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• W2091360030 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 125I-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 125I-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. 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 125I-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 125I-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. zona binding inhibitory factor-1 Earle's balanced salt solution glycodelin-A glycodelin-S bovine serum albumin phosphate-buffered saline hemizona binding assay hemizona binding index zona pellucida deglycosylated ZIF-1 glycans of ZIF-1 deglycosylated GdA glycans of glycodelin-A Human follicular fluid from women undergoing controlled ovarian stimulation (1Yao Y.Q. Yeung W.S.B. Ho P.C. Hum. Reprod. 1996; 11: 2674-2680Crossref PubMed Scopus (28) Google Scholar) and natural cycle (2Chiu P.C.N. Ho P.C. Ng E.H.Y. Yeung W.S.B. Mol. Reprod. Dev. 2002; 61: 205-212Crossref PubMed Scopus (23) Google Scholar) in vitro fertilization and embryo transfer treatment inhibits the binding of spermatozoa to the zona pellucida. The number of spermatozoa bound to the zona decreases significantly after exposing the spermatozoa to human follicular fluid. The inhibitory effect occurred in all the follicular fluid samples tested (1Yao Y.Q. Yeung W.S.B. Ho P.C. Hum. Reprod. 1996; 11: 2674-2680Crossref PubMed Scopus (28) Google Scholar, 2Chiu P.C.N. Ho P.C. Ng E.H.Y. Yeung W.S.B. Mol. Reprod. Dev. 2002; 61: 205-212Crossref PubMed Scopus (23) Google Scholar, 3Qiao J. Yeung W.S.B. Yao Y.Q. Ho P.C. Hum. Reprod. 1998; 13: 128-131Crossref PubMed Scopus (33) Google Scholar). Zona binding inhibitory factor-1 (ZIF-1),1 a glycoprotein isolated from human follicular fluid, is partly responsible for the zona binding inhibitory activity of follicular fluid (4Yao Y.Q. Chiu P.C.N. Ip S.M. Ho P.C. Yeung W.S.B. Hum. Reprod. 1998; 13: 2541-2547Crossref PubMed Scopus (26) Google Scholar). Glycodelin, previously known as placental protein 14 (PP14), is a member of the lipocalin superfamily. It is a glycoprotein consisting of 180 amino acids with three putative N-glycosylation sites (Asn-28, Asn-63, and Asn-85). There are two known isoforms of glycodelin, amniotic fluid glycodelin (glycodelin-A, GdA) and seminal plasma glycodelin (glycodelin-S, GdS). The two isoforms have identical protein backbones but have different glycosylation processes (5Morris H.R. Dell A. Easton R.L. Panico M. Koistinen H. Koistinen R. Oehninger S. Patankar M.S. Seppala M. Clark G.F. J. Biol. Chem. 1996; 271: 32159-32167Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 6Koistinen H. Koistinen R. Dell A. Morris H.R. Easton R.L. Patankar M.S. Oehninger S. Clark G.F. Seppala M. Mol. Hum. Reprod. 1996; 2: 759-765Crossref PubMed Scopus (98) Google Scholar, 7Dell A. Morris H.R. Easton R.L. Panico M. Patankar M. Oehninger S. Koistinen R. Koistinen H. Seppala M. Clark G.F. J. Biol. Chem. 1995; 270: 24116-24126Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 8Seppala M. Taylor R.N. Koistinen H. Koistinen R. Milgrom E. Endocr. Rev. 2002; 23: 401-430Crossref PubMed Scopus (213) Google Scholar). Only glycodelin-A has spermatozoa zona pellucida binding inhibitory activity (9Oehninger S. Coddington C.C. Hodgen G.D. Seppala M. Fertil. Steril. 1995; 63: 377-383Abstract Full Text PDF PubMed Scopus (179) Google Scholar), whereas glycodelin-S does not affect spermatozoa zona binding (5Morris H.R. Dell A. Easton R.L. Panico M. Koistinen H. Koistinen R. Oehninger S. Patankar M.S. Seppala M. Clark G.F. J. Biol. Chem. 1996; 271: 32159-32167Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 10Koistinen H. Koistinen R. Kamarainen M. Salo J. Seppala M. Lab. Invest. 