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First published online 20 March 2007
doi: 10.1242/jcs.004291
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Research Article |
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
Author for correspondence (e-mail: stanley{at}aecom.yu.edu)
Accepted 13 February 2007
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Summary |
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1,3-galactosyltransferase 1 (T-synthase), which initiates the synthesis of core-1-derived O-glycans, the only O-glycans on mouse ZP3. T-synF/F:ZP3Cre females in which T-synF was deleted at the beginning of oogenesis generated eggs lacking core-1-derived O-glycans. Nevertheless, T-synF/F:ZP3Cre females were fertile and their eggs bound sperm similarly to controls. In addition, T-syn–/– embryos generated from T-syn null eggs developed until 
E12.5. Thus, core-1-derived O-glycans are not required for blastogenesis, implantation, or development prior to midgestation. Moreover, T-syn–/–Mgat1–/– eggs lacking complex and hybrid N-glycans as well as core-1-derived O-glycans were fertilized. The combined data show that mouse ZP3 does not require terminal Gal or GlcNAc on either N- or O-glycans for fertilization.
Key words: Fertilization, Zona pelucida, O-glycans, N-glycans, T-synthase
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Introduction |
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). ZP proteins are synthesized and secreted exclusively by the oocyte during the 2-3 weeks of oogenesis prior to ovulation (Philpott et al., 1987). All three ZP proteins are heterogeneously glycosylated with N- and O-glycans (Philpott et al., 1987) but only ZP1 and ZP3 appear to possess O-glycans attached to Ser or Thr residues (Boja et al., 2003; Chalabi et al., 2006; Nagdas et al., 1994). ZP1 is not required to generate a functional ZP or for fertility in the mouse (Rankin et al., 1999). Soluble ZP3, but not soluble ZP1 or ZP2, competitively inhibits sperm binding to eggs in a dose-dependent manner, suggesting that ZP3 carries sperm receptor(s) (Bleil and Wassarman, 1980). The glycans of ZP3 were subsequently implicated in sperm binding using in vitro assays with ZP3 glycopeptides generated by Pronase digestion (Florman et al., 1984). These glycopeptides were almost devoid of amino acids suggesting that only ZP3 glycans are recognized by mouse sperm. Consistent with a potential direct or indirect role for glycans in sperm-egg binding in vivo is the finding that mouse eggs with a humanized zona containing human ZP2 and ZP3 in place of mouse ZP2 and ZP3 bind mouse sperm but do not bind human sperm (Rankin et al., 2003).
Support for specific roles for N- or O-glycans in mouse sperm-egg binding has also been obtained from glycosidase digestions. In vitro removal of terminal galactose (Gal) residues from O-glycans of mouse eggs by 
-galactosidase abolished the ability of ZP3 to inhibit sperm binding (Bleil and Wassarman, 1988; Florman and Wassarman, 1985). However, mice lacking 1,3-galactosyltransferase and thus Gal1
3Gal termini on ZP glycans are fertile (Thall et al., 1995). Removal of terminal N-acetylglucosamine (GlcNAc) from the zona by digestion of eggs with -N-acetylglucosaminidase also inhibited sperm binding (Shur and Hall, 1982), and terminal GlcNAc was thus proposed as a sperm receptor recognized by 1,4-galactosyltransferase 1 (4GalT-1) on the sperm head (Lopez et al., 1985; Miller et al., 1992). However, sperm lacking 4GalT-1 are able to fertilize ovulated eggs (Asano et al., 1997) with sperm binding actually increased (Lu and Shur, 1997). Fucose has also been proposed to play a role due to the inhibition of sperm binding by the LewisX and LewisA determinants (Johnston et al., 1998; Kerr et al., 2004). However, fertility in 1,3-fucosyltransferase 9 (Fut9)-null (Fut9–/–) mice whose eggs lack the LewisX determinant, is normal (Kudo et al., 2004). Finally, mannose present on N-glycans has been implicated in mouse sperm-egg recognition (Cornwall et al., 1991). However, treatment with N-glycanase, which should remove all N-glycans, did not affect sperm binding to mouse eggs (Florman and Wassarman, 1985).
