Since hyaluronan enhances sperm motility, we studied the effect of hyaluronan on CBF in vitro using digital video-microscopy. Exposure of cultured ciliated epithelial cells to hyaluronan had no effect on CBF. However, when the endogenous hyaluronan on the apical surface of these cells was removed using hyaluronidase (in the presence of protease inhibitors), exposure to 50-100 mg/mL hyaluronan with an average molecular size of ~200 kDa increased CBF by about 15% above baseline (49). This increase was independent of the commercial hyaluronan source. Hyaluronan digested for an extensive period of time with hyaluronidase (18 h) to yield disaccharides, however, had no effect on CBF (3).
The next question was whether RHAMM is involved in signaling cilia to beat faster upon exposure to medium-sized hyaluronan. In fact, functionally blocking anti-RHAMM antibodies completely abolished the ciliary response to hyaluronan. Cells exposed to non-specific, control rabbit anti-chicken IgG responded to hyaluronan with an increase in CBF indistinguishable from untreated controls. These experiments reveal a clear functional role of RHAMM in increasing CBF. RHAMM's exact function in this process is unclear, however, since it lacks a transmembrane signaling component. Future studies are necessary to unravel this interesting problem, especially since the few signaling pathways known to be activated by RHAMM (e.g., ERK) have not been reported to regulate CBF and are usually slower than the observed responses of CBF to mid-sized hyaluronan.
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