The role estrogens play in sex differentiation and sex changes of fish

Using genetically controlled all-female and all-male tilapia (Oreochromis niloticus), the role steroid hormones play in the sex differentiation was analyzed histologically, ultrastructurally, immunohistichemically and experimenntally. The results strongly suggest that endogenous estrogen acts as an ovarian inducer, and that the lack of steroid hormone including androgen is important for testicular differentiation. Moreover, the roles of steroid hormones in protogynous sex change of three-spotted wrasse (Halichoeres trimaculatus) and saddleback wrasse (Tharassoma duperrey) were examined. The results strongly support the hypothesis that the endogenous estrogen plays an important role in protogynous sex change.     

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.     

Increase in estrogen signaling in the early brain of orange-spotted grouper Epinephelus coioides: a mini-review

Despite neurosteroidogenic enzymes are playing important roles in the regulation of brain development and function, the potential link between brain and gonad by the action of steroid hormones during gonadal sex differentiation is still not clear in teleosts. In this mini-review, we summarized our understanding on the early brain development related to the synthesis of neurosteroids and receptor signaling during gonadal sex differentiation in protogynous orange-spotted grouper, Epinephelus coioides (functional females for the first 6 years of life and start to sex change around the age of 7 years) and protandrous black porgy (functional males for the first 2 years of life but begin to change sex during the third year). We found a similar profile in the increased expression of brain aromatase gene (aromatatse B or cyp19a1b), aromatase activity, estradiol (E₂), and estrogen signaling in the brain of both grouper and black porgy fish during gonadal sex differentiation. In contrast to mammals, teleost fish Cyp19a1b expressed in a unique cell type, a radial glial cell, which is acted as progenitors in the brain of developing and adult fish. In agreement with these pioneer studies, we demonstrated that the grouper cyp19a1b/Cyp19a1b was expressed in radial glial cells. Further, in vivo data in the grouper brain showed that exogenous E₂ upregulated Cyp19a1b immunoreactivity (ir) in radial glial cells. These data suggest the possible roles of Cyp19a1b and E₂ in early brain development which is presumably related to gonadal sex differentiation.     

Bi-directional sex change and its steroidogenesis in the wrasse, Pseudolabrus sieboldi

The protogynous wrasse, Pseudolabrus sieboldi, is a good model for studying the physiological mechanism of sex change in teleosts. We established a way to induce sex change bidirectionally by controlling the social conditions in captivity. This is the first report of a reversed sex change in a promiscuous species. After identifying all the steroid hormones produced in the ovarian follicles and testis, and clarifying their synthetic pathways, we examined the function of the sex steroids in the sex change of P. sieboldi. We concluded that the activation of 17β-HSD-III is the first step in inducing morphological and functional change from an ovary to a testis.     

Effects of aromatizable and nonaromatizable androgens on the sex inversion of red-spotted grouper (Epinephelus akaara)

The effects of aromatizable 17α-methyltestosterone (MT) and non-aromatizable 17α-methyldihydrotestosterone (MDHT) on sex inversion in red-spotted grouper, Epinephelus akaara, were investigated. Fish were implanted with MT, MDHT and MT+AI (aromatase inhibitor, AI) respectively for one month. The results showed that the three treated groups turned into transitional stage with intersex gonads, which contained atretic oocytes and spermatogenic germ cells at all stages of spermatogenesis. The controls did not change sex. The gonads of more than half MT-implanted fish were in early transitional stages of sex inversion, whereas those of more than half MDHT and MT+AI-implanted fish were in late transitional stages of sex inversion. No difference in serum estradiol-17β (E₂) levels between the controls and the treated groups were observed, whereas 11-ketotestosterone (11-KT) and testosterone (T) levels increased in all treated groups. Significantly lower gonadosomatic index (GSI) and gonadal aromatase activity were observed in the treated groups, which were in accordance with the lower mRNA expression of P450aromA. However, P450aromB mRNA expression increased in the MT group, while it did not change in the MDHT group. These results suggest that the sex inversion of red-spotted grouper by MT and MDHT implantation might be due to the suppression of P450aromA gene expression, and resulting in both the decrease of the ovarian estrogen –secretion, as well as the increase in the 11-KT levels. Furthermore, the main reason for MT being less effective than MDHT might be due to partial aromatization of MT to estrogen.     

