Expression and Transcript Localization of star, sf-1, and dax-1 in the Early Brain of the Orange-Spotted Grouper Epinephelus coioides

We investigated the developmental expression and localization of sf-1 and dax-1 transcripts in the brain of the juvenile orange-spotted grouper in response to steroidogenic enzyme gene at various developmental ages in relation to gonadal sex differentiation. The sf-1 transcripts were significantly higher from 110-dah (day after hatching) and gradually increased up to 150-dah. The dax-1 mRNA, on the other hand, showed a decreased expression during this period, in contrast to sf-1 expression. At the same time, the early brain had increased levels of steroidogenic gene (star). sf-1 and star hybridization signals were found to be increased in the ventromedial hypothalamus at 110-dah; however, dax-1 mRNA signals decreased in the early brain toward 150-dah. Furthermore, the exogenous estradiol upregulated star and sf-1 transcripts in the early brain of the grouper. These findings suggest that sf-1 and dax-1 may have an antagonistic expression pattern in the early brain during gonadal sex differentiation. Increased expression of steroidogenic gene together with sf-1 during gonadal differentiation strongly suggests that sf-1 may play an important role in the juvenile grouper brain steroidogenesis and brain development.


Introduction
Steroid synthesis is a biochemical process that occurs in the nervous system as well as the classical peripheral steroidogenic organs, notably in the brain [1]. These neurosteroids participate in a variety of physiological and behavioral processes during the early developmental period that are still unclear. The transcriptional regulation of neurosteroidogenic enzymes in the brain has been reported in a number of teleosts in relation to gonadal differentiation [2][3][4][5][6][7] and adult neurogenesis [8]. These findings provide evidence that neurosteroid production is under the control of brain steroidogenic enzymes. However, the expressive role of steroidogenic factors and their localization on the early brain steroidogenesis during gonadal differentiation remains unknown. In the steroidogenic pathway, the steroidogenic acute regulatory protein (Star) has been shown to be important in the regulation of cholesterol, which is transported from the outer to the inner mitochondrial membrane [9] and this is the rate-limiting step in the biosynthesis of steroids since cyp11a1, which metabolizes cholesterol, is located at the inner mitochondrial membrane. Characterization and molecular expression of star have been reported in different vertebrates during development, which includes chicken [10], rat and marmoset [11], and teleosts [2,[12][13][14][15].
Despite this, little is known about the developmental expression and transcript localization of steroidogenic genes in relation to orphan nuclear receptors (sf-1 and dax-1) in the developing brain.
SF-1 (Steroidogenic Factor-1, Ad4BP, NR5a1) is an orphan member of the nuclear hormone receptor superfamily and a homolog of the drosophila fushi tarazu factor-1 protein (FTZ-F1) [16,17]. SF-1/sf-1 plays a key role in the development and function of steroidogenic tissues within the hypothalamic-pituitary-gonadal axis and as an essential factor in sex differentiation of many species [3,[18][19][20][21][22][23][24][25][26][27]. It was originally identified in mammals as a tissue-specific transcriptional regulator of the cytochrome P450 hydroxylases, including cyp19a1a [28]. Since then, SF-1/sf-1 has been found to influence the transcription of several steroidogenic genes in human, adrenal, and gonadal tissues, including star [29], cyp11a1 [30], 3β-hsd [31], and cyp19a1a [32][33][34]. It remains unclear whether there is a cooperative mechanism of sf-1 regulation on cyp19a1b expression in the early brain development of the sex-changing fish. However, promoter motif analysis in air-breathing catfish (Clarias gariepinus) indicated that sf-1 binds to cis-acting regions in the upstream region of cyp19a1b, implying that sf-1 gene regulates cyp19a1b transcription via binding to the promoter of cyp19a1b in the brain [35]. Therefore, the majority of findings show that sf-1 is a critical element in the development and sexual differentiation of steroidogenic tissues, particularly the gonad.
Another orphan nuclear receptor is dax-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X-chromosome, gene 1). It functions as a negative co-regulator of many genes involved in the steroidogenic pathway [36][37][38][39][40]. dax-1 has been linked to female gonadal sex differentiation in chicken [41] and pig [42]. DAX-1 suppresses CYP19 expression in mice by binding to its promoter regions [43]. Furthermore, it has been reported that dax-1 inhibits sf-1 and foxl2-mediated cyp19a1a expression in medaka ovarian follicles [36], whereas SF-1 regulates the transcription of the DAX-1 gene in adult rats [44]. The role of dax-1 as a crucial factor for organ and sex development has been well documented in zebrafish (Danio rerio) [45][46][47][48]. In comparison to sf-1, there is little known about how dax-1 might regulate potential target genes during the early developmental period. It needs to be addressed for the precise role of sf-1 and dax-1 in the regulation of neurosteroidogenic enzyme genes in the early development of the brain and temporal correlation to gonadal sex differentiation. Therefore, we hypothesize that sf-1 and dax-1 play important roles in the steroidogenesis in the early brain.
Orange-spotted grouper (Epinephelus coioides), an economically important prime aquaculture species and a popular marine food fish widely cultured in Taiwan, Saudi Arabia, and southeast Asian countries. Grouper is a protogynous hermaphrodite species with several unique characteristic features that make them particular interest to study gene expression profiles that are crucial for early brain development and gonadal differentiation. The fish are sex differentiated to females when they are around 4-5-month-old juvenile, and then maintain for several years before changing to male [5,6]. Thus, this mono-female sex fish provides a suitable model to study early brain development [49]. The mRNA expression of cyp19a1a and sf-1 in the gonad of orange-spotted grouper after 17α-methyltestosteroneinduced precocious sex transition has been observed [50]. However, the developmental expression pattern of sf-1 and dax-1 in relation to other key steroidogenic enzyme genes, as well as its possible expressive role in the developing brain during gonadal differentiation, remain unknown. Therefore, we investigated the temporal expression profile of sf-1 and dax-1 in relation to the other key steroidogenic gene in the early ages of the grouper brain during gonadal differentiation. We used star as a key marker of the first step in the cascade of estrogen synthesis. Further, we localized the developmental transcript expression of star, sf-1, and dax-1 at 90-, 110-, and 150-dah (days after hatching) in the hypothalamic regions. Finally, we analyzed the effect of exogenous estradiol (E 2 ) on the mRNA expression of star, sf-1, and dax-1 in the grouper brain.

