The expression profiles of cyp19a1, sf-1, esrs and gths in the brain-pituitary during gonadal sex differentiation in juvenile Japanese eels.

Eels are gonochoristic species whose gonadal differentiation initiates at the yellow eel stage and is influenced by environmental factors. We revealed some sex-related genes were sex dimorphically expressed in gonads during gonadal sex differentiation of Japanese eel (Anguilla japonica); however, the expression of sex-related genes in the brain-pituitary during gonadal sex differentiation in eels is still unclear. This study aimed to investigate the sex-related gene expressions in the brain-pituitary and tried to clarify their roles in the brain and gonads during gonadal sex differentiation. Based on our previous histological study, the control eels developed as males, and estradiol-17ß (E2) was used for feminization. Our results showed that during testicular differentiation, the brain cyp19a1 transcripts and aromatase proteins were increased significantly; moreover, the cyp19a1, sf-1, foxl2s, and esrs (except gperb) transcripts in the midbrain/pituitary also were increased significantly. Forebrain gnrh1 transcripts increased slightly during gonadal differentiation of both sexes, but the gnrhr1b and gnrhr2 transcripts in the midbrain/pituitary were stable during gonadal differentiation. The expression levels of gths and gh in the midbrain/pituitary were significantly increased during testicular differentiation and were much higher in males than in E2-feminized females. These results implied that endogenous estrogens might play essential roles in the brain/pituitary during testicular differentiation, sf-1, foxl2s, and esrs may have roles in cyp19a1 regulation in the midbrain/pituitary of Japanese eels. For the GnRH-GTH axis, gths, especially fshb, may be regulated by esrs and involved in regulating testicular differentiation and development in Japanese eels.

Elevated estradiol-17ß levels inhibit final oocyte maturation via G protein-coupled estrogen receptor (Gper) in yellowfin porgy, Acanthopagrus latus.

Yellowfin porgy a protandrous teleost, exhibits asynchronous oocyte development and multiple spawning. Seasonal profiles of plasma estradiol-17ß (E2) levels showed a peak in three-year-old females during the spawning season, when batches of fully-grown oocytes undergo final oocyte maturation (FOM). Because E2 has been shown to inhibit FOM via the G protein-coupled estrogen receptor (Gper) in several teleost species, we investigated the role of this "paradoxical" increase in E2 during FOM in yellowfin porgy. In vivo treatment with a GnRH-agonist stimulated germinal vesicle breakdown (GVBD) and increased E2 plasma levels, and ovarian cyp19a1a transcripts, confirming the increase in E2 production at the time of FOM. Ovarian transcripts of gper peaked at the time of FOM, indicating an increase in ovarian responsiveness to Gper-mediated E2 effects. In vitro, E2 and the Gper agonist, G-1, inhibited the stimulatory effect of maturation-inducing steroids (MIS) on GVBD, while an aromatase inhibitor enhanced the MIS effect, in agreement with a physiological inhibitory role of E2 on FOM via Gper. Immunohistological studies showed that the Gper protein was specifically located on the oocyte plasma membrane. Ovarian membranes displayed high-affinity and limited-capacity specific [3H]-E2 receptor binding which was displaced by G-1, characteristic of Gper. Expression of gper increased at the time of FOM in mid-vitellogenic oocytes, but not in larger oocytes undergoing GVBD. These results suggest increases in both E2 production and E2 responsiveness via Gper upregulation in mid-vitellogenic oocytes, may maintain meiotic arrest in this oocyte stage class during the period when full-grown oocytes are undergoing FOM. This study indicates a critical involvement of E2 in the control of asynchronous oocyte maturation and the multiple spawning pattern in Sparidae.

Dmrt1 (doublesex and mab-3-related transcription factor 1) expression during gonadal development and spermatogenesis in the Japanese eel.

