2006 - Progress

 

Medaka

• Investigation of the gene expression pattern of the testis-ova induction-related genes.

Other

• Establishment of the six fishes ERs-transcriptional activity by 17b-estradiol using reporter gene assay.
• Written full shared research papers - See UKJ-related publications.

 

2006 - Objectives

 

Roach

• Define the molecular basis of of EE2 disruption of sexual development using the roach macroarray
• Establish QPCR assays for selected targets (possible candidates: aromatase, fox L2, vasa, sox9, amh, dmrt1, sf1) for validation of the macroarray results and a further understanding of the molecular mechanisms controlling testis-ova induction
• Using molecular approaches (macroarray and QPCR on specific gene targets) better understand the phenomenon of enhanced sensitivity to oestrogens following exposure during early life.

Medaka

• Isolate and characterise genes that are differentially expressed in the gonad on exposure to oestrogen to better define molecular mechanisms leading to disruption in germ cell differentiation/development.
• Apply macro array to examine suite of genes altered in testis-ova induction and establish and apply appropriate QPCR assays.
• Additionally…… Write up 2/3 full shared research papers

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2005 - Progress against Objectives

 

Development of macroarray for suites of genes controlling sexual function in roach and medaka

• More than 40 genes involved with reproduction have been cloned and sequenced and applied to a macro array. The roach macroarray has been developed and validated for use.
• Medaka genome project means gene targets can be selected for macroarray – established array with 750 genes (Japan)

Analysis of induction of testis-ova during exposure to EDCs during critical life windows of sensitivity (focus on ‘environmental oestrogens’)

• Exposures of roach to ethinyloestradiol and NP have been conducted and assessments made on critical windows of sensitivity. Significantly, exposure to an environmentally relevant concentration of ethinyloestradiol during early life induced all features of feminisation in wild fish, including germ cell disruption (true intersex) in later life.
• Demonstrate that exposure to oestrogen during early life sensitised the fish to oestrogen in later life.

Assessments of the molecular signaling pathways of effects for testis-ova induction in roach and medaka

• Detailed analyses of the effects of EE2 exposure during early life on the expression of ERs and aromatase mRNAs (gonad and brain). Macroarray studies ongoing.
• In medaka exposure to EE2 has resulted in testis-ova and differential display is being employed to identify the gene expression altered.

Development of roach and medaka ER reporter gene assays and assessment of chemical potencies through these receptors.

• Completed for roach and used to good effect to screen for potency of environmental oestrogens    

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2005 - Progress against Objectives

 

Development of macroarray for suites of genes controlling sexual function in roach and medaka

• More than 40 genes involved with reproduction have been cloned and sequenced and applied to a macro array. The roach macroarray has been developed and validated for use.
• Medaka genome project means gene targets can be selected for macroarray – established array with 750 genes (Japan)

Analysis of induction of testis-ova during exposure to EDCs during critical life windows of sensitivity (focus on ‘environmental oestrogens’)

• Exposures of roach to ethinyloestradiol and NP have been conducted and assessments made on critical windows of sensitivity. Significantly, exposure to an environmentally relevant concentration of ethinyloestradiol during early life induced all features of feminisation in wild fish, including germ cell disruption (true intersex) in later life.
• Demonstrate that exposure to oestrogen during early life sensitised the fish to oestrogen in later life.

Assessments of the molecular signaling pathways of effects for testis-ova induction in roach and medaka

• Detailed analyses of the effects of EE2 exposure during early life on the expression of ERs and aromatase mRNAs (gonad and brain). Macroarray studies ongoing.
• In medaka exposure to EE2 has resulted in testis-ova and differential display is being employed to identify the gene expression altered.

Development of roach and medaka ER reporter gene assays and assessment of chemical potencies through these receptors.

• Completed for roach and used to good effect to screen for potency of environmental oestrogens    

 

Collaborative Achievements up to 2005

• Full-length cDNA cloning of a suite of reproductive function control genes for roach
• Determined ontogeny of gonadal and brain expression of roach ERs mRNA and aromatase mRNAs
• Established effects of exposure to environmentally relevant concentrations of ethinyloestradiol on expression of ER and aromatase mRNAs and associated effects on sexual development

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