HSP60 Reduction Causes an Abnormal Genotype and Sex Distribution and Impairs Mitochondrial Activity in Mouse Spermatozoa

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor,

Thank you for this opportunity to review article entitled: HSP60 reduction impairs mitochondrial activity in mouse spermatozoa and causes abnormal genotype distribution

The review provides a well-structured overview of the study on HSP60's role in male fertility, emphasizing the connection between mitochondrial function and sperm quality. Here are some suggestions for improvement and clarity:

Consider providing a brief definition of mitochondrial chaperone complexes for readers who may not be familiar with the concept.

You might expand on the significance of studying male fertility specifically and how it relates to overall reproductive success.

It would be helpful to specify the number of mice used in the breeding experiments and the exact experimental conditions (e.g., diet, environment) to add context to your methodology.

Clarify how offspring characteristics were measured or evaluated—were they assessed based on physical traits, health, or reproductive success later on?

The discussion about active mitochondria and sperm velocity could be enhanced by relating these findings back to their potential implications for fertility outcomes.

Reinforce the broader implications of your findings in terms of how understanding HSP60’s role might inform strategies for addressing male infertility.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In their manuscript "HSP60 reduction impairs mitochondrial activity in mouse spermatozoa and causes abnormal genotype distribution" Hauffe et al. have investigated the effect of a heterozygous loss of the HSP60 gene on genotype and semen parameters in mice.

This is an interesting study, yet there are many open questions that have not been answered and should be adressed before publication.

Homozygous loss of HSP60 leads to embryonic lethality in mice and early postnatal death in humans, yet despite these dramatic effects, a heterozygous loss of HSP60 has been shown to be sufficient for normal development. The authors were now asking, if a 50% dose of HSP60 is sufficient for normal development, why does the organism invest energy in expression of higher doses of HSP60 under normal conditions? As the HSP60 protein is part of the mitochondrial complex HSP60/HSP10, known to interact with a multitude of proteins crucial for spermatozoa quality, the authors hypothesized that a heterozygous HSP60 expression would lead to decreased sperm quality.

For this question to answer, the authors bred the HSP60 heterozygosity into a C57Bl/6N background, as the established C57Bl/6J background has a known mutation in another mitochondrial gene and thus shows a mitochondrial dysfunction independently of HSP60.

Then, the authors analyzed breeding outcomes of heterozygous with wildtype mice regarding sex and genotype and analyzed the semen parameters. Here they could confirm the already published data, that a heterozygous deletion of HSP60 in one of the parents (no matter which) leads to a higher proportion of male than female born mice. The reason for this shift is not explained nor analyzed. As this shift occurs in both breeding conditions, and the sperm delivers the Y chromosome (and in the case of WT males bred with HET females the sperms all have a normal protein dosage of HSP60), this result should be investigated further and not left standing alone, especially as it has already been shown earlier.

Furthermore, the authors showed, that a heterozygous deletion of HSP60 in one of the parents (no matter which) leads to a shift of the expected Mendelian ratio towards wildtype mice. This is an interesting result that also should be analyzed further.

Unfortunately, the authors only regard the constellation when male HPS60 HET were crossed with female WT mice and thus conclude that the reason for the non-mendelian distribution must be the sperms. I think, this conclusion is not permissible, as it does not explain the result of the female HET crossed with male WT. Of course, it is possible, that two completely different reasons can have the same outcome (exactly the same non-mendelian distribution), but there is no proof of that. My first thought was, that there could be a partly embryonic lethality, which should be at least ruled out. For this, implantation and postimplantation development should be checked at several time points and  the litter size could be a first hint and should be compared to litter size when only wildtype mice are bred with each other.

The authors attribute the reduction in HET born mice after breeding of HET male mit WT female to the sperms carrying the HSP60- allel. But sperms carrying the HSP60- allel have not been analysed separately and have not been compared to sperms carrying the HSP60+ allel, so this conclusion cannot be drawn easily. Moreover, both haploid sperms stem from the same diploid cell (carrying the HET deletion) and thus should be equally equipped with proteins.

Several question arise here that have to be addressed: Do HSP60- sperms develop normally? When during spermatozoa development is HSP60 transcribed and translated? Does this even take place after haploid sperms have developed (thus beeing dependent on the haploid gene)?

One could argue that HSP60- develop normally, because the sperm numbers and sperm morphology are not different compared to WT, but then this would not explain the loss of heterozygous offspring.

