Molecular Human Reproduction vol.5 no.8 pp.

720725, 1999

Expression of genes encoding antioxidant enzymes in human and

mouse oocytes during the final stages of maturation

S.El Mouatassim1,2, P.Guerin2,3 and Y.Menezo1,2,4

1Laboratoire Marcel Merieux, Cytogenetique, Avenue Tony Garnier BP 7322, 69357 Lyon, 2INSA Biologie 406, Unite
biologie du developpement preimplantatoire, 20 Avenue A.Einstein, 69621 Villeurbanne cedex and 3Ecole Nationale
Veterinaire de Lyon, Unite biologie de la reproduction, CERREC, BP 83, 69280 Marcy letoile cedex France
4To whom correspondence should be addressed at: INSA Biologie 406, Unite biologie du developpement
preimplantatoire, 20 Avenue A.Einstein, 69621 Villeurbanne cedex France
The mRNA expression of five enzymes: catalase, Cu-Zn-superoxide dismutase (Cu-Zn-SOD), Mn-superoxide
dismutase (Mn-SOD), glutathione peroxidase (GPX), and -glutamylcysteine synthetase (GCS) each involved
in protection against free radicals was studied in human and mouse oocytes. In the mouse, oocytes were
collected at different stages of maturation in order to determine the storage of these transcripts. For the
human, germinal vesicle (GV) oocytes harvested during intracytoplasmic sperm injection (ICSI) procedures
and failed fertilized metaphase II (MII) oocytes were analysed. Human and mouse were compared in order to
determine whether the differential developmental capacity of mouse and human preimplantation embryos
in culture could be explained by the variations in the patterns of expression for these enzymes. mRNA
expression for these enzymes was examined using reverse transcriptionpolymerase chain reaction
(RTPCR). In the mouse, all transcripts (except for catalase) were detected, whatever the maturation stage.
No qualitative differences were detected between GV and MII oocytes. In human, all the enzymes (except for
catalase) were expressed in MII oocytes and Cu-Zn-SOD was particularly highly expressed. Transcripts
corresponding to GPX and Mn-SOD were not detected at GV stage but only at MII stage, suggesting that
storage could occur between GV and MII stages. However, using 39 end-specific primers for GPX and Mn-SOD,
instead of the oligo(dT)1218 primer, for the reverse transcription reaction, the transcripts for these antioxidants
enzymes have been detected in human oocytes at the GV stage. This suggests the presence of maturation-
specific polyadenylation of these transcripts. These enzymes can be considered as markers of cytoplasmic
maturation.
Key words: antioxidant enzymes/free oxygen radicals/gene expression/oocyte maturation/preimplantation
embryos

Introduction that variations in maternal mRNA synthesis or accumulation

Free oxygen radicals (FORs) are produced by embryo
metabolism (Nasr-Esfahani and Johnson, 1991; Goto et al.,
1993). Oxidative injury may be responsible for developmental
retardation and arrest of mammalian preimplantation embryos
in vitro (Noda et al., 1991; Chun et al., 1994). Increased
amounts of FORs have been detected during mouse embryo
development in vitro (Goto et al., 1993). A cell block, induced
by hypoxanthine in association with glucose (Loustradis et al.,
1987; Downs and Dow, 1991; Rieger, 1992), coincides with a
rise of superoxide anions and H2O2 concentration (Nasr-
Esfahani et al., 1990a; Nasr-Esfahani and Johnson, 1991; Noda
et al., 1991). Developmental arrest may also be induced by
traces of metallic ions (Fe21, Cu1), inducers of FOR formation
(Nasr-Esfahani et al., 1990b). Furthermore, metal chelators
such as EDTA or transferrin overcame the developmental
arrest in vitro (Nasr-Esfahani et al., 1990b, 1992; Legge and
Sellens, 1991; Nasr-Esfahani and Johnson, 1992).
Embryo protection against FOR depends, in part, upon an
endogenous pool of antioxidant enzymes (Harvey et al., 1995),
stored as mRNA in the oocyte during oogenesis. It appears
720
during oocyte maturation may affect the in-vitro development
of the embryo until zygotic gene activation (ZGA) (Piko and
Clegg, 1982; Telford et al., 1990). A drop below a critical
threshold may lead to developmental arrest.
Several antioxidant enzymes may protect the oocyte and
embryo against peroxidative damage: catalase, Cu-Zn-
superoxide dismutase (Cu-Zn-SOD), Mn-superoxide dismutase
(Mn-SOD), glutathione peroxidase (GPX), and -glutamyl-
cysteine synthetase (GCS) (Li et al., 1993). The addition of
Cu-Zn-SOD to the synthetic culture media results in an
increased blastocyst formation in rabbit (Li et al., 1993),
mouse (Noda et al., 1991; Nonogaki et al., 1992; Chun et al.,
1994) and cow (Lauria et al., 1994; Iwata et al., 1998).
Human and bovine embryo development in vitro is obtained
in complex media and/or with co-culture on somatic cell layers
(Heyman et al., 1987; Menezo et al., 1990). In contrast, mouse
embryo development is easier and more efficient in simple
culture media. These culture characteristics may partly reflect
differences between species in embryo sensitivity to FOR.
Early preimplantation embryo development is driven by oocyte
European Society of Human Reproduction and Embryology