Dmrt1, doublesex- and mab-3-related transcription factor-1, has been suggested to play critical roles in male gonadogenesis, testicular differentiation and development, including spermatogenesis, among different vertebrates. Vasa is a putative molecular marker of germ cells in vertebrates. In this study, we cloned the full-length dmrt1 cDNA from Japanese eel, and the protein comprised 290 amino acids and presented an extremely conserved Doublesex and Mab-3 (DM) domain. Vasa proteins were expressed in gonadal germ cells in a stage-specific manner, and were expressed at high levels in PGC and spermatogonia, low levels in spermatocytes, and were absent in spermatids and spermatozoa of Japanese eels. Dmrt1 proteins were abundantly expressed in spermatogonia B cells, spermatocytes, spermatids, but not in spermatozoa, spermatogonia A and Sertoli cells. To our knowledge, this study is the first to show a restricted expression pattern for the Dmrt1 protein in spermatogonia B cells, but not spermatogonia A cells, of teleosts. Therefore, Dmrt1 might play vital roles at the specific stages during spermatogenesis from spermatogonia B cells to spermatids in the Japanese eel. Moreover, the Dmrt1 protein exhibited a restricted localization in differentiating oogonia in the early differentiating gonad (ovary-like structure) of male Japanese eels and in E2-induced feminized Japanese eels. We proposed that dmrt1 may be not only required for spermatogenesis but might also play a role in oogenesis in the Japanese eel.

Gonadal development and expression of sex-specific genes during sex differentiation in the Japanese eel.

The process of gonadal development and mechanism involved in sex differentiation in eels are still unclear. The objectives were to investigate the gonadal development and expression pattern of sex-related genes during sex differentiation in the Japanese eel, Anguilla japonica. For control group, the elvers of 8-10cm were reared for 8months; and for feminization, estradiol-17ß (E2) was orally administered to the elvers of 8-10cm for 6months. Only males were found in the control group, suggesting a possible role of environmental factors in eel sex determination. In contrast, all differentiated eels in E2-treated group were female. Gonad histology revealed that control male eels seem to differentiate through an intersexual stage, while female eels (E2-treated) would differentiate directly from an undifferentiated gonad. Tissue distribution and sex-related genes expression during gonadal development were analyzed by qPCR. The vasa, figla and sox3 transcripts in gonads were significantly increased during sex differentiation. High vasa expression occurred in males; figla and sox3 were related to ovarian differentiation. The transcripts of dmrt1 and sox9a were significantly increased in males during testicular differentiation and development. The cyp19a1 transcripts were significantly increased in differentiating and differentiated gonads, but did not show a differential expression between the control and E2-treated eels. This suggests that cyp19a1 is involved both in testicular differentiation and development in control males, and in the early stage of ovarian differentiation in E2-treated eels. Importantly, these results also reveal that cyp19a1 is not a direct target for E2 during gonad differentiation in the eel.

Neuroendocrine gene expression reveals a decrease in dopamine D2B receptor with no changes in GnRH system during prepubertal metamorphosis of silvering in wild Japanese eel.

Silvering is a prepubertal metamorphosis preparing the eel to the oceanic reproductive migration. A moderate gonad development occurs during this metamorphosis from the sedentary yellow stage to the migratory silver stage. The aim of this study was to elucidate the molecular aspects of various endocrine parameters of BPG axis at different ovarian developmental stages in wild yellow and silver female Japanese eels. The GSI of the sampled female eels ranged between 0.18 and 2.3%, corresponding to yellow, pre-silver and silver stages. Gonad histology showed changes from previtellogenic oocytes in yellow eels to early vitellogenic oocytes in silver eels. Both serum E2 and T concentrations significantly increased with ovarian development indicating a significant activation of steroidogenesis during silvering. In agreement with previous studies, significant increases in pituitary gonadotropin beta subunits FSH-ß and LH-ß transcripts were also measured by qPCR, supporting that the activation of pituitary gonadotropin expression is likely responsible for the significant ovarian development observed during silvering. We investigated for the first time the possible brain neuroendocrine mechanisms involved in the activation of the pituitary gonadotropic function during silvering. By analyzing the expression of genes representative of the stimulatory GnRH control and the inhibitory dopaminergic control. The transcript levels of mGnRH and the three GnRH receptors did not change in the brain and pituitary between yellow and silver stages, suggesting that gene expression of the GnRH system is not significantly activated during silvering. The brain transcript levels of tyrosine hydroxylase, limiting enzyme of DA synthesis did not change during silvering, indicating that the DA synthesis activity was maintained. In contrast, a significant decrease in DA-D2B receptor expression in the forebrain and pituitary was observed, with no changes in DA-D2A receptor. The decrease in the pituitary expression of DA-D2BR during silvering would allow a reduced inhibitory effect of DA. We may raise the hypothesis that this regulation of D2BR gene expression is one of the neuroendocrine mechanisms involved in the slight activation of the pituitary gonadotropin and gonadal activity that occur at silvering.

17,20ß,21-Trihydroxy-4-pregnen-3-one biosynthesis and 20ß-hydroxysteroid dehydrogenase expression during final oocyte maturation in the protandrous yellowfin porgy, Acanthopagrus latus.

