Figure 3.

DNHD1 deficiency associated with sperm axoneme malformations and central pair loss

(A) Ultrastructure of spermatozoa determined by TEM from a fertile control and men with bi-allelic DNHD1 variants. Cross-sections of the sperm flagella in the control show a typical axoneme and peri-axoneme structure. The axoneme mainly comprises a ‘‘9 + 2’’ structure, including nine pairs of peripheral doublet microtubules (DMTs; green arrows) and a central pair of microtubules (CP; blue arrows). The peri-axoneme structure includes the fiber sheath, nine outer dense fibers (red arrows), and a helical mitochondrial sheath (MS; pink arrows). Scale bars: 200 nm.

(B) Quantification of different categories of flagellar ultrastructural defects. Total cross-section numbers for quantification in the control individual and men harboring bi-allelic DNHD1 variants (subjects F1-II-1, F2-II-1, and F3-II-1) were 135, 156, 84, and 102, respectively. Cross-sectional defects were classified into four categories: absence of CP, absence of DMTs, disorganization, and normal axoneme. The main defective types were an absence of CP (54.5%, 47.6%, and 10.9% for subjects F1-II-1, F2-II-1, and F3-II-1, respectively) and axoneme disorganization (30.1%, 26.2%, and 26.8% for subjects F1-II-1, F2-II-1, and F3-II-1, respectively).

(C and D) SPAG6 and SPEF2 immunofluorescence assays of human sperm. Anti-SPAG6 (red in C) and anti-SPEF2 (red in D) normally localized along the sperm flagella in the control sperm. However, expression of both SPAG6 and SPEF2 was almost absent in sperm obtained from men harboring bi-allelic DNHD1 variants. Anti-a-tubulin (green) marked the sperm flagella. The nuclei of spermatozoa were DAPI-labeled (blue). Scale bars: 5 mm.