EIF4G1 is a novel candidate gene associated with severe asthenozoospermia

Abstract

Background: Asthenozoospermia (AZS), also known as asthenospermia, is characterized by reduced motility of ejaculated spermatozoa and is detected in more than 40% of infertile patients. Because the proportion of progressive spermatozoa in severe AZS is <1%, severe AZS is an urgent challenge in reproductive medicine. Several genes have been reported to be relevant to severe asthenospermia. However, these gene mutations are found only in sporadic cases and can explain only a small fraction of severe AZS, so additional genetic pathogenies need to be explored.

Methods and results: By screening the variant genes in a patient with severe AZS using whole exome sequencing, we identified biallelic mutations c.2521C>T: p.(Pro841Ser) (NC_000003.11: g.184043412C>T) in exon13 and c.2957C>G: p.(Ala986Gly) (NC_000003.11: g.184045117C>G) in exon17 in the eukaryotic translation initiation factor 4 gamma 1 gene (EIF4G1, RefSeq: NM_004953.4, OMIM: 600495) of the patient. Both of the mutation sites are rare and potentially deleterious. Transmission electron microscopy analysis showed a disrupted axonemal structure with mitochondrial sheath defects. The EIF4G1 protein level was extremely low, and the mitochondrial marker cytochrome c oxidase subunit 4I1 (COXIV, OMIM: 123864) and mitochondrially encoded ATP synthase 6 (ATP6, OMIM: 516060) protein levels were also decreased in the patient's spermatozoa as revealed by WB and IF analysis. This infertility associated with this condition was overcome by intracytoplasmic sperm injections, as his wife became pregnant successfully.

Conclusion: Our experimental findings indicate that the EIF4G1 gene is a novel candidate gene that may be relevant to severe AZS.

Keywords: EIF4G1; biallelic mutation; severe asthenozoospermia; whole exome sequencing.

Figure 1

Morphological analysis in the sperm of the patient with severe asthenozoospermia (a) Family tree of the patient with severe asthenozoospermia. The black square represents the proband (II:3). (b) Morphological analysis of the sperm of patient by Papanicolaou staining. The black arrows indicate the abnormal sperm. Multiple images were taken, and representative images are presented. Scale bar: 10 μm. (c) Electron microscopic morphology of the sperm of control and the patient. (A) Longitudinal sections of sperm flagellum from the control subject. (B) Longitudinal sections of the sperm of the patient, showing damaged mitochondria and a disordered mitochondrial sheath. (C) Cross‐sections of the midpiece (MP) in the control sperm. (D) Cross‐sections of the MP in the patient's sperm. (E) Cross‐sections of the sperm flagellum in the PP of the control. (F) Cross‐sections of the sperm flagellum in the PP of the patient's sperm. Multiple images were taken, and representative images are presented. Scale bar: A = 1 μm, B = 0.5 μm, C‐F = 200 μm. Abbreviations: Ax, axoneme; CP, central pair; CR, circumferential rib; DA, dynein arm; DMT, doublet microtubule; FS, fibrous sheath; LC, longitudinal column; MP, midpiece; MS, mitochondrial sheath; N, nucleus; ODF, outer dense fiber; PP, principal piece; SC, segmented column.

Figure 2

Biallelic mutations in EIF4G1 identified in the patient with severe asthenozoospermia (a) Sanger sequencing confirmed the biallelic mutations in the EIF4G1 gene of the proband. The red rectangle indicates the mutation sites. (b) The locations of the biallelic mutation sites in EIF4G1. (Top) Genomic structure and (bottom) protein domains. Green boxes indicate coding exons, and rectangles filled with color represent the functional domains. (c) Conservative analysis of the amino acids encoded by the biallelic mutation sites in different species

Figure 3

EIF4G1 protein level in the patient and control. (a) EIF4G1 protein levels were determined with Western blots. (b) The density of each band was quantified with ImageJ. Ac‐tubulin was used as the loading control. The results were expressed as the mean ± SD of three independent experiments. Data were analyzed with spss 18.0 software. ***p < 0.001. (c) EIF4G1 protein expression was determined with an immunofluorescence assay. Multiple images were taken, and representative images are presented. Scale bar: 10 μm.

Figure 4

COXIV and ATP6 protein levels in the patient and control. (a) COXIV and ATP6 protein levels were determined with Western blots. (b) The density of each band was quantified with ImageJ. Ac‐tubulin was used as the loading control. The results were expressed as the mean ± SD of three independent experiments. Data were analyzed with spss 18.0 software. *p < 0.05, **p < 0.01. (c) COXIV and ATP6 protein expressions were determined by immunofluorescence assay. Multiple images were taken, and representative images are presented. Scale bar: 10 μm