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BOR - Papers in Press, published online ahead of print January 18, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.045351
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BIOLOGY OF REPRODUCTION 74, 857–864 (2006)
DOI: 10.1095/biolreprod.105.045351
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Molecular Biology of Ovarian Aromatase in Sex Reversal: Complementary DNA and 5'-Flanking Region Isolation and Differential Expression of Ovarian Aromatase in the Gilthead Seabream (Sparus aurata)1

Ten-Tsao Wong 3 , Shigeho Ijiri 4 , and Yonathan Zohar 2

Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202

ABSTRACT

To elucidate the involvement of aromatase in sex reversal, the gilthead seabream ovarian P450 aromatase (cyp19a1a) cDNA and its 5'-flanking region were isolated and characterized. Northern blot analysis revealed that only one cyp19a1a transcript (2.0 kb) is expressed in the ovary. Four cAMP-responsive elements were identified at the 5'-flanking region of seabream cyp19a1a indicating a high potential to respond to gonadotropin signaling. Studying the seasonal profile, two expression peaks of cyp19a1a transcripts in the ovarian tissues were found in July (about 15000 copies/ng total RNA) for ambisexual fish and in December (about 12000 copies/ng total RNA) for spawning females. Starting from September, transcript levels of cyp19a1a in the ovarian portions of the male-developing gonads gradually decreased. Furthermore, the ovarian portions of the female gonads expressed cyp19a1a at a significantly higher level than the ovarian portions of the male gonads after November. Taken together with levels of plasma estradiol in reversing females being significantly higher than those in developing males, the above results reinforce the importance of cyp19a1a in sex reversal. In vitro exposure of ovarian fragments to gonadotropins (hCG) at 1, 10, and 100 IU/ml significantly (P < 0.05) upregulated cyp19a1a expression. Additionally, expression of cyp19a1a displayed a stronger and significant correlation with the transcript expression of ovarian Lh receptor rather than Fsh receptor during the ambisexual stage. Our results indicate that the differential expression of cyp19a1a gene is associated with sex reversal and that gonadotropin signals (particularly Lh) may serve as major players in regulating the expression of cyp19a1a during the process of sex reversal.

aromatase, estradiol, follicle-stimulating hormone, luteinizing hormone, ovary, sex reversal

INTRODUCTION

Sex reversal, the transformation of an individual from one sex to the other in adulthood, is recognized in hermaphrodite fish. It has long been evident that estrogens are the key factors driving female development during the process of sex reversal. In the protandrous anemone fish, a decrease of 11-ketotestosterone (11-KT) was seen in the sex-reversing fish, and mature females had a higher level of 17ß-estradiol (E2), which indicated that the change in gonadal steroidogenesis is associated with sex reversal [1]. In the protandrous black porgy, high levels of plasma E2 during the prespawning and spawning seasons were correlated with natural sex change [2]. In the protandrous gilthead seabream, treatments with E2 induced various changes including the development of the ovarian parts in the ambisexual gonads and the inhibition of testicular development [3] and complete sex reversal [4]. In black porgy, E2 treatments induced a complete sex reversal [5], and aromatase inhibitor blocked the process of sex reversal [6]. Taken together, the above findings suggest the crucial role of estrogens in ovarian development during the process of sex reversal.

Cytochrome P450 aromatase (Cyp19) catalyzes the conversion of androgens to estrogens [7]. In nonmammalian vertebrates, estrogens are thought to be essential for ovarian development [8]. During embryonic and larval development, exogenous estrogens can feminize genetic males, and aromatase inhibitors were shown to block ovarian development in birds [9–12], reptiles [13–16], amphibians [17, 18], and fish [19–22]. Studies have shown that transcript levels of gonadal aromatase (cyp19a1a) are increased in association with aromatase enzyme activity during vitellogenesis in rainbow trout [23], medaka [24], tilapia [25], red seabream [26], and Japanese eel [27]. Moreover, plasma E2 has shown a good correlation with the transcript levels and enzyme activity of aromatase during oocyte development in red seabream [26]. Aromatase gene expression has, logically, been proposed as a key step of estrogen synthesis that is crucial for ovarian differentiation [28]. Thus, the change of aromatase expression is associated with the process of sex reversal.

