Tilapia sex determination: Where temperature and genetics meet.

This review deals with the complex sex determining system of Nile tilapia, Oreochromis niloticus, governed by the interactions between a genetic determination and the influence of temperature, shown in both domestic and wild populations. Naturally sex reversed individuals are strongly suggested in two wild populations. This can be due to the masculinising temperatures which some fry encounter during their sex differentiation period when they colonise shallow waters, and/or to the influence of minor genetic factors. Differences regarding a) thermal responsiveness of sex ratios between and within Nile tilapia populations, b) maternal and paternal effects on temperature dependent sex ratios and c) nearly identical results in offspring of repeated matings, demonstrate that thermosensitivity is under genetic control. Selection experiments to increase the thermosensitivity revealed high responses in the high and low sensitive lines. The high-line showed approximately 90% males after 2 generations of selection whereas the weakly sensitive line had 54% males. This is the first evidence that a surplus of males in temperature treated groups can be selected as a quantitative trait. Expression profiles of several genes (Cyp19a, Foxl2, Amh, Sox9a,b) from the gonad and brain were analysed to define temperature action on the sex determining/differentiating cascade in tilapia. The coexistence of GSD and TSD is discussed.

DMRT1 expression during gonadal differentiation and spermatogenesis in the rainbow trout, Oncorhynchus mykiss.

DMRT1 has been suggested to be the first conserved gene involved in sex differentiation found from invertebrates to human. To gain insight on its implication for fish gonadal differentiation, we cloned a DMRT1 homologue in the rainbow trout, Oncorhynchus mykiss (rtDMRT1), and showed that this gene is expressed during testicular differentiation, but not during ovarian differentiation. After 10 days of steroid treatment, expression was shown to be decreased in estrogen-treated male differentiating gonads but not to be restored in androgen-treated differentiating female gonads. This clearly reinforces the hypothesis of an important implication for DMRT1 in testicular differentiation in all vertebrates. In the adults a single 1.5 kb transcript was detected by Northern blot analysis in the testis, and its expression was found to be sustained throughout spermatogenesis and declined at the end of spermatogenesis (stage VI). Along with this expression in the testis we also detected by reverse transcriptase-polymerase chain reaction a slight expression in the ovary. We also obtained new DM-domain homologous sequences in fish, and their analysis suggest that at least four different genes bearing 'DM-domain' (DMRT genes) exist in fish just as in all vertebrate genomes.

Expression of cytochrome P450(11beta) (11beta-hydroxylase) gene during gonadal sex differentiation and spermatogenesis in rainbow trout, Oncorhynchus mykiss.

Androgens and especially 11-oxygenated androgens are known to be potent masculinizing steroids in fish. As a first step to study their physiological implication in gonadal sex differentiation in fish, we cloned a testicular cytochrome P450(11beta) (11beta-hydroxylase) cDNA in the rainbow trout, Oncorhynchus mykiss. We isolated a 1882 bp P450(11beta) cDNA (rt11betaH2, AF217273) which contains an open reading frame encoding a 552 putative amino acids protein. This sequence was highly homologous (98% in nucleotides and 96.5% in amino acids) to another rainbow trout P450(11beta) sequence (AF179894) and also to a Japanese eel P450(11beta) (68% in amino acids). Northern blot analysis detected a single transcript of 2 kb which was highly expressed in the testis (stage II) and to a lesser degree in the anterior kidney (containing the interrenal tissue). No signal was detected in the posterior kidney, brain, liver, skin, intestine and heart. In the testis this transcript was highly expressed at the beginning of spermatogenesis (stages I and II), followed by a decrease during late spermatogenesis (stages III to V). By semi-quantitative reverse transcription polymerase chain reaction, P450(11beta) expression during gonadal differentiation was estimated to be at least 100 times higher in male than in female gonads. This difference was first detected at 55 days post-fertilization (dpf), i.e. 3 weeks before the first sign of histological sex differentiation, and was sustained long after differentiation (127 dpf). Specific P450(11beta) gene expression was also demonstrated before testis differentiation (around 50 dpf) using virtual Northern blot, with no expression detected in female differentiating gonads. From these results, and also based on the already known actions of 11-oxygenated androgens in testicular differentiation in fish, it is now suggested that P450(11beta) gene expression is a key factor for the testicular differentiation in rainbow trout.

