Ovarian aromatase and estrogens

General and Comparative Endocrinology 165 (2010) 352–366

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Ovarian aromatase and estrogens: A pivotal role for gonadal sex differentiation and sex change in fish Yann Guiguen a,*, Alexis Fostier a, Francesc Piferrer b, Ching-Fong Chang c a

INRA, UR1037 SCRIBE, IFR140, Ouest-Genopole, F-35000 Rennes, France Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), Passeig Marítim, 37-49, 08003 Barcelona, Spain c Department of Aquaculture, National Taiwan Ocean University, Keelung 202, Taiwan b

a r t i c l e

i n f o

Article history: Received 11 December 2008 Revised 23 February 2009 Accepted 3 March 2009 Available online 14 March 2009 Keywords: Aromatase Cyp19a1a Sex differentiation Sex change Fishes Gonads Estrogens

a b s t r a c t The present review focuses on the roles of estrogens and aromatase (Cyp19a1a), the enzyme needed for their synthesis, in fish gonadal sex differentiation. Based on the recent literature, we extend the already well accepted hypothesis of an implication of estrogens and Cyp19a1a in ovarian differentiation to a broader hypothesis that would place estrogens and Cyp19a1a in a pivotal position to control not only ovarian, but also testicular differentiation, in both gonochoristic and hermaphrodite fish species. This working hypothesis states that cyp19a1a up-regulation is needed not only for triggering but also for maintaining ovarian differentiation and that cyp19a1a down-regulation is the only necessary step for inducing a testicular differentiation pathway. When considering arguments for and against, most of the information available for fish supports this hypothesis since either suppression of cyp19a1a gene expression, inhibition of Cyp19a1a enzymatic activity, or blockage of estrogen receptivity are invariably associated with masculinization. This is also consistent with reports on normal gonadal differentiation, and steroid-modulated masculinization with either androgens, aromatase inhibitors or estrogen receptor antagonists, temperature-induced masculinization and protogynous sex change in hermaphrodite species. Concerning the regulation of fish cyp19a1a during gonadal differentiation, the transcription factor foxl2 has been characterized as an ovarian specific upstream regulator of a cyp19a1a promoter that would co-activate cyp19a1a expression, along with some additional partners such as nr5a1 (sf1) or cAMP. In contrast, upstream factors potentially down-regulating cyp19a1a during testicular differentiation are still hypothetical, such as the dmrt1 gene, but their definitive characterization as testicular repressors of cyp19a1a would strongly strengthen the hypothesis that early testicular differentiation would need active repression of cyp19a1a expression. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction Fishes show an exceptional range of reproductive strategies with regards to the expression of their sexuality (Devlin and Nagahama, 2002). Along with this diversity many different physiological regulations of gonadal sex differentiation or sex change have been demonstrated or suggested, including for instance the participation of the brain in a hermaphrodite’s sex change (Grober and Sunobe, 1996; Black et al., 2004), and of external factors like temperature in species having Environmental Sex Determination (ESD) (Reinboth, 1980; Baroiller and D’Cotta, 2001). Despite this diversity of sex determination and sex differentiation processes, there is at least one well conserved factor common to nearly all teleost fish in the control of ovarian differentiation, which is the implication of estrogens and the enzyme needed for their synthesis, i.e., the

*

Corresponding author. Fax: +33 2 23 48 50 20. E-mail address: [email protected] (Y. Guiguen).

0016-6480/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2009.03.002

gonadal aromatase (Cyp19a1a, i.e., cytochrome P450, family 19, subfamily A, polypeptide 1a). This review focuses on this unique aspect, including some of the most relevant and recent information on this subject. For more broader information on sex determination and sex differentiation, readers should Reference: to one of the recent and comprehensive reviews dealing with either fish sex determination (Nagahama, 2002; Devlin and Nagahama, 2002; Penman and Piferrer, 2008), sex differentiation (Baroiller et al., 1999; Guiguen, 2000; Baroiller and Guiguen, 2001; Strüssmann and Nakamura, 2002; Nagahama, 2002; Piferrer and Guiguen, 2008) sex change (Frisch, 2004; Nakamura, 2005) and endocrine disruption of sex differentiation (Jobling et al., 1998; Nagahama et al., 2004; Orlando and Guillette, 2007; Cheshenko et al., 2008). Estrogens have long been regarded as important hormones for ovarian differentiation in non-eutherian vertebrates. For instance, administration of estrogens can reverse phenotypic males to females in marsupials (Coveney et al., 2001), birds (Scheib, 1983),

