Copper-zinc superoxide dismutase (CuZn-SOD) is
believed to play a major role in the first line of antioxidant defense
by catalyzing the dismutation of superoxide anion radicals to form
hydrogen peroxide and molecular oxygen. Recent studies have shown that missense mutations in this gene contribute, evidently through a
gain-of-function mechanism, to about 20% of familial amyotrophic lateral sclerosis. To define further the physiologic role of this enzyme, a model of mice deficient in this enzyme was generated using
gene targeting technology. Mice lacking this enzyme were apparently
healthy and displayed no increased sensitivity to hyperoxia. However,
they exhibited a pronounced susceptibility to paraquat toxicity. Most
surprisingly, female homozygous knock-out mice showed a markedly
reduced fertility compared with that of wild-type and heterozygous
knock-out mice. Further studies revealed that although these mice
ovulated and conceived normally, they exhibited a marked increase in
embryonic lethality. These data, for the first time, suggest a role of
oxygen free radicals in causing abnormality of female reproduction in
mammals.
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INTRODUCTION |
Reactive oxygen species
(ROS),1 which are produced as
by-products of normal metabolism, are capable of causing cellular
damage, leading to cell death and tissue injury (for review, see Ref. 1). Mammalian cells are equipped with both enzymatic and nonenzymatic antioxidant defense mechanisms to minimize the cellular damage resulting from interaction between cellular constituents and ROS (for
review, see Ref. 2). Despite the presence of these delicate cellular
antioxidant systems, an overproduction of ROS in both intracellular and
extracellular spaces often occurs upon exposure of cells or individuals
to radiation, hyperoxia, and certain chemicals. An unbalanced
production of ROS has been postulated to play a role in the
pathogenesis of a number of clinical disorders such as acute
respiratory distress syndrome, ischemia/reperfusion injury, atherosclerosis, neurodegenerative diseases, and cancer (for review, see Ref. 3). This understanding illustrates the importance of the
antioxidant defense system in maintaining normal cellular physiology.
However, due to the overlapping activity among some of the antioxidant
enzymes, it is generally difficult to define the role of each
individual antioxidant enzyme. We are interested in understanding the
physiologic relevance of copper-zinc superoxide dismutase (CuZn-SOD)
under normal physiologic conditions and in defending cells and animals
against the pathogenesis of ROS-mediated diseases. The results from
previous studies for defining the protective function of this
enzyme using cells and animals with augmented enzyme expression have
been controversial (4-9), since some of them develop an increased
susceptibility to certain oxidants relative to that of parental cells
and control animals. It is not clear whether the detrimental effect of
CuZn-SOD overexpression is a result of the associated free radical
generating activity of this enzyme or of its capability in enhancing
nitration of tyrosine by peroxynitrite (10-15). Therefore,
overexpression of this enzyme may not provide a suitable model to
address the nature of this enzyme in cellular antioxidant mechanisms.
To define further the role of CuZn-SOD in cellular antioxidant defense
mechanisms, we generated, by gene targeting technology, mice lacking
this enzyme.
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MATERIALS AND METHODS |
Targeted Disruption of the Mouse Sod1 Gene--
Eleven mouse
Sod1 genomic clones were isolated from a 129/SvJ genomic
library purchased from Stratagene (La Jolla, CA) by screening with a
rat Sod1 cDNA probe (16). An approximately 7.2-kb
SacI genomic fragment from clone 30 was found to contain the
entire mouse Sod1 gene with a sequence very similar to that published by Benedetto et al. (17). To inactivate the mouse Sod1 gene, the SmaI and HindIII
restriction sites flanking the SmaI-HindIII
fragment, which contains sequences from intron 1 to intron 4, were
converted into XhoI sites by linker ligation and then
inserted into the XhoI site in plasmid vector pPNT (see Ref.
18, Fig. 1a). Similarly, linker ligation was also used to
clone the EcoRI-SalI fragment containing the
3'-flanking sequence of the gene into the BamHI site in the
pPNT vector.