Elevated amh gene expression in the brain of male tilapia (Oreochromis niloticus) during testis differentiation.

Anti-müllerian hormone (AMH) is expressed in male embryos and represses development of müllerian ducts during testis differentiation in mammals, birds and reptiles. Amh orthologues have been identified in teleosts despite them lacking müllerian ducts. Previously we found sexually dimorphic aromatase activity in tilapia brains before ovarian differentiation. This prompted us to search for further dimorphisms in tilapia brains during sex differentiation and see whether amh is expressed. We cloned the tilapia amh gene and found that it contains 7 exons but no spliced forms. The putative protein presents highest homologies with Amh proteins of pejerrey and medaka as compared to other Perciformes. We analysed amh expression in adult tissues and found elevated levels in testes, ovary and brain. Amh expression was dimorphic with higher levels in XY male brains at 10-15 dpf, when the gonads were still undifferentiated and gonadal amh was not dimorphic. Male brains had 2.7-fold higher amh expression than gonads. Thereafter, amh levels decreased in the brain while they were up-regulated in differentiating testes. Our study indicates that amh is transcribed in male brains already at 10 dpf, suggesting that sexual differentiation may be occurring earlier in tilapia brain than in gonads.

Environmental effects on fish sex determination and differentiation.

Environmental factors affect the sex ratio of many gonochoristic fish species. They can either determine sex or influence sex differentiation. Temperature is the most common environmental cue affecting sex but density, pH and hypoxia have also been shown to influence the sex ratio of fish species from very divergent orders. Differential growth or developmental rate is suggested to influence sex differentiation in sea bass. Studies in most fish species used domestic strains reared under controlled conditions. In tilapia and sea bass, domestic stocks and field-collected populations showed similar patterns of thermosensitivity under controlled conditions. Genetic variability of thermosensitivity is seen between populations but also between families within the same population. Furthermore, in the Nile tilapia progeny testing of wild male breeders has strongly suggested the existence of XX males in 2 different natural populations. Tilapia and Atlantic silverside studies have shown that temperature sensitivity is a heritable trait which can respond to directional (tilapia) or frequency dependent selection. In tilapia, transitional forms within a genetic sex determination (GSD) and environmental sex determination (ESD) continuum seem to exist. Temperature regulates the expression of the ovarian-aromatase cyp19a1 which is consistently inhibited in temperature masculinized gonads. Foxl2 is suppressed before cyp19a1. Recent in vitro studies have shown that foxl2 activates cyp19a1, suggesting that temperature acts directly on foxl2 or further upstream. Dmrt1 up-regulation is correlated with temperature-induced male phenotypes. Temperature through apoptosis or germ cell proliferation could be a critical threshold for male or female sex differentiation.

Genetics of sex determination in tilapiine species.

We identified DNA markers linked to sex determining genes in six closely related species of tilapiine fishes. The mode of sex determination differed among species. In Oreochromis karongae and Tilapia mariae the sex-determining locus is on linkage group (LG) 3 and the female is heterogametic (WZ-ZZ system). In O. niloticus and T. zillii the sex-determining locus is on LG1 and the male is heterogametic (XX-XY system). A more complex pattern was observed in O. aureus and O. mossambicus, in which markers on both LG1 and LG3 were associated with sex. We found evidence for sex-linked lethal effects on LG1, as well as interactions between loci in the two linkage groups. Comparison of genetic and physical maps demonstrated a broad region of recombination suppression harboring the sex-determining locus on LG3. Sex-specific recombination suppression was found in the female heterogametic sex. Sequence analysis showed the accumulation of repetitive elements in this region. Phylogenetic analysis suggests that at least two transitions in the mode of sex determination have occurred in this clade. This variation in sex determination mechanisms among closely related species makes tilapias an excellent model system for studying the evolution of sex chromosomes in vertebrates.