The purpose of this study was to investigate the physiological maturation-inducing steroid (MIS) in the marine protandrous yellowfin porgy (Acanthopagrus latus). Female fish were injected with 2 doses of LHRH analog (10 and 40 µg per kg). Ovarian tissue was obtained at 6 h intervals for in vitro analysis of oocyte maturation. The most effective steroids for inducing in vitro maturation (germinal vesicle breakdown and GVBD) in cultured oocytes were 17,20ß-dihydroxy-4-pregnen-3-one (17,20ßP) and 17,20ß,21-trihydroxy-4-pregnen-3-one (20ß-S). 17,20ßP was less potent than 20ßS in inducing oocyte maturation. At higher concentrations, 11-deoxycortisol, 17α-hydroxy-progesterone, and 20ß-21-dihydroxy-4-pregnen-3-one also significantly induced oocyte maturation. A tritiated precursor [(3)H]-pregnenolone, was cultured in vitro together with the maturing ovarian tissue. The tritiated metabolites were purified and identified by solvent extraction, HPLC, TLC, acetylation reaction and recrystallization. HPLC, TLC and recrystallization analysis showed that significant levels of tritiated 11-deoxycortisol (a precursor of 20ß-S) and 20ß-S, but not 17,20ßP, were biosynthesized from [(3)H]-pregnenolone. Similar TLC profiles were obtained from the tritiated products that were isolated from the HPLC/TLC 20ß-S fraction and standard 20ß-S after the acetylation reaction. Constant specific radioactivity of tritiated 11-deoxycortisol and 20ß-S but not 17,20ßP by recrystallization was obtained in the tritiated metabolites isolated from HPLC and TLC fractions. The expression of 20ß-hydroxysteroid dehydrogenase (20ß-HSD) mRNA (a key enzyme that converts 11-deoxycortisol to 20ß-S) was significantly increased in maturing ovarian tissue. This study provides the first evidence that 20ß-S is converted from 11-deoxycortisol and is the possible MIS in yellowfin porgy.

Differential regulation of the expression of cytochrome P450 aromatase, estrogen and androgen receptor subtypes in the brain-pituitary-ovarian axis of the Japanese eel (Anguilla japonica) reveals steroid dependent and independent mechanisms.

This study aimed at investigating the role of sexual steroids in the regulation of the expression of the single aromatase gene and steroid receptor subtypes in the brain-pituitary-ovarian axis of the Japanese eel. Unlike other teleosts, which possess duplicated genes for aromatase, cyp19a1a and cyp19a1b, expressed in the gonads and in the brain, respectively, eel species possess a single cyp19a1. Phylogenetic analysis indicated that eel brain/gonadal cyp19a1 branches at the basis of both teleost gonadal cyp19a1a and brain cyp19a1b clades. Female eels treated with catfish pituitary homogenate (CPH) to induce sexual maturation showed an increase in the expression of cyp19a1 and aromatase enzymatic activity in the brain and in the ovaries. Treatments with sex steroids (estradiol-17ß, E(2) or testosterone, T) revealed that the increase in cyp19a1 expression in the brain may result from E(2)-specific induction. In contrast, the increase in cyp19a1 expression in the ovaries of CPH-treated eels is a result of steroid-independent control, probably from a direct effect of gonadotropins contained in the pituitary extract. Analysis of the expression of estrogen and androgen receptor subtypes, esr-α, esr-ß, ar-α and ar-ß, in eels treated with CPH or sex steroids revealed differential regulations. In CPH-treated eels, the expression of esr-α and ar-α was significantly increased in the brain, while the expression of ar-α and ar-ß was increased in the ovaries. No change was observed in esr-ß in any organ. Steroid treatments induced an upregulation by E(2) of esr-α, but not esr-ß expression, in the brain, pituitary and ovaries, while no autoregulation by T of its own receptors could be observed. These results reveal both steroid-dependent and -independent mechanisms in the regulation of cyp19a1 and steroid receptor subtype expression in the eel.

Involvement of Transforming Growth Factor Beta Family Genes in Gonadal Differentiation in Japanese Eel, Anguilla japonica, According to Sex-Related Gene Expressions.