protein and mRNA storage; to our knowledge, no information
is available on the presence of these transcripts during oocyte
maturation in humans. The objective of this study was to
analyse qualitatively the genetic expression of enzymes
involved in intracellular protection against FORs and their
storage at different stages of human and mouse oocyte matura-
tion. The mouse blastocyst content was also analysed as a
control for post-genomic activation embryo development.
Materials and methods
Collection of oocyte and embryo
Human oocytes were collected from an in-vitro fertilization (IVF)
centre (IRH/Laboratoire Marcel Merieux). Hormonal stimulation
was performed according to classical protocols involving a short
treatment with gonadotrophin-releasing hormone (GnRH) agonists
(decapeptyl or buserelin) followed by ovarian stimulation with urinary
or recombinant follicle stimulating hormone (FSH). In the absence
of fertilization, metaphase II (MII) oocytes were collected 24 h after
insemination. All the fertilization and embryo culture procedures
were performed under oil to allow better developmental potential and
to avoid oxidative stress. Germinal vesicle (GV) oocytes were
collected from intracytoplasmic sperm injection (ICSI) patients, when
maturation was not completed. Cumulus-enclosed oocytes (for ICSI)
or the unfertilized eggs were treated with hyaluronidase (Laboratoire
Choay, Paris, France) at a final concentration of 50 IU/ml. After
treatment for a few seconds, the oocytes were rinsed three times in
culture medium. Care was taken to ensure the total absence of corona
cells on the oocytes, by observation under an inverted microscope at
3200 magnification.
The mouse GV and MII oocytes were collected from females
(OF1) in which ovulation had been stimulated with i.p. injection of
5 IU equine chorionic gonadotrophin (eCG) and housed overnight
without a male. GV oocytes were obtained 14 h after eCG injection
by follicle puncture with a capillary glass tube. For MII oocyte
collection, eCG injection was followed 48 h later by human
chorionic gonadotrophin (HCG; 5 IU) administration. MII oocytes
were collected 20 h later by oviduct dissection. Cumulusoocyte
complexes were treated with bovine testis hyaluronidase (1 mg/ml)
in order to eliminate the cumulus cells. Denuded oocytes were
examined under light microscopy in order to assess the complete
elimination of cumulus and corona cells. Mouse blastocysts were
collected 96 h post-HCG injection by uterine flushing.
Table I. Antioxidant enzyme sequences used in reverse transcriptionpolymerase chain reaction (RTPCR)
Gene Primer
GCS Sense 59-CCTTCTGGCACAGCACGTTG-39
Antisense 59-TAAGACGGCATCTCGCTCCT-39
GPX Sense 59-CCTCAAGTACGTCCGACCTG-39
Antisense 59-CAATGTCGTTGCGGCACACC-39
Cu-Zn-SOD Sense 59-AAGGCCGTGTGCGTGCTGAA-39
Antisense 59-CAGGTCTCCAACATGCCTCT-39
Mn-SOD Sense 59-GCACATTAACGCGCAGATCA-39
Antisense 59-AGCCTCCAGCAACTCTCCTT-39
Catalase Sense 59-GCAGATACCTGTGAACTGTC-39
Antisense 59-GTAGAATGTCCGCACCTGAG-39
GCS 5 -glutamylcysteine synthetase; GPX 5 glutathione peroxidase (GPX); Cu-Zn-SOD 5 Cu-Zn-superoxide dismutase; and Mn-superoxide dismutase
(Mn-SOD).
Expression of antioxidants during oocyte maturation
Thermolysis of the oocytes and embryos
Single oocytes or blastocysts were placed in polymerase chain reaction
(PCR) tubes in 2 l of sterile diethylpyrocarbonate (DEPC)-treated
water and overloaded with one drop of mineral oil. Before use, the
oocytes underwent thermolysis for 1 min at 100C in order to release
nucleic acids (Kumazaki et al., 1994).
Reverse transcription (RT)
The reverse transcription reagents, RT buffer 13, 10 mmol/l dithio-
threitol (DTT), 0.5 mmol/l of each dNTP, 0.5 g oligo(dT)1218,
10 IU RNase inhibitor (Promega, Chrabonnieres, France) and 200 IU
superscript reverse transcriptase (Gibco-BRL, Cergy-Potoise, France)
were mixed on ice in a total volume of 20 l, and 18 l of the RT
mix was added to each single oocyte or blastocyst in tubes. RT
was carried out at 42C for 50 min followed by heating to 70C for
15 min to inactivate the reaction and storage at 4C. For each RT
reaction, a positive control was performed on 1 g of mouse liver
total RNA.
Polymerase chain reaction (PCR)
A total of 89 GV and 62 MII human oocytes were analysed for the
presence of transcripts encoding for GCS, GPX, Cu-Zn-SOD, Mn-
SOD and catalase. In the mouse, 54 GV, 57 MII oocytes and 50
blastocyts were analysed. Ten replicate RTPCR analyses for each
enzyme were performed on single oocytes or embryos.
PCR analyses were carried out in 50 l and contained cDNA (half
of the RT product), 2 mmol/l MgCl2, 50 mmol/l KCl, 10 mmol/l
TrisHCl (pH 8.3), 0.2 mmol/l each of dNTP, 0.4 mol/l of each
primer (Isoprim, Sable sur Sarthe, France) and 2 IU of Taq polymerase
(Perkin Elmer Cetus, Courtaboeuf, France). Primer sequences used
in this study are indicated in Table I. After an initial denaturation
step of 1 min at 94C, 35 amplification cycles were performed. Each
cycle included denaturation at 94C for 45 s, annealing at 56C
(GCS, GPX and Cu-Zn-SOD) or 55C (Mn-SOD and catalase) for 1
min and extension at 72C for 1 min. A final extension step of 5 min
at 72C was performed in order to complete the PCR reaction.
To confirm the identity of the antioxidant enzyme transcripts, each
RTPCR reaction product cleaved with an appropriate restriction
enzyme (Table I). In order to determine the presence of a specific
maturation polyadenylation, 39 end-specific primers of GPX,
Mn-SOD, Cu-Zn-SOD and -actin were used for RT instead of an
oligo(dT)1218 primer (Johnson et al., 1997). Each RT reaction was
split into two 10 l aliquots in order to analyse the expression of
antioxidant enzymes, in comparison with an internal control (-actin)
(Runesson et al., 1996). The PCR reactions were prepared as described
Reference Product Restriction Product
size (bp) enzymes size (bp)
(Yan and Meister, 1990) 346 Hinf1 260, 86
(Chambers et al., 1986) 197 Taq1 167, 30
(Ho and Crapo, 1987a) 246 Pst1 170, 76
(Ho and Crapo, 1987b) 241 Rsa1 156, 85
(Shaffer and Preston, 1990) 229 Sph1 163, 66
721
S.El Mouatassim, P.Guerin and Y.Menezo

above in order to separate batches containing 0.1 mol/l of 59-specific were observed in all mouse GV oocytes analysed. On the
end primers of the gene of interest and -actin respectively. The PCR other hand, the transcripts for GPX and Mn-SOD were never
conditions for antioxidant enzymes were used as described above. In detected at the GV stage in human.
order to eliminate the risk of genomic DNA contamination, RTPCR The expression of transcripts encoding antioxidant enzymes
was performed on mouse liver DNA using the same conditions as
of non-fertilized human and mouse oocytes at the MII stage
described above.
is shown in Figure 2. Transcripts for GCS, GPX, Cu-Zn-SOD,
Gel electrophoresis and Mn-SOD were detected in human and mouse MII oocytes.
After amplification, 20% of the RTPCR products was separated by To confirm the identity of the amplicons, each RTPCR product
agarose (2%) gel electrophoresis, stained by ethidium bromide and was digested with restriction enzymes (Table I). Figure 3
visualized under UV. displays an example of restriction enzyme digestion profiles
of GCS, GPX and Mn-SOD amplicons of human and mouse
oocyte at the MII stage with Hinf1, Taq1 and Rsa1 respectively.
Results
The predicted digestion product sizes were 260 and 82 for
A summary of the results is given in Table II. Gels correspond- GCS, 167 and 30 bp for GPX and 156 and 85 bp for Mn-SOD.
ing to transcripts encoding for the antioxidant enzymes of In both species, catalase transcripts were never observed
individual immature human and mouse oocytes at the GV whatever the maturation stage. The liver control was positive
stage are shown in Figure 1. The size of amplicons were for all the enzymes tested. Figure 4 summarizes the expression
346, 197, 246, 241 and 229 bp for GCS, GPX, Cu-Zn-SOD, patterns of the antioxidant enzyme transcripts within mouse
Mn-SOD and catalase mRNA respectively. blastocysts. Transcripts encoding GCS, GPX, Cu-Zn-SOD,
Transcripts encoding for GCS, GPX, Cu-Zn-SOD, Mn-SOD Mn-SOD, catalase and -actin were detected.
In human, GPX and Mn-SOD transcripts were not detected
at GV stage but only at the MII stage of oocyte maturation
Table II. Expression of genes encoding antioxidant enzymes in human and (Figures 1 and 2). Nevertheless, using 39 end-specific primers
mouse oocytes
of GPX, Mn-SOD and -actin instead of the oligo(dT)18 primer,
GCS GPX Cu-Zn-SOD Mn-SOD Catalase for RT reaction, transcripts of these antioxidant enzymes were
detected in human oocytes at the GV stage (Figure 5).
Human
Germinal vesicle 1 2 1 2 2
Metaphase II 1 1 1 1 2
Mouse Discussion
Germinal vesicle 1 1 1 1 2
Metaphase II 1 1 1 1 In mammals, oxidative stress interferes severely with in-vitro
Blastocyst 1 1 1 1 1 embryo development retardation and arrest. The series of
antioxidant enzymes studied protects gametes and embryos
GCS 5 -glutamylcysteine synthetase; GPX 5 glutathione peroxidase
(GPX); Cu-Zn-SOD 5 Cu-Zn-superoxide dismutase; and Mn-superoxide against FOR damage during maturation. The resulting impaired
dismutase (Mn-SOD). cellular function affects further embryo development (Beckman

Figure 1. mRNA expression analysis of antioxidant enzymes in human and mouse oocytes at the germinal vesicle (GV) stage, using reverse
transcriptionpolymerase chain reaction (RTPCR). Amplicons of 346, 197, 246, 241 and 229 bp represent the transcripts encoding for
-glutamylcysteine synthetase (GCS), glutathione peroxidase (GPX), Cu-Zn-superoxide dismutase (Cu-Zn-SOD), Mn-superoxide dismutase
(Mn-SOD) and catalase respectively. (A) Only the transcripts coding for GCS and Cu-Zn-SOD were detectable in the human oocyte at the
GV stage (lanes 1 and 5); GPX (lane 3) and Mn-SOD (lane 7) transcripts were not detected at the GV stage in the human oocyte. Liver
controls were positive for all the antioxidant enzymes tested (lanes 2, 4, 6, 8 and 10). (B) Transcripts encoding for GCS, GPX, Cu-Zn-SOD
and Mn-SOD (lanes 1, 3, 5 and 7 respectively) were detected at the GV stage in mouse oocytes. Transcripts encoding for catalase (lane 9)
were not observed in either species at the GV stage. Lane 11 5 RTPCR reaction blank; M 5 1 kb DNA ladder.

722
 Expression of antioxidants during oocyte maturation

Figure 2. mRNA expression analysis of antioxidant enzymes in non-fertilized human and mouse oocytes at the metaphase II (MII) stage,
using reverse transcriptionpolymerase chain reaction (RTPCR). Amplicons of 346, 197, 246, 241 and 229 bp represent the transcripts
encoding for -glutamylcysteine synthetase (GCS), glutathione peroxidase (GPX), Cu-Zn-superoxide dismutase (Cu-Zn-SOD), Mn-superoxide
dismutase (Mn-SOD) and catalase respectively. Except for catalase (lane 9), all the transcripts encoding for GCS, GPX, Cu-Zn-SOD and
Mn-SOD were detectable in human and mouse oocytes at the MII stage (lanes 1, 3, 5 and 7). Liver controls were positive for all the
antioxidant enzymes tested (lanes 2, 4, 6, 8 and 10). Lane 11 5 RTPCR reaction blank; M 5 1 kb DNA ladder.

Figure 3. Restriction enzyme digestion profiles of the polymerase
chain reaction (PCR) products of human (lanes 13) and mouse
(lanes 46) oocytes, at the metaphase II stage. The predicted
products of 260 and 82 bp (lanes 1 and 4) were obtained after
digestion of -glutamylcysteine synthetase (GCS) PCR products
with Hinf1. Restriction enzyme digestion of glutathione peroxidase
(GPX) and Mn-superoxide dismutase (Mn-SOD) PCR products
with Taq1 and RSA1 resulted respectively in the predicted
fragments of 167 and 30 bp (lanes 2 and 5), and 156 and 86 bp
(lanes 3 and 6). M 5 1 kb DNA ladder.
Figure 5. mRNA expression analysis of glutathione peroxidase
(GPX), Mn-superoxide dismutase (Mn-SOD) and Cu-Zn-superoxide
dismutase (Cu-Zn-SOD) antioxidant enzymes in human and mouse
oocytes at the germinal vesicle (GV) stage, using reverse
transcriptionpolymerase chain reaction (RTPCR) with 39 end-
specific primers instead of an oligo (dT)18 primer in the RT
reaction. Transcripts encoding for GPX, Mn-SOD and Cu-Zn-SOD
were detected at the GV stage (lanes 1, 3, and 5). Lanes 2, 4 and
6 5 -actin internal RTPCR control; lane 7 5 RTPCR reaction
blank; M 5 1 kb DNA ladder.

Figure 4. mRNA expression analysis of antioxidant enzymes in mouse embryos at the blastocyst stage, using reverse transcription
polymerase chain reaction (RTPCR). Amplicons of 346, 197, 246, 241 and 229 bp represent the transcripts encoding for -glutamylcysteine
synthetase (GCS), glutathione peroxidase (GPX), Cu-Zn-superoxide dismutase (Cu-Zn-SOD), Mn-superoxide dismutase (Mn-SOD) and
catalase respectively. All the transcripts encoding for GCS, GPX, Cu-Zn-SOD and Mn-SOD were detectable in mouse embryos at the
blastocyst stage (lanes 1, 3, 5, and 7). A catalase transcript was also observed in mouse embryo at the blastocyst stage (lane 9). Liver
controls were positive for all the antioxidant enzymes tested (lanes 2, 4, 6, 8 and 10). The fragment products of 540 bp represent the -actin
internal control of RTPCR (lanes 11 and 12). Lane 13 5 reaction blank; M 5 1 kb DNA ladder.

and Ames, 1997; Lopes et al., 1998). The presence of four mechanisms are conserved and are important for the final
out of five enzyme transcripts investigated at the MII stage steps of maturation and for early preimplantation embryo
in human and mouse oocytes suggests that these defence development. As these enzymes are polyadenylated at the MII
723
S.El Mouatassim, P.Guerin and Y.Menezo
stage, they are also good candidates for a translation during
the first embryo preimplantation stages.
Cu-Zn-SOD is expressed at a relatively high level in human
and mouse at GV and MII stage of oocyte maturation.
Transcripts coding for Cu-Zn-SOD are present in oocytes at
all stages of maturation, especially in humans. These results
are in accordance with previous results (Chun et al., 1994)
and confirm that this enzyme probably plays a crucial role in
protecting embryos against oxygen toxicity in vivo as well as
in vitro. In addition, Cu-Zn-SOD was highly expressed in
the mouse blastocyst. H2O2 can generate hydroxyl radicals via
the Fentons reaction. Catalase and GPX detoxify H2O2, the
product of SOD action. The three enzymes have obvious
complementary roles.
Catalase transcripts were not detected either in mouse or in
human oocytes, regardless of the stage of maturation. Low
levels of catalase mRNA were detected in the mouse blastocyst.
This confirms that these transcripts are rather detected in
embryos after genomic activation (Harvey et al., 1995). The
total amount of maternal transcripts stored in the embryo
decreases during embryo development (Croteau et al., 1995),
until activation of its genome. Consequently, the embryo may
be particularly vulnerable to FOR before this stage. However,
at this time in vivo, the embryo is present in the oviduct which
provides hypotaurine, a hydroxyl radical scavenger (Guerin
and Menezo, 1995; Bavister and Boatman, 1997), so the
preimplantation embryo can find complementary systems for
protection against peroxidative reactions in its environment.
GCS and Cu-Zn-SOD transcripts are present in human and
mouse oocytes and in the mouse blastocyst.
GPX transcripts are present in MII human oocytes and in
mouse oocytes and embryos. SOD and GPX represent the major
enzymes protecting mammalian cells, including spermatozoa,
against oxygen toxicity and lipid peroxidation (Alvarez et al.,
1987; Alvarez and Storey, 1989) and their role in early
conception is of major importance. These observations support
the theory that GPX activity, even though associated with
reduced glutathione generation, is more profitable for the
oocyte than catalase activity. Although both catalase and
glutathione peroxidase break down the hydrogen peroxide
(H2O2) produced by SOD, glutathione peroxidase catalyses
reactions for many other peroxides, including lipid peroxides,
thus providing better protection.
Transcripts encoding for GPX and Mn-SOD are present at
MII but are absent at the GV stage in human oocyte. This
may suggest that either their storage occurs between GV and
MII, or that specific RNA (re)adenylation of these transcripts
is initiated at this time. However, the use of 39 end-specific
primers instead of an oligo(dT)1218 in the RT reaction shows
that there is a last minute polyadenylation, as described for
some maternal mRNAs at the end of mouse oocyte maturation
(Paynton and Bachvarova, 1994). Ethical laws do not allow
the use of fresh MII oocytes. A putative secondary stimulatory
effect is related to post-ovulatory ageing of the oocytes,
although FOR generation should be balanced by the age-
related degradation of the mRNA (Piko and Clegg, 1982).
Final modifications of mRNA polyadenylation, in order to
regulate further expression/translation, have been already
