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BOR - Papers in Press, published online ahead of print October 17, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.064246
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BIOLOGY OF REPRODUCTION 78, 333–341 (2008)
DOI: 10.1095/biolreprod.107.064246
© 2008 by the Society for the Study of Reproduction, Inc.

Gamete Biology; 

Sexual Dimorphic Expression of Genes in Gonads During Early Differentiation of a Teleost Fish, the Nile Tilapia Oreochromis niloticus1

Shigeho Ijiri 3 4, Hiroyo Kaneko 3 4, Tohru Kobayashi 3 5, De-Shou Wang 3 4 6, Fumie Sakai 3 4, Bindhu Paul-Prasanth 3 4, Masaru Nakamura 7, and Yoshitaka Nagahama 2 3 4

Laboratory of Reproductive Biology,3 National Institute for Basic Biology, Okazaki 444-8585, Japan SORST,4 Kawaguchi, Saitama 332-0012, Japan National Research Institute of Aquaculture,5 Tamaki, Mie 519-0423, Japan School of Life Sciences,6 Southwest University, 400715 Chongqing, People's Republic of China Sesoko Station,7 Tropical Biosphere Research Center, University of the Ryukyus, Motobu-cho, Okinawa 905-0227, Japan

ABSTRACT

The Nile tilapia, a gonochoristic teleost fish with an XX/XY sex-determining system, provides an excellent model for studying gonadal sex differentiation because genetic all-females and all-males are available. In this study, we used quantitative real-time RT-PCR to determine the precise timing of the gonadal expression of 17 genes thought to be associated with gonadal sex differentiation in vertebrates. Gonads were isolated from all-female and all-male tilapia before (5–15 days after hatching [dah]) and after (25–70 dah) morphological sex differentiation. The transcript of aromatase (cyp19a1a), an enzyme responsible for producing estradiol-17beta, was expressed only in XX gonads at 5 dah, with a marked elevation in expression thereafter. In contrast, mRNA expression of steroid 11beta-hydroxylase (cyp11b2), an enzyme responsible for the synthesis of 11-ketotestosterone (11-KT, a potent androgen in fish), was found in XY gonads from 35 dah only. These results, combined with the presence of transcripts for other steroidogenic enzymes and estrogen receptors in XX gonads at 5–7 dah, are consistent with our earlier suggestion that estradiol-17beta plays a critical role in ovarian differentiation in tilapia, whereas a role for 11-KT in testicular differentiation is questionable. A close relationship between the expression of foxl2, but not nr5a1 (Ad4BP/SF-1), and that of cyp19a1a in XX gonads suggests an important role for Foxl2 in the transcriptional regulation of cyp19a1a. Dmrt1 exhibited a male-specific expression in XY gonads from 6 dah onward, suggesting an important role for Dmrt1 in testicular differentiation. Sox9 and amh (anti-Mullerian hormone) showed a testis-specific expression, being evident only in the later stages of testicular differentiation. It is concluded that the sex-specific expression of foxl2 and cyp19a1a in XX gonads and dmrt1 in XY gonads during early gonadal differentiation (5–6 dah) is critical for undifferentiated gonads to differentiate into either the ovary or testis in the Nile tilapia.

aromatase, DMRT1, fish, Foxl2, sex differentiation

INTRODUCTION

Sex determination, unlike many developmental processes, is characterized by a lack of conservation throughout the vertebrates. Although two sex-determining genes, SRY/Sry [1] and dmy [2, 3] have been identified in mammals and a teleost fish, the medaka Oryzias latipes, respectively, their gene structures are entirely different. Furthermore, dmy was found to be present only in two of more than 20 closely related species of medaka. Thus, the mechanisms by which sex is determined are extremely diverse in vertebrates. In contrast, factors operating during gonadal sex differentiation appear to be relatively conserved. For example, Sox9 has been implicated in testicular differentiation in mammals as one of the immediate gene products after SRY [4]. It has also been implicated in testicular development in birds [5, 6]. Dmrt1 and amh reportedly are involved in testicular differentiation in several vertebrate species [7–10]. Another good example of a conserved mechanism involved in sex differentiation is the important role of estrogens in ovarian differentiation in nonmammalian vertebrates including fish [11, 12], amphibians [13, 14], reptiles [15], and birds [9]. In contrast, there has been a controversy regarding the role of androgen or steroid receptors in the regulation of gonadal sex differentiation. However, the roles of these genes and factors in early gonadal sex differentiation do not appear to always be the same among different vertebrate groups. For example, in eutherian mammals, estrogens do not play an important role in early ovarian differentiation. It has also been reported that sox9 showed relatively strong expression at equivalent levels in both male and female gonads during the early sex differentiation of fish [16]. Thus, it is still too early to make any conclusions regarding the conserved and divergent mechanisms of sex determination and gonadal sex differentiation in vertebrates.