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reptiles (Merchant-Larios et al., 1997) and teleosts (Piferrer, 2001; Kobayashi et al., 2003; Kobayashi and Iwamatsu, 2005). Treatments with aromatase inhibitor (AI), that block aromatase activity, also resulted in the production of phenotypic males from females in birds (Elbrecht and Smith, 1992; Hudson et al., 2005), reptiles (Wibbels and Crews, 1994; Belaid et al., 2001) and fish (see below). Thus, endogenous estrogens are now considered to be key steroids and aromatase a key enzyme for ovarian differentiation, at least in reptiles (Pieau and Dorizzi, 2004) birds (Villalpando et al., 2000) and fish. Fish aromatase genes have also been the subject of some recent reviews, but not focusing only on sex differentiation, and readers can refer to these reviews (Piferrer and Blázquez, 2005; Cheshenko et al., 2008). One important point that should be mentioned here is that aromatase is a duplicated gene in all investigated teleost fish (Callard and Tchoudakova, 1997; Tchoudakova and Callard, 1998; Ijiri et al., 2000; Chiang et al., 2001) except in the eels, which belong to the ancient group of Elopomorphs (Jeng et al., 2005) in which the duplicate might have been lost during the course of evolution (Cheshenko et al., 2008). Thus, in most teleost fish, this gene duplication gave rise to two different genes, namely cyp19a1a and cyp19a1b (this gene nomenclature has been chosen according to the approved international nomenclature given for zebrafish, Danio rerio by the zebrafish Model Organism Database at http://zfin.org/). The cyp19a1a gene is also called the ‘‘gonadal aromatase” or ‘‘ovarian aromatase” (also referred as P450aromA, cyp19a or cyp19a1) as this gene is mainly expressed in the differentiating and adult gonad (mainly the ovary) of teleost fishes. The cyp19a1b gene is called the ‘‘neural aromatase” or ‘‘brain aromatase” (also referred as P450aromB, cyp19b, or cyp19a2) as this gene is highly expressed in the teleost brain in both males and females (Patil and Gunasekera, 2008) but its sexually dimorphic brain expression during gonadal sexual differentiation has not been established (Kallivretaki et al., 2007). Besides, a second brain aromatase gene (cyp19a1bII) has been found in rainbow trout, Oncorhynchus mykiss, and could be also functional (Dalla Valle et al., 2005). As this review focuses on the implication of aromatase on gonadal sex differentiation we restricted our review of the literature to the work on the ovarian gene (cyp19a1a) that is by far the most studied with regards to sex differentiation of the gonads. However, in Japanese medaka, Oryzias latipes (Patil and Gunasekera, 2008), rainbow trout (Blázquez et al., 2008) and Nile tilapia, Oreochromis niloticus (Kwon et al., 2001; Chang et al., 2005), the expression of the brain gene, cyp19a1b, has been investigated during gonadal differentiation and was found to be expressed at low levels both in male and female gonads. Furthermore, in Nile tilapia and Japanese fugu, Takifugu rubipres (Sudhakumari et al., 2003, 2005; Rashid et al., 2007), the cyp19a1b gene was expressed in the early developing testis and also induced following female-to-male androgen-induced sex change in the tilapia. However, the implications of this early cyp19a1b expression in the differentiating testis are still not known.

2. Fish ovarian aromatase 2.1. Aromatase enzyme and estrogens in fish The aromatase enzyme is part of an enzymatic complex which includes the cytochrome P450 aromatase, the product of the cyp19a1 gene, and a NADPH-dependent cytochrome P450 reductase known as an ubiquitous flavoprotein (Simpson et al., 1994). Aromatase is a microsomal enzyme localized in the smooth endoplasmatic reticulum of steroidogenic cells. Interestingly, it has been stressed that its high affinity for the substrates facilitates estrogen synthesis even when androgens are not synthesized in high concentrations (Conley and Hinshelwood, 2001). In fish, only

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a few kinetic studies have been performed, and mostly in tissues potentially expressing both aromatase genes (cyp19a1a and cyp19a1b). The enzyme has more or less similar affinities for androstenedione and testosterone, and the Michaelis–Menten affinity constant (Km) is usually in the nanomolar range (> M at 60 dph differentiating at 60 dh (35 mm TL). Not studied cyp19a1a as above and correlated with foxl2. Fshr suppressed by HT by 50 dph F: 28 dpf F > M already at (20 mm) 18–26 dpf (MS) M: 55 dpf LT F > HT F > LT M > HT M (>40 mm) (MS) Not studied Not measured HT induced apoptosis. All oocytes disappeared by 29 dph Not studied

F: 35 dph (17 mm) IT and 52 dph (22 mm) LT. M: 52 dph (25 mm) IT and 35 dph (19 mm) HT M: 42 dph F: 49 dph

Not studied

Not measured

Reference Kitano et al. (1999)

Yamaguchi et al. (2007) D’Cotta el al. (2001)

Kwon et al. (2002) Uchida et al. (2004)

LT = HT at 260 ddph LT > HT at 600 and 1100 ddph HT: 0.25 IT: bimodal

van Nes and Andersen (2006)

IT: cyp19a1a first increased at 28 dph, one week earlier than amh. Bimodal cyp19a1a levels correlated with IT sex ratio LT = HT at 0–60 dph

Fernandino et al. (2008)

Karube et al. (2007)

