(Received for publication, June 28, 1995)
From the
The Gal1
3Gal 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 (
1,3GT) encoding the
UDP-Gal:
-D-Gal-
1
3Gal-galactosyltransferase
enzyme responsible for Gal
1
3Gal synthesis and expression.
These mice develop normally and exhibit no gross phenotypic
abnormalities. The Gal
1
3Gal epitope is absent from the
vascular endothelium and other tissues in
1,3GT (-/-)
adult mice. By contrast,
1,3GT (-/-) mice, like
humans, develop naturally occurring anti-
-galactoside antibodies
normally absent in wild type mice. Female
1,3GT (-/-)
mice yield oocytes that are devoid of the Gal
1
3Gal epitope;
however, these mice are fully fertile. These observations indicate that
the Gal
1
3Gal moiety is not essential to sperm-oocyte
interactions leading to fertilization or to essentially normal
development. They further suggest that
1,3GT (-/-)
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 O-linked
oligosaccharides displayed by ZP3 ((2) ; reviewed in (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-related oligosaccharides containing
terminal Gal
1
3Gal moieties inhibit binding of sperm to eggs
in a dose-dependent manner(7) .
In the mouse, at least one
UDP-Gal:-D-Gal-
1
3Gal-galactosyltransferase
(
1,3GT) (
)is responsible for the synthesis of terminal
Gal
1
3Gal
1
4GlcNAc trisaccharides from common
lactosamine-terminated glycoconjugates(8, 9) . Mice
and other placental mammals express the
Gal
1
3Gal
1
4GlcNAc 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
1
3Gal 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
1
3Gal
epitopes(14, 15) . These antibodies are presumed to
arise through immunization by environmental antigens similar or
identical to the Gal
1
3Gal 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 (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
1
3Gal
1
4GlcNAc glycoconjugates expressed by the
vascular endothelium of the xenotransplant(20, 21) .
To directly address the role of Gal1
3Gal 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
(-/-) mice are deficient in the expression of
Gal
1
3Gal epitopes on oocytes but are as fertile as their
wild type litter mates, indicating that Gal
1
3Gal epitopes
are not essential to sperm-oocyte binding in this species. As with
humans, apes, and Old World monkeys,
1,3GT (-/-) mice
maintain naturally occurring anti-Gal antibodies but are deficient in
the expression of Gal
1
3Gal 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 (-/-) mouse may prove
useful as a small animal model for studying approaches that can
diminish anti-Gal-dependent hyperacute organ transplant rejection.
Figure 1:
Targeted disruption of the murine
1,3GT gene. a, restriction map of the
1,3GT allele,
the targeting vector, and the disrupted allele. Blackrectangles denote
1,3GT locus exons (thickestportions) and introns (thinnestportions). A neomycin resistance cassette (pgkNeo) was
used to disrupt the
1,3GT catalytic domain (found in the largest
1,3GT exon), resulting in a frameshift and premature stop codons
relative to both the Neo and
1,3GT translational reading frames.
The targeting vector contains 11 kilobases of 5` genomic DNA and 0.9
kilobases of 3` sequence flanking the neomycin resistance cassette used
to disrupt the
1,3GT catalytic domain exon (largest exon).
Restrictions sites are indicated by abbreviations (B, BstEII; N, NotI; M, MluI; P, PstI; S; SalI). Restriction
sites destroyed during vector construction procedures are in parentheses. Openarrows (
) denote the
transcriptional orientations of the
1,3GT locus, the pgkNeo
segment, and the pgkTK segment. Solidarrows (
) indicate positions corresponding to polymerase chain
reaction primers used to screen targeted ES lines. The position of the BstEII-PstI segment used in Southern blot analyses
described in b is indicated below the schematic of the
targeted allele (3` Probe). KB, kilobase(s). b, disruption of the
1,3GT locus. Homologous
recombination-mediated replacement of the wild type
1,3GT alleles
by the disrupted allele was confirmed by Southern blot analysis.
Genomic DNA isolated from the parental ES line, and from three ES
clones that gave rise to germ line transmission, is compared to tail
genomic DNA from the progeny of F1 heterozygous crosses of littermates.
The DNA was digested with PstI and probed with a DNA fragment
flanking the 3` end of the genomic sequence of the targeting vector (3` probe; see panela).
A targeted disruption of the murine
UDP-Gal:-D-Gal-
1
3Gal-galactosyltransferase
(
1,3GT) gene in embryonic stem cells (ES) was completed as shown
in Fig. 1a. F1 heterozygous (
1,3GT
(+/-)) littermates were intercrossed to yield viable progeny
with genotype frequencies (22% (-/-), 50% (+/-),
and 28% (+/+)) 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
(-/-) 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 (-/-) mice
and wild type control mice.
We used human Gal1
3Gal
antibodies to confirm that the
1,3GT (-/-) mice are
deficient in Gal
1
3Gal expression. In humans, at least 1% of
the circulating IgG class antibodies, and substantial amounts of
circulating IgM class antibodies, are directed against terminal
Gal
1
3Gal
1
4GlcNAc-containing
oligosaccharides(14, 17) . By contrast, human tissues
are essentially devoid of Gal
1
3Gal epitopes, because a
functional
1,3GT locus is apparently not present in
humans(12, 13) . In humans, ``naturally
occurring'' polyclonal antibodies directed against the
Gal
1
3Gal epitope (27) (termed ``anti-Gal''
antibodies) are presumed to occur as a consequence of continuous
immunization by gastrointestinal flora containing glycoconjugates with
terminal
-galactoside structures (16) . As noted
previously(10) , human anti-Gal antibody detects
-galactosidase-susceptible Gal
1
3Gal epitopes on a
variety of wild type murine cells, including vascular endothelium (Fig. 2, a and b). By contrast, the vascular
endothelium of
1,3GT (-/-) mice is devoid of
detectable terminal
-galactosides (Fig. 2c).