The Sod1 targeting vector, in which exon 5 was deleted, was
linearized by HindIII digestion and transfected into R1
embryonic stem cells (19). Clones resistant to G418 and ganciclovir
were screened by Southern blot analysis using a probe 5' external to the genomic sequence present in the targeting vector. Fifty-two clones
were identified from 666 clones screened to contain the expected
targeted Sod1 allele.
Targeted clones were microinjected into C57BL/6 blastocysts following
the standard procedure (20). Thirty chimeric mice with near 100%
chimerism were generated using Sod1 knock-out clones 5 and
8. Chimeric mice derived from either clone showed 100% transmission of
the 129/SvJ chromosomes.
Breeding of Sod1 Knock-out Mice--
The heterozygous CuZn-SOD
knock-out (Sod1+/
) mice were initially derived
from breeding between the chimeric mice and C57BL/6 mice. They,
therefore, are F1 hybrid between the 129SvJ and C57BL/6 inbred genetic
backgrounds. These Sod1+/
mice were interbred
to generate mice with three Sod1 genotypes (Sod1+/+, Sod1+/
, and
Sod1
/
). These F2 littermates were used in
expression studies including RNA and protein analysis. Since the female
Sod1
/
mice are not very fertile, breeding
was performed between F2 male Sod1
/
and
female Sod1+/
littermates as well as between
F2 Sod1+/+ male and female littermates to
generate a large number of wild-type and knock-out mice for various
pathologic and physiologic studies described in this report.
RNA Blot Analysis--
Total RNA was isolated from tissues by
the guanidinium isothiocyanate-CsCl method as described by Chirgwin
et al. (21). RNA was denatured with glyoxal and dimethyl
sulfoxide for electrophoresis on an agarose gel buffered with 10 mM sodium phosphate, pH 7.0 (22). Hybridization of the RNA
blot filter was performed according to the procedures described by
Thomas (23).
Assay for Superoxide Dismutase--
Activities of CuZn-SOD and
Mn-SOD were determined using a xanthine oxidase/cytochrome c
assay (25). However, acetylated cytochrome c instead of
cytochrome c was used in the reaction mixture to minimize
interference from cytochrome c reductases and oxidases
present in the tissue homogenates (26).
Detection of SOD Activity in a Native Polyacrylamide Gel
(24)--
One hundred micrograms of tissue protein were separated on a
non-denaturing polyacrylamide gel. The SOD activity was then visualized
by initially soaking the gel in 2.43 mM nitro blue tetrazolium for 20 min, followed by incubating in a solution of 50 mM potassium phosphate buffer, pH 7.8, containing 0.028 mM riboflavin, and 280 mM Temed.
Histological Analysis--
Wild-type and age-matched homozygous
knock-out mice were fixed by systemic perfusion with Bouin's fixative
through the left ventricle. The tissues were then embedded in paraffin,
sectioned, and stained with hematoxylin and eosin. Tissue sections were
examined under a light microscope.
Measurement of Estrous Cyclicity (27)--
Vaginal smears were
taken daily using a moisturized cotton tip and examined under a light
microscope. Entry into the estrous cycle is indicated by an increase in
the number of nucleated epithelial cells in vaginal samples.
Fecundity Index Determination--
Female mice of three
Sod1 genotypes were housed with either male
Sod1+/+ or Sod1
/
mice. Copulatory plugs were checked at around 9 a.m. each morning. Female mice with plugs were then housed individually and the birth date
and number of pups recorded.
Hyperoxic Exposure of Mice--
Ten-week-old
Sod1+/+ and Sod1
/
mice were used for exposure to >99% oxygen in several Plexiglas
chambers. The oxygen concentration varied less than 2%, and
CO2 concentration was maintained at less than 0.5% by
providing approximately 12 complete gas changes per h. During the
exposure, food and water were provided ad libitum, and the
animals were kept in a 12-h on, 12-h off light cycle at all times. The
numbers of surviving animals were counted three times each day.
