Gonadal development, aromatase activity and P450 aromatase gene expression during sex inversion of protogynous red-spotted grouper Epinephelus akaara (Temminck and Schlegel) after implantation of the aromatase inhibitor, fadrozole

In the present study, we implanted 2‐year‐old female red‐spotted grouper, Epinephelus akaara, with a non‐steroidal aromatase inhibitor (AI), fadrozole, in the breeding season and examined changes in gonadal histology, serum sex steroids, aromatase activities and P450 aromatase (P450arom) gene expression in gonads after AI implantation. Aromatase inhibitor at doses from 0.1 to 10.0 mg kg^?1 BW induced a sex inversion and completion of spermatogenesis up to the functional male phase, but doses of 1.0 and 10.0 mg kg^?1 BW AI produced more males than 0.1 mg kg^?1 BW AI. Serum estradiol‐17β (E₂) levels decreased, but 11‐ketotestosterone (11‐KT) levels increased significantly in all the AI‐implanted groups, whereas testosterone (T) levels increased significantly only in the 1.0 mg kg^?1 BW AI‐implanted group. Aromatase activities and P450arom gene expression in gonads were inhibited significantly in the AI‐implanted groups, which was in accordance with the decrease in serum E₂ levels. These results suggested the optimal dose of AI to induce sex inversion to be 1.0 mg kg^?1 BW. Furthermore, the sex inversion induced by AI may be attributed to the inhibition of P450arom gene expression and aromatase activity and the resultant decrease in the biosynthesis of endogenous E₂. Meanwhile, the elevated 11‐KT levels were also associated closely with the occurrence of sex inversion in protogynous red‐spotted grouper.     

Artificial sex reversal of white grouper (Epinephelus aeneus) utilizing aromatase inhibitor (Fadrozole)

The white grouper is a desirable aquaculture species that adapts to captivity, grows well and commands a high market price. However, little is known about reproductive biology or control of sex reversal of this protogynous hermaphrodite. In this study, female white groupers were implanted with one dose of 17α‐methyltestosterone (10 mg/kg body weight [BW], MT) and two doses of aromatase inhibitor, fadrozole (1 and 3 mg/kg BW, FD1 and FD3) once a month for 4 months (April–July). At the start of the study, the fish had gonads full of oocytes compared to the end of the experiment when the control group mature oocytes compared to the experimental groups MT, FD1 and FD3 that exhibited different stages of testicular tissue. Plasma levels of testosterone were significantly highest in the FD3 group and the highest 11‐Ketotestosterone levels were observed in the MT group. Plasma levels of oestradiol (E₂) were significantly lower in the FD implanted groups, compared to initial individuals and control groups. The use of aromatase inhibitor, FD for sex reversal both gives further insight into the mechanisms controlling sex differentiation and provides an alternative to steroid treatment.     

Sex reversal in Nile tilapia (Oreochromis niloticus Linnaeus) by androgen immersion

Two experiments were conducted to investigate the efficacy of three androgens applied through immersion treatments on the sex ratio of Nile tilapia (Oreochromis niloticus L.) fry. In experiment 1, 14 days post‐hatching (DPH) larvae were exposed to a single immersion treatment in 17α‐methyltestosterone (MT), 17α‐methyldihydrotestosterone (MDHT) or 17α‐ethynyltestosterone (ET) at 200, 600 and 1800 μg L^?1 over 4 h (130 larvae per treatment). In experiment 2, Nile tilapia larvae were exposed to the higher androgen concentration (1800 μg L^?1) applied as either a single immersion (14 DPH) or double immersion (10 and 14 DPH) over 4 h (125 larvae per treatment). Change in sex proportion within each experiment as well as between experiments was analysed by the chi‐square test. In experiment 1, MT, MDHT and ET were equally effective in significantly increasing the proportion of males when applied at 1800 μg L^?1 (86.0%, 90.0% and 86.7% respectively). At 200 μg L^?1 none of the androgens altered sex ratio. At 600 μg L^?1, only MDHT slightly, but significantly skewed the sex ratio towards males (73.0%). In experiment 2, a single immersion treatment at 14 DPH (1800 μg L^?1) significantly increased the proportion of males, but at this time the response was significantly hormone dependent (MDHT, 100.0%; MT, 91.6%; ET, 76.9%). When compared with a single immersion, two‐immersion treatments significantly increased the proportion of males in the MT‐treated group (from 91.6% to 98.3%), decreased the proportion of males in the MDHT group (from 100.0% to 93.4%) and had no significant effect the ET‐treated group (change from 76.9% to 82.5%). The overall comparison of the sex ratio among same treatments from different experiments (a single immersion in 1800 μg L^?1) was not significantly different.     

