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First published online 20 March 2007
doi: 10.1242/jcs.004291

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Fertilization in mouse does not require terminal galactose or N-acetylglucosamine on the zona pellucida glycans

Suzannah A. Williams^1,*, Lijun Xia^2,3,*, Richard D. Cummings^3,, Rodger P. McEver^2,3 and Pamela Stanley^1,

¹ Department of Cell Biology, Albert Einstein College of Medicine, New York, NY 10461, USA
² Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA
³ Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA

View larger version (38K): [in this window] [in a new window]   Fig. 1. Generation of the T-synF allele. (A) Gene targeting scheme. B, BamHI. (B) Southern analysis of BamHI-digested genomic DNA from ES clones after Cre-recombinase-mediated deletion of the Neo selection marker. (C) Southern blot analysis of BamHI-digested genomic DNA from mouse tails identifying T-synF mice. The probe for each Southern analysis was exon 2 of the T-syn gene.

View larger version (58K): [in this window] [in a new window]   Fig. 2. Generation of the T-syn allele and oocyte-specific deletion. (A) Diagram of T-syn and Mgat1 floxed and deleted alleles, and the ZP3Cre transgene with positions of primers used in genotyping. The T-syn-, Mgat1- and Cre-coding regions are shaded, and the promoter region for ZP3 is an open box. (B) Mouse genotype was determined using PCR of genomic tail DNA. Lane M, 1 kb plus markers. (C) O-glycan synthesis. T-synthase transfers Gal to GalNAc on Ser/Thr generating core 1. Subsequently GlcNAc may be added to the GalNAc to generate core 2. Core 3 and 4 structures have not been detected on mouse ZP3 (Boja et al., 2003; Chalabi et al., 2006). (D) Relationship of genotype of mutant females to genotype of eggs. (E) Diagram of complex N-glycan structure showing that the deletion of the Mgat1 gene generates oligomannosyl N-glycans. Open square, GalNAc; open circle, Gal; black square, GlcNAc; black diamond, sialic acid; triangle, fucose; black circle, mannose.

View larger version (60K): [in this window] [in a new window]   Fig. 3. Embryonic development from eggs lacking T-synthase. (A) T-syn–/– embryos were obtained from mating T-synF/F:ZP3Cre females to T-syn+/– males. At E12.5 mutant embryos exhibited angiogenic defects with hemorrhaging in the spinal and brain area (open arrowheads). (B) Embryos of each genotype were generated in approximately equal proportions from T-syn–/– eggs.

View larger version (128K): [in this window] [in a new window]   Fig. 4. Sperm binding to T-syn–/– eggs. (A,B) The zona of T-syn–/– eggs was marginally thinner and less uniform than wild-type zona. (C,D) Sperm binding to T-syn–/– and wild-type eggs was equivalent. Two-cell embryo controls had no sperm binding under these conditions. (E,F) Higher magnification shows comparable numbers of sperm bound to eggs of each genotype.

View larger version (50K): [in this window] [in a new window]   Fig. 5. Eggs from T-synF/F:ZP3Cre females lack core-1-derived O-glycans. (A,B) PNA-FITC bound to wild-type but not T-syn–/– eggs. Similar data were obtained in five experiments with eggs from 12 T-synF/F:ZP3Cre females. (C,D) Anti-Tn antibodies bound to the Tn antigen on T-syn–/– eggs, but poorly to wild-type eggs. Similar results were obtained in four experiments from 16 T-synF/F:ZP3Cre females. (E,F) L-PHA-FITC bound to mutant and wild-type eggs equivalently. Similar results were obtained in three experiments from seven T-synF/F:ZP3Cre females. (G,H) Western analyses of ZP1 and ZP3 from control and T-synF/F:ZP3Cre ovaries before and after digestion with N-glycanase.

View larger version (59K): [in this window] [in a new window]   Fig. 6. Eggs from T-synF/FMgat1F/F:ZP3Cre DM females lack core-1-derived O-glycans and complex and hybrid N-glycans. (A,B) Phase-contrast micrographs showing persistent cumulus cells attached to T-syn–/–Mgat1–/– eggs after hyaluronidase treatment. (C,D) Higher magnification of T-syn–/–Mgat1–/– eggs with less cumulus attached revealed a thin fragile zona. (E,F) PNA-FITC did not bind to T-syn–/–Mgat1–/– eggs. Similar data were obtained in three experiments with eggs from 12 T-synF/FMgat1F/F:ZP3Cre females. (G,H) L-PHA-FITC did not bind to T-syn–/–Mgat1–/– eggs. Similar data were obtained in four experiments from 16 T-synF/FMgat1F/F:ZP3Cre females. (I,J) Con A-Rho staining was enhanced in T-syn–/–Mgat1–/– eggs consistent with increased oligomannosyl N-glycans.

View larger version (48K): [in this window] [in a new window]   Fig. 7. Oocytes from T-synF/FMgat1F/F:ZP3Cre double mutant (DM) females lack core-1-derived O-glycans and complex and hybrid N-glycans. (A) The ZP of oocytes in T-synF/+Mgat1F/+:ZP3Cre control ovaries bound both L-PHA-FITC and PNA-FITC whereas the ZP on oocytes of DM ovaries (arrows) did not bind either lectin. Images are overexposed to reveal unstained oocytes present in DM ovaries and follicles are size-matched for developmental stage. (B) Monoclonal antibodies to ZP1, ZP2 and ZP3 bound to control and DM ZP. The ovaries were from three previously mated control and 3 DM females and represent six control and ten DM females. Asterisks indicate females that gave birth. Formalin fixation was used for lectin staining whereas both formalin (f) and Bouins (b) fixed ovaries were used for ZP staining.

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