Socorro et al. (2007)

Abbreviations and symbols: M, males; F, females; LT, low temperature; IT, intermediate temperature; HT, high temperature; dpf, days post-fertilization; dph, days posthatching; ddph, degree-days post hatching; TL, total length; When not accompanied by numbers, the symbols >, >> and >>> indicate that the reported level of statistical significance was P < 0.05, P < 0.01 and P < 0.001, respectively.

in females than in males. It was concluded that the follicle stimulating hormone (Fsh) signaling and foxl2 played an important role in the regulation of cyp19a1a in the Japanese flounder as well as in species where temperature is able to affect sex ratios (Yamaguchi et al., 2007), although the occurrence of available Fsh at these early stages of development remains to be further investigated (see below). In a mixed-sex population of Nile tilapia differences in cyp19a1a expression between sexes (females > males) were already evident before the histological differentiation of the ovary, which took place concomitantly with germ cells entering meiosis. Thereafter, cyp19a1a expression decreased after the differentiating stage (D’Cotta et al., 2001). In a monosex female population, high temperature decreased cyp19a1a expression both in females and in males, where levels were very low even at low temperature (D’Cotta et al., 2001). In another experiment with Nile tilapia, high temperature not only overrode genetic sex determination of XX females but also of YY males (Kwon et al., 2002). Treatment of genetic female Nile tilapia with an aromatase inhibitor (AI) at either low or high temperature resulted in masculinization. Unexpectedly, high temperature also resulted in the feminization of 35.5% of the genetic YY males, an effect that was completely suppressed by concomitant AI treatment (Kwon et al., 2002). The explanation for this outcome is not known, but because of their genotypic con-

stitution YY males may exhibit cyp19a1a responses unrelated to, or different from, those of normal males and females. All-female zebrafish embryos exposed to water temperatures of 28.5, 35 or 37 °C resulted in 100%, 31.2% and 0% females, respectively (Uchida et al., 2004), indicating a strong masculinizing effect of temperature in this species. Temperature, or treatment with an AI, induced oocyte apoptosis and differentiation of spermatogonia. This suggests that apoptosis may be part of the normal reorganization of the zebrafish gonad in future males, after the transient period in which all fish have an ovary-like structure. In the halibut, Hippoglossus hippoglossus, larvae were submitted from the onset of the first feeding to different temperature regimes at 7, 10, or 13 °C (van Nes and Andersen, 2006). The treatments ended after metamorphosis was completed. Thus, thermal treatment duration was inversely related to temperature to compensate for differences in growth rates. Although a small increase in the number of males was observed with increasing temperatures, differences from the 1:1 sex ratio were not statistically significant. However, cyp19a1a levels significantly increased in the fish exposed to 7 °C but not in those exposed to 10 or 13 °C and the trend continued until the end of the experiment (van Nes and Andersen, 2006). Thus, despite being a species with robust genetic sex determination, halibut is still able to respond to temperature with changes in cyp19a1a gene expres-

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sion in a manner similar to that of other species. In the pejerrey, Odontesthes bonariensis, an atherinid species with temperaturedependent sex determination, three temperatures, low (LT, 17– 18 °C), intermediate (IT, 24–25 °C) and high (HT, 28–29 °C) were assayed (Karube et al., 2007). They resulted in 0, 23–73 and 100% males, respectively. cyp19a1a mRNA levels increased before the first signs of histological differentiation of the ovaries, which took place around 35 dph in fish exposed to IT, and at 52 dph in fish exposed to LT. On the other hand, cyp19a1a was suppressed in the fish exposed to HT. In another study with the same species, it was shown that the number of fish with high levels of cyp19a1a at the IT correlated with the sex ratio observed after rearing at that temperature. Furthermore, the increase of cyp19a1a occurred not only prior to the first signs of histological sex differentiation but also one week before the increase in anti-Müllerian hormone (amh) expression, with an inverse relationship between cyp19a1a and amh levels (Fernandino et al., 2008). Effects of high temperature on cyp19a1a are evident only after a certain development stage has been reached. Socorro et al. (2007) measured cyp19a1a mRNA levels in European seabass larvae reared either at low (15 °C) or high (21 °C) temperature and found no differences in the period comprised between 0 and 60 dph. In a study of the expression of cyp19a1a during European seabass ontogenesis, it was found that the first significant differences between future males and females were not detected until 150 dph (Blázquez et al., 2008). Therefore, in this species cyp19a1a levels in the period 0–60 dph constitute basal levels that are unaffected by high temperature. Together, these results indicate that exposure to high temperature during the thermosensitive period suppresses cyp19a1a gene expression in these thermosensitive fish species, which presumably results in low aromatase activity and, in turn, low E2 levels. Thus, the lack of estrogen brought about by suppressed cyp19a1a gene expression and aromatase activity is responsible for the observed masculinization of genetic females when fish are exposed to high temperature. Further, apoptosis may be involved in the temperature effects on the gonads, at least in some species (Strüssmann et al., 1998; Ito et al., 2003). However, it seems unlikely that cyp19a1a is a direct target for temperature and thus the underlying mechanism connecting environmental temperature and cyp19a1a expression remains unclear, although recent evidence in the European seabass suggests that high temperature effects could be mediated by methylation of specific CpG sites in the cyp19a1a promoter (Piferrer et al., 2008). 3.3. Aromatase and sex change in hermaphrodite species Hermaphroditism in a species occurs when a substantial proportion of individuals in the population function as both sexes (Sadovy and Shapiro, 1987), either at the same time (synchronous hermaphrodite) or at a different times (sequential hermaphrodites) i.e., protogyny (in which some or all individuals function first as females and later as males) and protandry (sex change from male to female). Thus, hermaphrodite fishes form unique models to study sex differentiation and sex change. Different cues, varying from species to species, may induce sex changes, but the underlying physiology directing this process has received little attention. However, steroid production has been shown to mediate both the natural and induced sex change and has been thoroughly examined during gonadal sex change in many hermaphrodite species [for reviews see (Reinboth, 1982, 1983; Chan and Yeung, 1983)]. 3.3.1. The role of aromatase and estrogens in sex change In protandrous species, increasing serum E2 levels are often associated with male to female sex change, as in the Asian seabass,