1997; 76: 683-690PubMed Google Scholar). These data suggest that glycosylation determines the biological activities of these two isoforms. ZIF-1 is similar to glycodelin-A in a number of aspects. They have similar molecular size: 28 kDa for glycodelin-A (8Seppala M. Taylor R.N. Koistinen H. Koistinen R. Milgrom E. Endocr. Rev. 2002; 23: 401-430Crossref PubMed Scopus (213) Google Scholar, 12Chryssikopoulos A. Mantzavinos T. Kanakas N. Karagouni E. Dotsika E. Zourlas P.A. Fertil. Steril. 1996; 66: 599-603Abstract Full Text PDF PubMed Scopus (34) Google Scholar) and 32 kDa for ZIF-1 (4Yao Y.Q. Chiu P.C.N. Ip S.M. Ho P.C. Yeung W.S.B. Hum. Reprod. 1998; 13: 2541-2547Crossref PubMed Scopus (26) Google Scholar). Both are found in follicular fluid of women undergoing assisted reproduction treatment (2Chiu P.C.N. Ho P.C. Ng E.H.Y. Yeung W.S.B. Mol. Reprod. Dev. 2002; 61: 205-212Crossref PubMed Scopus (23) Google Scholar, 4Yao Y.Q. Chiu P.C.N. Ip S.M. Ho P.C. Yeung W.S.B. Hum. Reprod. 1998; 13: 2541-2547Crossref PubMed Scopus (26) Google Scholar, 8Seppala M. Taylor R.N. Koistinen H. Koistinen R. Milgrom E. Endocr. Rev. 2002; 23: 401-430Crossref PubMed Scopus (213) Google Scholar, 12Chryssikopoulos A. Mantzavinos T. Kanakas N. Karagouni E. Dotsika E. Zourlas P.A. Fertil. Steril. 1996; 66: 599-603Abstract Full Text PDF PubMed Scopus (34) Google Scholar) and inhibit the binding of human spermatozoa to zona pellucida (4Yao Y.Q. Chiu P.C.N. Ip S.M. Ho P.C. Yeung W.S.B. Hum. Reprod. 1998; 13: 2541-2547Crossref PubMed Scopus (26) Google Scholar, 9Oehninger S. Coddington C.C. Hodgen G.D. Seppala M. Fertil. Steril. 1995; 63: 377-383Abstract Full Text PDF PubMed Scopus (179) Google Scholar) in a dose-dependent manner. Neither of the glycoproteins affects the motility and spontaneous acrosome reaction of spermatozoa in vitro (2Chiu P.C.N. Ho P.C. Ng E.H.Y. Yeung W.S.B. Mol. Reprod. Dev. 2002; 61: 205-212Crossref PubMed Scopus (23) Google Scholar, 4Yao Y.Q. Chiu P.C.N. Ip S.M. Ho P.C. Yeung W.S.B. Hum. Reprod. 1998; 13: 2541-2547Crossref PubMed Scopus (26) Google Scholar, 9Oehninger S. Coddington C.C. Hodgen G.D. Seppala M. Fertil. Steril. 1995; 63: 377-383Abstract Full Text PDF PubMed Scopus (179) Google Scholar). ZIF-1 is also immunologically similar to glycodelin (13Tse J.Y.M. Chiu P.C.N. Lee K.F. Seppala M. Koistinen H. Koistinen R. Yao Y.Q. Yeung W.S.B. Mol. Hum. Reprod. 2002; 8: 142-148Crossref PubMed Scopus (38) Google Scholar). Our observations using N-terminal sequencing and protease-digested peptide mapping to be published elsewhere show that ZIF-1 has the same protein core as glycodelin-A. However, they are different in their oligosaccharide chains as demonstrated by fluorophore-assisted carbohydrate electrophoresis, lectin binding ability, and isoelectric focusing. 2P. C. N. Chiu, R. Koistinen, H. Koistinen, M. Seppala, K. F. Lee, and W. S. B. Yeung, unpublished data. These data suggest that ZIF-1 is a differentially glycosylated isoform of glycodelin. It is generally believed that follicular fluid stimulates fertilization. The physiological role of the paradoxical activity of ZIF-1 on spermatozoa zona pellucida binding inhibition is unclear. The presence of specific receptor/binding sites is a prerequisite for most physiological activities. Our previous data showed the presence of specific binding of iodinated ZIF-1 on human spermatozoa (2Chiu P.C.N. Ho P.C. Ng E.H.Y. Yeung W.S.B. Mol. Reprod. Dev. 2002; 61: 205-212Crossref PubMed Scopus (23) Google Scholar). To study further the binding and biological properties of these ZIF-1 binding sites, we perform binding kinetics experiment of ZIF-1 on human spermatozoa. We confirm these findings by investigating the ability of ZIF-1 binding to soluble extract of spermatozoa. Spermatozoa zona pellucida binding in mammals involves carbohydrate-mediated events (14Wassarman P.M. Cell. 1999; 96: 175-183Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 15Tulsiani D.R. Yoshida-Komiya H. Araki Y. Biol. Reprod. 