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Androgen-induced masculinization in rainbow trout results in a marked dysregulation of early gonadal gene expression profiles.

Baron D, Montfort J, Houlgatte R, Fostier A, Guiguen Y - BMC Genomics (2007)

Bottom Line: 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 (11beta-hydroxyandrostenedione, 11betaOHDelta4, 10 mg/kg of food for 3 months) on gonadal gene expression profiles of an all-female genetic population of trout.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: INRA, UR1037 SCRIBE, IFR140, Ouest-Genopole, F-35000 Rennes, France. daniel.baron@nantes.inserm.fr

ABSTRACT

Background: 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 (11beta-hydroxyandrostenedione, 11betaOHDelta4, 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.

Results: 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.

Conclusion: 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.

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Classification of gonad samples and histological analysis of some characteristic gonadal stages. (A) Dendrogram of the samples ranked using a hierarchical clustering. Gonad samples are colorized according to the sex i.e., red for females, blue for males and black for androgen-treated females. Correlation coefficients (R) of the last branches of a cluster are given. (B) Histology of the gonads from the female control group (a, b), the androgen-treated group (c, d), and the male control group (e, f) at 12 days (D12) and 90 days (D90) after the beginning of the androgen treatment. BV: blood vessel; FC: fibroblast like cell; Go (A1/A2/B): gonia type (A1/A2/B); I: Interstitial space; M: mitosis; Me: meiosis; N: nucleus; n: nucleolus; OV: ovocyte; P: pachytene stage of meiosis; (p)G: (pre)granulosa cell ; (p)S: (pre)sertoli cell ; Spc(I/II): spermatocyte I/II; Spt: spermatid; Str: ovarian stroma.
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Figure 1: Classification of gonad samples and histological analysis of some characteristic gonadal stages. (A) Dendrogram of the samples ranked using a hierarchical clustering. Gonad samples are colorized according to the sex i.e., red for females, blue for males and black for androgen-treated females. Correlation coefficients (R) of the last branches of a cluster are given. (B) Histology of the gonads from the female control group (a, b), the androgen-treated group (c, d), and the male control group (e, f) at 12 days (D12) and 90 days (D90) after the beginning of the androgen treatment. BV: blood vessel; FC: fibroblast like cell; Go (A1/A2/B): gonia type (A1/A2/B); I: Interstitial space; M: mitosis; Me: meiosis; N: nucleus; n: nucleolus; OV: ovocyte; P: pachytene stage of meiosis; (p)G: (pre)granulosa cell ; (p)S: (pre)sertoli cell ; Spc(I/II): spermatocyte I/II; Spt: spermatid; Str: ovarian stroma.

Mentions: This analysis was carried out on fish sampled at several stages of development from the onset of the free swimming period (Day 0 = D0), when fish first started to be fed with the androgen treatment until 110 days after the beginning of the treatment (D110). Unsupervised hierarchical clustering of samples (Fig 1A) reveals 3 main groups of correlated samples according to their global gene expression profiles i.e., late ovarian samples (D60 to D110, correlation coefficient R = 0.78), middle and late gonad samples of androgen-treated fish gonads (D27 to D110, R = 0.37) and middle and late testicular samples (D27 to D110, R = 0.26). All early samples (D0 to D12) cluster together with a weak correlation (R = 0.07), indicating that differences in the expression profiles of these early samples are rather large. Histological analysis of gonads at D12 (Fig. 1B, panels a, c, e) reveals characteristic features of differentiating gonads in control fish with the first appearance of ovarian meiosis and lamellar structures in females, or spermatogonia cysts in males. At this time-point (D12), gonads of androgen-treated females appear as a thin structure characterized by scattered germ cells in a predominant stroma of conjunctive tissue with fibroblast like cells. After 90 days of treatment (Fig. 1B, panels b, d, f) the gonads of androgen-treated females display a classical testicular organization with cysts of germ cells engaged at various stages of meioses whereas the control male gonads show the same organization but with only gonial mitosis. At D90, the control female gonads contain previtellogenic oocytes surrounded by flattened granulosa cells and oogonia within clearly organized ovarian lamellae.