The Nile tilapia Oreochromis niloticus is a gonochoristic teleost with a stable XX/XY sex-determination system. In this fish, all-female (XX) or all-male (XY) broods have been obtained by artificial fertilization of normal eggs (XX) and sex-reversed male sperm (XX), or normal eggs (XX) and super male sperm (YY), respectively. On the day of hatching (it takes approximately 4 days for the eggs to hatch at 26°C), primordial germ cells (PGCs), which are morphologically distinguishable from somatic cells, are located in the outer layer of the lateral plate mesoderm around the hind gut. At 3 days after hatching (dah), PGCs are located in the gonadal anlagen after the formation of the coelomic cavity in the lateral plate mesoderm, rather than actively migrating. Meiosis of germ cells begins 25–30 dah in XX gonads, but not before sex differentiation in XY gonads [17]. Under the light microscope, the first signs of gonadal sex differentiation appear in tilapia fry at 23–26 dah with the formation of the ovarian cavity in the XX gonad or the efferent duct in the XY gonad [18].

In this study, we determined the sex specificity and the precise timing of the expression of genes thought to be associated with gonadal sex differentiation in vertebrates, in XX and XY gonads during gonadal sex differentiation and development in tilapia, especially during the critical period of sex differentiation (5–10 dah). For this purpose, we have developed a dissection method to isolate undifferentiated gonads of tilapia from 5 dah onward and, the mRNA levels of 17 genes were determined using quantitative real-time RT-PCR. The genes examined include those encoding various factors shown to be involved in gonadal sex differentiation in vertebrates. The results of this study indicate that the differential expression of genes occurring in XX and XY gonads during the period 5–6 dah is critical for undifferentiated gonads to differentiate into either the ovary or testis in the Nile tilapia.

MATERIALS AND METHODS

Animals and Sampling

Nile tilapia (O. niloticus) were reared in fresh water (26°C) under a 14L:10D photoperiod in indoor tanks (500 L) for more than 1 yr until matured. The fish were fed commercial trout pellets ad libitum. Three matured females (XX) were kept in a 500-L tank with partitions so as to rear them separately under the same conditions. Under these conditions, females spawn repeatedly every 14–18 days. Eggs were stripped on the day of spawning and fertilized by the usual drying method. To obtain all-female (XX) or all-male (XY) populations, milt stripped from a sex-reversed male (XX) or a super male (YY), respectively, was used for artificial fertilization as described elsewhere [17]. Fertilized eggs cultured in a round-bottom glass tube (50 ml) were kept rotating by injecting circulating water at 26°C. Fry hatched at 4 days after the fertilization. At 5 dah, fry were transferred to a 40-L aquarium tank and fed commercial food for fish larvae. Sampling of gonads was carried out at 5, 6, 7, 10, 15, 25, 35, and 70 dah. The number killed from both sex groups is as follows: for each sampling point between 5 and 15 dah, 30–50 larvae; at 25 dah, at least 15 fry; at 35 dah, at least 7 fry; and at 70 dah, 3–5 fry. Fish were dissected and viscera were removed under a stereoscopic microscope. RNAlater reagent (Ambion, TX) was poured on the coelomic epithelium to stabilize the RNA in the gonads, and then the gonads were removed using fine forceps. A number of gonads were pooled in a tube with RNAlater reagent and stored at –80°C until used for RNA extraction. Sampling was repeated at least three times at each sampling point. All animal husbandry and experimentation was conducted in accordance with our Guide for Care and Use of Laboratory Animals and was approved by the Institutional Committee of Laboratory Animal Experimentation (National Institute for Basic Biology).

Histology

The body samples of tilapia fry were immersed in Bouin solution (Wako Pure Chemical Industries, Japan), dehydrated, and embedded in paraffin or Technovit resin (Kulzer, Germany). For light microscopy, 4-µm-thick (paraffin) or 2-µm-thick (resin) sections were cut and the paraffin or resin sections were stained with hematoxylin-eosin (Wako Pure Chemical Industries) or a 1% solution of toluidine blue (Wako Pure Chemical Industries), respectively.