1997; 57: 487-494Crossref PubMed Scopus (181) Google Scholar, 16Benoff S. Mol. Hum. Reprod. 1997; 3: 599-637Crossref PubMed Scopus (200) Google Scholar). It has been shown that sperm carbohydrate-binding proteins bind to glycoconjugates of the zona pellucida in different species and mediate gamete recognition (17Macek M.B. Shur B.D. Gamete Res. 1988; 20: 93-109Crossref PubMed Scopus (137) Google Scholar, 18O'Rand M.G. Gamete Res. 1988; 19: 315-328Crossref PubMed Scopus (88) Google Scholar, 19Chapman N.R. Barratt C.L. Mol. Hum. Reprod. 1996; 2: 767-774Crossref PubMed Scopus (45) Google Scholar). We hypothesize that ZIF-1 exerts its zona binding inhibitory activity via its carbohydrate moieties. In the second part of this report, we study the effects of deglycosylation on the biological activity of ZIF-1 and demonstrate that the oligosaccharide chains derived from ZIF-1 as well as monosaccharides can compete with ZIF-1 for the binding sites on human spermatozoa. The Ethics Committee of the University of Hong Kong approved the study protocol. Semen samples with normal sperm parameters (20World Health Organization Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. Cambridge University Press, Cambridge, UK1998Google Scholar) from men visiting the subfertility clinics of the Queen Mary Hospital, University of Hong Kong were used in this study. Spermatozoa were separated by Percoll (Amersham Biosciences, Uppsala, Sweden) density gradient centrifugation as described previously (21Yeung W.S.B. Ng V.K.H. Lau E.Y.L. Ho P.C. Hum. Reprod. 1994; 9: 656-660Crossref PubMed Scopus (58) Google Scholar). The resulting pellet was resuspended in Earle's balanced salt solution (EBSS, Flow Laboratories, Irvine, UK) supplemented with sodium pyruvate, penicillin-G, streptomycin sulfate, and 3% bovine serum albumin. The spermatozoa were washed and resuspended in EBSS containing 0.3% BSA (EBSS/BSA) before experimentation. Fifteen batches of human follicular fluid samples (20 samples per batch) were collected during oocyte retrieval from women undergoing assisted reproduction treatment in the Queen Mary Hospital, Hong Kong. Human menopausal gonadotropins after down-regulation with gonadotropin releasing hormone agonist and human chorionic gonadotropin were used for ovarian stimulation in these women. Only human follicular fluid samples without blood contamination and from follicles with a retrieved oocyte were used in this study. The cell debris in human follicular fluid was removed by centrifugation at 300 × g for 10 min. The samples were sterilized by filtration with a 0.22-μm filter unit (Millipore, Bedford), pooled, and stored at −20 °C until used. Before experimentation, human follicular fluid was thawed and diluted with EBSS/BSA to the desired concentration. The purification of ZIF-1 was performed as described previously (2Chiu P.C.N. Ho P.C. Ng E.H.Y. Yeung W.S.B. Mol. Reprod. Dev. 2002; 61: 205-212Crossref PubMed Scopus (23) Google Scholar). Briefly, human follicular fluid was passed successively through a Hi-Trap blue and a protein-G column (Amersham Biosciences). The flow-through fraction from these two columns was applied to a concanavalin A-Sepharose column (Amersham Biosciences). The fraction that bound to the concanavalin A column was eluted with 0.3 m α-d-methylglucoside and was separated into two parts using Amicon-10 concentrator (Amicon Inc., Beverly, CA) according to their molecular size. The part with molecular size >10 kDa was further purified with Mono Q and Superose columns as described (4Yao Y.Q. Chiu P.C.N. Ip S.M. Ho P.C. Yeung W.S.B. Hum. Reprod. 