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  1. Author Response:

    Reviewer #1:

    By presenting the detrimental effect of accumulative heterozygous mutations on the sperm head morphology, this report by Martinez and colleagues brings new attention to a wildly accepted paradigm in male germ cells that genetically haploid spermatids are phenotypically diploid, suggesting that multiple heterozygous mutations can lead to unexplained male infertility. The merit of this manuscript is the conceptual advance - oligogenic mutations as the possible cause of male infertility - the manuscript proposes, the strong rationale and reasoning of the motivation of the study, and development of a new tool to visually and quantitatively assess sperm head morphology which will benefit the field in general. The weakness that offsets these strengths is that the sperm phenotypes of the multiple heterozygous mice - while significant - are quite subtle in morphological changes and lack physiological phenotype. The study also does not provide data to support molecular mechanisms such as changes in the protein levels or localizations in their animal models. Due to these limitations, at currently presented the study remains rather descriptive and speculative. It would also be better to avoid excessive novelty claims.

    Thank you for highlighting the conceptual advance of our MS. Regarding the weakness you mentioned, we would like to nuance your statement on the lack of physiological phenotype. Indeed, we clearly show that accumulation of heterozygote mutations led to a significant decrease of sperm motility, mutated sperm being 3 times slower.

    Reviewer #2:

    Digenic and oligogenic inheritance are extensions of monogenic disease models, in which effects of variation at two loci (digenic) or a few loci (oligogenic) contribute to the overall phenotype of an individual. The existence of oligogenic inheritance has been appreciated in human genetics for decades, and has been especially well documented for rare disorders with extensive locus heterogeneity, such as retinal degeneration, a condition for which more than 250 loci have been identified (Kousi and Katsanis 2015). Male infertility, itself a collection of diverse and often severe disorders affecting sperm count and sperm morphology, is likely to be driven by as many or even more loci as retinal degeneration, and is thus likely to feature oligogenic inheritance in some familial cases. Indeed, hypogonadic hypogonadism is one of the earliest and best examples of a human disease displaying digenic inheritance. Nonetheless, numerous challenges abound in the identification of digenic or oligogenic causes of male infertility, and validated examples in humans and model organisms are badly needed. In this study, Martinez et al. demonstrate oligogenic inheritance of sperm abnormalities by breeding a series of KO strains known to feature multiple morphological abnormalities of the flagella (MMAF). This is a significant paper for both the sperm abnormality field and for the broader male infertility community. the experiments and analyses are straightforward and the manuscript is well written.

    My primary concerns are simply about the description of the experiments and analyses themselves.

    1. There are numerous references to the "% of abnormal cells", "% of head abnormalities", "% flagellum abnormalities" (Figures 1B, 2B, 3B, 4B, 5, 6 and elsewhere). There are no clear definitions of how a cell is classified as "abnormal" or a head is classified as "abnormal" or a flagellum is classified as "abnormal". Are these all defined from manual classification of images? This seems essential to know if someone would like to reproduce this experiment.

    We thank the reviewer for his remark and agree that this part was not sufficiently detailed. To allow an easy replication of the experiments, the material and methods now specify:

    “Morphology was visually assessed on a Nikon Eclipse 80i microscope equipped with a Nikon DS-Ri1 camera with NIS-ElementsD (version 3.1.) software by trained experimenters. At least 200 spermatozoa were counted per slide at a magnification of ×1000. Cells are classified as abnormal when they bear at least one morphological defect, either on the head or the flagellum. Normal head morphology is defined by a typical murine overall shape with a pointy hook tip, a well-defined flagellum insertion notch in continuation of a smooth central region, a prominent caudal bulge and a dorsal region without notches. Normal flagellum must be continuous, of regular size and caliber, without angulation or excessive curling. Examples of normal and abnormal morphologies are provided in Appendix 1-Figure 7.”

    We also added a new supplementary figure (Appendix 1-Figure 7) with light microscopy pictures of Harris-Schorr stained spermatozoa with typical and abnormal morphology. Legend section is modified accordingly.

