Gene Expression in Forebrain of Male Goldfish Following Exposure to Waterborne Sex Pheromones. Carassius auratus

Sex pheromones rapidly affect endocrine physiology and behaviour, but little is known about their effects on gene expression in the neuroendocrine tissues that mediate olfactory processing. In this study we exposed male goldfish for 6 h to waterborne 17,20betaP (4.3 nM) and PGF2α (3 nM), the main pre-ovulatory and post-ovulatory pheromones, respectively. Both treatments elevated milt volume (P = 0.001). Microarray hybridizations were used to male telencephalon samples following PGF2α treatment and identified 71 unique transcripts that were differentially expressed (q < 5%; 67 up, 4 down). Functional annotation of these regulated genes indicates that PGF2α pheromone exposure affects diverse biological processes including nervous system functions, energy metabolism, cholesterol/lipoprotein transport, translational regulation, transcription and chromatin remodelling, protein processing, cytoskeletal organization, and signalling. By using real-time RT-PCR, we further validated three candidate genes, ependymin-II, calmodulin-A and aldolase C, which exhibited 3 - 5 fold increase in expression following PGF2α exposure. Expression levels of some other genes that are thought to be important for reproduction were also determined using real-time RT-PCR. Expression of sGnRH was increased by PGF2α, but not 17,20betaP, whereas cGnRH expression was increased by 17,20betaP but not PGF2α. In contrast, both pheromones increase the expression of glutamate (GluR2a, NR2A) and gamma-aminobutyric acid (GABAA gamma2) receptor subunit mRNAs. This gene expression data link milt release and rapid modulation of neuronal transcription as part of the response to female sex pheromones. Overall design: A total of four microarray slides were hybridized to study the effects of PGF2α on gene expression in the telencephalon. Control samples (N = 4) were pooled to form our reference or technical replicates while treatment samples (N = 4), which formed our biological replicates, were ran separately. In addition, two dye swaps were performed for our biological samples for the treatments.

性信息素（sex pheromones）可快速影响内分泌生理与行为，但目前对其在介导嗅觉加工的神经内分泌组织中对基因表达的影响尚不清楚。本研究将雄性金鱼暴露于水溶性的17,20β-双羟孕酮（17,20betaP，4.3 nM）与前列腺素F2α（PGF2α，3 nM）环境中6小时，二者分别为主要的排卵前与排卵后性信息素。两种处理均提升了精液量（P=0.001）。我们采用微阵列杂交技术，对经PGF2α处理的雄性金鱼端脑样本进行检测，共鉴定出71个差异表达的独特转录本（校正后q值<5%；其中67个上调，4个下调）。对这些受调控基因的功能注释结果显示，PGF2α性信息素暴露可影响多种生物学过程，包括神经系统功能、能量代谢、胆固醇/脂蛋白转运、翻译调控、转录与染色质重塑、蛋白质加工、细胞骨架组织以及信号转导。我们进一步通过实时定量逆转录聚合酶链反应（real-time RT-PCR）验证了3个候选基因：室管膜蛋白-II（ependymin-II）、钙调蛋白-A（calmodulin-A）与醛缩酶C（aldolase C），经PGF2α暴露后其表达量提升了3~5倍。我们还通过实时定量RT-PCR检测了其他部分被认为与繁殖相关的重要基因的表达水平。s型促性腺激素释放激素（sGnRH）的表达可被PGF2α上调，但不受17,20betaP影响；而c型促性腺激素释放激素（cGnRH）的表达可被17,20betaP上调，但不受PGF2α影响。与之相反，两种性信息素均可提升谷氨酸受体（GluR2a、NR2A）与γ-氨基丁酸A型受体（GABAA gamma2）亚基的mRNA表达水平。该基因表达数据将精液释放与神经元转录的快速调控联系起来，作为雌性性信息素应答反应的一部分。实验设计概述：本研究共使用4张微阵列芯片，用于探究PGF2α对端脑基因表达的影响。对照组样本（N=4）混合作为参考样本或技术重复；处理组样本（N=4，作为生物学重复）则单独进行杂交。此外，我们还对处理组的生物学样本进行了2次染料交换实验。