Lates calcarifer (Guiguen et al., 1993), and the black porgy, Acanthopagrus schlegeli (Chang et al., 1994). In contrast, in protogynous hermaphrodites, E2 levels have been found to decrease during female to male sex change, as in rice-field eel (Yeung and Chan, 1987), saddleback wrasse, Thalassoma duperrey (Nakamura et al., 1989), stoplight parrotfish Sparisoma viride (Cardwell and Liley, 1991), black seabass, Centropristis striatus (Cochran and Grier, 1991), different grouper species (Shapiro et al., 1989; Johnson et al., 1998; Bhandari et al., 2005; Li et al., 2006, 2007; Nakamura et al., 2007), Mediterranean red porgy, Pagrus pagrus (Kokokiris et al., 2006), gilthead seabream (Wong et al., 2006) and blackeye goby, Coryphopterus nicholsii (Kroon and Liley, 2000). Expression of cyp19a1a has been recorded during natural sex change in hermaphrodite species and cyp19a1a is induced during protandrous sex change, as in the black porgy (Chang et al., 1995a) and decreases in transitional gonads undergoing sex change in protogynous species, as in rice-field eel (Liu et al., in press), gilthead seabream (Wong et al., 2006) and some groupers (Zhang et al., 2004; Li et al., 2006). Inhibition of estrogen production via AI treatments resulted in complete sex change in the three-spot wrasse, Halichoeres trimaculatus, and treatment with both AI and E2 prevented sex change in that species (Higa et al., 2003). Similar induction of functional sex change using AI treatments has also been reported in other protogynous species including groupers (Bhandari et al., 2003, 2004a,b; Li et al., 2005; Alam et al., 2006; Alam and Nakamura, 2007), and blackeye goby (Kroon et al., 2005). In contrast, estrogen treatments have been shown to induce male to female sex change in protandrous hermaphrodites, as in the Asian seabass (Anderson and Forrester, 2001). In the protogynous three-spot wrasse, estrogens have also been shown to induce sex change of initial phase adult males (i.e., males that differentiated first as males), showing that these males keep the potential to change sex into functional females and that estrogens can still trigger this process (Kojima et al., 2008). In the three-spotted wrasse, female to male sex change can be obtained spontaneously in vitro in the absence of estrogen supplementation (Todo et al., 2008). In the coral goby, Gobiodon erythrospilus, that naturally exhibits bidirectional sex change, experimental modulations of E2 levels can induce sex change either from male to female through E2 supplementation or through female to male through Cyp19a1a inhibition (Kroon et al., 2005). In the protandrous Asian seabass, estrogen synthesis potentialities were found to increase in transitional gonads during sex change (Guiguen et al., 1995). Treatment with AI in red-spotted grouper, Epinephelus akaraa inhibits cyp19a1a expression and Cyp19a1a enzyme activity, resulting in the decrease in the biosynthesis of endogenous estrogens and an induction of protogynous sex change (Li et al., 2006, 2007). Treatment with androgens (11-ketotestosterone or 17a-methyltestosterone) induced sex change in honeycomb grouper, Epinephelus merra (Bhandari et al., 2006a) and orange-spotted grouper, Epinephelus coioides (Yeh et al., 2003b) and these androgens may act through the inhibition of cyp19a1a gene expression and subsequent estrogen synthesis, as E2 levels were found to decrease during these androgen-induced female to male sex changes (Yeh et al., 2003a; Bhandari et al., 2006a). Altogether, these data suggest that Cyp19a1a and endogenous estrogens play a crucial role in natural sex change as their decrease triggers protogynous sex change and their increase triggers protandrous sex change. 3.3.2. The case of the protandrous black porgy Black porgy is a marine sparid fish and a protandrous monogynous hermaphrodite characterized by the presence of bisexual gonads (ovotestis), with only one type of female (monogynous i.e., secondary females are only derived from male sex change), and a natural male to female sex change occurring at around 2–3 years old. In black porgy, plasma E2 is maintained at low levels before