A next experiment adressed the actual expression of HSP60 protein in testis, cauda epididymis and spermatozoa, and here the authors could very nicely show, that indeed, heterozygosity leads to a roughly halving of the protein in testis, cauda and spermatozoa and that this had a diminishing effect on the expression of ATP synthase in spermatozoa. Why did the authors not quantify the other subunits of mitochondrial complexes that are visible in the Western blot? If they have been analysed by Western blot, they should be quantified as well. How many Western blots have been quantified, only one, or was the experiment repeated? Why have only three animals been analyzed compared to 6 animals in the other experiments?

Concerning the sperm motility parameters that have been analyzed, an isolated reduction in the curved-line velocity of rapid spermatozoa has been found. How is this result to be interpreted? Would a reduction in only this parameter explain a reduced ability to fertilize eggs? Is the curved line velocity a specific parameter for characterization of the sperm performance? Are there other examples for an isolated disability in this parameter while other parameters remain unchainched and does this lead to a phenotype?

Minor issues/comments:

1. How many generations were the mice backcrossed?

2. The offspring with female sex in the supplementary file is 118, but in table1 is written 117

3. In figure S1, the mitotracker signal in sperms derived from HSP+/- males all show the same reduction in the signal. This argues against specific differences in the haploid sperms attributed to their underlying genotype but promotes the hypothesis, that all these sperms should have the same phenotype. Despite these obvious differences in the mitotracker staining compared to WT sperms (arguing for reduction in mitochondrial activity) it is unclear, why only one of the tested sperm parameters is affected. Authors should include a paragraph into the discussion which helps the reader to understand how this diminished mitotracker staining is to be evaluated. Interestingly, no differences in HSP60 staining can be found in  spermatozoa derived from HET mice compared to WT.

4. line 362-367: The discussed expression changes in HSP60 HET females are no sufficient explanation for the observed genotype-sex interaction, as haploid MII oocytes are derived from the same diploid cell, thus all oocytes of HET females should be affected. Moreover, as WT females bred with HET males also show diminished ratio od heterozygous offspring and female offspring, this explanation is not sufficient.

5. Questions that should be included into the discussion: Are there known HSP60 mutations in humans? Is this associated with infertility of sperm abnormalities?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors, after reading your manuscript, here are my comments:

Some procedures are described with great precision (e.g., PCR primers, CASA parameters), while others are more general (e.g., "standard isopropanol-ethanol precipitation").

The use of abbreviations such as Nnt, Ctrl, WT, NCD, BSA, PBS, RIPA, SDS-PAGE, PVDF, OXPHOS, CASA, VSL, VCL, LIN, and BCF, which are generally standard in the field, could be defined upon first use to ensure clarity for all readers.

While the mitigation of Nnt deficiency is mentioned, a brief sentence explaining why this deficiency is problematic for mitochondrial function in the context of Hsp60 studies would be beneficial.

The "technical reasons" for the reduced number of Hsp60+/- samples regarding cauda weight and sperm number are not explained, raising questions about potential bias or issues during the experiment.

The concentration of formaldehyde for fixation is stated (4%), but the duration is not explicitly mentioned (it is implied to be after air-drying).

The dilutions of the primary and secondary antibodies are not provided, which is crucial for reproducibility. The final concentration of sperm is given, but the initial concentration of the filtered suspension is not.

The results indicate an association between Hsp60 heterozygosity and altered sperm mitochondrial function and offspring genotype frequency. However, it is speculative to directly attribute the genotype ratio distortion to this without more mechanistic evidence.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

Despite a substantial improvement of your manuscript, I would like to contribute with some ideas, adressing minor issues in the revised manuscript:

1. Lines 379-383: "The reason for this shift in offspring sex is still unclear. It raises the interesting question, whether a mitochondrial dysfunction in oocytes presents a selective disadvantage for a XX genotype post-fertilization. We propose further pre- and post-implantation studies, to assess at which stage female embryos are lost."

If a mitochondrial dysfunction in oocytes would lead to selective disadvantage, then why does this also occur when the mitochondrial dysfunction comes from sperms? Or do you mean a mitochondrial dysfunction in (preimplantation)embryos?

2. You stated that you included a new Figure 1D but I could not find it.

3. I would like to know how many western blots have been used for quantification. Besides using biological replicates it is always recommended to also do technical replicates. If you did not do technical replicates, you should state it in the M+M. Moreover the number of Western blots used for quantification should be stated in the figure legend. 

Author Response

Please see the attachment.

Author Response File: Author Response.docx