dax-1 mRNA Expression
Unlike sf-1 expression, dax-1 mRNA expression was significantly (p < 0.05) higher at 90-dah and subsequently declined from 110-to 150-dah in the telencephalon and mesencephalon, and 110-to 130-dah in the diencephalon ( Figure 2D-F). In 90-dah, the amounts of dax-1 transcripts in the telencephalon, mesencephalon, and diencephalon increased significantly (p < 0.05). When compared to the levels in the telencephalon at 90-dah, higher expression of dax-1 in the mesencephalon and diencephalon was 2.2-and 1.6-fold higher, respectively ( Figure 2D-F). Furthermore, higher expression of dax-1 mRNA was identified in the mesencephalon at 180-dah, which was 4-and 2.6-fold higher than the levels in the telencephalon and diencephalon at 180-dah, respectively ( Figure 2E).

Localization of sf-1, dax-1 and star Transcripts in the Brain of the Protogynous Orange-Grouper during Different Stages of Developmental
Three hypothalamic brain areas were selected for the in situ hybridization analysis corresponding to the series Sections 1, 2, and 3, the most enriched expression zone for the genes star, sf-1, and dax-1 ( Figure 1). The developmental mRNA distribution pattern of star, sf-1, and dax-1 in the hypothalamic areas of the grouper brain was demonstrated at 90-, 110-, and 150-dah (Figures 3-5). star, sf-1, and dax-1 transcripts were found in the ventral habenular nucleus, ventromedial thalamic nucleus, diencephalic ventricle, anteroventral part of the parvocellular preoptic nucleus, gigantocellular part of the magnocellular preoptic nucleus, and lateral tuberal nucleus, dorsal and lateral parts of the hypothalamic nucleus (

Effect of E 2 on star, sf-1 and dax-1 mRNA Expression in the Grouper Brain In Vivo
Exogenous E 2 stimulated star expression in all regions of the brain ( Figure 6A), but sf-1 expression was significantly (p < 0.05) higher in the mesencephalon and diencephalon but not in the telencephalon, according to the q-PCR results ( Figure 6B). In contrast to the telencephalon, E 2 significantly (p < 0.05) reduced dax-1 expression in the mesencephalon and diencephalon ( Figure 6C). Figure 6. In vivo effects of exogenous estradiol (E 2 , 1 µg/g BW, fish n = 8 (110-dah, day after hatching) for each group were given intramuscular injections on day 1 and 5). Brain tissues were collected on day 6 for the analysis of mRNA expression of star (A), sf-1 (B), and dax-1 (C), as measured by q-PCR analysis in the telencephalon, mesencephalon, and diencephalon. The results are expressed as the mean with standard deviation (SEM). The different letters indicate significant differences (p < 0.05) according to a one-way ANOVA followed by a S-N-K (Student-Newman-Keuls) multiple comparison test. An asterisk (*) represents significant (p < 0.05) differences between the control and E 2 -injected groups.

