Past studies from this laboratory have suggested that mouse sperm binding to the egg zona pellucida is mediated by a sperm galactosyltransferase (GalTase), which recognizes and binds to terminal N-acetylglucosamine (GIcNAc) residues in the zona pellucida (ShuT, B. D., and N. G. Hall, 1982, J. Cell Biol. 95:567-573; 95:574-579). We now present evidence that directly supports this mechanism for gamete binding. GalTase was purified to homogeneity by sequential affinity-chromatography on GIcNAc-agarose and a-lactalbumin-agarose columns. The purified enzyme produced a dose-dependent inhibition of sperm binding to the zona pellucida, relative to controls. To inhibit sperm/zona binding, GalTase had to retain its native conformation, since neither heat-inactivated nor Mn+÷-deficient GalTase inhibited sperm binding. GalTase inhibition of sperm/zona binding was not due to steric blocking of an adjacent sperm receptor on the zona, since GalTase could be released from the zona pellucida by forced galactosylation with UDPGal, and the resulting galactosylated zona was still incapable of binding sperm. In control experiments, when UDPGal was replaced with the inappropriate sugar nucleotide, UDPglucose, sperm binding to the zona pellucida remained normal after the adsorbed GalTase was washed away.The addition of UDPGal produced a dose-dependent inhibition of sperm/zona binding, and also dissociated preformed sperm/zona adhesions by catalyzing the release of the sperm GalTase from its GIcNAc substrate in the zona pellucida. Under identical conditions, UDPglucose had no effect on sperm binding to the zona pellucida. The ability of UDPGal to dissociate sperm/zona adhesions was both time-and temperature-dependent. UDPGal produced nearly total inhibition of sperm/zona binding when the zonae pellucidae were first galactosylated to reduce the number of GalTase binding sites.Finally, monospecific anti-GalTase IgG and its Fab fragments produced a dose-dependent inhibition of sperm/zona binding and concomitantly blocked sperm GalTase catalytic activity. Preimmune IgG or anti-mouse brain IgG, which also binds to the sperm surface, had no effect. The sperm GalTase was localized by indirect immunofluorescence to a discrete plasma membrane domain on the dorsal surface of the anterior head overlying the intact acrosome. These results, along with earlier studies, show clearly that sperm GalTase serves as a principal gamete receptor during fertilization.

From a systematic search of the UniGene and dbEST databanks, using human beta 4-galactosyltransferase (beta 4GalT-I), which is recognized to function in lactose biosynthesis, as the query sequence, we have identified five additional gene family members denoted as beta 4GalT-II, -III, -IV, -V, and -VI. Complementary DNA clones containing the complete coding regions for each of the five human homologs were obtained or generated by a PCR-based strategy (RACE) and sequenced. Relative to beta 4GalT-I, the percent sequence identity at the amino acid level between the individual family members, ranges from 33% (beta 4GalT-VI) to 55% (beta 4GalT-II). The highest sequence identity between any of the homologs is between beta 4GalT-V and beta 4GalT-VI (68%). beta 4GalT-II is the ortholog of the chicken beta 4GalT-II gene, which has been demonstrated to encode an alpha-lactalbumin responsive beta 4-galactosyltransferase (Shaper et al., J. Biol. Chem., 272, 31389-31399, 1997). As established by Northern analysis, beta 4GalT-II and -IV show the most restricted pattern of tissue expression. High steady state levels of beta 4GalT-II mRNA are seen only in fetal brain and adult heart, muscle, and pancreas; relatively high levels of beta 4GalT-VI mRNA are seen only in adult brain. When the corresponding mouse EST clone for each of the beta 4GalT family members was used as the hybridization probe for Northern analysis of murine mammary tissue, transcription of only the beta 4GalT-I gene could be detected in the lactating mammary gland. These observations support the conclusion that among the six known beta 4GalT family members in the mammalian genome, that have been generated through multiple gene duplication events of an ancestral gene(s), only the beta 4GalT-I ancestral lineage was recruited for lactose biosynthesis during the evolution of mammals.

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