Masculinization of the European sea bass (Dicentrarchus labrax) by treatment with an androgen or aromatase inhibitor involves different gene expression and has distinct lasting effects on maturation

The objective of this study was to contribute to our understanding of the role of sex steroids in fish sex differentiation and male maturation. Sexually undifferentiated sea bass were administered 17α-methyldihydrotestosterone (MDHT), estradiol-17β (E₂), fadrozole (Fz), cyproterone acetate (CPA) or tamoxifen (Tx). MDHT produced 100% males whereas E₂ and Tx resulted in 100% females. Fz produced 95% males while CPA did not alter sex ratios. E₂ treatment did not affect gonadal aromatase (cyp19a) expression levels, supporting the possibility that the feminizing effect of exogenous E₂ are not directly related to cyp19a regulation. Both MDHT and Fz decreased cyp19a expression. Moreover, androgen receptor (ar) expression levels increased during development in all but the MDHT group, suggesting that early exposure to an androgen down-regulates subsequent ar expression in males and that Fz does not interact with the androgen receptor. Together, these observations indicate that although MDHT and Fz result in a similar phenotype, the molecular pathways involved are likely different, and show that Fz masculinization is the consequence of inhibited ovarian differentiation rather than of a direct androgenic effect. Further, since CPA did not alter sex ratios when administered during the period of highest androgen sensitivity, we suggest that androgens are not required for initial testicular differentiation in the sea bass. MDHT and Fz did not alter the number of precocious males but reduced and increased, respectively, their gonadosomatic index (GSI). In addition, Fz had lasting effects on the GSI of precocious and non-precocious males, probably due to alterations of estrogen function in the testis.     

Cloning and sequence analysis of a vasa homolog in the European sea bass (Dicentrarchus labrax): tissue distribution and mRNA expression levels during early development and sex differentiation

Vasa is a protein expressed mainly in germ cells and conserved across taxa. However, sex-related differences and environmental influences on vasa expression have not been documented. This study characterized the cDNA of a vasa homolog in the European sea bass, Dicentrarchuslabrax (sb-vasa), a gonochoristic fish with temperature influences on gonadogenesis. The 1911 bp open reading frame predicted a 637-amino acid protein with the eight conserved domains typical of Vasa proteins. Comparisons of the deduced amino acid sequence with those of other vertebrates and invertebrates revealed the highest homology (68-85%) with those of other teleosts. An updated tree with the full-length sequences for Vasa proteins in 66 species belonging to six different phyla was constructed, establishing the evolutionary relationships of Vasa amino acid sequences. European sea bass vasa was highly expressed in gonads with little or no expression in other tissues. Real time RT-PCR quantification of the temporal expression of sb-vasa from early development throughout sex differentiation showed that mRNA levels were high in unfertilized eggs, decreased during larval development and increased again during the period of germ cell proliferation. Rearing of fish at high temperature resulted in further increased sb-vasa levels, most likely reflecting temperature effects on both somatic and gonadal growth. Differences in expression were also found well before sex differentiation and persisted until the end of the first year, with higher levels present in females. These differences in expression demonstrate the implication of vasa during the initial stages of fish sex differentiation and gametogenesis and suggest that, through its helicase activity, it might be implicated in the translational regulation of mRNAs involved in the specification and differentiation of gonadal-specific cell types.     

Androgen rather than estrogen up-regulates brain-type cytochrome P450 aromatase (cyp19a1b) gene via tissue-specific promoters in the hermaphrodite teleost ricefield eel Monopterus albus

CYP19A1 in the brain and pituitary of vertebrates is important for reproductive and non-reproductive processes. In teleosts, it is broadly accepted that estradiol (E₂) up-regulates cyp19a1b gene via a positive autoregulatory loop. Our present study, however, showed that E₂ did not up-regulate ricefield eel cyp19a1b in the hypothalamus and pituitary, whereas dihydrotestosterone (DHT) or testosterone (T) stimulated cyp19a1b expression only in the pituitary. Two tissue-specific promoters, namely promoter I and II directing the expression in the brain and pituitary respectively, were identified. Promoter I contained a non-consensus estrogen response element (ERE), and consequently did not respond to E₂. Promoter II contained an androgen response element (ARE) and consequently responded to DHT. Taken together, these results demonstrated a novel steroidal regulation of cyp19a1b gene expression and an alternative usage of tissue-specific cyp19a1b promoters in the brain and pituitary of a teleost species, the ricefield eel.     