Mapping of sox2 and sox14 in tilapia (Oreochromis spp.).

Sox genes encode transcription factors that are involved in a variety of embryonic developmental pathways. Sox2 and Sox14 are located on the same chromosomal arm in several mammalian and bird species and on the basis of comparative maps were suggested as candidate genes for the major sex-determining locus on tilapia LG3. We have sequenced the sox2 and sox14 genes in four tilapia species and mapped them to different chromosomes, LG17 and LG23 respectively. Although excluded as being one of the major sex-determining genes so far mapped in tilapia, sox14 did fall within a QTL region for growth, stress response, embryonic mortality and a minor effect on sex determination.

Environment and sex determination in farmed fish.

A plasticity of gonadal sex differentiation was reported in the 1930s following exogenous steroid treatments in fish, but demonstration that environmental factors (temperature, pH, density and social interactions) could influence the sex ratio in gonochoristic species has been relatively recent. In fish, as in reptiles and amphibians displaying environmental sex determination, the main environmental factor influencing sex seems to be temperature (TSD=Temperature Sex Determination). In most thermosensitive species (some Atherinids, Poecilids, Cichlids: tilapias, goldfish, a Siluriform, a flatfishellipsis) male to female ratio increases with temperature and/or ovarian differentiation is induced by low temperatures. Conversely, in some rare species (Dicentrarchus labrax, Ictalurus punctatus), high temperatures may produce female-biased sex ratios and/or low temperatures promote male-biased sex ratios. In the hirame Paralichthys olivaceus, both high and low temperatures induce monosex male populations while intermediate temperatures yield a 1:1 sex ratio (U-shape curve). Fish show particularities in their TSD patterns since mono-sex populations are generally not produced at extreme temperatures, suggesting the existence of strong temperature/genotype interactions. In reptiles, amphibians and fish displaying TSD, temperature treatments must be applied at a critical sensitive period, relatively similar to the hormone sensitive period. In gonochoristic fish, steroid hormones with estrogens in females and 11-oxygenated androgens in males, are probably key physiological steps in the regulation of gonadal sex differentiation. Cytochrome P450-aromatase, enzyme catalysing conversion of androgens to estrogens, seems to be a critical enzyme for ovarian differentiation. Molecular mechanisms of thermosensitivity have been addressed in two species tilapia Oreochromis niloticus and the hirame, where aromatase gene expression is down-regulated by masculinizing temperature treatments. Furthermore, in tilapia the gene expression of 11 beta-hydroxylase (a key enzyme involved in the synthesis of 11-oxygenated androgens) does not appear to be affected by temperature treatments.

Synthesis of gill Na(+)-K(+)-ATPase in Atlantic salmon smolts: differences in alpha-mRNA and alpha-protein levels.

Several parameters were analyzed to determine the mechanisms responsible for the enhancement of the gill Na(+)-K(+)-ATPase activity of Atlantic salmon smolts. A major alpha-subunit transcript of 3.7 kb was revealed by Northern blot in both parr and smolt gills when hybridized with two distinct cDNA probes. The alpha-mRNA abundance demonstrated an increase to maximal levels in smolts at an early stage of the parr-smolt transformation. This was followed by a gradual rise in alpha-protein levels, revealed by Western blots with specific antibodies and by an increase in gill Na(+)-K(+)-ATPase hydrolytic activity, both only reaching maximum levels a month later, at the peak of the transformation process. Parr fish experienced a decrease in alpha-mRNA abundance and had basal levels of alpha-protein and enzyme activity. Measurement of the binding of [(3)H]ouabain to Na(+)-K(+)-ATPase was characterized in smolts and parr gill membranes showing more than a twofold elevation in smolts and was of high affinity in both groups (dissociation constant = 20-23 nM). Modulation of the enzyme due to increased salinity was also observed in seawater-transferred smolts, as demonstrated by an increase in alpha-mRNA levels after 24 h with a rise in Na(+)-K(+)-ATPase activity occurring only after 11 days. No qualitative change in alpha-expression was revealed at either the mRNA or protein level. Immunological identification of the alpha-protein was performed with polyclonal antibodies directed against the rat alpha-specific isoforms, revealing that parr, freshwater, and seawater smolts have an alpha(3)-like isoform. This study shows that the increase in Na(+)-K(+)-ATPase activity in smolt gills depends first on an increase in the alpha-mRNA expression and is followed by a slower rise in alpha-protein abundance that eventually leads to a higher synthesis of Na(+)-K(+) pumps.