Discussion
This all-female brain of juvenile orange-spotted grouper (a protogynous species) provides a unique model to study early brain development. We have shown the developmental expression and localization of steroidogenic factors such as sf-1 and dax-1 transcripts in the early female brain of the orange-spotted grouper at different developmental ages in comparison to another key steroidogenic enzyme gene (star). The peak expression of sf-1 and star genes, as well as those cellular levels and effects of exogenous E 2 in the early brain, were the most notable findings of this study. In comparison to sf-1 expression, dax-1 expression was lower in the early development of the grouper brain. These intriguing findings suggest that sf-1 may have a regulatory role in steroidogenesis in the early brain of orange-spotted grouper.
sf-1 is an obvious candidate gene to explain the developmental expression pattern in neural estrogen synthesis in the early female brain, as it regulates the transcription of various target genes involved in steroidogenesis. The expression profiles of star, sf-1, and dax-1 are correlated to the time at which most of the other key steroidogenic genes (cyp11a1, hsd3b1, cyp17a1, and cyp19a1b) and estrogen receptors (erα, erβ1, erβ2, and gpr30) exhibited maximal expression in the early female grouper brain [4,6]. In contrast to this scenario, lower expression of dax-1 was found in the early brain during gonadal sex differentiation when sf-1 and other key steroidogenic enzymes (including Cyp19a1b activity) were high, indicating that the grouper brain has a functional peak of neurosteroidogenis that may be regulated by sf-1 and dax-1 (Figure 7). As a result, at 110-dah, decreased expression of dax-1 and increased expression of sf-1, as well as significantly increased expression of other key steroidogenic genes, may suggest that these orphan nuclear receptor genes play an important role in brain steroidogenesis during the early developmental period. Until now, available studies in the brain have shown that the expression and localization of orphan nuclear receptors in response to the development of the hypothalamus [19,51], rather than the regulation of brain steroid synthesis during early brain development, as compared to the gonad and interrenal organ [45,46,52]. Our in situ hybridization study revealed that sf-1 and star transcripts were highly expressed at 110-to 150-dah and 110-dah, respectively; while dax-1 exhibited higher transcripts at 90-dah and a decreasing trend towards 150-dah in cells of the ventral habenular nucleus, ventromedial thalamic nucleus, and ventromedial hypothalamus. In monosex rainbow trout, Oncorhynchus mykiss, sf-1 mRNA expression was localized in the mediobasal hypothalamus [7]. The expression of SF-1 in the hippocampus of marmosets and rats has been examined using immunohistochemistry [11]. Furthermore, in the marmoset and rat, all StAR-positive cells were also SF-1 positive, demonstrating that the expression of SF-1 and StAR has a functional relationship [11]. SF-1 and DAX-1 immunoreactive cells were discovered in the mouse VMH and pituitary throughout development [23]. Knockout mice deficient in SF-1 have profound defects in the VMH, strongly suggesting the presence of SF-1 target genes at this site [51]. Therefore, our findings revealed that the hypothalamus is a key site for the expression and function of sf-1 and dax-1. As a result, the intensive mRNA localization of star, sf-1, and dax-1 in the hypothalamic areas of the grouper brain agrees with these previous investigations, indicating that the functional linkage of these genes is plausible for steroidogenesis and brain development.
Furthermore, the current study highlights the in vivo effect of exogenous E 2 on the regulation of star, sf-1, and dax-1 in the grouper early brain. E 2 is the most biologically prominent and active member of the estrogen family of steroids, and it has a wide range of effects on the developing brain [53]. According to a recent study, the peak expression of numerous neurosteroidogenic genes in the brain of black porgy at the time of gonadal differentiation is mediated by both E 2 -independent and E 2 -dependent pathways [54]. E 2 exposure was found to up-regulate star mRNA in the brain and gonad of the self-fertilizing fish, Kryptolebias marmoratus [55]. On the other hand, the xenoestrogen, diethylstilbestrol exposure decreased in the total amount of sf-1 mRNA in the unborn rat testis but not in fetal ovaries [56]. Holt (1989) [57] proposed the possibility of E 2 , stimulating the transport of cholesterol to the inner mitochondrial membrane. This is consistent with our findings, which reveal that E 2 exposure increases the expression of sf-1 and star mRNA.
Our in vivo results showed that the exogenous E 2 up-regulated star (telencephalon, mesencephalon, and diencephalon) and sf-1 (mesencephalon and diencephalon) transcripts compared to their control counterparts. This upregulation could be due to E 2 binding with nuclear estrogen receptors (ers) and ers/sf-1 express in the same cells, or an estrogenresponsive element (ERE) located on the promoter region of the star and sf-1 in the brain. These proposals are unclear, and further research is needed to determine whether the star, sf-1, and ers co-express in the same cells. E 2 exposure, on the other hand, reduced dax-1 mRNA levels in the mesencephalon and diencephalon. The overexpression of sf-1 mRNA and the downregulation of dax-1 mRNA are strongly related to the results of brain steroidogenesis and E 2 exposure, owing to the opposing functions of dax-1 and sf-1 in steroidogenesis. As a result, for E 2 upregulation of steroidogenesis in the brain, sf-1 is more significant than dax-1. We previously reported that during early development, E 2 , aromatase enzyme activity, cyp19a1b, and estrogen receptors (er, er1, and er2) are all at their greatest levels in the brains of orange-spotted grouper and black porgy [2][3][4][5][6]. Thus, our findings imply that during the early developmental period, there is a local production of neurosteroids, particularly neuroestrogens, that is tightly regulated in the preparation for the peak of functional brain growth (neurogenesis). Therefore, this current and our previous studies [4][5][6] show the temporal expression and localization of sf-1 and dax-1 transcripts in the early brain along with neurosteroidogenic related genes (star, cyp11a1, cyp17a1, and cyp19a1b) in the mono-female sex-differentiated fish (Figure 7). sf-1 transcripts were increased in the early brain as other key steroidogenic genes and E 2 [2][3][4]58]; however, dax-1 had an antagonistic expression pattern during these time periods, suggesting that dax-1 may play a negative role in the early brain development of female grouper. Indeed, the star, sf-1, and dax-1 mRNAs were found in abundance in the cells of the ventromedial thalamic nucleus and the mediobasal hypothalamus, and these three genes were mostly found in the same regions, implying that their functions in steroidogenesis during brain development are closely related. Furthermore, exogenous E 2 also increased the expression of star, cyp19a1b, and sf-1 in the brain, indicating that these genes are primarily involved in E 2 synthesis. This finding led to a better understanding of the transcriptional regulation of sf-1 and dax-1 as molecular players for steroidogenesis during development (Figure 7). Therefore, the balance of sf-1 and dax-1 expression is critical in the regulation of brain development and sex differentiation.