The purpose of this study is to elucidate the involvement of ovarian aromatase during the process of sex reversal in the gilthead seabream at the molecular level. Hence, the cyp19a1a cDNAs and its 5'-flanking region were isolated and characterized. Based on the cDNA sequence information, a highly sensitive real-time fluorescence-based quantitative PCR assay (hereafter called quantitative PCR) was developed. This assay has provided the tools to investigate "stage-dependent" gene expression of cyp19a1a in the ovarian portions of gonads during the process of sex reversal and the influence of reproductive hormones on the regulation of cyp19a1a gene expression. The results generated in this study will contribute to a more complete understanding of the molecular mechanism involved in the process of sex reversal.

MATERIALS AND METHODS

Animals, Sample Collection, and Hormone Measurement

About 280 male gilthead seabream at the age of 2 yr were held in four tanks (2.4 m3) in our Aquaculture Research Center and exposed to simulated natural photoperiod conditions (15 h light in summer and 7 h light in winter) and temperatures ranging from 15°C (winter) to 23°C (summer), which mimics the conditions fish experience in the wild. To understand the change of gonadal development during sex reversal, monthly sampling was carried out from May through January in 2-yr-old seabream, a population that started as all male in February and in which 50%–60% of the fish reversed sex to female at the end of the year (examined in December). In accordance with the annual gonadal development of the gilthead seabream in our culture conditions, spawning starts in December and ends at the beginning of March. In males, after the spawning season, the testicular tissue regressed, while the ovarian portion proliferated, resulting in the appearance of the ambisexual gonad from May through August. At monthly intervals, eight fish (ambisexual, male or female stages) were anesthetized in 200-ppm 2-phenoxyethanol. On removal of the gonad, the ovarian portion of the gonad was carefully separated from the connective tissue and the testicular portion and immediately frozen in liquid nitrogen. Pieces of gonads were also fixed in phosphate buffered 4% formaldehyde for further histological analysis of their developmental stages. Samples used to analyze tissue distribution of cyp19a1a transcripts were collected from ambisexual fish in July, except the ovary and testis from spawning fish (collected in December) and the gonad from reversing females (collected in October). For brain, pituitary, and spleen, whole organ was used for RNA isolation. Plasma E2 was measured using commercially available solid phase 125Iodine radioimmunoassay (DPC, Los Angeles, CA) that measure total amount of E2 in unextracted and heparinized plasma. All animal husbandry and experimentation were conducted in accordance with our Institutional Animal Care and Use Protocols and adhered to the National Research Council's Guide for Care and Use of Laboratory Animals.

Oligonucleotide Primers and Nucleotide Sequencing and Analyses

The particular primers used in this study are listed in Table 1, and the positions at which they hybridize to cyp19a1a are shown in Figure 1A. The nucleotide sequences of the cloned cDNA inserts and 5'-flanking region were determined by dye-terminator automatic sequencing (ABI 373 DNA Sequencer STRETCH; Applied Biosystems, Foster City, CA). The potential identity of the peptide encoded by seabream cyp19a1a was determined by homology search with the Position Specific Iterated-BLAST (PSI-BLAST) method [29]. All the above analyses were performed with the Internet server of the DNA Data Bank of Japan (DDBJ; http://www.ddbj.nig.ac.jp/). Putative transcriptional factor binding sites were located on the 5'-flanking region of cyp19a1a using analyses posted on two Web sites: http://pdap1.trc.rwcp.or.jp/ and http://www.motif.genome.ad.jp/.


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  		TABLE 1. Oligonucleotide primers used in this study.


Figure 1
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  		FIG. 1. The cDNA (A) and the 5'-flanking region (B) of cyp19a1a. In A, the hybridization regions for the primers used in this study are indicated by a, b, c, d, e, and f; the consensus sequence of the polyadenylation signal is also presented. In B, the locations of cis-acting elements are labeled. CRE, cAMP response element; SF1, steroidogenic factor 1; ERE, estrogen response element.

Isolation of cyp19a1a cDNAs from Gilthead Seabream Ovary

Ovarian cDNA was synthesized using 1 µg of ovarian mRNA with oligo (dT) primers and SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. An aliquot of the first-strand cDNA was amplified with the degenerate primers DARO1 and DARO2 (Table 1). PCR was performed in 25 µl of reaction mixture containing 200 µM dNTP, 1 µM of each degenerate primer, and 1x Advantage-2 polymerase mix (BD Biosciences, Palo Alto, CA). The PCR conditions were 94°C for 30 sec, 58°C for 30 sec, and 72°C for 1 min for the initial five cycles; during the remaining 30 cycles the annealing temperature was decreased from 58 to 52°C. Fragments of about 480 bp were purified from 1.5% agarose gel with QIAquick gel extraction kit (Qiagen, Valencia, CA), cloned to pGEM-T vector (Promega, Madison, WI), and sequenced.