Immortalization by large T-antigen of the adult epididymal duct epithelium

The teleost brain is characterized by exceptionally high levels of aromatase, the enzyme that converts androgens into estrogens, and by its continuous growth throughout life. Gonadal estrogens have been implicated in sex differentiation and the control of reproduction in adult fish, but the role of neural estrogens during early development is far from clear. The present study describes the isolation and characterization of the cDNA sequence from brain aromatase (P450aromB) in the European sea bass (Dicentrarchus labrax L.), a well established model for neuroendocrine research in fish. P450aromB was cloned from a brain cDNA library and encoded a predicted protein of 505 residues, with a calculated molecular weight of 57.2 kDa. Comparisons of the deduced amino acid sequence to that of the ovarian aromatase (P450aromA) in the same species revealed 62% identity, lower than the 84% identity shared between sea bass and tilapia brain aromatases. Phylogenetic analysis showed the occurrence of a gene duplication for P450arom in the teleost lineage after its divergence from the tetrapods. Moreover, the low percentage of identity between brain and ovarian forms within the same species suggests that both genes evolved separately right after the appearance of the teleosts. Tissue-specific expression of P450aromA and P450aromB mRNA was studied in adult sea bass. P450aromB was preferentially expressed in brain of both males and females but also present at much lower levels in testis, ovary and head kidney, an organ known for its steroidogenic capabilities in fish. However, P450aromA expression was restricted to testis and ovary. A semiquantitative PCR was developed to measure P450aromB mRNA levels. Analysis of the expression of P450aromB in the brain of juvenile sea bass showed that females exhibited higher mRNA levels than males at 200 days post fertilization (dpf), by the time of gonadal sex differentiation. A switch in expression occurred thereafter, between 200 and 250 dpf, with males exhibiting higher levels than females. This situation was maintained by 300 dpf and is in agreement with measured levels of enzymatic activity in adults. These changes and sex-related differences in expression profiles may imply differences in the functionality of the enzyme between males and females, suggesting an important role for P450aromB in sea bass sex differentiation. However, due to the continuous growth of the teleost brain throughout life, a role in neurogenesis for brain aromatase should also be considered.     

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.     

Genetic, endocrine, and environmental components of sex determination and differentiation in the European sea bass (Dicentrarchus labrax L.)

The European sea bass (Dicentrarchus labrax L.) is a differentiated gonochoristic marine teleost of the family Moronidae (closely related to the hermaphrodites of the family Serranidae), where many juvenile males exhibit intratesticular oocytes, suggesting a certain sexual lability. Like most fish, the sea bass does not have recognizable heterochromosomes or sex-linked markers but there are clear parental effects on the sex ratios. The data available so far indicate that the proportion of females resulting from individual crossings may range from as little as 1 to about 70%. Sex differentiation proceeds in a caudo-cranial fashion and starts when fish reach 8-9 cm standard length (usually about 200 days post-hatching, dph, under typical rearing conditions), with females differentiating first. Both forms of aromatase have been cloned in this species and their temporal expression has been studied. Brain aromatase is detectable already in the larval stages but its involvement in sex differentiation is not yet clear. The ovarian form increases after 100 dph before ovarian differentiation, with high levels in females and basal levels in males. Thus, ovarian aromatase seems to be involved in female differentiation. On the other hand, androgen receptor (AR) gene expression levels show the opposite pattern, with higher levels in males than in females. It is not yet known whether androgens are necessary for testicular differentiation or rather they are the result of it. Of the several environmental factors tested, temperature is the only one that has been shown to be able to clearly influence sex ratios. Larval and juvenile sea bass reared in captivity at high temperature usually develop as males. Recent research suggests that the high incidence of males under aquaculture conditions is due to the high water temperature used, and that the effects of temperature would be mediated by an inhibition of aromatase mRNA expression and activity in genotypic females. However, other effects of temperature mediated through alterations in developmental rates cannot be discarded. This paper reviews the current knowledge on sex determination and differentiation in the sea bass and suggests some directions for future research.     

Developmental expression and estrogen responses of endocrine genes in juvenile yellow perch (Perca flavescens)

The present study examines the expression of growth-regulating genes (gh, prl, smtl and igf1b), the estrogen receptors (esr1 and esr2a) and aromatase (cyp19a1a) in developing yellow perch. To gain an initial understanding into the endocrine control of growth preceding and involved with sexual size dimorphism (SSD), where females have been reported to grow faster and larger than males, young of the year fish were sampled for length, weight and tissues at several time points (102-421 days post-hatch (dph)). Positive growth was seen in both sexes over the sampling interval, but SSD was not manifested. Using real-time quantitative PCR, we found that pituitary growth hormone (gh) and liver insulin-like growth factor-1b (igf1b) mRNA levels were significantly affected by dph and levels were found to be correlated with growth in both sexes. Liver cyp19a1a, esr1 and esr2a mRNA levels were significantly influenced by dph, whereas there was a significant dph * sex interaction on liver esr2a mRNA levels with males having higher levels than females at 379 and 421 dph. Ovarian cyp19a1a decreased with dph, but there were no changes in esr1 or esr2a mRNA levels. Dietary treatment of juvenile (∼300 dph) females with 20 mg/kg diet 17β-estradiol resulted in significantly higher liver esr1 mRNA levels and a sustained hepatosomatic index (I_H). Across all data sets liver esr2a mRNA levels showed the most significant positive correlation with liver igf1b mRNA levels. These findings show that growth is accompanied by increases in pituitary gh, liver igf1b and liver esr1 and esr2a mRNAs in juvenile yellow perch.     