Experimental Fish
Orange-spotted female grouper (E. coioides) were used in the present study. The experimental fish were acclimated to the pond condition of the University culture station in a seawater and natural light system (salinity of 33 ppt; temperatures ranged from 20-24 • C). The fish were fed ad libitum with a commercial food (Fwu Sou Feed Co., Taichung, Taiwan). All procedures and investigations were approved by the National Taiwan Ocean University Institutional Animal Care and Use Committee and were performed in accordance with standard guiding principles.
Three batches of orange-spotted groupers were used for the experiment. For the gene expression analysis, fish (n = 8 per age group) were obtained at 90-, 110-, 130-, 150-, and 180-dah. To investigate the localization of genes in hypothalamic brain regions, fish with 90-, 110-, and 150-dah were obtained. To investigate the effects of exogenous E 2 on gene expression, 110-dah fish were obtained. The fish were anesthetized with 0.05% ethylene glycol monophenyl ether before being decapitated. The brain was dissected in the same manner as previously described for the orange-spotted grouper and black porgy [4,6,54,59,60]. Brain samples, including telencephalon (including the olfactory bulb, telencephalon, and part of the preoptic area; located between the anterior commissure and the optic chiasm), mesencephalon, and diencephalon (including mesencephalon, thalamus, epithalamus, subthalamus, and hypothalamus), were immediately frozen in liquid nitrogen and stored at −80 • C until RNA isolation. Another batch of whole-brain tissues was fixed in a 4% paraformaldehyde solution for histological/cellular analysis.

