Multi-genome comparisons reveal gain-and-loss evolution of anti-Mullerian hormone receptor type 2 as a candidate master sex-determining gene in Percidae.

BACKGROUND: The Percidae family comprises many fish species of major importance for aquaculture and fisheries. Based on three new chromosome-scale assemblies in Perca fluviatilis, Perca schrenkii, and Sander vitreus along with additional percid fish reference genomes, we provide an evolutionary and comparative genomic analysis of their sex-determination systems. RESULTS: We explored the fate of a duplicated anti-Mullerian hormone receptor type-2 gene (amhr2bY), previously suggested to be the master sex-determining (MSD) gene in P. flavescens. Phylogenetically related and structurally similar amhr2 duplicates (amhr2b) were found in P. schrenkii and Sander lucioperca, potentially dating this duplication event to their last common ancestor around 19-27 Mya. In P. fluviatilis and S. vitreus, this amhr2b duplicate has been likely lost while it was subject to amplification in S. lucioperca. Analyses of the amhr2b locus in P. schrenkii suggest that this duplication could be also male-specific as it is in P. flavescens. In P. fluviatilis, a relatively small (100 kb) non-recombinant sex-determining region (SDR) was characterized on chromosome 18 using population-genomics approaches. This SDR is characterized by many male-specific single-nucleotide variations (SNVs) and no large duplication/insertion event, suggesting that P. fluviatilis has a male heterogametic sex-determination system (XX/XY), generated by allelic diversification. This SDR contains six annotated genes, including three (c18h1orf198, hsdl1, tbc1d32) with higher expression in the testis than in the ovary. CONCLUSIONS: Together, our results provide a new example of the highly dynamic sex chromosome turnover in teleosts and provide new genomic resources for Percidae, including sex-genotyping tools for all three known Perca species.

Multi-genome comparisons reveal gain-and-loss evolution of the anti-Mullerian hormone receptor type 2 gene, an old master sex determining gene, in Percidae.

The Percidae family comprises many fish species of major importance for aquaculture and fisheries. Based on three new chromosome-scale assemblies in Perca fluviatilis, Perca schrenkii and Sander vitreus along with additional percid fish reference genomes, we provide an evolutionary and comparative genomic analysis of their sex-determination systems. We explored the fate of a duplicated anti-Mullerian hormone receptor type-2 gene (amhr2bY), previously suggested to be the master sex determining (MSD) gene in P. flavescens. Phylogenetically related and structurally similar amhr2 duplications (amhr2b) were found in P. schrenkii and Sander lucioperca, potentially dating this duplication event to their last common ancestor around 19-27 Mya. In P. fluviatilis and S. vitreus, this amhr2b duplicate has been lost while it was subject to amplification in S. lucioperca. Analyses of the amhr2b locus in P. schrenkii suggest that this duplication could be also male-specific as it is in P. flavescens. In P. fluviatilis, a relatively small (100 kb) non-recombinant sex-determining region (SDR) was characterized on chromosome-18 using population-genomics approaches. This SDR is characterized by many male-specific single-nucleotide variants (SNVs) and no large duplication/insertion event, suggesting that P. fluviatilis has a male heterogametic sex determination system (XX/XY), generated by allelic diversification. This SDR contains six annotated genes, including three (c18h1orf198, hsdl1, tbc1d32) with higher expression in testis than ovary. Together, our results provide a new example of the highly dynamic sex chromosome turnover in teleosts and provide new genomic resources for Percidae, including sex-genotyping tools for all three known Perca species.

Corticosteroid plasma kinetics and gonadal receptor gene expression during the reproductive cycle in female Eurasian Perch: Investigation of the roles of corticosteroids in vitellogenesis.

