74
75 Fig. 1. Scores scatter plots obtained by PCA (A) and PLS-DA (B) of 1H NMR spectra of polar extracts of testes at different ages. Animal age in months is indicated in the legends above the plots.
biologicaly relevant effect sizes (ES>0.7) with respect to the results attained for 3 months-of- age group testis samples [23]. Alternatively, estimated metabolic changes are presented as bar graphs of normalized integrals with indicated statistical significancies in Supplementary Fig. S3-S6.
Testicular metabolic profiles show differential response to aging
The testicular metabolome seems to be highly sensitive to aging and we observed differences in various classes of metabolites including amino acid content (Fig. 3, Supplementary Fig. S3).
The most noticeable changes were observed early on. There is a clear increase in branched- chain (BCAA) and aromatic amino acids (Phe, Tyr) in the testes of 6 months-of-age group when compared to the 3 months-of-age group. On the contrary, Glu concentrations in testes decreased in the same period. In addition, changes were observed in lipid metabolism, with an increase registered for most metabolites in this group, especially noticeable in the testes from 12 and 24 months-of-age (Fig. 3, Fig. S4) groups. In the metabolic pathways associated with nucleic acids and their derivatives, similar to what was detected for amino acids, we observed sharp changes after the 3 months-of-age for the majority of the studied metabolites in the testes (Fig. 3, Fig. S5). Energy related metabolism in testes was also effected by age (Fig. 3, Fig. S6). Lactate, which is indispensable for a successful spermatogenesis and acts as a fuel for germ cells [26], succinate, indicative of tricarboxylic acid (TCA) cycle activity, and creatine show age-dependent changes in testes. Antioxidant-defense-related metabolites generally tend to change with age and most changes were observed for 12 and 24 months-of- age testes. We found that both radical scavenging and non-scavenging mechanisms were
76 Fig. 2. Scores scatter plots obtained by PCA (A) and PLS-DA (B) analysis of 1H NMR spectra of polar testicular extracts at 3 and 6 months-of-age group samples. LV1 loadings extracted from PLS-DA are presented in fig. C.
Loadings are colored according to variable importance to the projection (VIP) and some assignments are indicated.
Abbreviations: CMP, cytidine monophosphate; GPC, glycerophosphocholine; GSSH, oxidized glutathione; IMP, inosine monophosphate; PC, phosphocholine; three letter code is used for amino acids terminology. N=8 for each group.
affected in testes, as evidenced in changes of small metabolites with antioxidant properties (betaine, glutathione, taurine, hypotaurine). Triethylamine testicular content increased at 12 months-of-age, and reflects the gut microbiota changes caused by advancing age. Most metabolites present in testes show greater variability in the groups of 12 and 24 months-of- age groups than those in younger age groups. According to what is suggested by the PCA (Fig. 1), we observed two distinct metabolic signatures within each of these age groups. While some of the samples in these two groups resemble metabolically the groups of 6 and 9 months, the others are distinctly different. The biggest contribution to the separation of these two groups into two subclasses was attributed to the changes in testicular lipid (increase in GPC, carnitine, myo-inositol, and glycerol) and antioxidant (decrease in betaine, GSSG, DMG, creatine, and
77 increase in hypotaurine) metabolites. Finally, the identified metabolites were used for pathway analysis. The most relevant pathways perturbed by aging are presented in Fig. 3.
Fig. 3. Testicular metabolome changes associated with aging as compared with testicular tissue of 3 months-of- age animals. Heatmap of effect size (A). Animal age in months is indicated (6m - 6 months-of-age; 9m - 9 months- of-age; 12m - 12 months-of-age; 24m - 24 months-of-age). Pathway analysis of identified metabolites in the different age groups (B). Abbreviations: ATP adenosine triphosphate, BCAA branched chain amino acids, CMP cytidine monophosphate, GPC glycerophosphocholine, GSSG glutathione disulfide, IMP inosine monophosphate, PC phosphocholine, U unknown, three letter code used for amino acids. N=8 for each group.
Lactate dehydrogenase and monocarboxylate transporter 4 present distinct age- related expression in testis
Due to the paramount relevance of lactate for the testicular germ cells and the observed increase of its testicular content in 9 and 12 months old animals, we evaluated the expression of lactate dehydrogenase (LDH) and monocarboxylate transporter (MCT4) in that tissue (Fig.