Dosage-dependent differences in the effect of aromatizable and nonaromatizable androgens on the resulting phenotype of coho salmon (Oncorhynchus kisutch)

This study was conducted to investigate whether aromatization to estrogen could be the cause for the paradoxical feminization of gonads of sexually-undifferentiated fish after treatment with androgen at either high doses or for long periods. The aromatizable androgen 17-methyltestosterone (MT) and the nonaromatizable androgen 17-methyldihydrotestosterone (MDHT) were administered to groups of newly hatched coho salmon (Oncorhynchus kisutch) in a single 2h immersion at concentrations ranging from 6.25 to 6,400µg/l. The effects of treatment were evaluated by determining the resultant proportion of males in each experimental group. The effects of steroid administration on the final mean weight, length and condition factor were also determined. An increase in all these three variables was observed in the groups treated with the higher doses of MT. Regarding the resultant sexual phenotype, the response to both androgens was similar at the majority of doses tested. However, at the highest dose, the proportion of females increased with respect to that of males for MT, but not for MDHT. Since the major difference between the two androgens tested is their capacity to be aromatized, it seems that aromatization to estrogen, rather than inhibition of the biosynthesis of endogenous androgen, may explain the paradoxical feminization encountered.     

Efficacy of 17 α‐methyl testosterone and letrozole on sex reversal of protogynous grouper,Epinephelus tauvina (Forskal, 1775) and spawning performance of sex‐reversed males

This study aimed to test the efficacy of 17 α‐methyl testosterone (17 α‐MT) alone and in combination with letrozole, an aromatase inhibitor, for the induction of sex reversal in protogynous greasy grouper, Epinephelus tauvina. Further, the long‐lasting effects of these treatments and spawning performance of sex‐reversed males were also investigated. Greasy grouper with oocytes in the perinucleolus stage were implanted with 5 mg 17 α‐MT kg^?1 body weight (T₁), 5 mg 17 α‐MT and 0.2 mg letrozole kg^?1 body weight (T₂) and 5 mg 17 α‐MT with 0.4 mg letrozole kg^?1 body weight (T₃) and no androgens/enzyme inhibitor implanted (C). The 17 α‐MT alone and in combination of letrozole‐induced sex reversal in greasy grouper, whereas untreated control fish (C) showed normal ovarian development. However, T₂ and T₃ group showed 100% sex reversal and completion of spermatogenesis up to functional male phase in 2 and 3 months, respectively, whereas T₁ group resulted in only 66.67% functional male with motile spermatozoa after 4 months. Sex‐reversed males successfully fertilized the eggs during induced spawning. There were significant differences on fertilization and hatching rates between T₂ group (79.00 ± 4.36%; 77.67 ± 2.87%, respectively) and T₁ group (57.67 ± 3.17%; 63.87 ± 2.91%, respectively). The result suggested that 17 α‐MT (5.0 mg kg^?1 BW) in combination with letrozole (0.2 mg kg^?1 BW) has the potential to produce 100% sex‐reversed male in short period in greasy grouper, which might greatly help in seed production of greasy grouper.     

Evaluation of a 40 day Assay for Testing Endocrine Disrupters: Effects of an Anti-Estrogen and an Aromatase Inhibitor on Sex Ratio and Vitellogenin Concentrations in Juvenile Zebrafish (Danio rerio)

In the present study, the effects of the anti-estrogen ZM 189,156 and the aromatase inhibitor fadrozole were evaluated in a 40-day juvenile assay developed for screening of endocrine disrupting chemicals. Juvenile zebrafish were exposed from 20 to 60 days post hatch (dph) to ZM 189, 154 (100 μg l⁻¹ and 200 μg l⁻¹) and fadrozole (10, 32, and 100 μg l⁻¹). VTG concentrations were measured at 38 dph by the use of a direct non-competitive sandwich ELISA and sex ratios were determined at 60 dph by histological examination of the gonads. A small but significant increase in VTG concentrations was observed in fish exposed to ZM 189, 156 (100 and 200 μg l⁻¹) and fadrozole (10 and 100 μg l⁻¹) compared to control groups. In fish exposed to ZM 189, 156 and fadrozole, the percentage of females declined and the number of undifferentiated fish increased. These findings show that exposure of juvenile zebrafish to an aromatase inhibitor or an anti-estrogen during early development inhibits differentiation and development of female gonads. The data presented, furthermore, show that the 40-day juvenile assay may be suitable for screening endocrine disrupting chemicals acting as anti-estrogens and aromatase inhibitors.     