Androgen-induced masculinization in rainbow trout results in a marked dysregulation of early gonadal gene expression profiles.

Baron D, Montfort J, Houlgatte R, Fostier A, Guiguen Y - BMC Genomics (2007)

Classification of gonad samples and histological analysis of some characteristic gonadal stages. (A) Dendrogram of the samples ranked using a hierarchical clustering. Gonad samples are colorized according to the sex i.e., red for females, blue for males and black for androgen-treated females. Correlation coefficients (R) of the last branches of a cluster are given. (B) Histology of the gonads from the female control group (a, b), the androgen-treated group (c, d), and the male control group (e, f) at 12 days (D12) and 90 days (D90) after the beginning of the androgen treatment. BV: blood vessel; FC: fibroblast like cell; Go (A1/A2/B): gonia type (A1/A2/B); I: Interstitial space; M: mitosis; Me: meiosis; N: nucleus; n: nucleolus; OV: ovocyte; P: pachytene stage of meiosis; (p)G: (pre)granulosa cell ; (p)S: (pre)sertoli cell ; Spc(I/II): spermatocyte I/II; Spt: spermatid; Str: ovarian stroma.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2099445&req=5

Figure 1: Classification of gonad samples and histological analysis of some characteristic gonadal stages. (A) Dendrogram of the samples ranked using a hierarchical clustering. Gonad samples are colorized according to the sex i.e., red for females, blue for males and black for androgen-treated females. Correlation coefficients (R) of the last branches of a cluster are given. (B) Histology of the gonads from the female control group (a, b), the androgen-treated group (c, d), and the male control group (e, f) at 12 days (D12) and 90 days (D90) after the beginning of the androgen treatment. BV: blood vessel; FC: fibroblast like cell; Go (A1/A2/B): gonia type (A1/A2/B); I: Interstitial space; M: mitosis; Me: meiosis; N: nucleus; n: nucleolus; OV: ovocyte; P: pachytene stage of meiosis; (p)G: (pre)granulosa cell ; (p)S: (pre)sertoli cell ; Spc(I/II): spermatocyte I/II; Spt: spermatid; Str: ovarian stroma.
Mentions: This analysis was carried out on fish sampled at several stages of development from the onset of the free swimming period (Day 0 = D0), when fish first started to be fed with the androgen treatment until 110 days after the beginning of the treatment (D110). Unsupervised hierarchical clustering of samples (Fig 1A) reveals 3 main groups of correlated samples according to their global gene expression profiles i.e., late ovarian samples (D60 to D110, correlation coefficient R = 0.78), middle and late gonad samples of androgen-treated fish gonads (D27 to D110, R = 0.37) and middle and late testicular samples (D27 to D110, R = 0.26). All early samples (D0 to D12) cluster together with a weak correlation (R = 0.07), indicating that differences in the expression profiles of these early samples are rather large. Histological analysis of gonads at D12 (Fig. 1B, panels a, c, e) reveals characteristic features of differentiating gonads in control fish with the first appearance of ovarian meiosis and lamellar structures in females, or spermatogonia cysts in males. At this time-point (D12), gonads of androgen-treated females appear as a thin structure characterized by scattered germ cells in a predominant stroma of conjunctive tissue with fibroblast like cells. After 90 days of treatment (Fig. 1B, panels b, d, f) the gonads of androgen-treated females display a classical testicular organization with cysts of germ cells engaged at various stages of meioses whereas the control male gonads show the same organization but with only gonial mitosis. At D90, the control female gonads contain previtellogenic oocytes surrounded by flattened granulosa cells and oogonia within clearly organized ovarian lamellae.

Bottom Line: 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 (11beta-hydroxyandrostenedione, 11betaOHDelta4, 10 mg/kg of food for 3 months) on gonadal gene expression profiles of an all-female genetic population of trout.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: INRA, UR1037 SCRIBE, IFR140, Ouest-Genopole, F-35000 Rennes, France. daniel.baron@nantes.inserm.fr

ABSTRACT

Background: 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 (11beta-hydroxyandrostenedione, 11betaOHDelta4, 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus