Figure 1.
Total RNA was extracted from occipital cortex from four males and four females for each primate species; human (Homo sapiens), macaque (Macaca fascicularis) and marmoset (Callithrix jacchus). Transcribed cDNA (n = 24) was hybridized to human cDNA microarrays (n = 24) (Human 46k cDNA, KTH Microarray Center, Stockholm) in male-female pairs within each species. Yellow circles represent RNA samples from females (F) and males (M). Arrows symbolize microarray hybridizations, where the tip of the arrow indicates that the sample was labeled with Cy5 dye and the base of the arrow that the sample was labeled with Cy3 dye.
Figure 2.
Volcano-plots of cDNA microarray data in three primate species.
The figure shows an overview of the microarray results in each of the three primate species; human (Homo sapiens), macaque (Macaca fascicularis), marmoset (Callithrix jacchus). The y-axis denotes √(PenF) i.e. significance level, and the x-axis denotes log2(F/M) i.e. fold change expression difference between females and males. The horizontal lines; a, b, c, illustrate the PenF thresholds corresponding to Table 1. The vertical lines represent log2 fold changes of 0.5 (i.e. 1.4 on decimal base), marked to facilitate comparisons between the three graphs. The green lines tangent the maximum √(PenF) for any given fold change. The slope of this line is inversely proportional to √a0 (the penalty constant), and a gentle slope is reflecting a relatively high technical and biological variation.
Table 1.
Number of sexually dimorphic genes in occipital cortex of human, macaque and marmoset.
Figure 3.
Sequence identity between selected marmoset transcripts and their orthologous human cDNA clones present on the microarrays.
The figure shows that the 4 highest ranked clones with respect to sexual dimorphism in marmoset (Callithrix jacchus, PenF≥5.0, Table 1 and Figure 2) do not have a higher sequence identity distribution than a random sample of low ranked genes (n = 185). This demonstrates that the low number of sexually dimorphic genes in marmoset is not mainly due to an effect of sequence identity difference between the species.
Figure 4.
A conserved sex signature in occipital cortex gene expression among human, macaque and marmoset.
The presence of a conserved sex signature of occipital cortex gene expression in human (Homo sapiens), macaque (Macaca fascicularis) and marmoset (Callithrix jacchus) is here shown by a heatmap. Rows represent genes and each column a male Vs. female hybridization. Yellow illustrates female up-regulation, blue illustrates male up-regulation, and black illustrates no difference in gene expression between the sexes. The red map on the left shows background subtracted mean intensity values (Ab) for each gene and species. The intensities in marmoset are not lower than in the other species, demonstrating that the lack of sex differences in marmoset was not due to weak binding to the human cDNA microarrays. The two top genes (XIST, HSBP1) display conserved sexually dimorphic expression patterns among all three species while the following 83 genes display a conserved sex-specific expression pattern in humans and macaques. Selection criteria: FDR≤0.05.
Figure 5.
Expression of the occipital cortex conserved sexually dimorphic genes in other human tissues.
Many of the genes in the identified conserved sex signature in primate occipital cortex are highly expressed in the nervous system, and expressed less in sexual tissues. To generate the figure, we used normalized expression data for human tissues from SOURCE [19]. Normalized gene expression presents the relative expression level of a gene (defined as a UniGene Cluster) in different tissues and is normalized for the number of clones from each tissue that are included in UniGene. Rows denote genes and columns denote tissues. Rank 1 means that expression of a gene is highest in the indicated tissue, symbolized by deep red color, and lower ranks are represented by steps of less intense red, as indicated by the color code bar at the top of the figure. Gray color denotes that expression level of the gene in tissue was not available or not detected (NA or ND). 55 out of the 85 genes in the conserved sex signature have a rank equal to or higher than 5 in nervous tissues (65%). 81 of the occipital cortex sex signature genes are highly expressed in tissues present in the head or neck (95%, rank ≥5), while 23 of the genes are highly expressed in any of the sexual tissues (27%, rank ≥5). Genes appear in the same order as in Figure 4.
Figure 6.
Non-synonymous versus synonymous substitutions in conserved sexually dimorphic genes.
We estimated non-synonymous and synonymous ratios (dN/dS ratios) in a large set of human-chimp-rhesus three-way alignments. The dN/dS ratios of genes in the conserved sexual signature in humans and macaques (genes in Figure 4, Cons.Sex, n = 48 found in the three-way alignment) were compared to genes specifically expressed in brain [20] (Brain, n = 935), and to all other genes found in the three-way alignment (Genome, n = 8624). The genes in the conserved sex signature had dN/dS ratios significantly lower than the genome reference (median of Cons.Sex = 0.06, median of Genome = 0.16, permutation test of the medians; p = 0.0003), and dN/dS ratios in the same range as other brain expressed genes (median = 0.08). The heights of the bars represent median dN/dS ratios. p-values; *** p≤0.001.
Figure 7.
Non-synonymous versus synonymous substitutions in male-biased and female-biased genes.
Using a large set of human-chimp-rhesus three-way alignments, we compared ratios of non-synonymous versus synonymous substitutions (dN/dS) between genes that were identified as male up-regulated (Male-biased) and female up-regulated (Female-biased) in human and/or macaque in the single species analysis (genes with PenF≥5, Table 1 and Figure 2). 136 of the male-biased genes and 52 of the female-biased genes were found in the three-way alignments. Male-biased genes had dN/dS ratios three times higher than female-biased genes (median of male-biased = 0.09, median of female biased = 0.03, permutation test of the medians; p = 0.006). Non-biased brain-expressed genes had intermediate dN/dS ratios (median = 0.08). The heights of the bars represent median dN/dS ratios. p-values; ** p≤0.01, *** p≤0.001.
Figure 8.
Confirmation of conserved sex expression signature with RT q-PCR.
mRNA levels of two genes with male-biased expression and three genes with female-biased expression in human (Homo sapiens) and macaque (Macaca fascicularis), but not in marmoset (Callithrix jacchus), as quantified with RT q-PCR. The expression patterns observed in the array experiment (Figure 4) were confirmed for each gene, with the exception of MAP1A, which was not differentially expressed between males and females in human. Least square means and standard errors of the mean are given. Female expression levels were used as point of reference. p-values; * p≤0.05, ** p≤0.01, *** p≤0.001.
Table 2.
Overrepresented ontology classes in the conserved sex signature in primate occipital cortex.