Statistical Analysis--
One-way analysis of variance was
used to examine differences in each measurement performed on wild-type,
heterozygous, and homozygous knock-out mice. If a significant
difference was observed (p < 0.05), then pairwise
comparisons among mice were made using Duncan's test. Fecundity
indices of mice were analyzed by one-sided Fisher's exact test.
Survival of wild-type and knock-out mice exposed to >99% oxygen or
following intraperitoneal administration of paraquat at 10 mg/kg body
weight was analyzed using the Kaplan-Meier method.
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RESULTS AND DISCUSSION |
Generation and Characterization of Sod1 Knock-out Mice--
As
shown in Fig. 1a, exon 5 of
the mouse Sod1 gene (which encodes the C-terminal of the
protein from amino acid residues 120-154 that constitute both the
structure and function of the active site channel (28)) and some of the
flanking intron sequences were replaced by a neomycin resistance
cassette (neo). Insertion of the neo in the mouse
Sod1 gene creates a new PstI restriction site,
resulting in a shorter PstI genomic fragment from the
targeted allele (~12.5 kb) than that from the wild-type allele
(~16.5 kb). Mice heterozygous (Sod1+/
) for
the targeted allele were interbred to generate homozygous knock-out
(Sod1
/
) mice. An example of DNA blot
analysis of mouse DNA is shown in Fig. 1b. In addition to
the ~16.5-kb wild-type and the ~12.5-kb targeted genomic fragments,
the 5' external probe containing exon 1 sequence also hybridized with a
PstI fragment of 6.6 kb. This is believed to result from
cross-hybridization between the probe and the mouse Sod1
pseudogene(s) (29).

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Fig. 1.
Generation and characterization of
CuZn-SOD-deficient mice. a, schematic diagram showing the
genomic and partial restriction map of the mouse Sod1 locus
(top), the targeting vector (middle), and the
predicted structure of the targeted locus (bottom).
Numbered black boxes represent exons. Striped box
represents the 5' external sequence used as a hybridization probe.
neo, neomycin resistance gene cassette; TK,
herpes thymidine kinase gene cassette. B, BamHI; E, EcoRI; H, HindIII;
P, PstI; S, SacI;
Sa, SalI; Sm, SmaI. The approximate sizes of hybridizing PstI genomic fragments of
the wild-type allele and the targeted allele are indicated at the top and bottom of the figure, respectively.
kb, kilobase pairs. b, DNA blot analysis of mouse
offspring. Mouse tail DNA was digested with PstI and probed
with the 5' external probe shown in a. +/+, +/ , and /
represent wild-type, heterozygous, and homozygous knock-out mice,
respectively. c, RNA blot analysis of total cellular RNA
isolated from tissues of Sod1+/+,
Sod1+/ , and Sod1 /
mice. Twenty-five micrograms of total RNA from each tissue were separated on agarose gel for blot analysis. The RNA blot was initially hybridized with a rat CuZn-SOD cDNA probe (top panel),
and then re-hybridized with a rat glyceraldehyde-3-phosphate
dehydrogenase (GAPDH, bottom panel) cDNA to
check the potential variation in sample loading. d, a native
polyacrylamide gel showing activity staining for SOD in tissues of
Sod1+/+, Sod1+/ , and
Sod1 / mice (24). The types of tissues and
Sod1 genotypes are shown at the top of the gel.
The positions of CuZn-SOD and Mn-SOD migration are indicated to the
left.
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Inactivation of the functional mouse Sod1 gene by gene
targeting was then demonstrated by expression study. RNA blot analysis revealed an approximate 40-60% reduction of Sod1 mRNA
in tissues of Sod1+/
mice compared with that
of wild-type (Sod1+/+) mice (Fig.