1998; 13: 2541-2547Crossref PubMed Scopus (26) Google Scholar). The concentration of purified ZIF-1 was measured with a protein assay kit (Bio-Rad, Hercules, CA). The yield of ZIF-1 obtained was 30–100 μg/liter. Glycodelin-A and glycodelin-S were purified from amniotic fluid and seminal plasma, respectively. Seminal plasma was diluted 1:4 (v/v) with 50 mmTris-HCl-buffered saline containing 9 g/liter NaCl, pH 7.7 (TBS), before purification. Triton X-100 (0.1%, v/v) was mixed with amniotic fluid or diluted seminal plasma and passed through a monoclonal anti-glycodelin antibody (clone F43-7F9)-Sepharose column as described previously (22Riittinen L. Narvanen O. Virtanen I. Seppala M. J. Immunol. Methods. 1991; 136: 85-90Crossref PubMed Scopus (48) Google Scholar). The column was then washed successively with TBS, 1m NaCl, containing 1% isopropanol and 10 mmammonium acetate before the bound glycodelin was eluted with 0.1% trifluoroacetic acid. The purified protein was dialyzed against 100 mm sodium phosphate, pH 7.2, and its concentration was measured. Glycodelin-S was further purified using anion exchange column as described (13Tse J.Y.M. Chiu P.C.N. Lee K.F. Seppala M. Koistinen H. Koistinen R. Yao Y.Q. Yeung W.S.B. Mol. Hum. Reprod. 2002; 8: 142-148Crossref PubMed Scopus (38) Google Scholar). A kit,N-Glycosidase F Deglycosylation (Bio-Rad), was used to deglycosylate ZIF-1 or glycodelin-A. Fifty microliters of ZIF-1 or glycodelin-A (1 μg/μl in PBS) was denatured in 1 μl of 5% SDS and 1.5 μl of 1:10 dilution of β-mercaptoethanol at 95 °C for 5 min. Four microliters of 10% Nonidet P-40 was added to entrap excess SDS after the reaction mixture was cooled to room temperature. The denatured protein was incubated in an equal volume of releasing buffer supplied with the kit containing 2 μl of N-glycosidase F for 24 h at 37 °C. Deglycosylated protein was obtained after three successive protein precipitations, each with 3 volumes of cold 100% ethanol followed by centrifugation for 5 min at 5000 ×g. The deglycosylated ZIF-1 (deglyco-ZIF-1) or glycodelin-A (deglyco-GdA) obtained was dried in a vacuum concentrator (Virtis, New York), redissolved in 20 μl of PBS and further purified by gel filtration using Superdex-75 column in a SMART system (Amersham Biosciences). The purity of the deglycosylated proteins was checked by SDS-PAGE, and their concentrations were determined. Oligosaccharide chains from ZIF-1 and glycodelin-A were isolated as reported (23Verostek M.F. Lubowski C. Trimble R.B. Anal. Biochem. 2000; 278: 111-122Crossref PubMed Scopus (33) Google Scholar). In brief, the glycoproteins were denatured as described above except that no Nonidet P-40 was added. The denatured protein was precipitated by the addition of 4 volumes of 80% acetone at −20 °C, incubation of the mixture at −20 °C for 30 min and centrifugation at 13,000 × g at 4 °C for 20 min. The pellet obtained was redissolved and deglycosylated using theN-Glycosidase F Deglycosylation kit (Bio-Rad). Deglycosylated protein and oligosaccharide chains were precipitated in 80% acetone at −20 °C for 1 h, and the supernatant was discarded. The pellet was first triturated (23Verostek M.F. Lubowski C. Trimble R.B. Anal. Biochem. 2000; 278: 111-122Crossref PubMed Scopus (33) Google Scholar) in 50 μl of ice-cold 60% aqueous methanol and subsequently dispersed in 1 ml of the same solution. The supernatant was obtained by centrifugation at 13,000 × 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 μl of water. The percentage recovery of oligosaccharide chains using this method was greater than 90% (23Verostek M.F. Lubowski C. Trimble R.B. Anal. Biochem. 