3.1 Metabolomic profiles showed sex differences and dramatic changes during sexual maturation
LC-HRMS analyses revealed 6554 significant features of metabolic fingerprints in ESI- mode and 7105 features in ESI + mode. PCA analyses in both negative and positive modes showed significant differences between PSM vs. SM, POF vs. OF, OF vs. SM, and POF vs. SM (Figs. 1 and S2; Table S2). The goodness of fit for these models resulted in R2X = 0.69 for male sexual maturation (PSM vs. SM), R2X = 0.715 for female sexual maturation (POF vs. OF), and sex differences in mature adults (OF vs. SM: R2X = 0.751) and immature adults (POF vs. PSM: R2X = 0.917). In each comparison, the goodness of prediction (Q2) was greater than 0.5, indicating that the model was acceptable for metabolomic analysis (Table S2). PLS-DA plots also confirmed significant differences between PSM vs. SM, POF vs. OF, OF vs. SM, and POF vs. SM (Figs. S3 and S4). The goodness of fit for each comparison (PSM vs. SM; POF vs. OF; POF vs. PSM; OF vs. SM) resulted in R2X > 0.5, R2Y > 0.8, and Q2 > 0.5 (Table S2).
Discriminant analyses revealed dramatic upregulations in sea lamprey-specific bile acids in the comparisons between sexes and male sexual maturation (Table 1). For example, PSM contained 244-fold (↑) PZS compared to POF, SM contained 155-fold (↑) PZ and 53-fold (↑) ACA compared to OF, and SM contained 314-fold (↑) ACA and 201-fold (↑) PZ compared to PSM. Other bile acids, including cholic acid, lithocholic acid and chenodeoxycholic acid-3-sulfate were also upregulated hundreds of folds in SM compared to PSM or OF. Changes in nucleotide and amino acid metabolisms were apparent in all comparisons. SM showed increases in adenine (↑ 77-fold) but decreases in creatine (↓ 81-fold) compared to OF. On the other hand, OF showed downregulations of fatty acid metabolism compared to POF (11-dehydrothromboxane: ↓ 4-fold; palmitic acid: ↓ 2-fold).
Table 1
List of metabolic pathways and the most discriminant metabolites in the comparison of different sea lamprey groups between sexes and maturation states
Group | Metabolism pathway | Metabolite | Regulation | Fold Change | p-value | q-value |
PSM vs SM | Secondary bile acid biosynthesis | Lithocholic acid | ↑ | 152.9 | 9.66E-11 | 2.55E-08 |
- | Petromyzonol sulfate | ↑ | 212.3 | 1.38E-10 | 3.37E-08 |
- | Chenodeoxycholic acid 3-sulfate | ↑ | 264.2 | 1.83E-09 | 3.68E-07 |
- | Petromyzonol | ↑ | 201.0 | 4.45E-09 | 6.49E-07 |
Purine metabolism | Adenine | ↑ | 46.8 | 2.21E-07 | 4.95E-05 |
Amino acid metabolism | 3-Hydroxypropenoate | ↓ | 8.4 | 1.63E-06 | 2.11E-04 |
Primary and Secondary bile acid biosynthesis | Cholic acid | ↑ | 134.0 | 2.44E-04 | 2.72E-03 |
Secondary bile acid biosynthesis | 5α-Cholic acid | ↑ | 313.9 | 1.07E-04 | 4.26E-03 |
- | D-Xylulosonic acid | ↑ | 11.2 | 1.64E-04 | 6.07E-03 |
Tyrosine metabolism | Tyrosine | ↑ | 1.9 | 2.61E-02 | 7.76E-02 |
Amino acid metabolism | Arginine | ↓ | 2.2 | 5.64E-02 | 1.08E-01 |
Lysine degradation | Lysopine | ↓ | 1.5 | 6.98E-02 | 1.18E-01 |
Amino acid metabolism - Glutathione metabolism | Ornithine | ↑ | 1.5 | 1.04E-01 | 1.44E-01 |
Arginine and proline metabolism | Octopine | ↓ | 1.2 | 4.52E-01 | 3.02E-01 |
Heme catabolism (bile pigment) | Biliverdin | ↑ | 1.4 | 5.14E-01 | 3.25E-01 |
Purine metabolism | Hypoxanthine | ↑ | 1.2 | 7.15E-01 | 3.97E-01 |
Histidine metabolism | Formimino-L-glutamic acid | ↑ | 1.2 | 7.29E-01 | 4.01E-01 |
Pyrimidine metabolism | Cytosine | ↑ | 37.2 | 4.70E-05 | 1.10E-03 |
Amino acid metabolism | Glutamic acid | ↑ | 1.7 | 4.50E-03 | 2.60E-02 |
