Temporal sex specific brain gene expression pattern during early rat embryonic development

Abstract

Background: The classical concept of brain sex differentiation suggests that steroid hormones released from the gonads program male and female brains differently. However, several studies indicate that steroid hormones are not the only determinant of brain sex differentiation and that genetic differences could also be involved. Methods: In this study, we have performed RNA sequencing of rat brains at embryonic days 12 (E12), E13, and E14. The aim was to identify differentially expressed genes between male and female rat brains during early development. Results: Analysis of genes expressed with the highest sex differences showed that Xist was highly expressed in females having XX genotype with an increasing expression over time. Analysis of genes expressed with the highest male expression identified three early genes, Sry2, Eif2s3y, and Ddx3y. Discussion: The observed sex-specific expression of genes at early development confirms that the rat brain is sexually dimorphic prior to gonadal action on the brain and identifies Sry2 and Eif2s3y as early genes contributing to male brain development.

Keywords: RNA sequencing; differentiation; neuronal; sex chromosome; sexual dimorphism.

FIGURE 1

Venn diagrams illustrate the number of differentially expressed genes (DEGs) in male rats, categorized by their developmental stage. DEGs were identified based on an adjusted p-value <0.05, following False Discovery Rate (FDR) correction. DEGs common to multiple stages are also annotated.

FIGURE 2

Venn diagrams illustrate the number of differentially expressed genes (DEGs) in female rats, categorized by their developmental stage. DEGs were identified based on an adjusted p-value <0.05, following False Discovery Rate (FDR) correction. DEGs common to multiple stages are also annotated.

FIGURE 3

Hierarchical clustering and heat map of the differentially expressed genes. (A) The expression patterns at the three studied developmental stages were visualized using standardized gene expression values (z-scores). (B) 3D scatter plot illustrates the results of a Principal Component Analysis (PCA) performed on gene expression data from male and female embryos at different developmental stages.

FIGURE 4

Y chromosome genes differentially expressed at all three developmental stages in male rat brains. The reads per million of DEGs at all three developmental stages are shown. Statistical analysis was performed using One way ANOVA followed by Tukey’s multiple comparison post-test (*p < 0.05 and **p < 0.01) (n = 3, mean ± SEM).

FIGURE 5

X chromosome genes were differentially expressed at all three developmental stages in female rat brain (closed circles). The reads per million of DEGs at all three developmental stages are shown For male brains (open circles) only three of the genes showed differences in reads per million between developmental stages. (A) Xist, (B) Kdm6a, (C) Eifs2s3x, (D) Enpp1, (E) Pbdc2. Statistical analysis was performed using One way ANOVA followed by Tukey’s multiple comparison post-test. Different letters were used to denote the level of significance between stages, and asterisk was used to denote statistical differences between sexes at p < 0.05 (n = 3, mean ± SEM).

FIGURE 6

GO enrichment at E12 to E14. Bubble plots were constructed to visually represent Gene Ontology (GO) annotations for each embryonic stage utilizing DEGs identified at each stage, providing a comprehensive overview of the functional categories that are enriched during development. Enriched biological processes (BP), cellular components (CC), and molecular functions (MF) are presented using color gradient to represent the percentage of sex bias for given terms. Red color indicates male dominated enrichment while green color indicates female dominated enrichment.