Rat membrane cofactor protein (MCP; CD46) is expressed only in the acrosome of developing and mature spermatozoa and mediates binding to immobilized activated C3.
The rat analogue of the complement regulator membrane cofactor protein (MCP; CD46) was recently cloned and analysis at the mRNA level suggested that expression was restricted to testis. In light of the proposed roles of human MCP in sperm-egg interaction, we undertook to analyze rat MCP expression at the protein level in order better to address its putative role in fertilization. Recombinant fusion proteins comprising antibody Fc and specific domains of rat MCP were generated and used to develop a monoclonal antibody, MM.1, specific for rat MCP. Immunohistochemistry using these reagents confirmed the reported testis-specific expression of MCP in sexually mature rats and demonstrated that MCP was expressed only by spermatozoa and their immediate precursors in spermiogenesis, spermatids. Prepubertal male rats did not express MCP, and there was no evidence of MCP expression at any site in the embryo. Spermatozoal MCP expression was restricted to the inner acrosomal membrane, exposed only after fixation or induction of the acrosome reaction. Acrosome-reacted but not unreacted spermatozoa bound methylamine-activated C3 immobilized on plastic. The retention of MCP at this subcellular site, which is probably crucial to sperm-egg interaction, and the functional demonstration of binding to activated C3 strengthen suggestions from human studies that MCP may play an important role in fertilization. The reagents and results described here will enable studies of the role of spermatozoal MCP in sperm-egg interaction using a relevant animal model system.
Mizuno
Masashi
M
Complement Biology Group, Department of Medical Biochemistry and Immunology, University of Wales College of Medicine, Cardiff CF14 4XN, United Kingdom.
Harris
Claire L
CL
Johnson
Peter M
PM
Morgan
B Paul
BP
D000177:Acrosome / Q000378:metabolism*; D020101:Acrosome Reaction / Q000502:physiology; D000367:Age Factors; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D003176:Complement C3 / Q000378:metabolism*; D006224:Cricetinae; D007150:Immunohistochemistry; D008297:Male; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D013087:Spermatids / Q000378:metabolism*; D013091:Spermatogenesis / Q000235:genetics / Q000502:physiology; D013094:Spermatozoa / Q000378:metabolism*
D015703:Antigens, CD; C497860:Cd46 protein, rat; D003176:Complement C3; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins; D012333:RNA, Messenger