The combined biochemical data implicate sugar recognition in mouse sperm-egg binding but do not lead to a unified hypothesis. Indeed in most instances, genetic ablation in vivo of sugars identified as critical determinants of sperm-egg binding by in vitro biochemical assays, does not lead to infertility. Most recently, oocyte-specific deletion of the mannoside acetylglucosaminyltransferase 1 (Mgat1) gene revealed that mouse eggs lacking terminal Gal and GlcNAc residues on N-glycans are efficiently fertilized, although they have a fragile, thin zona (Shi et al., 2004), and bind fewer sperm than control eggs using a classic in vitro sperm binding assay (Hoodbhoy et al., 2005). Whereas this genetic approach ruled out terminal Gal or GlcNAc on N-glycans as necessary for fertilization or sperm binding, O-glycans remained unaltered. Mouse ZP3 has at least five O-glycans and mouse ZP1 has multiple O-glycans (Boja et al., 2003). The predominant form of O-glycan on mouse ZP3 has the core 2 structure (Dell et al., 2003) that is generated from a core 1 O-glycan. Core-1-derived O-glycans arise from the extension of the Tn antigen (GalNAc1-Ser/Thr) by the action of core 1 1,3-galactosyltransferase (T-synthase) which adds Gal to generate the T antigen or core 1 O-glycans (Gal1-3GalNAc1-Ser/Thr) (Ju et al., 2002a; Ju et al., 2002b). The addition of a branching 1,6-linked GlcNAc to the GalNAc (N-acetylgalactosamine) of a core 1 O-glycan generates core 2 which may be extended with Gal and N-acetyllactosamine. Mice lacking core 2 N-acetylglucosaminyltransferase L (C2GnT-L) are fully fertile (Ellies et al., 1998), but two other core 2 GlcNAcTs exist (Bierhuizen et al., 1993; Yeh et al., 1999) and may rescue the C2GnT-L deficiency in eggs. No core 3, core 4 or sialyl-GalNAc O-glycans were detected on mouse ZP1 or ZP3 by mass spectrometry (Chalabi et al., 2006; Dell et al., 2003; Easton et al., 2000).
In this paper we investigate whether terminal Gal or GlcNAc residues on O-glycans of the ZP are required for fertilization in the mouse using a genetic approach. A logical choice for inhibiting O-glycan synthesis would be to prevent the addition of the initiating GalNAc, but UDP-GalNAc:polypeptide GalNAc-transferases are encoded by many different genes. On the other hand, T-synthase acts after GalNAc-transferase to generate core-1-derived O-glycans and has no obvious homologs in mammalian genomes (Ju et al., 2002a; Ju et al., 2002b). It is, therefore, a suitable target for producing eggs lacking extended O-glycans. However, T-syn–/– (also known as C1galt1–/–; MGI) embryos die by embryonic day (E)14 from angiogenic defects (Xia et al., 2004), and thus oocyte-specific deletion of the T-syn gene was performed to generate mouse eggs lacking core-1-derived O-glycans by inhibition of their synthesis rather than biochemical removal post ovulation. This results in the generation of an intact mutant zona on oocytes and eggs, allowing functional analysis in a biologically relevant environment. We show here that mouse eggs lacking core-1-derived O-glycans are efficiently fertilized and bind sperm well. Additional removal of complex and hybrid N-glycans in double mutant T-synF/FMgat1F/F:ZP3Cre females also resulted in eggs that were fertilized. Thus neither Gal nor GlcNAc on N- or O-glycans of the mouse ZP function as essential sperm receptors.
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Results |
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) were transfected with an expression vector carrying Cre recombinase to obtain ES cells in which the T-syn allele was floxed (T-synF) and the Neo gene was deleted as described in the Materials and Methods (Fig. 1A). ES cells with a T-synF allele were identified by Southern analysis following BamHI digestion of genomic DNA. An extra BamHI site introduced immediately upstream of exon 1 of the T-synF allele gave a 5.9 kb band following Cre-mediated deletion. Thus analysis of T-synF/+ ES cells showed the 5.9 kb band and a 10 kb wild-type band, whereas analysis of parental ES cells showed the 10 kb wild-type band and a 7.7 kb band corresponding to the tri-loxP T-syn allele containing the Neo gene (Fig. 1B). Chimeras that transmitted the T-synF allele were used to generate T-synF/+ heterozygotes. T-synF/+ females were mated to T-synF/+:ZP3Cre males to obtain T-synF/F:ZP3Cre females and control females.
