Background: The mechanism of sex determination in zebrafish is largely unknown and neither sex chromosomes nor a sex-determining gene have been identified. This indicates that sex determination in zebrafish is mediated by genetic signals from autosomal genes. The aim of this study was to determine the precise timing of expression of six genes previously suggested to be associated with sex differentiation in zebrafish. The current study investigates the expression of all six genes in the same individual fish with extensive sampling dates during sex determination anddifferentiation.

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Prostaglandins are important regulators of reproductive function in fish. Analgesics like aspirin and ibuprofen are prostaglandin inhibitors and have been detected in freshwater systems at ng/L-μg/L levels. We investigated whether ibuprofen would affect prostaglandin and sex steroid hormone levels in adult zebrafish (Danio rerio) and if expression levels of genes involved in steroidogenesis and prostaglandin synthesis were affected. Zebrafish were exposed to moderate concentrations of ibuprofen (21, 201 or 506 μg/L) for 7 days in a semi-static test system. Ibuprofen concentrations were close to nominal levels and decreased by a maximum of 12-13% over 24 h. Prostaglandin E(2) (PGE(2)) levels in whole body homogenates of males and ovaries of females decreased in a monotonic dose-response relationship whereas male 11-ketotestosterone levels and ovarian 17β-estradiol levels remained unchanged. Ibuprofen did not have an influence on vitellogenin levels, female gonadosomatic index or cumulative egg production and no dose-response relationship in ovarian and testicular expression levels of the investigated genes was observed. This study shows that ibuprofen reduces PGE(2) levels in male and female zebrafish but has no consistent effects on other investigated reproductive parameters.

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BackgroundInvestigating gonadal gene expression is important in attempting to elucidate the molecular mechanism of sex determination and differentiation in the model species zebrafish. However, the small size of juvenile zebrafish and correspondingly their gonads complicates this type of investigation. Furthermore, the lack of a genetic sex marker in juvenile zebrafish prevents pooling gonads from several individuals. The aim of this study was to establish a method to isolate the gonads from individual juvenile zebrafish allowing future investigations of gonadal gene expression during sex determination and differentiation.MethodsThe laser capture microdissection technique enables isolation of specific cells and tissues and thereby removes the noise of gene expression from other cells or tissues in the gene expression profile. A protocol developed for laser microdissection of human gonocytes was adjusted and optimised to isolate juvenile zebrafish gonads.ResultsThe juvenile zebrafish gonad is not morphologically distinguishable when using dehydrated cryosections on membrane slides and a specific staining method is necessary to identify the gonads. The protocol setup in this study allows staining, identification, isolation and subsequent RNA purification and amplification of gonads from individual juvenile zebrafish thereby enabling gonadal gene expression profiling.ConclusionThe study presents a protocol for isolation of individual juvenile zebrafish gonads, which will enable future investigations of gonadal gene expression during the critical period of sex differentiation. Furthermore, the presented staining method is applicable to other species as it is directed towards alkaline phosphatase that is expressed in gonocytes and embryonic stem cells, which is conserved among vertebrate species.