Primer Design

All target genes examined in the present study were isolated previously by our group. The primer sets for quantitative real-time RT-PCR were designed using Primer Express software 2.0 (Applied Biosystems, CA). Although total RNA was treated with DNase, each primer set was chosen with one primer flanking an intron-exon boundary to prevent genomic amplification, except for foxl2, which is intronless. The primer sets are listed in Table 1.


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  		TABLE 1 Primer sets used for quantitative real-time RT-PCR.

RNA Extraction and One-Step Real-Time RT-PCR

Total RNA was extracted using a column-based RNA extraction kit specialized for small quantities of RNA (RNAeasy Micro Kit; Qiagen, The Netherlands). DNase treatment was also carried out on the column according to the manufacturer's instructions. RNA sample (1 µl) was used for quantification with a NanoDrop spectrometer (NanoDrop Technologies, DE). Quantified RNA samples were used for quantitative RT-PCR with a one-step RT-PCR system. Real-time RT-PCR was carried out on an ABI Prism7000 (Applied Biosystems). Reactions were performed in 20 µl including 10 ng of total RNA using a one-step real-time RT-PCR kit according to the manufacturer's instructions (SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit; Invitrogen, CA). A melting-curve analysis was performed for each sample in order to check the single amplification. Data from real-time RT-PCR were expressed as the mean ± SEM of at least three independent samples.

Statistics

Significant differences in the data within a group (XX or XY) were tested by ANOVA with post-hoc test (Tukey/Kramer). Simple linear regression analyses were also employed in a particular time period to test for a trend, such as an increase or a decrease. The data sets for XX and XY at a particular time point were compared with a Student t-test.

RESULTS

Gonadal Development

As we reported previously [18], hematoxylin-eosin staining has revealed that the first signs of morphological differentiation of the gonads, evidenced by the formation of the ovarian cavity in the XX gonad or the efferent duct in the XY gonad, occur around 23–26 dah (Fig. 1).


Figure 1
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  		FIG. 1 Gonadal differentiation of XX and XY tilapia. Undifferentiated gonad 7 dah XX fry (A). B) XX gonad at 30 dah. Arrow indicates elongation of the gonad for the formation of the ovarian cavity. C) XY gonad at 30 dah. Arrow indicates a slit-like space for the formation of the efferent duct. XX gonad at 70 dah (D) and XY gonad at 70 dah (E). Bars = 10 µm (A–C); 50 µm (D, E).

Steroidogenic Enzymes cyp11a1, hsd3b, cyp17a1, hsd17b1, cyp11b2, and cyp19a1a

The expression of cyp11a1 (cytochrome P450, subfamily XIA, polypeptide 1; previously known as P450scc, cholesterol side-chain cleavage enzyme) was weak in both XX and XY gonads at 5–7 dah. The levels in XX gonads rapidly increased from 10 to 15 dah (7–15 dah: y = –7146 + 1143x, r = 0.99, n = 12, P < 0.0001), but decreased at 35–70 dah (25–70 dah: y = 15 735 – 181x, r = 0.62, n = 11, P = 0.04). The levels in XY gonads remained low from 5 to 25 dah, but markedly increased from 35 to 70 dah (25–70 dah: y = –4166 + 246x, r = 0.84, n = 11, P = 0.001). The cyp11a1 level at 70 dah was higher in the XY than XX gonads (Fig. 2A).


Figure 2
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  		FIG. 2 Changes in mRNA levels of (A) cyp11a1 (P450scc; cholesterol side-chain cleavage), (B) hsd3b (3β-HSD; 3β-hydroxysteroid dehydrogenase/{Delta}4–{Delta}5 isomerase), (C) cyp17a1 (P450c17; steroid 17{alpha}-hydroxylase/C17–20 lyase type1), (D) hsd17b1 (17β-HSD1; 17β -hydroxysteroid dehydrogenase type1), (E) cyp11b2 (P45011 β; steroid 11β-hydroxylase), and (F) cyp19a1a (ovarian type of aromatase) in XX and XY gonads during gonadal development from 5 to 70 dah. A magnified view of cyp19a1a from 5 to 7 dah (labeled days post-hatching) is also presented (G). Each value represents the mean ± SEM for three or four groups. Closed circles represent means for XX gonads. Open squares represent means for XY. ND represents nondetected level. Different letters indicate statistical differences at P < 0.05 as determined by Tukey-Kramer test. Upper- or lowercase letters indicate comparisons within XX or XY groups, respectively. Asterisks indicate statistical differences determined by Student t-test.