To improve the quality of reproduction in Eurasian perch, Perca fluviatilis L., which is a promising candidate for Eurasian freshwater aquaculture that is currently cultivated in recirculating aquaculture systems (RAS), investigating the hormones that mediate and affect reproduction in this species is indispensable. The literature defines a group of four major corticosteroids (11-deoxycorticosterone, 11-deoxycortisol, corticosterone and cortisol) that might mediate critical stages of reproduction in female perch. Unfortunately, neither the basic roles nor the kinetics of these four corticosteroids throughout the reproductive cycle of female perch have been well defined to date. In this study, we therefore elucidated the plasma kinetics of these four corticosteroids during the reproductive cycle of domesticated female perch while monitoring the expression of the different receptors and enzymes that mediate their production and possible functions. Additionally, we performed an in vitro experiment during late vitellogenesis to investigate the possible direct roles of these steroids during that stage. Our results revealed that these four corticosteroids were detectable throughout the reproductive cycle, and the levels of most of them (11-deoxycorticosterone, 11-deoxycortisol, and cortisol) fluctuated significantly depending on the stage of reproduction. 11-Deoxycorticosterone and 11-deoxycortisol exhibited their highest levels, 1.8 ng/ml and 58 ng/ml, respectively, at the beginning of the reproductive cycle. By the end of the reproductive cycle, 11-deoxycortisol and cortisol plasma levels exhibited a surge, reaching 58 ng/ml and 150 ng/ml, respectively. During the perch reproductive cycle, the corticosteroid receptor complex is not regulated only at the hormone level, as the expression levels of all corticosteroid receptor genes showed a progressive and similar decline. In vitro exposure of vitellogenic oocytes to some of these corticosteroids (11-deoxycorticosterone and 11-deoxycortisol) induced an increase in yolk globule diameter and a decrease in the density of yolk globules, which indicates the involvement of both of these hormones in yolk globule coalescence. Taken together, these results implicate corticosteroids in the reproductive cycle, although the related cellular mechanisms remain to be investigated.

Domestication may affect the maternal mRNA profile in unfertilized eggs, potentially impacting the embryonic development of Eurasian perch (Perca fluviatilis).

Domestication is an evolutionary process during which we expect populations to progressively adapt to an environment controlled by humans. It is accompanied by genetic and presumably epigenetic changes potentially leading to modifications in the transcriptomic profile in various tissues. Reproduction is a key function often affected by this process in numerous species, regardless of the mechanism. The maternal mRNA in fish eggs is crucial for the proper embryogenesis. Our working hypothesis is that modifications of maternal mRNAs may reflect potential genetic and/or epigenetic modifications occurring during domestication and could have consequences during embryogenesis. Consequently, we investigated the trancriptomic profile of unfertilized eggs from two populations of Eurasian perch. These two populations differed by their domestication histories (F1 vs. F7+-at least seven generations of reproduction in captivity) and were genetically differentiated (FST = 0.1055, p<0.05). A broad follow up of the oogenesis progression failed to show significant differences during oogenesis between populations. However, the F1 population spawned earlier with embryos presenting an overall higher survivorship than those from the F7+ population. The transcriptomic profile of unfertilized eggs showed 358 differentially expressed genes between populations. In conclusion, our data suggests that the domestication process may influence the regulation of the maternal transcripts in fish eggs, which could in turn explain differences of developmental success.

An alternative developmental table to describe non-model fish species embryogenesis: application to the description of the Eurasian perch (Perca fluviatilis L. 1758) development.