Reproductive performance of common dentex, Dentex dentex, broodstock held under different photoperiod and constant temperature conditions

The effect of photoperiod and temperature on the timing and the quality of spawning, and on associated endocrine changes in circulating 17β-oestradiol, 11-ketotestoserone (11-KT) and vitellogenin (Vtg) were investigated in common dentex, Dentex dentex, undergoing their second reproductive cycle. The possibility was also explored of using the measurement of steroids in the culture water of broodstock tanks, rather than in individual blood plasma samples, as a potentially useful tool for assessing the physiological state of a fish without disturbing them. One group of fish was exposed to a simulated natural seasonal cycle and ambient temperature (CONTROL). The other two groups were exposed to simulated seasonal photoperiod cycles (12 month-long) but which were phase-shifted either three months before (ADVANCED) or after (DELAYED) the natural cycle. Temperature was kept at 19.4±0.9 °C all-year-round. In the CONTROL, spawning started in mid-April and lasted until mid-June, while in the ADVANCED group, spawning started 4 months earlier and in the DELAYED group 2 months later than the CONTROL. The total egg production, egg quality, hatching rate, relative fecundity, and spawning index of the experimental groups were similar to the controls. The differences in spawning time induced by photothermal manipulation were associated with a difference in the timing of peak concentrations of plasma E₂, 11-KT and Vtg. In all three groups, the amounts of conjugated 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and free and sulfated 11-KT which could be extracted from the water during the spawning period were significantly higher than those found in the preovulatory period. However, the differences were mostly less than 2-fold suggesting that, at least under the conditions employed in this study, the method was of limited use for non-intrusive detection of gonadogenesis and spawning (as had been hoped). The observed differences in spawning time and in the seasonal changes of sex steroids and Vtg confirm and extend the findings on marine fish. This revised version was published online in August 2006 with corrections to the Cover Date.     

Promoter analysis of two aromatase genes in the serial-sex changing gobiid fish, Trimma okinawae

Despite numerous endocrine studies on sex change in teleost, no general mechanism that mediates sex change has emerged. The gobiid fish, Trimma okinawae, can change sex in both directions repeatedly. This phenomenon of sex change in goby assigns it as an excellent animal model to elucidate the understanding mechanisms of sex change. In hermaphrodite fishes, estrogen plays a particularly important role in natural and experimentally induced sex change. To investigate the role of estrogen in the serial-sex changing fish T. okinawae, we cloned and analyzed the 5′-flanking regions of P450arom genes from goby genome DNA. Both regions have consensus sequences of TATA, CRE and ERE. Ad4 binding site was restricted in the region of P450aromA. These findings indicate that different regulators control the expression of the two P450arom genes.     

Precocious sex change and spermatogenesis in the underyearling Malabar grouper Epinephelus malabaricus by androgen treatment

The Malabar grouper Epinephelus malabaricus is an important candidate species for commercial aquaculture in tropical and subtropical areas. In nature, this species requires more than 10 years to change sex from female to male and have active spermatogenic tissues in the testis. Thus, it is essential to find a means of producing sperm for seed production. This is the first report of artificial sex change in underyearling E. malabaricus . Female E. malabaricus with immature ovaries at 144 days post-hatch (DPH) were fed a diet with 17α-methyltestosterone (MT) at 50 μg g⁻¹ diet for 6 months. Sex change occurred in most of the treated fish, which had testis with all stages of spermatogenic germ cells including spermatozoa. In contrast, most of the control fish had immature ovaries. These results, which reveal that germ cells in the underyearling grouper have the ability to produce spermatozoa in response to exogenous androgen, demonstrate that sex change can be artificially induced during ovarian development.     

Isolation, cloning, sequencing of brain type aromatase and its expression in male and female Wrasse, Pseudolabrus sieboldi