Histidine metabolism | Methylhistidine | ↑ | 2.8 | 1.00E-02 | 5.10E-02 |
Amino acid metabolism | Glutamine | ↑ | 4.4 | 7.40E-05 | 3.80E-03 |
Amino acid metabolism | Glutamic acid | ↑ | 1.5 | 1.60E-03 | 3.30E-02 |
- | N3,N4-Dimethyl-L-arginine | ↓ | 7.1 | 2.70E-03 | 1.90E-02 |
TCA cycle | Citric acid | ↓ | 2.0 | 6.20E-04 | 7.10E-03 |
Biosynthesis of ansamycins | AminoDHQ | ↓ | 2.1 | 3.03E-03 | 2.00E-02 |
Amino acid metabolism | 2-Aminoadipic acid | ↑ | 2.1 | 2.10E-03 | 1.70E-02 |
Phenylalanine metabolism | 2-Oxo-4-pentenoic acid | ↓ | 2.9 | 2.30E-03 | 4.30E-02 |
POF vs OF | Lysine biosynthesis | N-acetyl-LL-2,6-diaminopimelic acid | ↑ | 12.2 | 7.22E-07 | 9.39E-06 |
- | N,N-dimethylarginine | ↓ | 6.4 | 7.83E-07 | 1.01E-05 |
- | 7a,12a-Dihydroxy-3-oxo-4-cholenoic acid | ↓ | 11.0 | 4.13E-05 | 1.64E-04 |
Cysteine and methionine metabolism | Methionine | ↑ | 4.0 | 1.26E-03 | 2.34E-03 |
- | Tryptophan | ↑ | 3.0 | 2.21E-03 | 3.76E-03 |
- | D-Xylulosonic acid | ↑ | 6.6 | 3.22E-04 | 6.17E-03 |
- | N-(3-Carboxypropyl)-L-glutamine | ↓ | 19.5 | 1.96E-03 | 1.46E-02 |
- | Prostaglandin C1 | ↓ | 2.8 | 2.60E-03 | 1.79E-02 |
Arachidonic acid metabolism | 11-Dehydrothromboxane B2 | ↓ | 4.4 | 5.21E-03 | 2.06E-02 |
Valine, leucine, and isoleucine biosynthesis | Acetyl lactic acid | ↑ | 4.3 | 5.40E-03 | 2.10E-02 |
Amino acid metabolism | Creatine | ↓ | 4.9 | 2.60E-02 | 1.2E-01 |
Arginine and proline metabolism | Creatinine | ↓ | 15.2 | 2.20E-02 | 1.1E-02 |
| Alanine, aspartate, and glutamate metabolism | Aspartic acid | ↓ | 9.5 | 1.40E-02 | 8.20E-02 |
| - | Glutamyl-glutamic acid | ↓ | 2.2 | 7.90E-02 | 2.20E-01 |
| Pyrimidine metabolism | 3-Ureidopropionic acid | ↓ | 2.2 | 1.10E-01 | 2.70E-01 |
| Phenylalanine, tyrosine, and tryptophan biosynthesis | Pretyrosine | ↓ | 13.0 | 1.40E-03 | 2.50E-02 |
| Fatty acid metabolism | Palmitic acid | ↓ | 1.7 | 3.60E-02 | 1.90E-01 |
| Pyrimidine metabolism | Methylmalonic acid | ↓ | 2.1 | 2.30E-01 | 4.40E-01 |
| Pentose phosphate pathway | (2R)-2,3-Dihydroxypropanoic acid | ↓ | 1.8 | 2.54E-02 | 1.60E-01 |
| Fatty acid degradation | Glutaric acid | ↓ | 1.8 | 2.40E-02 | 1.60E-01 |
| - | Petromyzonol | ↑ | 155.1 | 3.54E-09 | 3.16E-07 |
| Purine metabolism | Adenine | ↑ | 76.7 | 2.04E-07 | 2.08E-05 |
| Riboflavin metabolism | Riboflavin | ↑ | 4.9 | 8.20E-06 | 1.63E-04 |
| TCA cycle | Isocitric acid | ↓ | 2.1 | 6.50E-04 | 4.89E-03 |
OF vs SM | Bile secretion | Carnitine | ↓ | 20.9 | 2.12E-03 | 6.67E-03 |
| Primary and Secondary bile acid biosynthesis | Cholic acid | ↑ | 93.7 | 4.27E-03 | 1.09E-02 |
| Pyrimidine metabolism | Uridine | ↓ | 4.6 | 5.96E-03 | 1.57E-02 |
| Glycine, serine, and threonine metabolism | Creatine | ↓ | 81.0 | 9.32E-03 | 1.82E-02 |
| Secondary bile acid biosynthesis | 5a-Cholic acid | ↑ | 53.4 | 9.86E-03 | 2.25E-02 |
| Biosynthesis of unsaturated fatty acids | Behenic acid | ↓ | 1.4 | 6.50E-02 | 6.94E-02 |
| - | N-glycoloyl-beta-D-glucosamine | ↑ | 81.0 | 7.23E-02 | 7.54E-02 |
| Biosynthesis of amino acids | N-acetyl-LL-2,6-diaminopimelic acid | ↓ | 117.2 | 3.7E-15 | 1.4E-12 |
| | Norleucine | ↑ | 1.7 | 1.8E-03 | 1.5E-02 |
| Biosynthesis of amino acids | Tryptophan | ↑ | 2.8 | 6.5E-04 | 7.5E-03 |
POF vs PSM | Tyrosine metabolism | Tyramine | ↑ | 1.7 | 3.2E-02 | 1.0E-01 |
| | Petromyzonol sulfate | ↑ | 244.4 | 1.1E-02 | 5.1E-02 |
| Riboflavin metabolism | Riboflavin | ↑ | 4.9 | 9.2E-07 | 5.2E-05 |
| Purine metabolism | Adenine | ↑ | 10.2 | 8.7E-02 | 2.6E-01 |
| | 3,6-Dideoxy-L-galactose | ↑ | 2.5 | 8.8E-03 | 6.2E-02 |