BACKGROUND: Fish correspond to the most diversified phylum among vertebrates with a large variety of species. Even if general features are distinguishable during the embryogenesis, several differences in term of timing, organ implementation or step progression always occur between species. Moreover, the developmental timing of wild non-model fish often presents variability within a species. In that context, it is necessary to define a model of developmental table flexible enough to describe fish development by integrating this variability and allow intra- and inter-specific comparisons. The elaboration of a model passes by the definition of new stages that could be easily observable on individuals. The present study aims at proposing such a model and describing accurately the Eurasian perch (Perca fluviatilis) embryogenesis using microscopic techniques among which time lapse video and histological studies. The Eurasian perch belongs to the Percidae family that includes 235 species classified in 11 genera. It is a member of the Perca gender and inhabits the Northern part of Europe and Asia. RESULTS: At 13 °C, P. fluviatilis development elapses for 15 days from the fertilization to the first oral feeding. The staging division first took into account the cellular status to define periods, then the acquisition of new abilities by the embryo to further define stages. It allowed distinguishing two main stages during the cell cleavage period depending on the synchronization of the cell divisions, two stages during the gastrulation period depending on the cell speed migration and five stages during the organogenesis according to the acquisition of key abilities as proposed in the saltatory theory. During each stage, organs implementation was carefully followed with a particular attention for the visual and digestive systems. In addition, our study shows that embryos hatch at various developmental stages while they all begin to feed at a fixed date, 15 days after the fertilization whatever the spawn and the hatching date. These data give arguments to propose the first oral feeding as the best definition of the embryonic-to-larval transition. CONCLUSIONS: The present model of developmental table combines flexibility and accuracy allowing detailed description of non-model fish species and intra- and inter-specific comparisons.

Müllerian inhibiting substance in the caudate amphibian Pleurodeles waltl.

Müllerian inhibiting substance (MIS, also known as anti-Müllerian hormone), is a key factor of male sex differentiation in vertebrates. In amniotes, it is responsible for Müllerian duct regression in male embryos. In fish, despite the absence of Müllerian ducts, MIS is produced and controls germ cell proliferation during gonad differentiation. Here we show for the first time the presence of MIS in an amphibian species, Pleurodeles waltl. This is very astonishing because in caudate amphibians, Müllerian ducts do not regress in males. Phylogenetic analysis of MIS P. waltl ortholog revealed that the deduced protein segregates with MIS from other vertebrates and is clearly separated from other TGF-ß family members. In larvae, MIS mRNA was expressed at higher levels in the developing testes than in the ovaries. In the testis, MIS mRNA expression was located within the lobules that contain Sertoli cells. Besides, expression of MIS was modified in the case of sex reversal: it increased after masculinizing heat treatment and decreased after estradiol feminizing exposure. In addition to the data obtained recently in the fish medaka, our results suggest that the role of MIS on Müllerian ducts occurred secondarily during the course of evolution.

Temporal and spatial SOX9 expression patterns in the course of gonad development of the caudate amphibian Pleurodeles waltl.

The SOX family of transcription factors is thought to regulate gene expression in a wide variety of developmental processes. Namely, SOX9 expression is conserved in vertebrate sex determination or differentiation. Nevertheless, information about caudate amphibians is lacking. In this study, we provide data on Pleurodeles waltl, a species that displays a ZZ/ZW genetic mode of sex determination and a temperature-dependent mechanism of female-to-male sex reversal. Phylogenetic analysis of SOX9 P. waltl ortholog reveals that the deduced protein segregates from the group of anuran and could be more closely related to amniote than to anamniote. However, SOX9 lacks the PQA-rich domain present in amniotes. In larvae, SOX9 is expressed in both sexes in gonad-mesonephros complexes as soon as stage 42, before gonad differentiation. At stage 54(60d) at which testis differentiation is already in progress, analyses of isolated gonads reveal a male-enriched expression of SOX9, which was quantified by real-time PCR. At the end of metamorphosis (stage 56), SOX9 shows a nuclear localization only in the testis. In adults, SOX9 is still expressed in testes and ovaries. In the ovary, SOX9 is found in oocytes from stage I to stage VI but it is never detected in the nucleus. Our results suggest that in P. waltl, like in non mammalian vertebrates, SOX9 could play a role during the late phase of gonad differentiation rather than in sex determination. Its role in germ cells of the adult ovary has still to be elucidated.

Differential RNA-binding activity of the hnRNP G protein correlated with the sex genotype in the amphibian oocyte.

