Mitochondrial defects caused by PARL deficiency lead to arrested spermatogenesis and ferroptosis
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This manuscript reports an important finding that spermatogenic defects in Parl KO mice, a genetic model for Leigh syndrome, may result from mitochondrial defects leading to ferroptosis. The finding, if confirmed, would be of great significance because male germ cell ferroptosis has not been well characterized. However, the criteria for determining male germ cell ferroptosis were vague, and the supporting data were inadequate.
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Abstract
Impaired spermatogenesis and male infertility are common manifestations associated with mitochondrial diseases, yet the underlying mechanisms linking these conditions remain elusive. In this study, we demonstrate that mice deficient for the mitochondrial intra-membrane rhomboid protease PARL, a recently reported model of the mitochondrial encephalopathy Leigh syndrome, develop early testicular atrophy caused by a complete arrest of spermatogenesis during meiotic prophase I, followed by degeneration and death of arrested spermatocytes. This process is independent of neurodegeneration. Interestingly, genetic modifications of PINK1, PGAM5, and TTC19 – three major substrates of PARL with important roles in mitochondrial homeostasis – fail to reproduce or modify this severe phenotype, indicating that the spermatogenic arrest arises from distinct molecular pathways. We further observed severe abnormalities in mitochondrial ultrastructure in PARL-deficient spermatocytes, along with prominent electron transfer chain defects, disrupted coenzyme Q (CoQ) biosynthesis, and metabolic rewiring. These mitochondrial defects are associated with a germ cell-specific decrease in GPX4 expression leading arrested spermatocytes to ferroptosis – a regulated cell death modality characterized by uncontrolled lipid peroxidation. Our results suggest that mitochondrial defects induced by PARL depletion act as an initiating trigger for ferroptosis in primary spermatocytes through simultaneous effects on GPX4 and CoQ – two major inhibitors of ferroptosis. These findings shed new light on the potential role of ferroptosis in the pathogenesis of mitochondrial diseases and male infertility warranting further investigation.
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Author Response
Reviewer 1 (Public Review):
- In Figure 2, electron microscopy images represent n=1 cell, making it hard to know how generalizable the mitochondrial phenotypes are. It would be useful to see a quantitative summary of a larger dataset indicating how frequently the mitochondrial defects are seen.
As requested, we performed quantitative analysis of mitochondrial ultrastructure in a larger dataset (n=163 analyzed in WT and n=206 in the KO) confirming that this finding is very consistent. This additional quantitative analysis that we included in the revised manuscript confirms a very significant and diffuse alteration of mitochondrial ultrastructure in Parl-/- vs WT spermatocytes (p=0.0002).
- In Figure 3, representative images are shown for a single field from n=1 animal. It is hard to decisively conclude that the phenotype …
Author Response
Reviewer 1 (Public Review):
- In Figure 2, electron microscopy images represent n=1 cell, making it hard to know how generalizable the mitochondrial phenotypes are. It would be useful to see a quantitative summary of a larger dataset indicating how frequently the mitochondrial defects are seen.
As requested, we performed quantitative analysis of mitochondrial ultrastructure in a larger dataset (n=163 analyzed in WT and n=206 in the KO) confirming that this finding is very consistent. This additional quantitative analysis that we included in the revised manuscript confirms a very significant and diffuse alteration of mitochondrial ultrastructure in Parl-/- vs WT spermatocytes (p=0.0002).
- In Figure 3, representative images are shown for a single field from n=1 animal. It is hard to decisively conclude that the phenotype of Pink1-/-;Pgam5-/- and Ttc19-/- testes is completely normal based on this limited data. There may be other tubules outside the field of view that are abnormal, or more subtle changes in cell ratios. This conclusion would be significantly strengthened by cell counting (e.g. # round spermatids per Sertoli cell per tubule and # spermatocytes per Sertoli cell per tubule) or other quantitation. Likewise, the similarities in phenotype between Parl-/-, Parl-/-;Pink2-/-, and Parl-/-;Pgam5-/- should be more thoroughly documented. At least some additional images should be shown.
