Mitochondrial defects caused by PARL deficiency lead to arrested spermatogenesis and ferroptosis
- Department of Pathobiology, Comparative Pathology Core, School of Veterinary Medicine, University of Pennsylvania, United States
- University of Angers, France
- Unité Mixte de Recherche (UMR) MITOVASC, Centre National de la Recherche Scientifique (CNRS) 6015, Institut National de la Santé et de la Recherche Médicale (INSERM) U1083, University of Angers, France
- Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide-Consejo Superior de Investigaciones Científicas-Junta de Andalucía, Spain
- CIBERER, Instituto de Salud Carlos III, Spain
- Neuromuscular Reference Center, Department of Neurology, CHU Angers, France
Figures
Figure 1 with 2 supplements
Severe testis atrophy in Parl-/- mice is caused by arrested spermatogenesis.
(A) Reduced testicular size and weight in 5-week-old Parl-/- mice (n = 5) compared to WT littermates (n = 6; unpaired two-tailed t-test, p-value<0.0001). The reduction in testicular weight is not …
Figure 1—figure supplement 1
Quantitative morphometry, cell composition, and meiotic stage evaluation in 5-week-old WT and Parl-/- seminiferous tubules.
(A) Tubule diameters (n = 3 mice for each genotype; 8–12 tubules considered per mouse; p=0.0002). (B) Number of total cells/tubule (n = 3 mice for each genotype; 8–12 tubules considered per mouse;. …
Figure 1—figure supplement 2
Nestin expression in Leydig cells.
Immunohistochemistry for GFP identifies diffuse signal in the Leydig cell population (arrows) of reporter mice with transgenic GFP expression under the Nes promoter (n = 3). Scale bar, 50 µm. …
Figure 2 with 1 supplement
Impaired spermatogenesis in Parl-/- testis is associated with early mitochondrial morphological abnormalities and progressive degeneration of arrested spermatocytes.
(A) Toluidine blue-stained semithin sections of testis from 5-week-old WT and Parl-/- mice. Seminiferous tubules from Parl-/- mice show extensive degenerative changes in arrested spermatocytes …
Figure 2—figure supplement 1
Ultrastructural abnormalities of mitochondria and other cell compartments are restricted to arrested spermatocytes and absent in other testis cell types.
(A) Degenerating/dying spermatocytes from 5-week-old Parl-/- mice are mainly observed across the adluminal compartment of the seminiferous tubule (left panel, asterisks). At higher magnification …
Figure 3 with 1 supplement
Mice with genetic manipulation of the PARL substrates PINK1, PGAM5, and TTC19 do not reproduce or modify Parl-/- testis phenotype.
(A) Immunoblots of testis mitochondria from 6-week-old WT and Parl−/− mice with antibodies for the established PARL substrates PINK1, PGAM5, TTC19, DIABLO, STARD7, and CLPB. Severe accumulation of …
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Figure 3—source data 1
Original images for Figure 3A.
- https://cdn.elifesciences.org/articles/84710/elife-84710-fig3-data1-v2.pdf
Figure 3—figure supplement 1
Similar to what has been described in Parl-/- mice, immunohistochemistry for AIF-1 confirms spermatogenesis arrest with complete absence of spermatids in Parl-/-/Pink1-/-, Parl-/-/Pgam5-/-, and Parl-/-/Pink1-/-/Pgam5-/- mice.
On the contrary, as observed in WT mice, normal seminiferous tubules in Pink1-/-, Pgam5-/-, Ttc19-/- and Pink1-/-/Pgam5-/- mice are populated by AIF-1-positive spermatids at different levels of …
Figure 4 with 1 supplement
Severe mitochondrial electron transfer defects in Parl-/- testis mitochondria.
(A) Immunoblots of testis lysates from 6-week-old WT and Parl−/− mice with antibodies for PARL, ATPB, TOMM20, and ACTB (n = 3 for each genotype). ACTB is the loading control. (B) Quantification of …
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Figure 4—source data 1
Original images for Figure 4A.