Survival of ovarian somatic cells during sex change in the protogynous wrasse, Halichoeres trimaculatus

The three-spot wrasse (Halichoeres trimaculatus), which inhabits the coral reefs of Okinawa, changes sex from female to male. Sex change in this species is controlled by a social system. Oocytes disappear completely from the ovary, and male germ cells and somatic cells comprising testicular tissue arise a new during the sex change process. However, little is known of the fate and origin of the gonadal tissue-forming cells during sex change. In particular, the fate of ovarian somatic cells has not been determined, although the ovarian tissue regresses histologically. To approach this question, we analyzed apoptosis and cell proliferation in the sex-changing gonads. Unexpectedly, we found that few apoptotic somatic cells were present during sex change, suggesting that ovarian somatic cells might survive during the regression of the ovarian tissue. On the other hand, cell proliferation was detected in many granulosa cells surrounding the degenerating oocytes, a few epithelial cells covering ovigerous lamella and a few somatic cells associated with gonial germ cells at an early stage of sex change. Then, we found that proliferative ovarian somatic cells remained in the gonads late in the sex change process. Based on these results, we concluded that some functional somatic cells of the ovary are reused as testicular somatic cells during the gonadal sex change in the three-spot wrasse.     

Effects of estrogens on Tilapia aurea: Implications for production of monosex genetic male tilapia

Three naturally occurring estrogens, estriol, estrone and 17-Beta estradiol, were each administered perorally at three concentrations for 3- or 5-week periods to fry of Tilapia aurea, 8–11 mm long. Estrogen treatment neither affected survival nor altered growth of fry. Post-treatment stocking in outdoor concrete tanks revealed no apparent differences in survival between hormone-treated and untreated fish after 99–148 days. Gonadal examination of treated and untreated groups after growth trials revealed no aberrant sex ratios in the groups treated with estrogen, but in all treatment groups there were significant proportions of males with a female-like urogenital papilla. Greater proportions of such ‘atypical’ males occurred at the higher estrogen dosage levels and in the longer treatment period. No atypical males were found in the control groups. Sexing of fish that died after treatment indicated no difference in the death rate of the sexes. Histological examination of gonads revealed no alteration of normal male and female germ cell development; pseudo-oocytes were present in both treated and untreated groups. No ovotestis condition was observed. Male and female homogamety among different tilapia species and a method for producing monosex genetic males from matings of sex-reversed brood stock are discussed.     

Effects of estradiol-17β and 17α-methyltestosterone on gonadal differentiation in the coho salmon, Oncorhynchus kisutch

Methods are described for controlling sex differentiation in the coho salmon, Oncorhynchus kisutch. Eyed eggs and alevins were immersed in solutions of estradiol-17β and 17α-methyltestosterone and fed these steroids in the diet for a period of 10 weeks postswim-up. In seven out of 10 groups that received estradiol all fish resembled normal females when sampled at 4 months posthatch. In fish treated with methyltestosterone at all except the lowest dose (25 and 50 μg/l) the gonads resembled neither normal males nor normal females; these gonads were composed largely of connective tissue with only occasional germ cells. Various proportions of male, female and intersex gonads were observed in groups which received androgen or estrogen only in the diet. The implications of the work for salmon culture include the production of sterile fish and increasing the proportion of female salmon in hatchery populations.     

Dimorphic expression of sex-related genes in different gonadal development stages of sterlet, <Emphasis Type="Italic">Acipenser ruthenus</Emphasis>, a primitive fish species

Molecular mechanism of sex determination and differentiation of sturgeon, a primitive fish species, is extraordinarily important due to the valuable caviar; however, it is still poorly known. The present work aimed to identify the major genes involved in regulating gonadal development of sterlet, a small species of sturgeon, from 13 candidate genes which have been shown to relate to gonadal differentiation and development in other teleost fish. The sex and gonadal development of sterlets were determined by histological observation and levels of sex steroids testosterone (T), 11-ketotestosterone (11-KT), and 17β-estradiol (E2) in serum. Sexually dimorphic gene expressions were investigated. The results revealed that gonadal development were asynchronous in 2-year-old male and female sterlets with the testes in early or mid-spermatogenesis and the ovaries in chromatin nucleolus stage or perinucleolus stage, respectively. The levels of T and E2 were not significantly different between sexes or different gonadal development stages while 11-KT had the higher level in mid-spermatogenesis testis stage. In all the investigated gonadal development stages, gene dmrt1 and hsd11b2 were expressed higher in male whereas foxl2 and cyp19a1 were expressed higher in female. Thus, these genes provided the promising markers for sex identification of sterlet. It was unexpected that dkk1 and dax1 had significantly higher expression in ovarian perinucleolus stage than in ovarian chromatin nucleolus stage and in the testis, suggesting that these two genes had more correlation with ovarian development than with the testis, contrary to the previous reports in other vertebrates. Testicular development-related genes (gsdf and amh) and estrogen receptor genes (era and erb) differentially expressed at different testis or ovary development stages, but their expressions were not absolutely significantly different in male and female, depending on the gonadal development stage. Expression of androgen receptor gene ar or rspo, which was supposed to be related to ovarian development, presented no difference between gonadal development stages investigated in this study whenever in male or female.     