Search for genes involved in the temperature-induced gonadal sex differentiation in the tilapia, Oreochromis niloticus.

In the tilapia, Oreochromis niloticus, sex is determined by genetic factors (XX/XY) but temperature can also influence the gonadal sex differentiation. Elevated temperatures of 35 degrees C can generate functional male phenotypes if applied before and during sexual differentiation. The genes and mechanisms by which temperature acts on the cascade leading to sex differentiation have been investigated. Two strategies have been followed: 1) Search for novel genes by differential display, and 2) Expression studies of candidate genes. Genetically all-female and all-male progenies were reared at 27 degrees C (natural temperature) and at 35 degrees C (masculinizing treatment) and gonads dissected. Using differential display, we isolated a 300 bp cDNA (MM20C) from temperature-masculinized females. Virtual northern analysis revealed a 1.2 kb transcript in 35 degrees C treated females and males, but hardly any expression in natural females (27 degrees C). Semi-quantitative RT-PCR established a several-fold increase in MM20C expression in 35 degrees C masculinized fry. Elevated expression was observed in natural males (27 degrees C) with higher levels detected in those reared at 35 degrees C. Furthermore, we have analyzed as a candidate gene the P450 11beta-hydroxylase, an important androgen steroidogenic enzyme. Low levels of expression were found in natural males. This coincides with low concentrations of 11 ketotestosterone in the gonads before and during gonadal sex differentiation. Higher expression levels of 11beta-hydroxylase were detected in male gonads at 35 degrees C but levels in phenotypic males were similar to those found for natural females. Previous results reported that expression of aromatase is repressed by masculinizing treatments. Our study demonstrated that masculinizing-temperature can also stimulate the expression of other gene(s).

Comparison between parr and smolt Atlantic salmon (Salmo salar) α subunit gene expression of Na(+)/K (+) ATPase in gill tissue.

Increases in branchial Na(+)/K(+) ATPase activity during seawater adaptation of euryhaline fish species, have been well documented. During the parr-smolt transformation of salmonids this activity increases two to five fold and is used as an indicator of the transformation. In order to improve the understanding of differences in enzyme activity found between Atlantic salmonSalmo salar parr and smolt fish, we investigated the gene expression of the Na(+)/K(+) ATPase α-subunit(s) in gill tissue. Gill mRNAs were analyzed and quantified at distinct time points using Northern and Dot blot techniques. We amplified by PCR, a conserved region of the cDNA encoding the Na(+)/K(+) ATPase α-subunit of the rainbow troutOncorhynchus mykiss. The PCR products (670 bp) were cloned and all independent clones showed a sequence corresponding to the α subunit of the Na(+)/K(+) ATPase. The fragments obtained appeared as a heterogenous population of three sequences showing, when compared between each other, 86 to 93% identity. This suggests that different allelic forms of the α-subunit are expressed in gill tissue. Hybridization studies performed with these PCR probes revealed two mRNA species, a major 3.7 kb transcript and a minor transcript of 1.8 kb. Enhanced 3.7 kb transcript levels are concurrent with elevated enzyme activity in smolts during the March and April parrsmolt transformation of Atlantic salmon. Interestingly, our study disclosed that smolt fish only displayed a two-fold increase in transcript levels when compared to parr whereas enzyme activity showed a 4 to 5 fold increase. This suggests that the increase in the 3.7 kb mRNA content of gill tissue is probably not the only mediator leading to the rise in enzyme activity during parr-smolt transformation.

