Polypeptide encoded by mouse ZP3 exon-7 is necessary and sufficient for binding of mouse sperm in vitro

Zev Williams, Eveline S. Litscher, Luca Jovine, Paul M. Wassarman

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

Fertilization in mice is initiated by species-specific binding of sperm to mZP3, one of three mouse zona pellucida (ZP) glycoproteins. At nanomolar concentrations, purified egg mZP3 binds to acrosome-intact sperm heads and inhibits binding of sperm to eggs in vitro. Although several reports suggest that sperm recognize and bind to a region of mZP3 encoded by mZP3 exon-7 (so-called, sperm combining-site), this issue remains controversial. Here, exon-swapping and an IgG(Fc) fusion construct were used to further evaluate whether mZP3 exon-7 is essential for binding of sperm to mZP3. In one set of experiments, hamster ZP3 (hZP3) exon-6, -7, and -8 were individually replaced with the corresponding exon of mZP3. Stably transfected embryonal carcinoma (EC) cell lines carrying the recombinant genes were produced and secreted recombinant glycoprotein was purified and assayed for the ability to inhibit binding of sperm to eggs. While EC-hZP3, a recombinant form of hZP3 made by EC cells, is unable to inhibit binding of mouse sperm to eggs in vitro, the results suggest that substitution of mZP3 exon-7 for hZP3 exon-7, but not mZP3 exon-6 or -8, can impart inhibitory activity to EC-hZP3. In this context, a fusion construct consisting of human IgG(Fc) and mZP3 exon-7 and -8 was prepared, an EC cell line carrying the recombinant gene was produced, and secreted chimeric glycoprotein, called EC-hulgG(Fc)/mZP3(7), was purified and assayed. It was found that the chimeric glycoprotein binds specifically to plasma membrane overlying sperm heads to a similar extent as egg mZP3 and, at nanomolar concentrations, inhibits binding of mouse sperm to eggs in vitro. Collectively, these observations provide new evidence that sperm recognize and bind to a region of mZP3 polypeptide immediately downstream of its ZP domain that is encoded by mZP3 exon-7. The implications of these findings are discussed.

Original languageEnglish (US)
Pages (from-to)30-39
Number of pages10
JournalJournal of Cellular Physiology
Volume207
Issue number1
DOIs
StatePublished - Apr 2006
Externally publishedYes

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Spermatozoa
Exons
Peptides
Cricetinae
Embryonal Carcinoma
Embryonal Carcinoma Stem Cells
Eggs
Glycoproteins
Sperm Head
Ovum
Fusion reactions
Immunoglobulin G
Genes
Cells
In Vitro Techniques
Cell Line
Zona Pellucida
Acrosome
Cell membranes
Fertilization

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology

Cite this

Polypeptide encoded by mouse ZP3 exon-7 is necessary and sufficient for binding of mouse sperm in vitro. / Williams, Zev; Litscher, Eveline S.; Jovine, Luca; Wassarman, Paul M.

In: Journal of Cellular Physiology, Vol. 207, No. 1, 04.2006, p. 30-39.

Research output: Contribution to journalArticle

Williams, Zev ; Litscher, Eveline S. ; Jovine, Luca ; Wassarman, Paul M. / Polypeptide encoded by mouse ZP3 exon-7 is necessary and sufficient for binding of mouse sperm in vitro. In: Journal of Cellular Physiology. 2006 ; Vol. 207, No. 1. pp. 30-39.
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abstract = "Fertilization in mice is initiated by species-specific binding of sperm to mZP3, one of three mouse zona pellucida (ZP) glycoproteins. At nanomolar concentrations, purified egg mZP3 binds to acrosome-intact sperm heads and inhibits binding of sperm to eggs in vitro. Although several reports suggest that sperm recognize and bind to a region of mZP3 encoded by mZP3 exon-7 (so-called, sperm combining-site), this issue remains controversial. Here, exon-swapping and an IgG(Fc) fusion construct were used to further evaluate whether mZP3 exon-7 is essential for binding of sperm to mZP3. In one set of experiments, hamster ZP3 (hZP3) exon-6, -7, and -8 were individually replaced with the corresponding exon of mZP3. Stably transfected embryonal carcinoma (EC) cell lines carrying the recombinant genes were produced and secreted recombinant glycoprotein was purified and assayed for the ability to inhibit binding of sperm to eggs. While EC-hZP3, a recombinant form of hZP3 made by EC cells, is unable to inhibit binding of mouse sperm to eggs in vitro, the results suggest that substitution of mZP3 exon-7 for hZP3 exon-7, but not mZP3 exon-6 or -8, can impart inhibitory activity to EC-hZP3. In this context, a fusion construct consisting of human IgG(Fc) and mZP3 exon-7 and -8 was prepared, an EC cell line carrying the recombinant gene was produced, and secreted chimeric glycoprotein, called EC-hulgG(Fc)/mZP3(7), was purified and assayed. It was found that the chimeric glycoprotein binds specifically to plasma membrane overlying sperm heads to a similar extent as egg mZP3 and, at nanomolar concentrations, inhibits binding of mouse sperm to eggs in vitro. Collectively, these observations provide new evidence that sperm recognize and bind to a region of mZP3 polypeptide immediately downstream of its ZP domain that is encoded by mZP3 exon-7. The implications of these findings are discussed.",
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