Mutations in ARL2BP, a protein required for ciliary microtubule structure, cause syndromic male infertility in humans and mice

Abstract

Cilia are evolutionarily conserved hair-like structures with a wide spectrum of key biological roles, and their dysfunction has been linked to a growing class of genetic disorders, known collectively as ciliopathies. Many strides have been made towards deciphering the molecular causes for these diseases, which have in turn expanded the understanding of cilia and their functional roles. One recently-identified ciliary gene is ARL2BP, encoding the ADP-Ribosylation Factor Like 2 Binding Protein. In this study, we have identified multiple ciliopathy phenotypes associated with mutations in ARL2BP in human patients and in a mouse knockout model. Our research demonstrates that spermiogenesis is impaired, resulting in abnormally shaped heads, shortened and mis-assembled sperm tails, as well as in loss of axonemal doublets. Additional phenotypes in the mouse included enlarged ventricles of the brain and situs inversus. Mouse embryonic fibroblasts derived from knockout animals revealed delayed depolymerization of primary cilia. Our results suggest that ARL2BP is required for the structural maintenance of cilia as well as of the sperm flagellum, and that its deficiency leads to syndromic ciliopathy.

Fig 1. Pedigrees and genetic findings.

(A) Pedigrees of the patients analyzed in this study. DNA was available only for subjects P1, P2, and P3. (B) Exome-wide homozygosity mapping for autosomal chromosomes, using the AutoMap tool. The autozygous region containing the gene ARL2BP is highlighted in red. (C and D) Sanger validation of the WES findings, showing the presence of a homozygous splice site mutation in patient P1 (C, NM_012106.3:c.207+1G>A, leading to p.Asp35PhefsTer8), and a frameshift deletion in patients P2 and P3 (D, NM_012106.3:c.33_36delGTCT: p.Phe13ProfsTer15), alongside with relevant control sequences (ctrl).

Fig 2. Findings on retinal imaging of patients P1 and P2.

(A and B) Fundus photos of patients P1 and P2, respectively. P1 presents a pale optic disc, vascular thinning and retinal atrophy in the posterior pole and along the vascular arcades. Scarce pigment deposition can be found along the superior vascular arcade (as shown by the arrowheads). Similarly, P2 displays a pale optic disc, vascular thinning and marked retinal changes, with opalescent areas of the retinal tissue in the periphery and central atrophic lesion. Scarse pigment deposition can also be found adjacent to the optic nerve head and periphery (arrowheads). (C and D) Fundus autofluorescence showing in P1 multiple hypoautofluorescent spots in the periphery corresponding to RPE atrophy, which in contrast is largely diffused in P2. Moreover, both patients present a central hyperautofluorescent ring, typical of RP. (E and F) SD-OCT revealing in both patients diffuse retinal thinning, absent photoreceptors and enhanced visualization of the choroidal vessels. Panels A-D: right eye followed by left eye. Panels E and F: right eye above and left eye below.

Fig 3. ARL2BP localizes to the sperm tail in humans and mice.

(A) Staining for ARL2BP (green) and DNA (blue) in a sperm sample from a control individual (top) and from patient P1 (bottom). (B) WT (+/+) and KO (-/-) murine sperm stained for ARL2BP (green) and Acetylated Tubulin (AcTu, red). Lack of ARL2BP staining in the KO serves as a negative control. Scale Bar = 10μm.

Fig 4. Effect of the splice site mutation in patient P1.

(A) Agarose gel showing the RT-PCR amplified product of ARL2BP spanning exon 2 to 4 using cDNA obtained from sperm-derived RNA (P1, cDNA from patient; ctrl, cDNA from a healthy control; P1 RT- and ctrl RT-, reverse transcription reaction lacking the RT enzyme with templates from P1 and a ctrl, respectively; H2O, control reaction with no cDNA template) (B) Chromatogram of the PCR product from patient P1, from panel (A). (C) In-scale schematic representation of the splicing pattern resulting from the presence of the mutation c.207+1G>A that leads to the skipping of exon 3 and the joining of exon 2 with exon 4, with subsequent shifting of the reading frame and creation of a premature stop codon in exon 4 (red). The asterisk denotes the location of the mutation. The in-frame exons are in black, whereas the striped portion of exon 4 indicates the out-of-frame region.