For rapid amplification of cDNA ends (RACE), ovarian 5'- and 3'-RACE cDNAs were used following a published protocol [30]. Gene-specific primers, WTAQ26 for 5'-RACE and WTAQ25 for 3'-RACE and the universal primer mix (UPM) primer (adapter primer from kit) were used for RACE amplifications. PCR products were purified, subcloned, and sequenced.

Isolation and Analysis of the 5'-Flanking Region of cyp19a1a Gene

A seabream Genome Walker library for a PCR-based method [30] was used for the isolation of the 5'-termini of the cyp19a1a gene. The first "genome walking" PCR was carried out using GsbOA1 and Ap1 primer (adapter-specific primer 1 from kit) followed by a nested second amplification with GsbOA2 and Ap2 primer. The PCR amplicons were subcloned and sequenced.

Syntheses of RNA Standards and Riboprobe

For RNA standard syntheses, plasmids containing cDNAs encoding relevant open-reading frame of seabream Cyp19a1a, Fsh receptor (Fshr), and Lh receptor (Lhcgr) [31] were linearized and used as templates for gene-specific RNA standard syntheses using a SP6/T7 transcription kit (Roche, Indianapolis, IN). RNA standards were purified through a size exclusion column (ChromaSpin-400; BD Biosciences), and the amount of each RNA standard was determined using RiboGreen RNA quantification kit (Molecular Probe, Eugene, OR). The same protocol described above was followed for cyp19a1a antisense riboprobe syntheses except a 32P-UTP mixture was used instead of UTP.

Northern Blot Analysis

Ten micrograms of ovarian mRNA were electrophoresed through a 1.1% agarose gel containing formaldehyde and then transferred onto a nylon membrane. The membrane was prehybridized at 60°C in 50% formamide, 5x Denhart's, 5x SSC, 1% SDS, and 100 µg/ml of yeast RNA for 2 h. After 16 h of hybridization with a 32P-labeled seabream cyp19a1a antisense riboprobe (1 x 106 dpm/ml), the membrane was then washed in 2x SSC, 0.1% SDS at 68°C for 30 min, 0.5x SSC, 0.1% SDS at 68°C for 30 min, and 0.1x SSC, 0.1% SDS at 68°C for 30 min. After air-drying, the membrane was exposed to a phosphor storage screen and visualized with a Storm 840 PhosphoImage Analyzer (Amersham Biosciences, Piscataway, NJ).

Quantification of Gene Expression at Transcript Levels

Gene expression of cyp19a1a, fshr, and lhcgr in gonad and other seabream tissues were determined at the transcript levels using quantitative PCR assays. RNA standards and total RNA, isolated from each sample using Tri-reagent (MRC, Cincinnati, OH), a modified acid-phenol extraction method, were reverse-transcribed into cDNA using random hexamers and MMLV reverse transcriptase (Promega). Twenty nanograms of cDNA were used for each quantitative PCR assay. PCR was carried out via the ABI Prism 7700 Sequence Detection System using SYBR Green PCR Core Reagent (Applied Biosystems) and gene-specific primers, WTAQ25 and WTAQ26 for cyp19a1a, WTAQ51, and WTAQ52 for fshr and WTAQ37 and WTAQ38 for lhcgr (Table 1). Copy number in unknown samples was determined by comparing CT (threshold cycle) values [32] to the gene-specific standards. The results were normalized to the amount of 18s RNA (amplified by TSB18SF and TSB18SR primers) in each sample.

In Vitro Ovarian Fragment Incubation

Ambisexual gonads were used from four fish in July, at which time most of oocytes in the ovarian portion were at primary growth stages. The ovarian portion, separated from the connective tissue and testicular portion as described above, was cut into 1–2-mm-thick fragments and washed for 30 min twice with culture medium, Leibovitz's L-15 medium (Invitrogen) supplemented with 0.2% BSA (Sigma, St. Louis, MO) and 10 mM Hepes (pH 7.4). Duplicate ovarian fragments from each ambisexual gonad were incubated in 24-well tissue-culture plates with the fresh culture medium containing 100 U of penicillin and 100 µg of streptomycin/ml (Invitrogen) at 20°C while shaking gently (60 rpm). Human chorionic gonadotropin (hCG; Sigma) was added to the medium at concentrations of 0, 0.1, 1, 10, and 100 IU/ml. After 3 h of incubation, tissues were harvested and stored at –80°C before analysis.