A proteomic approach has enabled the identification of an orthologue of the splicing factor hnRNP G in the amphibians Xenopus tropicalis, Ambystoma mexicanum, Notophthalmus viridescens and Pleurodeles walt, which shows a specific RNA-binding affinity similar to that of the human hnRN G protein. Three isoforms of this protein with a differential binding affinity for a specific RNA probe were identified in the P. walt oocyte. In situ hybridization to lampbrush chromosomes of P. waltl revealed the presence of a family of hnRNP G genes, which were mapped on the Z and W chromosomes and one autosome. This indicates that the isoforms identified in this study are possibly encoded by a gene family linked to the evolution of sex chromosomes similarly to the hnRNP G/RBMX gene family in mammals.

Evidence for a conserved role of retinoic acid in urodele amphibian meiosis onset.

Pleurodeles waltl is a urodele amphibian displaying a ZZ/ZW genetic mode of sex determination. Gonad differentiation can later be modulated by hormone treatment. To investigate germ cell differentiation, we analyzed the expression of the meiosis marker PwDmc1 and show that germ cells enter meiosis in late larval life in females, and 2 months after metamorphosis in males. Organotypic cultures of gonad-mesonephros complexes demonstrated that retinoic acid triggers meiosis entry in P. waltl. In vivo analyses of both PwRaldh2 and PwCyp26b1 expressions, the enzymes required for RA synthesis and degradation respectively, indicate that meiosis onset depends on PwCyp26b1 repression in the gonad during normal or steroid-induced sex-reversed development. Taken together, our results show that RA-dependent meiosis entry could be a conserved mechanism of germ cell differentiation in vertebrates and provide evidence for crosstalk between steroid and RA signaling in the course of sex differentiation. Developmental Dynamics 238:1389-1398, 2009. (c) 2009 Wiley-Liss, Inc.

Lifelong testicular differentiation in Pleurodeles waltl (Amphibia, Caudata).

BACKGROUND: In numerous Caudata, the testis is known to differentiate new lobes at adulthood, leading to a multiple testis. The Iberian ribbed newt Pleurodeles waltl has been studied extensively as a model for sex determination and differentiation. However, the evolution of its testis after metamorphosis is poorly documented. METHODS: Testes were obtained from Pleurodeles waltl of different ages reared in our laboratory. Testis evolution was studied by several approaches: morphology, histology, immunohistochemistry and RT-PCR. Surgery was also employed to study testis regeneration. RESULTS: In this species, the testis is linked to the lung. This association consists of connective tissue derived from the mesorchium and the coelomic epithelium surrounding the lung and takes place at the end of larval life. This tissue contains lobules including primordial germ cells with a typical large and polylobular nucleus. The anterior part of the testis remains thin and undifferentiated while the posterior part differentiates in a large first testis lobe where spermatogenesis occurs during the first year of life. The undifferentiated status of the anterior part is attested by the lack of expression of the testis marker Dmrt1 and the meiosis entry marker Dmc1. Three-year-old Pleurodeles waltl possess multiple testes made up of two lobes. The second lobe appears at the caudal extremity of the first one from residual primordial germ cells located near or even inside efferent ducts in the glandular tissue that usually appears following spermatozoa extrusion. Surprisingly, in the case of surgical elimination of the anterior part of the testis, de novo spermatogenesis is stopped in the first lobe which becomes restricted to the glandular tissue. Following first testis lobe removal, the anterior part of the testis regenerates a new testis lobe, a process stimulated in the presence of DHT. CONCLUSION: Pleurodeles waltl constitute an original gonochoristic vertebrate model in which testis differentiation is observed up to adulthood.

Female-enriched expression of ERalpha during gonad differentiation of the urodele amphibian Pleurodeles waltl.

