Binding characteristics of sperm with recombinant human zona pellucida glycoprotein-3 coated beads.

BACKGROUND & OBJECTIVE An inability or decreased ability of spermatozoa to bind to the zona pellucida (ZP), an extracellular glycoproteinaceous matrix surrounding egg, is one of the plausible causes of idiopathic infertility. It will be clinically useful to distinguish this condition from other causes of infertility. An assay system, investigating binding of human sperm with ZP glycoprotein may prove useful in this regard. We attempted to develop a simple assay system to analyse the binding of capacitated human spermatozoa to human zona pellucida glycoprotein-3 (ZP3) using baculovirus-expressed recombinant human ZP3 coated beads. METHODS Recombinant baculovirus-expressed ZP3 was purified, labelled with biotin and coated on streptavidin sepharose beads. An in vitro assay system was optimized to study binding of capacitated human sperm to ZP3 coated beads. RESULTS A higher percentage of baculovirus-expressed recombinant human ZP3 coated beads showed significant (P<0.05) binding of capacitated human sperm as compared to beads coated with fetuin. An inhibition in the binding of sperm to ZP3 coated beads was observed in presence of cold recombinant human ZP3. Further, prior incubation of ZP3 coated beads with monoclonal antibodies (MAbs) against ZP3 but not against ZP2 resulted in the decrease in number of sperm bound to bead. INTERPRETATION & CONCLUSION An in vitro assay system to study the binding of human sperm to ZP3- primary sperm receptor was established, which may be useful to determine the functional competence of spermatozoa.

[1]  A. Grootenhuis,et al.  Recombinant human zona pellucida protein ZP3 produced by chinese hamster ovary cells induces the human sperm acrosome reaction and promotes sperm-egg fusion. , 1994, Biology of reproduction.

[2]  W K Whitten,et al.  The culture of mouse embryos in vitro. , 1971 .

[3]  P. Wassarman,et al.  Mammalian sperm-egg interaction: Identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm , 1980, Cell.

[4]  Jingdong Tian,et al.  Gamete Interactions in Xenopus laevis: Identification of Sperm Binding Glycoproteins in the Egg Vitelline Envelope , 1997, The Journal of cell biology.

[5]  D. Wolf,et al.  Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid , 1986 .

[6]  S. Gupta,et al.  Production of monoclonal antibodies against recombinant human zona pellucida glycoproteins: utility in immunolocalization of respective zona proteins in ovarian follicles. , 2008, Journal of reproductive immunology.

[7]  L. Lipshultz,et al.  Conditions influencing human sperm capacitation and penetration of zona-free hamster ova. , 1984, Fertility and sterility.

[8]  S R M REYNOLDS,et al.  Physiology of reproduction. , 1948, Annual review of physiology.

[9]  D. C. Joseph.  Methods in mammalian embryology , 1971 .

[10]  G. Doncel,et al.  Mannose-binding sites on human spermatozoa and sperm morphology. , 1996, Fertility and sterility.

[11]  G. Fontenot,et al.  Cloning and characterization of zona pellucida genes and cDNAs from a variety of mammalian species: the ZPA, ZPB and ZPC gene families. , 1994, DNA sequence : the journal of DNA sequencing and mapping.

[12]  B. Rogers.  The sperm penetration assay: its usefulness reevaluated. , 1985, Fertility and sterility.

[13]  P. Talbot,et al.  Ultrastructural observations on binding and membrane fusion between human sperm and zona pellucida-free hamster oocytes. , 1982, Fertility and sterility.

[14]  M. Monk,et al.  Four zona pellucida glycoproteins are expressed in the human. , 2004, Human reproduction.

[15]  L. Zaneveld,et al.  Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. , 1984, Journal of reproduction and fertility.

[16]  H. Baker,et al.  Evaluation and assessment of semen for IVF/ICSI. , 2002, Asian Journal of Andrology.

[17]  S. Oehninger,et al.  Relationship between zona pellucida-induced acrosome reaction, sperm morphology, sperm-zona pellucida binding, and in vitro fertilization. , 2003, Fertility and sterility.

[18]  S. Gupta,et al.  Baculovirus expressed C‐terminal fragment of bonnet monkey (Macaca radiata) zona pellucida glycoprotein‐3 inhibits ZP3‐mediated induction of acrosomal exocytosis , 2005, Molecular reproduction and development.

[19]  S. Oehninger.  Biochemical and functional characterization of the human zona pellucida. , 2003, Reproductive biomedicine online.

[20]  R. Sharma,et al.  Bonnet monkey (Macaca radiata) ovaries, like human oocytes, express four zona pellucida glycoproteins , 2008, Molecular reproduction and development.

[21]  S. Kadunganattil,et al.  Relevance of glycosylation of human zona pellucida glycoproteins for their binding to capacitated human spermatozoa and subsequent induction of acrosomal exocytosis , 2008, Molecular reproduction and development.

[22]  K. Takahashi,et al.  Hypoosmotic swelling test of sperm. , 1990, Archives of andrology.

[23]  S. Gupta,et al.  Baculovirus-expressed recombinant human zona pellucida glycoprotein-B induces acrosomal exocytosis in capacitated spermatozoa in addition to zona pellucida glycoprotein-C. , 2005, Molecular human reproduction.

[24]  P. Wassarman,et al.  Interaction of mouse sperm with purified sperm receptors covalently linked to silica beads. , 1989, Journal of cell science.

[25]  K. Langer,et al.  Heterologous ovum penetration test and seminal parameters in fertile and infertile men. , 1983, Journal of andrology.

[26]  鍔本 浩志.  Expression of recombinant human zona pellucida protein 2 and its binding capacity to spermatozoa , 2007 .

[27]  A. Romeu,et al.  Carbohydrate analysis of the zona pellucida and cortical granules of human oocytes by means of ultrastructural cytochemistry. , 2004, Human reproduction.

[28]  D. Hornby,et al.  The polypeptide backbone of recombinant human zona pellucida glycoprotein-3 initiates acrosomal exocytosis in human spermatozoa in vitro. , 1998, The Biochemical journal.

[29]  Satyandra K. Gupta,et al.  Effects of Native Human Zona Pellucida Glycoproteins 3 and 4 on Acrosome Reaction and Zona Pellucida Binding of Human Spermatozoa , 2008, Biology of reproduction.

[30]  L. Furlong,et al.  Binding of recombinant human proacrosin/acrosin to zona pellucida glycoproteins. II. Participation of mannose residues in the interaction. , 2005, Fertility and sterility.

[31]  W. Yeung,et al.  Native human zona pellucida glycoproteins: purification and binding properties. , 2008, Human reproduction.

[32]  J. D. Neill,et al.  The Physiology of reproduction , 1988 .

[33]  C. Coddington,et al.  The hemizona assay (HZA): Development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential , 1989, Fertility and sterility.

[34]  M. Tanokura,et al.  Recombinant Porcine Zona Pellucida Glycoproteins Expressed in Sf9 Cells Bind to Bovine Sperm but Not to Porcine Sperm* , 2005, Journal of Biological Chemistry.