Androgen-induced masculinization in rainbow trout results in a marked dysregulation of early gonadal gene expression profiles
© Baron et al; licensee BioMed Central Ltd. 2007
Received: 04 April 2007
Accepted: 04 October 2007
Published: 04 October 2007
Fish gonadal sex differentiation is affected by sex steroids treatments providing an efficient strategy to control the sexual phenotype of fish for aquaculture purposes. However, the biological effects of such treatments are poorly understood. The aim of this study was to identify the main effects of an androgen masculinizing treatment (11β-hydroxyandrostenedione, 11βOHΔ4, 10 mg/kg of food for 3 months) on gonadal gene expression profiles of an all-female genetic population of trout. To characterize the most important molecular features of this process, we used a large scale gene expression profiling approach using rainbow trout DNA microarrays combined with a detailed gene ontology (GO) analysis.
2,474 genes were characterized as up-regulated or down-regulated in trout female gonads masculinized by androgen in comparison with control male or female gonads from untreated all-male and all-female genetic populations. These genes were classified in 13 k-means clusters of temporally correlated expression profiles. Gene ontology (GO) data mining revealed that androgen treatment triggers a marked down-regulation of genes potentially involved in early oogenesis processes (GO 'mitotic cell cycle', 'nucleolus'), an up-regulation of the translation machinery (GO 'ribosome') along with a down-regulation of proteolysis (GO 'proteolysis', 'peptidase' and 'metallopeptidase activity'). Genes considered as muscle fibres markers (GO 'muscle contraction') and genes annotated as structural constituents of the extracellular matrix (GO 'extracellular matrix') or related to meiosis (GO 'chromosome' and 'meiosis') were found significantly enriched in the two clusters of genes specifically up-regulated in androgen-treated female gonads. GO annotations 'Sex differentiation' and 'steroid biosynthesis' were enriched in a cluster of genes with high expression levels only in control males. Interestingly none of these genes were stimulated by the masculinizing androgen treatment.
This study provides evidence that androgen masculinization results in a marked dysregulation of early gene expression profiles when compared to natural testicular or ovarian differentiation. Based on these results we suggest that, in our experimental conditions, androgen masculinization proceeds mainly through an early inhibition of female development.
The embryonic gonad has the potential to develop into a fully functional organ able to produce the gametes necessary for sexual reproduction. Sex differentiation is a crucial step in this developmental process and is considered as the differentiation from a bipotential gonadal primordium towards a testis or an ovary. In teleostean fish, sex differentiation can be controlled by in vivo treatments with sex steroids (reviewed in ) as in reptiles and amphibians and to some extent in birds (reviewed in [2–4]). In fish, these steroid treatments are often able to induce fully functional sex-inversed phenotypes and these treatments have been widely used to produce all-male or all-female populations of fish for aquaculture purposes . Many studies have been focused on the role of these hormones during gonadal sex differentiation highlighting for instance the crucial role of estrogens in ovarian differentiation . However, most of the studies performed thus far were focused on a very small number of well characterized genes, proteins or hormones and mostly on natural gonadal differentiation.
Rainbow trout, Oncorhynchus mykiss, has a male heterogametic XY genetic system and we experimentally produced XX and YY males allowing the production of genetically all-male and all-female populations . These all-male or all-female populations provide a unique opportunity to work on numerous animals for which the normal gonadal development as testis or ovary is known a priori. Using the extensive collection of expressed sequenced tags (ESTs) obtained through sequencing projects in trout as a resource [7, 8], we designed and built a DNA microarray in order to characterize, on a genome-wide scale, the mechanisms by which 11β-hydroxyandrostenedione (11βOHΔ4), a natural androgen in fish [9, 10] is able to masculinize the embryonic ovary.