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sex change (Chang et al., 1994; Lee et al., 2008), and high plasma E2 levels are correlated with natural sex change in 2–3 years old fish during late pre-spawning and spawning periods (Chang et al., 1994). The cyp19a1a transcript levels and Cyp19a1a protein (Fig. 3) are significantly higher in undifferentiated gonads as compared to early male differentiated gonads in black porgy (Wu et al., 2008a; Wu et al., 2008b). In the bisexual gonads of 1 year old fish low expressions of the Cyp19a1a protein are detected both in the testis and ovary (Fig. 3), but during natural sex change cyp19a1a expression increases in the ovarian part of the ovotestis (Fig. 3). Ovarian cyp19a1a transcripts remain low during the non-spawning and pre-spawning seasons even though the ovarian tissue develops (Lee et al., 2008; Wu et al., 2008a,b). However, during the period of sex change, ovarian cyp19a1a transcripts significantly increase in fish undergoing sex change (Wu et al., 2008a,b). Exogenous E2 (4–6 mg/kg of food) successfully induces male to female sex change in 0–1 year old black porgy (Chang et al., 1995b; Chang and Lin, 1998) by triggering testicular tissue regression in the bisexual gonads, together with an increase in plasma E2 levels

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and gonadal aromatase activity (Chang and Lin, 1998; Lee et al., 2000, 2001; Wu et al., 2008b). The E2-induced females developed full ovaries with only primary oocytes but without vitellogenic oocytes. Longer E2 administration for at least 5 months in 1 year old male fish resulted in sex change, with the development of vitellogenic oocytes in the female part of the gonad (Chang and Lin, 1998). Finally, AI-administration is able to block natural sex change and results in an all-male population at 3 years old (Lee et al., 2002). These data indicate that Cyp19a1a and E2 play an important role both in natural and controlled sex change in the black porgy.

4. Ovarian aromatase gene regulation during sex differentiation The gene cascade leading to the female specific expression of cyp19a1a, and the subsequent feminization of the indifferent gonads, has been poorly investigated in fish (see Fig. 4 for a scheme of the potential regulations of fish cyp19a1a). Among the putative

Fig. 3. Immunohistochemical staining against aromatase (Cyp19a1a) antiserum in the gonad of protandrous black porgy during sex differentiation and sex change. Left Panel: (a–c) Different ages (months) of juvenile fish (a = 3 months, b = 4.5 months, c = 6 months) (Wu et al., 2008b). Fish gonad differentiated at 5 months age. Stronger staining in 3 months (undifferentiated) and 4.5 months gonad (differentiating) than in 6 months gonad (differentiated). Middle Panel: (d–f) Bisexual gonad in 1 year old fish. (d) Faint staining in bisexual gonad with ovarian tissue and testicular tissue separated by connective tissue in May (non-spawning period). (e) Ovarian tissue in bisexual gonad with primary oocytes and oogonia in May (non-spawning period). Follicle cells around primary oocytes could be stained. (f) Testicular tissue in bisexual gonad in October (prespawning period). Interstitial cells could be stained. Right Panel: Staining in the various stages of ovarian tissue during natural sex change in 2–3 years old fish. (g) Early primary oocyte in May (post-spawning period). (h) Late primary oocyte in October (non-spawning period). (i) Early vitellogenic oocyte in sex-changing fish in November (prespawning period). CT, connective tissue; EG, early oogonia; Fc, follicular cell; Ic, interstitial cell; OG, oogonia; OT, ovarian tissue; PO, primary oocyte; Sc, somatic cells; SC, spermatocyte; SG, spermatogonia; TT, testicular tissue; VO, vitellogenic oocyte.

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Fig. 4. Schematic representation of the known (solid arrows) or suggested (dashed arrows) regulations of fish cyp19a1a during gonadal differentiation in females (upper part of the figure, $) or males (lower part of the figure, #). Dotted line arrows indicate an absence of positive regulation due to a decrease in the upstream regulators needed at these steps. + indicates positive regulation,  indicates negative regulation.

