DNALI1 deficiency causes male infertility with severe asthenozoospermia in humans and mice by disrupting the assembly of the flagellar inner dynein arms and fibrous sheath

Abstract

The axonemal dynein arms (outer (ODA) and inner dynein arms (IDAs)) are multiprotein structures organized by light, intermediate, light intermediate (LIC), and heavy chain proteins. They hydrolyze ATP to promote ciliary and flagellar movement. Till now, a variety of dynein protein deficiencies have been linked with asthenospermia (ASZ), highlighting the significance of these structures in human sperm motility. Herein, we detected bi-allelic DNALI1 mutations [c.663_666del (p.Glu221fs)], in an ASZ patient, which resulted in the complete loss of the DNALI1 in the patient's sperm. We identified loss of sperm DNAH1 and DNAH7 rather than DNAH10 in both DNALI1^663_666del patient and Dnali1^-/- mice, demonstrating that mammalian DNALI1 is a LIC protein of a partial IDA subspecies. More importantly, we revealed that DNALI1 loss contributed to asymmetries in the most fibrous sheath (FS) of the sperm flagellum in both species. Immunoprecipitation revealed that DNALI1 might interact with the cytoplasmic dynein complex proteins in the testes. Furthermore, DNALI1 loss severely disrupted the transport and assembly of the FS proteins, especially AKAP3 and AKAP4, during flagellogenesis. Hence, DNALI1 may possess a non-classical molecular function, whereby it regulates the cytoplasmic dynein complex that assembles the flagella. We conclude that a DNALI deficiency-induced IDAs injury and an asymmetric FS-driven tail rigid structure alteration may simultaneously cause flagellum immotility. Finally, intracytoplasmic sperm injection (ICSI) can effectively resolve patient infertility. Collectively, we demonstrate that DNALI1 is a newly causative gene for AZS in both humans and mice, which possesses multiple crucial roles in modulating flagellar assembly and motility.

Fig. 1. 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 DNALI1^663_666del patient are stained with anti-DNALI1 and anti-βTUBULIN (β-TUB). E Light microscopy revealed spermatozoa with normal morphology from the DNALI1^663_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.

Fig. 2. Location of IDA components in the human flagellum.

A A schematic diagram of the positions of the dynein proteins docks on the A-tubule of the microtubule doublet (green) in the 96 nm axonemal repeat, according to the prior structural information on Chlamydomonas and Sea urchin sperm flagella. B–E IF illustrating various dyneins components’ locations on the human sperm flagella. A loss of DNALI1 leads to the mislocation of DNAH1(B) and DNAH7(C). Asterisks indicate the miss of DNAH1 and DNAH7, respectively. F IF staining against DRC1 (a component of the N-DRC structure adjacent to IDA). G The spermatozoa from male infertile patients with indicated protein deficiency are stained with anti-DNALI1.

Fig. 3. Dnali1^−/− males display asthenozoospermia (ASZ).

A A summary of the CRISPR/Cas9 knockout targeting approach. B The PCR identification of mouse genotypes. Primer sites are shown in (A). F\R1 is used for wildtype sequence and F\R2 for knockout sequence. C Western blotting verifying that testis samples from Dnali1^−/− mice do not produce a band of the expected size (28 kDa). β-TUBULIN (β-TUB) was used as a normalization control. D IF analysis of spermatozoa from the Dnali1^+/+ and Dnali1^−/− mice using an anti-DNALI1 antibody. Asterisks indicate the complete loss of DNALI1. E Testes from 9-week-old Dnali1^+/+ and Dnali1^−/− mice. F Mean testis weight adjusted with body weight, Data are presented as means ± SEM (n = 8 vs. 5; NS, P > 0.05, Student’s t-test). G H&E-stained spermatozoa from the Dnali1^+/+ and Dnali1^−/− mice. H Mean frequencies of the motile sperm in the Dnali1^+/+ and Dnali1^−/− mice, (n = 5 vs. 5; ****P < 0.0001, Student’s t-test). I Frequencies of sperm with aberrant morphological profiles in the Dnali1^+/+ and Dnali1^−/− mice (n = 5 vs. 5; NS, P > 0.05, Student’s t-test). J and K H&E-stained testis (J) and epididymis (K) sections from the Dnali1^+/+ and Dnali1^−/− mice.

Fig. 4. 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 βTUBULIN (β-TUB). The red lines indicate the area where the fluorescence intensity are measured. D Quantitative co-localization analysis of AKAP3 and β-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. 5. DNALI1 localizes in the cytoplasm of spermatids and regulates the assembly of structural proteins in the flagellum.

A DNALI1 staining at distinct murine developmental phases of the male germ cells, as evidenced by IF. Z zygotene, P Pachytene, D Diplotene, RS round spermatid, ES elongated spermatids. B IF analysis of round and elongated spermatids from the Dnali1^+/+ mice. C The expression and location of DNAH1 during spermiogenesis. The DNAH1 protein is expressed, but cannot be assembled into the flagella. Arrows indicate the localization of DNAH1 in the flagella, and the asterisks indicate the absence. D The AKAP4 staining during spermiogenesis. The AKAP4 expression and assembly efficiency are significantly reduced. E IF analysis of AKAP3 during spermiogenesis. Arrows mark the abnormal distribution of AKAP3.

Fig. 6. DNALI1 may serve as a portion of the cytoplasmic dynein complex that regulates the transport and assembly of flagellar components.

A–C GO analysis of the DNALI1 binding proteins in mice testes. D A schematic of the cellular distributions of the DNALI1 binding proteins. The DNALI1 binding proteins are enriched in the Dynactin and Dynein complexes. A large number of flagellum component proteins are identified and may serve as cargos transmitted by the dynein complex.

Fig. 7. Dnali1^−/− mice have distinct hydrocephalus.

A Representative lateral images of 8-week-old Dnali1^−/− mice with a domed cranial vault, compared to the Dnali1^+/+ mice. B The top view of the whole skulls from the Dnali1^+/+ and Dnali1^−/− mice. C The coronal mice brain sections stained with H&E. High magnification images reveal that cilia are present on intact Dnali1^−/− ependyma. D3V dorsal 3rd ventricle, LV lateral ventricle. The asterisk indicates the enlarged ventricle.