Posttranslational modification by ubiquitination marks defective or outlived intracellular proteins for proteolytic degradation by the 26S proteasome. The ATP-dependent, covalent ligation and formation of polyubiquitin chains on substrate proteins requires the presence and activity of a set of ubiquitin activating and conjugating enzymes. While protein ubiquitination typically occurs in the cell cytosol or nucleus, defective mammalian spermatozoa become ubiquitinated on their surface during post-testicular sperm maturation in the epididymis, suggesting an active molecular mechanism for sperm quality control. Consequently, we hypothesized that the bioactive constituents of ubiquitin-proteasome pathway were secreted in the mammalian epididymal fluid (EF) and capable of ubiquitinating extrinsic substrates. Western blotting indeed detected the presence of the ubiquitin-activating enzyme E1 and presumed E1-ubiquitin thiol-ester intermediates, ubiquitin-carrier enzyme E2 and presumed E2-ubiquitin thiol-ester intermediates and the ubiquitin C-terminal hydrolase PGP 9.5/UCHL1 in the isolated bovine EF. Thiol-ester assays utilizing recombinant ubiquitin-activating and ubiquitin-conjugating enzymes, biotinylated substrates, and isolated bovine EF confirmed the activity of the ubiquitin activating and conjugating enzymes within EF. Ubiquitinated proteins were found to be enriched in the defective bull sperm fraction and appropriate proteasomal deubiquitinating and proteolytic activities were measured in the isolated EF by specific fluorescent substrates. The apocrine secretion of cytosolic proteins was visualized in transgenic mice and rats expressing the enhanced green fluorescent protein (eGFP) under the direction of ubiquitin-C promoter. Accumulation of eGFP, ubiquitin and proteasomes was detected in the apical blebs, the apocrine secretion sites of the caput epididymal epithelia of both the rat and mouse epididymal epithelium, although region-specific differences exist. Secretion of eGFP and proteasomes continued during the prolonged culture of the isolated rat epididymal epithelial cells in vitro. This study provides evidence that the activity of the ubiquitin system is not limited to the intracellular environment, contributing to a greater understanding of the sperm maturation process during epididymal passage.

High content of the platelet activating factor (PAF) and its plasma membrane receptor (PAFr) in semen is thought to benefit fertility in farm animals and humans. We used flow cytometric, biochemical, and immunocytochemical analysis to examine PAFr levels alone (Trial 1, n 5 156 bulls) or in a dual assay with sperm defect marker ubiquitin (UBI; Trial 2, n 5 88 bulls), in semen samples from 160 yearling bulls undergoing Breeding Soundness Evaluations (BSE). In both trials, we observed increased PAFr levels in semen samples with high content of white blood cells (WBC). Consequently, PAFr levels within such semen samples correlated negatively with several subjective parameters of BSE, including palpation, satisfaction of evaluation, and scrotal circumference. Due to a high WBC content, increased semen sample dilution had to be applied for microscopic evaluation. There was a negative correlation between semen PAFr and conventional sperm morphology, while the increased levels of PAFr correlated positively with sperm UBI content. Immunofluorescence microcopy revealed high expression of PAFr on the surface of leukocytes and morphologically normal spermatozoa, while reduced immunoreactivity was observed in defective spermatozoa immunoreactive to anti-UBI antibodies. A single PAFr band of appropriate mass was observed in Western blots of ejaculated spermatozoa, while testicular and epididymal spermatozoa also displayed several larger bands indicative of posttranslational processing or modification. Collectively, these data suggest that high levels of semen PAFr in young bulls are indicative of semen contamination with WBC. In the future, objective protein marker-based semen analyses in young bulls will likely require additional parameters distinguishing between marker expression in the spermatozoa and in the contaminating WBC. While identification of high sperm PAFr levels may support fertility, this assay alone is not reliable, due to the expression of PAFr in WBC that contaminate semen samples.