Statistical Analyses

Gonadal-somatic index (GSI), levels of plasma E2, and data obtained via quantitative PCR for the transcription levels were presented as the mean and SEM. The results were analyzed for each developmental stage (categorized by month) and for hCG treatment (categorized by the dosage). For statistical analyses, data were first tested for equality of variance using the Bartlett test and for normality using the Kolmogorov and Smirnov tests. Since data for the cyp19a1a profile, E2 profile, and hCG experiment did not pass either one or both tests, nonparametric ANOVA (Kruskal-Wallis test) was applied, followed by a Tukey-Kramer multiple comparison test for the seasonal expression profile and a Dunnett (control) test for the hCG experiments. A Student t-test was applied for the comparison of the GSI between ambisexual fish, male-developing fish, and female-developing fish. Associations of cyp19a1a expression with either fshr or lhcgr expression at the transcript level during the ambisexual stage were analyzed using the Pearson correlation coefficient and multiple linear regression. The statistical significance of r is tested using a Student t-test, and the significance was accepted at P < 0.05.

RESULTS

Isolation and Characterization of cyp19a1a and Its 5'-Flanking Region from Gilthead Seabream

Twelve clones from the RACE were isolated. primecleotide sequences were determined for all clones. The entire cDNA sequence was edited from the nucleotide sequences in 5'-RACE and 3'-RACE clones and determined to be the gilthead seabream ovarian P450 aromatase (GenBank accession number AF399824). The 1895-bp cyp19a1a cDNA contained a 1557-bp open-reading frame that predicted a 58.9-kDa protein of 519 residues (Fig. 1A). The 5'-untranslated region (5'-UTR) before the first ATG initiation codon is relatively short (54 bp), which is consistent with the finding in cytochrome P450 steroidogenic enzymes that have been isolated from fish. The 3'-UTR contained one consensus polyadenylation signal (AATAAA) at 1869 bp. Similar to other cyp19a1a derived from the teleost ovary, the seabream cyp19a1a had a second potential translation initiation site at 85 bp; however the first ATG codon resulted in better similarity to the consensus sequence proposed by Kozak [33]. The deduced amino acid sequence of cyp19a1a shared only 60% identity with the gilthead seabream brain form of aromatase (Cyp19a1b; unpublished results). However, this deduced sequence had high homology with the Cyp19a1a from other fish including black porgy (96%; GenBank accession number AY273211–1), red seabream (94%) [26], European sea bass (87%) [34], Japanese flounder (83%) [35], and medaka (81%) [36] and 75%–80% overall sequence identity with other fish Cyp19a1a. Less than 53% homology was seen when compared with mammalian aromatases, including human (49%) [37, 38].

Using genome walking PCR, the amplicon (1148 bp) corresponding to the 5'-flanking region of the cyp19a1a gene were isolated (GenBank accession number AY779630; Fig. 1B). The TATA box is located 30 bp upstream of the transcription initiation site predicted from the cyp19a1a cDNA sequence. Moreover, the 5'-flanking region of cyp19a1a contains the sequences of four putative cAMP-responsive elements (CRE), three steroidogenic factor 1 (SF1) binding sites, and two estrogen-response-element half sites (ERE-halves).

Northern Blot Analysis and Tissue-Specific Expression of cyp19a1a

The size and number of cyp19a1a transcript(s) expressed in the ovary were examined by Northern blot analysis. Only one transcript was evident in the ovary (Fig. 2A). The size of the transcript (2.0 kb) calculated by RNA molecular weight markers corresponds to the size of cDNAs isolated using the RACE method. To examine the tissue distribution of the cyp19a1a transcript, quantitative PCR assays were undertaken using total RNA isolated from selected tissues. In addition to the ovary, transcripts of cyp19a1a were also expressed at different levels in the brain, pituitary, gill, thyroid, retina, heart, head-kidney, trunk-kidney, spleen, intestine, testis, and ambisexual gonad but not in liver or muscle (Fig. 2B).


Figure 2
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  		FIG. 2. Northern blot analysis of the cyp19a1a mRNA in the ovarian portion (A) and tissue distribution of the cyp19a1a (B). Pit, Pituitary; TG, thyroid gland; HK, head-kidney; TK, trunk-kidney; OFSF, ovary from spawning female; TFSM, testis from spawning male; GFRF, gonad from reversing female; ND, not detected.