1c). Furthermore, no CuZn-SOD mRNA could be found in the
tissues from Sod1
/
mice, indicating that the
truncated CuZn-SOD or CuZn-SOD-neo fusion mRNA was degraded rapidly
in these tissues. Reduction of CuZn-SOD activity in tissues of
Sod1+/
and Sod1
/
mice was also confirmed by activity staining on a native polyacrylamide gel (Fig. 1d) and the enzyme assay (Table
I). It should be noted that whereas no
CuZn-SOD activities were found in brain and liver of
Sod1
/
mice, a very low level of CuZn-SOD
activity was present in the lung samples. This activity presumably
represents the activity of extracellular superoxide dismutase, as
expression of this SOD isoform is relatively high in the lungs compared
with other tissues (30). A decrease in CuZn-SOD activity apparently had
no effect on the activity of other cellular antioxidant enzymes such as manganese superoxide dismutase (Mn-SOD) (Fig. 1d and Table
I), catalase, and glutathione peroxidase and the enzymes that
participate in the recycling of oxidized glutathione including
glutathione reductase and glucose-6-phosphate dehydrogenase in these
tissues from Sod1+/+,
Sod1+/
, and Sod1
/
mice (data not shown).
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Table I
SOD activities in tissues of Sod1+/+,
Sod1+/ , and Sod1 / mice.
Values are means ± SD, and n = 5 for all tissue
samples. NA, no CuZn-SOD activities were detected.
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Mice Lacking CuZn-SOD Are Phenotypically Normal and Show No
Increased Sensitivity to Hyperoxia, but Exhibit a Pronounced
Susceptibility to Paraquat--
The ratio of the three Sod1
genotypes of mouse progeny obtained from interbreeding of
Sod1+/
mice was in agreement with Mendelian
inheritance, indicating that there was no lethality in development of
Sod1
/
mice. Male and female
Sod1
/
mice grew normally and were apparently
healthy upon observation to 16 months of age. Histologic survey at the
light microscopic level performed on five
Sod1
/
mice at 4.5 months of age showed no
evidence for abnormalities in various tissues including the brain,
heart, intestine, kidney, liver, lung, testis, uterus, and ovary (data
not shown). Since CuZn-SOD is highly expressed in erythrocytes (31) and
is believed to play a protective role against the superoxide-mediated
damage in these cells, a survey of hematologic profile was performed to
assess the effect of CuZn-SOD deficiency on homeostasis of these cells.
No differences were found in the numbers of red cells, reticulocytes,
and differential leukocyte counts including lymphocytes, monocytes,
neutrophils, eosinophils, and platelets of
Sod1+/+, Sod1+/
, or
Sod1
/
mice (data not shown).
Since the protective role of CuZn-SOD against hyperoxia has been
implicated in earlier studies on rats and CuZn-SOD transgenic mice (32,
33), we next determined whether a deficiency in pulmonary CuZn-SOD
activity would render animals more susceptible to hyperoxic exposure.
As shown in Fig. 2a, a
virtually identical survival curve with a median survival time of
3.4 ± 0.1 (± S.E.) days was found in both
Sod1+/+ and Sod1
/
mice, indicating that the function of this enzyme in lung defense against the damage from increased production of superoxide anion radicals during hyperoxia is very limited. However, the
Sod1
/
mice were extremely sensitive to
paraquat at a dose of 10 mg/kg body weight with a median survival time
of 1.4 ± 0.3 (S.E.) days (Fig. 2b, p < 0.0001 compared with Sod1+/+ or
Sod1+/
mice). Remarkably, the
Sod1
/
mice became listless at about 30 min
after intraperitoneal administration of paraquat, while
Sod1+/+ and Sod1+/
mice
were phenotypically normal even at the end of 7 days of observation.

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Fig. 2.
