Oocyte Gal alpha 1,3Gal epitopes implicated in sperm adhesion to the zona pellucida glycoprotein ZP3 are not required for fertilization in the mouse.

The Galα13Gal structure is displayed on the zona pellucida glycoprotein ZP3 on murine oocytes. This trisaccharide has been implicated in sperm-zona pellucida adhesive events thought to be essential to fertilization in the mouse. To determine directly if this molecule is required for fertilization, we have generated mice that are deficient in a gene (α1,3GT) encoding the UDP-Gal:β-D-Gal-α13Gal-galactosyltransferase enzyme responsible for Galα13Gal synthesis and expression. These mice develop normally and exhibit no gross phenotypic abnormalities. The Galα13Gal epitope is absent from the vascular endothelium and other tissues in α1,3GT (−/−) adult mice. By contrast, α1,3GT (−/−) mice, like humans, develop naturally occurring anti-α-galactoside antibodies normally absent in wild type mice. Female α1,3GT (−/−) mice yield oocytes that are devoid of the Galα13Gal epitope; however, these mice are fully fertile. These observations indicate that the Galα13Gal moiety is not essential to sperm-oocyte interactions leading to fertilization or to essentially normal development. They further suggest that α1,3GT (−/−) mice will find utility for exploring approaches to diminish anti-Gal-dependent hyperacute xenograft rejection, which presents a major barrier to the use of porcine and other non-primate organs for xenotransplantation in humans.

Fertilization in mammals involves an adhesive interaction between sperm and the zona pellucida, a glycoprotein-containing shell that surrounds the oocyte. Sperm receptor activity of the murine oocyte resides in the zona pellucida glycoprotein ZP3 (1). Sperm recognition of murine ZP3 depends upon Olinked oligosaccharides displayed by ZP3 (Ref. 2; reviewed in Refs. [3][4][5]. Treatment of purified egg ZP3 and ZP3-derived O-linked oligosaccharides with ␣-galactosidase eliminates sperm receptor activity (6). These observations have been taken to imply that terminal ␣-galactosides on ZP3 glycoconjugates are critical for sperm binding activity (6). This notion is supported by more recent observations demonstrating that structurally defined bi-and tetraantennary blood group I-re-lated oligosaccharides containing terminal Gal␣133Gal moieties inhibit binding of sperm to eggs in a dose-dependent manner (7).
In the mouse, at least one UDP-Gal:␤-D-Gal-␣133Gal-galactosyltransferase (␣1,3GT) 1 is responsible for the synthesis of terminal Gal␣133Gal␤134GlcNAc trisaccharides from common lactosamine-terminated glycoconjugates (8,9). Mice and other placental mammals express the Gal␣133Gal␤134GlcNAc trisaccharide products of ␣1,3GT on a variety of glycoproteins and in a variety of tissues (10,11). Aside from the postulated role of Gal␣133Gal moiety in murine fertilization, the function(s) of this structure are not known.
By contrast, humans, apes, and Old World monkeys lack the ability to synthesize these oligosaccharide moieties, because the genetic homologues of the murine ␣1,3GT locus are pseudogenes incapable of encoding a functional ␣1,3GT (12,13). Consequently, these latter species are reciprocally replete with immunoglobulins of all classes directed against terminal Gal␣133Gal epitopes (14,15). These antibodies are presumed to arise through immunization by environmental antigens similar or identical to the Gal␣133Gal epitope (16). In humans these natural antibodies (termed anti-Gal) comprise approximately 1% of circulating IgG, as well as a significant fraction of circulating IgM class antibodies (14,17). Anti-Gal antibodies are clinically important in the context of the proposed use of porcine and other non-primate mammalian organs to circumvent the shortage of human organs for transplantation purposes (reviewed in Refs. 18 and 19). Anti-Gal antibodies serve to initiate hyperacute rejection of xenografts derived from such mammalian species, via complement-mediated cytolytic events involving terminal Gal␣133Gal␤134GlcNAc glycoconjugates expressed by the vascular endothelium of the xenotransplant (20,21).
To directly address the role of Gal␣133Gal containing oligosaccharides in fertilization in the mouse, we have used a gene disruption approach in embryonic stem cells (22) to generate mice homozygous for a null ␣1,3GT allele. ␣1,3GT (Ϫ/Ϫ) mice are deficient in the expression of Gal␣133Gal epitopes on oocytes but are as fertile as their wild type litter mates, indicating that Gal␣133Gal epitopes are not essential to spermoocyte binding in this species. As with humans, apes, and Old World monkeys, ␣1,3GT (Ϫ/Ϫ) mice maintain naturally occurring anti-Gal antibodies but are deficient in the expression of Gal␣133Gal epitopes on vascular endothelium and other tissues. These observations imply that the inactivated ␣1,3GT gene represents the only functional murine ␣1,3GT locus, and they suggest that the ␣1,3GT (Ϫ/Ϫ) mouse may prove useful as a small animal model for studying approaches that can diminish anti-Gal-dependent hyperacute organ transplant rejection.