724
described in Xenopus, mouse and cow (Meric et al., 1996;
Verrotti and Strickland 1997; Brevini-Gandolfi et al., 1999).
A strong correlation between nuclear and cytoplasmic
maturation can be established in humans, since these enzymes
seem to be true markers of cytoplasmic maturation.
Unfavourable embryo culture conditions may result in altera-
tion of embryo metabolism and intracellular production of
FORs. FORs may interfere with the embryo redox status
causing oxidative stress which may alter essential cellular
functions such as gene expression (Wasserman and Fahl, 1997).
Expression of many genes can be up-regulated or down-
regulated by FOR. Antioxidant enzyme gene expression is
stimulated by an oxidative stress (Maitre et al., 1993; Barnett
and Bavister, 1996). The intracellular redox potential can
modulate the activity of some transcription factors and FORs
may activate the antioxidant defence genes (Shaffer and
Preston, 1990; Schultz, 1993). It would be of great interest to
investigate the expression (i.e. transcription and translation)
of genes encoding antioxidant enzymes in embryos after
oxidative stress, in relation with their developmental potential.
Acknowledgments
This research was supported by grants from ARCEFAR and AKZO-
Organon. We also thank Dr Heddi Abdelaziz and Chaque
Khatchadourian for supplying the restriction enzymes.
References
Alvarez, J.G. and Storey, B.T. (1989) Role of glutathione peroxidase in
protecting mammalian spermatozoa from loss of motility caused by
spontaneous lipid peroxidation. Gamete Res., 23, 7790.
Alvarez, J.G., Touchstone, J.C., Blasco, L. et al. (1987) Spontaneous lipid
peroxidation and production of hydrogen peroxide and superoxide in human
spermatozoa. Superoxide dismutase as major enzyme protectant against
oxygen toxicity. J. Androl., 8, 338348.
Barnett, D.K. and Bavister, B.D. (1996) Inhibitory effect of glucose and
phosphate on the second cleavage division of hamster embryos: is it linked
to metabolism? Hum. Reprod., 11, 177183.
Bavister, B.D. and Boatman, D.E. (1997) The neglected human blastocyst
revisited. Hum. Reprod., 12, 16071610.
Beckman, K.B. and Ames, B.N. (1997) Oxidative decay of DNA. J. Biol.
Chem., 272, 1963319636.
Brevini-Gandolfi, T.A., Favetta, L.A., Muri, L. et al. (1999) Changes in
poly(A) tail length of maternal transcripts during in vitro maturation of
bovine oocytes and their relation with developmental competence. Mol.
Reprod. Dev., 52, 427433.
Chambers, I., Frampton, J., Goldfarb, P. et al. (1986) The structure of the
mouse glutathione peroxidase gene: the selenocysteine in the active site is
encoded by the termination codon, TGA. EMBO J., 5, 12211227.
Chun, Y.S., Kim J.H., Lee H.T. et al. (1994) Effect of superoxide dismutase
on the development of preimplantation mouse embryos. Theriogenology,
41, 511520.
Croteau, S., Menezo, Y. and Benkhalifa, M. (1995) Transforming growth
factors- and - expression in fertilized and parthenogenetic pre-
implantation mouse embryos: RNA detection with fluorescent in situ
hybridization. Dev. Growth. Differ., 37, 433440.
Downs, S.M. and Dow, M.P.D. (1991) Hypoxanthine-maintained 2-cell block
in mouse embryos: dependence on glucose and effect of hypoxanthine
phosphoribosyltransferase inhibitors. Biol. Reprod., 44, 10251039.
Goto, K., Noda, Y., Mori T. et al. (1993) Increased generation of reactive
oxygen species in embryos cultured in vitro. Free Radic. Biol. Med., 15,
6975.
Guerin, P. and Menezo, Y. (1995) Hypotaurine and taurine in gamete and
embryo environments: de novo synthesis via cysteine sulfinic acid pathway
in oviduct cells. Zygote, 3, 333343.
 Expression of antioxidants during oocyte maturation