- https://cdn.elifesciences.org/articles/84710/elife-84710-fig4-data1-v2.pdf
Figure 4—figure supplement 1
Unaltered TFAM expression in Parl-/- spermatocytes.
Normalized quantification of TFAM immunofluorescence in SCP1-positive primary spermatocytes does not reveal significant expression differences between 5-week-old WT and Parl-/- mice (n = 3 mice for …
Figure 5
Severe loss of COX4 associated with increased expression of glucose intracellular transporter in Parl-/- spermatocytes.
(A) Quantitative immunofluorescence shows decreased expression of COX4 in SCP-1-positive spermatocytes from 5-week-old Parl-/- mice compared to WT littermates (n = 3 for each genotype, 500–1000 …
Figure 6 with 1 supplement
Severe alteration in coenzyme Q (CoQ) biosynthesis and redox state in Parl-/- testis.
(A) Concentration (left) and CoQ red/ox ratio (right) of total CoQ (Q9 + Q10) measured by HPLC in the testes of 5-week-old WT and Parl-/- mice (n = 5 for each genotype). Total CoQ levels are …
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Figure 6—source data 1
Original images for Figure 6B.
- https://cdn.elifesciences.org/articles/84710/elife-84710-fig6-data1-v2.pdf
Figure 6—figure supplement 1
Severe loss of COQ4 in Parl-/- spermatocytes.
Normalized quantification of COQ4 immunofluorescence in SCP1-positive primary spermatocytes shows significantly higher levels of expression in 5-week-old WT compared to Parl-/- mice (n = 3 mice for …
Figure 7 with 3 supplements
Massive ferroptosis activation in Parl-/- arrested spermatocytes.
(A) Immunoblot of total testis lysates obtained from 5-week-old WT and Parl-/- mice using antibodies for GPX4, TOMM20, and ACTB (n = 3 for each genotype). ACTB is the loading control. GPX4 …
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Figure 7—source data 1
Original images for Figure 7A.
- https://cdn.elifesciences.org/articles/84710/elife-84710-fig7-data1-v2.pdf
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Figure 7—source data 2
Original images for Figure 7B.
- https://cdn.elifesciences.org/articles/84710/elife-84710-fig7-data2-v2.pdf
Figure 7—figure supplement 1
Unremarkable levels of apoptosis activation in degenerated Parl-/- testis.
Cleaved-caspase-3 immunohistochemistry on testis from 5-week-old Parl-/- mice and WT littermates (n = 3 for each genotype). The maturation defect and degenerative changes of PARL-deficient …
Figure 7—figure supplement 2
Lack of effect of PARL proteolytic activity on GPX4 expression in vitro and testis-specific induction of ferroptosis in PARL-deficient mice.
(A) Mitochondria isolated from brain, liver, and testis of 6-week-old WT and Parl-/- mice (n = 3 for each genotype) were immunoblotted with antibodies for GPX4 and HSP60. HSP60 is the loading …
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Figure 7—figure supplement 2—source data 1
Original images for Figure 7—figure supplement 2A.
- https://cdn.elifesciences.org/articles/84710/elife-84710-fig7-figsupp2-data1-v2.pdf
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Figure 7—figure supplement 2—source data 2
Original images for Figure 7—figure supplement 2B.
- https://cdn.elifesciences.org/articles/84710/elife-84710-fig7-figsupp2-data2-v2.pdf
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Figure 7—figure supplement 2—source data 3
Original images for Figure 7—figure supplement 2C.
- https://cdn.elifesciences.org/articles/84710/elife-84710-fig7-figsupp2-data3-v2.pdf
Figure 7—figure supplement 3
Increased activation of p53 in PARL-deficient spermatocytes undergoing ferroptosis.
(A) Immunoblot analysis of testis total lysates from 7-week-old WT and Parl-/- mice (n = 3 for each genotype) with antibodies for p53 and ACTB. ACTB is the loading control. Parl-/- testes shows …
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Figure 7—figure supplement 3—source data 1
Original images for Figure 7—figure supplement 3A.