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) when the ZP3 promoter becomes active 2-3 weeks prior to ovulation (Philpott et al., 1987). To confirm that T-syn had been deleted in ovulated eggs, pups from T-synF/F:ZP3Cre and T-synF/+:ZP3Cre females were genotyped using the primers described in Fig. 2A that identify the wild-type, floxed and deleted T-syn alleles (Fig. 2B). The reaction catalyzed by T-synthase to form core 1 and core 2 O-glycans is shown in Fig. 2C and the genotypes of females carrying floxed alleles and their oocytes after gene deletion are shown in Fig. 2D.
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Core-1-derived O-glycans are not required for development before E12.5
Mutant T-syn–/– embryos lacking T-synthase generated from T-syn+/– matings have hemorrhages in the brain and spinal region at E11-E13 and die by E14 (Xia et al., 2004). Such embryos might be rescued during blastogenesis and implantation by maternal T-synthase transcripts that are expected to be present in heterozygous eggs (Su et al., 2004). To investigate potential roles for T-synthase in pre-implantation development, timed matings between T-synF/F:ZP3Cre females and T-syn+/– males were performed. Two females were dissected at E11.5 and five females at E12.5. Mutant embryos had the same overall phenotype of hemorrhaging and defective angiogenesis at E11.5 (data not shown), which was more severe at E12.5 (Fig. 3A) as described previously (Xia et al., 2004). In addition, mutant embryos were obtained at the expected ratio of 1:1 (Fig. 3B) providing strong evidence that blastocysts lacking both maternal and zygotic T-synthase are able to develop, implant and progress to E12.5.
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Sperm bind efficiently to T-syn–/– eggs
T-synF/F:ZP3Cre females were as fertile as controls (Table 1). However, fertilization requires only one sperm to bind and traverse the zona. The zona on T-syn–/– eggs was marginally thinner and slightly looser in appearance than the ZP of wild-type eggs (Fig. 4A,B). To determine if sperm binding to mutant zona was altered by the removal of terminal Gal and GlcNAc residues on O-glycans, classic in vitro assays of sperm binding were performed. Ovulated eggs denuded of cumulus cells by hyaluronidase treatment were incubated with sperm in the presence of two-cell embryo controls that do not bind sperm. Sperm binding of T-syn–/– eggs was indistinguishable from that of wild-type eggs, under conditions in which two-cell embryos showed no sperm binding (Fig. 4C-F). Thus, the ZP surrounding the eggs of T-synF/F:ZP3Cre females binds sperm equivalently to wild-type ZP. In three experiments using eggs ovulated from two to three control or T-synF/F:ZP3Cre females in each experiment, sperm binding to wild-type and T-syn–/– eggs was always equivalent.
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T-syn–/– eggs lack core-1-derived O-glycans
Considering the fertility of T-synF/F:ZP3Cre females and the robust binding of sperm to their eggs, it was important to confirm that core-1-derived O-glycans were absent. T antigen (Gal3GalNAc1Ser/Thr) generated by T-synthase (Fig. 2C) was detected by binding of fluoresceinated peanut agglutinin (PNA-FITC). The Tn antigen (GalNAc1Ser/Thr), the precursor of the T antigen, was detected using anti-Tn antibody (Fig. 5A-D). Eggs from T-syn+/+:ZP3Cre females stained brightly with PNA-FITC (Fig. 5A), whereas eggs from T-synF/F:ZP3Cre females bound only background levels of PNA-FITC (Fig. 5B). Consistent with the absence of the T antigen and a lack of T-synthase activity in mutant eggs, T-syn–/– eggs bound anti-Tn antibody, whereas wild-type or heterozygous eggs did not (Fig. 5C,D). Thus core-1-derived O-glycans were essentially absent from T-syn–/– eggs. However, as expected, complex N-glycans were not affected, as shown by the fact that T-syn–/– eggs bound Phaseolus vulgaris leukoagglutinin-FITC (L-PHA-FITC) equivalently to wild-type eggs (Fig. 5E,F). The combined data provide strong evidence that T-synthase was expressed in wild-type eggs and was not active in T-syn null eggs. Further evidence of this was obtained by western analysis of mutant oocytes.