2000; 278: 111-122Crossref PubMed Scopus (33) Google Scholar). Therefore, the amount of oligosaccharide chains used in each experiment was expressed as the amount of native glycoprotein required for producing that quantity of oligosaccharide chains. Fifty microliters of PBS was also processed through all the above procedures and served as control (PBS control). The hemizona binding assay was performed as described (24Yao Y.Q. Yeung W.S.B. Ho P.C. Hum. Reprod. 1996; 11: 1516-1519Crossref PubMed Scopus (23) Google Scholar). Unfertilized oocytes from our intracytoplasmic sperm injection program were bisected into two identical hemizonae by a micromanipulator. In the assay, each hemizona was incubated with 2 × 105 spermatozoa/ml in a 100-μl droplet of EBSS/BSA under mineral oil for 3 h at 37 °C in an atmosphere of 5% CO2 in air. We used a sperm concentration higher than those in a typical HZA because more spermatozoa would bind to the zona pellucida in this condition and, therefore, the variation of the assay would be smaller. Loosely attached spermatozoa were removed by pipetting the hemizonae through a micropipette of internal diameter 200 μm. The numbers of tightly bound spermatozoa on the outer surface of the hemizonae were counted. The hemizona binding index (HZI) was defined as, HZI=number of spermatozoa bound in test dropletnumber of spermatozoa bound in control droplet×100Equation 1 Fifty micrograms of ZIF-1/gylcodelin-A in 0.02 ml of 0.05 m PBS (pH 7.4) was mixed with 2 mCi of carrier-free Na125I (20 μl, Amersham Biosciences, England) in a small conical vial. Freshly prepared chloramine T (100 μg in 0.02 ml of 0.05 m PBS, pH 7.4) was then added and mixed thoroughly. After 60 s, sodium metabisulfite (300 μg in 0.05 ml of 0.05 m PBS, pH 7.4) was used to stop the reaction. Free 125I was removed by passing the mixture through a 10-ml disposable desalting column. The first radioactive peak containing iodinated ZIF-1/glycodelin-A was collected. Percoll-processed spermatozoa (n = 5) were divided into eight portions. They were incubated with 0, 0.01, 0.1, 1, 10, 25, or 50 μg/ml125I-labeled ZIF-1/glycodelin-A in a 1-ml volume or in unlabeled ZIF-1/glycodelin-A at 37 °C in an atmosphere of 5% CO2 for 3 h. After incubation, the spermatozoa were washed with fresh EBSS/BSA. HZA were performed on these treated spermatozoa. Two million spermatozoa in 1 ml of EBSS/BSA were incubated with different concentrations (from 0.03 to 3000 pmol/ml) of 125I-ZIF-1 at 37 °C for 3 h. The binding was terminated by the addition of 15 ml of ice-cold PBS followed by centrifugation at 150 × g for 10 min. The spermatozoa were further washed twice with fresh EBSS. A gamma counter (Model 5500B, Beckman) was used to count the radioactivity associated with the spermatozoa. Specific binding of ZIF-1 was determined by subtracting the counts bound in the presence of a 100-fold concentration of unlabeled ZIF-1 from the counts bound in the absence of unlabeled protein. The determinations of total binding and nonspecific binding were done in triplicate. After equilibration of 2 × 106spermatozoa (n = 3) to the desired temperature (4, 25, or 37 °C), 125I-ZIF-1 (300 pmol/ml) was added at the appropriate temperature and mixed rapidly. The mixture was incubated for 0, 5, 10, 20, 30, 60, 90, 120, 150, 180, or 210 min. A large volume of ice-cold buffer was added to terminate the binding. Nonspecific binding was determined by the inclusion of 30 nmol/ml cold ZIF-1. Bound and free ligands were separated by centrifugation at 150 ×g for 10 min. The radioactivity associated with the treated spermatozoa after washing twice in EBSS/BSA was measured. Two million human spermatozoa in 1 ml of EBSS/BSA were incubated with 125I-ZIF-1 (300 pmol/ml) at 37 °C for 150 min. A 100-fold excess of unlabeled ZIF-1 (30 nmol/ml) was then added. After a further incubation for 0, 5, 10, 20, 30, 60, 90, 120, 180, 210, 240, 270, 300, or 360 min, 15 ml of ice-cold buffer was added to stop