Using this genome-wide approach we characterized 2,474 genes (2372 microarray and 102 real-time RT-PCR gene expression profiles) with a clear differential temporal expression profile in females masculinized by androgen. We classified these genes in 13 different clusters of correlated temporal expression profiles, and searched within these clusters for significant enrichment in Gene Ontology (GO) terms. This strategy allowed us to define a few very clear biological trends potentially explaining how androgen induces masculinization of female fish. Our results clearly demonstrate that masculinization with androgen proceeds through a marked dysregulation of gene expression profiles, including a quick down-regulation of the ovarian pathway. Surprisingly, most of the genes over-expressed during natural testicular differentiation were not restored by the androgen-induced masculinization suggesting that the inhibition of female gonadal development is the main required step sufficient for building a testis.
The complete dataset is available through the National Center for Biotechnology Information (NCBI), in the Gene Expression Omnibus database  under the GSE7018 accession number. After statistical filtering, 2,474 expression profiles (2372 microarray and 102 real-time RT-PCR gene expression profiles, data available as supplemental material in Additional file 1) were identified as being characteristic for either natural differentiation (ovarian or testicular differentiation) or androgen-induced masculinization (trans-differentiating gonads). Among these 2,474 expression profiles, 73% (1,805) were associated with genes with significant homologies with well characterized proteins in Swissprot or Prodom databases (the complete list of clones and their annotations is available as supplemental material in Additional file 2).
Biological sample clustering and histology
Global analysis of gene expression profiles
Annotation of gene clusters using Gene Ontology (GO)
The top 5 most significant Gene Ontology (GO) terms significantly enriched in K-means clusters 1 to 5
Term (GO ID)
1 to 5
regulation of progression through cell cycle (74)
mitotic cell cycle (278)
macromolecule biosynthesis (9059)
protein biosynthesis (6412)
cytoplasmic membrane-bound vesicle (16023)
telomere maintenance (723)
ion homeostasis (50801)
cellular respiration (45333)
translation regulator activity (45182)
aerobic respiration (9060)
pyrophosphatase activity (16462)
GTPase activity (3924)
rRNA binding (19843)
nuclear hormone receptor binding (35257)
replication fork (5657)
interphase of mitotic cell cycle (51329)
hydrogen-transporting ATPase activity (46961)
ATP biosynthesis (6754)
cell growth (16049)
transcription factor binding (8134)
chromatin assembly (31497)
ribonucleoprotein complex (30529)
cytoplasm organization and biogenesis (7028)
cytosolic large ribosomal subunit (5842)
ribosome biogenesis and assembly (42254)
The top 5 most significant Gene Ontology (GO) terms significantly enriched in K-means clusters 6 to 13
Term (GO ID)
cell surface (9986)
T cell activation (42110)
immune response (6955)
lipid binding (8289)
neuron development (48666)
extracellular matrix (sensu Metazoa) (5578)
actin binding (3779)
muscle contraction (6936)
phosphate transport (6817)
structural molecule activity (5198)
condensed chromosome (793)
response to endogenous stimulus (9719)
DNA repair (6281)
magnesium ion binding (287)
calcium ion binding (5509)
organ morphogenesis (9887)
plasma membrane (5886)
organ development (48513)
structural constituent of cytoskeleton (5200)
urogenital system development (1655)
lipid binding (8289)
response to temperature stimulus (9266)
protein kinase binding (19901)
metallopeptidase activity (8237)
peptidase activity (8233)
endopeptidase activity (4175)
transcription from RNA polymerase II promoter (6366)
ligase activity, forming carbon-nitrogen bonds (16879)
metallopeptidase activity (8237)
mRNA metabolism (16071)
glutamine family amino acid biosynthesis (9084)
regulation of RNA metabolism (51252)
steroid biosynthesis (6694)
sex differentiation (7548)
transcription, DNA-dependent (6351)
hormone metabolism (42445)
DNA binding (3677)
Validation and enrichment of DNA microarray data by real-time RT-PCR