Hsd3b (hydroxy-delta-5-steroid dehydrogenase, 3 beta; previously known as 3β-hydroxysteroid dehydrogenase/{Delta}4–{Delta}5 isomerase) and cyp17a1 (cytochrome P450, family 17, subfamily A, polypeptide 1; previously known as P450c17type1, steroid 17{alpha}-hydroxylase/C17–20 lyase type1) were expressed at similar levels (relatively high levels) in XX and XY gonads at 5–7 dah. Hsd3b levels in XX gonads increased from 5 to 25 dah (y = –7061 + 1875x, r = 0.90, n = 22, P < 0.0001) and were maintained at high levels thenceforth. Cyp17a1 levels in XX gonads increased from 5 to 15 dah (y = –24 233 + 5604x, r = 0.97, n = 20, P < 0.0001), and were maintained at high levels thereafter. Levels of both hsd3b and cyp17a1 in XY gonads remained relatively low during this period, but increased after 25–70 dah (hsd3b: y = –60 501 + 3092x, r = 0.78, n = 10, P = 0.008; cyp17a1: y = –23 778 + 2036x, r = 0.86, n = 10, P = 0.001). Both hsd3b and cyp17a1 levels at 70 dah were higher in the XY gonads than those in the XX gonads (Fig. 2, B and C).

Hsd17b1 (hydroxysteroid (17-beta) dehydrogenase 1; previously known as 17β-hydroxysteroid dehydrogenase type1) was expressed in XX gonads from 5 to 35 dah. At 70 dah, hsd17b1 dropped to low levels. In XY gonads, hsd17b1 was detected at 5 dah, and then dropped to barely detectable levels from 6 dah onward (Fig. 2D).

Cyp11b2 (cytochrome P450, family 11, subfamily B; previously known as P45011β, steroid 11β-hydroxylase) was not detected in either XX or XY gonads until 25 dah. In XY gonads, its expression first becomes apparent at 35 dah. Cyp11b2 was not detected in XX gonads throughout the sampling period (Fig. 2E).

Cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a; previously known as cyp19a1, P450arom, ovarian type of aromatase) levels in XX gonads increased rapidly from 5 to 15 dah (y = –19 287 + 3733x, r = 0.98, n = 21, P < 0.0001), and remained high thereafter. In contrast, cyp19a1a levels in XY gonads were barely detectable throughout the sampling period (Fig. 2F). The level of cyp19a1a mRNA was significantly higher in XX gonads than in XY gonads even at 5 dah (Fig. 2G).

Transcription Factors foxl2, nr5a1, and nr0b1

Foxl2 (forkhead box L2) was expressed at higher levels in XX gonads than in XY gonads from 5 dah throughout the experimental period (Fig. 3, A and B). The levels in XX gonads increased linearly until 35 dah (y = –778 + 187x, r = 0.91, n = 24, P < 0.0001) and remained high at 70 dah. The foxl2 levels in XY gonads were consistently very low throughout the sampling period (Fig. 3A).


Figure 3
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  		FIG. 3 Changes in mRNA levels of (A) foxl2, (C) nr5a1 (Ad4BP/SF-1), (D) nr0b1 (DAX1), (E) dmrt1, and (F) sox9 in XX and XY gonads during gonadal development from 5 to 70 dah. A magnified view of foxl2 from 5 to 7 dah is also presented (B). Each value represents the mean ± SEM for three or four groups. Closed circles represent means for XX gonads. Open squares represent means for XY. Different letters indicate statistical differences at P < 0.05 as determined by Tukey-Kramer test. Upper- or lower-case letters indicate comparisons within XX or XY groups, respectively. Asterisks indicate statistical differences determined by Student t-test.

In the early developmental period from 5 to 7 dah, nr5a1 (nuclear receptor subfamily 5, group A, member 1; previously known as Ad4BP, SF-1) levels were not significantly different between XX and XY gonads. Nr5a1 was expressed at higher levels in XX gonads than in XY gonads at 10–25 dah, whereas levels were higher in the testis than ovary at 70 dah (Fig. 3C).

Nr0b1 (nuclear receptor subfamily 0, group B, member 1; previously known as DAX1) was expressed at similar levels in both XX and XY from 5 to 10 dah. In XX gonads, these levels were relatively high at 5–35 dah, then decreased at 70 dah. The levels became lower in XY gonads than XX gonads from 10 to 35 dah. At 70 dah, the level in XY gonads became higher than XX gonads (Fig. 3D).

dmrt1 and sox9

Dmrt1 (doublesex and mab-3 related transcription factor 1) was expressed initially (5 dah) at similar levels (low levels) in the XX and XY gonads, but its expression in the XY gonads was higher than XX at 6 dah (P = 0.011). Thereafter, levels rapidly increased until 10 dah (5–10 dah: y = –1121 + 394x, r = 0.83, n = 17, P < 0.0001), and then gradually increased until 70 dah (10–70 dah: y = 2409 + 46x, r = 0.74, n = 19, P = 0.0003). In contrast, dmrt1 expression in XX gonads was consistently low throughout the period from 5 to 70 dah (Fig. 3E).

