hsd17b1 is a key gene for ovarian differentiation of the Siberian sturgeon.

This is the first work using gonads from undifferentiated, genetically-sexed Siberian sturgeon describing expression changes in genes related to steroid synthesis and female and male sex differentiation. One factor identified as relevant for ovarian differentiation was the gene coding for the enzyme Hsd17b1, which converts estrone into estradiol-17ß. hsd17b1 was highly activated in female gonads at 2.5 months of age, around the onset of sex differentiation, preceding activation of two other genes involved in estrogen production (cyp19a1 and foxl2). hsd17b1 was also strongly repressed in males. Two known foxl2 paralogs are found in Siberian sturgeon-foxl2 and foxl2l-but only foxl2 appeared to be associated with ovarian differentiation. With regard to the male pathway, neither 11-oxygenated androgens nor classic male genes (amh, dmrt1, sox9, and dhh) were found to be involved in male sex differentiation, leaving open the question of which genes participate in early male gonad development in this ancient fish. Taken together, these results indicate an estrogen-dependence of female sex differentiation and 11-oxygenated androgen-independence of male sex differentiation.

Siberian sturgeon multi-tissue reference transcriptome database.

MOTIVATION: Siberian sturgeon is a long lived and late maturing fish farmed for caviar production in 50 countries. Functional genomics enable to find genes of interest for fish farming. In the absence of a reference genome, a reference transcriptome is very useful for sequencing based functional studies. RESULTS: We present here a high-quality transcriptome assembly database built using RNA-seq reads coming from brain, pituitary, gonadal, liver, stomach, kidney, anterior kidney, heart, embryonic and pre-larval tissues. It will facilitate crucial research on topics such as puberty, reproduction, growth, food intake and immunology. This database represents a major contribution to the publicly available sturgeon transcriptome reference datasets. AVAILABILITY: The database is publicly available at http://siberiansturgeontissuedb.sigenae.org Supplementary information:  Supplementary data are available at Database online.

RNA-Seq transcriptome data of undifferentiated and differentiated gonads of Siberian sturgeon.

RNA-Seq transcriptome data from twenty Siberian sturgeon gonads at different developmental stages is described: ten undifferentiated gonads, six gonads of immature males and four gonads from immature females. Siberian sturgeon, Acipenser baerii, is long-lived, late-maturing fish farmed in 50 countries but its production remains on a craftsman scale when compared to industrial species. Sturgeon genetic and physiological studies are less developed than for industrial fish. The data presented hereafter enables fundamental studies on the regulatory mechanisms of sturgeon gonad development, which can further be applied both in aquaculture and in fundamental research.

Multi-tissue Siberian sturgeon RNA sequencing data.

Siberian sturgeon, Acipenser baerii, is a commercially valuable fish for flesh and caviar production and a threatened species. We produced transcriptomic data for ten tissues with relevance to puberty, reproduction, early development, growth and food intake. The data includes RNA-Seq read sets of brain, pituitary, anterior-kidney, kidney, stomach, liver, heart, embryonic, pre-larval, and immature gonad sequences. Tissues were collected from sex differentiated fish (17 to 42 months of age, 66 to 85 cm) RNA was extracted and sequenced. Our purpose is to facilitate fundamental studies of sturgeon physiology to wild and aquaculture populations management.

De novo transcriptome analysis to search for sex-differentiation genes in the Siberian sturgeon.