Correlation between real-time RT-PCR and DNA microarray data
steroidogenic acute regulatory protein
doublesex- and mab-3-related transcription factor 1
nuclear receptor subfamily 5, group A, member 1 b
apolipoprotein E b
benzodiazepine receptor, peripheral
bone morphogenetic protein 7
growth differentiation factor 9
germ cell-less homolog (Drosophila)
very low density lipoprotein receptor
heat shock protein 90 beta
aldolase b, fructose-bisphosphate
mago-nashi homolog, proliferation-associated
tissue inhibitor of metalloproteinase 2
transform er-2 alpha
Our global approach based on gene expression profiling clearly reveals that, in our experimental conditions (11βOHΔ4, 10 mg/kg of food for 3 months), the androgen masculinization does not induce a natural physiological response since the transcriptome of testicular trans-differentiating gonads is quite different from the one observed during natural testicular differentiation. These differences might be due to the non physiological dosage of androgen used in our experiment. A similar study using a lower dosage may help to clarify this issue, but this study was first designed with an androgen dosage that is commonly used in rainbow trout aquaculture conditions. Whether the observed gene expression dysregulations are the reflection of a direct action of the androgens on the gonad, an indirect retro-control on the hypothalamus-pituitary axis, or a conjunction of both, remains to be elucidated. However, the synthesis of Gonadotropin Releasing Hormone (GnRH) and of pituitary hormones is established very early during rainbow trout ontogenesis  with at least the synthesis of Follicle Stimulating Hormone FSH . Thus indirect feedback effects cannot be totally excluded.
Due to the lack of specific Gene Ontology (GO) annotation for rainbow trout we linked the best blast hits of each clone sequence with a cross-species GO annotation. This strategy relies on the accuracy of the blast homology search and also on the resulting accuracy of the GO annotations with regards to their use in a fish species. However, even if this could lead to potential errors on a gene per gene scale, the global analysis and stringent statistical screen that we carried out enabled us to unambiguously assign most clusters with a clear biological theme. Among these GO categories, some were considered as biologically informative – i.e., not too general like for instance GO "physiological process" – and robust as they contain a sufficient number of different genes to support a potential biological meaning. We focused our analysis on these biologically informative GO categories.
With regards to the effects on the gonad, our analysis first reveals that female development is highly affected by the androgen treatment, with a down-regulation of most of the genes involved in early oogenesis stages. However within this analysis we did not characterize any cluster of early female-specific up-regulated genes potentially involved in ovarian differentiation. Expression profiles of some female-specific candidate genes  (e.g. foxl2a and foxl2b, cyp19a1, fst, inha) were introduced in our analysis and all these genes were strongly and quickly inhibited by the masculinizing androgen treatment. But when pooled with our DNA microarrays dataset they did not form a tight cluster. This is probably because no additional similar expression profile was found within the DNA microarray dataset. This very small number of early female-specific genes is in agreement with the small number of candidate genes known to be involved in the ovarian differentiation pathway  in comparison with the relatively high number of genes that are known to characterize testicular differentiation . In agreement with this view, our analysis clearly characterizes a cluster displaying testicular-specific gene expression profiles, containing both genes known to be involved in testicular differentiation (e.g. amh, sox9, dmrt1, gata4, lhx9)  and some potential new players revealed by our analysis. Interestingly the expression levels of all these genes are not restored by the androgen masculinizing treatment, and this could indicate that they are probably not necessary for early testicular differentiation in rainbow trout.