Fig 5. Loss of ARL2BP leads to decreased sperm count and abnormal sperm structure in mice.

(A) The average weight of testis between WT (+/+) and KO (-/-) mice are comparable, according to unpaired, two-tailed t test (n = 10). Data are represented as the mean ± SEM. (B) H&E sections of WT (+/+) and KO (-/-) testis. Residual bodies (Rb), PS = primary spermatocyte, SS = secondary spermatocyte, ES = elongating spermatid, SC = sertoli cells. Scale Bar = 20μm. (C) Graph presenting the epididymal sperm cell counts of WT (+/+) and KO (-/-) mice. Data are represented as the mean ± SEM. ***P = 0.0002, according to unpaired, two-tailed t test (n = 3). (D) Light microscopy images displaying WT (+/+) sperm with normal sperm structure, while KO (-/-) sperm show abnormal sperm heads, shorter sperm tails, detached head and tails, and retained cytoplasm. Scale Bar = 10μm.

Fig 6. ARL2BP loss results in impaired sperm tail development.

(A) Immunoblot of WT (+/+) and KO (-/-) testis and sperm lysates probed for the indicated sperm tail markers: axonemal markers (Glutamylated tubulin (GT335) and β-tubulin) and accessory structure markers (Pyruvate dehydrogenase (Pyr. Deh.), Outer Dense Fiber 1 (ODF1), and A-kinase anchoring protein 4 (pre-processed = Pro-AKAP4 and phosphorylated, processed = Ph. AKAP4). Molecular weights in kilodaltons (kDa) are displayed on the left. α-tubulin is used as the loading control. (B) WT (+/+) and KO (-/-) murine testes sections stained with PNA lectin (acrosomes, red) and AKAP4 (green), or (C) mitotracker (mitochondria, red) and GT335 (green). The nuclei are stained with DAPI (blue). Scale Bar = 20μm. (D and E) Sperm stained with the indicated sperm tail markers in WT (+/+) and KO (-/-) murine sperm. GT335 –axoneme, green; AKAP4 –fibrous sheath, green; Retinititis Pigmentosa GTPase Regulator (RPGR–axoneme, red); ODF1 –outer dense fibers, green; Heat Shock Protein 60kDa (HSP60 –mitochondrial sheath, green), and Sperm Flagellar Protein 2 (SPEF2 –axoneme, red). Arrows point to sperm tails. Scale Bar = 10μm.

Fig 7. Abnormal acrosomes with loss of ARL2BP.

(A-D) WT (+/+) and KO (-/-) adult murine testes sections displaying staining of DAPI (nuclei, blue), PNA lectin (acrosomes, red) and GT335 (green, sperm tails in stages IV-VI and XII). Scale bar = 10μm. Inserts show magnified image. Arrows point to abnormal acrosomes.

Fig 8. Loss of ARL2BP results in disorganized axoneme and accessory structures.

