Testicular ACE regulates sperm metabolism and fertilization through the transcription factor PPARγ - PubMed (original) (raw)

. 2024 Jan;300(1):105486.

doi: 10.1016/j.jbc.2023.105486. Epub 2023 Nov 20.

Shabir A Bhat 1, DuoYao Cao 2, Suguru Saito 2, Ellen A Bernstein 2, Erika Nishi 2, Juliet D Medenilla 3, Erica T Wang 3, Jessica L Chan 3, Margareta D Pisarska 4, Warren G Tourtellotte 5, Jorge F Giani 6, Kenneth E Bernstein 6, Zakir Khan 7

Affiliations

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

Tomohiro Shibata et al. J Biol Chem. 2024 Jan.

Abstract

Testis angiotensin-converting enzyme (tACE) plays a critical role in male fertility, but the mechanism is unknown. By using ACE C-domain KO (CKO) mice which lack tACE activity, we found that ATP in CKO sperm was 9.4-fold lower than WT sperm. Similarly, an ACE inhibitor (ACEi) reduced ATP production in mouse sperm by 72%. Metabolic profiling showed that tACE inactivation severely affects oxidative metabolism with decreases in several Krebs cycle intermediates including citric acid, cis-aconitic acid, NAD, α-ketoglutaric acid, succinate, and L-malic acid. We found that sperms lacking tACE activity displayed lower levels of oxidative enzymes (CISY, ODO1, MDHM, QCR2, SDHA, FUMH, CPT2, and ATPA) leading to a decreased mitochondrial respiration rate. The reduced energy production in CKO sperms leads to defects in their physiological functions including motility, acrosine activity, and fertilization in vitro and in vivo. Male mice treated with ACEi show severe impairment in reproductive capacity when mated with female mice. In contrast, an angiotensin II receptor blocker (ARB) had no effect. CKO sperms express significantly less peroxisome proliferators-activated receptor gamma (PPARγ) transcription factor, and its blockade eliminates the functional differences between CKO and WT sperms, indicating PPARγ might mediate the effects of tACE on sperm metabolism. Finally, in a cohort of human volunteers, in vitro treatment with the ramipril or a PPARγ inhibitor reduced ATP production in human sperm and hence its motility and acrosine activity. These findings may have clinical significance since millions of people take ACEi daily, including men who are reproductively active.

Keywords: ATP; PPARγ; fertilization; male fertility; metabolism; sperm; testicular ACE (tACE).

Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

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Conflict of interest statement

Conflict of interest The authors declare that there is no conflict of interest regarding the publication of this article.

Figures

Figure 1

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 means ± SEM. ∗_p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. CKO, C-domain KO; tACE, testis angiotensin-converting enzyme.

Figure 2

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

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 1_A_ and 2_A_. 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, 4′,6-diamidino-2-phenylindole; NS, no significance.

Figure 4

Figure 4

The role PPARγ i n 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 to analyze group comparisons. ∗_p < 0.05, ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. CKO, C-domain KO; NS, no significance; PPARγ, peroxisome proliferators–activated receptor gamma; qRT-PCR, quantitative real time PCR; tACE, testis angiotensin-converting enzyme.

Figure 5

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 enzyme.

Figure 6

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 represents 10 μm. Right: the fertilization rate was calculated as the number of two cell embryos divided by the number of total oocytes. K, measurement of in vivo fertilization rate. Table shows pregnancy rates, embryos per female mice, and total number of embryos at E14.0 after artificial insemination. G and H, groups of mice were treated with 40 mg/l ramipril or 600 mg/l losartan in drinking water for one week before sperm isolation. For PPARγ blockade, isolated sperm were incubated with 10 mM GW9662 for 12 h A_–_H, data are presented as means ± SEM. A one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons, and data are presented as means ± SEM. NS, no significance. ∗∗p < 0.01 and ∗∗∗p < 0.001. CKO, C-domain KO; POP, prolyl oligopeptidase; PPARγ, peroxisome proliferators–activated receptor gamma; tACE, testis angiotensin-converting enzyme.

Figure 7

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.

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