4). Testicular expression levels of LDH decreased in the oldest rats, with the 12 months-of- age group showing a 0.7-fold variation and the 24 months-of-age group samples showing a 0.9-fold variation when compared with the values obtained for 3 months-of-age group. We also observed a decrease in the amounts of MCT4 in the testes of the older rats (0.6 and 0.15-fold variation to control, for rats of 9 and 12 months-of-age, respectively).
78 Fig. 4. LDH and MCT4 protein levels (fold variation to 3 months-of-age) in testes of male rats of different ages.
Animal age in months (3 m - 3 months-of-age; 6 m - 6 months-of-age; 9 m - 9 months-of-age; 12 m - 12 months-of- age; 24 m - 24 months-of-age) is indicated. Results are expressed as mean ± standard error of the mean (SEM), with N=8 for each group. Statistical significances of multiple comparisons were marked by the following letter code:
each age group was assigned a letter (3 m=a, 6 m=b, 9 m=c, 12m=d, 24m=e). Letters above the bars denote significance between respective groups. Upper-case letters indicate statistical significance at the 0.05 level and lower-case significance at the 0.001 level.
Aging induces changes in testicular oxidative stress markers
As we observed significant changes in testicular content of metabolites with antioxidant properties, we evaluated specific oxidative stress markers that seem to reach a definitive level at 6 months-of-age (Fig. 5). Indeed, there is an increase in protein carbonylation (DNPHZ) levels in the testis from 6 months-of-age (1.5-fold variation to 3 months-of-age samples) to 24 months-of-age (1.6-fold variation to 3 months-of-age samples) rats (Fig. 5). We also observed a significant increase in testicular levels of protein nitrotyrosine groups with aging (NitroT).
Except for the group of 12 months-of-age, testicular nitrotyrosine group levels were augmented in all samples as compared to 3 months-of-age samples (Fig. 5).
Fig. 5. Effect of age on rat testicular tissue oxidative damages. The figure shows changes in lipid peroxidation (4- HNE), protein nitration (Nitro-Tyr) and carbonylation (DNPHZ) levels. Animal age in months is indicated (3m - 3 months-of-age; 6m - 6 months-of-age; 9m - 9 months-of-age; 12m - 12 months-of-age; 24m - 24 months-of-age).
Results are expressed as mean ± standard error of the mean (SEM), with N=8 for each group. Statistical significances of multiple comparisons were marked by the following letter code: each age group was assigned a
79 letter (3m=a, 6m=b, 9m=c, 12m=d, 24m=e). Letters above the bars denote significance between respective groups.
Upper-case letters indicate statistical significance at the 0.05 level and lower-case significance at the 0.001 level.
The levels of lipid oxidation marker, 4-hydrononenal (4-HNE) were increased until the 9 months-of-age. In the samples from the older rats there was no significant difference between testicular 4-HNE as compared to those of the testis of 3 months-of-age rats.
The expression of mitochondrial complexes is decreased in the testis of aged rats
Mitochondria is a key player in the maintenance of cellular homeostasis and produces approximately 95% of cellular ATP. The electron transport chain (ETC) is one of the main cellular generators of reactive oxygen species (ROS) and includes four multi-subunit complexes (complexes I–IV), responsible for oxidative phosphorylation. The analysis of the oxidative phosphorylation complexes protein levels showed that both total protein expression levels and individual complexes are affected by aging (Fig. 6). Interestingly, the peak of mitochondrial complexes expression was observed in the testis of 9 months-of-age rats.
Fig. 6. Effect of aging on rat testicular mitochondrial complexes expression at different ages. Animal age in months (3m - 3 months-of-age; 6m - 6 months-ofage; 9m - 9 months-of-age; 12m - 12 months-of-age; 24m - 24 months-of- age). CI: NADH dehydrogenase (ubiquinone), 1 beta subcomplex, subunit 8; CII: succinate dehydrogenase complex, subunit B, iron sulfur; CIII: ubiquinol-cytochrome c reductase, core protein II; CIV: mitochondrial encoded cytochrome c oxidase I; CV: ATP synthase alpha-subunit. Results are expressed as mean ± standard error of the mean (SEM), with N=8 for each group. Statistical significances of multiple comparisons were marked by the
80 following letter code: each age group was assigned a letter (3m=a, 6m=b, 9m=c, 12m=d, 24m=e). Letters above the bars denote significance between respective groups. Upper-case letters indicate statistical significance at the 0.05 level and lower-case significance at the 0.001 level.