MPM-2 reactive sperm tail phosphoproteins: Isolation, localization and role during zygotic development

• Main Author: Long, Charles Roy
• Language: ENG
• Published: ScholarWorks@UMass Amherst 1996
• Subjects: Veterinary services|Cellular biology|Livestock
• Online Access: https://scholarworks.umass.edu/dissertations/AAI9638993

MPM-2, a monoclonal antibody specific for phosphorylated epitopes of mitotic proteins, has been used to identify unique 75-85 kD proteins of the mammalian sperm tail, named MPM-2 reactive sperm tail phosphoproteins (MSP). Due to the loss of MPM-2 reactivity following fertilization in bovine and rabbit zygotes, additional antibodies were produced to non-phosphorylated epitopes of MSP. One rabbit polyclonal sera (rMSPab) and three mouse monoclonal antibodies were produced. These antibodies were used to immunolabel rabbit and bull whole sperm cells and resulted in nearly identical patterns and labelled the sperm tail similar to MPM-2. Immunoelectron microscopy of whole rabbit sperm and rabbit testicular sections, showed MPM-2 reactivity primarily over the outer dense fibers and the capitulum of the sperm tail. Immunolabel with rMSPab revealed labelling of the outer dense fibers, fibrous sheath and the material surrounding the centrioles of the connecting piece. Immunoblots labelled with rMSPab indicated that reactivity is predominate at a 70-85 kD region of whole rabbit, bull, boar and mouse sperm proteins, with accessory labelled bands at 62, 64 and 55 kD. Immunoblots of extracted boar sperm proteins with MSP monoclonal antibodies displayed reactive bands at 70-85 kD as well as an additional cross reactive band at 55 kD, suggesting that the 55 kD protein is immunologically related. Zygote labelling with MSP antibodies revealed that MSP remains associated with the sperm tail throughout the first cell cycle and is centered in the sperm aster. Labelling of rabbit tissue proteins (spleen, liver, heart, lung, intestine, uterus, ovary and brain) on immunoblot with rMSPab and MPM-2 revealed that MSP is unique to mammalian sperm and all tissues contain immunologically related proteins, strongly reactive at 51-53 kD region. Injection of MSP antibodies into mature bovine oocytes, followed by sperm penetration, inhibited sperm derived microtubule aster formation. Taxol treatment of injected oocytes and zygotes suggested that MSP antibody blocked aster formation, but not by preventing microtubule nucleation. These data suggest that MSP is a structural component of the sperm tail outer dense fibers, specifically dephosphorylated following fertilization and blocking MSP with specific antibodies results in the failure of the sperm aster to develop but not by interfering with microtubule nucleation. We hypothesize that MSP is a possible intermediate filament type protein, forms a structural component of the sperm tail and MSP disassembly is a prerequisite for microtubule aster formation around the paternal centrosome.