The goal of figure 3 is to indicate that WT, Pink1-/-;Pgam5-/- and Ttc19-/- have no gross morphological abnormality and have preserved sperm production in sharp contrast with Parl/-, Parl-/-;Pink1-/-, and Parl-/-;Pgam5-/- and the TKO that show total lack of sperm in the tubular lumen, indicating that the loss of Parl alone or in combination drives this phenotype. To strengthen these conclusions we performed additional work. We stained testis sections from all strains with an antibody for AIF-1, a marker of post-mitotic spermatids/spermatozoa included in Fig3-figure supplement 1. This additional experiment clearly confirms that production of differentiated germ cells occurs only in WT, Pink1-/-;Pgam5-/- and Ttc19-/-, but not in Parl-/, Parl-/-;Pink1-/-, and Parl-/-;Pgam5-/-. These results are consistent with the reproductive capacity of these mouse lines (the first group is fertile, the second is infertile). We acknowledge we cannot rule out minimal subclinical differences in reproductive fitness between the fertile mouse groups, but this is beyond the goal of our study.
- In Figure 4, it looks like there is a significant decrease in CIV-driven respiration in Parl knockouts, but the text describes this as "did not significantly enhance" - that is, the absence of an increase. This result is difficult to interpret without further explanation.
We recognize this might be confusing but it is specified in the text that CIV driven (TMPD+ascorbate) respiration- relying on endogenous cytochrome c- is diminished (line 195) in Parl-/- testis mitochondria. This test reflects cytochrome c oxidase respiratory capacity/activity. We performed then an additional experiment just after the previous where we add exogenous cytochrome c in the cuvette to test the integrity of the outer mitochondrial membrane and checked if CIV-driven respiration increases or not after,compared to before, the addition of cytc. Exogenous cytochrome c does not cross intact mitochondrial outer membranes, so the test is performed to verify the good quality of mitochondrial preparations and/or pathological changes by looking if of the outer membrane integrity, not the function of CIV. CIV driven respiration increases only modestly after compared to before the addition of cytc and to a similar extent in both WT and Parl-/- indicating a good quality of the mitochondrial preparations and that the outer mitochondrial membrane of these mitochondria is overall well preserved in both WT and KO.
- In Figure 5B, there is some variation in band intensity between replicates. Quantifying the band intensity relative to the loading control would help to increase confidence in the conclusion that coQ levels are reduced.
We performed this quantification, as suggested by the reviewer, and added the quantification in figure 5B. Quantification of the band intensity relative to the loading control confirms a significant difference between WT and KO. Moreover, we performed quantitative immunofluorescence of COQ4 in SCP-1 positive cells included now in Fig 5-figure supplement 1, which confirms a significantly decreased expression of COQ4 in Parl-/- primary spermatocytes.
- GPX4 is not a Parl substrate, and no explanation is provided for why it might be reduced in Parl-/- testes. This makes the result and model difficult to interpret.
We thank the reviewer for pointing this out. We acknowledged this limitation in the discussion. We mentioned in the discussion that decreased GPX4 levels have been observed in other conditions (chemical inhibition, pathological conditions, etc.) and no mechanism has so far been demonstrated to our knowledge, but some evidence raises a possible link with CoQ deficiency that we discussed. Potential mechanisms including protein degradation are likely although unproven. This remains an important and intriguing issue to address in future studies.
- Since Parl knockout induces necrosis in the brain, necrosis could be a contributing factor to cell death in spermatocytes alongside ferroptosis. No data is presented that can exclude this possibility.
Ferroptosis is actually considered, by some authors, a form of regulated necrosis (Seibt TM FRBM 2019). Therefore, we can affirm that PARL deletion leads to regulated necrosis in testis via ferroptosis through specific ferroptosis pathways that do not appear to be activated in the brain, or at least not overtly. Importantly, there is no recognized marker or specific molecular pathway for generic «accidental» necrosis that can be tested to differentiate between the 2 different cell death modalities.