Experiment 2: Localization of sf-1, dax-1 and Star Gene Expression in the Hypothalamus of Orange-Spotted Grouper
We used in situ hybridization in distinct hypothalamic brain regions to determine the anatomical localization of cells that express the star, sf-1, and dax-1 genes (see detailed methods below).

Experiment 3: In Vivo Effects of E 2 on the Expression of mRNAs Encoding sf-1, dax-1, and Star in the Brain of Grouper
In order to further investigate the effects of E 2 on the mRNAs expression of sf-1, dax-1 and star, 110-dah female grouper for each group (BW = 32.3 ± 1.8 g, BL = 13.5 ± 0.25 cm) were divided into two groups, with n = 8 fish in each of the following experimental groups: control (vehicle alone, coconut oil; Sigma, St. Louis, MO, USA) and E 2 treatment (1 µg/g BW; Sigma). On day 1 and 5, the fish received an intramuscular injection (two injections in total). Fish telencephalon, mesencephalon, and diencephalon were collected 24 h (day 6) after the 2nd injection (day 5) and stored at −80 • C until further use. Q-PCR was used to examine changes in the mRNA expression of the corresponding genes in the brains of control and E 2 -treated groups.

RNA Extraction, First-Strand cDNA and Molecular Cloning of Genes in the Grouper Brain
Total RNA was extracted from the telencephalon, mesencephalon, and diencephalon (90-to 180-dah) using the TRIzol reagent method (Gibco BRL; Grand Island, NY, USA) according to the manufacturer's instructions. The quality and concentrations of RNA were assessed by spectrophotometry and checked by running an aliquot (1 µg) on a 1.8% agaroseformaldehyde gel. Single-stranded cDNA was constructed from total RNA using Invitrogen reagents (Invitrogen, Carlsbad, CA, USA), oligo (DT) 12-18 primers and Superscript II reverse transcriptase (Gibco BRL) in a 20 µL reaction volume with an incubation at 42 • C for 60 min, 37 • C for 15 min, and 70 • C for 15 min. The resulting cDNAs were used as a template for subsequent PCR amplification. The PCR reaction was performed in a final volume of 25 µL reaction containing 2.5 µL of 10× reaction buffer (200 mM Tris-HCl, pH 8.4, 500 mM KCl), 1µL of 10 mM dNTP, 1 µL of 2 mM MgCl 2 , 0.5 µL each of 10 µM forward and reverse primer, respectively (Mission Biotech Co., Ltd., Taipei, Taiwan), 0.2 µL superscript enzyme (Invitrogen, Carlsbad, CA, USA), and 1 µL cDNA. The reaction conditions for degenerate PCR were as follows: 94 • C for 5 min, 35 cycles of 94 • C for 30 s, 50 • C for 30 s, 72 • C for 30 s and 72 • C for 7 min. Each PCR product was electrophoresed on 1.5% agarose gel and the fragment showing the predicted molecular weight was then excised using Gel-MTM Gel Extraction system kit (Viogene, Bio 101, La Jolla, CA, USA). Extracted cDNAs were ligated into the pGEM-T Easy vector (Promega, Madison, WI, USA) and transformed into Escherichia coli competent cells following the manufacturer's instruction. The Plasmid containing the insert was sequenced and compared with the NCBI database using BLAST. The genes star (GenBank accession no. GU929702), sf-1 (JQ320496), and dax-1 (GU929703) were cloned from the grouper brain, which was deduced from the conserved regions of other teleosts (Figures S1-S3).

Preparation of Brain Tissue for In Situ Hybridization
For in situ hybridization experiments, 90-, 110-, and 150-old female grouper brains were collected to define star, sf-1, and dax-1 mRNA signals in the brain. For in situ hybridization, we chose three hypothalamic areas with the most enriched expression zones for the genes star, sf-1, and dax-1 (Figure 1). Groupers were anesthetized by immersion in ethylene glycol monophenyl ether (0.05%). Grouper brains were fixed overnight in a 4% paraformaldehyde in PBS (phosphate-buffered saline) at 4 • C. Upon fixation, the skull was removed and the brain was carefully dissected out and stored overnight in fresh fixative at 4 • C. The brain was then rinsed several times in PBS. Later, the fixed brain was dehydrated in a series of alcohols, embedded in paraffin, and then cross-sections (5-6 µm) were collected on APTES-treated slides (3-aminopropyltriethoxysilane, Sigma) diluted in acetone.

