Immunofluorescence Reveals Ubiquitination of Retained Distal Cytoplasmic Droplets on Ejaculated Porcine Spermatozoa

The purpose of the present study was twofold: 1) to determine if antibodies raised against ubiquitin would recognize antigens associated with the porcine cytoplasmic droplet (CD), and 2) to determine if the same antibody would identify ubiquitinated substrates on the surface of morphologically abnormal boar spermatozoa. Permeabilization with the detergent Triton X-100 (0.05%) showed virtually all CDs to be ubiquitin positive. Distal droplets (DDs) retained in situ on boar spermatozoa were readily labeled following Triton permeabilization, whereas DDs present on non-permeabilized cells were not. Negative control preparations lacked the ubiquitin staining on the DD. The use of microtubes for fixation and incubation provided clearer images as well as better sperm cell distribution and density than an initial slide-mounted technique. Immunoblotting indicated that larger amounts of ubiquitinated proteins were present in extracts from sperm cells from an ejaculate with an abnormally high percentage of retained DDs (52% DDs) compared to a morphologically normal sample (6% DDs). The primary antibody recognized both mono-ubiquitin of bovine origin (8.5 kd) and human ubiquitin conjugate (35 kd), as demonstrated by Western blot. Preabsorption of the anti-ubiquitin antibody with purified bovine ubiquitin was successful in preventing diaminobenzidine staining of sperm extract from the high DD ejaculate. The presence of antigens recognized by anti-ubiquitin antibodies in the boar sperm CD, coupled with the possibility that superfluous ubiquitin species are detrimental to embryonic development by targeting critical paternally contributed zygotic organelles, raises concerns that retained DDs may be more detrimental to fertility than previously suspected.