A comparative analysis of expression and processing of the rat epididymal fluid and sperm-bound forms of proteins D and E.
The mammalian epididymis secretes numerous proteins important for sperm maturation. Among these are proteins D and E, which belong to the CRISP family (cysteine-rich secretory proteins) and are the product of the Crisp-1 gene. These proteins have been the focus of a number of studies and have been implicated in sperm/egg fusion. Protein D and protein E have been purified to apparent homogeneity in several laboratories. Polyclonal antibodies raised against each protein typically cross-reacted with both proteins, suggesting that they were immunologically similar, if not identical. Our laboratory has previously reported the generation of a monoclonal antibody (mAb 4E9) that recognizes only protein E. Using mAb 4E9, the localization of protein E was shown to be domain specific on the sperm surface and there is processing of the protein in the fluid, with only the lowest molecular weight form associating with sperm. Subsequent purification and amino acid sequencing of protein D confirmed that proteins D and E are nearly identical and differ only by presence of the 4E9 epitope on protein E. Here we report the generation of antibodies to regions of amino acid sequence identity in proteins D and E. Using these antibodies, we demonstrate that protein D associates with the sperm head and that a portion of this protein may be proteolytically processed. In addition, we demonstrate that the proteolytic processing of protein E occurs in the carboxy terminal region of this protein. The data also suggest that a portion of protein D may also undergo processing, similar to that of protein E. Finally, we use these antibodies to demonstrate that proteins D and E are differentially expressed by the epididymal epithelium. Taken together, these data suggest that proteins D and E may have individual roles in sperm function.
Department of Genetics, Cell Biology, and Development, University of Minnesota, 321 Church Street SW, Minneapolis, MN 55455, USA.
Kenneth P
KP
Roberts
Kathy M
KM
Ensrud
David W
DW
Hamilton
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D001826:Body Fluids / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004822:Epididymis / Q000378:metabolism*; D000939:Epitopes / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics*; D007150:Immunohistochemistry; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics*; D008566:Membranes / Q000378:metabolism; D018613:Microscopy, Confocal; D019151:Peptide Library; D051381:Rats; D017207:Rats, Sprague-Dawley; D029589:Seminal Plasma Proteins / Q000096:biosynthesis* / Q000235:genetics*; D013094:Spermatozoa / Q000378:metabolism*
D000911:Antibodies, Monoclonal; C486320:Crisp3 protein, rat; D000939:Epitopes; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D019151:Peptide Library; D029589:Seminal Plasma Proteins; C470441:protein D, rat