Pseudolabrus sieboldi, wrasse being a diurnal spawner provides a good opportunity to study the endocrine mechanism of estrogen formation in brain and gonads. Moreover, an extremely large amount of E2 was produced in serum and testis of wrasse. It is assumed that the presence of E2 may play a major role in diurnal gametogenesis in male fish. In this study brain type aromatase have been isolated, cloned and sequenced from the brain of wrasse. Further, the expression pattern of brain type aromatase in gonads and adult tissue of male and female fish have been analyzed. In addition, the diurnal expression pattern of brain type aromatase in both male and female fish brain during spawning season have been analyzed. The P450arom (br) was isolated, cloned and sequenced from both male and female bamboleaf wrasse. The P450arom (br) gene (1877 sequenced nucleotide) contains an ORF of 1470 bp, a 5′-UTR of 18 bp and at least 407 bp in 3′-UTR. The amino acid sequence homology in the coding region of wrasse P450arom (br) is high compared to that of medaka, Oryzias latipes (80%), rainbow trout type 2, Oncorhynchu mykiss (78.2%), fugu, Takifugu ribripes (78%) rainbow trout type 1, (76%), goldfish, Carassius auratus (66.8%) and zebrafish, Danio rerio (66.2%). Expression study reveals that P450arom (br) mRNA were most abundant in brains of both male and female fish throughout the day during the spawning season. RT-PCR study revealed that P450arom (br) was expressed in skin, anal fin and tail fin of both male and female wrasse. P450arom (br) was not detected at any time of the spawning day in either ovary or testis of wrasse.     

Effect of 17 α-methyltestosterone on gonadal differentiation in pikeperch, Stizostedion lucioperca L.

Morphological and histological studies demonstrated that 17 α-methyltestosterone (MT) affects sex differentiation and can be used to control the phenotypic sex of pikeperch. Stizostedion lucioperca L. Treatment (for 21 days) of pikeperch (initially 2.2 g wet weight) with MT at 30 mg kg^-1 diet induced germ cells inversion in approximately 46.67% of individuals (although 96.67% of the fish were classified as males). An increase in treatment duration (60 and 90 mg kg^-1) increased the percentage of female, intersex and sterile fish. Slower growth rates of all the fish treated with MT in comparison to the control groups were demonstrated. However, no significant differences in conditions and mortalities between the MT treated fish and controls were observed.     

Presence of 11-ketotestosterone in pre-differentiated male gonads of Odontesthes bonariensis

The involvement of androgens during sex differentiation period was investigated in the pejerrey Odontesthes bonariensis, by classic biochemical studies and gonadal histology. We studied in particular whether the enzyme activities involved in 11-oxygenated androgen production were active in a gonadal/peritoneum complex (GPC) of very small larvae exposed to masculinizing temperatures previous to morphological sex differentiation (5 weeks post-hatching). The GPC was incubated with 17-hydroxyprogesterone (³H-17P), and the presence of 11-KT as major metabolite in early gonads undergoing masculine pathway after temperature treatment exposure is reported. 11-KT was identified by thin-layer chromatography and high-pressure liquid chromatography. The present results show that 11-KT is produced at very early stages of testis development in pejerrey, being this androgen one of the main mediators of the masculinization induced by temperature treatment at the gonad level.     

Nucleotide sequence and mRNA expression analysis of <Emphasis Type="Italic">β</Emphasis>-actin gene in the orange-spotted grouper <Emphasis Type="Italic">Epinephelus coioides</Emphasis>

The full-length cDNA, encoding the orange-spotted grouper β-actin and spanning 1920 bp including a poly (A) tail, was cloned from its brain cDNA library. The open reading frame encodes a protein of 375 amino acids. Sequence analysis indicated that it contained the typical structural features of cytoplasmic actins, and showed higher homology with other vertebrate β-actin than any other members of the actin family. The partial genomic sequence indicated that the organization of the β-actin gene in the orange-spotted grouper might also be conserved. Northern blot analysis indicated that it was expressed at high levels in the brain, spleen, adipose tissue, ovary, and liver, but at low levels in the gill filament and heart, and at a very low level in the kidney. The expression of β-actin gene in the skeletal muscle was barely detectable. These results indicated that the expression of the orange-spotted grouper β-actin gene showed significant variation in different tissues. Therefore, caution should be taken when using β-actin gene as an internal control in the normalization of gene expression among tissues. Whereas, semi-quantitative RT-PCR analysis indicated that treatment with 17α–methyltestosterone (MT) had little effect on the mRNA expression of β-actin gene in the in vitro incubated hypothalamus, pituitary, and ovary fragments of the orange-spotted grouper, suggesting β-actin can be used as an internal control for RT-PCR analysis of MT effects on gene expression in these tissues.     

