Ubiquitin-dependent Sperm Quality Control Mechanism Recognizes Spermatozoa with DNA Defects As Revealed by Dual Ubiquitin-TUNEL Assay

Defective mammalian spermatozoa become ubiquitinated during epididymal passage, a mechanism that may mark the abnormal spermatozoa for proteolytic destruction (Sutovsky et al., 2001a: J Cell Sci 114:1665-1675). It is not known how such spermatozoa are recognized by the epididymal ubiquitination pathway and whether there is a selection against certain types of sperm defects. We examined the relationship between sperm ubiqutination, lifelong sperm morphology and sperm DNA defects using a single chanel, ubiquitin-activated flow cytometric assay, and a dual, ubiquitin-TUNEL assay. Semen samples from nine service sires of good-to-average fertility were screened. A positive correlation was found between sperm ubiquitination and the average frequency of morphological semen abnormalities from field evaluations performed throughout the reproductive life of individual sires. Sample correlation coefficients were r=0.65 for primary (head and tail) and r=0.60 for total semen abnormalities in the single channel assay. In a dual assay, we found a high, positive correlation (r=0.93) between the ubiquitin-positive sperm and the TUNEL positive sperm. Substantial correlations (r=0.47-0.64) were observed when the measurements from these two respective assays were compared for individual sires. While anti-ubiquitin antibodies recognized most of the TUNEL-positive sperm cells, the TUNEL-positive spermatozoa represented only a subset (approximately 20-40%) of all ubiquitin-positive cells. It appears that the ubiquitin-dependent sperm quality control, residing in the epididymal epithelium, has the ability to detect spermatozoa with apoptotic or necrotic DNA, while spermatozoa with defects other than DNA fragmentation are also recognized and ubiquitinated.