Seasonal Gonadal Development of the Gilthead Seabream

In seasonal gonadal development (Fig. 3A) and as previously shown by Zohar et al. [39], the testicular portion of the ambisexual gonad is packed with spermatagonia and remains latent, the ovarian portion of the ambisexual gonad undergoes previtellogenetic growth, and the oogonia start to undergo folliculogenesis. By September and thereafter, the gonads appear to be differentiated and can be recognized as either developing ovaries in the sex-reversing fish or developing testes in those animals that will remain as males. After September, the sex-reversing fish undergo vitellogenesis (growth of the ovarian portion populated with more secondary growth oocytes), while the others (male-developing fish) undergo spermatogenesis (increase of the testicular portion and differentiated spermatocytes). A significant increase of the GSI was first seen in sex-reversing (female-developing) fish in September (Fig. 3B), while no significant change was observed in male-developing fish in the same month when compared with ambisexual fish (May–August). After October, rapid gonadal growth was seen in both sex-reversing and male-developing fish. Comparing the GSI of male-developing fish with that of the female-developing fish, a significant difference was only found in the month of September.


Figure 3
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  		FIG. 3. Schematic patterns of gonadal development (A) and changes in the monthly gonadal-somatic index (GSI) of the gilthead seabream (B). When comparing the GSI of male-developing fish with that of the female-developing fish (month by month from September to January) using a Student t-test, a significant difference was found only in the month of September (indicated by an asterisk).

Seasonal Profiles of cyp19a1a Transcript Expression and Plasma E2 Levels

To understand the involvement of cyp19a1a in the process of sex reversal, seasonal changes in abundance of cyp19a1a transcripts in the ovarian portions of gonads were investigated. In the expression profile of cyp19a1a (Fig. 4), higher levels of cyp19a1a were seen in the ovarian portion of the ambisexual gonad starting in May and peaking in July (about 15 000 copies/ng total RNA). Levels of cyp19a1a transcripts in the ovarian portions dropped to 5000 copies/ng total RNA in August and remained at the same level in developing females until November (late vitellogenesis). In December, the beginning of the spawning season, the cyp19a1a transcript level in the ovarian portions of developing females increased to 12000 copies/ng total RNA. In the ovarian portion of the male-developing gonad, a decreasing trend of cyp19a1a expression was seen from September to November; thereafter, levels of cyp19a1a remained low in December and January (Fig. 4). Starting from September, in each month, we compared the transcript level of cyp19a1a in the ovarian portion of male-developing gonads with that in the female-developing gonads. Levels of cyp19a1a transcript are lower in the ovarian portions of male-developing gonads; a significant difference was found in the months of December and January (Fig. 4).


Figure 4
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  		FIG. 4. Changes in the seasonal expression, at the transcript level, of cyp19a1a in the ovarian portion of the gilthead seabream gonad. Data are shown as the mean and SEM. The number of samples per data point is 8. Nonparametric ANOVA was performed (P < 0.0001). Data points not sharing a letter (a, b, c) are significantly different by Tukey-Kramer multiple comparison test.

Levels of plasma E2 were low in the ambisexual fish (Fig. 5). Higher levels of plasma E2 were found in reversing females, starting from September, peaking in November at the level of 1100 pg/ml and maintaining high in December and January. In developing males, levels of plasma E2 were significantly lower than those of reversing females. It first dropped to its lowest point (<10 pg/ml) in September, the initial month that fish can be recognized as developing males, and slightly increased but remained at a lower level (<100 pg/ml) thereafter.


Figure 5
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  		FIG. 5. Seasonal changes of plasma estradiol in the gilthead seabream from ambisexual stage to spawning season. Data are shown as the mean and SEM. The number of samples per data point is 8. Nonparametric ANOVA was performed (P < 0.0001). Data points not sharing a letter (a, b, c, d) are significantly different by Tukey-Kramer multiple comparison test.