upstream regulators of fish cyp19a1a, Foxl2 is of special interest as it is one of the very few conserved ovarian differentiation genes across vertebrate evolution, with a female-specific expression in the differentiating ovary of mammals (Baron et al., 2005a), birds (Govoroun et al., 2004), and fish (Baron et al., 2004; Nakamoto et al., 2006; Wang et al., 2007). In rainbow trout and Nile tilapia, this gene is temporally co-expressed with cyp19a1a during the initial steps of ovarian differentiation (Vizziano et al., 2007; Wang et al., 2007; Ijiri et al., 2008), and in Japanese medaka, Japanese flounder and Nile tilapia, its expression is co-localized with cyp19a1a in some somatic cells of the differentiating ovaries (Nakamoto et al., 2006; Yamaguchi et al., 2007; Wang et al., 2007). In rainbow trout, estrogens strongly and quickly up-regulate both foxl2a and its divergent paralog, foxl2b (Baron et al., 2004; Vizziano-Cantonnet et al., 2008). The fact that Foxl2 is able to up-regulate Cyp19a1 in both mammals and fish (Pannetier et al., 2006; Yamaguchi et al., 2007; Wang et al., 2007), and that estrogens up-regulate foxl2 in fish (Baron et al., 2004; Wang et al., 2007), suggests a positive feedback loop regulating these two genes. This positive loop would explain the important sexually dimorphic expression of these genes during early gonad differentiation in fish (Vizziano et al., 2007). However, at least in rainbow trout, the estrogen up-regulation of foxl2 is not able by itself to restore cyp19a1a expression, suggesting either that foxl2 up-regulation is not able to counterbalance the estrogen inhibition of the cyp19a1a gene and/or that foxl2 needs an additional partner to be able to induce cyp19a1a expression (Vizziano-Cantonnet et al., 2008). Such a co-activation has been demonstrated in Nile tilapia in which the interaction of Nr5a1 (Sf1) with Foxl2 regulates cyp19a1a in a sexspecific manner (Wang et al., 2007). In both Japanese medaka and rainbow trout, Nr5a1 (Sf1) has also been demonstrated as a positive upstream regulator of cyp19a1a (Watanabe et al., 1999; Kanda et al., 2006). The fact that, in rainbow trout, nr5a1 is down-regulated both by estrogens (Vizziano-Cantonnet et al., 2008) and androgens (Vizziano et al., 2008; Baron et al., 2008) fits in well with this hypothesis. However, even if the regulatory loop between foxl2a and cyp19a1a is impaired by the estrogen treatment in rainbow trout, the estrogenic signal provided by the exogenous steroid treatment is still sufficient to induce the female pathway. In this context, it is interesting to note that after the treatment when gonads are feminized by estrogens, cyp19a1a is slowly restored, supporting the idea that cyp19a1a up-regulation may be inhibited by the exogenous estrogen treatment.

The Nuclear receptor Dax1 (Nr0b1) is another transcription factor that has been identified as a potential regulator of Cyp19a1, through its repressive action on the Nr5a1-mediated transactivation of the Cyp19a1 promoter (Wang et al., 2001). In fish, Nr0b1 has also been found to down-regulate both nr5a1- and foxl2-mediated cyp19a1a expression in Japanese medaka ovarian follicles (Nakamoto et al., 2007). In rainbow trout, the up-regulation of nr0b1, either by estrogens or androgens, is always associated with a simultaneous down-regulation of both nr5a1 and cyp19a1a (Vizziano et al., 2008; Baron et al., 2008; Vizziano-Cantonnet et al., 2008), suggesting a similar down-regulation of cyp19a1a by a Nr5a1-mediated transactivation of the Cyp19a1 promoter (Wang et al., 2001). In the protandrous black porgy, a similar correlation between these three genes was also found with low nr5a1 and high nr0b1 transcripts found in the inactive ovarian part of the ovotestis before sex change, and during natural sex change ovarian cyp19a1a expression increases along with an increase of nr5a1 and a nr0b1 decrease (Wu et al., 2008a). Based on the existence of conserved cAMP responsive elements in the promoter of teleost fish cyp19a1a gene, the implication of cAMP on the regulation of the cyp19a1a promoter was investigated in vitro using a Japanese flounder cyp19a1a construction. cAMP was found to dose-dependently induce the cyp19a1a promoter. As cyp19a1a expression is known to be controlled by gonadotropins, at least in adult ovaries (Kagawa et al., 2003; Montserrat et al., 2004; Wong et al., 2006), it was then suggested that Fsh may play an important in the cyp19a1a control during ovarian differentiation through the synthesis of cAMP second messenger (Yamaguchi et al., 2007). In contrast, the luteinizing hormone (Lh) does not seem to be involved in E2 regulation during early sex differentiation since in vitro E2 secretion by developing ovaries was found to be stimulated by partially purified Lh only after sex differentiation in rainbow trout (Fitzpatrick et al., 1993). Besides, similar low Lh levels were found in male and female blood during the early sex differentiation period of seabass by Moles and colleagues (Moles et al., 2005). In the same work, Fsh subunit beta mRNA (fshb) were detected in both female and male pituitary during this period. Besides, pituitary cells immunoreactive for Fsh have been detected just before and during the sex differentiation period in rainbow trout (Saga et al., 1993; Feist and Schreck, 1996), platyfish, Xiphophorus maculatus (Magliulo-Cepriano et al., 1994), pejerrey (Miranda et al., 2001), the cichlid fish Cichlasoma dimerus (Pandolfi et al., 2006), and mummichog, Fundulus heteroclitus, (Shimizu et al.,

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2008). However, the actual release of Fsh by the embryonic pituitary and its availability for the embryonic gonad remain poorly known. Thus, the over-expression of fshb in the differentiating ovary is worth to be considered as a possible paracrine regulator (Baron et al., 2005b).

