Gene Bionetworks Involved in Epigenetic Transgenerational Inheritance of Environmentally Altered Sexual Selection: Role of Epigenetics in Evolutionary Biology. Rattus norvegicus

Sexual selection involves mate preference behavior and is a critical determinant for natural selection and evolutionary biology. Previously an environmental compound (fungicide vinclozolin) was found to promote epigenetic transgenerational inheritance of modified mate selection characteristics in all progeny for three generations after exposure of a gestating female. The current study investigated gene networks involved in various regions of the brain that correlated with the mate preference behavior altered in F3-Vinclozolin lineage animals. Statistically significant correlations of differentially expressed gene clusters and modules were identified to associate with specific mate preference behaviors. This novel systems biology approach identified critical gene networks involved in mate preference behavior and demonstrated the ability of environmental factors to promote epigenetic transgenerational inheritance of this altered evolutionary biology determinant. Combined observations elucidate the potential molecular control of mate preference behavior and suggests environmental epigenetics can have a role in evolutionary biology. We used Affymetrix Rat Gene 1.0 ST microarrays to determine genes expressed differentially in F3 Vinclozolin lineage male or female rats' 6 brain areas - amygdala (Amy), hippocampus (Hipp), olfactory bulb (OlfB), cingulate cortex (CngCtx), entorhinal cortex (EnCtx), and preoptic area-anterior hypothalamus (POAH) - due to Vinclozolin treatments of their grand-grandmothers (F0). Overall design: For each of 6 brain areas of male or female rats (female: amygdala (F-Amy), cingulate cortex (F-CngCTX), enterorhinal cortex (F-EnCTX), hippocampus (F-Hipp), olfactory bulbs (F-OlfB), and preoptic area-anterior hypothalamus (F-POAH); male: amygdala (M-Amy), cingulate cortex (M-CngCTX), enterorhinal cortex (M-EnCTX), hippocampus (M-Hipp), olfactory bulbs (M-OlfB), and preoptic area-anterior hypothalamus (M-POAH)), RNA samples from 2 treatment groups - F3 Control lineage (Con) or F3 Vinclozolin lineage (Vin) - were compared to each other. Each of treatment groups contained 4-6 biological replicas for each brain region. RNA for each replica was isolated from an individual animal in order to compare to individual animal mate preference behavior studied with the same rats before sacrifice. Totally, 132 RNA samples from 24 animals (6 male F3 Control, 6 male F3 Vinclozolin,6 female F3 Control, and 6 female F3 Vinclozolin) were isolated and studied.

性选择（Sexual selection）涵盖配偶偏好行为，是自然选择与进化生物学的核心决定因素。此前研究发现，一种环境化合物——杀菌剂乙烯菌核利（vinclozolin）——可在妊娠雌性动物暴露后，促使其所有后代连续三代获得经修饰的配偶选择特征的表观遗传跨代遗传（epigenetic transgenerational inheritance）。本研究针对F3代乙烯菌核利谱系动物中发生改变的配偶偏好行为，探究了其与大脑多个区域内基因调控网络的关联。研究鉴定出与特定配偶偏好行为显著相关的差异表达基因簇与模块。本研究采用的新型系统生物学（systems biology）方法，鉴定出了参与配偶偏好行为的关键基因网络，并证实环境因素可促使这一进化生物学核心决定因素的表观遗传跨代遗传。综合上述研究结果，阐明了配偶偏好行为潜在的分子调控机制，并提示环境表观遗传学（environmental epigenetics）在进化生物学中具有潜在作用。本研究使用Affymetrix大鼠基因1.0 ST芯片（Affymetrix Rat Gene 1.0 ST microarrays），检测了因曾祖母（F0代）接受乙烯菌核利处理而形成的F3代乙烯菌核利谱系雌雄大鼠的6个大脑区域——杏仁核（amygdala, Amy）、海马体（hippocampus, Hipp）、嗅球（olfactory bulb, OlfB）、扣带回皮层（cingulate cortex, CngCtx）、内嗅皮层（entorhinal cortex, EnCtx）以及视前区-前下丘脑（preoptic area-anterior hypothalamus, POAH）——中的差异表达基因。实验设计：针对雌雄大鼠的6个大脑区域（雌性：杏仁核F-Amy、扣带回皮层F-CngCTX、内嗅皮层F-EnCTX、海马体F-Hipp、嗅球F-OlfB以及视前区-前下丘脑F-POAH；雄性：杏仁核M-Amy、扣带回皮层M-CngCTX、内嗅皮层M-EnCTX、海马体M-Hipp、嗅球M-OlfB以及视前区-前下丘脑M-POAH），本研究对两组处理组——F3代对照谱系（Con）与F3代乙烯菌核利谱系（Vin）——的RNA样本进行了比较分析。每个处理组的每个大脑区域均设置4~6个生物学重复样本。每个重复样本的RNA均提取自单只大鼠，以便与处死前已完成配偶偏好行为检测的同一只大鼠进行关联分析。本研究共从24只大鼠（6只F3代对照雄性、6只F3代乙烯菌核利雄性、6只F3代对照雌性、6只F3代乙烯菌核利雌性）中提取并分析了132份RNA样本。