Sox9 (SRY-box containing gene 9) was expressed at similar levels in XX and XY gonads during the period 5–25 dah. The levels were significantly higher in XY gonads than XX gonads at 35–70 dah (Fig. 3F).

amh

At 5–10 dah, amh (anti-Mullerian hormone; also known as Mullerian-inhibiting substance [Mis] or Mullerian inhibiting factor [Mif]) expression was low in gonads of both sexes. At 15 dah, amh expression differed between males and females, with higher levels and further sharp increases in XY gonads until 35 dah (10–35 dah: y = 9426 + 3045x, r = 0.78, n = 19, P < 0.0001). The levels in XX gonads remained low throughout the sampling period (Fig. 4).


Figure 4
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  		FIG. 4 Changes in mRNA levels of amh (Anti-Mullerian hormone) in XX and XY gonads during gonadal development from 5 to 70 dah. Each value represents the mean ± SEM for three or four groups. Closed circles represent means for XX gonads. Open squares represent means for XY. Different letters indicate statistical differences at P < 0.05 as determined by Tukey-Kramer test. Lowercase letters indicate comparisons within XX or XY groups. Asterisks indicate statistical differences determined by Student t-test.

esr1, esr2a, and esr2b

Esr1 (estrogen receptor 1; previously known as ER{alpha}) was expressed at relatively high levels in XX and XY gonads at 5–6 dah. Esr1 expression tended to increase in XY gonads (25–70 dah: y = –1206 + 207x, r = 0.74, n = 10, P = 0.014), but not XX gonads (P = 0.71), at 70 dah. There were no significant differences in esr1 expression between XX and XY gonads throughout the experimental period (Fig. 5A). Both esr2a (estrogen receptor 2a; previously known as ERβ1) and esr2b (estrogen receptor 2b; previously known as ERβ2) were expressed at similar (relatively high) levels in XX and XY gonads throughout the sampling period, except for higher levels of esr2a in the XY gonad at 70 dah and esr2b at 35 and 70 dah (Fig. 5, B and C).


Figure 5
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  		FIG. 5 Changes in mRNA levels of (A) esr1 (estrogen receptor 1), (B) esr2a, (C) esr2b, (D) ar1 (androgen receptor 1), and (E) ar2 in XX and XY gonads during gonadal development from 5 to 70 dah. Values represent the mean ± SEM for three or four groups. Different letters indicate statistical differences at P < 0.05 as determined by Tukey-Kramer test. Upper- or lowercase letters indicate comparisons within XX or XY groups, respectively. Asterisks indicate statistical differences determined by Student t-test.

ar1 and ar2

Ar1 (androgen receptor 1; previously known as AR{alpha}) was expressed at similar (relatively low) levels in XX and XY gonads during the early period of sex differentiation from 5 to 7 dah. The ar1 levels in both sexes tended to increase until 15 dah (XX: y = 192 + 114x, r = 0.79, n = 16, P = 0.0003; XY: y = 68 + 156x, r = 0.75, n = 16, P = 0.0008). The levels in XX gonads showed a significant increase from 25 to 70 dah, but the levels in XY gonads changed little after 15 dah. Ar1 levels were significantly higher in XX gonads than XY gonads at 70 dah (Fig. 5D). The expression levels of ar2 (androgen receptor 2; previously known as ARβ) mRNA were also relatively low from 5 to 7 dah in both sexes. The levels in both sexes tended to increase gradually throughout the experimental period. Ar2 expression levels did not show sexual dimorphism throughout the experiment (Fig. 5E).

DISCUSSION

We determined the expression patterns of 17 genes encoding steroidogenic enzymes, steroid receptors, transcription factors, and amh during gonadal differentiation and development in tilapia. The wide variances at the later stages may indicate the intragroup differences in the developmental status of the gonads. In the case of tilapia, such differences become more prominent with the advancement in growth. Further, this study used a pool of 30–50 gonads as one sample at early stages like 5–15 dah for the extraction of RNA, while a lesser number of gonads were used at stages after 25 dah especially because the amount of RNA from fewer gonads was sufficient for measurement. Therefore, the magnitude of differences between the individuals may become larger at later stages, and this may have a greater influence on the data from these stages. Even though wide variances appear in later time periods, the results clearly indicate that overall expression patterns of these genes change dynamically, depending on the genetic sex and developmental stage of tilapia. Our findings support the conclusion that in tilapia, foxl2 and cyp19a1a play a crucial role in ovarian differentiation, and dmrt1 does in testicular differentiation.