Aromatase plays a key role during normal and temperature-induced sex differentiation of tilapia Oreochromis niloticus.

In the tilapia Oreochromis niloticus, sex is determined genetically (GSD), by temperature (TSD) or by temperature/genotype interactions. Functional masculinization can be achieved by applying high rearing temperatures during a critical period of sex differentiation. Estrogens play an important role in female differentiation of non-mammalian vertebrates. The involvement of aromatase, was assessed during the natural (genetic all-females and all-males at 27 degrees C) and temperature-induced sex differentiation of tilapia (genetic all-females at 35 degrees C). Gonads were dissected between 486--702 degree x days. Aromatase gene expression was analyzed by virtual northern and semi-quantitative RT-PCR revealing a strong expression during normal ovarian differentiation concomitant with high levels (465 +/- 137 fg/g) of oestradiol-17 beta (E2-17 beta). This was encountered in gonads after the onset of ovarian differentiation (proliferation of both stromal and germ cells prior to ovarian meiosis). Genetic males exhibited lower levels of aromatase gene expression and E2-17 beta quantities (71 +/- 23 fg/ g). Aromatase enzyme activity in fry heads established a sexual dimorphism in the brain, with high activity in females (377.9 pmol/head/hr) and low activity in males (221.53 pmol/head/hr). Temperature induced the masculinization of genetic females to a different degree in each progeny, but in all cases repression of aromatase expression was encountered. Genetic males at 35 degrees C also exhibited a repression of aromatase expression. Aromatase brain activity decreased by nearly three-fold in the temperature-masculinized females with also a reduction observed in genetic males at 35 degrees C. This suggests that aromatase repression is required in the gonad (and perhaps in the brain) in order to drive differentiation towards testis development. Mol. Reprod. Dev. 59:265-276, 2001.

Steroid enzyme gene expressions during natural and androgen-induced gonadal differentiation in the rainbow trout, Oncorhynchus mykiss.

In fish, according to Yamamoto's model, androgens would drive testis differentiation and estrogens ovarian differentiation. In order to study the implication of steroid enzymes in rainbow trout gonadal differentiation, we examined the expression of some steroid enzyme genes during natural differentiation (cholesterol side chain cleavage = P450scc, 17-hydroxylase/lyase = P450c17, 3beta-hydroxysteroid dehydrogenase = 3betaHSD) and androgen-induced differentiation (P450scc, P450c17, 3betaHSD, aromatase = P450aro, and 11beta-hydroxylase = P45011beta). Expressions of P450scc, 3betaHSD, and P450c17 were all detected in male and female gonads at 55 days post-fertilization (dpf), i.e., two weeks before histological differentiation. There were no differences in their expression level respective to the sex. The androgen treatment was carried out by administration of 11beta-hydroxyandrostenedione (11betaOHDelta4) in genetic all-female populations and the resulting sex ratios were found to be 100% male even at a low dosage of 1 mg/kg of food. Following 11betaOHDelta4 treatment, only the expression of P450c17 was found to be sustained when compared with the female untreated control. In contrast, P450scc was clearly up-regulated and 3betaHSD and P450aro down-regulated by the androgen treatment. P45011beta gene expression remained low in gonads of androgen-treated females, as it did in control untreated females. These results together demonstrate that steroidogenesis in rainbow trout is potentially active in pre-differentiating gonads of both sexes, and that one of the masculinizing actions of androgens in the species may be to down-regulate the female-specific gonadal P450aro gene expression. However, in vivo androgen treatment in genetic females does not induce the same pattern of steroid gene expression as in genetic males. These data suggest that exogenous androgens might induce a male differentiation process with P450aro inhibition being one of the steps required. However, this process would not involve endogenously produced 11-oxygenated androgens.