134
Supplementary Material
Senescence and declining reproductive potential: insight into molecular mechanisms through testicular metabolomics
Ivana Jaraka,b,c*, Susana Almeidab*, Rui A. Carvalhoa, Mário Sousab, Alberto Barrosd,e, Marco G. Alvesb, Pedro F. Oliveirab,d,e
* Both authors contributed equally
a Department of Life Sciences, Faculty of Sciences and Technology and Centre for Functional Ecology (CFE), University of Coimbra, Coimbra, Portugal
b Laboratory of Cell Biology and Unit for Multidisciplinary Research in Biomedicine (UMIB), Department of Microscopy, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal
c Health Sciences Research Centre (CICS–UBI), University of Beira Interior, Covilhã, Portugal
d i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal
e Department of Genetics, Faculty of Medicine, University of Porto, Porto, Portugal
Corresponding authors:
Pedro F. Oliveira, PhD
Department of Microscopy, Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar (ICBAS), Rua de Jorge Viterbo Ferreira no. 228, 4050-313 Porto, Portugal. Tel: +351- 220-428000; E-mail: pfobox@gmail.com
Marco G. Alves, PhD
Department of Microscopy, Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar (ICBAS), Rua de Jorge Viterbo Ferreira no. 228, 4050-313 Porto, Portugal. Tel: +351- 220-428000; E-mail: alvesmarc@gmail.com
135 Supplementary Fig. S1: Average 1H NMR spectra of polar testicular extracts at different age. High field part of the spectra (5.2-8.9 ppm) was vertically expanded 30x. Signals of some major metabolites are indicated according to Table 1: 1 acetate, 3 adenosine, 5 alanine, 8 betaine, 9 carnitine, 13 creatine, 22 glycerol, 24 glycine, 26 hypotaurine, 28 IMP, 30 lactate, 35 phenyalanine, 36 phosphocholine, 43 tyrosine.
136
137 spectra of polar testicular extracts at a) 3 months vs 9 months; b) 3 months vs 12 months; c) 3 months vs 24 months. LV1 loadings extracted from PLS-DA are presented in the bottom. 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.
Supplementary Fig. S3: Changes in amino acid metabolism. Rats age (in months) is presented on x axes and normalized peak areas (by probabilistic quotient) are presented on y axes (mean ± SEM) and used to estimate relative metabolite concentrations. Statistical significances of multiple comparisons were marked by the 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 FDR q<0.01 and lower-case significance at the q<0.001.
138 Supplementary Fig. S4: Changes in lipid metabolism. Rats age (in months) is presented on x axes and normalized peak areas (by probabilistic quotient) are presented on y axes (mean ± SEM) and used to estimate relative metabolite concentrations. Statistical significances of multiple comparisons were marked by the 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 significance at the FDR q<0.01 and lower-case significance at the q<0.001.
Supplementary Fig. S5: Changes in nucleotide metabolism. Rats age (in months) is presented on x axes and normalized peak areas (by probabilistic quotient) are presented on y axes (mean ± SEM) and used to estimate
139 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 significance at the FDR q<0.01 and lower-case significance at the q<0.001.
Supplementary Fig. S6: Changes in miscellaneous metabolites. Rats age (in months) is presented on x axes and normalized peak areas (by probabilistic quotient) are presented on y axes (mean ± SEM) and used to estimate relative metabolite concentrations. Statistical significances of multiple comparisons were marked by the 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 significance at the FDR q<0.01 and lower-case significance at the q<0.001.