In fishes, like amphibians, gonadal sex differentiation is widely susceptible to estrogens and androgens [11, 12, 19], suggesting that sex steroids play an important role in gonadal sex differentiation in these animals. In this study, we have shown that the terminal enzyme necessary for producing estrogen, cyp19a1a, is expressed only in XX female gonads prior to morphological differentiation; the expression was seen as early as 5 dah, followed by sharp increases. As in other teleosts, the Nile tilapia has another gene (cyp19a1b) encoding the aromatase found in the brain [20]. However, cyp19a1b expression in the gonads at 5–7 dah was much weaker than cyp19a1a expression, suggesting a limited role for cyp19a1b in the ovarian synthesis of estrogens early on in sex differentiation (data not shown).

To prove the critical role of estrogen in ovarian differentiation, it is important to show that XX gonads prior to or during early ovarian differentiation contain not only Cyp19a1a, but also all other steroidogenic enzymes necessary for the synthesis of estrogens. In fact, cyp11a1, hsd3b, cyp17a1, and hsd17b1 were also expressed in undifferentiated XX gonads as early as 5 dah; cyp11a1, hsd3b, and cyp17a1 levels then gradually increased in both XX and XY gonads. Our earlier immunocytochemical studies confirmed the present findings at the protein level [21, 22]. Taken together with earlier reports on the complete sex reversal of XY tilapia fry by following estrogen treatment, the results obtained from tilapia are consistent with the notion that estrogen is produced in XX gonads during the period 5–7 dah and plays a crucial role in ovarian differentiation [21].

Numerous studies on fish including tilapia have shown that treatment of XX larvae with androgens induced sex reversal toward testicular development [11, 12, 19, 23, 24]. However, no direct experimental evidence has been provided of an essential role for endogenous androgens in initial testicular differentiation in fish. In tilapia, mRNAs of most steroidogenic enzymes were constantly detected in XY gonads at 5–7 dah. However, unlike in XX gonads, in XY gonads there were no further increases in these levels at 10–25 dah. Another important observation in this study is that the expression of cyp11b2 (P45011β; steroid 11β -hydroxylase), which contributes to the synthesis of 11-ketotestosterone (11-KT), the most potent androgen in teleost fish, from testosterone, is not detected in either XX or XY gonads at 5–25 dah. These findings may indicate that 11-KT is not produced in tilapia gonads until 25 dah and, thus, in tilapia, androgens do not appear to play a major role in testicular differentiation. In contrast, at 70 dah, the mRNA levels of four steroidogenic enzymes including cyp11b2 are higher in XY gonads than in XX gonads. Because spermatogenesis is initiated during this period, increased steroidogenic activity of XY gonads may be involved.

For sex steroids to have their effects during gonadal development, their receptors must also be present. We have previously isolated two types of estrogen receptor, termed Esr1 and Esr2 (Esr2a), from tilapia gonads [25], and more recently, a third type (Esr2b) [26]. In the present study, all three types (ear1, esr2a, and esr2b) were constantly expressed at relatively high levels from our first sampling point at 5 dah in both sexes with no sexual dimorphic expression until 35 dah. Equal expression of esr1 between undifferentiated XX and XY gonads was reported from medaka [27] and rainbow trout [28]. In chicken, in which estrogen is a key inducer of ovarian development, Esr1 is expressed in gonads of both sexes early in embryogenesis [29, 30]. Taken together, these results indicate that estradiol synthesized in female gonads mediates female sex differentiation by stimulating the development of undifferentiated XX gonads through estrogen receptors. Esr expression in XY gonads early during differentiation explains the susceptibility of males to feminization by exogenous estradiol.

Despite the pivotal role of Cyp19a1a in female sexual differentiation, how the expression of cyp19a1a is regulated within the developing gonads remains to be determined. In mammals, Nr5a1 (also known as Ad4BP or SF-1) is an important activator of steroidogenic enzymes, including aromatase. Our earlier studies have also shown that Nr5a1 plays an important role in the transcriptional regulation of cyp19a1a in the vitellogenic follicle of tilapia [31, 32]. In the present study, nr5a1 was consistently highly expressed in both XX and XY gonads during gonadal sex differentiation, the levels being higher in XX gonads than in XY gonads at 10–25 dah. This expression pattern of nr5a1 differs from that of cyp19a1a, which exhibits a rapid increase at 5–15 dah, suggesting that Nr5a1 does not play a major role in the transcriptional regulation of cyp19a1a during ovarian differentiation in tilapia. It is of interest that at 70 dah, nr5a1 was expressed at higher levels in male gonads than in female gonads. This may be correlated with the greater expression of cyp11a1, hsd3b, cyp17a1, and cyp11b2 seen in XY gonads during this stage.