The sturgeon family includes many species that are lucrative for commercial caviar production, some of which face critical conservation problems. The purpose of this study was to identify genes involved in gonadal sex differentiation in sturgeons, contributing to our understanding of the biological cycle of this valuable species. A high-quality de novo Siberian sturgeon gonadal transcriptome was built for this study using gonadal samples from undifferentiated fish at 3, 5, and 6 months of age; recently sex-differentiated fish at 9 months of age; and immature males and females at 14-17 months of age. Undifferentiated fish were sexed after validation of forkhead box L2 (foxl2) and cytochrome P450, family 19, subfamily A, and polypeptide 1a (cyp19a1a) as sex markers, and the transcriptomes of the 3-month-old undifferentiated fish, 5-6-month-old future females, and 5-6-month-old putative males were compared. The ovarian program was associated with strong activation of genes involved in estrogen synthesis (cyp19a1, foxl2, and estradiol 17-beta-dehydrogenase 1), stem-cell niche building and regulation, and sex-specific nerve cell development. The genes related to the stem-cell niche were: (1) the family of iroquois-class homeodomain proteins 3, 4, and 5 (irx3, irx4, irx5-1, irx5-2, and irx5-3), which are essential for somatic-germ cell interaction; (2) extracellular matrix remodeling genes, such as collagen type XXVIII alpha 1 chain and collagen type II alpha 1 chain, matrix metalloproteinases 24-1 and 24-2, and NADPH oxidase organizer 1, which, along with the somatic cells, provide architectural support for the stem-cell niche; and (3) mitogenic factors, such as lim homeobox 2, amphiregulin, G2/M phase-specific E3 ubiquitin-protein ligase, and connector enhancer of kinase suppressor of ras 2, which are up regulated in conjunction with the anti-apoptotic gene G2/M phase-specific E3 ubiquitin-protein ligase suggesting a potential involvement in regulating the number of germ cells. Genes related to sex-specific nerve cell developments were: the neurofilament medium polypeptides, the gene coding for serotonin receptor 7, 5-hydroxytryptamine receptor 7; neurotensin, isoform CRA-a, the neuron-specific transmembrane protein Delta/Notch-like epidermal growth factor-related receptor; and insulinoma-associated protein 1. The putative testicular program was poorly characterized by elements of the immune response. The classic markers of maleness were not specifically activated, indicating that testicular differentiation occurs at a later stage. In sum, the ovarian program, but not the testicular program, is in place by 5-6 months of age in the Siberian sturgeon. The female program is characterized by estrogen-related genes with well-established roles in gonadal differentiation, but also by several genes with no previously-described function in the ovarian development of fish. These newly-reported genes are involved in stem-cell niche building and regulation as well as sex-specific nerve development.

Sex hormone-binding globulin b expression in the rainbow trout ovary prior to sex differentiation.

Salmonids have two sex hormone-binding globulin (Shbg) paralogs. Shbga is mainly expressed in the liver, while Shbgb is secreted by the granulosa cells of the rainbow trout ovary. Coexpression of shbgb and the gonadal aromatase cyp19a1a mRNAs been observed in granulosa cells, suggesting a physiological coordination between Shbgb expression and estrogen synthesis. As estrogens are essential for female sex determination in the fish ovary, we propose that Shbgb participates in early ovarian differentiation, either by binding with estrogen or through another mechanism that remains to be discovered. To elucidate this potential role, monosex populations of female trout were studied during the molecular ovarian differentiation period (28-56 dpf). shbgb mRNA expression was measured using qPCR and compared with expression of genes for other ovarian markers (cyp19a1a, foxl2, follistatin, and estrogen receptors). shbgb transcript expression was detected during the final stages of embryonic development (21-26 dpf) and during molecular ovarian differentiation (32-52 dpf) after hatching (which occurred at 31 dpf). In situ hybridization localized shbgb transcription to the undifferentiated ovary at 42 dpf, and shbgb and cyp19a1a mRNA showed similar expression patterns. These results suggest that Shbgb is involved in early ovarian differentiation, supporting an important role for the salmonid shbgb gene in sex determination.

Identification of the molecular sex-differentiation period in the Siberian sturgeon.