140
Antibody Source KDa Dilution Vendor Catalog #
MCT4 Rabbit 43 1:1000 Santa Cruz Biotechnology,
Heidelberg, Germany sc-50329 LDH Rabbit 37–38 1:10000 Abcam, Cambridge, MA, USA ab52488 OXPHOS Mouse 20, 30, 39, 47
and 53 1:5000 MitoSciences MS604
Tubulin Mouse 51 1:5000 Sigma-Aldrich T9026
DNP Rabbit – 1:5000 Sigma–Aldrich, St. Louis, MO,
USA D9656
Nitro-
tyrosine Rabbit – 1:5000 Cell Signaling Technology, Leiden, Netherlands 9691
4-HNE Goat – 1:5000 Merck Millipore, Temecula,
USA AB5605
Mouse Goat – 1:5000 Sigma–Aldrich, St. Louis, MO,
USA A3562
Rabbit Goat – 1:5000 Sigma–Aldrich, St. Louis, MO,
USA A3687
Goat Rabbit – 1:5000 Sigma–Aldrich, St. Louis, MO,
USA A4187
MCT4: monocarboxylate transporter 4; LDH: lactate dehydrogenase; DNP: 2,4-dinitrophenylhydrazone; 4-HNE: 4- hydroxynonenal; OXPHOS − oxidative phosphorylation.
141 extracts (s: singlet, d: doublet, t: triplet, m: multiplet, dd: doublet of doublets).
Compound Assignment δ 1H ppm
(multiplicity)
1 acetate αCH3 1.91 (s)
2 adenine C8H ring
C2H ring
8.18 (s) 8.22 (s) 3 adenosinea
C1'H ribose C8H ring C2H ring
4.28 4.43 4.79 6.10 (d) 8.23 (s) 8.35 (s)
4 ADP/ATPa C'4H ribose
C3'H ribose C1'H ribose C2H ring NH ring
4.41 4.48 6.14 8.26 (s) 8.55 (s)
5 alanine βCH3
αCH
1.47 (d) 3.77 (t)
6 arginine γCH2
βCH2
δCH2
αCH
1.65 (m) 1.91 (m) 3.24 3.76
7 aspartate βCH
β'CH αCH
2.68 (dd) 2.81 (dd) 3.91
8 betaine CH3
αCH2
3.26 (s) 3.90 (s)
9 carnitine γCH2
αCH2
2.44 3.43
10 choline CH3
CH2 (N) CH2 (OH)
3.20 (s) 3.51 (m) 4.06 (m)
11 citrate α,γCH
α',γ'CH
2.50 (d) 2.65 (d)
12 CMP C2'H ribose
C1'H ribose C5H ring C6H ring
4.37 (m) 5.99 (d) 6.14 (d) 8.10 (d)
13 creatine CH3
CH2
3.03 (s) 3.92 (s)
14 creatinine CH3
CH2
3.04 (s) 4.05 (s)
15 cytidine 4.30
5.90 (d) 6.05 (d) 7.84 (d) 16 dimethylglycinea αCH
CH3
2.91 (s) 3.72 (s) 17 ethanolamine CH2 (NH2)
CH2 (OH)
3.14 (m) 3.82 (m)
142 (multiplicity)
18 formate CH 8.46 (s)
19 glutamate βCH
β'CH γCH2
αCH
2.05 2.13 2.35 3.73
20 glutamine βCH2
γCH2
αCH
2.14 2.45 3.77 21 glutathione disulfide βCH2 Glu
γCH2 Glu βCH2 Cys βCH2 Cys αCH Gly
CH2 Cys
2.17 (q) 2.55 (m) 2.97 (m) 3.30 (m) 3.78 (m) 4.75 (m)
22 glycerol C1H2
C3H2
C2H
3.56 (dd) 3.65 (dd) 3.78 (m) 23 glycerophosphocholine CH3
CH2 (N) αCH2
CH2 (O)
3.23 (s) 3.69 (m) 3.92 (m) 4.32 (m)
24 glycine CH2 3.56 (s)
25 3-hydroxybutyratea γCH3
αCH βCH
1.19 (d) 2.38 4.16 26 hypotaurine CH2 (NH2)
CH2 (S)
2.64 (t) 3.36 (t)
27 hypoxanthine C2H
C8H
8.19 (s) 8.21 (s) 28 IMP
C1'H ribose C8H ring C2H ring
4.03 4.37 4.51 6.14 (d) 8.26 (s) 8.59 (s)
29 isoleucine δCH3
β'CH3
γCH γ'CH βCH αCH
0.94 (t) 1.01 (d) 1.26 1.49 1.98 3.67
30 lactate βCH3
αCH 1.33 (d)
4.11 (q)
31 leucine δCH3, γ'CH3
γCH, βCH2
αCH
0.96 (d) 1.70 3.73
32 lysine γCH2
δCH2
βCH2
εCH2
αCH
1.44 1.72 1.91 3.01 3.76
143 (multiplicity)
33 myo-inositol C5H C1H, C3H C4H, C6H C2H
3.29 (t) 3.53 (dd) 3.61 (m) 4.06 (t) 34 nicotinamidea C5H ring
C4H ring C6H ring C2H ring
7.60 (m) 8.25 (m) 8.71 (dd) 8.94 (dd) 35 phenylalanine C2H, C6H ring
