Purification and characterization of 26S proteasomes from human and mouse spermatozoa

We purified by fractionation on 10-40% glycerol gradients, 26S proteasomesfrom normal human spermatozoa. These proteasomes, which participate in theATP-dependent degradation of ubiquitinated proteins, share a similarsedimentation coefficient to those purified from other human tissues.Fluorogenic peptide assays reveal they have chymotrypsin, trypsin andpeptidyl-glutamyl-like peptide hydrolysing activities; the chymotrypsinactivity is ablated by the specific 26S proteasome inhibitor MG132.Confirmation that these large proteases are 26S proteasomes is provided bydetection of the 20S proteasome subunits HC2, XAPC7, RN3 and Z andregulatory ATPases MSS1, TBP1, SUG1 and SUG2 by Western analyses withmonoclonal antisera. These antigens are found only in the gradientfractions enriched in proteolytic activities. We have also shown that,although mature spermatozoa from mice have considerably reduced amounts ofa ubiquitin-conjugating enzyme (E2) and ubiquitin-protein conjugates incomparison with less mature germ cells, they retain relatively high valuesof 26S proteasome activity. This suggests that proteasomes may have furtherroles to play in normal sperm physiology.