Transcriptional response across estrous in the mouse dorsal hippocampus is dysregulated by Cnih3 mutation

The hippocampus is a critical brain region for coordinating learning, memory, and behavior. In adult females, the estrus cycle alters these functions through the activity of steroids hormones, with well-characterized effects on cellular physiology and behavior. However,the molecular genetic basis of these outcomes has not been systematically explored. In order to better understand the role of sex and the estrous cycle in the hippocampus, we profiled the transcriptome of hippocampi from female mice in each stage of the estrus cycle, along with those of males. We identify only subtle sex differences in gene expression between the sexes on average, yet comparing males to individual estrous stages reveals ~100 genes deviating from male expression patterns at one point in their cyclic fluctuations across estrous cycle. These estrus-responsive genes are especially enriched in oligodendrocytes and glycinergic neurons, and are potentially regulated downstream of estrogen receptor repressed pathways. To further understand our previous observations of female- and estrous-specific behavioral outcomes in knockout of Cnih3, we performed the same profiling in the knockout strain. Surprisingly, Cnih3 knockouts showed far broader transcriptomic differences between estrous cycle stages and males, despite very subtle within-group-across-genotype expression changes. We show that Cnih3 knockout drives expression changes in opposing directions between males and all points of the estrous cycle, extensively accentuating the magnitude of sex-differential hippocampal gene expression compared to wild-types. We thus provide a novel resource characterizing estrous-specific gene expression patterns in the adult hippocampus, which can provide insights into mechanisms of sex differential neuropsychiatric functions and dysfunctions, and identify a role for Cnih3 in buffering the female brain against the transcriptional effects of estrous. RNA-seq of dorsal hippocampus from wild-type C57/B6 and Cnih3 knockout (Frye 2021) adult male mice (n≥5 per genotype) and female mice at each estrous stage (n=4-7 females per stage)

海马体（hippocampus）是协调学习、记忆与行为的关键脑区。对于成年雌性个体而言，发情周期（estrus cycle）通过类固醇激素的活性调控上述功能，其对细胞生理与行为的影响已得到充分阐释。然而，上述效应的分子遗传基础尚未得到系统性探究。

为深入阐明性别与发情周期在海马体中的作用，我们对处于发情周期各阶段的雌性小鼠，以及雄性小鼠的海马体转录组（transcriptome）进行了分析表征。研究发现，总体而言雌雄个体间的基因表达仅存在细微的性别差异；但将雄性与单个发情阶段的雌性进行比较时，可见约100个基因在发情周期的周期性波动中，于某一节点偏离雄性的表达模式。这些响应发情周期的基因在少突胶质细胞（oligodendrocytes）和甘氨酸能神经元（glycinergic neurons）中显著富集，且可能受雌激素受体抑制通路的下游调控。

为进一步阐释我们此前在Cnih3基因敲除（knockout）小鼠中观察到的雌性特异性及发情周期特异性行为表型，我们对该敲除品系开展了相同的转录组分析。令人意外的是，尽管同一基因型组内不同个体间的表达差异极细微，但Cnih3敲除小鼠的发情周期阶段与雄性间的转录组差异显著扩大。我们证实，Cnih3基因敲除会使雄性与所有发情阶段雌性的基因表达呈现相反的变化方向，相较野生型（wild-types）小鼠大幅增强了海马体性别差异基因的表达幅度。

据此，本研究提供了一项全新的研究资源，对成年海马体中发情周期特异性的基因表达模式进行了系统表征，可为性别差异性神经精神功能及功能异常的机制研究提供新视角，并揭示了Cnih3在缓冲雌性大脑免受发情周期相关转录影响中的作用。本数据集包含野生型C57/B6和Cnih3基因敲除型（Frye 2021）成年雄性小鼠（每组样本量n≥5），以及处于各发情阶段的雌性小鼠（每阶段样本量n=4~7）的背侧海马体RNA测序（RNA-seq）数据。