Induction of sex reversal in blue drum (Nibea mitsukurii) and gynogenetic yellow drum (Nibea albiflora) by oral administration of letrozole

The objective of this study was to test the efficiency of an aromatase inhibitor, letrozole (LZ), to induce masculinization in blue drum (Nibea mitsukurii) and gynogenetic yellow drum (N. albiflora). Two experiments (Exp‐1 and Exp‐2) were performed to investigate the effect of LZ on the growth, sex ratio and gonad development in these two fishes. In Exp‐1, blue drum were treated with oral administration of letrozole at different doses (0, 1, 10, 100 mg/kg). In Exp‐2, gynogenetic yellow drum were orally administered different doses of LZ (0, 10, 100 mg/kg) and a dose of 10 mg/kg 17α‐methyltestosterone (MT). The treatments in both experiments were performed from 25 to 85 days post hatch (dph). As a result, all the LZ‐treated fish were phenotypic males despite of slower growth than the control during the treatment period. Free spermatozoa were observed from 150 dph onwards and the gonadosomatic index of the LZ‐treated fish did not differ significantly from that in the control at maturity. However, the sex‐reversed fish induced by MT exhibited a high proportion (33.4%) of testicular abnormalities. Our results showed that oral administration of LZ was efficient in inducing sex reversal in drums, and thereby facilitated their mono‐sex culture.     

Viral encephalopathy and retinopathy is endemic in wild groupers (genus Epinephelus spp.) of the Algerian coast

This work describes betanodavirus infection in two species of groupers (family Serranidae) from the Algerian coast: the dusky grouper Epinephelus marginatus and the golden grouper Epinephelus costae. At necropsy, characteristic clinical signs, external injuries, clouded eyes and brain congestion, generally associated with viral encephalopathy and retinopathy (VER) infection were observed. The partial sequences of RNA1 and RNA2 from two viral strains were obtained, and the phylogenetic analysis revealed the presence of the red-spotted grouper nervous necrosis virus (RGNNV) genotype closely related to strains previously detected in groupers in the same geographic area. Results obtained in this study support the hypothesis that VER disease is endemic in the Algerian grouper population.     

Effect of temperature decrease on oocyte development,sex steroids,and gonadotropin β-subunit mRNA expression levels in female Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis>

To improve understanding of the mechanism of early ovarian development in eels, the effects of water temperature decrease on oocyte development, plasma levels of sex steroids [estradiol 17β (E2), testosterone (T), 11-ketotestosterone (11-KT)], and gonadotropin β-subunit [follicle-stimulating hormone (FSHβ), luteinizing hormone (LHβ)] messenger RNA (mRNA) expression levels were investigated. A total of 27 female Japanese eels Anguilla japonica were divided into initial, control, and test (water temperature decrease) groups. Starting on 22 September 2009, eels in the test group were reared in a tank with gradual temperature decrease from 25°C to 15°C over 39 days, while the control group was maintained at 25°C. The test group accumulated more oil droplets in their oocytes than did the other groups. Levels of sex steroids, especially 11-KT, were higher in the test group. In contrast, FSHβ and LHβ mRNA expression levels were lower in the test group. These results suggest that water temperature decrease only induced an early stage of ovarian development that was partly affected by an 11-KT increase. For further maturation, other environmental factors related to induction of gonadotropin increase appear to be needed.     

Sex steroid level and sexual dimorphism expression of genes in gonads of the great sturgeon Huso huso Linneaus, 1758 during maturity developmental stages

Little is known about molecular mechanisms involved in gonad differentiation in sturgeon species. In non‐mammalian vertebrates, sox9, dmrt1, cyp17a1 and ar are male specific genes in testicular differentiation and are highly conserved. In order to understand the mechanism underlying testicular development in sturgeon, we investigated sex steroids level of 11‐ketotestosterone (KT) and testosterone (T) and relative expression of sox9, dmrt1, cyp17a1 and ar in great sturgeon gonads during different stages of sexual maturity. The results showed all studied genes had dimorphic patterns in males. In immature gonads (stage I), only cyp17a1 expressed in male gonads, while other genes did not express, and KT and T levels were low. Dmrt1 showed dimorphic expression pattern in male gonads at maturity stage II, III and IV. Furthermore, sox9 and ar mRNA presented significant dimorphic expression pattern in male gonads only at maturity stage 4. Plasma androgens levels were significantly higher in males compared to females during maturity stages II, III and IV. The results showed that among these four genes, only cyp17a1 expressed in male gonads at maturity stage I (immature), suggesting that this gene may be applicable as a sex marker in recently differentiated male great sturgeon. Sexually dimorphic patterns in other studied genes (sox9, dmrt1 and ar) suggesting that these genes may be important for testicular development and differentiation in premature great sturgeon. The obtained results provide a foundation for further research on sex differentiation and developing strategies for the sexing of sturgeon for aquaculture.