The gonochoristic feature with environmental sex determination that occurs during the yellow stage in the eel provides an interesting model to investigate the mechanisms of gonadal development. We previously studied various sex-related genes during gonadal sex differentiation in Japanese eels. In the present study, the members of transforming growth factor beta (TGF-ß) superfamily were investigated. Transcript levels of anti-Müllerian hormone, its receptor, gonadal soma-derived factor (amh, amhr2, and gsdf, respectively) measured by real-time polymerase chain reaction (qPCR) showed a strong sexual dimorphism. Transcripts were dominantly expressed in the testis, and their levels significantly increased with testicular differentiation. In contrast, the expressions of amh, amhr2, and gsdf transcripts were low in the ovary of E2-feminized female eels. In situ hybridization detected gsdf (but not amh) transcript signals in undifferentiated gonads. amh and gsdf signals were localized to Sertoli cells and had increased significantly with testicular differentiation. Weak gsdf and no amh signals were detected in early ovaries of E2-feminized female eels. Transcript levels of amh and gsdf (not amhr2) decreased during human chorionic gonadotropin (HCG)-induced spermatogenesis in males. This study suggests that amh, amhr2, and especially gsdf might be involved in the gene pathway regulating testicular differentiation of Japanese eels.

Sex differentiation and sex change in the protandrous black porgy, Acanthopagrus schlegeli.

Protandrous black porgy fish, Acanthopagrus schlegeli, have a striking life cycle with a male sex differentiation at the juvenile stage and male-to-female sex change at 3 years of age. We had characterized the sex differentiation and sex change in this species by the integrative approaches of histology, endocrine and molecular genetics. The fish differentiated in gonad at the age around 4-months and the gonad further developed with a bisexual gonad for almost for 3 years and sex change at 3 year of age. An antagonistic relationship in the testicular and ovarian tissues was found during the development of the gonadal tissue. Male- (such as sf-1, dmrt1, dax-1 and amh) and female- (such as wnt4, foxl2 and cyp19a1a) promoting genes were associated with testicular and ovarian development, respectively. During gonadal sex differentiation, steroidogenic pathway and estrogen signaling were also highly expressed in the brain. The increased expression of sf-1 and wnt4, cyp19a1a in ovarian tissue and decreased expression of dax-1 in the ovarian tissue may play important roles in sex change from a male-to-female. Endocrine factors such as estradiol and luteinizing hormone may also involve in the natural sex change. Estradiol induced the expression of female-promoting genes and resulted in the precocious sex change in black porgy. Our series of studies shed light on the sex differentiation and sex change in protandrous black porgy and other animals.

The presence and ancestral role of gonadotropin-releasing hormone in the reproduction of scleractinian coral, Euphyllia ancora.

The objectives of this study were to investigate the presence of immunoreactive GnRH (irGnRH) in scleractinian coral, Euphyllia ancora, study its seasonal variation, and evaluate its biological activity. irGnRH was detected and quantified in coral polyps. The biological activity of coral irGnRH was tested on pituitary cells from black porgy by evaluating its ability to stimulate LH release. Coral extracts (10(-9)-10(-5) M irGnRH) as well as mammalian (m) GnRH agonist (10(-10)-10(-6) M) had a similar dose-dependent effect on LH release. Furthermore, GnRH receptor antagonist dose-dependently inhibited the stimulation of LH release in response to coral extracts (10(-5) M irGnRH) and mGnRH agonist (10(-6) M). Peak levels of irGnRH (10-fold increase) were observed during the spawning period in a 3-yr investigation. Significantly higher aromatase activity and estradiol (E2) levels were also detected during the period of spawning compared with the nonreproductive season. In in vivo experiments, mGnRH agonist time- and dose-dependently stimulated aromatase activity as well as the concentrations of testosterone and E2 in free and glucuronided forms in coral. In conclusion, our data indicate that irGnRH does exist in coral, with its ability to stimulate LH release in fish. Seasonal variations of coral irGnRH, with a dramatic increase during the spawning period, concomitant to that in aromatase and E2, as well as the ability of mGnRH agonist to stimulate coral aromatase, steroidogenesis, and steroid glucuronization suggest that irGnRH plays an important role in the control of oocyte growth and mass spawning in corals.

Expression of aromatase in radial glial cells in the brain of the Japanese eel provides insight into the evolution of the cyp191a gene in Actinopterygians.

