng et al., 2012). The inhibition of the aromatase enzyme activity during the period of sex differentiation results in genotypic females developing as phenotypic males (Piferrer and Blázquez, 2005; Piferrer and Guiguen, 2008). These results emphasize the importance of the aromatase enzyme and estrogens as a pivotal role to the ovarian fate.

6 Juan I. Fernandino, Ricardo S. Hattori, Carlos A. Strüssmann et al.

In pejerrey, the expression of cyp19a1a was studied in relation to the gonadal sex differentiation process as well as to the rearing temperature. Gonadal aromatase showed to be over-expressed at FPT compared to MPT-reared larvae (Karube et al., 2007), whereas at MixPT some fish showed a female- and other a male-like expression pattern. The pharmacological inhibition of aromatase activity induced a male skewed sex ratio in this species (Fernandino et al., 2008a), confirming the importance of this gene on ovarian development.

3.2. Dmrt1

One of the genes initially characterized in the process of sex determination and gonadal differentiation was the transcription factor DMRT1 (Doublesex Male abnormal-3 Related Transcription Factor-1). This transcription factor contains a DNA binding motif of zinc finger (DM domain) and it was first identified in nematodes and fruit flies (Shen and Hodgkin, 1988; Burtis and Baker, 1989). This gene is considered a key factor in male gonadal development from invertebrates to humans (Ferguson-Smith, 2007; Herpin and Schartl, 2011). In humans, for example, deletion of a copy of DMRT1 is associated with male sex reversal in XY phenotypic females (Raymond et al., 1999a). Moreover, male DMRT1 null mice exhibit severe defects in postnatal growth of the testis, while females are normal and fertile (Raymond et al., 2000). In chickens, dmrt1 expression is higher in ZZ embryos (males) than in ZW (female) from the early stages of development of the genital ridge (Raymond et al., 1999b). A higher expression in testicles during gonadal development has been also described in reptiles (Sreenivasulu et al., 2002; Murdock and Wibbels, 2003; Valenzuela, 2010), amphibians (Shibata et al., 2002) and several teleost fish species (Marchand et al., 2000; He et al., 2003; Kobayashi et al., 2004; Guo et al., 2005; Raghuveer and Senthilkumaran, 2009; Cao et al., 2012; Masuyama et al., 2012). In pejerrey, dmrt1 showed a dimorphic expression pattern during early larval development displaying high expression at MPT, low at FPT and a bimodal expression at MixPT (Fernandino et al. 2008a). At this temperature regime, individuals with high dmrt1 expression and conversely low cyp19a1a expression are considered to be those differentiating males whereas the opposite pattern is taken as an indication of female differentiation (Fernandino et al., 2008a).

3.3. Amh

Another gene that has generated great interest is the AMH (anti-Müllerian hormone, also known as Müllerian inhibiting Substance or Mis). AMH is a glycoprotein which belongs to the TGF-β family of growth and differentiation factors (Josso et al., 2001). In amniotes AMH is responsible of the regression of the Müllerian duct in male embryos, the control and evolution of the genital tract, and the regulation of follicular development (Josso et al., 2006). Although teleost fish do not have Müllerian ducts (Wrobel, 2003), the expression of amh and its receptor (amhrII) have been detected in both sexes early in development during the gonadal differentiation period (Rodríguez-Marí et al., 2005; Nakamura et al., 2006; Klüver et al., 2007; Vizziano et al., 2007). The function of the amh/amhrII system was then established Atherinopsid Fishes and Temperature-Dependent Sex Determination 7 using a mutant for the receptor amhrII in medaka (hotei mutant), in which it was demonstrated that this system inhibits the proliferation of germ cells (Morinaga et al., 2007). Therefore, the male pathway regulates the germ cell proliferation in early stages of the developing gonad. In pejerrey the amh expression profile, together with its regulation by gonadal steroids and glucocorticoids, was analyzed during the gonadal sex differentiation period. Amh is first expressed at low levels in the undifferentiated gonads of both sexes but becomes dimorphic, with higher expression in putative male gonads compared to females, during the process of gonadal differentiation (Fernandino et al., 2008b; Hattori et al., 2008; Hattori et al., 2009). Thus, amh shows the opposite expression pattern compared to cyp19a1a. These studies also demonstrated that the onset of cyp19a1a expression at the MixPT preceded that of amh. Moreover, larvae treated with estradiol during the critical time of sex differentiation consistently show low amh expression levels similar to those found at the FPT (Fernandino et al., 2008b). On the other hand, cortisol administration caused masculinization of pejerrey larvae (see subsequent section) and it was associated with amh and cyp19a1a profiles resembling those at the MPT (Hattori et al., 2009).