Down regulation (↓): SM < PSM; OF < POF; SM < OF; PSM < POF. Up regulation (↑): SM > PSM; OF > POF; SM > OF; PSM > POF. –: Not identified in KEGG. OF: ovulatory females; POF: preovulatory females; PSM: prespermiating males; SM: spermiating males. |
3.2 Males reduced sugar and energy metabolisms and altered amino acid metabolisms in favor of bile acid biosynthesis during sexual maturation
Pathway analyses revealed that the most prominent changes between SM and PSM were the metabolites in primary and secondary bile acid biosynthesis (Table 1); e.g., lithocholic acid (↑ 153-fold) and cholic acid (↑ 134-fold). Interestingly, lamprey-specific bile acids were upregulated at least 2 times more than those common bile acids (Table 1); e.g., ACA (5α-cholic acid ↑ 314-fold), PZ (↑ 201-fold), and PZS (↑ 212-fold). Several amino acid metabolic pathways were affected during male sexual maturation (impact > 0.15, Table S5); e.g., tyrosine (↑ 2-fold), arginine (↓ 2-fold), glutamate (↑ 2-fold), and glutamine (↑ 4-fold). Significantly changed metabolites are listed in Table 1, and their associated primary metabolic pathways are shown in Fig. S5. In general, metabolic pathway maps indicated that SM reduced TCA cycle-related activities and carbohydrate and energy metabolisms, and at the same time increased biosynthesis of bile acids, cofactors, and vitamins (Fig. S5).
On the contrary, most metabolic pathways were downregulated in OF compared to POF, including lipid metabolism (e.g., palmitate ↓ 2-fold; Tables 1 and S5, and Fig. S6). Notably, two eicosanoid metabolites were downregulated (Table 1); i.e., prostaglandin C1 (↓ 3-fold) and 11-dehydrothromboxane B2 (↓ 4-fold). Other notable changes were amino acid metabolisms (Table 1); e.g., methionine (↑ 4-fold), tryptophan (↑ 3-fold), creatine (↑ 5-fold), and pretyrosine (↓ 13-fold). Interestingly, D-aspartate but not L-aspartate were downregulated (↓ 10-fold, Table 1).
Sex differences are most prominent in the pathways involved in biosynthesis of bile acids, cofactors, and vitamins (Tables 1 and S5, and Fig. S7: OF vs. SM and Fig. S8: POF vs. PSM), as males contained more bile acids than females (Tables S3 and S4); e.g., PZS: ↑ 244-fold in PSM vs. POF; PZ: ↑ 155-fold in SM vs. OF (Tables 1 and S5).
Since bile acid biosynthetic pathways showed the most dramatic changes between sexes and during male sexual maturation, we used targeted analyses to confirm the results from untargeted analyses. Indeed, SM plasma contained the highest levels of most bile acids, except TCDCA (Tables S3 and S4). PZS was the most abundant bile acid in SM and PSM, with concentrations of 19 µg/ml and 2 µg/ml, respectively (Table S3). Upregulations of bile acids were seen during sexual maturation (PSM vs. SM and POF vs. OF, Tables S3 and S4), and the most dramatic changes were observed between sexes (males > females; OF vs. SM and POF vs. PSM; Tables S3 and S4), consistent with the untargeted analysis results. A putative sea lamprey bile acid biosynthetic pathway is shown in Fig. 2. Higher levels of bile acids in SM vs. OF (Fig. 2 and Tables S3 and S4) were exemplified by PZS (↑ 388-fold), ACA (↑ 138-fold), and 3kACA (↑ 83-fold). For pre-spawning adults, higher levels of bile acids in PSM vs POF (Tables S3 and S4) were found in PZS (↑ 388-fold), 3kPZS (↑ 255-fold), and PAMS-24 (↑ 69-fold). ACA was probably important for sea lamprey sexual maturation since it was the most upregulated metabolite in PSM vs. SM (↑ 87-fold; Table S4) and POF vs. OF (↑ 15-fold; Table S4).