Among the gene clusters specifically up-regulated in females following masculinization with androgens, extracellular matrix, muscle markers/cytoskeleton and meiosis were characterized as the 3 main gene annotations. Simultaneous up-regulation of extracellular matrix protein genes expression and down-regulation of matrix proteinase genes was detected in gonads of androgen-treated females. At the same time the histological analysis of these gonads showed that they contain a predominant stroma of conjunctive tissue with fibroblast like cells. Matrix protein synthesis and the concomitant decrease in matrix proteinase activity have been well described as a characteristic fibrotic response of an excessive Transforming Growth Factor-beta (TGFβ) production [18, 19]. Of special interest in that context is the up-regulation of transforming growth factor-β1 (tgfb1) in gonads of androgen-treated animals. In rat, TGFβ induced morphological changes in Leydig cells, accompanied by an increased secretion of fibronectin, laminin and collagen IV . In fibroblasts treated with TGFβ1 a similar over-expression of genes associated with matrix formation has been detected including many different matrix protein genes, like SPARC (Secreted Protein, Acidic and Rich in Cysteine), MGP (matrix Gla protein), and TGFβ1 itself , that we also detected as up-regulated in gonads following androgen treatment. It could then be hypothesized that this late androgen up-regulation of tgfb1 in trout gonads triggers a fibrotic response. Surprisingly, these effects are detected transiently and rather late after the application of the androgen treatment (but concomitantly with tgfb1 up-regulation). Whether this reflects a total dysregulation or an exacerbation of a testicular-specific event remains to be analyzed. However, extracellular matrix deposition is known as a major event for the testicular organization. For instance, LAMA5 (Laminin α5) has been characterized as a structural protein involved in the formation of the basement membrane of the testicular cords  and this protein was found to be anti-correlated with Anti-Müllerian Hormone (AMH) . In trout gonads, amh expression is not restored to male levels in androgen-treated females. This may produce a disrupted expression of some structural proteins, like lama5. In the same manner, sparc is highly up-regulated in androgen-treated females. In mouse Sparc gene expression has been identified in pre-Sertoli cells at the time of sex differentiation  and this protein has also been postulated to play a crucial role in both Leydig and Sertoli cells differentiation by affecting their morphology . Structural proteins including matrix proteins are then of major importance for a complete and functional testicular differentiation and their up-regulation in trout following an androgen treatment inducing testicular transdifferentiation may be the consequence of a dysregulation of some major regulators of their synthesis like amh or tgfb1.
We also detected a high number of genes associated with cytoskeletal reorganization and muscle development that were up-regulated by the treatment. Some of them (e.g. cnn1, myh11, myl6, tagln) are even considered as characteristic smooth muscle markers. This expression of muscle markers in the testis is likely in relation with the peritubular myoid cells that surround the seminiferous tubules . These myoid cells are known to express muscle markers like for instance, tpm1 (tropomyosin 1, alpha) , smooth muscle alpha-actin , and smooth muscle myosin . Differentiation of these cells is androgen dependent  and they contribute to the testicular secretion of extracellular matrix components  along with the Sertoli cells . It is therefore suggested that the masculinizing androgen treatment may induce the differentiation and subsequently a disturbed androgen-dependent proliferation of these peritubular myoid cells. These cells are also probably involved in the important extracellular matrix synthesis that occurs concomitantly with this differentiation.
In our experiment, the androgen treatment also induced a precocious spermatogenesis as revealed both by the histological analysis and by the increased expression levels of some genes involved in testicular meiosis. In fish, androgens and particularly, 11-oxygenated androgens, are strongly involved in spermatogenesis regulation  and they have been shown to directly induce spermatogenesis in vitro in some species . Similarly, in mammals, three independent studies using Sertoli cell-specific AR-knockout mice (mice knockout for the androgen receptor, AR) demonstrated that the action of androgen is an absolute requirement for the completion of spermatogenesis, particularly in the process of meiosis [34–36].
This study gives a first comprehensive survey of gene expression during androgen-induced masculinization in female rainbow trout. Our data provide supportive evidences that this treatment results in a marked dysregulation of gene expression levels when compared to natural testicular or ovarian differentiation. In our experimental condition the androgen treatment induces the complete down-regulation of female specific genes, but not the complete restoration of the male-specific gene expression patterns. Instead, some disturbed responses were characterized by an exacerbation of extracellular matrix synthesis and muscle type cell differentiation and proliferation (myoid cells) followed by a precocious meiosis of germ cells. All together, we suggest that androgen masculinization acts mainly through an early inhibition of female development rather than through a direct induction of testicular differentiation.