The cyp19a1 gene that encodes aromatase, the only enzyme permitting conversion of C19 aromatizable androgens into estrogens, is present as a single copy in the genome of most vertebrate species, except in teleosts in which it has been duplicated. This study aimed at investigating the brain expression of a cyp19a1 gene expressed in both gonad and brain of Japanese eel, a basal teleost. By means of immunohistochemistry and in situ hybridization, we show that cyp19a1 is expressed only in radial glial cells of the brain and in pituitary cells. Treatments with salmon pituitary homogenates (female) or human chorionic gonadotrophin (male), known to turn on steroid production in immature eels, strongly stimulated cyp19a1 messenger and protein expression in radial glial cells and pituitary cells. Using double staining studies, we also showed that aromatase-expressing radial glial cells exhibit proliferative activity in both the brain and the pituitary. Altogether, these data indicate that brain and pituitary expression of Japanese eel cyp19a1 exhibits characteristics similar to those reported for the brain specific cyp19a1b gene in teleosts having duplicated cyp19a1 genes. This supports the hypothesis that, despite the fact that eels also underwent the teleost specific genome duplication, they have a single cyp19a1 expressed in both brain and gonad. Such data also suggest that the intriguing features of brain aromatase expression in teleost fishes were not gained after the whole genome duplication and may reflect properties of the cyp19a1 gene of ancestral Actinopterygians.

Differential expression and regulation of gonadotropins and their receptors in the Japanese eel, Anguilla japonica.

Eel species have a striking life cycle with a blockade of puberty until the oceanic migration. We report the first molecular data on eel gonadotropin receptors. The partial sequences cloned covered two-third of the open reading frame and included most of the extracellular and transmembrane domains. Phylogenetic analysis partitioned the two eel gonadotropin receptors into the two teleost FSHR and LHR clusters, respectively. Real-time quantitative RT-PCR was used to quantify the expression of eel gonadotropins and their receptors. Similar levels of pituitary FSH-beta and LH-beta transcripts were found in the immature previtellogenic female eels. In contrast, ovarian FSHR mRNA level was at 100- to 185-fold higher than that of LHR. This revealed that FSHR rather LHR would mediate gonadotropin stimulation of the early stages of ovarian growth. Chronic treatment with fish pituitary homogenates, applied to induce eel sexual maturation, stimulated pituitary LH-beta but suppressed FSH-beta transcripts. In the ovaries, both FSHR and LHR mRNA were significantly increased in experimentally matured eels. Treatments with sexual steroids showed a stimulatory effect of estradiol-17beta (E(2)) on pituitary LH-beta mRNA levels, while FSH-beta transcripts were suppressed by E(2) or testosterone (T). In contrast, neither E(2) nor T-treatment had any significant effect on ovarian FSHR nor LHR transcripts. This suggests that steroid feedbacks may be responsible for the opposite regulation of pituitary gonadotropins in experimentally matured eels, but are not involved in the regulation of gonadotropin receptors. In conclusion, these are the first data on the sequence, expression and regulation of gonadotropin receptors in the eel. They provide new foundation for basic and applied research on eel reproduction.

Identification of 17,20beta,21-trihydroxy-4-pregnen-3-one as an oocyte maturation-inducing steroid in black porgy, Acanthopagrus schlegeli.

The identity of the maturation-inducing steroid (MIS) in black porgy, Acanthopagrus schlegeli, a marine protandrous teleost, is unknown. Previous studies demonstrated that two teleost MISs, the progestins 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S) and 17,20beta-dihydroxy-4-pregnen-3-one (DHP) can induce maturation of black porgy oocytes in vitro. The purpose of the present study was to identify the major progestin produced during oocyte maturation (OM) in black porgy and investigate whether its secretion increases during this process. Females were injected twice with a LHRH analog to induce OM. Ovarian follicles undergoing OM were incubated in vitro with tritiated [3H]pregnenolone precursor and the tritiated products were extracted, purified, and identified by HPLC, TLC, acetylation, and recrystallization. Significant amounts of tritiated products were biosynthesized from [3H]pregnenolone that co-migrated with 20beta-S but not with DHP on HPLC and TLC. Similar TLC profiles were obtained with the tritiated products isolated from the HPLC/TLC 20beta-S fraction and standard 20beta-S after the acetylation reaction. The identity of the tritiated products as 20beta-S was confirmed by recrystallization. 20beta-S had a slightly higher potency than DHP in the inducing in vitro final oocyte maturation. Plasma 20beta-S concentrations increased significantly during the oocyte maturation after injection with a LHRH analog. The present data suggest that 20beta-S is the MIS in black porgy.

Seasonal profiles of brain and pituitary gonadotropin-releasing hormone and plasma luteinizing hormone in relation to sex change of protandrous black porgy, Acanthopagrus schlegeli.