5. Hypothesis on aromatase and gonadal differentiation 5.1. Maintenance of somatic cells and ovarian differentiation is only supported by sustained high estrogen levels (an auto-regulation loop) Cyp19a1 is classically described as a somatic cell enzyme in all vertebrates studied to date (Kanamori et al., 1988; Petrino et al., 1989). Maintenance of Cyp19a1 expression and estrogen production in the gonad is then directly linked with maintenance of the differentiation status of these somatic cells. Factors potentially affecting this differentiation state may then in turn affect the production of estrogens and the ovarian differentiation. Studies in knock-out mice for the estrogen receptors (ERabKO) or Cyp19a1 (ArKO) also reveal that, in mice, estrogens are needed to maintain this ovarian somatic cell differentiation (Britt et al., 2001, 2002; Britt and Findlay, 2003). Thus, sustained high estrogen levels may be a conserved feature across vertebrate evolution for maintaining ovarian differentiation. It may then be hypothesized that in fish any de-differentiation of the Cyp19a1a-producing cells would in turn have a negative impact on their ability to produce estrogens. As these estrogens are needed in order to maintain their differentiation, this may produce a negative feedback loop that will further enhance this de-differentiation. Metabolites of E2, like catecholestrogens could also be involved in this negative feedback (Chourasia and Joy, 2008). Recent experiments in fish showing that masculinization can be obtained by AI treatment of already differentiated females (Nakamura et al., 2003; Bhandari et al., 2006c; Ogawa et al., 2008) clearly illustrate the crucial role of a sustained high estrogen synthesis in maintaining ovarian differentiation (or preventing a spontaneous testicular differentiation). 5.2. Germ cell proliferation, gonadal estrogen production and ovarian differentiation may be tightly linked Experimental germ cell depletion during the early steps of gonadal differentiation has recently been shown to produce masculinization of gonads in Japanese medaka genetic females (Shinomiya et al., 2001; Kurokawa et al., 2007) and also in zebrafish (Slanchev et al., 2005; Siegfried and Nüsslein-Volhard, 2008). These masculinizing effects are supposed to be in relation to the intimate cross-talk between germ cells and somatic cells during the early sex differentiation steps of the gonad (Tanaka et al., 2008). From these observations it has been suggested that somatic cells in the gonad would autonomously activate a male phenotype, and that germ cells would allow the activation of the female phenotype by sending signal(s) to both repress male predisposition and maintain feminization (Tanaka et al., 2008). This assumption fits well with the above discussion suggesting that ovarian somatic cells need to maintain their fully differentiated phenotype in order to produce estrogens that in turn will support their differentiation. Any disruption of this germ cell/somatic cell cross-talk would then disrupt ovarian somatic cell differentiation, leading to an absence of estrogen synthesis and a subsequent masculinization. But more subtle regulations involving not only the complete loss of germ cells, but instead some differential germ cell proliferation rates, could then be also suspected as important triggers of gonadal sex differentiation (see Fig. 5). These germ cell proliferation rates could eventually be affected by endogenous estrogen levels as suggested by some

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authors (Pannetier et al., 2006). In this respect, the bioavailability of active E2 from the hydrolysis of its sulfate counterpart would also be worth considering (Reed et al., 2005). In agreement with the hypothesis that germ cell proliferation could affect gonadal differentiation, Kurokawa and collaborators (2007) point to the fact that this germinal/somatic cell cross-talk could also be involved in the natural gonadal differentiation of zebrafish as germ cell apoptosis always precedes testicular differentiation in this species (Uchida et al., 2002, 2004). In this respect, it is interesting to note that proliferation of germ cells has been often found to be more active during ovarian differentiation, than during testicular differentiation (Lebrun et al., 1982; Hano et al., 2005; Saito et al., 2007; Lewis et al., 2008) and that masculinizing high temperatures in thermosensitive fish species (OspinaÁlvarez and Piferrer, 2008) have an opposite effect by stimulating germ cell apoptosis (Strüssmann et al., 1998; Ito et al., 2003). It is also quite intriguing to see that the Japanese medaka sex determining gene, dmrt1Y (dmy) (Matsuda et al., 2002), has been found to be an inhibitor of germ cell proliferation (PaulPrasanth et al., 2006; Herpin et al., 2007) and is only expressed during testicular differentiation (Kobayashi et al., 2004). In the same context, the Japanese medaka hotei mutation affecting the anti-Müllerian hormone receptor (amhr2) leads to an increase in germ cell number and some male to female sex reversions (Morinaga et al., 2007). However, and apparently in contradiction with these results, anti-Müllerian hormone (amh) has also been found to be required for proliferation of germ cells in the same species (Shiraishi et al., 2007). Another tgfb member, the gonadal soma-derived growth factor (gsdf), was also described in rainbow trout as a potential stimulator of primordial germ cell and spermatogonia proliferation (Sawatari et al., 2007). However, like amh, gsdf is also expressed in both sexes, albeit at much higher levels during testicular differentiation (Vizziano et al., 2007; Baron et al., 2007; Lareyre et al., 2008). Thus, any sex specific effect of these tgfb members on germ cell proliferation should either be a function of their local protein abundance or of their concerted action with additional partners. For instance, follistatin (fst) has been found to be expressed at much higher levels during ovarian differentiation in rainbow trout (Baron et al., 2005b; Vizziano et al., 2007), and is also known to maintain germ cell survival in the mouse ovary (Yao et al., 2004). Thus any over-expression in the female gonads of