Increased susceptibility in
Sod1 / mice to paraquat but not hyperoxia.
a, survival analysis of Sod1+/+ and
Sod1 / mice under hyperoxia. The survival
times of age-matched Sod1+/+ and
Sod1 / mice of both genders under >99%
oxygen were measured. b, survival curves of age-matched male
Sod1+/+, Sod1+/ , and
Sod1 / mice following intraperitoneal
administration of paraquat at a dose of 10 mg/kg body weight.
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Female Mice Lacking CuZn-SOD Exhibit a Marked Increase in
Post-implantation Embryo Death--
During the study, we intended to
generate a large number of Sod1
/
mice for
various pathologic and physiologic studies by interbreeding between
Sod1
/
mice. To our surprise, the
reproductive performance of female Sod1
/
mice was inferior to that of female Sod1+/
mice. As shown in Table II, while 10 female Sod1+/
mice gave birth to 26 litters
(mean litter size 7.5 ± 2.5) in a period of 3 months, only 16 litters (mean litter size 1.6 ± 1.0) were yielded from an equal
number of Sod1
/
female mice. Of these 16 litters, 6 litters contained only 1 pup, 3 litters contained 2 pups,
and one litter contained 4 pups. It should be noted that the drastic
reduction in reproduction of Sod1
/
females
is not a result of a defect in the development of
Sod1
/
fetuses, since of the 194 pups derived
from the breeding between Sod1+/
female and
Sod1
/
male mice, 52% were heterozygous and
48% homozygous for the targeted Sod1 allele.
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Table II
Number of progeny produced from breeding of female Sod1 knock-out
mice
Ten female +/ and / mice of 2 months of age were used in breeding
for a period of 3 months.
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To understand further this unexpected observation, the reproductive
performance of the female mice with three Sod1 genotypes was
closely followed. As shown in Table III,
male Sod1
/
mice were as fertile as
Sod1+/+ males, and female
Sod1+/+ and Sod1+/
mice
were similarly fertile when bred with either
Sod1+/+ or Sod1
/
male
mice. However, the fecundity index (number of litters/number of
copulations) and size of the litters of
Sod1
/
females were much less than those of
Sod1+/+ and Sod1+/
females.
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Table III
Reproductive performance of Sod1+/+, Sod1+/ ,
and Sod1 / female mice in breeding with Sod1+/+ and
Sod1 / male mice
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The mechanism underlying the poor reproductive performance of
Sod1
/
females was further investigated.
Examination of vaginal smears indicated that all types of mice had
similar estrous cycles, with an average length of 4 to 5 days. The
frequency of female mice that became receptive to males was also
measured. For 6 weeks, eight Sod1+/+,
Sod1+/
, and Sod1
/
female mice mated 17, 20, and 18 times with vasectomized males, respectively. Apparently, the reduced fertility in
Sod1
/
mice was not a result of altered
estrous cycles. The numbers of ova ovulated by these three types of
females at each estrous cycle were also found to be equivalent (Table
IV). In addition, female
Sod1
/
mice exhibited a normal ovarian
histology including the number, size, or morphology of antral follicles
and corpora lutea compared with that of Sod1+/+
and Sod1+/
mice (data not shown). These
results suggested that the reduced fertility in
Sod1
/
female mice might result from a defect
in implantation of embryos to the wall of the uterine horns or
premature death of the fetuses.
To evaluate further these two possibilities, we then examined the
number and viability of fetuses in Sod1
/
female mice bred with Sod1
/
male mice
between 10.5 and 14.5 days postcoitum. As shown in Table
V, of 15 mice examined, only 2 of them
were not pregnant. However, 75 of 91 implanted embryos in a variety of
sizes were found dead and in the process of being resorbed. Compared
with the normal 12.5-day fetuses, most of those dead and resorbed
embryos were loosely attached to the wall of the uterine horns without a developed placenta or yolk sac (data not shown). Judging from the
sizes of these embryos, embryonic death might have occurred at various
times before 10 days of pregnancy. In control experiments, 14 of 15 female Sod1+/
mice were pregnant from breeding
with Sod1
/
males. A total of 122 implanted
embryos was found, and of them only 9 were dead. Although the average
number of successfully implanted embryos in
Sod1
/
females (6.3 ± 4.4), including
both the live and dead ones, was less than that in
Sod1+/
females (8.2 ± 3.0), the
difference was not statistically significant (p = 0.2).