    1. In order to be of most value to the community, it would be helpful to provide the individual-level data behind Figures 5,6,7, indexed by genotype. Currently the supplementary tables just contain the summary statistics for each group. Further, for the individual level data, it would be good to decode the labels from "two genes" and "three genes" to the actual genotypes, since there are multiple genotypes in those groups. These data could be used for fitting genetic models to each of the traits (e.g. to estimate additive effects, epistatic effects, etc).

    As requested by the reviewer, raw data behind figures 5 to 7 and data by genotype for the “two genes” and “three genes” groups have been added as source data files.

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  2. Evaluation Summary:

    Oligogenic inheritance is likely to be an important mode of disease risk for many male infertility traits, but few validated examples exist. This clear documentation of oligogenic effects on mouse sperm is significant for both the sperm abnormality field and for the broader male infertility community.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    By presenting the detrimental effect of accumulative heterozygous mutations on the sperm head morphology, this report by Martinez and colleagues brings new attention to a wildly accepted paradigm in male germ cells that genetically haploid spermatids are phenotypically diploid, suggesting that multiple heterozygous mutations can lead to unexplained male infertility. The merit of this manuscript is the conceptual advance - oligogenic mutations as the possible cause of male infertility - the manuscript proposes, the strong rationale and reasoning of the motivation of the study, and development of a new tool to visually and quantitatively assess sperm head morphology which will benefit the field in general. The weakness that offsets these strengths is that the sperm phenotypes of the multiple heterozygous mice - while significant - are quite subtle in morphological changes and lack physiological phenotype. The study also does not provide data to support molecular mechanisms such as changes in the protein levels or localizations in their animal models. Due to these limitations, at currently presented the study remains rather descriptive and speculative. It would also be better to avoid excessive novelty claims.

    Was this evaluation helpful?
  4. Reviewer #2 (Public Review):

    Digenic and oligogenic inheritance are extensions of monogenic disease models, in which effects of variation at two loci (digenic) or a few loci (oligogenic) contribute to the overall phenotype of an individual. The existence of oligogenic inheritance has been appreciated in human genetics for decades, and has been especially well documented for rare disorders with extensive locus heterogeneity, such as retinal degeneration, a condition for which more than 250 loci have been identified (Kousi and Katsanis 2015). Male infertility, itself a collection of diverse and often severe disorders affecting sperm count and sperm morphology, is likely to be driven by as many or even more loci as retinal degeneration, and is thus likely to feature oligogenic inheritance in some familial cases. Indeed, hypogonadic hypogonadism is one of the earliest and best examples of a human disease displaying digenic inheritance. Nonetheless, numerous challenges abound in the identification of digenic or oligogenic causes of male infertility, and validated examples in humans and model organisms are badly needed. In this study, Martinez et al. demonstrate oligogenic inheritance of sperm abnormalities by breeding a series of KO strains known to feature multiple morphological abnormalities of the flagella (MMAF). This is a significant paper for both the sperm abnormality field and for the broader male infertility community. the experiments and analyses are straightforward and the manuscript is well written.

    My primary concerns are simply about the description of the experiments and analyses themselves.
    1. There are numerous references to the "% of abnormal cells", "% of head abnormalities", "% flagellum abnormalities" (Figures 1B, 2B, 3B, 4B, 5, 6 and elsewhere). There are no clear definitions of how a cell is classified as "abnormal" or a head is classified as "abnormal" or a flagellum is classified as "abnormal". Are these all defined from manual classification of images? This seems essential to know if someone would like to reproduce this experiment.

    2. In order to be of most value to the community, it would be helpful to provide the individual-level data behind Figures 5,6,7, indexed by genotype. Currently the supplementary tables just contain the summary statistics for each group. Further, for the individual level data, it would be good to decode the labels from "two genes" and "three genes" to the actual genotypes, since there are multiple genotypes in those groups. These data could be used for fitting genetic models to each of the traits (e.g. to estimate additive effects, epistatic effects, etc).

    Was this evaluation helpful?