4. THE ROLE OF CORTISOL AND ANDROGENS IN GONADAL MASCULINIZATION

In teleost fish the production of glucocorticoids (GCs) by the interrenal gland, located in the anterior kidney, is primarily regulated by the adrenocorticotropic hormone (ACTH), considered to be its main secretagogue (Mommsen et al., 1999). ACTH release in turn is regulated by the hypothalamic peptide corticotrophin releasing hormone (CRH). The mechanism of action of GCs involves their passage through the plasma membrane and subsequent binding to cytoplasmic receptors (GRs). The hormone-receptor complex is transported into the nucleus where it binds to a specific DNA segment, the glucocorticoid response site (GRE), resulting in additional production of mRNA and subsequent synthesis of specific proteins in the cell (Bury and Sturm, 2007). The main GC in teleost fish is cortisol, which plays an important role in the modulation of the adaptive intermediary metabolism (Vijayan et al., 1994), ionic regulation (Sakamoto and McCormick, 2006), and immune function (Salas-Leiton et al., 2012). Pejerrey reared at high temperatures eventually display signs of thermal stress. These include alterations in body shape, reduced swimming and feeding activity, changes in their pigmentation pattern, loss of weight and progressive germ cell degeneration (Ito et al., 2008). The thermal profile of the natural habitat of pejerrey (Gómez et al., 2007) also suggests that this species prefers cold to temperate waters and then high temperatures could act as a stress factor. Within this context, studies were conducted to critically asses the association between high temperature and stress (cortisol levels) during gonadal differentiation and masculinization in pejerrey. These studies showed that body cortisol levels during the critical period of sex determination were positively correlated with rearing temperature (Hattori et al., 2009). Also, pejerrey larvae reared at MixPT and treated with cortisol and dexamethasone, a cortisol agonist, showed significantly male-biased sex-ratios in relation to the control. It is 8 Juan I. Fernandino, Ricardo S. Hattori, Carlos A. Strüssmann et al. important to note that similar results were observed in the Japanese flounder Paralichthys olivaceus and in medaka, evidencing that GCs also play key role(s) in gonadal fate in other species (Hayashi et al., 2010; Yamaguchi et al., 2010). The exact mode of action of cortisol during pejerrey masculinization still needs to be scrutinized. It is well known that GCs are responsible for triggering the apoptotic cascade in mammals (Schmidt et al., 2004). In fact, treatment of pejerrey larvae with cortisol induced a significant increase in the incidence of apoptosis (Hattori et al., 2009), as observed in animals reared at the MPT (Strüssmann et al., 2008; Fernandino et al., 2011). Another possible pathway is the modulation of steroidogenesis. For instance, cortisol levels in pejerrey larvae were correlated with those of the main bioactive androgen in fish, 11-ketotestosterone (11- KT), and testosterone (T) and, cortisol administration caused inhibition of the expression of cyp19a1a (Hattori et al., 2009). Interestingly, relatively high levels of T and 11-KT were observed at MixPT even before the appearance of a sexually dimorphic (high/low) pattern of cyp19a1a expression. These results may have a bearing on the controversial issue of the involvement of 11-oxygenated androgens in fish gonadal sex determination/differentiation. Thus, it is currently believed that estrogens are essential for female sex differentiation whereas androgens are products or the consequence of testicular differentiation (Ijiri et al., 2008; Vizziano et al., 2008; Guiguen et al., 2010). However, high levels of 11-oxygenated androgens or the expression of enzymes involved in their synthesis have been detected during the critical period of sex determination/differentiation in some teleosts (Miura et al., 2008) and exposure to androgens during the early stages of fish development usually produces male-biased progeny (Devlin and Nagahama, 2002). The high 11-KT levels during the sex determination period of pejerrey, even before the first evidences of molecular and morphological evidences of gonadal differentiation (Ito et al., 2005; Fernandino et al., 2008a,b; Fernandino et al., 2011), suggests that testicular formation is the result of a proactive process and not merely the alternative outcome for ovarian differentiation (Figure 2). Taken together, these results are indications that GCs can act at earlier stages of gonadal differentiation and point to their strong effects on steroidogenesis at this critical period in life.

Figure 2. Proposed mechanism of the sex differentiation process in pejerrey Odontesthes bonariensis.

Atherinopsid Fishes and Temperature-Dependent Sex Determination 9

CONCLUSION

The fact that pejerrey sex determination shows a marked sensitiveness to temperature, such that all-female and all-male populations can be reliably produced at environmentally relevant temperatures, and the wealth of information on the morphological, endocrine, and molecular mechanisms involved in the determination/differentiation process make this species an excellent model to study the physiology of gonadal fate determination in fish. Looking ahead, the recent discovery of the amhy gene in the pejerrey genome (Hattori et al., 2012) may provide even further avenues to explore the interactions between the environment and genotypic factors on the canalization of gonadal development. Further studies on those processes can undoubtedly contribute to the clarification of the complex gonadal fate framework in fish and possibly in other vertebrates.

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