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.