Assessment of spermatogenesis and plasma sex steroids in a seasonal breeding teleost: a comparative study in an area of influence of a tributary, downstream from a hydroelectric power dam, Brazil

River damming and building of hydroelectric power plants interrupt the reproductive migration routes and change the major physicochemical parameters of water quality, with drastic consequences for populations of migratory fishes. The goal of this study was to evaluate proliferation and cell death during spermatogenesis and serum profiles of sex steroids in Prochilodus argenteus, from the S?o Francisco River, downstream from the Três Marias Dam. A total of 257 adult males were caught quarterly during a reproductive cycle in two sites: the first 34?km of the river after the dam (site 1) and the second 34–54?km after the dam (site 2), after the confluence with a tributary, the Abaeté River. Seasonal changes in the testicular activity associated with morphometric analyses of germ cells as well as proliferation and testicular apoptosis support a more active spermatogenesis in fish from site 2, where higher levels of sex steroids and gonadosomatic index (GSI) were also found. In site 1, fish presented low serum levels of testosterone, 17β-estradiol and 17α-hydroxyprogesterone and a low GSI during gonadal maturation. Spermatogonial proliferation (PCNA) and apoptosis (TUNEL) were more elevated in fish from site 1, but spermatocytes were mainly labelled in fish from site 2. Overall, these data demonstrate changes in testicular activity and plasma sex steroids in a neotropical teleost fish living downstream from a hydroelectric dam, supplying new data on fish reproduction in regulated rivers. Moreover, morphometric analyses associated with sex steroids profiles provide reliable tools to assess fish spermatogenesis under environmental stress conditions.     

Studies on feminization,sex determination,and differentiation of the Southern catfish, <Emphasis Type="Italic">Silurus meridionalis</Emphasis>—a review

The sex ratio of the feral Southern catfish was reported to be about 1:1, while the fish obtained by artificial fertilization were always female. Hence, we examined the possible influence of the micro-environment during artificial insemination (pH of the ovarian fluid and concentration of the semen) and early development (feed, hatching temperature, and water) on the sex ratio of Southern catfish fry. In order to examine the possibility of the occurrence of gynogenesis during artificial propagation, cytological observations on the insemination processes and the artificial induction of gynogenesis were also performed. However, no male fish were obtained even in these experiments, excluding the possibilities of these micro-environmental changes on catfish sex ratio and the occurrence of gynogenesis during artificial propagation. Female-to-male sex reversal was achieved by treatment with fadrozole (an aromatase inhibitor) and tamoxifen (an estrogen receptor antagonist). Histological analyses on the gonadal development of both female and induced male fish were subsequently performed. Moreover, several genes involved in sex differentiation, such as dmrt1, foxl2, and cyp19, and three subunits of gonadotropin (gth), i.e., gthα, lhβ, and fshβ, were isolated. Their expression patterns were studied under normal gonadal development and sex reversal conditions. The results revealed that dmrt1, foxl2, and cyp19a were closely related to catfish sex differentiation, and the gth subunits were possibly related to ovarian differentiation and oocyte development. Taken together, we hypothesized that estrogen was highly responsible for the ovarian differentiation and feminization of catfish fry under artificial propagation, although the mechanism involved remains elusive.     

Treatment with an aromatase inhibitor induces complete sex change in the protogynous honeycomb grouper (Epinephelus merra)

To study the mechanism of protogynous sex change in honeycomb grouper, the wild fish of both sexes and transitionals were collected for one year, and changes in gonadal structures and serum levels of sex hormones in each individual were examined. The onset of sex change was associated with low serum estradiol - 17β (E2) levels. In order to clarify whether E2 deprivation causes sex change, maturing females were implanted with the non-steroidal aromatase inhibitor (Fadrozole, AI). AI caused complete sex change from female to functional males in two and half months. The present results suggest that E2 is an important endogenous factor which regulates protogynous sex change in the honeycomb grouper.     