In the amphibian Pleurodeles waltl, estradiol treatment of genetically male larvae (ZZ) induces male-to-female sex reversal whereas heat treatment of genetically female larvae (ZW) inhibits estradiol synthesis and leads to female-to-male sex reversal. No data are available on estrogen receptors in this species. In the present study, we have isolated a unique full-length pwERalpha cDNA and its 5'-flanking region whose promoter activity was confirmed by transfection assays. RT-PCR studies performed in adult animals using ERalpha-specific primers, revealed that pwERalpha mRNA was present mainly in reproductive tissues: gonads, fat body and oviduct. PwERalpha transcript was also detected in liver, suggesting its implication in vitellogenesis control as in numerous oviparous species. The level of pwERalpha transcripts was also studied during gonad differentiation by quantitative real-time PCR. At stage 54(30d) pwERalpha expression in gonads of ZW larvae was significantly higher than in ZZ ones. This sex-specific discrimination was confirmed when gonad-mesonephros-interrenal complexes (GMI), taken at the same stage, were subjected to whole mount in situ hybridization. In comparison, the female-enriched expression of P450 aromatase, which was studied as a control of ovary differentiation, was observed earlier (stage 54). In ZW larvae reared at 32 degrees C, a condition leading to sex reversal, pwERalpha mRNA level at stage 54(30d) was lower than in control females. Taken together, these results showing a female-enriched and thermosensitive expression of pwERalpha suggest an important role for this receptor in gonad differentiation of the urodele amphibian Pleurodeles waltl.

Freemartin in the amphibian Pleurodeles waltl: parabiosis between individuals from opposite sex triggers both germ and somatic cells alterations during female gonad development.

Wild type embryos of the newt Pleurodeles waltl were used to realize parabiosis, a useful model to study the effect of endogenous circulating hormones on gonad development. The genotypic sex of each parabiont (ZZ male or ZW female) was determined early from the analysis of the sex chromosome borne marker peptidase-1. In ZZ/ZZ and ZW/ZW associations, gonads develop according to genetic sex. In ZZ/ZW associations, the ZZ gonads differentiate as normal testes while ZW gonads development shows numerous alterations. At the beginning of sex differentiation, these ZW gonads possess a reduced number of germ cells and a reduced expression of steroidogenic factor 1 and P450-aromatase mRNAs when compared to gonads from ZW/ZW associations. During gonad differentiation, conversely to the control situation, these germ cells do not enter meiosis as corroborated by chromatin status and absence of the meiosis entry marker DMC1; the activity of the estradiol-producing enzyme P450-aromatase is as low as in ZZ gonads. At adulthood, no germ cells are observed on histological sections, consistently with the absence of VASA expression. At this stage, the testis-specific marker DMRT1 is expressed only in ZZ gonads, suggesting that the somatic compartment of the ZW gonad is not masculinized. So, when exposed to ZZ hormones, ZW gonads reach the undifferentiated status but the ovary differentiation does not occur. This gonad is inhibited by a process affecting both somatic and germ cells. Additionally, the ZW gonad inhibition does not occur in the case of an exogenous estradiol treatment of larvae.

Cerebral and gonadal aromatase expressions are differently affected during sex differentiation of Pleurodeles waltl.

In vertebrates, sex is determined essentially by two means, genetic factors located on sex chromosomes and epigenetic factors such as temperature experienced by the individual during development. Steroids, especially estrogens, are clearly involved in gonadal differentiation in non-mammalian vertebrates. In this regard, the expression of the estrogen-producing enzyme, aromatase, has been shown to be temperature-sensitive in species where temperature can reverse sex differentiation, especially in our model, the amphibian Pleurodeles waltl. We investigated here the regulation of aromatase expression in the brain during sex differentiation in Pleurodeles. We first isolated a brain isoform of aromatase mRNA which differs in its 5' untranslated region from the isoform previously isolated from adult gonads. In adult Pleurodeles, the brain isoform is mainly expressed in brain tissue while the other isoform is gonad specific. Thus, regulation of aromatase expression in P. waltl could occur by alternative splicing of non-coding exon 1 as previously described in mammals. We then investigated aromatase expression in the brain of male and female larvae and found no differences with regard to sex. Measures of aromatase activity in the brain also showed no differences between sexes at larval stages whereas activity markedly increases in the ovary concomitant with the start of gonadal differentiation. These results support the hypothesis that aromatase could be a target of a temperature-sensitive sex-reversing effect in the gonads but not in the brain.

