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.     

The mRNA expression of P450 aromatase, gonadotropin beta-subunits and FTZ-F1 in the orange-spotted grouper (Epinephelus Coioides) during 17alpha-methyltestosterone-induced precocious sex change

The orange-spotted grouper Epinephelus coioides is a protogynous hermaphroditic fish, but the physiological basis of its sex change remains largely unknown. In the present study, the 2-year-old orange-spotted grouper was induced to change sex precociously by oral administration of 17alpha-methyltestosterone (MT, 50 mg/Kg diet, twice a day at daily ration of 5% bodyweight) for 60 days. The serum testosterone levels were significantly elevated after MT treatment for 20 and 40 days as compared to control, but the levels of serum estradiol (E(2)) remained unchanged. The expression of P450aromA in the gonad significantly decreased after MT treatment for 20, 40, and 60 days. Accordingly, the enzyme activity of gonadal aromatase was also lower. The expression of FSHbeta subunit in the pituitary was significantly decreased after MT treatment for 20 days, but returned to the control levels after 40 and 60 days; however, the expression of LHbeta subunit was not altered significantly by MT treatment. The expression of FTZ-F1 in the gonad also decreased significantly in response to MT treatment for 40 and 60 days, but its expression in the pituitary was not altered significantly. Interestingly, when tested in vitro on ovarian fragments, MT had no direct effect on the expression of P450aromA and FTZ-F1 as well as the activity of gonadal aromatase, suggesting that the inhibition of gonadal P450aromatase and FTZ-F1 by MT may be mediated at upper levels of the brain-pituitary-gonadal axis. Taken together, these results indicated that FSH, P450aromA, FTZ-F1, and serum testosterone are associated with the MT-induced sex change of the orange-spotted grouper, but the cause-effect relationship between these factors and sex change in this species remains to be characterized.     

Characterization of estrogen receptor beta2 and expression of the estrogen receptor subtypes alpha, beta1, and beta2 in the protandrous black porgy (Acanthopagrus schlegeli) during the sex change process

Estrogens play an important role in many physiological processes in both female and male vertebrates, mediated by specific nuclear receptor, estrogen receptors (ERs). We have isolated a third ER (ERbeta2), which was found to contain 2004 nucleotides including an open reading frame that encodes 667 amino acids. We have also cloned ERalpha and ERbeta1 from the published information (GenBank accession nos. AY074780 and AY074779) and investigated the expression pattern of these ER subtypes in the gonads during gonad sex change of black porgy by quantitative polymerase chain reaction. Maturity stages can be divided into five stages during the sex change process from immature male to female (immature male, mature male, male of mostly testis, male of mostly ovary and mature female). The expression of ERalpha mRNA was highest in the ovary of mature female, followed by the testis of mature male and testicular portion of mostly testis. ERbeta1 expression was higher in the mature testis and ovary than in the gonads of other maturity stages. In contrast to that, ERbeta2 was highest in the ovary of mature female, and significantly lower levels of ERbeta2 expression were observed in the gonads of the other maturity stages. The present study describes the molecular characterization of ERbeta2, and documents the expression changes of three ER subtypes during sex change process of the protandrous black porgy.     

Estradiol-17beta triggers luteinizing hormone release in the protandrous black porgy (Acanthopagrus schlegeli Bleeker) through multiple interactions with gonadotropin-releasing hormone control.

We investigated the mechanism of estradiol-17beta (E2) action on stimulation of LH (=gonadotropin II) release in the black porgy fish (Acanthopagrus schlegeli Bleeker) using an in vivo approach and primary cultures of dispersed pituitary cells in vitro. In vivo, E2 but not androgens (testosterone [T] and 11-ketotestosterone [11-KT]) significantly stimulated plasma LH in a dose-dependent manner. Estradiol-17beta also increased brain content of seabream GnRH. GnRH antagonist prevented E2 stimulation of LH release in vivo, indicating that the effect of E2 on LH was mediated by GnRH. In vitro, sex steroids (E2, T, 11-KT) alone had no effect on basal LH release in the cultured pituitary cells, but GnRH significantly stimulated LH release. Estradiol-17beta potentiated GnRH stimulation of LH release, an effect that was inhibited by GnRH antagonist, and 11-KT, but not T, also potentiated GnRH stimulation of LH release. The potentiating effect of 11-KT on GnRH-induced LH release in vitro was stronger than that of E2. These data suggest that E2 triggers LH release in vivo by acting both on GnRH production at the hypothalamus and on GnRH action at the pituitary. In contrast, 11-KT may only stimulate GnRH action at the pituitary. The E2) induction of LH release, through multiple interactions with GnRH control, supports a possible central role of E2in the sex change observed in the protandrous black porgy.     