Animals and samplings
Research involving animal experimentation has been approved by the authors' institution (authorization no. 35-14). It conforms to principles for the use and care of laboratory animals and is in compliance with French and European regulations on animal welfare (European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes, ETS no. 123, January 1991). All-male and all-female rainbow trout populations were obtained at the INRA experimental fish farm (Sizun, France) as previously described . Treatment with androgens (female treated Group 'F11β') was carried out at the onset of the first feeding [Day 0 = D0 at 55 days post-fertilization (55 dpf)], on an all-female population. The androgen, 11β-hydroxyandrostenedione (11βOHΔ4, Sigma, St. Louis, MO, USA), was administered by adding it to the food (10 mg/kg food) during 3 months starting from the first feeding and this treatment has been shown to produce 100% sex-inversions . In each group, 20 to 100 gonads were sampled and pooled in duplicates corresponding to the various stages of development: onset of the free swimming period after complete yolk resumption (Day 0 = D0), D0+7 days (D7), occurrence of oocyte meiosis (D12), beginning of ovarian lamellar structures development (D27), occurrence of previtellogenic oocytes (D60), D90 and D110. They were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same time-points for histological analysis, which was performed as previously described .
Total RNA extraction
Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France) as previously described . The total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip® kit (Agilent Technologies, Stockport, UK) according to the manufacturers' instructions.
DNA microarrays preparation
DNA Microarrays construction
Gene expression analyses were carried out using home-made Nylon DNA microarrays using a previously described technology . These DNA microarrays were built using as templates cDNA clones provided by the INRA-AGENAE program . All these clones were PCR-amplified at the INRA Resources Centre for Animal Genomics (CRB GADIE, Jouy en Josas, France) using primers designed on the plasmid sequences flanking the cDNA inserts (M13RP1 5'-GTGGAATTGTGAGCGGATAAC and M13RP2 5'-GCAAGGCGATTAAGTTGGG). 35 cycles of PCR amplifications were carried out in 100 μl of 1× buffer containing 25 mM MgCl2, 250 μM dNTP, 100 μM of each primer, and 2.5 Units of Taq polymerase (Promega, Madison, WI). For each cDNA clone, two 100 μl PCR reactions were pooled, desiccated and resuspended in 50 μl of distilled water. They were then spotted as previously described  onto Hybond-N+ 2 × 7 cm2 membranes (Amersham Pharmacia Biotech, Cleveland, OH, USA) attached to glass slides using an 8-pin print head (Pin-and-Ring™ technology) on the GMS 417™ (Affymetrix, MWG-Biotech, Ebersberg, Germany). Spotted DNA was then denatured and UV – cross-linked onto nylon filters. All DNA microarrays used in this study were made at the same time and under the same conditions. These trout microarrays contained 9,216 DNA spots representing 9,120 trout cDNA clones and a set of 96 controls. Among these cDNA clones, 7,584 were issued from a pooled-tissues library and 1,536 from a testis library . Negative controls consisted of 80 spots of an Arabidopsis thaliana cytochrome c554 clone which is devoid of similarity with trout DNA sequences, 8 spots of poly(dA)80 and 8 spots of PCR reaction without template.
DNA microarray hybridizations
Microarrays were hybridized with two types of 33P-labeled probes. The first one was an oligonucleotide with a sequence common to all spotted PCR-products (vector hybridization) in order to determine the amount of target DNA accessible to hybridization in each spot. After stripping, a second hybridization was performed with complex probes made from 1 μg of retrotranscribed total RNA [40–42]. Protocols for probes preparation, hybridizations and washes are available online . After stringent washes, arrays were exposed to phosphor-imaging plates and scanned with a FUJI BAS 5000 at 25 μm resolution. Hybridization signals were quantified using ArrayGauge software (Fuji Ltd, Tokyo, Japan).
