Testicular ACE regulates sperm metabolism and fertilization through the transcription factor PPARγ

Figure 1. The effect of tACE on sperm metabolites.A, heatmap showing metabolites that are significantly changed (p-value < 0.01) among two groups: CKO versus WT and WT+Ram versus WT. Mean z-scores were created for each protein using GraphPad Prism version 7.04. The mean data for all metabolites are listed in Table S1. WT+Ram groups of mice were treated with 40 mg/l ramipril (Ram) for 1 week before sperm isolation. B, volcano plots showing the metabolites that are significantly changed in CKO versus WT, WT+Ram versus WT and CKO versus WT+Ram. The blue dots represent downregulated metabolites. The differentially expressed metabolites were sorted with the criteria of p-value < 0.01 and Fold Change > 5. C, differential cellular levels of ATP, ADP, AMP, and adenosine in WT, CKO, and WT+Ram sperm. A–C, metabolite array was performed using mass spectrometry (n = 5/group). D, biochemical analysis of ATP production in WT and CKO sperm. Indicated groups of mice were treated with either ramipril (40 mg/l) or losartan (600 mg/l) for a week before sperm isolation. A–D, a one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons, and data are presented as…

Figure 2. The effect of tACE on mitochondrial protein in sperm.A, heat map showing the level of mitochondrial proteins in WT and CKO sperm measured by the MitoPlex assay. Mean z-scores were created for each protein using GraphPad Prism version 7.04. Data are from the analysis of sperm (5 mice/group). The mean data for all proteins are listed in Table S2. B, volcano plot showing differential level of proteins between CKO and WT sperm. The blue dots represent downregulated mitochondrial proteins. The significantly different proteins are sorted according to the criteria of p-value < 0.05. C, measurement of selected mitochondrial proteins by Western blot analysis. Data are presented as means ± SEM (n = 10/group). ∗p < 0.05; ∗∗∗p <0.001 determined by two-tail student t test. D, KEGG pathway and GO analyses of mitochondrial proteins using the MitoPlex array. A significant difference between WT and CKO sperm is determined by the p-value less than 0.05. CKO, C-domain KO; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; tACE, testis angiotensin-converting enzyme.

Figure 3. The measurement of the Krebs cycle in CKO and WT sperm.A, measurement of intermediate metabolites and mitochondrial proteins of the Krebs cycle in sperm using metabolite and MitoPlex arrays. The Krebs cycle was analyzed using ingenuity pathway analysis. Blue shading represents decreased levels of metabolites (p = 0.05) or mitochondrial proteins (p = 0.01). Data for this analysis have been taken from the mass spectrometry analysis shown in Figures 1A and 2A. B, the total amount of protein per million sperm measured by the BCA assay. C and D the mitochondrial content/size of sperm is measured by staining them with MitoSOX dye (Mitotracker Red) and DAPI (blue). C, stained samples were analyzed by flow cytometry. Left: representative histograms. Right: graph showing mean fluorescent intensity (MFI). Each dot represents data from one mouse. D, samples were examined by microscopy (5 μm scale bar). A–D, two-sided unpaired Student t test was used to analyze comparisons, and one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. BCA, bicinchoninic acid; CKO, C-domain KO; DAPI,…

Figure 4. The role PPARγ in the metabolic effect of tACE in sperm.A, Western blot showing PPARγ level in WT and CKO sperm (left). Quantitative data (relative levels after β-actin-corrected) (n = 10/group). B, the measurement of the PPARγ mRNA level using qRT-PCR in CKO and WT sperm ± WT mice treated with ramipril (40 mg/l) for one week. C, heat map illustrating mass spectrometry metabolites array data obtained from WT and CKO sperm after 12 h treatment with 10 μM GW9662. The significantly changed metabolites were sorted according to the criteria of p-value < 0.05 and Fold Change >2 among the two groups. The mean data for all metabolites are shown in Table S3. D, measurement of ATP levels in sperm after 12 h treatment with or without GW9662 (10 μM; n = 12/group). E, measurement of ATP levels in sperm following 12 h treatment with or without Pioglitazone (10 μM; n = 6/group). F, Western blot showing the level of mitochondrial proteins in WT and CKO sperm after 12 h treatment with 10 μM GW9662 (n = 6/group). A–F, data are presented as means ± SEM. Two-sided unpaired Student t test was used to analyze comparisons, and one-way ANOVA with Bonferroni’s correction for multiple comparisons was used…

Figure 5. tACE induces mitochondrial respiration.A, oxygen consumption rates (OCR) of WT or CKO sperm measured with an Agilent MitoXpress oxygen consumption assay. The graph shows the trace of OCR in sperm under basal conditions and in response to mitochondrial effectors oligomycin, FCCP, and antimycin A/rotenone. B, the rates of ATP production from glycolysis (ATPGlyco.) and oxidative phosphorylation (ATPOxPhos.) in WT and CKO sperm. C, OCR of maximal respiration (n = 10/group). D, extracellular acidification rate (ECAR) of WT or CKO sperm. The data show the trace of ECAR in sperm under basal conditions and in response to glucose, oligomycin, and 2-deoxy-d-glucose (2DG). E and F, ECAR of glycolysis (E) and glycolytic capacity (F) (n = 12 per group). A–F, groups of samples were treated with 10 μM GW9662 for 12 h before analysis as indicated. Data are presented as means ± SEM. An one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons. NS, no significance. ∗∗p < 0.01 and ∗∗∗p < 0.001. CKO, C-domain KO; ECAR, extracellular acidification rate; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; tACE, testis angiotensin-converting…

Figure 6. The role of tACE in sperm physiological functions.A, the total number of sperm per cauda in WT and CKO mice. B, measurement of sperm length using image-J software. Each dot represents a sperm. C, measurement of sperm motility. Sperm motility video was shown in Video S1. D, measurement of sperm acrosin activity. C and D, groups of samples were treated for 12 h with 10 mM GW9662 as indicated (n = 12/group). E and F, measurement of sperm motility (E), and acrosin activity (F) ± treated for 12 h with either 10 μM ramipril or 100 μM losartan (n = 9/group). G–I, measurement of ATP production, motility, and acrosin activity. Groups of WT mice were treated with the drugs i.p. (1 dose/day) for 5 days before sperm isolation as follows: bradykinin 2 receptor (B2R) antagonist HOE-140 at 100 μg/kg/day, neurokinin 1 (NK1) receptor antagonist L-733060 at 20 mg/kg/day, and POP inhibitor KYP-2047 at 10 mg/kg/day (n = 6/group). The drugs were continued during the experiment (10 μM HOE-140, 10 μM L-733060 and 50 μM KYP-2047). J, In vitro fertilization rate of CKO and WT sperm. Left: representative image of embryos at different stages (Pronuclear, 2-cell, 4-cell, and Morulae). The scale bar…

Figure 7. The metabolic and physiologic effect tACE in human sperm. Human sperm were treated for 12 h with either 10 μM GW9662 or 10 μM ramipril or 100 μM losartan and then (A) production of ATP, (B) motility, and (C) acrosine activity were determined as described in the Experimental procedures. Untreated samples were used as a control. Sperm representative motility video is shown in Video S2. Data are presented as means ± SEM (n = 13/group). An one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. tACE, testis angiotensin-converting enzyme.