Reproductive tract secretions and bull spermatozoa contain different clusterin isoforms that cluster cells and inhibit complement-induced cytolysis

Clusterin from bull rete testis fluid (RTF), cauda epididymal fluid (CEF), and octyl-beta-D-glucopyranoside extract of cauda epididymal sperm (CES) wa s identified and characterized using monoclonal and polyclonal antibodies ( Abs) developed against ram clusterin and a beta-subunit-specific oligopepti de of porcine clusterin. One-dimensional sodium dodecyl sulphate-polyacryla mide gel electrophoresis and western blotting showed that bovine RTF cluste rin had dimeric and monomeric molecular weights (M(r)s) of approximate to 9 4 kDa and of 42 and 43 kDa, respectively. Clusterin in CEF and CES had simi lar dimeric M(r)s (74 kDa). Reduced CEF clusterin appeared as three monomer s (M-r = 40, 39, and 38 kDa), whereas reduced CES clusterin appeared only a t M-r 40 kDa. Enzymatic deglycosylation resulted in similar M(r)s of cluste rin from RTF, CEF, and CES. The M, of RTF clusterin decreased from 94 kDa t o 51 kDa, indicating a carbohydrate content of 45%. After deglycosylation, the M-r of the CEF clusterin decreased from 74 kDa to two distinct bands at 57 and 50 kDa (with carbohydrate contents of 31 and 32%, respectively), su ggesting that two isoforms of the heterodimeric protein are present because of the two isoforms of the alpha-subunit. Under nonreduced conditions, a b eta-subunit-specific Ab reacted with M, of 36-38 kDa, indicating the existe nce of free clusterin beta-subunits in CES. RTF, CEF, and CES extracts all caused mouse fibroblastic L-cell aggregation. CEF cell aggregation was inhi bited by Hyb-17 Ab but not by other Abs. Both RTF and CEF caused a dose-dep endent inhibition of complement-induced cytolysis, although RTF clusierin w as more potent than CEF clusterin. We conclude that several isoforms of clu sterin occur in the bull reproductive tract and that the variation in carbo hydrate content among these isoforms may affect the biological or functiona l activity of the protein.