Direct evidence for the involvement of carbohydrate sequences in human sperm-zona pellucida binding |
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Authors: | Ozgur K; Patankar MS; Oehninger S; Clark GF |
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Institution: | Department of Obstetrics and Gynaecology, Eastern Virginia Medical School, Norfolk 23507-1980, USA. |
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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.
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