RSPH6A is required for sperm flagellum formation and male fertility in mice

Abstract

The flagellum is an evolutionarily conserved appendage used for sensing and locomotion. Its backbone is the axoneme and a component of the axoneme is the radial spoke (RS), a protein complex implicated in flagellar motility regulation. Numerous diseases occur if the axoneme is improperly formed, such as primary ciliary dyskinesia (PCD) and infertility. Radial spoke head 6 homolog A (RSPH6A) is an ortholog of Chlamydomonas RSP6 in the RS head and is evolutionarily conserved. While some RS head proteins have been linked to PCD, little is known about RSPH6A. Here, we show that mouse RSPH6A is testis-enriched and localized in the flagellum. Rsph6a knockout (KO) male mice are infertile as a result of their short immotile spermatozoa. Observation of the KO testis indicates that the axoneme can elongate but is disrupted before accessory structures are formed. Manchette removal is also impaired in the KO testis. Further, RSPH9, another radial spoke protein, disappeared in the Rsph6a KO flagella. These data indicate that RSPH6A is essential for sperm flagellar assembly and male fertility in mice.This article has an associated First Person interview with the first author of the paper.

Keywords: Axoneme; CRISPR/Cas9; Flagella; Radial spoke protein; Spermatozoa.

Fig. 1.

Characterization of mouse Rsph6a. (A) Schematic drawing of the flagella axoneme (end piece) and radial spoke structure. (B) The expression of mouse Rsph genes in various organs examined by RT-PCR. Rsph6a is testis-enriched but weak expression is also detected in the thymus and lung. β-actin was used as an expression control. BR, brain; TH, thymus; LU, lung; HE, heart; SP, spleen; LI, liver; KI, kidney; TE, testis; OV, ovary; UT, uterus. (C) Western blot analysis of RSPH6A. RSPH6A was detected in the testis, but not in the thymus or lung. GAPDH was detected as a loading control, and acetylated tubulin as a marker for stable microtubules including cilia and flagella. (D) The expression of mouse Rsph6a on indicated postnatal days in the testis was examined by RT-PCR. Rsph6a begins expression at postnatal day 18. β-actin was used as an expression control. (E) Immunofluorescence analysis of spermatozoa from WT mice labeled with antibodies against RSPH6A (red). Fluorescence is seen along the entire sperm flagella. (F) Fractionation of mouse spermatozoa. RSPH6A was found in the SDS-soluble fraction. SLC2A3, acetylated tubulin and AKAP4 were detected as makers for Triton-soluble, SDS-soluble and SDS-resistant fractions, respectively.

Fig. 2.

Interaction of RS head proteins. RSPH6A interaction with other RS head proteins was examined by co-immunoprecipitation of the RSPH6A-FLAG and RSPH-PA complex using anti-FLAG antibody-conjugated beads. Input: whole cell lysate from transfected cells. IP: immunoprecipitation with FLAG-conjugated beads. The left lower panel shows α-tubulin as a loading control. RSPH6A can bind to all other RS head proteins in HEK293T cells.

Fig. 3.

Generation of Rsph6a mutant mice using the CRISPR/Cas9 system. (A) Structure of the Rsph6a and CRISPR/Cas9 targeting scheme. Rsph6a has two variants and both were targeted in exon 1. Black underline indicates gRNA sequence. Red characters indicate PAM (protospacer adjacent motif) sequence. (B) Wave pattern sequence of Rsph6a in WT and Rsph6a^+4/+4 mice. (C) Genotyping Rsph6a^+4/+4 mice by MscI digestion. The MscI recognition site (5′-TGGCCA-3′) was disrupted due to the 4 bp insertion. In WT mice, the PCR product was cut into two similar-sized sequences (resulting in one band of ∼250 bp) after MscI digestion. (D) The 4 bp insertion in the first exon in Rsph6a^+4/+4 mice caused a P67S mutation resulting in a premature stop codon introduced 13 amino acids later. (E) Protein expression of RSPH6A in Rsph6a^WT/+4 and Rsph6a^+4/+4 testis and cauda epididymal spermatozoa. Coomassie Brilliant Blue (CBB) staining shows equal loading.

Fig. 4.