EXPERIMENTAL PROCEDURES
Generation of ␣1,3GT (Ϫ/Ϫ) Mice-A genomic clone of the ␣1,3GT locus was isolated from the 129SV mouse strain and restricted with NotI-MluI, and the resulting 12-kilobase fragment was cloned into pGEM-5 (Stratagene). A neomycin resistance cassette, pgkNeo, was inserted into the SalI site within the catalytic domain, and a 500-base pair BstEII-NotI fragment was subsequently removed from a position * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Associate Investigator of the Howard Hughes Medical Institute.
ϳ900 base pairs 3Ј of the pgkNeo insertion. A thymidine kinase cassette (pgkTK) (23) was then inserted into the targeting vector at a position corresponding to the BstEII site. The Neo insertion disrupts the largest coding exon in the ␣1,3GT gene (8); fusion of pgkNeo sequences to this exon yields premature termination codons in all three exonic reading frames. D3 embryonic stem cells (ES) (24) were electroporated and selected by standard methods (25). Homologous recombination of the targeting vector with the native allele was detected in individual ES clones by a nested polymerase chain reaction strategy (Fig. 1a, solid arrowheads). Polymerase chain reaction-positive ES clones were expanded and were subjected to extensive restriction analysis by Southern blotting of genomic DNA. ES clones containing a single, homologously integrated targeted allele were used to generate chimeric mice via blastocyst injection, as described previously (26). Progeny from two ES lines (1G7 and 1F3) were used for subsequent experiments.

RESULTS AND DISCUSSION
A targeted disruption of the murine UDP-Gal:␤-D-Gal-␣133Gal-galactosyltransferase (␣1,3GT) gene in embryonic stem cells (ES) was completed as shown in Fig. 1a. F1 heterozygous (␣1,3GT (ϩ/Ϫ)) littermates were intercrossed to yield viable progeny with genotype frequencies (22% (Ϫ/Ϫ), 50% (ϩ/Ϫ), and 28% (ϩ/ϩ)) corresponding to a Mendelian inheritance pattern, indicating that homozygosity for the null ␣1,3GT allele is compatible with essentially normal intrauterine development. Mice that are homozygous for the null allele do not differ in size or appearance from their wild type litter mates. The major organs of the ␣1,3GT (Ϫ/Ϫ) animals are grossly and histologically normal, as are the levels of a variety of serum analytes. Total and differential blood leukocyte counts, red cell counts, and platelet counts are not significantly different between the ␣1,3GT (Ϫ/Ϫ) mice and wild type control mice.
Studies in vitro indicate that terminal ␣-galactosides displayed by O-linked glycans on the mouse zona pellucida glycoprotein ZP3 are required for the binding of sperm to the oocyte (2)(3)(4). These glycoconjugates are easily demonstrated on the zona pellucida of wild type oocytes (Fig. 3b), using a lectin (BSIB4) that specifically recognizes these molecules (30). By contrast, oocytes obtained from ␣1,3GT (Ϫ/Ϫ) females do not stain with this lectin (Fig. 3e). The same result was also observed by staining oocytes with human anti-Gal (data not shown). The loss of the ability to detect oocyte ␣-galactosides is not due to a blocking effect of maternal anti-Gal immunoglobulins bound to the oocyte, since anti-mouse immunoglobulins did not interact with these oocytes (data not shown). These observations directly demonstrate that the ␣1,3GT locus determines oocyte expression of terminal ␣-galactosides. Table I summarizes breeding studies completed to determine if fertility is affected by absence of zona pellucida terminal ␣-galactosides consequent to nullizygosity at the ␣1,3GT locus. In matings between ␣1,3GT (Ϫ/Ϫ) females and fertile wild type males of the same genetic background, we observed fertility rates and litter sizes equivalent to those observed in control matings involving ␣1,3GT (Ϫ/ϩ) and ␣1,3GT (ϩ/ϩ) females. These observations demonstrate that absence of zona pellucida terminal ␣-galactosides is compatible with normal fecundity and indicate that terminal ␣-galactosides do not represent an essential component of the mouse oocyte sperm receptor(s). This conclusion leaves open the possibility that Gal␤13 4GlcNAc-terminated blood group I-related oligosaccharides capable of blocking sperm-egg binding (7) are instead responsible for sperm-egg adhesion during fertilization. Absence of an essential role for terminal Gal␣133Gal structure in fertilization is also consistent with an alternative hypothesis that murine sperm-egg adhesion during fertilization is accomplished through an interaction between terminal N-acetylglucosamine moieties on the oocyte and surface-localized ␤(1,4)galactosyltransferase on murine spermatids (31).
circulating anti-Gal antibodies, represent the only available experimental animal recipient for such studies; cost and logistical considerations associated with the care of these large animals can represent a substantial impediment to experimental progress in this area. The ␣1,3GT (Ϫ/Ϫ) mice we describe here may represent a useful alternative small animal for this work, since it can be anticipated that the naturally occurring anti-Gal antibodies in an ␣1,3GT (Ϫ/Ϫ) murine graft recipient will lead to hyperacute graft rejection of a transplanted organ taken from an ␣1,3GT (ϩ/ϩ), Gal␣133Gal-positive, but otherwise syngeneic donor mouse. The extensive experience with organ transplants in mice (33), the well defined histocompatibility loci in this species (34), and highly developed systems for murine transgenesis represent additional advantages of this system for the study of anti-Gal-dependent hyperacute organ transplant rejection. Studies are currently in progress to study hyperacute transplant rejection utilizing these ␣1,3GT (Ϫ/Ϫ) mice.

TABLE I
Comparison of the fecundity of ␣1,3GT null mice with wild type mice Chimeras that transmitted the inactive allele were derived from ES lines 1G7 and 1F3. Male chimeras were mated with female F1(C57BI/6J ϫ DBA/2J) mice to yield F1(129SV ϫ C57BI/6J ϫ DBA/2J) offspring. The percentages of different genotypes in heterozygous F1 crosses were 28%, 50%, and 22%, for wild type, heterozygous, and null, respectively. Null crosses were performed with male and female littermates. Wild type crosses with null mice were performed with proven fertile wild type animals of the same F2(129SV ϫ C57BI/6J ϫ DBA/2J) genetic background. Numbers in parentheses indicate the number of pups born.