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65-80 kDa, reflecting glycan heterogeneity, whereas ZP3 from T-synF/F:ZP3Cre ovaries migrated with an apparent molecular mass of 63-72 kDa, consistent with the loss of O-glycans from mutant oocytes (Fig. 5G). Prolonged exposure of the membrane did not reveal any ZP3 bands of higher molecular masses from T-synF/F:ZP3Cre ovaries indicating that all of the ZP3 was affected by the loss of T-synthase. Removal of N-glycans using N-glycanase reduced the molecular mass of wild-type ZP3 to 35-45 kDa and of mutant ZP3 to 32 kDa, which is the predicted mass of ZP3 with no N-glycans and only five GalNAc residues at O-glycan sites (Boja et al., 2003). The lack of heterogeneity of ZP3 from mutant ovaries and its molecular mass after N-glycanase treatment provides strong evidence that no O-glycans were extended by T-synthase in mutant oocytes. Similar conclusions were obtained from western analyses of ZP1. ZP1 from wild-type ovaries migrated with an apparent molecular mass of 95-155 kDa whereas ZP1 from mutant ovaries migrated from 75-140 kDa (Fig. 5H). Prolonged exposure of the membrane did not reveal any ZP1 from mutant ovary of the highest molecular mass of wild-type ZP1. Removal of N-glycans by digestion with N-glycanase caused wild-type ZP1 to migrate in a band of 65-90 kDa and ZP1 from mutant oocytes in a tight band of 60 kDa (Fig. 5H), consistent with the predicted molecular mass of ZP1 lacking N-glycans and having only GalNAc at O-glycan sites (Boja et al., 2003). Three females of each genotype gave similar western results. ZP2 from oocytes or eggs of wild-type and T-synF/F:ZP3Cre females had the same apparent molecular mass (data not shown) consistent with the predicted absence of O-glycans on ZP2 (Boja et al., 2003). The combined data show that inactivation of the gene encoding T-synthase gives rise to oocytes with glycoproteins (exemplified by ZP1 and ZP3) that are essentially devoid of core 1 and core 2 O-glycans.
Fertility of females with oocyte-specific deletion of T-syn and Mgat1
The absence of Gal and GlcNAc residues on core-1-derived O-glycans of T-syn–/– eggs did not alter sperm binding or fertility. However, complex and hybrid N-glycans generated by GlcNAc-T1 encoded by the Mgat1 gene also have Gal and GlcNAc residues which may compensate for their loss of core-1-derived O-glycans (Fig. 2E). To investigate this question, T-synF/FMgat1F/F:ZP3Cre females were generated and mated with C57BL/6 males. T-synF/+Mgat1F/+:ZP3Cre and T-syn+/+Mgat1+/+:ZP3Cre females were used as controls. The genotypes of their respective oocytes are shown in Fig. 2D. The results in Table 2 show that T-synF/FMgat1F/F:ZP3Cre double mutant (DM) females were fertile but their fertility was severely reduced compared to control females. Only three of 10 DM females produced a litter (Table 2) and no DM female produced more than a single litter, despite being with a male for up to 6 months. These single litters were produced at the same time after mating as the first litters of control females, suggesting that ovulation was initially unaffected. However, the number of pups produced by DM females was smaller (Table 2). Genotyping using the primers shown in Fig. 2A confirmed the absence of floxed T-syn and Mgat1 alleles in all pups from T-synF/FMgat1F/F:ZP3Cre and T-synF/+Mgat1F/+:ZP3Cre females (see Fig. 2B). This demonstrates that expression of the ZP3Cre transgene was able to delete two floxed alleles as efficiently as a single floxed allele such as T-syn or Mgat1 (Shi et al., 2004). Superovulation of 4-week-old females resulted in 50% fewer eggs from DM females compared with wild-type females. Moreover, superovulation of the DM females (Table 2) after the termination of mating resulted in no eggs from any of the 10 females, whereas wild-type and heterozygous females ovulated 49.8±9.8 eggs (n=6) and 44.0±12.8 eggs (n=4), respectively. This strongly suggests that the reduced fertility of DM females was not due to defective fertilization but to compromised oogenesis, a result confirmed by finding a reduced number of developing follicles in ovaries from these DM females at 3-6 months of age.