the dissociation. The spermatozoa were then washed and their associated radioactivity was determined. Competition binding analysis was used to investigate the affinity of ZIF-1 binding sites to glycodelin and other lipocalins. The binding of labeled ZIF-1 (300 pmol/ml) to human spermatozoa (2 × 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. The lipocalins used included glycodelin-A, glycodelin-S, bovine β-lactoglobulin A (Sigma), and human retinol-binding protein (Sigma). Another zona binding inhibitory factor that we purified from the human follicular fluid, ZIF-2 (4Yao Y.Q. Chiu P.C.N. Ip S.M. Ho P.C. Yeung W.S.B. Hum. Reprod. 1998; 13: 2541-2547Crossref PubMed Scopus (26) Google Scholar), was also used in this competition assay. Cell-bound radioactivity was determined after washing the treated spermatozoa twice with EBSS/BSA. Each individual experiment was repeated three times. Percoll-processed spermatozoa (n = 5) were divided into eleven portions. Each portion was incubated in 0, 0.01, 0.1, 1, or 10 μg/ml ZIF-1, deglycosylated ZIF-1 (deglyco-ZIF-1), glycans of ZIF-1 (ZIF-1-glyco), glycodelin-A (GdA), deglycosylated glycodelin-A (deglyco-GdA), glycans of glycodelin-A (GdA-glyco), or EBSS/BSA (control) at 37 °C in an atmosphere of 5% CO2 in air for 3 h. After incubation, the spermatozoa were washed with fresh EBSS/BSA. HZA were performed on the treated spermatozoa. 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 also studied these parameters of the spermatozoa treated above. The acrosomal status of the spermatozoa was evaluated by fluorescein isothiocyanate-Pisum sativum agglutinin (Sigma) as described (2Chiu P.C.N. Ho P.C. Ng E.H.Y. Yeung W.S.B. Mol. Reprod. Dev. 2002; 61: 205-212Crossref PubMed Scopus (23) Google Scholar). Hoechst staining was used to determine the viability of the spermatozoa. The acrosomal status and viability of 300 spermatozoa were determined in randomly selected fields under a fluorescence microscope (Zeiss, Germany) with ×1000 magnification. The filter set used for Hoechst staining consisted of an excitation filter G365, a chromatic beam splitter FT395, and a barrier filter LP420, whereas that for fluorescein isothiocyanate-Pisum sativumagglutinin staining consisted of an excitation filter BP 450–490, a chromatic beam splitter FT510, and a barrier filter LP520. The acrosomal status of spermatozoa was classified into: (i) intact acrosome: complete staining of the acrosome; (ii) reacting acrosome: Hoechst-negative and partial or patchy staining of the acrosome; and (iii) reacted acrosome: Hoechst-negative and complete staining of the equatorial segment only or no staining of the whole sperm head. The proportions of spermatozoa with reacted acrosomes were determined. Hobson Sperm Tracker System (HST, Hobson Tracking Systems Ltd., Sheffield, UK) was used to determine sperm motility. The system consisted of a phase-contrast microscope, closed circuit television video camera, a microcomputer, a tracking screen, and HST software (Hobson Tracking Systems Ltd.). The set-up parameters of the system were: (i) framing rate, 50 Hz; (ii) aspect ratio, 1.39; (iii) calibration, ×10.0; (iv) refresh time, 5 s; (v) thresholds, +20/−20; (vi) filter weights, 1 and −1, 2 and 0, 3 and 0, 4 and 0; (vii) minimum chamber depth, 20 μm; (viii) minimum trail point, 25; (ix) maximum trail point, 250; and (x) trail draw time, 3 s. The motility of spermatozoa was analyzed on a warmed microscope stage at 37 °C with a Cell-VU disposable semen analysis chamber (Fertility Technologies, Inc., Natick, MA). Five hundred spermatozoa per specimen in randomly selected fields were evaluated to determine 1) curvilinear velocity (VCL, μm/s), 2) mean straight line velocity (VSL, μm/s), 3) average path velocity (VAP, μm/s), 4) mean linearity (LIN, VSL/VCL), 5) amplitude of lateral head displacement (ALH, μm), 6) head beat cross-frequency (BCF, Hz), and 7) percentage of motile sperm (MOT). The binding of 125I-ZIF-1 (300 pmol/ml) to human spermatozoa (2 × 106/ml) was measured in the presence of increasing concentration (from 0.3 pmol/ml to 30 nmol/ml) of deglyco-ZIF-1, ZIF-1-glyco, deglyco-GdA, GdA-glyco, or EBSS/BSA (control). After incubation at 37 °C for 3 h, the treated spermatozoa were washed with fresh EBSS/BSA and the cell-bound radioactivity was measured. Each experiment was repeated three times. Sperm extracts was isolated as described (25Shi Y.L. Ma X.H. Mol. Reprod. Dev. 1998; 50: 354-360Crossref PubMed Scopus (25) Google Scholar). Spermatozoa were separated from the seminal plasma by centrifugation at 5000 rpm for 5 min, washed three times with 0.01m PBS, pH 7.4, and resuspended in 0.01 mTris-HCl buffer containing 0.5 m KCl, 1 mmEDTA, 0.2 mm phenylmethylsulfonyl fluoride, 5% glycine, pH 8.3. Triton X-100 (1% v/v) was then added to the sperm suspension and stirred at 4 °C overnight. The insoluble material was discarded after centrifugation at 15,000 rpm for 40 min. The supernatant containing mainly proteins of the plasma membrane and acrosome (25Shi Y.L. Ma X.H. Mol. Reprod. Dev. 1998; 50: 354-360Crossref PubMed Scopus (25) Google Scholar) was dialyzed against 100 mm sodium phosphate, pH 7.2. The protein concentration in the solution was determined by using a commercial kit (Bio-Rad). Human zona pellucida was separated from the oocytes using glass micropipettes under a microscope (26Franken D.R. Kruger T.F. Oehninger S.C. Andrologia. 1994; 26: 277-281Crossref PubMed Scopus (18) Google Scholar) and heat-solubilized at 70 °C for 90 min in distilled water with pH adjusted to 9 with Na2CO3 (27Dunbar B.S. Wardrip N.J. Hedrick J.L. Biochemistry. 1980; 19: 356-365Crossref PubMed Scopus (187) Google Scholar). The purified sperm extracts was divided into six identical portions (10 μg/ml). Each portion was incubated with 1 μg/ml125I-ZIF-1 in the presence of zona pellucida protein at concentrations of 0, 0.01, 0.05, 0.1, or 0.2 zona pellucida/μl, or with 1 μg/ml 125I-glycodelin-A at 37 °C for 3 h. After incubation, the mixtures were analyzed by native-gel electrophoresis, and the radioactive bands were visualized by exposing the gel to BIO-MAX film (Kodak, New York). Spermatozoa (n = 5) were incubated in 0.03, 3, and 30 pmol/ml ZIF-1, glycodelin-A, or EBSS/BSA (as control) at 37 °C under 5% CO2 in air for 3 h. After washing with fresh EBSS/BSA, the treated spermatozoa were incubated with 1 μg/ml progesterone or EBSS/BSA (as control) for 30 min. The acrosomal status of the spermatozoa was then evaluated as described above. All the data were expressed as means ± S.E. The data were analyzed by SigmaStat statistical software (SigmaPlot 2000 & Enzyme Kinetics Analysis Module 1.0, Jandel Scientific, San Rafael, CA). A paired Student's t test was used to compare the number of spermatozoa bound to zona pellucida between matching hemizona. After glycosidase digestion and purification by gel filtration, both ZIF-1 and glycodelin-A showed a single band of size 19 kDa (Fig. 1) corresponding to the size of deglycosylated glycodelin-A. Our unpublished data showed that the sequence for the first 25 amino acids from the N-terminal of deglycosylated ZIF-1 was identical to that reported for glycodelin-A (11Julkunen M. Seppala M. Janne O.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8845-8849Crossref PubMed Scopus (168) Google Scholar). The iden" @default.
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• W2091360030 title "Binding of Zona Binding Inhibitory Factor-1 (ZIF-1) from Human Follicular Fluid on Spermatozoa" @default.
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