Gamete Biology

Differential Ubiquitination of Stallion Sperm Proteins: Possible Implications for Infertility and Reproductive Seasonality

Abstract

Antibodies against ubiquitin (Ubi), a universal proteolytic marker, show increased cross-reactivity with defective spermatozoa in men and bulls. We investigated sperm ubiquitination in the stallion, a seasonally polyestrous mammal. Immunofluorescence and immunoelectron microscopy demonstrated that anti-Ubi antibodies bind to the surface of both membrane intact and aldehyde-fixed spermatozoa. Cross-reactivity to the ubi-conjugating enzyme E2 was also detected in sperm. Immunohistochemistry showed that ubiquitinated spermatozoa first were detected in the caput epididymis, coincident with a strong accumulation of Ubi and Ubi-C-terminal hydrolase, PGP 9.5, in the apical stereocilia of the epididymal epithelium. Testicular spermatozoa did not display significant Ubi cross-reactivity. Similarly, lesser accumulation of Ubi-cross-reactive substrates was identified in the accessory sex glands. Semen samples were collected from three fertile and one subfertile stallion between December and February and probed for Ubi by flow cytometry and immunoblotting. Flow cytometric analysis showed that sperm from the subfertile stallion had higher Ubi levels than the other 3 stallions. Additionally, immunoblot analysis of sperm proteins from the subfertile stallion showed two unique Ubi-cross-reactive bands that were not present in sperm extracts from the three fertile stallions. To screen for a possible role for Ubi in seasonal changes in sperm production, semen samples from two fertile stallions were collected in March, June, September and December and subjected to a flow cytometric Ubi-assay. The lowest levels of Ubi-labeled sperm were found in March, approximately coincident with the onset of the natural horse breeding season. A progressive increase in sperm Ubi levels were found during summer and fall, with a peak in December. These data suggest that stallion sperm are differentially ubiquitinated during epididymal maturation and that this ubiquitination may reflect changes in sperm numbers and semen quality. The association between changes in sperm ubiquitination and seasonal changes in sperm production will be subjected to further studies in a larger cohort of animals.