The absence of both RIBC1 and RIBC2 induces decreased sperm motility and litter size in male mice

Abstract

Background: RIBC1 (RIB43A domain with coiled-coils 1) and RIBC2 (RIB43A domain with coiled-coils 2) are homolog proteins of RIB43a which is localized to microtubules in the cilia and flagella of unicellular organisms. Cryo-electron microscopy and artificial intelligence studies showed that RIBC1 and RIBC2 are microtubule inner proteins (MIPs) localized in the inner lumen of the doublet microtubules (DMTs) in mouse sperm flagella. However, the function of RIBC1 and RIBC2 in mammalian reproduction and sperm flagella is still unknown.

Objective: To clarify the function of RIBC1 and RIBC2 in mouse spermatozoa.

Materials and methods: We generated Ribc1 knockout (KO), Ribc2 KO, and Ribc1 and Ribc2 double-knockout (Ribc1/2 DKO) mice using the CRISPR/Cas9 system and analyzed their phenotypes.

Results: We revealed that the loss of either RIBC1 or RIBC2 alone did not affect male fertility, but the absence of both caused a decrease in pup numbers. Sperm motility analysis showed that Ribc1 KO spermatozoa had reduced velocity, but Ribc2 KO sperm velocities were comparable with WT mice. However, Ribc1/2 DKO sperm velocities were significantly lower than those from Ribc1 KO mice. No structural abnormalities in the axonemal structure at the transmission electron microscope (TEM) level and no abnormalities in the flagellar waveform pattern were observed in Ribc1/2 DKO spermatozoa.

Discussion and conclusion: Both RIBC1 and RIBC2 are not significant for maintaining the axonemal structure in mouse spermatozoa, but both proteins function cooperatively in sperm motility. This result may indicate that minor structural changes due to RIBC protein absence are not detected at the TEM level, and RIBC2 function depends on RIBC1 in sperm motility. We think that reduced litter size in Ribc1/2 DKO mice is caused by reduced sperm motility due to minor structural abnormalities caused by the loss of two RIBC proteins.

Keywords: RIBC1; RIBC2; doublet microtubules; microtubule inner proteins; sperm motility.

FIGURE 1

Both Ribc1 and Ribc2 are expressed in mouse sperm flagellum. Expression profiles of RIBC1 and RIBC2 in multiple human tissues from MRGDV2 RNA‐Seq datasets. The minimum TPM value was set to 0 and the maximum TPM value to 50. (A) Expression profiles of Ribc1 and Ribc2 in multiple mouse tissues from MRGDV2 RNA‐Seq datasets. The minimum TPM value was set to 0, and the maximum TPM value was set to 50. (B,C) Expression profiles of RIBC1 and RIBC2 in human testis from MRGDV2 RNA‐Seq datasets. Ud Spg, undifferentiated spermatogonia; Dif Spg, differentiated spermatogonia; Sc, Spermatocytes; St, Spermatids; SC, Sertoli cells. (D) Expression profiles of Ribc1 and Ribc2 in mouse testis from MRGDV2 RNA‐Seq datasets. Ud Spg, Undifferentiated spermatogonia; Dif Spg, differentiated spermatogonia; Le Sc, Leptotene spermatocytes; Pa Sc, Pachytene spermatocytes; Rd St, round spermatids; SC, Sertoli cells. (E) Western blot analysis using sperm head and tail fractions. Both RIBC1 and RIBC2 were detected in the tail fraction. IZUMO1 and acetylated tubulin were detected as markers for sperm heads and tails, respectively. (F) Western blot analysis using fractionated proteins of mouse spermatozoa. RIBC1 was found in the Triton‐soluble and SDS‐soluble fractions, whereas RIBC2 was found in the SDS‐soluble and SDS‐resistant fractions. SLC2A3, acetylated tubulin, and AKAP4 were used as markers for the Triton‐soluble, SDS‐soluble, and SDS‐resistant fractions, respectively. Number of litters born per plug detected. The average litter size was decreased in all KO mouse lines compared with WT. However, only Ribc1/2 DKO male mice showed statistically significant differences with WT. P = 0.069 (WT vs. Ribc1 KO), P = 0.359 (WT vs. Ribc2 KO), and *P = 0.017 (WT vs. Ribc1/2 DKO), one‐way ANOVA followed by Tukey's multiple comparison test (G). SDS, sodium dodecyl sulfate; KO, knockout; WT, wild type.

FIGURE 2

No obvious abnormalities are found in the testis and spermatozoa of Ribc1 KO, Ribc2 KO, and Ribc1/2 DKO mice. (A) Gross morphology of testes in WT, Ribc1 KO, Ribc2 KO, and Ribc1/2 DKO male mice. (B) Testis weight of WT, Ribc1 KO, Ribc2 KO, and Ribc1/2 DKO male mice. No differences were observed (one‐way ANOVA followed by Tukey's multiple comparisons test, error bars represent SD). ns, not significant. (C) He‐PAS staining of testis sections in WT, Ribc1 KO, Ribc2 KO, and Ribc1/2 DKO male mice. (D) HE staining of cauda epididymis in WT, Ribc1 KO, Ribc2 KO, and Ribc1/2 DKO male mice. Sperm morphology from the cauda epididymis in WT, Ribc1 KO, Ribc2 KO, and Ribc1/2 DKO male mice (E). KO, knockout; WT, wild type; SD, standard deviation.

FIGURE 3

Sperm velocity is impaired in Ribc1 KO and Ribc1/2 DKO mice, but not in Ribc2 KO mice. (A) Percentage of motile spermatozoa in WT, Ribc1 KO, Ribc2 KO, and Ribc1/2 DKO mice. Different letters (a vs. b, and A vs. B) indicate significant differences according to one‐way ANOVA followed by Tukey's multiple comparisons test (P < 0.05, error bars represent SD). (B) Average path velocity (VAP) of motile spermatozoa in WT, Ribc1 KO, Ribc2 KO, and Ribc1/2 DKO mice. Different letters (a vs. b, a vs. c, b vs. c, A vs. B, A vs. C, and B vs. C) indicate significant differences according to one‐way ANOVA followed by Tukey's multiple comparisons test (P < 0.05, error bars represent SD). (C) Straight‐line velocity (VSL) of motile spermatozoa in WT, Ribc1 KO, Ribc2 KO, and Ribc1/2 DKO mice. Different letters (a vs. bc, a vs. c, bc vs. ab, ab vs. c, A vs. B, A vs. C, and B vs. C) indicate significant differences according to one‐way ANOVA followed by Tukey's multiple comparisons test (P < 0.05, error bars represent SD). (D) Curvilinear velocity (VCL) of motile spermatozoa in WT, Ribc1 KO, Ribc2 KO, and Ribc1/2 DKO mice. Different letters (a vs. b, a vs. c, b vs. c, and A vs. B) indicate significant differences according to one‐way ANOVA followed by Tukey's multiple comparisons test (P < 0.05, error bars represent SD). (E) Flagellar waveform patterns of motile spermatozoa in WT, Ribc1 KO, Ribc2 KO, and Ribc1/2 DKO mice analyzed 10 or 120 min after incubation. The motility was videotaped at 200 frames/s. Single frames throughout one beating cycle were superimposed. Ultrastructure of sperm tails in WT, Ribc1 KO, Ribc2 KO, and Ribc1/2 DKO mice. Cross‐sections of the midpiece and the principal piece were observed using TEM (F). KO, knockout; WT, wild type; SD, standard deviation; TEM, transmission electron microscope.