Deficiency in a special dynein DNAH12 causes male infertility by impairing DNAH1 and DNALI1 recruitment in humans and mice

Reviewer #1 (Public Review):

Summary:

Even though this is not the first report that the mutation in the DNAH12 gene causes asthenoteratozoospermia, the current study explores the sperm phenotype in-depth. The authors show experimentally that the said mutation disrupts the proper axonemal arrangement and recruitment of DNALI1 and DNAH1 - proteins of inner dynein arms. Based on these results, the authors propose a functional model of DNAH12 in proper axonemal development. Lastly, the authors demonstrate that the male infertility caused by the studies mutation can be rescued by ICSI treatment at least in the mouse. This study furthers our understanding of male infertility caused by a mutation of axonemal protein DNAH12, and how this type of infertility can be overcome using assisted reproductive therapy.

Strengths:
This is an in-depth functional study, employing multiple, complementary methodologies to support the proposed working model.

Weaknesses:

The study strength could be increased by including more controls such as peptide blocking of the inhouse raised mouse and rat DNAH12 antibodies, and mass spectrometry of control IP with beads/IgG only to exclude non-specific binding. Objective quantifications of immunofluorescence images and WB seem to be missing. At least three technical replicates of western blotting of sperm and testis extracts could have been performed to demonstrate that the decrease of the signal intensity between WT and mutant was not caused by a methodological artifact.

Reviewer #2 (Public Review):

Summary:

The authors first conducted whole exome sequencing for infertile male patients and families where they co-segregated the biallelic mutations in the Dynein Axonemal Heavy Chain 12 (DNAH12) gene.
Sperm from patients with biallelic DNAH12 mutations exhibited a wide range of morphological abnormalities in both tails and heads, reminiscing a prevalent cause of male infertility, asthenoteratozoospermia. To deepen the mechanistic understanding of DNAH12 in axonemal assembly, the authors generated two distinct DNAH12 knockout mouse lines via CRISPR/Cas9, both of which showed more severe phenotypes than observed in patients. Ultrastructural observations and biochemical studies revealed the requirement of DNAH12 in recruiting other axonemal proteins and that the lack of DNAH12 leads to the aberrant stretching in the manchette structure as early as stage XI-XII. At last, the authors proposed intracytoplasmic sperm injection as a potential measure to rescue patients with DNAH12 mutations, where the knockout sperm culminated in the blastocyst formation with a comparable ratio to that in WT.

Strengths:

The authors convincingly showed the importance of DNAH12 in assembling cilia and flagella in both human and mouse sperm. This study is not a mere enumeration of the phenotypes, but a strong substantiation of DNAH12's essentiality in spermiogenesis, especially in axonemal assembly.

The analyses conducted include basic sperm characterizations (concentration, motility), detailed morphological observations in both testes and sperm (electron microscopy, immunostaining, histology), and biochemical studies (co-immunoprecipitation, mass-spec, computational prediction). Molecular characterizations employing knockout animals and recombinant proteins beautifully proved the interactions with other axonemal proteins.

Many proteins participate in properly organizing flagella, but the exact understanding of the coordination is still far from conclusive. The present study gives the starting point to untangle the direct relationships and order of manifestation of those players underpinning spermatogenesis. Furthermore, comparing flagella and trachea provides a unique perspective that attracts evolutional perspectives.

Weaknesses:

Seemingly minor, but the discrepancies found in patients and genetically modified animals were not fully explained. For example, both knockout mice vastly reduced the count of sperm in the epididymis and the motility, while phenotypes in patients were rather milder. Addressing the differences in the roles that the orthologs play in spermatogenesis would deepen the comprehensive understanding of axonemal assembly.