C4H ring C3H, C5H ring
7.32 (m) 7.38 (m) 7.42 (m) 36 phosphocholine CH3
CH2 (N) CH2 (O)
3.22 (s) 3.59 (m) 4.17 (m) 37 phosphoethanolamine CH2 (NH2)
CH2 (OH)
3.22 3.99 (m)
38 proline γCH2,
β'CH βCH δ´CH2
δCH2
αCH
2.00 2.08 2.33 3.34 3.43 4.13
39 succinate CH2 2.41 (s)
40 taurine CH2 (NH2) CH2 (S)
3.26 (t) 3.43 (t)
41 threonine γCH3
αCH βCH
1.33 (d) 3.58 4.24
42 trimethylaminea CH3 2.89 (s)
43 tyrosine C3H, C5H ring
C2H, C6H ring
6.88 (d) 7.18 (d)
44 uracil C5H ring
C6H ring
5.80 (d) 7.53 (d) 45 uridine
C2'H ribose C1'H ribose C5H ring C6H ring
4.13 4.22 4.35 5.89 (d) 5.91 (d) 7.87 (d)
46 valine γCH3
γ'CH3
βCH αCH
0.99 (d) 1.04 (d) 2.26 3.62
47 U1 8.53 (s)
144
Supplementary Material
Seminal plasma metabolites and aging: Impact on male reproductive potencial.
Susana Almeida, Ivana Jarak, Rui Carvalho; Soraia Pinto, Mario Sousa, Marco Alves, Pedro Oliveira.
Supplementary Table S1: Figure S1. Significance values and Pearson’s correlation coefficient values of identified NMR-derived metabolites in seminal plasma. r=Pearson’s correlation coefficient; P=Significance. Legend: U1- metabolite we could not identify; GlcNac - N-Acetylglucosamine; GPC - glycerophosphocholine; r=Pearson’s correlation coefficient; P=Significance. The relative quantification of NMR-derived metabolites was evaluated by computing Pearson correlation coefficients (r) assuming Gaussian distribution and a confidence interval of 95%. All P values < 0.05 were considered statistically significant.
145 Supplementary Table S2: Significance values and Pearson’s correlation coefficient values of NMR-derived metabolites in seminal plasma of the samples from males of the samples from males of the normozoospermic (N=35), oligozoospermic (N=11) and asthenozoospermic (N=8) groups according to the age of the individual.
Legend: U1- metabolite we could not identify; GlcNac - N-Acetylglucosamine; GPC - glycerophosphocholine;
r=Pearson’s correlation coefficient; P=Significance; *P<0,05. The relative quantification of NMR-derived metabolites was evaluated by computing Pearson correlation coefficients (r) assuming Gaussian distribution and a confidence interval of 95%. All P values < 0.05 were considered statistically significant.
146 Supplementary Table S3: Significance values and Pearson’s correlation coefficient values of NMR-derived metabolites in seminal plasma of the samples from males of the different group ages (G1 -group 1: 30 to 35 years;
G2 - group 2: 35 to 40 years; G3 - group 3: above 40 years) according to the age of the individual. Legend: U1- metabolite we could not identify; GlcNac - N-Acetylglucosamine; GPC - glycerophosphocholine; r=Pearson’s correlation coefficient; P=Significance; *P<0,05. The relative quantification of NMR-derived metabolites was evaluated by computing Pearson correlation coefficients (r) assuming Gaussian distribution and a confidence interval of 95%. All P values < 0.05 were considered statistically significant.