Sperm flagellar 2 (SPEF2) is essential for sperm flagellar assembly in humans

Abstract

Spermiogenesis is a complex and tightly regulated process, consisting of acrosomal biogenesis, condensation of chromatin, flagellar assembly, and disposal of extra cytoplasm. Previous studies have reported that sperm flagellar 2 (SPEF2) deficiency causes severe asthenoteratozoospermia owing to spermiogenesis failure, but the underlying molecular mechanism in humans remains unclear. Here, we performed proteomic analysis on spermatozoa from three SPEF2 mutant patients to study the functional role of SPEF2 during sperm tail development. A total of 1262 differentially expressed proteins were detected, including 486 upregulated and 776 downregulated. The constructed heat map of the differentially expressed proteins showed similar trends. Among these, the expression of proteins related to flagellar assembly, including SPEF2, sperm associated antigen 6 (SPAG6), dynein light chain tctex-type 1 (DYNLT1), radial spoke head component 1 (RSPH1), translocase of outer mitochondrial membrane 20 (TOM20), EF-hand domain containing 1 (EFHC1), meiosis-specific nuclear structural 1 (MNS1) and intraflagellar transport 20 (IFT20), was verified by western blot. Functional clustering analysis indicated that these differentially expressed proteins were specifically enriched for terms such as spermatid development and flagellar assembly. Furthermore, we showed that SPEF2 interacts with radial spoke head component 9 (RSPH9) and IFT20 in vitro, which are well-studied components of radial spokes or intra-flagellar transport and are essential for flagellar assembly. These results provide a rich resource for further investigation into the molecular mechanism underlying the role that SPEF2 plays in sperm tail development and could provide a theoretical basis for gene therapy in SPEF2 mutant patients in the future.

Keywords: SPEF2; flagellar assembly; male infertility; protein interaction; proteomics.

Figure 1

SPEF2 mutation and changes in sperm flagella proteome. (a) Volcano plot showing proteins with altered expression from the proteomics analysis of SPEF2 mutants and normal controls (NCs). P: SPEF2-mutant samples. A total of 486 upregulated (red) and 776 downregulated (green) proteins were found. Red spot, upregulated protein; green spot, downregulated protein; black spot, unchanged protein. (b) Heat map of differentially expressed sperm proteins. NC and SPEF2-mutation samples were compared to determine the changes in the selected proteins in three repeated experiments; the similar trends observed indicate that the protein quantification is consistent. Blue indicates low expression. Red indicates high expression. SPEF2: sperm flagellar protein 2.

Figure 2

Functional analysis of the dysregulated proteins identified in SPEF2-mutant spermatozoa. Scatterplot of differentially expressed proteins commonly identified in SPEF2-mutated spermatozoa. The axonemal/peri-axonemal structural or assembly proteins were significantly reduced, including proteins involved in outer dynein arms (ODAs), inner dynein arms (IDAs), fibrous sheath (FS), radial spokes (RSs), and central apparatus assembly. The common components of cytoplasmic dynein-1 and dynein-2 were reduced (e.g., DYNLT1, DYNLL1, and DYNLRB2), while IFT-related proteins were elevated (e.g., IFT20, IFT27, IFT54, and IFT144). In addition, testis-specific proteins involved in the ubiquitination process (e.g., IQUB, UBTD2, and ZNRF4) were also downregulated. DYNLT1: dynein light chain tctex-type 1; DYNLL1: dynein light chain 1; DYNLRB2: dynein light chain roadblock-type 2; IFT20: intraflagellar transport protein 20; IFT27: intraflagellar transport protein 27; IFT54: intraflagellar transport protein 54; IFT144: intraflagellar transport protein 144; IQUB: IQ and ubiquitin-like domain-containing protein; UBTD2: ubiquitin domain-containing protein 2; ZNRF4: E3 ubiquitin-protein ligase ZNRF4.

Figure 3

Confirmation of the differentially expressed proteins in SPEF2-mutant spermatozoa. The expression of the randomly selected proteins in the spermatozoa of the normal control (NC) and SPEF2-mutant individuals. The western blot results confirmed the increased expression of IFT20 and the decreased expression of SPEF2, SPAG6, RSPH4A, RSPH1, TOM20, SPACA1, EFHC1, DYNLT1 and MNS1 in the spermatozoa of the SPEF2-mutant individuals compared with those of the NC. ACTIN and GAPDH were used as loading controls. Data are representative of three independent assays. SPEF2: sperm flagellar protein 2; SPAG6: sperm-associated antigen 6; RSPH4A: radial spoke head protein 4; RSPH1: radial spoke head 1; TOM20: translocase of outer mitochondrial membrane 20; SPACA1: sperm acrosome associated 1; EFHC1: EF-hand domain containing 1; DYNLT1: dynein light chain tctex-type 1; MNS1: meiosis-specific nuclear structural protein 1; ACTIN: actin beta; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IMT: intramanchette transport; IFT: intra-flagellar transport.

Figure 4

Protein interaction network built from the proteomic dataset using STRING network analysis. The predicted results revealed that SPEF2 can interact with multiple axonemal proteins (SPAG6, RSPH9, SPAG17, etc.), as well as a few IFT proteins (IFT20, IFT27, etc.). In addition, proteins related to ubiquitination process (e.g., UBA1, USP5, and USP14), intramanchette transport (e.g., ODF3 and KIF9), fibrous sheath (AKAP3, AKAP4, and SPA17), and cytoplasmic dynein (e.g., DYNLT1 and DYNLL1) were predicted to interact with SPEF2 or SPAG6. SPAG6: sperm-associated antigen 6; RSPH9: radial spoke head 9; SPAG17: sperm-associated antigen 17; IFT20: intraflagellar transport protein 20; IFT27: intraflagellar transport protein 27; UBA1: ubiquitin-like modifier-activating enzyme 1; USP5: ubiquitin carboxyl-terminal hydrolase 5; USP14: ubiquitin carboxyl-terminal hydrolase 14; ODF3: outer dense fiber protein 3; KIF9: kinesin-like protein KIF9; AKAP3: A-kinase anchor protein 3; AKAP4: A-kinase anchor protein 4; SPA17:, sperm surface protein Sp17; DYNLT1: dynein light chain tctex-type 1; DYNLL1: dynein light chain 1; SPEF2: sperm flagellar protein 2.

Figure 5

The interaction between RSPH9 and SPEF2 was identified by co-IP assay in vitro. HEK293T cells were co-transfected with vectors encoding FLAG-SPEF2 and HA-RSPH9. HEK293T cells were transfected with the vector encoding HA-RSPH9 as a negative control. Cell lysates were immunoprecipitated (IP) with an anti-FLAG antibody. The resultant protein samples were separated by SDS-PAGE and analyzed by immunoblotting (IB) with antibodies against FLAG and HA. Data are representative of three independent assays. RSPH9: radial spoke head component 9; SPEF2: sperm flagellar protein 2; FLAG: DYKDDDDK peptide; HA: haemagglutinin; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis.