Rhbg is dispensable for spermatogenesis but fine-tunes sperm motility trajectory during capacitation in mice

Rhesus (Rh) family B glycoprotein (RHBG) is a conserved ammonium transporter of the Rhesus family with established roles in systemic acid-base regulation. Although Rh family members are expressed in the male reproductive tract, the physiological role of RHBG in spermatogenesis and sperm function remains unclear. To investigate its role in male reproduction, Rhbg-/- mice were generated using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene editing, and male fertility and reproductive phenotypes were assessed by breeding assays, histological analyses of the testes and epididymides, sperm morphology evaluation, computer-assisted sperm analysis during capacitation, and transmission electron microscopy of flagellar ultrastructure. Rhbg-/- male mice showed largely preserved fertility and produced offspring numbers comparable to those of wild-type controls, although a modest reduction in litter size was observed. Consistently, testicular architecture, spermatogenesis, and epididymal morphology were normal, and transmission electron microscopy confirmed intact flagellar ultrastructure. Computer-assisted sperm analysis further showed normal progressive motility under both basal and capacitating conditions. However, under hyperactivation-associated conditions, Rhbg-/- sperm exhibited a mild reduction in total motility, accompanied by subtle decreases in straight-line velocity and straightness. In addition, a slight increase in tail-coiling abnormalities was observed, whereas overall sperm morphology remained largely normal. Together, these findings indicate that Rhbg is not essential for spermatogenesis or male fertility in mice, but its absence causes minor, context-dependent alterations in sperm trajectory efficiency during capacitation without affecting core motility or ultrastructural integrity. Thus, RHBG may contribute to the fine regulation of sperm motility behavior rather than acting as a major determinant of reproductive capacity.