Sexual development prior to gonadal sex differentiation is regulated by various molecular mechanisms. In fish, a "molecular sex-differentiation period" has been identified in species for which sex can be ascertained prior to gonadal sex differentiation. The present study was designed to identify such a period in a species for which no genetic sex markers or monosex populations are available. Siberian sturgeons undergo a slow sex-differentiation process over several months, so gonad morphology and gene expression was tracked in fish from ages 3-27 months to identify the sex-differentiation period. The genes amh, sox9, and dmrt1 were selected as male gonad markers; cyp19a1a and foxl2a as female gonad markers; and cyp17a1 and ar as markers of steroid synthesis and steroid receptivity. Sex differentiation occurred at 8 months, and was preceded by a molecular sex-differentiation period at 3-4 months, at which time all of the genes except ar showed clear expression peaks. amh and sox9 expression seemed to be involved in male sexual development whereas dmrt1, a gene involved in testis development in metazoans, unexpectedly showed a pattern similar to those of the genes known to be involved in female gonadal sex differentiation (cyp19a1 and foxl2a). In conclusion, the timing of and gene candidates involved with molecular sex differentiation in the Siberian sturgeon were identified.

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 ((3)H-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.

Expression of dmrt1 and sox9 during gonadal development in the Siberian sturgeon (Acipenser baerii).

The sex differentiation period of the Siberian sturgeon was investigated through expression profiling of two testicular markers (dmrt1 and sox9). At the molecular level, a clear sexual dimorphism of dmrt1 and sox9 was observed in 3-year-old fish with immature gonads, in which males showed higher expression of these genes. Among 16-month-old sturgeons cultured in Uruguay, gonad morphology analyses showed one group of fish with undifferentiated gonads and a second group which had started their histological differentiation into ovaries or testes. dmrt1 showed a significantly higher expression in testes of recently differentiated fish, but this was not the case for sox9. In undifferentiated fish, we observed two clearly different groups in terms of expression: one group of fish over-expressing male markers (dmrt1, sox9) and another group of fish showing very low expression of these genes. This suggests that fish undergoing male differentiation can be identified by their profiles of gene expression before they undergo morphological differentiation.

Expression and phylogeny of candidate genes for sex differentiation in a primitive fish species, the Siberian sturgeon, Acipenser baerii.

The molecular mechanisms underlying testis differentiation in basal actinopterygian fish remains poorly understood. The sex differentiation period was investigated in the Siberian sturgeon, Acipenser baerii, by expression profiling of Sertoli cell transcription factors (dmrt1, sox9) that control testis differentiation in vertebrates; Leydig cell factors (cyp17a1, star) affecting androgen production; the androgen receptor (ar); a growth factor controlling testis development (igf1); and a gene coding for a gonadotropin hormone (lh). Two genes were characterised for the first time in the Siberian sturgeon (dmrt1, cyp17a1), while the others came from public databases. Sturgeon gonad development is very slow, with a late sexual differentiation time during their juvenile stage, and are still immature at 3 years of age. Immature fish showed a sex-dimorphic pattern; all the genes studied displayed a higher expression level in male gonads. We took advantage of the presence of juvenile fish with pre- and post-differentiated gonads (16 and 18 months old) to characterise them at the molecular level. The post-differentiated fish displayed a sex dimorphism of gene expression in their gonads for all genes studied, with the exception of sox9. The trends in undifferentiated fish lead us to propose that sturgeons undergoing male differentiation express high levels of Sertoli cell factors (dmrt1, sox9) and of genes involved in the production and receptivity of androgens (cyp17a1, star and ar) together with lh. Expression profiles and phylogenetic studies suggest that these genes are potential regulators of testis development in the Siberian sturgeon.

Sexual dimorphism in the brain aromatase expression and activity, and in the central expression of other steroidogenic enzymes during the period of sex differentiation in monosex rainbow trout populations.