- The severe spermatogenesis phenotype implies that Parl knockout males should be infertile, but the fertility status is not described in the manuscript. It may be difficult to test fertility in these animals due to the neurodegeneration phenotype; if so, this can be clarified. If it is feasible to test fertility, demonstration of a fertility phenotype would significantly strengthen the conclusion that loss of Parl leads to spermatogenic arrest.
We specify in the text that Parl-/- mice are sterile due to total lack of sperm production caused by arrested spermatogenesis, as evidenced by detailed histological analysis and AIF1 staining. This is not due to the neurodegeneration since Parl-Ncre knockout have normal production of sperm as presented in the paper. Fertility in Parl-/- cannot be tested in vitro since these mice have no sperm due to the complete block of spermatogenesis, nor in vivo since they die young due to neurodegeneration. With these limitation Parl-/- males and WT females are kept together and in no single exception since the beginning of the colonies a pregnancy has ever been observed. Parl-/- mice are sterile.
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eLife assessment
This manuscript reports an important finding that spermatogenic defects in Parl KO mice, a genetic model for Leigh syndrome, may result from mitochondrial defects leading to ferroptosis. The finding, if confirmed, would be of great significance because male germ cell ferroptosis has not been well characterized. However, the criteria for determining male germ cell ferroptosis were vague, and the supporting data were inadequate.
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Reviewer #1 (Public Review):
In this manuscript Radaelli et al investigate the effects of knocking out Parl, encoding a mitochondrial rhomboid protease, on spermatogenesis. Parl knockout has been used as a genetic model for Leigh syndrome, which in humans can be caused by mutations in several different components of the mitochondrial respiratory chain. This study describes the nature of the spermatogenesis defect found in Parl mutant mice, evaluates double mutants for Parl and other factors known to act with Parl in the context of neurodegeneration, and investigates the changes to mitochondrial function that occur in mutant testes. The authors conclude that Parl-/- males have a severe spermatogenesis defects with arrest at the spermatocyte stage, and that Parl function in spermatogenesis depends on different factors compared to neurons. …
Reviewer #1 (Public Review):
In this manuscript Radaelli et al investigate the effects of knocking out Parl, encoding a mitochondrial rhomboid protease, on spermatogenesis. Parl knockout has been used as a genetic model for Leigh syndrome, which in humans can be caused by mutations in several different components of the mitochondrial respiratory chain. This study describes the nature of the spermatogenesis defect found in Parl mutant mice, evaluates double mutants for Parl and other factors known to act with Parl in the context of neurodegeneration, and investigates the changes to mitochondrial function that occur in mutant testes. The authors conclude that Parl-/- males have a severe spermatogenesis defects with arrest at the spermatocyte stage, and that Parl function in spermatogenesis depends on different factors compared to neurons. Detailed characterization of mitochondrial function in mutant testis shows a variety of defects, including lower overall levels of coenzyme Q (coQ) and a higher ratio of reduced to oxidized coQ. They also conclude that ferroptosis is responsible for spermatocyte cell death in Parl mutants based on the presence of increased transferrin receptor, reduced GPX4 and increases in the ferroptosis end-product 4-hydroxynonenal (HNE).
The conclusions of this manuscript are well supported by (a) strong genetics including phenotype analysis in multiple double knockout mouse strains to show that Parl acts through different pathways in spermatogenic cells compared to neurons; (b) a clear spermatogenesis phenotype as shown by histology and immunostaining; (c) demonstration of mitochondrial defects during spermatogenesis using electron microscopy and respirometry of testis mitochondria; and (d) evidence for a mechanism of spermatocyte death by ferroptosis based on changes in transferrin receptor protein 1, coQ, GPX4, and HNE. Overall, this study advances understanding of the effects of mitochondrial dysfunction on spermatogenesis and may shed light on patient phenotypes in Leigh syndrome. The study will be useful in the fields of fertility and mitochondrial biology. There are a few places where the conclusions are not robustly supported by the data, especially inadequate quantification of some of the phenotype data and some cases where the data presented is not consistent with the model proposed:
- In Figure 2, electron microscopy images represent n=1 cell, making it hard to know how generalizable the mitochondrial phenotypes are. It would be useful to see a quantitative summary of a larger dataset indicating how frequently the mitochondrial defects are seen.
