Identification of apolipoprotein A1 and immunoglobulin as components of a serum complex that mediates activation of human sperm motility.
Serum is superior to other body fluids in activating the progressive motility of human spermatozoa and is used in connection with sperm separation for fertilization in vitro. The major activating capacity is localized to a macromolecular fraction, purified to homogeneity by a four-step protocol with ion-exchange chromatography, chromatofocusing, exclusion FPLC (elution corresponding to a molecular mass of about 250 kDa), and Blue Sepharose chromatography (no binding but elimination of albumin). The pure protein, at a concentration of 20-70 nmol/L, activated the motility to the same extent as serum. SDS-polyacrylamide gel electrophoresis under nonreducing conditions showed one band corresponding to a molecular mass of about 180 kDa. In the presence of mercaptoethanol, two bands are obtained corresponding to 50 kDa and about 25 kDa, respectively. Without the Blue Sepharose step, the sample after reduction revealed an additional band at about 67 kDa, suggesting that the fraction is then in complex also with albumin. Amino acid sequence analysis of the Blue Sepharose eluate identified three protein chains--those of apolipoprotein A1 and immunoglobulin heavy and light chains--suggesting that the preparation is an apolipoprotein A1-immunoglobulin complex. Antiserum raised toward the pure preparation in a rabbit inhibited human sperm motility, when added directly to spermatozoa. Pretreatment of human serum with rabbit antiserum significantly reduced its ability to activate sperm motility. The sperm activating capacity of the protein complex was destroyed by heating at 100 degrees C for 5 min, suggesting that the activity was dependent on intact protein conformations. Albumin, apolipoprotein A1, and immunoglobulins by themselves had only minor effects on sperm motility.(ABSTRACT TRUNCATED AT 250 WORDS)
Akerlöf
E
E
Department of Clinical Chemistry, Karolinska Institutet, Huddinge, Sweden.
Jörnvall
H
H
Slotte
H
H
Pousette
A
A
D000595:Amino Acid Sequence; D000818:Animals; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000097:blood*; D002845:Chromatography; D006801:Humans; D007106:Immune Sera / Q000737:chemistry; D007136:Immunoglobulins / Q000302:isolation & purification*; D008297:Male; D008969:Molecular Sequence Data; D011817:Rabbits; D013081:Sperm Motility
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D007106:Immune Sera; D007136:Immunoglobulins