Expression of aromatase and steroidogenic factor 1 in the lung of the urodele amphibian Pleurodeles waltl.

We report here the results of the analysis of aromatase and steroidogenic factor 1 (Sf1) expression in adult lung of the urodele amphibian Pleurodeles waltl. Using RT-PCR experiments, we show the expression of the estrogen-synthesizing enzyme, aromatase, in this organ. In the lung, no significant difference between males and females was observed in the level of aromatase mRNAs. Aromatase mRNA levels were also identical to those found in the brain or the testis, but the levels were 2-fold lower than in the ovary. Aromatase activity measurements revealed the presence of an active form of aromatase in the lung, which was similar in males and females. There was no difference in the level of aromatase activity between lung, brain, and testis, but a higher activity was measured in the ovary (13.7-fold compared with testis). Therefore, the differences in aromatase mRNA level between the ovary and the other organs did not mirror the differences in aromatase activity, suggesting the involvement of posttranslational events. Aromatase was also expressed in the lung of the anuran amphibian Xenopus laevis. In Pleurodeles lung, Sf1 mRNAs were also detected. There was no difference between males and females in the level of these mRNAs. The Sf1 mRNA levels were not significantly different from those measured in the brain, but a significant 2.1-fold higher level of expression was found in the gonads. These results demonstrate clearly the expression of steroidogenic markers in the adult lung of amphibians, but the biological significance of this remains to be determined.

Ethanol stimulates proliferation, ERalpha and aromatase expression in MCF-7 human breast cancer cells.

It is well documented that alcohol is associated with an increased risk factor for breast carcinogenesis although the underlying mechanisms are not clearly understood. It has been reported that in vitro, the culture of estrogen receptor (ER) expressing breast cancer cells in ethanol containing medium was associated with an increase in the proliferation rate, in the ERalpha content as well as in ER transcriptional activity. Since these changes are not observed in ER negative breast cancer cells, and since alcohol intake has been associated to an increased level of circulating estrogens, we have postulated that aromatase expression could be increased following ethanol exposure. The results of our studies show a 1.3-fold increase in cell proliferation after 6 days of culture of MCF-7 cells in the presence of 0.1% ethanol. This enhanced proliferation is confirmed by the use of clonogenic assays which show a 1.5-fold increase in clonal growth in the presence of 0.1% ethanol. No statistically significant changes were observed in the presence of higher ethanol concentration (0.3%). After a 6-day exposure to 0.1% ethanol, RT-PCR analyses reveal a 1.7-fold increase in ERalpha mRNA that was not significant, whereas western blot analyses show a significant 3.3-fold increase in ERalpha content. At the same stage, RT-PCR studies demonstrate a 2.4-fold increase in aromatase mRNA level which is confirmed at the protein level by western blots performed after immuno-precipitation of the enzyme. Taken together, these results are in agreement with the involvement of ER signalling in ethanol-induced stimulation of breast cancer cell proliferation and could help to understand why alcohol consumption is associated with breast cancer risk.

Analysis of the effects of different alcohols on MCF-7 human breast cancer cells.

Alcohol consumption is known to be an increased risk factor for breast cancer, but the underlying molecular mechanisms are not well understood. We have recently shown that the exposure of MCF-7 breast cancer cells to 0.1% ethanol enhanced their proliferation and increased their content in both estrogen receptor-alpha (ERalpha) and aromatase. The aim of the present work was to determine if the effects of ethanol could be mimicked by other short-chain aliphatic alcohols such as methanol and 1-butanol. Our results show that these compounds do not stimulate MCF-7 cell proliferation. An increase in ERalpha content was observed by Western blot in methanol-treated cells, but this parameter was not affected in butanol-treated cells. Neither of these two alcohols induced an increase in aromatase mRNA level. So despite a similarity in molecular structure, these primary alcohols do not exert the same effects. Taken together, these results suggest that the increase in aromatase expression might be a key event required for the enhanced proliferation observed in the presence of ethanol.