- In Figure 3, representative images are shown for a single field from n=1 animal. It is hard to decisively conclude that the phenotype of Pink1-/-;Pgam5-/- and Ttc19-/- testes is completely normal based on this limited data. There may be other tubules outside the field of view that are abnormal, or more subtle changes in cell ratios. This conclusion would be significantly strengthened by cell counting (e.g. # round spermatids per Sertoli cell per tubule and # spermatocytes per Sertoli cell per tubule) or other quantitation. Likewise, the similarities in phenotype between Parl-/-, Parl-/-;Pink2-/-, and Parl-/-;Pgam5-/- should be more thoroughly documented. At least some additional images should be shown.
- In Figure 4, it looks like there is a significant decrease in CIV-driven respiration in Parl knockouts, but the text describes this as "did not significantly enhance" - that is, the absence of an increase. This result is difficult to interpret without further explanation.
- In Figure 5B, there is some variation in band intensity between replicates. Quantifying the band intensity relative to the loading control would help to increase confidence in the conclusion that coQ levels are reduced.
- GPX4 is not a Parl substrate, and no explanation is provided for why it might be reduced in Parl-/- testes. This makes the result and model difficult to interpret.
- Since Parl knockout induces necrosis in the brain, necrosis could be a contributing factor to cell death in spermatocytes alongside ferroptosis. No data is presented that can exclude this possibility.
- The severe spermatogenesis phenotype implies that Parl knockout males should be infertile, but the fertility status is not described in the manuscript. It may be difficult to test fertility in these animals due to the neurodegeneration phenotype; if so, this can be clarified. If it is feasible to test fertility, demonstration of a fertility phenotype would significantly strengthen the conclusion that loss of Parl leads to spermatogenic arrest.
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Reviewer #2 (Public Review):
This study characterized the mice deficient for PARL and concluded that mitochondrial defects lead to ferroptosis and spermatogenic cell death. In mammalian germ cells, the existence of ferroptosis is not known so far. Interestingly, a study using C. elegans recently established the occurrence of germ cell ferroptosis (Perez et al., Dev Cell 2020: PMID: 32652074). Thus, if the conclusion of this study is valid, this study can be a timely demonstration of germ cell ferroptosis in mammals. I understand the potential value of this study. However, in this study, although several indirect data were provided, I do not think the results firmly established the occurrence of germ cell ferroptosis. Further, some major technical barriers prevent the interpretation of these results. In general, perturbations in …
Reviewer #2 (Public Review):
This study characterized the mice deficient for PARL and concluded that mitochondrial defects lead to ferroptosis and spermatogenic cell death. In mammalian germ cells, the existence of ferroptosis is not known so far. Interestingly, a study using C. elegans recently established the occurrence of germ cell ferroptosis (Perez et al., Dev Cell 2020: PMID: 32652074). Thus, if the conclusion of this study is valid, this study can be a timely demonstration of germ cell ferroptosis in mammals. I understand the potential value of this study. However, in this study, although several indirect data were provided, I do not think the results firmly established the occurrence of germ cell ferroptosis. Further, some major technical barriers prevent the interpretation of these results. In general, perturbations in mitochondria dynamics could be expected to disrupt spermatogenesis. It would be necessary to establish germ-cell ferroptosis to explain the specific phenotype of the PARL mutants. Overall, I appreciate the potential impact; but I am not fully convinced by the main conclusion reported in this study.
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