- https://cdn.elifesciences.org/articles/84710/elife-84710-fig7-figsupp3-data1-v2.pdf
Figure 8 with 2 supplements
Hallmarks of ferroptosis in Parl-/- primary spermatocytes.
(A) Quantitative immunofluorescence shows severely reduced GPX4 expression in SCP-1-positive primary spermatocytes from 5-week-old Parl-/- mice compared to WT littermates (n = 3 mice for each …
Figure 8—figure supplement 1
Unchanged GPX4 expression in Parl-/- Sertoli cells.
Quantitative immunofluorescence in WT and Parl-/- tubules shows very low expression of GPX4 in Vimentin-positive Sertoli cells of both genotypes. Normalized quantification of GPX4 immunofluorescence …
Figure 8—figure supplement 2
Increased transferrin receptor expression in Parl-/- spermatocytes undergoing ferroptosis.
Normalized quantification of TfR1 immunofluorescence in SCP1-positive spermatocytes shows significantly higher levels of expression in 5-week-old Parl-/- mice compared to WT littermates (n = 4 mice …
Figure 9
Cartoon illustrating the identified mechanisms underlying the spermatogenesis defect of Parl-/- mice and the induction of spermatocyte ferroptosis.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | B6.129P2(Cg)-Parltm1.1Bdes/Ieg | PMID:16839884 | RRID:IMSR_EM:02075 | |
| Genetic reagent (M. musculus) | B6.129P2-Parltm1Bdes/Ieg | PMID:16839884 | RRID:IMSR_EM:02076 | |
| Genetic reagent (M. musculus) | Tg(Nes-cre)1Kln; Parltm1Bdes/Parltm1Bdes | PMID:10471508 | RRID:MGI:6280694 | |
| Genetic reagent (M. musculus) | Parltm1.1Bdes/Parltm1.1Bdes; Pgam5tm1d(EUCOMM)Wtsi/ Pgam5tm1d(EUCOMM)Wtsi | PMID:30578322 | RRID:MGI:6280688 | |
| Genetic reagent (M. musculus) | Parltm1.1Bdes/Parltm1.1Bdes; Pink1tm1.1Wrst/Pink1tm1.1Wrst | PMID:20049710 | RRID:MGI:6280687 | |
| Genetic reagent (M. musculus) | C57BL/6J-Ttc19em1Bds | PMID:30578322 | RRID:MGI:6280684 | |
| Cell line (M. musculus) | WT, Parl-/-, Parl-/-+ParlWT, Parl-/-+ParlS275A | PMID:30578322 | RRID:MGI:2159769 | |
| Antibody | Anti-SCP-1 (rabbit monoclonal) | Abcam | Cat#: ab175191 | IHC 1:200, IF 1:500 |
| Antibody | Anti-AIF1 (rabbit polyclonal) | Wako | Cat#: 019-19741; RRID:AB_839504 | IHC 1:200 |
| Antibody | Anti-COQ4 (rabbit polyclonal) | ProteinTech | Cat#: 16654-1AP; RRID:AB_2878296 | IF 1:800, IHC 1:200, WB 1:1000 |
| Antibody | Anti-GPX4 (rabbit polyclonal) | Sigma | Cat#: HPA047224; RRID:AB_2679990 | IHC 1:100, IF 1:500 |
| Antibody | Anti-HNE (rabbit polyclonal) | Alpha Diagnostic International | Cat#: HNE11-S; RRID:AB_2629282 | IF 1:10,000, IHC 1:3000 |
| Antibody | Anti-TFR1 (rabbit monoclonal) | Abcam | Cat#: ab214039; RRID:AB_2904534 | IF 1:3000, IHC 1:1000 |