Three molecular variants of GnRH in the brain (sbGnRH, sGnRH, and cGnRH-II) and two forms in the pituitary (sbGnRH and sGnRH) were detected in protandrous black porgy, Acanthopagrus schlegeli using chromatographic and immunological methods. In juvenile fish, brain sbGnRH, sGnRH, and cGnRH-II levels increased in May and reached their highest levels in July and August (the nonspawning season) and in January through March (the spawning season). In fish aged 1 yr and older, high levels of brain sbGnRH and sGnRH were detected in September, November, and February-March, but the levels of brain cGnRH-II remained constant. A gradual increase in pituitary sbGnRH was detected in juvenile fish from July to March. In fish aged 1+ yr, pituitary sbGnRH levels were high in September and March-May, but low in January-February. A close correlation between pituitary sbGnRH and plasma LH levels was found in juvenile fish and in those aged 1+ yr. In fish aged 2+ yr, significantly lower levels of plasma LH was detected during the nonspawning period in fish that changed sex compared with the fish that remained as males. Higher plasma LH levels were detected in the sex-changing fish from artificially sex-reversed female to male. FSH receptor and LH receptor transcripts were higher in bisexual testicular tissue than in ovarian tissue in 2+-yr-old fish. Direct effects of hCG on sex change were studied and the results show that exogenous hCG did not stimulate gonadal aromatase activity in 2+-yr-old fish. Therefore, it is suggested that high and basal levels of plasma LH during the nonspawning season correlate with the development of male and female gonad, respectively, in black porgy. This important role of the neuroendocrine system in sex change (for male direction) is proposed in hermaphroditic fish.

Oocyte maturation-inducing steroids in protandrous black porgy, Acanthopagrus schlegeli.

The study objectives aimed to investigate the maturation-inducing steroid (MIS) in marine protandrous black porgy, Acanthopagrus schlegeli. The characteristics of oocyte maturation were also described. Females were injected with two successive doses of LHRH analog (LHRH-A, 10 and 50 microg/kg of fish). The ovarian tissue was obtained at 6-h intervals for in vitro oocyte maturation. Both 17,20 beta-dihydroxy-4-pregnen-3-one (DHP) and 17,20 beta,21-trihydorxy-4-pregnen-3-one (20 beta-S) were the most effective steroids to induce in vitro maturation (e.g. germinal vesicle breakdown, GVBD) in oocytes cultured for either 24 h or 1 min. 20 beta-S had a better potency than DHP in inducing oocyte maturation. 17-hydroxyprogesterone, 11-deoxycortisol, and 20 beta-21-dihydroxy-4-pregnen-3-one also significantly induced oocyte maturation at high concentrations. The process of oocyte maturation (after the injection of LHRH analog) was founded to be divided into four stages: hormone-insensitive stage (insensitive to gonadotropin and MIS); MIS-insensitive (respond to gonadotropin, but not MIS); MIS-sensitive (respond to MIS); and spontaneous stage (GVBD in the hormone-free condition), respectively. Cycloheximide blocked GVBD at the MIS-insensitive stage, control (hormone-free), and hormone-induced GVBD at the MIS-sensitive stage in a dose-dependent effect.

Aromatase inhibitors block natural sex change and induce male function in the protandrous black porgy, Acanthopagrus schlegeli Bleeker: possible mechanism of natural sex change.

The objectives of the present study were to investigate the effects of oral administration of aromatase inhibitors on sex change, milt volume, 11-ketotestosterone (11-KT), and LH in plasma; aromatase activity in gonad, pituitary, and brain in the protandrous fish, black porgy (Acanthopagus schlegeli Bleeker). Two-year-old functional male black porgy were divided into two groups; one was fed a control diet and the other was fed a diet mixed with aromatase inhibitors (AIs; fadrozole and 1,4,6-androstatriene-3,17-dione, each 10 mg/kg feed) for 8.5 mo. A significantly higher gonadosomatic index was observed in the AI group. Fish treated with AIs showed complete suppression of natural sex change. Significantly higher levels of plasma 11-KT, LH, and milt volume were shown in the AI group than the controls. Lower aromatase activity in the gonad, pituitary, forebrain, midbrain, and hindbrain in concordance with the suppression of sex change was observed in the AI group. The data show that aromatase is directly involved in the mechanism of natural sex change of protandrous black porgy. AIs also enhanced male function in concordance with the elevated plasma levels of 11-KT and spermiation in milt volume.