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Eggs from DM females lack core-1-derived O-glycans and complex and hybrid N-glycans
To confirm that both the O- and N-glycans of the ZP had been altered in DM females, lectins were used to detect the T antigen and complex N-glycans on ovulated eggs. PNA-FITC bound well to wild-type eggs whereas eggs from DM females bound little PNA-FITC, confirming the absence of the T antigen (Fig. 6E,F), as observed with T-syn–/– eggs (Fig. 5A,B). L-PHA-FITC binding was also absent (Fig. 6G,H), and concanavalin A-Rhodamine (Con A-Rho) binding to DM eggs was enhanced (Fig. 6I,J). This confirmed the lack of complex and hybrid N-glycans and the consequent increase in oligomannosyl N-glycans on DM eggs. Therefore, the O- and N-glycans of eggs from DM females were altered as expected. However, properties of the zona of DM eggs were also altered. Cumulus cells remained attached to most DM eggs (Fig. 6B,D) but not wild-type eggs (Fig. 6A,C) after 3-5 minutes of hyaluronidase digestion. Prolonged incubation, of up to 20 minutes, in hyaluronidase did not remove the cumulus cells from DM eggs. Mild agitation by pipetting resulted in the zona tearing away from DM eggs without cumulus cells being removed, and revealed the thinness of the DM zona (Fig. 6D). A fragile, thin, zona was to be expected in eggs lacking Mgat1, as previously described (Shi et al., 2004).
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; Shur et al., 2006; Wassarman, 2005; Wassarman et al., 2005). In this paper we addressed this question by targeted inactivation of T-synthase, the glycosyltransferase that initiates the synthesis of the core 1 and core 2 O-glycans, the only mucin O-glycans detected on mouse ZP3 by mass spectrometry (Boja et al., 2003; Chalabi et al., 2006). T-synthase is essential for embryonic development beyond E14 (Xia et al., 2004). Therefore, we generated the conditional T-synF allele and used a ZP3Cre transgene for oocyte-specific deletion to obtain eggs lacking core-1-derived O-glycans. This approach enabled direct assessment of the in vivo function of mutant ZP in fertilization.
Given the predicted importance of O-glycans on ZP3 for sperm binding, it was surprising to find that fertilization was not in the least impaired in T-synF/F:ZP3Cre females. We eliminated the possibility that another glycosyltransferase substituted for T-synthase by showing that mutant eggs did not bind PNA, which recognizes the product of T-synthase, but did bind anti-Tn antibody, which recognizes the GalNAc1Ser/Thr substrate of T-synthase. More importantly, western analyses showed that the full complement of ZP1 and ZP3 glycoproteins was affected by the mutation in T-syn–/– eggs. It has been proposed that, because of the heterogeneous nature of the zona matrix that leads to differences in the glycans present on the outer surface of the ZP compared to the inner (Aviles et al., 2000), only ZP3 proteins on the outer surface of the ZP need to have O-glycans at Ser 332 and 334 in order for them to function as sperm receptors (Williams et al., 2006); a model that we were able to test. Inactivation of the T-syn gene early in oogenesis left no ZP1 or ZP3 with a wild-type complement of core-1-derived O-glycans and thus ovulated eggs had no core-1-derived O-glycans on the outer layer of their ZP. These results provide convincing evidence that Gal (Bleil and Wassarman, 1988), GlcNAc (Miller et al., 1992; Shur and Hall, 1982) and/or fucose in the context of Lewis antigens (Johnston et al., 1998; Kerr et al., 2004) on O-glycans are dispensable for fertilization and thus for sperm binding. Gal or GlcNAc on N-glycans did not compensate for their loss from O-glycans because, when complex and hybrid N-glycans were also removed by deletion of the Mgat1 gene, DM females were fertile. However, oogenesis was severely compromised since none of the DM females ovulated after stimulation with exogenous gonadotrophins at 3-6 months of age. The defective oogenesis in DM females was much more severe than the partially compromised developmental competence of ovulated eggs previously observed in Mgat1F/F:ZP3Cre females that lack only complex and hybrid N-glycans during oogenesis (Shi et al., 2004). Nevertheless, the fact that a proportion of DM females with eggs shown to possess the expected glycosylation-defective phenotype gave rise to at least one litter leads to the conclusion that terminal Gal or GlcNAc residues on ZP glycoproteins are not essential for fertilization in the mouse. Indeed, there may not be a single sperm receptor. The recently described ZP3-independent sperm-binding ligand (Rodeheffer and Shur, 2004) may play a significant role in fertilization and, may compensate for the lack of complex O- and N-glycans in DM females.
The results described in this paper also have implications for a second model of the molecular basis of the specificity of mouse sperm-egg recognition which is based on the supramolecular structure of the mouse zona (Dean, 2004). This model proposes that the overall conformation of the ZP is different in different species and is responsible for taxon-specific sperm binding. Such a ZP conformation would need to be quite robust to account for the fertilization of mouse eggs with a thin, fragile ZP lacking both core-1-derived O-glycans and complex and hybrid N-glycans.