Synthesis of Star, sf-1 and dax-1 RNA Probes
The PCR product of the target gene from the plasmid DNA containing respective inserts of the genes in the pGEM-T Easy vector was generated with 50 U DNA polymerase (New England Biolabs, Ipswich, MA, USA) for DNA amplification with in situ hybridization primers ( Table 1). The PCR products were purified using a kit (PCR-Advanced Clean-Up Kit, Viogene). In vitro transcription was carried out using this purified DNA as a template. Digoxigenin-labeled sense and anti-sense riboprobes were synthesized from a fragment of 751 bp, 570 bp, and 443 bp encoding orange-spotted grouper brain star, sf-1, and dax-1, respectively, by using T7 and SP6 polymerase (Promega). The DNA (1µg) templates were incubated for 3 h at 37 • C in the thermocycler PCR machine (Applied Biosystems, Foster City, CA, USA) in a solution containing transcription buffer (5X), 0.1 M dithiothreitol (DTT), DIG-rNTP mix (10X) (Roche, Penzberg, Germany), an RNase inhibitor (40 U/µL) (Promega) and T7 or SP6 RNA polymerase (20 U/µL) and adjusted to 20 µL final volume with sterile DEPC H 2 O. The extra template was removed by digesting it with 4 µL of DNase I (10 U/µL) at 37 • C for 30 min. After incubation, the probes were precipitated with 100 µL of LiCl (7.5 M) and 900 µL of isopropanal at −20 • C overnight. The pellets were collected by centrifuging the solution at 12,000 rpm at 4 • C for 30 min and the pellets were re-suspended with 2 µL RNase inhibitor and 98 µL sterile DEPC H 2 O. The probe quality was checked by spectrophotometry at 260 nm. Table 1. Primer sequence used for q-PCR and in situ hybridization analysis for the genes star, sf-1, and dax-1 in the orange-spotted grouper. The nucleotide (nt) length of RNA probes for in situ hybridization: star, 751 nt; sf-1, 570 nt; dax-1, 443 nt.

Q-PCR Analysis
Transcripts of grouper star [4], sf-1, and dax-1 were quantified by quantitative realtime PCR analysis in the telencephalon, mesencephalon, and diencephalon at different developmental ages (90-to 180-dah). Q-PCR primers were designed for the analysis of star, sf-1, dax-1, and β-actin with the assistance of Primer Express Software (Applied Biosystems) ( Table 1). The expression levels of brain β-actin were not significantly different among development ages ( Figure S5). β-actin was used as a housekeeping gene in the q-PCR analysis to calibrate the expression levels of brain genes. Gene quantification of the standards, samples, and control was simultaneously conducted in a q-PCR machine (iQTM Multicolor Real-Time PCR Detection System; Bio-Rad Co., Hercules, CA, USA) with iQTM SYBR green (Bio-Rad Co.), which is a dsDNA minor-groove binding dye, forward and reverse primers and water for the reaction mix in a MicroAmp ® 96-well reaction plate. The respective standard curve of the log (transcript concentrations) vs. CT (the calculated fractional cycle number at which the PCR-fluorescence product is detectable above a threshold) was −0.995.

Statistical Analysis
All values (n = 8) are represented as the mean ± SEM (standard error of mean), and the data were analyzed by one-way ANOVA followed by an S-N-K (Student-Newman-Keuls) multiple comparison test to compare the differences (p < 0.05) in the developmental expression of the genes in different parts of the brain at different ages. Student t-test was also conducted to compare the significant differences (p < 0.05) between control and E 2 -injected fish.

Conclusions
In conclusion, the current study found developmental changes in the mRNA expression and localization of orphan nuclear receptors such as sf-1 and dax-1 in relation to neurosteroidogenic enzyme gene in the early brain of the orange-spotted grouper. It has been suggested that sf-1 and dax-1 play an important role with key enzymes in steroidogenesis in the early brain of the orange-spotted grouper. Furthermore, our findings revealed that E 2 increased star and sf-1 mRNA expression in the telencephalon and diencephalon, which is critical for early brain development and steroidogenesis (Figure 7). Overall, this research found that the orange-spotted grouper brain has all the necessary mechanisms for neurosteroidogenesis and, as a result, local E 2 synthesis, which may regulate early brain development during gonadal sex differentiation.