However, they lose important functions during their lifespan in the circulation. We separated rainbow trout Oncorhynchus mykiss erythrocytes into young and old fractions using fixed angle-centrifugation and analyzed transcriptome changes using RNA sequencing RNA-seq technology and quantitative real-time PCR. We found differentially expressed between young and old erythrocyte fractions; of these showed higher transcript levels in young, while only 34 protein-coding genes had higher transcript levels in old erythrocytes.
In particular genes involved in ion binding, signal transduction, membrane transport, and those encoding various enzyme classes are affected in old erythrocytes. The transcripts with higher levels in old erythrocytes were associated with seven different GO terms within biological processes and nine within molecular functions and cellular components, respectively.
Our study furthermore found several highly abundant transcripts as well as a number of differentially expressed genes DEGs for which the protein products are currently not known revealing the gaps of knowledge in most non-mammalian vertebrates. Our data provide the first insight into changes involved in aging on the transcriptional level and thus opens new perspectives for the study of maturation processes in fish erythrocytes.
As in all vertebrates apart from mammals, the erythrocytes in fish are nucleated Nikinmaa, Throughout their lifespan, which is between 80 and days Avery et al.
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They are also capable of transcription and translation throughout their lifespan, although, again, the capability decreases with age. Young erythrocytes respond much more readily to external stimulation by, e. In fish that have recently suffered anemia, the proportion of young RBCs is larger compared to unstressed fish Lane, Associated with the decreased capability to respond to external stimulation, several properties of the erythrocytes are different in young and old erythrocytes.
For example, the shape changes from circular to elliptical during maturation Tavares-Dias, , the membrane of young erythrocytes is more fluid Lecklin et al. We have divided the erythrocytes in young and old cohorts on the basis of the old erythrocytes having higher density than young ones Lane et al. Since old erythrocytes have higher mean cellular hemoglobin concentration MCHC than young ones Lane et al. Thereafter RNA sequencing was carried out and differences in transcriptomes in the young and old cohort evaluated.
Since the water in the fish tanks of the hatchery is pumped from a nearby bay of the Finnish Archipelago, the water temperature in the tanks follows natural rhythms and is on the rise at this time of the year K. Malmberg, personal communication. Fish were netted from the tanks, quickly killed by a blow on the head and blood was sampled by caudal puncture into heparinized syringes, transferred into sterile falcon tubes, and stored on ice.
Diversity of Globin Function: Enzymatic, Transport, Storage, and Sensing
Fish were weighed and their length was measured. Density centrifugation of erythrocyte samples followed essentially the procedure described previously Murphy, ; Speckner et al. The tubes were cut into three equally sized parts: the top part, containing the youngest and least dense erythrocyte class fraction, the middle part which was discarded, and the lower part, containing the oldest and most dense erythrocytes.
The success of age separation was evaluated by determining the mean cellular hemoglobin concentration MCHC of both fractions using conventional methods centrifugation for haematocrit and cyanmethaemoglobin method for hemoglobin concentration; Speckner et al.
11.3 Circulatory and Respiratory Systems
Base calling on the reads was done using the Bcl2fastq2 software version 1. The quality control of raw sequencing reads was performed with FastQC www. The read alignment was performed against the reference genome using TopHat v2.
The number of uniquely aligned reads was between 2. The sample correlation values Spearman's metrics were between 0. Sample pairing young and old erythrocyte age classes of one individual was taken into account when building the linear model for statistical testing. The association between reads and known genes and the number of reads associated with each gene was assessed using the subreads package v1.
Vertebrate Red Blood Cells
Gos are organized hierarchically in terms of biological processes, cellular components and molecular functions. We selected eight genes which on the basis of earlier research have important functions in fish erythrocytes and designed specific primers Table S1 using the Primer3 software Koressaar and Remm, ; Untergasser et al. Each qPCR plate contained non-template controls to detect potential contamination in reaction mixes. Data were analyzed with the QuantStudio software. Reaction efficiency for each gene was calculated using a standard curve generated from a serial dilution of the pooled samples.
Standard curve reactions were performed in duplicate. Data are shown relative to the respective mixed blood samples and log2 transformed. Significant differences were assessed first between raw Ct-values of the young, old and originalsamples, and second between the raw Ct-values of the young and old erythrocyte samples using a one-way repeated measures ANOVA after equal variances Brown—Forsythe and normality Shapiro—Wilk test of data were tested using the Real Statistics Resource Pack software Release 4.