Foxl2 belongs to the forkhead family (transcription factors containing winged helix DNA binding motif [forkhead domain]) and is another transcription factor shown to be involved in the transcriptional regulation of cyp19a1a expression [33]. With our recent in vitro assays, we have shown that Foxl2 binds to the promoter region of cyp19a1a directly through its forkhead domain and activates the gene's transcription [34]. It is important to note that two transcription factors, foxl2 and nr5a1, both of which are thought to be involved in the regulation of steroidogenesis, differ not only in the levels of their transcripts, but also in their expression patterns. Foxl2 expression was apparent in XX gonads as early as 5 dah, with a linear increase during the next 35 days; there were barely detectable levels of the foxl2 transcript in XY gonads during this period. Thus, the expression pattern of foxl2, but not nr5a1, precisely correlated with that of cyp19a1a, at least until 15 dah. In contrast to the linear increase in the levels of foxl2, cyp19a1a was sustained at constant levels after 15 dah. It is highly probable that cyp19a1a levels would have reached the threshold levels by this time, because another activating factor, nr5a1 was also found to be stable from 15 to 35 dah. It is assumed that there is no influence from other as-yet-unknown regulating factors. We have also reported that the disruption to endogenous Foxl2 in XX tilapia caused by the overexpression of a dominant negative mutant induces varying degrees of testicular development, with occasional sex reversal from ovary to testis [34]. Taken together, these results are consistent with the hypothesis that Foxl2 is an important regulator of cyp19a1a expression in female gonads early during sex differentiation. Further studies are required to determine whether Foxl2 is involved in the transcriptional regulation of other steroidogenic enzymes that are expressed early during sex differentiation.

The Drosophila doublesex and Caenorhabditis elegans mab-3 genes encode transcription factors characterized by a conserved DNA-binding domain, the DM domain. A vertebrate homologue, Dmrt1, has been shown to be expressed exclusively in the urogenital system and is implicated in testicular differentiation in various vertebrate species [7, 11, 35]. The most direct evidence for an important role of Dmrt1 (the DM domain) has come from our recent discovery that the sex-determining gene of medaka, dmy, was derived from a duplicate copy of the autosomal dmrt1 [36]. Consistent with our earlier study showing its exclusive expression in the adult testis [37], the present study demonstrated a restricted expression of dmrt1 in the XY gonads, first evident at 6 dah. This restricted pattern of expression in the tilapia XY gonad persisted through subsequent stages, with gradual increases until 70 dah. The critical role of Dmrt1 in testicular differentiation in tilapia was confirmed by our earlier findings that estrogen-induced sex reversal in XX tilapia was accompanied by the disappearance of dmrt1 expression in gonads of the treated fish [21]. The specific expression of dmrt1 in male gonads during the sex differentiation period was also indicated in trout [38, 39]. In birds with a ZZ:ZW system, characterized by female heterogamety (ZW), dmrt1 is the best candidate for a male-determining gene to be identified so far [9]. Dmrt1 is expressed specifically in the gonads of chicken embryos, being more highly expressed in males than females prior to and during gonadal sex differentiation [40]. The timing of dmrt1 expression during testicular differentiation and development varies among fish species. There is a significant delay between the first appearance of dmy and that of dmrt1 in medaka XY gonads during differentiation of the testes, showing that Dmrt1 expression occurs much later than Dmy expression [41]. Further studies on the transcriptional regulation of dmrt1 and the identification of target(s) of Dmrt1 are required to determine the roles and actions of Dmrt1 in testicular differentiation in tilapia.