1,3GT activity, normally present in murine spleen cells, is not
detectable in
1,3GT (-/-) splenocytes (data not
shown).
Figure 2:
1,3GT (-/-) mice are
deficient in vascular endothelial cell Gal
1
3Gal epitopes and
generate naturally occurring anti-Gal antibodies. Vessels of the lung
were stained with human anti-Gal antibodies. The characteristic strong
staining of
1,3GT (+/+) lung vessels is shown in a. Pretreatment of sections containing
1,3GT
(+/+) lung vessels with
-galactosidase eliminates
reactivity (b) and, as shown previously(29) , confirms
the specificity of the anti-Gal antibody for terminal
-galactosides. In contrast to
1,3GT (+/+) lung
vessels, the vessels of
1,3GT (-/-) mice do not stain
with the anti-Gal antibody (c) (original magnification,
480). d, anti-Gal titers in sera
1,3GT
(-/-) mice (Null) were compared
1,3GT
(+/+) (WT) mice by direct (Direct)
hemagglutination of rabbit erythrocytes (4) and indirect
agglutination (Indirect) using an anti-mouse IgG reagent.
Indirect hemagglutination was also completed using
1,3GT
(-/-) sera that had been preincubated with the immobilized
synthetic trisaccharide Gal
1
3Gal
1
4GlcNAc (Gal
) or Fuc
1
2Gal
1
4GlcNAc (Fuc
) glycoconjugates (*, p < 0.001 direct; p < 0.0001 indirect).
The reciprocal relationship between the absence of
Gal1
3Gal epitopes and the presence of anti-Gal antibody
observed in humans and some other primates (12) is
recapitulated in the
1,3GT (-/-) mice. Sera from
1,3GT (-/-) mice directly agglutinate
Gal
1
3Gal-positive (14, 28) rabbit
erythrocytes, whereas sera from
1,3GT (+/+) mice, as
expected, do not and thus are devoid of anti-Gal antibody activity. The
rabbit erythrocyte hemagglutinating activity present in
1,3GT
(-/-) sera can be removed by preincubation of the sera with
immobilized synthetic Gal
1
3Gal
1
4GlcNAc
structures, whereas removal of hemagglutinating activity does not occur
when the sera are preabsorbed with Fuc
1
2Gal
1
4GlcNAc structures (Fig. 2d). Sera prepared from
1,3GT (-/-) mice contain antibodies that also bind to
murine laminin, a glycoprotein containing terminal
-galactosides(29) , but do not bind
-galactosidase-treated laminin. By contrast, sera from
1,3GT
(+/+) mice do not bind to murine laminin (data not shown).
These observations indicate that the
1,3GT (-/-) mice
maintain naturally occurring anti-Gal antibodies and indicate that
these mice are therefore essentially deficient in the expression of
terminal Gal
1
3Gal moieties.
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 (-/-) 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.
Figure 3:
Oocytes from 1,3GT (-/-)
mice are deficient in Gal
1
3Gal epitopes. Live wild type
mouse oocytes (a-c) and
1,3GT (-/-)
oocytes (d and e) were stained with fluoresceinated B. simplicifolia isolectin B4 (BSIB4-FITC), a terminal
-galactoside-specific lectin(15) . a and d show phase contrast images corresponding to confocal fluorescence
images seen in b and e, respectively. Wild type
oocytes (b) show strong BSIB4-FITC binding of both the zona
pellucida and the oocyte. The specific interaction of BSIB4-FITC with
-galactosides is shown by loss of staining in the presence of
methyl
-galactopyranoside (15) (c). By contrast,
1,3GT (-/-) oocytes (e) lack detectable BSIB4-FITC
binding. Ten oocytes from both
1,3GT (+/+) and
1,3GT (-/-) females were examined in three separate
experiments (original magnification,
100).
Table 1summarizes 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 (-/-) 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 (-/+) and
1,3GT (+/+)
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
1
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
1
3Gal 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) .
In humans, naturally occurring anti-Gal
antibodies of the type found in the 1,3GT (-/-) mice
present a major obstacle to the use of porcine and other non-primate
organs for human xenotransplantation. These antibodies bind to terminal
-galactosides on vascular endothelial cells of these mammals (17, 18) and mediate hyperacute xenograft rejection (19, 21) through complement-dependent endothelial cell
cytotoxicity. Early attempts to completely block these interactions in vivo met with limited success(21) . More recent
work involving transgene-directed overexpression of complement
inhibitors in the xenograft has shown substantial promise as a means to
mitigate anti-Gal-dependent hyperacute xenograft rejection (32) . Currently, Old World monkeys, which are naturally
deficient in the Gal
1
3Gal epitope but reciprocally replete
with 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 (-/-) 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 (-/-) murine graft recipient will lead to
hyperacute graft rejection of a transplanted organ taken from an
1,3GT (+/+), Gal
1
3Gal-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 (-/-) mice.