The Gonadotropin Signals and the cyp19a1a Expression

The effects of gonadotropins on the expression of cyp19a1a were investigated in the ovarian portion of the ambisexual gonads from four fish in July. Using short-term in vitro ovarian fragment incubation (3 h), hCG at 1, 10, and 100 IU/ml significantly (P < 0.05) enhanced the transcript expression of cyp19a1a in a dose-response manner (Fig. 6). To further understand the differential involvement of gonadotropin signals at the gonadal level in the regulation of cyp19a1a expression during sex reversal, we investigated the respective associations in gene expression between cyp19a1a and each gonadotropin receptor, fshr and lhcgr, which have also been cloned and characterized in our lab [31]. Understanding the expression association between aromatase and each respective gonadotropin receptor provides us a physiological perspective on the differential involvement of gonadotropin signaling in the regulation of aromatase expression. Our results show that transcript expression of cyp19a1a is better associated with the expression of lhcgr (n = 32, r = 0.567, P < 0.001) rather than fshr (n = 32, r = 0.4, P = 0.023) during the ambisexual stage (Fig. 7; May–August). When the data were categorized by month (Table 2), they show that in May neither fshr (r = 0.259, P = 0.534) nor lhcgr (r = 0.355, P = 0.388) transcript expression were found significantly correlated with cyp19a1a expression. This nonsignificant correlation of cyp19a1a with fshr continued in June (r = 0.109, P = 0.798) and July (r = 0.334, P = 0.418). In August, a greater but not statistically significant correlation of fshr and cyp19a1a transcript expression was found (r = 0.683, P = 0.062). In contrast, the association of the ovarian lhcgr and cyp19a1a transcript expression became strong and significant in June (r = 0.811, P = 0.015) and then remained at an intermediate level in July (r = 0.759, P = 0.029). The strongest association between lhcgr and cyp19a1a expression (r = 0.92, P = 0.001) was seen in August, the last month of the ambisexual stage. The difference between the combination analysis (Fig. 7) and month-by-month analysis suggested that season (month) and/or both fshr and lhcgr have effects on cyp19a1a expression. To further address this question, multiple linear regression was applied. Regression equation: cyp19a1a = 66 + 1.52 fshr + 3.69 lhcgr + 5504 May – 479 June + 7624 July (August was set as a reference level, and adjusted R2 = 66.0%). Results (Table 3) from this analysis further demonstrated that expression of cyp19a1a is better associated with the expression of lhcgr (coefficient = 3.69, P < 0.001) rather than fshr (coefficient = 1.52, P = 0.3) during the ambisexual stage. Moreover, season also has a significantly different effect on the expression of cyp19a1a.


Figure 6
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  		FIG. 6. The effects of hCG on the transcript expression of cyp19a1a in the ovarian portion of the ambisexual gonad in vitro. Data (n = 4) are shown as the mean and SEM. In hCG incubation, categorized by dosage, nonparametric ANOVA was performed followed by the Dunnett (control) test. The asterisk indicates a significant (P < 0.05) difference from control.


Figure 7
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  		FIG. 7. Associations of cyp19a1a expression with either fshr (A) or lhcgr (B) expression at the transcript level during the ambisexual stage (n = 32, May–August). The linear regression line, Pearson correlation coefficient (r), and P-value from a Student t-test of r are presented.


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  		TABLE 2. Association of cyp19a1a expression with gonadotropin receptors' mRNA expression in the ovarian portion of the ambisexual gonads (May through August).


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  		TABLE 3. Association of cyp19a1a expression with the combination of multiple factors, fshr, lhcgr, and month.

DISCUSSION

It has been demonstrated that two forms of aromatases (Cyp19a1a and Cyp19a1b) exist in teleosts such as goldfish [40, 41] and zebrafish [42, 43]. The expression of cyp19a1a was detected mainly in the ovary, while cyp19a1b was expressed in extragonadal tissues, brain, pituitary, and retina. The deduced amino acid sequence of gilthead seabream cyp19a1a displays a high degree of overall homology with ovary-derived aromatases from other teleosts. The phylogenetic analysis of aromatase in fish indicates that the ovarian aromatase branch is clearly segregated from the brain aromatase branch [27, 44]. In fish, aromatase activity or transcript expression was detected in other tissues in addition to the ovary, including brain, pituitary, retina, kidney, and testis [40–43, 45, 46]. Using quantitative PCR assays, we detected that in addition to the high degree of expression in the ovary of spawning and sex-reversing females, transcript of cyp19a1a was also found in brain, pituitary, gill, thyroid, retina, heart, head-kidney, trunk-kidney, spleen, intestine, and testis from spawning males. Although the abundance is low and the physiological significance is not well understood in extragonadal tissues, the tissue distribution of cyp19a1a in the gilthead seabream is intriguing.

