The estrous cycle surpasses sex differences in regulating the transcriptome in the rat medial prefrontal cortex and reveals an underlying role of early growth response 1

Abstract

Background: Males and females differ in cognitive functions and emotional processing, which in part have been associated with baseline sex differences in gene expression in the medial prefrontal cortex. Nevertheless, a growing body of evidence suggests that sex differences in medial prefrontal cortex-dependent cognitive functions are attenuated by hormonal fluctuations within the menstrual cycle. Despite known genomic effects of ovarian hormones, the interaction of the estrous cycle with sex differences in gene expression in the medial prefrontal cortex remains unclear and warrants further investigations.

Results: We undertake a large-scale characterization of sex differences and their interaction with the estrous cycle in the adult medial prefrontal cortex transcriptome and report that females with high and low ovarian hormone levels exhibited a partly opposed sexually biased transcriptome. The extent of regulation within females vastly exceeds sex differences, and supports a multi-level reorganization of synaptic function across the estrous cycle. Genome-wide analysis of the transcription factor early growth response 1 binding highlights its role in controlling the synapse-related genes varying within females.

Conclusions: We uncover a critical influence of the estrous cycle on the adult rat medial prefrontal cortex transcriptome resulting in partly opposite sex differences in proestrus when compared to diestrus females, and we discovered a direct role for Early Growth Response 1 in this opposite regulation. In addition to illustrating the importance of accounting for the estrous cycle in females, our data set the ground for a better understanding of the female specificities in cognition and emotional processing.

Fig. 1

A sexually biased transcriptome in the rat medial prefrontal cortex (mPFC) and effect of the estrous cycle. a A principal component analysis separates males and females along the second axis while proestrus and diestrus females are clustered separately along the first axis. b The hierarchical clustering of the 70 genes showing the most variance (rlog-transformed) revealed more similarity of diestrus females to males over proestrus females. c, e-g Representation of the log2 fold-change over the averaged normalized read counts, with significantly different genes at the false discovery rate (FDR) 5 % threshold highlighted in red. d The sexually dimorphic genes in the rat mPFC do not show a sex-chromosome bias, and are distributed among all chromosomes. To account for differences in number of genes per chromosome, data were normalized to the total number of genes detected in our study on each chromosome. h The Venn diagram representing the number of differentially expressed genes (FDR 5 %) in all pairwise comparisons depicts a relatively small overlap between genes affected by the estrous cycle within females, and those sexually biased in either stage of the cycle. The area of each circle is proportional to the number of genes it contains. In (a–c, e-g), values from the R package DESeq2 were used

Fig. 2

Sexually biased genes in the rat medial prefrontal cortex (mPFC) are associated with cellular communication and translation. a Enrichment map depicting the clusters of differentially modulated pathways between females and males identified by the gene-set enrichment analysis. The area of each node, representing a gene set (functional pathway), corresponds to the number of genes of the gene set it contains, and the edge thickness is proportional to the number of genes overlapping between the two connected nodes. Pathways related to the interaction with the extracellular matrix and its downstream signaling were widely associated with males, while only pathways related to translation and oxidative phosphorylation were associated with the female phenotype. b Illustration of the averaged read coverage for the male (blue), proestrus (pink), and diestrus (green) groups for two genes up-regulated (left) or down-regulated (right) in females when compared to males. c A substantial proportion of the genes differentially expressed in proestrus or diestrus when compared to males are specific to each cycle stage. d Enrichment map depicting the cluster of pathways identified by gene-set enrichment analysis as differentially regulated in the proestrus versus males (inner circle of each node), and diestrus versus males comparisons (outer ring of each node). Green and blue edges correspond to the proestrus versus males, and diestrus versus males datasets, respectively

Fig. 3

Widespread functional reorganization of the rat medial prefrontal cortex transcriptome throughout the estrous cycle. a Enrichment map depicting the clusters of differentially modulated pathways between proestrus and diestrus females identified by the gene-set enrichment analysis. The area of each node, representing a gene set (functional pathway), corresponds to the number of genes of the gene set it contains, and the edge thickness is proportional to the number of genes overlapping between the two connected nodes. Pathways related to translation, degradation, oxidative phosphorylation, and transcription are associated with diestrus females, whereas extracellular matrix interactions, as well as insulin and synaptic signals transduction are associated with proestrus females. b treemap representation of Cellular Compartment terms from the Gene Ontology database, showing a marked enrichment of neuronal and synaptic genes. The size of each rectangle is proportional to the -log10 of the p-value (the bigger the rectangle, the more significant the enrichment). c The enrichment analysis of pathways from the Kyoto Encyclopedia of Genes and Genomes reveals a widespread alteration of signal transduction pathways, including neuronal and synaptic, as well as translation processes

Fig. 4

Estrous cycle-dependent transcriptomic regulation exceeds sex differences and primarily targets synaptic function. A gene clustering analysis identified nine distinct profiles of gene regulation between males (left side), proestrus (center), and diestrus females (right side). At the top left corner of each plot is detailed the total number of genes as well as the percentage of all differentially expressed genes represented in the cluster. In each cluster, the enrichment in Biological Processes from the Gene Ontology (GO) database was analyzed for the differentially expressed genes represented in the cluster. The first four hits (if four or more hits were found) ranked by p-value are depicted on the right of each plot and highlight the enrichment of synapse-related genes in proestrus females from clusters 1 and 2, translation processes in diestrus females from clusters 3 and 4, and interaction with the extracellular matrix from cluster 6. The full list of enriched GO terms can be found in Additional file 10: Table S5

Fig. 5

Egr1 binds to synapse-related genes in a sex-specific and estrus cycle-specific manner. a The majority of differential Egr1 binding was observed in proestrus when compared to either males (pink circle) or diestrus females (green circle). b, c treemap representation of Cellular Compartment terms from the Gene Ontology database, showing a marked enrichment of receptors complexes, synapse, and zones of cell–cell communication localizations in proestrus when compared to males (b) or diestrus (c). The size of each rectangle is proportional to the -log10 of the p-value (the bigger the rectangle, the more significant the enrichment). d–f The enrichment analysis of pathways from the Kyoto Encyclopedia of Genes and Genomes on the nearest genes to a differential Egr1 binding locations reveals an alteration of signal transduction pathways between males and females in proestrus (d) and diestrus (e), as well as within females (f)