Harvey, M.B., Arcellana-Panlilio, M.Y., Zhang, X. et al. (1995) Expression Schultz, R.M. (1993) Regulation of zygotic gene activation in the mouse.
of genes encoding antioxidant enzymes in preimplantation mouse and cow Bioessays, 15, 531538.
embryos and primary bovine oviduct cultures employed for embryo Shaffer, J.B. and Preston, K.E. (1990) Molecular analysis of an acatalasemic
coculture. Biol. Reprod., 53, 532540. mouse mutant. Biochem. Biophys Res. Commun., 173, 10431050.
Heyman, Y., Vincent, C., Garnier, V. et al. (1987) Transfer of frozenthawed Telford, N.A., Watson, A.J. and Schultz, G.A. (1990) Transition from maternal
embryos in sheep. Vet. Rec., 120, 8385. to embryonic control in early mammalian development: a comparison of
Ho, Y.S. and Crapo, J.D. (1987a) Sequences of cDNA and deduced amino several species. Mol. Reprod. Dev., 26, 90100.
acid of rat copper-zinc-containing superoxide dismutase. Nucleic Acids Verroti, A.C. and Strickland, S. (1997) Oocyte selection of maturations
Res., 15, 6746. affecting cytoplasmic polyadenylation of maternal mRNAs. Mol. Reprod.
Ho, Y.S. and Crapo, J.D. (1987b) Nucleotide sequences of cDNAs coding Dev., 46, 482488.
for rat manganese-containing superoxide dismutase. Nucleic Acids Res., Wasserman, W.W. and Fahl, W.E. (1997) Functional antioxidant responsive
15, 10070. elements. Proc. Natl Acad. Sci. USA, 94, 53615366.
Iwata, H., Akamatsu, S., Minami, N. et al. (1998) Effect of antioxidant on Yan, N. and Meister, A. (1990) Amino acid sequence of rat kidney gamma-
the development of bovine IVM/IVF embryos in various concentration of glutamylcysteine synthetase. J. Biol. Chem., 265, 15881593.
glucose. Theriogenology, 50, 365375.
Johnson, M.D., Baty, D.W., Behr, B. et al. (1997) Genetic expression Received on February 22, 1999; accepted on May 13, 1999
of hexokinase and glucose phosphate isomerase in late-stage mouse
preimplantation embryos: transcription activities in glucose/phosphate-
containing HTF and glucose/phosphate-free P1 media. Mol. Hum. Reprod.,
3, 351357.
Kumazaki, T., Hamada, K. and Mitsui, Y. (1994) Detection of mRNA
expression in a single cell by direct RTPCR. Biotechniques, 16, 10171019.
Lauria, A., Luvoni, G.C., Parravicini, E. et al. (1994) Effect of superoxide
dismutase (SOD) on early stages of bovine embryogenesis in vitro. [Abstr.]
Theriogenology, 41, 234.
Legge, M. and Sellens, M.H. (1991) Free radical scavengers ameliorate the
2-cell block in mouse embryo culture. Hum. Reprod., 6, 867871.
Li, J., Foote, R.H. and Simkin, M. (1993) Development of rabbit zygotes
cultured in protein-free medium with catalase, taurine, or superoxide
dismutase. Biol. Reprod., 49, 3337.
Lopes, S., Jurisicova, A. and Casper, R.F. (1998) Gamete-specific DNA
fragmentation in unfertilized human oocytes after intracytoplasmic sperm
injection. Hum. Reprod., 13, 703708.
Loutradis, D., John, D. and Kiessling, A.A. (1987) Hypoxanthine causes a
2-cell block in random-bred mouse embryos. Biol. Reprod., 37, 311316.
Maitre, B., Jornot, L., and Junod, A.F. (1993) Effects of inhibition of catalase
and superoxide dismutase activity on antioxidant enzymes mRNA levels.
Am. J. Physiol., 265, L636L643.
Meric, F., Searfoss A.M., Wormington, M. and Wolffe, A.P. (1996) Masking
and unmasking maternal mRNA. The role of polyadenylation, transcription,
splicing and nuclear history. J. Biol. Chem., 29, 3080430810.
Menezo, Y., Guerin, J.F. and Czyba, J.C. (1990) Improvement of human early
embryo development in vitro by coculture on monolayers of Vero cells.
Biol. Reprod., 42, 301306.
Nasr-Esfahani, M.H. and Johnson, M.H. (1991) The origin of reactive oxygen
species in mouse embryos cultured in vitro. Development, 113, 551560.
Nasr-Esfahani, M.H. and Johnson, M.H. (1992) How does transferrin overcome
the in vitro block to development of the mouse preimplantation embryo?
J. Reprod. Fertil., 96, 4148.
Nasr-Esfahani, M.H., Aitken, R.J. and Johnson, M.H. (1990a) Hydrogen
peroxide levels in mouse oocytes and early cleavage stage embryos
developed in vitro or in vivo. Development, 109, 501507.
Nasr-Esfahani, M.H., Aitken, R.J. and Johnson, M.H. (1990b) The effect of
iron and iron chelators on the in-vitro block to development of the mouse
preimplantation embryo: BAT6 a new medium for improved culture of
mouse embryos in vitro. Hum. Reprod., 5, 9971003.
Nasr-Esfahani, M.H., Winston, N.J. and Johnson, M.H. (1992) Effects of
glucose, glutamine, ethylenediaminetetraacetic acid and oxygen tension on
the concentration of reactive oxygen species and on development of the
mouse preimplantation embryo in vitro. J. Reprod. Fertil., 96, 219231.
Noda, Y., Matsumoto, H., Umaoka, Y. et al. (1991) Involvement of superoxide
radicals in the mouse two-cell block. Mol. Reprod. Dev., 28, 356360.
Nonogaki, T., Noda, Y., Narimoto, K. et al. (1992) Effects of superoxide
dismutase on mouse in vitro fertilization and embryo culture system.
J. Assist. Reprod. Genet., 9, 274280.
Paynton, B.V. and Bachvarova, R. (1994) Polyadenylation and deadenylation
of maternal mRNAs during oocyte growth and maturation in the mouse.
Mol. Reprod. Dev., 37, 172180.
Piko, L. and Clegg, K.B. (1982) Quantitative changes in total RNA, total
poly(A) and ribosomes in early mouse embryos. Dev. Biol., 89, 362378.
Rieger, D. (1992) Relationships between energy metabolism and development
of early mammalian embryos. Theriogenology, 37, 8690.
Runesson, E., Bostrom, E.K., Janson, P.O. et al. (1996) The human preovulatory
follicule is a source of the chemotactic cytokine interleukin-8. Mol. Hum.
Reprod., 2, 245250.

725