Whereas the genetic ablation strategy clearly demonstrates that complex O- and N-glycans terminating in Gal or GlcNAc are superfluous for fertilization, considerable in vitro biochemical evidence indicates a requirement for these glycans for sperm-egg binding. Purified solublized zona proteins were used for the competitive in vitro binding studies. However, solubilizing zona glycoproteins alters their conformation from the structure assembled into the zona matrix, potentially exposing protein or glycan determinants which may function in vitro but not in vivo. In addition, the zona matrix is heterogeneously glycosylated (Aviles et al., 2000) and purified ZP proteins generated from whole zona will contain glycoproteins that are not on the zona surface and thus would be unavailable for sperm binding in the oviduct. The data presented here demonstrate the importance of performing in vivo modifications using genetic deletion analysis to arrive at definitive conclusions regarding biological functions.
In summary, the mouse models we describe allow analyses of tissue-specific roles for core-1-derived O-glycans and complex O- and N-glycans. Following oocyte-specific deletion of T-syn, females were fully fertile even though their eggs lacked core-1-derived O-glycans. In addition, embryos lacking maternal and zygotic T-synthase progressed at a normal rate through blastogenesis and early embryonic development to E12.5. Thus, core-1-derived O-glycans are dispensable for sperm binding, fertilization, and for development to mid-gestation. Eggs that lack both core-1-derived O-glycans and complex and hybrid N-glycans and thus have no terminal Gal or GlcNAc on their ZP are also fertilized, demonstrating that these residues are not essential for functional sperm-egg recognition in the mouse.
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) (Fig. 1A) were transiently transfected with an expression vector encoding Cre recombinase (a gift from Brian Sauer, Stowers Institute for Medical Research, Kansas City, MO) to delete the Neo cassette. To screen for ES cells with Cre-mediated recombination, individual clones were transferred into a 96-well plate after transfection. When the clones were confluent, cells were frozen, and also divided into two 96-well plates in medium with and without G418. After 3 days, Cre-mediated deletion of the Neo gene was considered to have taken place in clones that did not survive G418 selection. PCR genotyping was used for further screening as described previously (Xia et al., 2004). About 15% of ES clones had deleted only the Neo gene. Genomic DNA from these ES cells was digested with BamHI, subjected to Southern analysis and probed with exon 2 DNA from the T-syn gene. ES cells with a floxed T-syn gene shown to also have a normal karyotype were microinjected into C57BL/6J blastocysts, which were implanted into pseudopregnant mice. Six of ten chimeras transmitted the floxed T-syn allele to their offspring. T-synF/+ mice were bred to generate homozygous T-synF/F mice in a mixed 129/SvlmJ and C57BL/6J genetic background. Southern analysis of genomic DNA from mouse tail biopsies was performed following digestion with BamHI and hybridization using exon 2 of the T-syn gene.
Mice with floxed T-syn and Mgat1 alleles and a ZP3Cre transgene
Female mice with a T-synF allele were crossed with male mice of a mixed background carrying a Cre recombinase transgene under the control of the ZP3 promoter (Shi et al., 2004). Subsequent matings generated T-synF/F:ZP3Cre females and T-synF/+:ZP3Cre females in which inactivation of the T-syn gene occurs at the start of oogenesis when ZP3 is expressed (Lewandoski et al., 1997; Philpott et al., 1987) (Fig. 2A). To obtain homozygote and heterozygote double mutant (DM) females carrying floxed Mgat1 allele(s) (Shi et al., 2004) in addition to floxed T-syn allele(s), Mgat1F/F mice, described previously (Shi et al., 2004), were crossed to obtain T-synF/FMgat1F/F:ZP3Cre and T-synF/+Mgat1F/+:ZP3Cre females. T-syn+/+:ZP3Cre and T-syn+/+Mgat1+/+:ZP3Cre females were generated as controls.
