Seasonal and sex-dependent gene expression in emu (Dromaius novaehollandiae) fat tissues

Abstract

Emu (Dromaius novaehollandiae) farming has been gaining wide interest for fat production. Oil rendered from this large flightless bird's fat is valued for its anti-inflammatory and antioxidant properties for uses in therapeutics and cosmetics. We analyzed the seasonal and sex-dependent differentially expressed (DE) genes involved in fat metabolism in emus. Samples were taken from back and abdominal fat tissues of a single set of four male and four female emus in April, June, and November for RNA-sequencing. We found 100 DE genes (47 seasonally in males; 34 seasonally in females; 19 between sexes). Seasonally DE genes with significant difference between the sexes in gene ontology terms suggested integrin beta chain-2 (ITGB2) influences fat changes, in concordance with earlier studies. Six seasonally DE genes functioned in more than two enriched pathways (two female: angiopoietin-like 4 (ANGPTL4) and lipoprotein lipase (LPL); four male: lumican (LUM), osteoglycin (OGN), aldolase B (ALDOB), and solute carrier family 37 member 2 (SLC37A2)). Two sexually DE genes, follicle stimulating hormone receptor (FSHR) and perilipin 2 (PLIN2), had functional investigations supporting their influence on fat gain and loss. The results suggested these nine genes influence fat metabolism and deposition in emus.

Figure 1

Gene ontology classification of seasonally differentially expressed genes for each sex. The two series of stacked bar plots correspond to all female and male seasonally differentially expressed transcripts. Sidak corrected p-values from χ² tests for each domain and their categories within are in brackets where those significantly different between sexes are indicated with an asterisk. Those without p-values were equal to 1 and, therefore, were not significantly different between sexes.

Figure 2

Seasonal differential expression between time points for both sexes, females, and male emus. Volcano plots of month-wise comparisons for each sex condition are displayed. Genes surviving cut offs of − 1 > log₂(fold change) > 1 correspond to down-regulated genes (dark and light blue) and up-regulated genes (red and pink), and Benjamini–Hochberg corrected p-value < 0.01 (dark blue and red), or Benjamini–Hochberg corrected p-value between 0.01 and 0.05 (light blue and pink) are displayed. The number of genes associated with these classes is coloured for each condition of p-value and log₂(fold change) (LFC) cutoffs within each comparison. Grey points indicate genes that either did not survive the thresholds and, therefore, were not significant, or those that were deemed non-substantial as determined by DESeq2, as described in the text.

Figure 3

Sexually differential expression pairwise comparisons between sexes for each time point. Volcano plots of sex-wise comparisons for each month are displayed. LFC is the log₂(fold change) of gene expression. Other plot details are the same as in Fig. 2.

Figure 4

Triacylglycerol synthesis pathway for female seasonal differential expression. Emu triacylglycerol synthesis is represented by the Gallus gallus pathway, which is fuelled by the fatty acid synthesis pathway to provide fatty acyl-CoA metabolites. Metabolites (grey oval nodes) are catalysed (black solid arrows) by gene products (rectangle nodes). Dashed arrows represent a process that involves additional steps not displayed in the diagram. Gene products are split into thirds and correspond to the month-wise comparisons from the female seasonal differential expression analysis. Each third is coloured by their respective gene’s Benjamini–Hochberg corrected p-value in a gradient from more significant (red) to less significant (blue). White represents genes that have non-substantial p-values calculated by DESeq2, while grey represents no annotation available for emu or chicken. Three seasonally DE genes (DGAT2, LPL, and MOGAT2) have bar plots of expression levels in median log₂(TPM) by month featuring median absolute deviation error bars. Numerical Benjamini–Hochberg corrected p-values are shown above the horizontal brackets of the bars that indicate the month-wise comparison, where an asterisk to the right represents statistical significance (p < 0.01).

Figure 5

Triacylglycerol synthesis pathway and male seasonal differential expression. Emu triacylglycerol synthesis is represented by the Gallus gallus pathway. Gene products are coloured by their respective gene’s Benjamini–Hochberg corrected p-values from the male seasonal differential expression analysis. Three genes (DGAT2, LPL, and MOGAT2) have bar plots of expression levels. MOGAT2 was seasonally DE in males. Other figure details are the same as in Fig. 4.