Using genetic monosex male and female rainbow trout populations, the potential sex differences in the central expression of estrogen receptors (esr1, esr2a, esr2b), brain aromatase (cyp19a1b) and some other steroidogenic enzymes was studied over the period of sex differentiation (from 35 to 63 dpf: days post-fertilization) using quantitative polymerase chain reaction (q-PCR). In addition, aromatase activity was evaluated during this period. The results indicated that brain aromatase (cyp19a1b) expression and activity showed a clear and significant sexually dimorphic pattern with higher levels in male brain between 35 and 53 dpf before the time of gonad morphological differentiation. At that time the expression of a key enzyme involved in the conversion of cholesterol into steroids, the cyp11a1 (p450scc), as well as the estrogen receptors were also sexually dimorphic. The dimorphism was lost from 56 dpf onwards. Transcription factors such as nr5a1b (sf1) and nr0b1 (dax1), but not foxl2a were also higher in males than in females. These results demonstrate that, before or during the early period of morphological gonad differentiation, the brain exhibits a clear sexual dimorphism with respect to the expression and activity of aromatase as well as of certain enzymes and factors involved in steroid synthesis as p450scc and sf1. The results suggest a higher potentiality to produce estrogens by male brains during sex differentiation time.

Molecular characterization of cyp11a1 and cyp11b1 and their gene expression profile in pejerrey (Odontesthes bonariensis) during early gonadal development.

Sex steroids are known to be involved in gonadal differentiation in fish, but whether androgens are early mediators of testis differentiation remains unclear. We studied the sex-related developmental variations in the gene expression of two key enzymes involved in steroids and androgen synthesis (cyp11a1 and cyp11b1) in trunks and isolated gonads of pejerrey (Odontesthes bonariensis) larvae during and after the sex determination period. Also, and in order to have a better characterization of this process we studied the expression of Sertoli (dmrt1, amh, sox9) and Leydig (nr5a1 or sf-1) cell markers as well as a gene with higher expression in females (cyp19a1a). No clear differences were observed in the expression of cyp11a1 and cyp11b1 during the temperature-sensitive window in the trunk of pejerrey larvae. Nevertheless, a clear increase of cyp11b1 was observed in isolated gonads taken from fish reared at the male producing temperature. In these gonads we also confirmed the trends of genes with higher expression in males (dmrt1, amh) and females (cyp19a1a) as previously described in larval trunks of pejerrey. Our results showed that the expression of cyp11b1 was positively associated with the morphological differentiation of the testis. Nevertheless the involvement of 11-oxygenated androgens during the temperature-sensitive window could not be clearly established.

Estrogen treatment up-regulates female genes but does not suppress all early testicular markers during rainbow trout male-to-female gonadal transdifferentiation.

In non-mammalian vertebrates, estrogens are key players in ovarian differentiation, but the mechanisms by which they act remain poorly understood. The present study on rainbow trout was designed to investigate whether estrogens trigger the female pathway by activating a group of early female genes (i.e. cyp19a1, foxl2a, foxl2b, fst, bmp4, and fshb) and by repressing early testicular markers (i.e. dmrt1, nr0b1, sox9a1 and sox9a2). Feminization was induced in genetically all-male populations using 17alpha-ethynylestradiol (EE2, 20 mg/kg of food during 2 months). The expression profiles of 100 candidate genes were obtained by real-time RT-PCR and 45 expression profiles displayed a significant differential expression between control populations (males and females) and EE2-treated populations. These expression profiles were grouped in five temporally correlated expression clusters. The estrogen treatment induced most of the early ovarian differentiation genes (foxl2a, foxl2b, fst, bmp4, and fshb) and in particular foxl2a, which was strongly and quickly up-regulated. Simultaneously, Leydig cell genes, involved in androgen synthesis, as well as some Sertoli cell markers (amh, sox9a2) were strongly repressed. However, in contrast to our initial hypothesis, some genes considered as essential for mammalian and fish testis differentiation were not suppressed during the early process of estrogen-induced feminization (dmrt1, nr0b1, sox9a1 and pax2a) and some were even strongly up-regulated (nr0b1, sox9a1and pax2a). In conclusion, estrogens trigger male-to-female transdifferentiation by up-regulating most ovarian specific genes and this up-regulation appears to be crucial for an effective feminization, but estrogens do not concomitantly down-regulate all the testicular differentiation markers.