Francesc Piferrer

Ovarian aromatase and estrogens: A pivotal role for gonadal sex differentiation and sex change in fish

The present review focuses on the roles of estrogens and aromatase (Cyp19a1a), the enzyme needed for their synthesis, in fish gonadal sex differentiation. Based on the recent literature, we extend the already well accepted hypothesis of an implication of estrogens and Cyp19a1a in ovarian differentiation to a broader hypothesis that would place estrogens and Cyp19a1a in a pivotal position to control not only ovarian, but also testicular differentiation, in both gonochoristic and hermaphrodite fish species. This working hypothesis states that cyp19a1a up-regulation is needed not only for triggering but also for maintaining ovarian differentiation and that cyp19a1a down-regulation is the only necessary step for inducing a testicular differentiation pathway. When considering arguments for and against, most of the information available for fish supports this hypothesis since either suppression of cyp19a1a gene expression, inhibition of Cyp19a1a enzymatic activity, or blockage of estrogen receptivity are invariably associated with masculinization. This is also consistent with reports on normal gonadal differentiation, and steroid-modulated masculinization with either androgens, aromatase inhibitors or estrogen receptor antagonists, temperature-induced masculinization and protogynous sex change in hermaphrodite species. Concerning the regulation of fish cyp19a1a during gonadal differentiation, the transcription factor foxl2 has been characterized as an ovarian specific upstream regulator of a cyp19a1a promoter that would co-activate cyp19a1a expression, along with some additional partners such as nr5a1 (sf1) or cAMP. In contrast, upstream factors potentially down-regulating cyp19a1a during testicular differentiation are still hypothetical, such as the dmrt1 gene, but their definitive characterization as testicular repressors of cyp19a1a would strongly strengthen the hypothesis that early testicular differentiation would need active repression of cyp19a1a expression.

Endocrine sex control strategies for the feminization of teleost fish

Abstract In many species of cultured finfish, females exhibit higher growth rates than males and attain larger sizes. In addition, in some species, males mature before reaching marketable size. Together, this results in a larger dispersion of sizes and an overall reduction in production. Therefore, there is great interest from the private sector to produce all-female stocks. This review concentrates on the use of oestrogens for sex control, discussing the advantages of producing monosex female stocks for finfish aquaculture, and pointing out those cases in which hormonal sex reversal technology is worth applying. The biological basis on which hormonal sex manipulation rests, the process of sex differentiation—which, compared to that of other vertebrates, is quite labile in teleost fish—is described in order to understand the effects of treatments. Sex control is typically achieved by exposing sexually undifferentiated fish to exogenous steroids in order to direct the process of sex differentiation towards the desired sex. These treatments finish months or years before marketing and steroid residues disappear in less than a month after the end of treatment. The currently available methods to produce monosex female stocks, the direct and the indirect methods, are explained, comparing their respective advantages and disadvantages. Feminizing treatments are also used to produce all-male stocks in some species. Thus, this review concentrates on the use of oestrogens for sex control, either in the direct method of feminization or in the indirect method of masculinization. So far, oestrogens have been applied to at least 56 different species, using 12 different oestrogenic substances (three natural and nine synthetic). Special attention is given to the method of administration, including immersion and dietary treatment, and to the variables of the hormonal treatment itself: steroids used, dose, timing and duration of treatments. The importance of correct treatment timing in relation to the degree of gonadal development is emphasized and the outcome of the treatment evaluated in terms of survival, gonadal morphology and sex ratios, growth performance and deformities. Next, the current methods to produce all-female or essentially all-female stocks are presented for 35 different species, including eels, salmonids, cyprinids, poecilids, cichlids, gouramies and flatfishes. A section on regulatory issues discusses the advantages of using the indirect method of feminization, when feasible, and emphasizes the convenience of using the natural oestrogen estradiol-17β rather than synthetic oestrogens. A guideline for the development of monosex technology in new species is presented. The overall goal is to emphasize the use of the indirect method, which means that fish that reach the marketplace have never been exposed to steroids. If this method is not feasible, as it happens in many species, an alternative is the use of the direct method, applied in an optimized protocol, to achieve maximum treatment efficiency with minimum exposure to steroids.

