Abstract
Previous studies showed that zona binding inhibitory factor-1 (ZIF-1) was the glycoprotein mainly responsible for the spermatozoa zona binding inhibitory activity of human follicular fluid. ZIF-1 has a number of properties similar to glycodelin-A. A binding kinetics experiment in the present study demonstrated the presence of two binding sites of ZIF-1 on human spermatozoa. These binding sites were saturable, reversible, and bound to (125)I-ZIF-1 in a time-, concentration-, and temperature-dependent manner. Glycodelin-A shared one common binding site with ZIF-1 on spermatozoa, and it could displace only 70% of the (125)I-ZIF-1 bound on human spermatozoa. ZIF-1 and glycodelin-A formed complexes with the soluble extract of human spermatozoa. Coincubation of solubilized zona pellucida proteins reduced the binding of ZIF-1 to two complexes of the extract, suggesting that the ZIF-1 binding sites and zona pellucida protein receptors on human spermatozoa were closely related. ZIF-1, but not glycodelin-A, significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. The carbohydrate moieties derived from ZIF-1 reduced the binding of native ZIF-1 on human spermatozoa as well as the zona binding inhibitory activity of the glycoprotein, although the intensity of the effects are lower when compared with the native protein. These effects are not due to the action of the molecules on the motility, viability, and acrosomal status of the treated spermatozoa. Deglycosylated ZIF-1 had no inhibitory effect on both ZIF-1 binding and zona binding capacity of spermatozoa. We concluded that the carbohydrate part of ZIF-1 was critical for the functioning of the glycoprotein.
Highlights
Previous studies showed that zona binding inhibitory factor-1 (ZIF-1) was the glycoprotein mainly responsible for the spermatozoa zona binding inhibitory activity of human follicular fluid
The carbohydrate moieties derived from ZIF-1 reduced the binding of native ZIF-1 on human spermatozoa as well as the zona binding inhibitory activity of the glycoprotein, the intensity of the effects are lower when compared with the native protein
Equilibrium Studies—The mean number of spermatozoa bound to the control hemizona was 164.7 Ϯ 1.6. 125I-ZIF-1 and 125I-glycodelin-A had the same zona binding inhibitory activity as their native form (Fig. 2)
Summary
Semen Samples—The Ethics Committee of the University of Hong Kong approved the study protocol. The supernatant was obtained by centrifugation at 13,000 ϫ g at 4 °C for 20 min This extraction process was repeated twice, and the methanol supernatant containing the peptide N-glycosidase F-released oligosaccharide chain (ZIF-1-glyco/GdA-glyco) was pooled, dried in a vacuum concentrator (Virtis, New York), and redissolved in 20 l of water. The binding of labeled ZIF-1 (300 pmol/ml) to human spermatozoa (2 ϫ 106/ml) was determined in the presence of an increasing concentration (from 0.3 pmol/ml to 30 nmol/ml) of unlabeled ZIF-1, lipocalins, or EBSS/BSA at 37 °C for 3 h. To confirm that the effects of oligosaccharide chains or deglycosylated protein cores from ZIF-1 and glycodelin-A on spermatozoa zona pellucida binding are not due to their effects on sperm motility, viability, and acrosomal status, we studied these parameters of the spermatozoa treated above. A paired Student’s t test was used to compare the number of spermatozoa bound to zona pellucida between matching hemizona
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