(A1-A3) WT testes. Panel A1 showing multiple heads with normal nuclei (N) and acrosomes (Ac). Tail cross-sections (+). Scale Bar = 1 μm. (A2 and A3) tail cross-sections with central pair (CP) microtubules, outer dense fibers (ODF), and fibrous sheath (FS). Scale Bar = 500 nm. (B1) Tail cross-sections from WT testes (>20, +) with normal FS, ODF, CP, and mitochondrial sheath (MS). Scale Bar = 1 μm. (B2) Higher magnification of (B1) Scale Bar = 500 nm. (C1) KO spermatid heads mostly normal with some aberrantly shaped (arrow). Tail cross-sections show disorganized FS components. Scale Bar = 1 μm. (C2) Higher magnification of box in (C1) shows disorganized microtubules (arrowhead) with associated ODF material (*). Some microtubules are incomplete (circle). Scale Bar = 500 nm. (D) Step 9 spermatids with normal manchettes (arrowhead) in WT (D1) and KO (D2). Scale Bars = 1 μm. (E1) Abnormal spermatid in KO (bracket) with microtubules, ODF, and FS present but disorganized (Higher mag in E2, E3 Bar = 1 μm, and E4 Bar = 500 nm). Residual bodies (Rb) in the lumen. Bar = 10 μm. (E2) Abnormal head and neck region with parallel microtubules and disorganized tail accessory structures. Microtubules (bracket and pink arrowheads), putative ODF (yellow arrowheads), and putative FS (*). Tail cross-section top/center with single, unorganized microtubules (<). Bar = 1 μm. (F1-F4) KO testes show disorganized microtubules and aggregation of mitochondria (M) that fail to organize into a tight spiral. Bar = 5 microns; (F3 and F4) Bar = 2 microns. (F2) M and microtubules are disorganized. In the neck region, basal plate (BP) and capitulum (C) are seen. Bar = 1 μm.

Fig 9. Laterality defects and enlarged lateral ventricles in the adult murine brain with absence of ARL2BP.

(A) WT (+/+) mouse displaying situs solitus (normal axis patterning), and KO (-/-) mouse exhibiting situs inversus (complete reversal). L = Left and R = Right. (B) Percentages of KO animals with left/right-asymmetry abnormalities. (C) Graph displaying the non-Mendelian distribution of KO animals identified from Het x KO crosses of full-term litters (total of 100 animals with 23 KO’s, chi square value of 39.19, p>0.001), or collected at embryonic day 13.5 (e13.5) (total of 7 animals with 0 KO’s, chi square value of 7, 0.01>p>0.005), compared to the Mendelian distribution seen at embryonic day 7.5 (e7.5) (total of 24 animals with 16 KO’s, chi square value of 0.96). (D) Comparison of ventricular volumes between WT (+/+) and KO (-/-) brains from 3D-recontruction of CT images. Left p = 0.0002, Right p = 0.0087; according to unpaired, two-tailed t test (n = 3). Data are represented as the mean ± SEM. (E) Micro-CT scans of WT (+/+) and KO (-/-) murine brains at postnatal day 70, in Sagittal (Sag), Axial (Ax), and Coronal (Cor) planes. Arrows mark the lateral ventricles. Ac. = Actual #, Ex. = Expected #; LV = left ventricle, RV = right ventricle.

Fig 10. Cells lacking ARL2BP undergo slower ciliary depolymerization.

(A) Mouse embryonic fibroblasts (MEFs) from WT (+/+) and KO (-/-) littermates stained to show DAPI (nuclei, blue), ARL13B (cilia, green), and γ-tubulin (basal body, red). (B) Violin plot displaying the length measurements from WT (+/+) and KO (-/-) MEFs. n = 200. P = 0.0001, according to unpaired, two-tailed t test (n = 200). (C) MEFs from WT (+/+) and KO (-/-) littermates after serum starvation (cilia induction) for 48 hours, followed by serum addition (cilia depolymerization) for 2 hours stained for Acetylated tubulin (AcTu–cilia, red) and pericentrin (basal body, green). (D) Dot plot displaying the percentage of ciliated cells between WT (+/+) and KO (-/-) MEFs after 48 hours of serum starvation and at indicated time points after re-addition of serum. Average percentage of ciliated cells is to the right of each dot set. Data are represented as the mean ± SEM. For 2hrs, p = 0.0174, other time points not statistically significant according to unpaired, two-tailed t test (n (each dot representing 200≥100 cells) = 3 or 4). (E) Immunoblot of WT (+/+), Heterozygous (+/-), and KO (-/-) MEF cell lysates probed for ARL2BP, with GAPDH as the loading control. Molecular weights in kilodaltons (kDa) are displayed on the left.