| Antibody | Anti-p53 (rabbit polyclonal) | Leica/Novocastra | Cat#: NCL-L-p53-CM5p; RRID:AB_2895247 | IHC 1:300 |
| Antibody | Anti-p53 (mouse monoclonal) | Cell Signaling Technology | (1C12) Mouse mAb #2524; RRID:AB_331743 | WB 1:1000 |
| Antibody | Anti-Wilm’s tumor 1 (rabbit monoclonal) | Abcam | Cat#: ab89901; RRID:AB_2043201 | IF 1:1500 |
| Antibody | Anti-cKit (rabbit polyclonal) | Agilent/DAKO | Cat#: A4502; RRID:AB_2335702 | IHC 1:50 |
| Antibody | Anti-γH2AX (rabbit monoclonal) | Cell Signaling Technology | Cat#: 2577; RRID:AB_2118010 | IHC 1:1000 |
| Antibody | Anti-COX4 (rabbit polyclonal) | ProteinTech | Cat#:11242–1-AP; RRID:AB_2085278 | IF1:3000 |
| Antibody | Anti-GLUT1 (rabbit monoclonal) | Cell Signaling Technology | Cat#:73015 | IHC 1:600 |
| Antibody | Anti-TOMM20 (rabbit polyclonal) | ProteinTech | Cat#: 73015; RRID:AB_2207530 | IF 1:4000 |
| Antibody | Anti-TFAM (rabbit polyclonal) | Abcam | Cat#: ab307302 | IF 1:3000 |
| Antibody | Anti-PARL (rabbit polyclonal) | PMID:16839884 | Cat#: N/A | WB 1/1000 |
| Antibody | Anti-actin (mouse monoclonal) | Sigma | Cat#: A5441; RRID:AB_476744 | WB 1:200,000 |
| Antibody | Anti-HSP60 (mouse monoclonal) | BD Biosciences | Cat#: 611562; RRID:AB_399008 | WB 1:50,000 |
| Antibody | Anti-ATP5B (mouse monoclonal) | Abcam | Cat#: ab14730; RRID:AB_301438 | WB 1:50,000 |
| Antibody | Anti-TOMM20 (rabbit polyclonal) | Santa Cruz | Cat#: sc-11415; RRID:AB_2207533 | WB 1:5000 |
| Antibody | Anti-PINK1 (rabbit polyclonal) | Cayman | Cat#: 10006283; RRID:AB_10098326 | WB 1:500 |
| Antibody | Anti-PGAM5 (rabbit polyclonal) | Sigma | Cat#: HPA036979; RRID:AB_10960559 | WB 1:250 |
| Antibody | Anti-TTC19 (rabbit polyclonal) | Sigma | Cat#: HPA052380; RRID:AB_2681806 | WB 1:2000 |
| Antibody | Anti-CLPB (rabbit polyclonal) | ProteinTech | Cat#: 15743-1-AP; RRID:AB_2847900 | WB 1:1000 |
| Antibody | Anti-STARD7 (rabbit polyclonal) | ProteinTech | Cat#: 15689-1-AP; RRID:AB_2197820 | WB 1:2000 |
| Antibody | Anti-DIABLO (rabbit polyclonal) | Cell Signaling Technology | Cat#: 15108; RRID:AB_2798711 | WB 1:1000 |
| Antibody | Anti-GPX4 (mouse monoclonal) | R&D Systems | Cat#: MAB5457; RRID:AB_2232542 | WB 1:1000 |
| Antibody | Anti-GPX4 (mouse monoclonal) | Santa Cruz | Cat#: sc-166570; RRID:AB_2112427 | WB 1:1000 |
| Antibody | Anti-HNE (mouse monoclonal) | R&D Systems | Cat#: 198960; RRID:AB_664165 | WB 1:500 |
| Antibody | Anti-citrate synthase (mouse monoclonal) | Abcam | Cat#: ab96600; RRID:AB_10678258 | WB 1:1000 |
| Software, algorithm | GraphPad Prism software | GraphPad Prism (https://graphpad.com) | RRID:SCR_015807 | |
| Software | ImageJ software | ImageJ (http://imagej.nih.gov/ij/) | RRID:SCR_003070 |