Temperature-Dependent Sex Determination in Fish Revisited: Prevalence, a Single Sex Ratio Response Pattern, and Possible Effects of Climate Change

Background In gonochoristic vertebrates, sex determination mechanisms can be classified as genotypic (GSD) or temperature-dependent (TSD). Some cases of TSD in fish have been questioned, but the prevalent view is that TSD is very common in this group of animals, with three different response patterns to temperature. Methodology/Principal Findings We analyzed field and laboratory data for the 59 fish species where TSD has been explicitly or implicitly claimed so far. For each species, we compiled data on the presence or absence of sex chromosomes and determined if the sex ratio response was obtained within temperatures that the species experiences in the wild. If so, we studied whether this response was statistically significant. We found evidence that many cases of observed sex ratio shifts in response to temperature reveal thermal alterations of an otherwise predominately GSD mechanism rather than the presence of TSD. We also show that in those fish species that actually have TSD, sex ratio response to increasing temperatures invariably results in highly male-biased sex ratios, and that even small changes of just 1–2°C can significantly alter the sex ratio from 1∶1 (males∶females) up to 3∶1 in both freshwater and marine species. Conclusions/Significance We demonstrate that TSD in fish is far less widespread than currently believed, suggesting that TSD is clearly the exception in fish sex determination. Further, species with TSD exhibit only one general sex ratio response pattern to temperature. However, the viability of some fish populations with TSD can be compromised through alterations in their sex ratios as a response to temperature fluctuations of the magnitude predicted by climate change.

DNA methylation of the gonadal aromatase (cyp19a) promoter is involved in temperature-dependent sex ratio shifts in the European sea bass.

Sex ratio shifts in response to temperature are common in fish and reptiles. However, the mechanism linking temperature during early development and sex ratios has remained elusive. We show in the European sea bass (sb), a fish in which temperature effects on sex ratios are maximal before the gonads form, that juvenile males have double the DNA methylation levels of females in the promoter of gonadal aromatase (cyp19a), the enzyme that converts androgens into estrogens. Exposure to high temperature increased the cyp19a promoter methylation levels of females, indicating that induced-masculinization involves DNA methylation-mediated control of aromatase gene expression, with an observed inverse relationship between methylation levels and expression. Although different CpGs within the sb cyp19a promoter exhibited different sensitivity to temperature, we show that the increased methylation of the sb cyp19a promoter, which occurs in the gonads but not in the brain, is not a generalized effect of temperature. Importantly, these effects were also observed in sexually undifferentiated fish and were not altered by estrogen treatment. Thus, methylation of the sb cyp19a promoter is the cause of the lower expression of cyp19a in temperature-masculinized fish. In vitro, induced methylation of the sb cyp19a promoter suppressed the ability of SF-1 and Foxl2 to stimulate transcription. Finally, a CpG differentially methylated by temperature and adjacent to a Sox transcription factor binding site is conserved across species. Thus, DNA methylation of the aromatase promoter may be an essential component of the long-sought-after mechanism connecting environmental temperature and sex ratios in vertebrate species with temperature-dependent sex determination.

Aromatase activity in the European sea bass (Dicentrarchus labrax L.) brain. Distribution and changes in relation to age, sex, and the annual reproductive cycle

Cytochrome P450 aromatase activity (AA) was measured in different tissues of the sea bass (Dicentrarchus labrax L.) using a tritiated water release assay that was previously optimized and validated for this species. In adult fish entering the reproductive season, AA was highest on a per mg protein basis, in the brain (2.04+/-0.4 pmol/mg prot/h; mean+/-SEM), followed by the ovary (0.59+/-0.1) and was detectable in visceral fat (0.21+/-0.05), liver (0.08+/-0.009), and head kidney (0.03+/-0.004). However, AA was negligible in the rest of the tissues tested: heart, testis, muscle, and spleen. Consistent with results obtained in other species, dissection of the brain into its major constitutive parts revealed that AA was concentrated in areas implicated in the control of reproduction, including the olfactory bulb, telencephalon, and hypothalamus (range: 2.6-16.2 pmol/mg prot/h), as well as the pituitary gland (6.2-9.3 pmol/mg prot/h). Lower AA was noted in the optic bulb, cerebellum, and medulla. However, in contrast to some previously published reports concerning the content and distribution of neural aromatase in fish, males consistently exhibited higher AA than females. In one-year-old juvenile fish completing the process of gonadal sex differentiation, brain AA (0.63 pmol/mg prot/h) was similar in both sexes and ten times lower than that measured in the brain of first time spawners (6.52 pmol/mg prot/h), in this case with males showing an overall higher (24%) activity than females. When surveyed throughout the year, brain AA dramatically changed during the reproductive cycle. Maximum average values of approximately 7 pmol/mg prot/h were obtained that coincided with the spawning season. The peak in brain AA was preceded by two and one months by the peak of plasma testosterone and the peak of the gonadosomatic index, respectively. This is the first measurement of the distribution of the activity of a steroidogenic enzyme in the sea bass, an established model in comparative endocrinology. Together, these results demonstrate sex- and seasonally-related variations in AA and establish the basis for further comparative studies of certain androgen-mediated actions through locally formed estrogen in both central and peripheral targets.