The 5'-flanking region of cyp19a1a exhibited four CREs, two ERE-halves, and three SF1 binding sites. In mammals, a consensus SF1 binding site [47] was demonstrated as a regulator for the transcription of steroidogenic enzymes, including aromatase. This consensus sequence was also observed in the 5'-flanking region of cyp19a1a in medaka [36] and other hermaphrodites [44] and identified as a functional promoter [48, 49]. SF1 appears to be a common transcriptional factor of cyp19a1a. The existence of ERE-halves was also seen in the 5'-flanking region of cyp19a1a in medaka [36] and zebrafish [50]. However, the exact function of the ERE-half is still unclear. It has been well documented that the transcription of mammalian aromatase is stimulated by gonadotropin via the cAMP second messenger in follicular granulosa cells [51, 52]. Similarly, there are CREs in the 5'-flanking region of the medaka [36], the Atlantic stingray [53], and the zebrafish [50] aromatase genes. Given the presence of four CREs in the 5'-flanking region of the gilthead seabream cyp19a1a, the transcription of cyp19a1a has the potential for regulation by gonadotropins via cAMP.

Results from our short-term in vitro tissue incubations showed that hCG enhanced the expression of cyp19a1a in the ovarian portions of the ambisexual gonads, indicating that gonadotropin signals are involved in the regulation of ovarian cyp19a1a. In rat granulosa cells, the expression of aromatase mRNA is induced by Fsh both in vivo [54] and in vitro [55]. In teleosts, gonadotropin has been reported to induce aromatase activity in ovarian follicles of goldfish [56] and medaka [57]. However, it remained unclear whether, like in mammals, fish Fsh could stimulate the activity of ovarian aromatase until a recent report by Kagawa et al. [58]. Surprisingly, using highly purified red seabream gonadotropins, the biological activity of red seabream Fsh was revealed to be much lower than that of Lh in inducing aromatase activity and E2 production of vitellogenic follicles in vitro [58]. Our results from the hCG experiment are in good agreement with data obtained from rats, medaka, and red seabream. In granulosa cells isolated from immature rats, the induction of aromatase activity by FSH alone is less than 2-fold [55]. In medaka ovarian follicles, about a 2-fold increase in aromatase activity (E2 production) was induced by pregnant mare serum gonadotropin alone [57]. In red seabream ovarian follicles, less than a 2-fold increase of aromatase mRNA was induced by seabream Lh. In the same experiment, seabream Fsh had no effect on the level of aromatase mRNA [58]. Moreover, the changes in transcript levels of pituitary lhb but not of fshb were observed to be associated with oocyte development in the same species [26]. Thus, unlike in mammals, Lh, rather than Fsh, may have a higher potential to regulate cyp19a1a expression in red seabream.

To further address the differentiated involvement of gonadotropin signals in the regulation of cyp19a1a expression during sex reversal, we investigated the correlation of gene expression between cyp19a1a and gonadotropin receptors. Our data revealed a strong association of transcript expression of cyp19a1a with lhcgr, but not fshr, in the ovarian tissues of the ambisexual gonads. This suggests that Lh signaling is more potent than Fsh signaling in promoting ovarian aromatase expression, similar to the findings in red seabream. Together with the results from multiple linear regression analysis, it further strengthens our conclusion that expression of cyp19a1a has a stronger association with the expression of lhcgr during the ambisexual stage. It has also been documented that expression of fshb and lhb in the pituitary of the gilthead seabream displays a sexually dimorphic pattern [59]; thus, developing females expressed higher levels of lhb transcripts than developing males. Moreover, Lh was also found to be expressed in and secreted from developing oocytes [30]. These data, together with the four CREs found in the 5'-flanking region of cyp19a1a, suggest that Lh signaling may play an important role in the regulation of aromatase activity during the process of sex reversal in the gilthead seabream.

It has been reported that aromatase inhibitor blocked sex reversal of the protandrous black porgy, resulting in a 100% male population [6], and promoted female-to-male change in G. erythrospilus [60]. These findings imply that the suppression of aromatase expression may play an important role in blocking male-to-female sex change and promoting male development. In Trimma okinawae, aromatase immunoreactivities were found to be much stronger in the ovarian interstitial, thecal, and granulosa cells of female-phase fish, while weak immunoreactivity was found only in the ovarian interstitial cells, but not the testis, in the gonads from male-phase fish [61]. Studying the seasonal profiles, starting from September, transcript levels of cyp19a1a in the ovarian portions of the male-developing gonads gradually decreased. Furthermore, females' ovaries expressed cyp19a1a at a significantly higher level than the ovarian portion of male gonads in December and January. Together with the rapid growth of the ovarian tissue in female-developing gonads, levels of plasma E2 in reversing females increase dramatically and are significantly higher than those in developing males. Higher levels of plasma E2 in developing and spawning females are more correlated with the growth of ovarian tissue in these fish (up to 8% of GSI). In males, slight increases of plasma E2 were also found in November and January. However, these increases may not be derived from the gonads. Instead, the increase in plasma E2 may come from extragonadal tissues, particularly the brain. Gonadectomy of male seabream in November did not significant alter plasma E2 levels (Klenke et al., unpublished results), and approximately 2.5-fold higher levels of cyp19a1b transcripts were found in the brains of spawning males compared to ambisexual fish (unpublished results).