Characterization of testicular expression of P450 17α-hydroxylase, 17,20-lyase in zebrafish and its perturbation by the pharmaceutical fungicide clotrimazole

The aim of the present study was to characterize P450 17α-hydroxylase/17,20-lyase (cyp17a1) expression in zebrafish and to assess the effect of the pharmaceutical clotrimazole, a known inhibitor of various cytochrome P450 enzyme activities, on testicular gene and protein expression of this enzyme as well as on the testicular release of 11-ketotestosterone (11-KT), a potent androgen in fish. We first showed that cyp17a1 is predominantly expressed in gonads of zebrafish, notably in male. In vivo, clotrimazole induced a concentration-dependent increase of cyp17a1 gene expression and Cyp17-I protein synthesis in zebrafish testis. Using zebrafish testicular explants, we further showed that clotrimazole did not directly affect cyp17a1 expression but that it did inhibit 11-KT release. These novel data deserve further studies on the effect of azole fungicides on gonadal steroidogenesis.     

Fish with thermolabile sex determination (TSD) as models to study brain sex differentiation

As fish are ectothermic animals, water temperature can affect their basic biological processes such as larval development, growth and reproduction. Similar to reptiles, the incubation temperature during early phases of development is capable to modify sex ratios in a large number of fish species. This phenomenon, known as thermolabile sex determination (TSD) was first reported in Menidia menidia, a species belonging to the family Atherinopsidae. Since then, an increasing number of fish have also been found to exhibit TSD. Traditionally, likewise in reptiles, several TSD patterns have been described in fish, however it has been recently postulated that only one, females at low temperatures and males at high temperatures, may represent the “real” or “true” TSD. Many studies regarding the influence of temperature on the final sex ratios have been focused on the expression and activity of gonadal aromatase, the enzyme involved in the conversion of androgens into estrogens and encoded by the cyp19a1a gene. In this regard, teleost fish, may be due to a whole genome duplication event, produce another aromatase enzyme, commonly named brain aromatase, encoded by the cyp19a1b gene. Contrary to what has been described in other vertebrates, fish exhibit very high levels of aromatase activity in the brain and therefore they synthesize high amounts of neuroestrogens. However, its biological significance is still not understood. In addition, the mechanism whereby temperature can induce the development of a testis or an ovary still remains elusive. In this context the present review is aimed to discuss several theories about the possible role of brain aromatase using fish as models. The relevance of brain aromatase and therefore of neuroestrogens as the possible cue for gonadal differentiation is raised. In addition, the possible role of brain aromatase as the way to keep the high levels of neurogenesis in fish is also considered. Several key examples of how teleosts and aromatase regulation can offer more insight into basic mechanisms of TSD are also reviewed.     

Two cytochrome P450 aromatase genes in the hermaphrodite ricefield eel Monopterus albus: mRNA expression during ovarian development and sex change

Previously, the ricefield eel (Monopterus albus) was speculated to have only one cytochrome p450 aromatase gene. In this study, however, the cDNAs encoding two distinct cytochrome p450 aromatases, cyp19a1a and cyp19a1b, were isolated. The genomic organizations of both cyp19 genes were conserved when compared with other teleosts. Northern blot detected an abundant expression of cyp19a1a in the ovary, and cyp19a1b in the hypothalamus. RT-PCR coupled with Southern blot showed that cyp19a1a was expressed predominantly in the gonads of both sexes, with higher levels in the ovary than testis, while cyp19a1b was expressed in all the tissues examined in the male, but only in the brain and pituitary in the female. The levels of cyp19a1a mRNA in the ovary were increased significantly during vitellogenesis, but decreased significantly at mature stage. The levels of cyp19a1b mRNA in the brain and pituitary did not vary significantly during vitellogenesis. As ovarian development shifted from vitellogenesis to maturation, the levels of cyp19a1b mRNA was decreased significantly in the brain, but increased significantly in the pituitary. During natural sex change from female to male, the levels of cyp19a1a mRNA in the gonad were significantly decreased. The levels of cyp19a1b mRNA in the hypothalamus were significantly increased at the early intersexual phase, whereas the expression levels in the pituitary were significantly decreased at the intersexual phases. Taken together, these results showed a novel sexual dimorphism of cyp19a1b mRNA tissue distribution, and both CYP19 genes were associated with the ovarian development and natural sex change of the ricefield eel.