To Örebro University

Testis differentiation in zebrafish involves juvenile ovary to testis transformation initiated by an apoptotic wave. The molecular regulation of this transformation process is not fully understood. NF-kappa B is activated at an early stage of development and has been shown to interact with steroidogenic factor-1 in mammals, leading to the suppression of anti-Mullerian hormone (Amh) gene expression. Because steroidogenic factor-1 and Amh are important for proper testis development, NF-kappa B-mediated induction of anti-apoptotic genes could, therefore, also play a role in zebrafish gonad differentiation. The aim of this study was to examine the potential role of NF-kappa B in zebrafish gonad differentiation. Exposure of juvenile zebrafish to heat-killed Escherichia coli activated the NF-kappa B pathways and resulted in an increased ratio of females from 30 to 85%. Microarray and quantitative real-time-PCR analysis of gonads showed elevated expression of NF-kappa B-regulated genes. To confirm the involvement of NF-kappa B-induced anti-apoptotic effects, zebrafish were treated with sodium deoxycholate, a known inducer of NF-kappa B or NF-kappa B activation inhibitor (NAI). Sodium deoxycholate treatment mimicked the effect of heat-killed bacteria and resulted in an increased proportion of females from 25 to 45%, whereas the inhibition of NF-kappa B using NAI resulted in a decrease in females from 45 to 20%. This study provides proof for an essential role of NF-kappa B in gonadal differentiation of zebrafish and represents an important step toward the complete understanding of the complicated process of sex differentiation in this species and possibly other cyprinid teleosts as well.

The sex differentiation mechanisms in zebrafish (Danio rerio) remains elusive, partly due to the absence of sex chromosomes but also the process appears to depend on the synchrony of multiple genes and possibly environmental factors. Zebrafish gonadal development is initiated through the development of immature oocytes. Depending on multiple signaling cues, in about half of the individuals, the juvenile ovaries degenerate or undergo apoptosis to initiate testes development while the other half maintains the oogenic pathway. We have previously shown that activation of NFkappaB and prostaglandin synthase 2 (ptgs2) results in female biased sex ratios. Prostaglandin synthase and prostaglandins are involved in multiple physiological functions including cell survival and apoptosis. In the present study we show that inhibition of ptgs2 by meloxicam result in male biased sex ratios. On further evaluation, we observed that exposure with the prostaglandin D2 (PGD2) analogue BW-245C induced SRY-box containing gene 9a (sox9a) and resulted in male biased sex ratios. On the other hand, prostaglandin E2 (PGE2) treatment resulted in female biased sex ratios and involved activation of NFkappaB and the beta-catenin pathway as well as inhibition of sox9. Exposure to the beta-catenin inhibitor, PNU-74654, resulted in up-regulation of ptgds and male biased sex ratios which further confirmed the involvement of beta-catenin in the female differentiation pathway. In this study we show that PGD2 and PGE2 can program the gonads to either the testis or ovary differentiation pathways, indicating that prostaglandins are involved in the regulation of zebrafish gonadal differentiation.