Fig. 5. A hypothesis linking germ cell proliferation with Cyp19a1a and estrogens maintenance of ovarian differentiation. Stimulation of germ cell proliferation (or anti-apoptotic action) would sustain the differentiation of somatic steroid-producing cell resulting in higher steroid and estrogen production and ovarian differentiation. Germ cell apoptosis, for instance induced by masculinizing high temperature (t°) regimes in TSD fish species, or inhibition of germ cell proliferation would have an opposite effect, resulting in less estrogen production and testicular differentiation.

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positive regulators of germ cell proliferation or anti-apoptotic germ cell factors could also lead to the same effects on germ cell numbers, and in turn on gonadal differentiation (i.e., promote ovarian differentiation). The importance of estrogens in these proliferative or apoptotic regulations within the differentiating gonad should now be worth considering.

tiation and protandrous sex change (positive regulation hypothesis), or by preventing testicular differentiation and protogynous sex change (negative regulation hypothesis), or by doing both at the same time.

6. Conclusions

The authors would like to acknowledge Chantal Cauty and Ahmad Gharaei for their help with trout cyp19a1a in situ hybridization and Miranda Maybank for English improvement of the manuscript.

From the above review of the literature, it is now evident that the cyp19a1a gene, Cyp19a1a enzyme and estrogens are pivotal for the regulation of gonadal sex differentiation and sex change in most teleost fishes. However, despite the quite high number of studies that support this pivotal role of Cyp19a1a and estrogens, especially concerning the control of ovarian differentiation in fish, there are still some results indicating that this is not an absolute rule. For instance, in the Japanese medaka, expression of cyp19a1a has not been detected in the differentiating ovary but only later on at the onset of ovarian gametogenesis (Suzuki et al., 2004), and some AI and estrogen antagonist treatments failed to induce functional masculinization, leading to the hypothesis that ovary formation in the Japanese medaka is not dependent on an endogenous estrogen synthesis (Kawahara and Yamashita, 2000). In the channel and blue catfish, Ictalurus punctatus and I. furcatus, some cases of unexplained paradoxical feminizing effects of non-aromatisable androgens have been reported (Davis et al., 1990, 1992) and they are hardly explicable only by the key role of Cyp19a1a. In the protandrous black porgy it has also been reported that the long-term administration of low doses of E2 clearly enhanced testicular development, as revealed by the growth of large and active testis, together with production of high levels of plasma 11ketotestosterone (Chang et al., 1995b; Wu et al., 2008b), suggesting that aromatase expression is important and necessary for testicular development and also possibly for testicular differentiation in this species. Many questions still remain to be answered, like for instance, the identification of upstream physiological inhibitors of cyp19a1a expression in males that have still not been formally identified to date. The new discovery that doublesex and mab-3 related transcription factor 1 (dmrt1) may be needed for the down-regulation of cyp19a1a during testicular differentiation in the Nile tilapia (Wang and Nagahama, 2008) is one additional indication that estrogens may actually be needed to repress male differentiation. This inhibition of the synthesis of the inhibitor (estrogen) would then allow testicular differentiation to proceed (Fig. 6). To fully support this hypothesis more work is needed on estrogens and other potentially active metabolites of estrogens, the mode of mediation of their effects and their downstream targets during gonadal differentiation in teleost species. This may then in turn shed new light on whether estrogens act by promoting ovarian differen-

Ovarian phenotype

Female genotype

cyp19a1a Male genotype

Estrogens

Testicular phenotype

Fig. 6. A working hypothesis on a pivotal role for cyp19a1a gene fish sex differentiation. Under a female genotype, the cyp19a1a gene would be up-regulated in the differentiating gonad, resulting in the production of the aromatase enzyme and of estrogens that will either induce the ovarian phenotype or repress the testicular phenotype (or both). In contrast, the male genotype, by triggering an early testicular differentiation pathway, would block the expression of cyp19a1a. This blockage would prevent any endogenous estrogen production in the differentiating gonads, inducing in turn a testicular phenotype.

Acknowledgments

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