Although there is no significant GSI change during the ambisexual stage, the expression of cyp19a1a is higher in the ambisexual stage and reaches its highest peak in July. These high levels of cyp19a1a expression did not reflect on the plasma E2 levels due to the small mass of the gonads in the ambisexual fish (no more than 1% of GSI). The expression of cyp19a1a may have a more local effect in the development of the ambisexual gonads. By September (the first month that gonadal sex can be distinguished), the GSI of female-developing fish is significantly higher than those of ambisexual fish. This significant increase comes from the continuous growth of the ovarian tissue. In contrast to female-developing fish, although the recognizable growth of the testicular tissue was observed in male-developing fish, the large degeneration of the ovarian portion of the ambisexual gonad occurred simultaneously, which led to no gain of the GSI of male-developing fish in September. In gilthead seabream, a portion of ovarian tissue is always present in the male-developing and spermiating gonads. Maintenance of this ovarian tissue enables males to enter the next sex-reversal cycle. The study of cyp19a1a expression in the ovarian portions of male-developing gonads has provided an understanding of the differential regulation of cyp19a1a in a unique reproductive situation where testicular and ovarian tissues reside in the same gonad and regulate each other. Our results revealed that a decrease in cyp19a1a transcript levels occurs in the ovarian portions of male-developing gonads. However, when compared with levels found in the ovarian portion of developing females, a significant difference was found only in December and January. Levels of cyp19a1a transcripts in the ovarian tissues of developing females may be diluted because of the rapid growth of ovary, which results in a GSI change from 1% to 8% during this period. In contrast, the rapid increase of GSI seen in the developing males is due to the growth of the testicular portion but not the ovarian portion, which degenerates in the developing males. Thus, when the mass of the ovarian tissues is considered, the total amount of ovarian cyp19a1a transcript would be much higher in developing females compared to developing males. This suggests that a downregulation of cyp19a1a expression in the ovarian tissue is associated with male development and a block in sex reversal.

The isolation and characterization of cyp19a1a cDNA and its 5'-flanking region has provided additional tools and information toward understanding the molecular basis of sex reversal in protandrous hermaphrodites. Our results indicate that the regulation of cyp19a1a gene expression is associated with the process of sex reversal. Additionally, they also suggest that Lh may serve as a major upstream signal in regulating the expression of the cyp19a1a gene during the process of sex reversal in the gilthead seabream. Results from our previous work indicated that sex reversal in the gilthead seabream is controlled by group dynamics and social activities. Specifically, old males have a much higher potential to conduct sex reversal than younger males do, and the presence of females inhibits the sex reversal of males [62]. Our current hypothesis is that changes in social activities and environmental cues are received by the brain, which initiates the different signal cascades through the pituitary (differential expression of Fsh and Lh) down to the gonadal level (differential expression of Fshr and Lhcgr), regulating the expression of cyp19a1a during the sex-reversal process.

ACKNOWLEDGMENTS

We are grateful to Steven Rogers and Eric Evans at the Aquaculture Research Center, University of Maryland Biotechnology Institute, for maintaining the experimental fish. Thanks are also extended to Katherine Kight and John Stubblefield for editing this manuscript.

FOOTNOTES

1 Supported by a Maryland Sea Grant Fellowship to T.-T.W. and award NA46RG0091 to Y.Z. Contribution 05–127 from the Center of Marine Biotechnology, University of Maryland Biotechnology Institute. Back

2 Correspondence: Yonathan Zohar, 701 E. Pratt St., Baltimore, MD 21202. FAX: 410 234 8896; zohar{at}umbi.umd.edu Back

3 Current address: Department of Animal Sciences, Purdue University, West Lafayette, IN 47907. Back

4 Current address: Department of Reproductive Biology, National Institute of Basic Biology, Nishigonaka 38, Myodaiji, Okazaki 444-8585 Aichi, Japan. Back

Received: 18 July 2005.

First decision: 7 September 2005.

Accepted: 9 January 2006.

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