Molecular cloning and expression of caspase-3 in the protandrous cinnamon clownfish, Amphiprion melanopus, during sex change

The caspase-3 appears to be a key protease in the apoptotic pathway. We identified caspase-3 complementary DNAs from the ovaries of the protandrous cinnamon clownfish (Amphiprion melanopus), and investigated its mRNA and proteins, and activity levels during the sex change (I, mature male; II, male at 90 days after removing of the female; and III, mature female). The nucleotide sequence of the caspase-3 cDNA was 969 base pairs in length with open reading frames encoding peptides of 282 amino acids. The caspase-3 mRNA and protein, and activity levels in stages of the mature gonad are higher than those of the development gonad stage. To understand the effect of gonadotropin-releasing hormone (GnRH) on gonad apoptosis, we examined expression of genes caspase-3 mRNA and activity level in immature cinnamon clownfish gonads after GnRH analogue (GnRHa). The findings support the hypothesis that caspase-3 expression is associated with both testicular and ovarian development, and suggests that it may play a role in the control of ovarian development in cinnamon clownfish. Also, we demonstrate that GnRH agonists stimulate caspase-3 production which can in turn stimulate apoptosis. The present study provides a framework for better understanding of the role of caspase-3 during sex change processes in fish.     

Effects of Gender and Sex Hormones on Disease Susceptibility of Channel Catfish to Edwardsiella ictaluri

The use of monosex populations for aquaculture is becoming widely used for several species. The current studies determined if there were any differences between male and female channel catfish, Ictalurus punctatus, in disease susceptibility to Edwardsiella ictaluri, one of the most important pathogenic bacteria in catfish culture. Disease challenge experiments were carried out on fingerling channel catfish fed 17β‐estradiol or testosterone before the challenge, and on all male and on sibling all female fingerlings. All male populations were produced by mating YY males with normal XX females. Sibling females were produced by hormonally sex reversing a subpopulation of six of the all male families. Weight gain or specific growth rate did not differ in fish fed testosterone or estrogen. Fish fed the highest dose of estrogen (50 mg/kg) had a significant higher mortality (P < 0.05), while mortality was similar in catfish fed 10 and 50 mg/kg of testosterone compared to controls. There were no differences in mortality between sibling males and females. These data indicate no increased disease susceptibility to E. ictaluri between males and females or due to exogenous sex hormones. Production of all male catfish for culture can proceed without concern for disease susceptibility to E. ictaluri.     

Effects of individual and binary mixtures of estrogens on male goldfish (Carassius auratus)

Adverse effects of five typical environmental estrogens, namely estrone (E₁), 17β-estradiol (E₂), 4-n-octylphenol (4-n-OP), 4-n-nonylphenol (4-n-NP) and bisphenol A (BPA) on adult male goldfish (Carassius auratus) were investigated both individually and in binary mixtures, using serum vitellogenin (VTG) induction and gonadosomatic index (GSI) as the endpoints. Doses of individual and binary mixtures of estrogens were chosen at broad ranges. Five individual estrogens induced common dose-dependent increases of serum VTG in the experimental fish when injection doses of the estrogen series were comparatively low. The levels of VTG induction in fish descended after peaked at a certain dose of the individual estrogen. Significant GSI decreases were observed in fish treated by all dose series of E₁ and E₂, and comparatively high doses of 4-n-OP, 4-n-NP and BPA when compared with that of solvent control (SC). Effects of binary mixtures of the five typical estrogens on VTG induction in male goldfish were in additive manner at low-effect doses, but divergences occurred at high dose levels, with the predicted effects by additive manner exceeding those were observed. All of GSI of fish treated by the binary mixtures were about or lower than 10^?3 %. Serious atrophy of gonads was observed in all the mixture treatment groups when compared with that of SC. These findings highlight the potential reproductive risk of fish resulted from existing mixtures of hormones in the aquatic environment, and they have important implications for environmental estrogen hazard assessment.     

Sex ratios of nigorobuna Carassius auratus grandoculis reared in paddy fields with fluctuating temperatures during larval and juvenile growth stages

The sex ratios of the nigorobuna Carassius auratus grandoculis reared in paddy fields during the sex differentiation periods of larval and juvenile stages were examined, since the phenotypic expression of sex in the fish is thermolabile, so that sex determination is under the control of genetic factors and temperature. After 2 days from hatching, the larvae were introduced into five paddy fields at five different densities in late May. After 37 or 41 days from hatching the juveniles were collected when the water was drained from the paddy fields, and reared in laboratory tanks supplied with running freshwater pumped from the lake shore until the age of 144–164 days. The mean water temperatures in the paddy fields were 22.4–24.1 ºC, and the maximum water temperature during the day frequently exceeded 30 ºC during the supposed critical period of sex determination of the fish. The sex ratios in the five paddy fields did not show a male-bias. Instead, all fields produced either balanced or slightly female-bias sex ratios. These results suggest that temporary high and fluctuating temperatures in paddy fields with low mean temperatures have hardly any masculinization effects on genotypic females of C. auratus grandoculis during sex differentiation stages.