In order to validate and enrich the DNA microarray dataset, expression of 102 genes involved in early gonad development  was measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). For cDNA synthesis, 1 μg of total RNA was denatured in the presence of random hexamers (0.5 μg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI, USA) as described by the manufacturer. Real-time PCR was carried out as previously described  using the iCycler iQTM (Bio-Rad, Hercules, CA, USA) and the SYBER Green PCR master Mix (Eurogentec, Seraing, Belgium). For each target gene, all the samples were analyzed on the same plate in the same PCR assay. PCR data were processed as previously described, each transcript level being normalized by division with the expression values of the constitutive elongation factor 1α (ef1a), which was used as an internal standard . Data were then included in the microarray data matrix for clustering analysis (see next paragraph).
First, non-linear effects such as background, print-tip effects or saturation were corrected by LOWESS , using a channel by channel procedure . Each array was individually normalized to the median profile of all arrays. We used the print-tip LOWESS version implemented in the statistical software package R . Data were further corrected for the amount of spotted cDNA. This step is necessary as it has been shown that the signal intensity is proportional to the amount of probe on the surface of the array [40, 47]. This effect can be observed both for glass and Nylon surfaces. This effect is corrected by the use of a reference in dual channel arrays, and by an independent measurement of the spotted amount of DNA probe in single channel arrays. On Nylon membranes, this effect is linear and can be corrected by dividing the signal by the amount of probe . Briefly, sample signal intensity of each spot ("S") was divided ("S/V") by the corresponding signal intensity of the same spot obtained with the vector hybridization ("V"). To minimize experimental differences between different complex probe hybridizations, 'S/V' values from each hybridization were divided by the corresponding median value of 'S/V' (quantile normalization).
A triple filtering procedure was then applied to the microarray dataset. The first consisted of filtering background signals due to low amount of spotted DNA. When a "V" spot signal was too weak (vector signal < 3× vector local background), the data of the corresponding cDNA clone was discarded (missing data). The second filtering procedure was applied to eliminate non informative genes that were not measured (sample signal < 3× sample local background) in more than 20% of the samples. Finally, genes exhibiting little variation (coefficient of variation < 0.1) across all arrays were excluded from the analysis [48, 49]. After these three filtering steps, 2,372 genes were retained for further analysis.
All data (2372 microarray and 102 real-time RT-PCR gene expression profiles) were then log2-transformed and were analyzed by unsupervised and supervised clustering methods. Hierarchical clustering (Cluster program ) investigated the relationships between the genes and between the samples by using centroid linkage clustering with Pearson's uncentered correlation as similarity metric on data that were median-centered on genes. Gene clusters were distinguished using the non-hierarchical unsupervised learning k-means algorithm implemented in the Cluster program . It was run on log2-transformed and gene median-centered data with a maximum cycles parameter of 100. The optimal minimal 'k' number of clusters, corresponding to the stability of the k-means clustering, was empirically set at 13. Indeed, with smaller k numbers, some clusters merged together whereas with greater k numbers, the size of some clusters decreased (less than 50 genes to truly empty clusters). Results (colorized matrix) of hierarchical and k-means clustering analyses were visualized using the Java TreeView program . Functional annotation of genes was performed using Gene Ontology  and the GoMiner program . Significance of over- or under-representation was calculated using Fisher's exact test at 0.05% risk.
Authors are grateful to Béatrice Loriod and Laurence Loï for their help with spotting PCR products, Benoît Ballester and Gildas Bleas and the Sigenae team for their help with bioinformatics. We also thank Francois Piumi (CRB, Jouy-en-Josas) for his help with PCR amplifications and the experimental facility staff of the INRA-SCRIBE laboratory for their help with fish rearing. Grateful acknowledgement is made to Marja Steenman for English correction. This work was supported by funds from the "Institut National de la Recherche Agronomique" (INRA), and from PNETOX and CIPA-OFIMER-INRA (IFOP) grants. The first author (DB) received a fellowship from the "Ministère de la Recherche et de l'Enseignement Supérieur".
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