In cases where tests for equal variances and normality failed we used a non-parametric test Friedman's test. Post-hoc analyses were done with the Holm-Sidak test. In this study, an average number of 12,, reads mean young erythrocytes: 11,,; mean old erythrocytes 12,, were generated. Only uniquely mapped reads average 4,,; mean young erythrocytes 3,,; mean old erythrocytes 4,, were used for the downstream analysis, resulting in 51, unigenes. The 15 most abundant genes those among the top 10 abundant genes in any sample account for 9. The products of two abundant transcripts in the list are currently not known.
Table 1. Read percentages for the 15 most abundant genes in the erythrocyte samples.
Seven transcripts were associated with the various GO terms related to hemoglobin function GO hemoglobin complex; GO oxygen transporter activity; GO iron ion binding; GO heme binding; GO oxygen transport; GO oxygen binding. There was a large shift in transcript abundance between young and old erythrocyte samples Figure 1. The total number of DEGs was ; Positive log 2 -fold changes of transcripts in old erythrocytes did not exceed 2.
Figure 1. Heat map of age-related changes in gene expression of erythrocytes RBCs with hierarchical clustering complete linkage method with Euclidean distance measure of all biological replicates.
Red indicates highest expression and blue indicates lowest expression. Table 2. List of genes with significantly higher transcript levels in old vs. The largest three subcategories in the biological group were cellular processes IDs , metabolic processes IDs and single-organism processes IDs. In the molecular function category IDs were associated with ion binding, and in cellular component category, 4 enriched GO terms were found: intracellular IDs , intracellular part IDs , membrane-bounded organelle IDs and intracellular organelle IDs were enriched terms in the cellular component.
Figure 2. Pie charts were generated at level 2 for biological process A,D , and level 3 for molecular function B,E and cellular component C,F by Blast2GO using transcripts with higher levels in young erythrocytes Table S2 ; pie charts A—C and 34 transcripts with higher levels in old erythrocytes Table 2 ; pie charts D—F as input.
The input list of the transcripts with higher levels in old erythrocytes contained 34 IDs Table 2 , for 18 IDs a total of 25 Gene ontology GO annotation terms were identified where 7 were within biological process Figure 2D , and 9 each in cellular component Figure 2F and molecular function Figure 2E. For 16 IDs no GO terms could be found.
GO terms were enriched for cellular process 8 IDs , single-organism process 5 IDs and metabolic process 4 IDs in the biological process category. In the metabolic function category, no enriched GO terms were found and with each of the GO terms 1—3 IDs were associated. Intracellular components 7 IDs , membrane-bounded organelle 4 IDs and intracellular organelle 6 IDs were enriched terms in the cellular component. We selected eight genes whose products have important functions in fish erythrocytes for a comparison of expression levels between young and old erythrocyte fractions with the original erythrocyte sample with qPCR.
The levels of expression of the genes are shown in Figure 3. Figure 3. Comparative gene expression levels between young and old erythrocytes RBCs. Maturation of fish erythrocytes in the circulation is associated with marked changes in many aspects of their physiology. We analyzed these changes on the transcriptomic level in young and old erythrocytes in order to get insights into the yet unknown pathways involved in aging.
Measuring cellular steady-state levels as done by RNA-seq does not take into account alterations in either of these processes Hayles et al. Our results revealed that aging in fish erythrocytes is accompanied by a decrease in transcript levels of a number of genes. It was shown earlier that the rate of transcription and the cellular RNA content decreases during maturation of nucleated non-mammalian erythrocytes Grasso et al. High transcriptional and translational activity is required in young, immature erythrocyte because they gradually undergo changes in cell shape, membrane rigidity and other properties until maturation e.
In contrast, in old, mature erythrocytes the need for gene transcription and protein synthesis is much lower and the necessary machinery, such as ribosomes is very low Lane et al. Figure 1. Hemoglobin molecule with four globin chains, two alpha-like and two beta-like chains each bearing a heme group, responsible for the reversible binding of oxygen.
The figure was generated by Pymol 0. Figure 2.
A monomer of lamprey hemoglobin generated by Pymol 0. The figure shows the globin with its a -chain segments and the heme group. Perutz MF. Species adaptation in a protein molecule. Adv Protein Chem ; Berenbrink M. Evolution of vertebrate haemoglobins: Histidine side chains, specific buffer value and Bohr effect.
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