These results indicated that the efficiency of embryo implantation in
the former mice was equivalent to that in the latter mice. However, the
rate of post-implantation embryonic death that occurred in
Sod1
/
females was significantly higher than
that in Sod1+/
females (83 ± 23%
versus 7.0 ± 7.0%, respectively; p < 0.0001).
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Table V
Post-implantation embryonic lethality in female
Sod1+/ and Sod1 / mice in breeding with
male Sod1 / mice
Pregnant female mice were sacrificed between 10.5 and 14.5 days
postcoitum, and the number and viability of embryos were determined.
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During the course of this study, generation and characterization
of a line of mice lacking the entire Sod1 locus have also been reported (34). Although we inactivated the Sod1 gene by a different approach, our results on the apparently normal phenotype of
the Sod1
/
mice are in agreement with those
reported previously. However, our study has focused on some other
antioxidant functions of this enzyme that were not described previously
(34). Our data reveal that although the role of CuZn-SOD in defending
lungs against lethal exposure of hyperoxia is negligible, it is
essential in protecting animals against paraquat toxicity. These data
indicate that the protective role of CuZn-SOD is dependent on the
cellular site of oxygen radical generation. The mitochondrion is known to be a major subcellular site producing oxygen radicals under normal
physiologic conditions (for review, see Ref. 1), and the rate of
radical production is further enhanced in mitochondria of hyperoxic
lungs (35-37). This understanding and our results suggest that Mn-SOD
may play a more critical role than does CuZn-SOD in antioxidant defense
mechanism(s) under normal physiologic conditions, and in defending
against lung injury resulting from hyperoxic insults. This notion is
supported by the recent findings that mice lacking Mn-SOD die at very
young ages (38-39). However, CuZn-SOD is apparently critical for
animals to survive under a lethal exposure to paraquat, a bipyridyl
herbicide capable of generating oxygen radicals through the redox
cycling mechanism. This reaction is believed to be catalyzed by the
enzyme NADPH-dependent cytochrome P-450 reductase,
primarily located in the endoplasmic reticulum. Our data suggest
that both the cytosolic and microsomal enzymes may be the primary
targets of superoxide radicals generated during paraquat toxicity. This
conclusion is in agreement with an earlier study reported by Phillips
and colleagues (40) that Drosophila deficient in CuZn-SOD is
hypersensitive to paraquat.
The most intriguing observation made in this study is the reduced
fertility of female mice lacking this enzyme. Apparently, this defect
is associated with CuZn-SOD deficiency in the female mice and unrelated
to the Sod1 genotypes of the fetuses. Since the female
Sod1
/
mice exhibited a normal estrous cycle
and generated comparable numbers of ova compared with those of
Sod1+/+ and Sod1+/
females, the reduced fertility might not have been a result of a gross
defect in the hypothalamic-pituitary axis in these mice. However,
post-implantation embryonic loss did occur, and this could certainly be
endocrine-related. Interestingly, male CuZn-SOD-deficient fruit flies
are sterile, and females show a markedly reduced fertility (40). These
and our results suggest that CuZn-SOD plays a critical role in female
reproduction. The exact mechanism(s) underlying the observed reduced
fertility in female CuZn-SOD-deficient mice as well as its implication
in human reproductive dysfunction remain to be defined.
We thank Dr. Richard Mulligan of
Massachusetts Institute of Technology for the gift of plasmid
pPNT, Dr. Andras Nagy of Mount Sinai Hospital at Toronto for the gift
of R1 embryonic stem cells, and Syntex Inc. (Palo Alto, CA) for
supplying ganciclovir.