To distinguish between mice carrying floxed or deleted T-syn allele(s), and the ZP3Cre transgene, separate PCR genotyping was performed using tail genomic DNA. Primers TS-1 (5'-gataaatgtcttacagaagg-3') and TS-2 (5'-ttatgttggctggaatctgc-3') detected the wild-type (T-syn+) and the floxed T-synF alleles, and primers TS-1 and TS-3 (5'-aatactgtcctgggctatactacagtg-3') detected the deleted T-syn allele. For TS-1 and TS-2 primers, PCR reactions of 25 µl contained 2.5 µl 10x PCR buffer (not containing MgCl2) (Invitrogen, Carlsbad, CA), 1.5 µl 50 mM MgCl2 (Invitrogen), 0.5 µl 10 mM dNTPs (Invitrogen), 1 µl 10 mM primers, 3 IU Taq polymerase (Roche, Indianapolis, IN) and 1.5 µl DNA. For TS-1 and TS-3 primers, reactions of 25 µl contained 2.5 µl of 10x PCR buffer already containing 20 mM MgCl2, 0.5 µl 50 mM MgCl2, 0.5 µl 10 mM dNTPs, 0.5 µl 10 mM primers, 3 IU Taq polymerase (Roche), and 0.5 µl DNA. Deleted Mgat1 was detected using primers Mgat1 Del 1 (5'-ctgctccaggacaagagcca-3') and Mgat1 Del 2 (5'-gagacctgcttactgcagcc-3'). These reactions of 25 µl contained 2.5 µl of 10x PCR buffer containing 20 mM MgCl2, 0.5 µl 10 mM dNTPs, 0.5 µl 10 mM primers, 1.5 IU Taq polymerase (Roche), and 1 µl DNA. All PCR reactions except for deleted Mgat1 were performed as follows: preheating (94°C, 2 minutes), 40 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute, followed by one cycle of 72°C for 5 minutes. Deleted Mgat1 PCR reactions were as described above with an annealing temperature of 65°C and 35 cycles. Mgat1 genotyping for the floxed allele and PCR reactions for the ZP3Cre transgene were performed as described previously (Shi et al., 2004).
Fertility of T-synF/F:ZP3Cre and T-synF/FMgat1F/F:ZP3Cre females
To determine fertility, T-synF/F:ZP3Cre, T-synF/+:ZP3Cre and T-syn+/+:ZP3Cre females and T-synF/FMgat1F/F:ZP3Cre, T-synF/+Mgat1F/+:ZP3Cre and T-syn+/+Mgat1+/+:ZP3Cre females were mated to C57BL/6 males. Time to first litter and litter size were determined, and all pups born from mothers carrying a T-synF or Mgat1F allele were genotyped to determine deletion of the floxed gene. At the termination of breeding (3-6 months of age), double mutant females were superovulated by intraperitoneal injection of 5 IU pregnant mare serum gonadotrophin (Sigma-Aldrich, St Louis, MO) followed 46-48 hours later by 5 IU human chorionic gonadotrophin (Sigma-Aldrich). Fourteen hours later, eggs were collected and counted, and the ovaries were fixed for 7-10 hours in Bouins or 10% buffered formalin (Sigma-Aldrich) for subsequent immunohistochemistry. All fixations were carried out at room temperature.
Preimplantation development in embryos lacking core-1-derived O-glycans
In order to generate T-syn–/– embryos from eggs that lacked core-1-derived O-glycans and T-syn– sperm, T-synF/F:ZP3Cre females were mated with T-syn+/– males and embryonic development was assessed at E11.5 and E12.5. Noon of the following day after pairs were placed together at 4 pm was taken as 0.5 days post-coitum. Hind leg genomic DNA was used to genotype the embryos, as described previously (Xia et al., 2004).
Sperm binding
To examine sperm binding, 3-week-old female mice were superovulated as described above. Eggs were collected from the oviduct and treated with 0.3 mg/ml hyaluronidase (Sigma-Aldrich) to remove cumulus cells (denuded eggs) in the presence of protease inhibitors (Roche). Cauda epididymi from a male of proven fertility were dissected, minced in 1 ml IVF-30 medium (Vitrolife, Denver, CO) and allowed to `swim out'. After 15 minutes, 10 µl sperm were added to a 250 µl droplet of IVF-30 medium and capacitated for 1 hour. Denuded eggs (20-30) and 2-cell embryo controls (3-5) were added and incubated for 30 minutes at 37°C in a 5% CO2 incubator with pre-equilibrated IVF-30 medium. Two-cell embryos were used as negative controls because they have a ZP that is modified at fertilization to prevent further sperm binding. They were generated by IVF the previous day with superovulated wild-type eggs and incubated with capacitated sperm for 6 hours as described. Sperm were allowed to bind to eggs and two-cell embryos for 30 minutes, after which they were gently washed by pipetting until less than five sperm were bound per two-cell embryo, fixed in 50 µl of 2% paraformaldehyde for 1 hour, washed again, and photographed.