The Sry-related gene sox9 has been shown to have a conserved, sexually dimorphic expression pattern in vertebrate embryos, its expression being upregulated male-specifically in mammals and birds [5, 42, 43] and maintained in males but downregulated in females in reptiles [44, 45]. Sox9 is first expressed at low levels in the undifferentiated gonads of both sexes, but becomes dramatically upregulated in the Sertoli cells immediately after the expression of Sry. Based on these expression patterns, together with other functional data, sox9 is thought to be a strong candidate as a genuine target of Sry during testicular differentiation in mammals [4]. However, this does not appear to be the case for the tilapia sox9 (282 within 294 amino acids are identical with the medaka sox9a2). In this fish, sox9 expression was relatively intense in both XX and XY gonads at 5–25 dah, with the first distinct sexual dimorphic difference at 35 dah, being upregulated only in the XY gonads. Similar expression patterns also seem to occur in medaka gonads early during differentiation. In medaka, the level of testicular type sox9 (sox9a2) expression in somatic cells is equally high in both sexes at the time when dmy expression first occurs during early gonadal differentiation. However, during the period from 10 to 30 dah, sox9 expression continues only in the Sertoli cell lineage in male gonads, with a marked reduction in the XX gonad [16]. Based on these findings, it is concluded that in tilapia and medaka, Sox9 does not play a major role in early sex determination and differentiation, but may be involved in the later development of testicular tubules. Further studies are needed to determine the precise role of Sox9 in gonadal formation and development in fish. Thus, it appears that even the role of Sox9 is not completely conserved among vertebrates.

Amh is a glycoprotein belonging to the transforming growth factor β superfamily. A major function of Amh in mammals is to mediate regression of Mullerian ducts in males. Targeted mutagenesis has shown that Amh is not required for testicular determination in the mouse [8]. Importantly, unlike other vertebrates, fish do not have Mullerian ducts. In tilapia, amh mRNA starts to be expressed in undifferentiated gonads of both sexes, and the expression is upregulated in male gonads, but not in female gonads. The sexual dimorphic expression of Amh in fish gonads during sex differentiation was reported in Japanese flounder, Paralichthys olivaceus [46]. Amh expression starts in supporting cells of undifferentiated gonads of both sexes and is upregulated in male gonads but downregulated in female gonads. Similarly, in the chicken, the onset of gonadal Amh expression begins just prior to sexual differentiation in both sexes, and is consistently higher in males [9]. Thus, Amh mRNA expression is upregulated in the supporting cells of the differentiating testis in most vertebrates, suggesting that Amh plays an important role in testicular differentiation in vertebrates as well as in the regression of Mullerian ducts.

The Nr0b1 (DAX1: dosage-sensitive sex reversal, adrenal hypoplasia congenital critical region on the X chromosome) acts as a negative regulator of steroidogenesis through a function that inhibits the transcriptional activity of Nr5a1 (Ad4BP/SF-1) [47]. In the present study, however, the nr0b1 expression level was higher in the XX gonads than XY gonads from 15 to 35 dah, an expression pattern similar to that of nr5a1. Thus, there is no negative correlation between nr0b1 and nr5a1 expression during gonadal sex differentiation. These results do not seem to support the notion that Nr0b1 is involved in the regulation of gonadal sex differentiation by inhibiting the transcriptional activation of genes encoding steroidogenic enzymes.

In summary, considerable changes in the abundance of transcripts occur in the gonads of the Nile tilapia during the course of sex determination and differentiation (Fig. 6). Increases in the expression of foxl2 and cyp19a1a in XX gonads from 5 dah suggest an important role for Foxl2 and Cyp19a1a in ovarian differentiation. Dmrt1 exhibited a male-specific expression in XY gonads from 6 dah onward, suggesting an important role for Dmrt1 in testicular differentiation. Thus, the differential expression of genes occurring in XX and XY gonads during the period 5–6 dah is critical for undifferentiated gonads to differentiate into either the ovary or testis in the Nile tilapia. Whether any genes (sex-determining gene?) are expressed in either gonad prior to this period is an important question.


Figure 6
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  		FIG. 6 Schematic representation of changes in mRNA levels of cyp11a1 (P450scc), hsd3b (3β-HSD), cyp17a1 (P450c17type1), hsd17b1 (17β-HSDtype1), cyp19a1a (ovarian type of P450arom), cyp11b2 (P45011β), amh, nr5a1, foxl2, nr0b1, dmrt1, sox9, esr (estrogen receptor) 1, 2a, and 2b, and ar (androgen receptor) 1 and 2 in the tilapia gonad during gonadal sex differentiation.

FOOTNOTES

1Supported in part by a Grant-in-Aid for Research from SORST, JST (Japan Science and Technology Corporation), and the Ministry of Education, Science, Sports and Culture of Japan. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. Back

Correspondence: 2Yoshitaka Nagahama, Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan. FAX: 81 564 55 7556; e-mail: nagahama{at}nibb.ac.jp

Received: 16 July 2007.

First decision: 19 August 2007.

Accepted: 17 October 2007.

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