Fig 2: The LCs position of the Dnali1-/- spermatozoa is asymmetrical and the IDA components are incomplete.A and B The flagellar cross-sectional structure of Dnali1+/+ and Dnali1-/-, as evidenced by TEM. The red arrow indicates an obvious IDA. The numbers represent the quantity of peripheral microtubules. A yellow dash marks the LCs, which are diagonally positioned in the Dnali1+/+ spermatozoa. C The co-staining and the co-localization analysis of AKAP3 and ßTUBULIN (ß-TUB). The red lines indicate the area where the fluorescence intensity are measured. D Quantitative co-localization analysis of AKAP3 and ß-TUB. E IF staining against AKAP4. F–I IF staining depicting the locations of each DNAH proteins in the Dnali1+/+ and Dnali1-/- mice. The asterisks indicate the miss of DNAH1 and DNAH7, respectively.

Fig 3: The DNALI1 loss leads to the asymmetrical development of sperm fibrous sheaths in humans.A Pedigree analysis of the family affected by bi-allelic DNALI1 mutations, as identified by WES. The black-filled squares represent the infertile males of this family. B The WES-identified mutations are further confirmed via Sanger sequencing. The black box marks the missing sequence. C DNALI1 is missing in spermatozoa from the patient. D Spermatozoa from a fertile control male and the DNALI1663_666del patient are stained with anti-DNALI1 and anti-ßTUBULIN (ß-TUB). E Light microscopy revealed spermatozoa with normal morphology from the DNALI1663_666del patient and a control male. F TEM images of the flagellar cross-sectional structure of a control human and patient. The red arrow represents an obvious IDA. The numbers highlight the quantity of peripheral microtubules. A yellow dash represents the LCs, which are diagonally positioned in the control spermatozoa. G TEM cross-sections of the principal piece of sperm are quantified and categorized according to the LC symmetry. H and I Immunofluorescence (IF) staining against AKAP4 and GAPDHS. The asterisk indicates a significant decrease in AKAP4 protein expression.