Egg collection and cytochemistry
Three-week-old females were superovulated as described above. Eggs were collected into M2 medium (Sigma-Aldrich) and cumulus cells removed with 0.3 mg/ml hyaluronidase with protease inhibitors (Roche). Eggs were fixed in 2% paraformaldehyde in M2 medium for 1 hour. All blockings, washings and lectin dilutions were in phosphate-buffered saline (PBS) pH 7.2 containing 2% bovine serum albumin (BSA) and performed at room temperature. After blocking for 1 hour, T antigen was detected with 20 µg/ml peanut agglutinin conjugated to fluorescein isothiocyanate (PNA-FITC; Vector Labs, Burlingame, CA) by incubation for 1 hour followed by washing and photography. Tn antigen was detected by incubation in anti-Tn monoclonal antibody (a generous gift from Henrik Clausen, University of Copenhagen, Denmark) for 1 hour. Eggs were then washed, and incubated with goat anti-mouse FITC-conjugated antibody (Zymed, San Francisco, CA), washed and photographed. Binding of Phaseolus vulgaris leukoagglutinin-FITC (L-PHA-FITC; Vector Labs) at 20 µg/ml was also determined as described above. Double mutant eggs obtained with deleted T-syn and Mgat1 were examined for binding of LPHA-FITC and concanavalin A-Rhodamine (Con A-Rho, 2.5 µg/ml; Vector Labs) in addition to PNA-FITC.
N-Glycanase digestion and western analysis
Ovaries were isolated in dissection buffer [40 mM Tris, 150 mM NaCl, complete protease inhibitors (Roche)] and immediately homogenized using a pestle in a 1.5 ml microcentrifuge tube containing 400 µl dissection buffer with 0.1% SDS. Protein concentration was determined using Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) with BSA standards. Ovary samples (2.5 µg protein) were digested for 12 hours at 37°C with 1000 units of N-glycanase (New England Biolabs, Beverley, MA). Protein was separated on a 7% Tris gel using SDS-PAGE under reducing conditions and transferred to a polyvinylidenefluoride membrane which was probed with monoclonal antibodies to ZP1 (Rankin et al., 1998), ZP2 (East et al., 1984) or ZP3 (East et al., 1985) as previously described (Shi et al., 2004).
Lectin staining of ovarian sections
Ovaries were fixed in 10% buffered formalin (Sigma-Aldrich) for 6-8 hours, washed in 70% ethanol and embedded in paraffin. Sections of 5 µm were dewaxed with Histoclear (Sigma-Aldrich) and rehydrated. For lectin staining, sections were preincubated for 1 hour in PBS-2% BSA and incubated with 20 µg/ml PNA-FITC or L-PHA-FITC 20 µg/ml. After 1 hour, sections were washed with PBS-2% BSA and photographed.
ZP immunohistochemistry
ZP immunohistochemistry was performed as previously described (Shi et al., 2004). Briefly, ovaries were fixed in 10% buffered formalin or Bouins for 6-8 hours, washed in 70% ethanol, embedded in paraffin, and 5 µm sections cut. Sections were dewaxed, rehydrated and incubated in methanol containing 0.3% hydrogen peroxide for 30 minutes. Slides were washed for 3 minutes in water, 3 minutes in Tris-buffered saline [TBS; 0.1 M Tris (pH 7.5) and 0.3 M NaCl] with 0.05% Tween 20 (TBST), and incubated in TBS containing 15% normal rabbit serum (NRS; Vectastain Elite ABC kit, Vector Labs) for 30 minutes in a humidified chamber. Sections were incubated with undiluted hybridoma medium containing monoclonal antibodies to ZP1, ZP2 and ZP3 for 1 hour or TBS-15% NRS as a control. After washing three times for 3 minutes with TBST, sections were incubated with rabbit anti-rat immunoglobulin G biotinylated secondary antibody (Vectastain Elite ABC kit; 50 µl in 10 ml of TBS-15% NRS) for 30 minutes, washed, and incubated with ABC solution (Vectastain Elite ABC kit) for 30 minutes. After three washes with PBS containing 0.05% Tween 20, sections were stained using a DAB kit (Vector Labs) and counterstained with Hematoxylin before dehydration and mounting.
Statistical analyses
Statistical analyses were determined using two-tailed t-tests using Microsoft Excel Data Analysis package.
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Present address: Department of Biochemistry, Emory University, Atlanta, GA 30322, USA
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