Fertilizing ability and testicular structure of Rsph6a^+4/+4 mutant male mice. (A) Number of litters born per plug detected. N=3 males each for WT and Rsph6a^+4/+4, mated with two WT females per male. **P<0.01, Student's t-test. (B) Testes of Rsph6a^+4/WT and Rsph6a^+4/+4 mice. (C) Testicular weights of Rsph6a^+4/WT and Rsph6a^+4/+4 mice were not significantly different (P=0.64, Student's t-test). N=3 males each for Rsph6a^+4/WT and Rsph6a^+4/+4. (D) PAS staining of testicular sections. Lower panels are magnified images of the boxes indicated in the figures above. Development of normal spermatocytes (red arrowheads) and spermatids (black arrowheads) can be seen in stage II-III tubules in both control and mutant mice. In stage VII-VIII seminiferous tubules of Rsph6a^+4/WT mice, properly elongated spermatid tails are present in the lumen (white arrowheads), but similar elongated flagella are missing in Rsph6a^+4/+4 tubules. Sections of Rsph6a^+4/WT show no mature elongated spermatids remaining in the stage IX tubules but they are retained in Rsph6a^+4/+4 mice (red arrows). Stage XII tubules of Rsph6a^+4/WT mice have normal, hook-shaped heads (black arrows) whereas Rsph6a^+4/+4 XII tubules have abnormal, club-shaped heads (white arrows).

Fig. 5.

Histological evaluation of the epididymis and spermatozoa. (A) PAS staining of cauda epididymis in Rsph6a^+4/WT and Rsph6a^+4/+4 mice. (B) Observation of spermatozoa obtained from cauda epididymis. Rsph6a^+4/WT spermatozoa possessed full-length flagella and normal head shapes, while Rsph6a^+4/+4 spermatozoa had truncated flagella and abnormal head shapes. (C) Immunofluorescence analysis of spermatozoa from control and mutant mice labeled with antibodies against TOM20 (red). TOM20 (mitochondria) localizes to the midpiece of control spermatozoa. Fluorescence in Rsph6a^+4/+4 mouse spermatozoa was undetectable (black arrowhead) or spotty (white arrowhead). (D) Immunofluorescence analysis of spermatozoa from control and mutant mice labeled with antibodies against CABYR (red). The fibrous sheath protein was localized along the principal piece of control spermatozoa. Signal in Rsph6a^+4/+4 mouse spermatozoa was undetectable (black arrowhead) or spotty (white arrowhead).

Fig. 6.

Immunofluorescence of α-tubulin in Rsph6a^+4/WT and Rsph6a^+4/+4 mice. Immunofluorescence analysis of spermatids from control and mutant mice labeled with antibodies against α-tubulin (green). α-tubulin localizes to the manchette of both control and Rsph6a^+4/+4 mouse spermatids. Head elongation is shown progressively from left to right based on nuclear and manchette shapes. White arrow indicates abnormal caudal movement of the manchette. Hoechst staining (blue) indicates the nucleus.

Fig. 7.

Ultrastructural analysis of the Rsph6a KO testicular spermatozoa. There is no difference in manchette formation (A,B: stage X) or axoneme structures that are not surrounded by mitochondrial or fibrous sheath (C: stage VI; D: stage VI) between control and Rsph6a^+4/+4 mutant spermatozoa. However, compared with control mice (E: stage VIII), axoneme structures surrounded by a mitochondrial sheath and mitochondrial sheath formation were disrupted in Rsph6a^+4/+4 mutant mice (F,G: stage VIII). M, manchette.

Fig. 8.

RSPH9 localization in control and Rsph6a KO mice. (A) Immunofluorescence analysis of spermatids from control and Rsph6a^+4/+4 mutant mice labeled with antibodies against RSPH9 (red). RSPH9 signal was seen along the flagellum in Rsph6a^+4/WT mice. In contrast, the signal decreased in the flagella of Rsph6a^+4/+4 mutant mice. α-tubulin (green) stains both manchette and flagellum. Hoechst staining (blue) indicates the nucleus. W/o 1st Ab, Rsph6a^+4/WT mouse spermatids processed without RSPH9 antibody staining. (B) Protein expression of RSPH9 in Rsph6a^WT/+4 and Rsph6a^+4/+4 testis and cauda epididymal spermatozoa. Acetylated tubulin signal decreased in the KO spermatozoa because the tail was short. In contrast, RSPH9 signal disappeared in the Rsph6a^+4/+4 spermatozoa. CBB staining confirms equal loading.