Role of aromatase and androgen receptor expression in gonadal sex differentiation of ZW/ZZ-type frogs,Rana rugosa

To elucidate the mechanisms of amphibian gonadal sex differentiation, we examined the expression of aromatase and androgen receptor (AR) mRNAs for days 17-31 after fertilization. The effects of inhibitors and sex steroid hormones were also examined. In ZZ males, expression of AR decreased after day 19, while aromatase expression was low throughout the sampling period. Males treated with 17beta-estradiol (E2) showed increasing aromatase expression after day 21, and formed ovaries. AR antagonist treatment also induced high-level aromatase expression and ovarian differentiation. In males co-treated with an aromatase inhibitor and E2, the undifferentiated gonads developed into testes despite high-level aromatase expression. Males treated with androgen and E2 before and during an estrogen sensitive period, respectively, also formed testes. In ZW females, AR expression persisted at a low-level, while aromatase expression increased after day 18. Short-term treatment with an aromatase inhibitor was ineffective in preventing ovarian differentiation, whereas long-term treatment resulted in testes developing from ovarian structure. Compared with the ZZ males and ZW females, WW females did not exhibit detectable expression of AR, suggesting that the active AR gene(s) itself, or a putative gene regulating AR gene expression, is located on Z chromosomes. From the time lag of aromatase expression between ZW females and ZZ males treated with E2 and the effect of AR antagonist, it was found that in males elevated AR expression suppresses aromatase expression directly or indirectly. Consequently, endogenous androgens, accumulated by blocking estrogen biosynthesis, induced testicular differentiation. The gonadogenesis of males is dependent on sex hormone, whereas that of females has evolved to hormone-independence.     

Differential expression of neural and gonadal aromatase enzymatic activities in relation to gonadal development in Japanese eel, Anguilla japonica

The objectives of the present study were to investigate the enzymatic characteristics of brain aromatase in Japanese eel, Anguilla japonica, as well as the correlations between aromatase activities in various tissues (brain regions, pituitary, and gonads) and ovarian development. Eel brain aromatase exhibited a K(m) of 75 nM and a V(max) of 1.14 fmol/min mg protein (91.5 fmol/h mg protein), indicating an enzymatic activity much lower than in other teleosts but similar to that in mammals. This supports the hypothesis of the occurrence of a single aromatase gene in eels (representative of an ancient group of teleosts, Elopomorphs), as in mammals, but unlike what is observed in more recent teleosts. Aromatase inhibitors could significantly suppress brain and pituitary aromatase activity. There was no significant sex difference in aromatase activity in the forebrain, midbrain, hindbrain, or pituitary, but there was in the gonads, where aromatase could be detected in the ovaries but not in the testes, in accordance with the role of this enzyme in ovarian differentiation. Comparison with another teleost, black porgy (Percomorph), under the same experimental conditions, further confirmed the low activity of aromatase in the eel. We investigated variations in brain, pituitary, and gonad aromatase activity in relation to ovarian development in control female eels (gonadosomatic index, GSI, 0.1-1.6%) as well as in eels treated with pituitary extract (experimental ovarian maturation; GSI up to 25%). Differential expression of neural and gonadal aromatase was observed in relation to the course of gonadal development. Pituitary aromatase activity increased with GSI at all stages. Brain (specially forebrain) aromatase activity significantly increased in early vitellogenic control eels (GSI>0.8%) and in treated eels. The low activity of eel aromatase may be related to the characteristic life cycle of the eel, in which there is a long delay of the onset of puberty before oceanic reproductive migration.     

Molecular characterization of gonadotropin subunits and gonadotropin receptors in black porgy,Acanthopagrus schlegeli: Effects of estradiol-17β on mRNA expression profiles

The cDNAs of three gonadotropin (GTH) subunits (GTHα, FSHβ, and LHβ) and two GTH receptors (FSHR and LHR) from pituitary and gonads of black porgy were cloned. The nucleotide sequences of the GTHα, FSHβ, and LHβ cDNA were 354, 363, and 414 base pairs (bps) in length with open reading frames (ORF) encoding peptides of 117, 120, and 137 amino acids, respectively. The FSHR and LHR cDNA was 2118 and 2076 bps in length with ORFs encoding peptides of 705 and 691 amino acids, respectively. To study the mechanism of the estradiol-17β (E₂) action, we examined the expression pattern of GTH subunit mRNAs in pituitary and GTH-receptor mRNAs in gonads, and the changes of plasma E₂ level when E₂ treatment was applied to immature black porgy. E₂ treatment increased mRNA expression levels of the genes and plasma E₂ levels, indicating that E₂ stimulated the increases in GTH subunit and GTH-receptor mRNAs. These data indicate that E₂ plays an important regulatory role in the brain–pituitary–gonad axis of immature black porgy. We provide the molecular characterization and expression of the GTH subunits and GTH receptors during sex change in the protandrous black porgy.     

Regulation of plasma gonadotropin II secretion by sex steroids, aromatase inhibitors, and antiestrogens in the protandrous black porgy, Acanthopagrus schlegeli Bleeker

Plasma gonadotropin II (GTH II) concentrations were significantly higher (approx. 15-20-fold) in estradiol-17beta (E(2)) treated (1.0 microg or 2.5 microg g(-1) body weight) female black porgy from days 4 to 12 compared with the control. E(2) (1 microg g(-1) wt.) had a stronger stimulation on plasma GTH II in early recrudescent phase (low GSI) males (11-fold) than in high GSI and late spermiating males (2.6-fold, P< 0.05). No effect of androgens (testosterone, T; 5alpha-dihydrotestosterone, DHT) on plasma GTH II levels was observed either sex. The levels of plasma GTH II were stimulated in 1,4,6-androstatriene-3,17-dione (ATD, 1 microg g(-1), 2 microg g(-1) body wt.) and fadrozole-treated (1 microg g(-1), 3 microg g(-1) body wt.) groups compared to control. Tamoxifen (1 microg g(-1), 3 microg g(-1) body wt.) but not enclomiphene could stimulate high GTH II levels in plasma. In another experiment of ATD in combination with T, T treatment further attenuated the ATD stimulation of plasma GTH II levels. We concluded that GTH II secretion is positively regulated by an estrogen-specific effect in female and male black porgy. Gonadal stage had significant effects on the responsiveness of GTH II to E(2) stimulation in males. A negative aromatase-dependent feedback control of plasma GTH II levels was also suggested in the protandrous black porgy, Acanthopagrus schlegeli.     

Effects of gonadotropin-releasing hormone analog (GnRHa) on steroidogenic factor-1 (SF-1) and estrogen receptor beta (ERbeta) gene expression in the black porgy (Acanthopagrus schlegeli)

We examined effects of GnRHa on expression of steroidogenic factor-1 (SF-1) and estrogen receptor beta (ERbeta) in the pituitary and gonad of protandrous black porgy (Acanthopagrus schlegeli). Fish were intraperitoneally injected with 0.2 microg GnRHa/g fish and then pituitary, gonad and plasma were sampled at 0, 6, 12, 24 and 48 h after injection. In gonad, the mRNA levels of the SF-1 were high at 6 h post injection, and then continuously decreased until 24 h; high expression of ERbeta mRNA levels was only observed at 12 h. In contrast, pituitary SF-1 mRNA levels were very low during the experimental period. GnRHa stimulation caused a significant increase of plasma testosterone (T) and estradiol-17beta (E(2)) after 24 h. We suggest that SF-1 and ERbeta play an important role in the development of gonad and these genes are involved with sex change in fish.     

Aromatase inhibitor induces complete sex change in the protogynous honeycomb grouper (Epinephelus merra)

The protogynous hermaphrodite fish change sex from female to male at the certain stages of life cycle. The endocrine mechanisms involved in gonadal restructuring throughout protogynous sex change are not clearly understood. In the present study, we implanted maturing female honeycomb groupers with nonsteroidal aromatase inhibitor (AI), Fadrozole (0, 1, and 10 mg/fish) and examined changes in gonadal structures and serum levels of sex steroid hormones 2(1/2) months after implantation. The ovaries of control females had oocytes undergoing active vitellogenesis, whereas AI caused females to develop into functional males. These males had testes, which were indistinguishable in structure from those of normal males, but bigger in size, and completed all stages of spermatogenesis including accumulation of large amount of sperm in the seminiferous tubules. AI significantly reduced the serum levels of estradiol-17beta (E2) and increased levels of testosterone (T), 11-ketotestosterone (11-KT), and 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP). Further, AI suppressed in vitro production of E2, and stimulated the production of T and 11-KT in the ovarian fragments of mature female. In the honeycomb grouper, suppression of both in vitro and in vivo production of E2 and degeneration of oocytes by AI suggests that AI induces complete sex change through inhibition of estrogen biosynthesis, and perhaps, subsequent induction of androgen function.     

Aromatase pathway mediates sex change in each direction

The enzyme aromatase controls the androgen/oestrogen ratio by catalysing the irreversible conversion of testosterone into oestradiol (E2). Therefore, the regulation of E2 synthesis by aromatase is thought to be critical in sexual development and differentiation. Here, we demonstrate for the first time that experimental manipulation of E2 levels via the aromatase pathway induces adult sex change in each direction in a hermaphroditic fish that naturally exhibits bidirectional sex change. Our results demonstrate that a single enzymatic pathway can regulate both female and male sexual differentiation, and that aromatase may be the key enzyme that transduces environmental, including social, cues to functional sex differentiation in species with environmental sex determination.