Induction of triploidy and gynogenesis in teleost fish with emphasis on marine species

The induction of triploidy and gynogenesis by chromosome set manipulation has traditionally been studied more intensively in freshwater than in marine fish. In the last years, however, several studies have applied these manipulations in about a dozen marine species, including mainly sparids, moronids and flatfishes. This paper focuses on the methodologies used to induce, verify, and assess performance of both triploids and gynogenetics of these marine species. Since many of them are batch spawners and have small and fragile eggs and larvae, peculiarities relating to broodstock management, gamete quality and mortality assessment during early larval stages are also taken into account. However, data show that if handling is correct and the treatments are optimized, triploid and gynogenetic rates of 100% can be easily achieved. Survival of triploids with respect to the controls is about 70–80%, whereas in gynogenetics it is generally low and more variable, depending on the species considered. In the marine fish investigated so far, triploidy has not resulted in significantly higher growth rates. On the other hand, the induction of gynogenesis has resulted in the production of both all-female and mix-sex stocks. Throughout the paper, special reference is made to the European sea bass (Dicentrarchus labrax L.), a species of both basic and applied interest, for which a comprehensive study has been carried out on the induction, verification and performance of triploids and gynogenetics.

Cloning, sequence analysis, tissue distribution, and sex-specific expression of the neural form of P450 aromatase in juvenile sea bass (Dicentrarchus labrax).

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.

Fish Gonadogenesis. Part I: Genetic and Environmental Mechanisms of Sex Determination

Many species of fish produced in aquaculture or for the ornamental fish trade exhibit sexual dimorphism in growth, age at maturity, secondary sexual characters, or other traits of interest. This has led to a desire to produce populations of only one sex for commercial ongrowing. Although direct sex reversal via manipulation of sex differentiation is used commercially (e.g., in tilapia aquaculture), in most cases there is a need to understand the sex determination system to some extent and manipulate this to produce monosex fish. Sex determination is the genetic or environmental process that establishes the sex (gender) of an organism, whereas sex differentiation is the process by which an undifferentiated gonad is transformed into an ovary or a testis. Fish are the most diverse group of vertebrates in terms of sex determination, and the number of fish species of interest to aquaculture keeps increasing. Together, these aspects explain the growing interest to understand how sex determination and differentiation produce the sex ratio. This review concentrates on recent research using the tools of molecular biology to broaden our understanding of the different aspects related to fish sex determination, both in model fish species and in species of commercial importance.

Gonadal differentiation in coho salmon, Oncorhynchus kisutch, after a single treatment with androgen or estrogen at different stages during ontogenesis

Abstract The labile period for hormonal treatment for purposes of controlling sexual differentiation in coho salmon was deternined by administration of estradiol-17β (E2) and 17α-methyltestosterone (MT) through immersion for 2 h at a concentration of 400 μg/l, to groups of coho salmon sampled weekly from the late eyed-egg stage until the third week of feeding. Histological samples of the gonadal region were prepared from a control group at each weekly treatment interval using a plastic embedding process. It was found that a single estrogen treatment significantly increased the proportion of phenotypic females when administered between 8 days pre-hatch and 13 days post-hatch, while a single androgen treatment significantly increased the proportion of phenotypic males when administered between 6 and 13 days post-hatch. The maximum response for E2-treated groups (84% females) occurred as a result of treatment 1 day before 50% hatch while the maximum response for MT-treated groups (73.1% males) occurred as a result of treatment 1 week later. Thus, the labile period during which the fish were able to respond to a single administration of exogenous steroid was determined to last 3 weeks, and coincided with the time of hatching and the first days of the alevin stage. Sexual differentiation was first observed at 27 days post-hatching, 21–28 days after the time of maximum steroid sensitivity and coinciding approximately with the time of first feeding.

Fish Gonadogenesis. Part II: Molecular Biology and Genomics of Sex Differentiation

The combined result of the mechanisms of sex determination and sex differentiation is the sex ratio of a population. Because sex-related growth dimorphism in fish is quite common, sex ratios influence population reproductive capacity and contribute to size variation and growth patterns before and after sexual maturation. This may have practical consequences for aquaculture and explains the interest in sex control to favor the sex with superior growth, better food conversion efficiency, later sexual maturation, or to prevent reproduction if both sexes mature before harvest. This review concentrates on recent research using molecular biology tools to broaden our understanding of the different aspects related to fish sex differentiation, both in model fish species and economically important species. The contribution of genomics to this field is mainly through the use of microarrays for the analysis of the transcriptome, to identify expression signatures associated with the development of a particular phenotype, or genes involved in the process of sex differentiation, both under normal conditions and after exposure to a particular natural or aquaculture environment.