Direct evidence for the involvement of carbohydrate sequences in human sperm-zona pellucida binding

Abstract

Several lines of evidence indicate that mammalian fertilization is initiated via a binding process that is dependent upon the recognition of oligosaccharide sequences associated with zona pellucida (ZP) glycoproteins. Here, specific chemical and enzymatic methods were employed to modify human ZP and to test their effects on sperm binding in the hemizona assay system (HZA). Periodate oxidation of human ZP under very mild conditions (10 min, 0 degrees C, 1 mM sodium m-periodate) that attacks only terminal sialic acid resulted in a 30% loss of human sperm binding in the HZA [hemizona index (HZI) = 70.2 +/- 10.9, n = 22; P < 0.05]. Periodate oxidation under mild conditions (1 h, 23 degrees C, 10 mM sodium m-periodate) caused a 40% decrease in binding (HZI = 60.8 +/- 10.3; n = 24; P< 0.01). Treatment of human ZP with neuraminidase caused a substantial increase in sperm binding to human ZP (HZI = 297 +/- 45, n = 22; P < 0.01). These findings indicate that there are sialic acid dependent binding sites coexisting with binding sites that are obscured by sialic acid. To determine the periodate sensitivity of these obscured sites, hemizona were first digested with neuraminidase and subsequently subjected to mild periodate oxidation. The combined enzymatic and chemical treatments caused a 79% decrease in sperm binding compared to control hemizona (HZI = 20.7 +/- 4.4, n = 16; P < 0.001). Human sperm-ZP interaction was also increased by digestion of human ZP with endo-beta-galactosidase (HZI = 710 +/- 232, n = 14; P < 0.01), indicating that potential binding sites for spermatozoa are also obscured by lactosaminoglycan sequences. These studies support a definitive role for the involvement of ZP-associated glycans in the binding of human spermatozoa to oocytes.

Binding of Zona Binding Inhibitory Factor-1 (ZIF-1) from Human Follicular Fluid on Spermatozoa

“…The recognition and interaction between complementary molecules present on the spermatozoa and the zona pellucida is the first step in this cascade. There is compelling evidence that carbohydratebinding proteins on the sperm surface mediate gamete recognition of different species by binding with high affinity and specificity to complex glycoconjugates on the zona pellucida (14 -16, 19, 34), including human (16,19,35,36). In the past two decades, the presence of several carbohydrate-binding proteins, such as galactosyltransferase (37), fucosyltransferase (38), ␣-mannosidase (39, 40), fucose-binding protein (41), selectin-like molecules (42), ␤-hexosaminidase (43), FA-1 (44), and others, on the sperm plasma membrane and their complementary sugar molecules on the zona pellucida have been suggested to be involved in sperm-egg interaction.…”

Structural Analysis of Murine Zona Pellucida Glycans

“…In the human, there is evidence suggesting that a sperm surfaceassociated neuraminidase exists that is activated by a specific uterine glycoprotein (21). Removal of sialic acid from the surface of the human ZP results in a 3-fold increase in sperm binding to this matrix in the hemizona assay system (22). By comparison, neuraminidase treatment of murine eggs results in only a 30 -40% increase in binding (23).…”

Glycodelin-A interacts with fucosyltransferase on human sperm plasma membrane to inhibit spermatozoa-zona pellucida binding

“…It is generally accepted that the specific interaction between spermatozoa and zona pellucida is a carbohydrate-mediated event (Wassarman, 1999;Ozgur et al, 1998;Chapman and Barratt, 1996). In the past two decades, several carbohydratebinding proteins on the sperm plasma membrane such as ␤1,4-galactosyltransferase (Nixon et al, 2001), ␣-mannosidase (Tulsiani et al, 1989), ␤-N-acetylglucosaminidase (Godknecht and Honegger, 1991) and selectin-like receptors (Oehninger, 2001) have been proposed to be involved in such interaction.…”

“…There is ample evidence that sperm surface carbohydrate-binding proteins mediate gamete recognition and interact with high affinity and specificity to the zona pellucida glycoproteins (Chapman and Barratt, 1996;Wassarman, 1999;Ozgur et al, 1998). Despite the fact that several carbohydratebinding proteins have been proposed to be responsible for spermatozoa-zona pellucida binding (Nixon et al, 2001;Leyton et al, 1992;Cheng et al, 1994), the exact identity of these sperm surface protein(s), especially those for humans, remains unclear.…”