Molecular Human Reproduction, Vol. 5, No. 8, 720-725,
August 1999
© 1999 European Society of Human Reproduction and Embryology
Expression of genes encoding antioxidant enzymes in human and mouse oocytes during the final stages of maturation
1 Laboratoire Marcel Mérieux, Cytogénétique, Avenue Tony Garnier BP 7322, 69357 Lyon, 2 INSA Biologie 406, Unité biologie du développement préimplantatoire, 20 Avenue A.Einstein, 69621 Villeurbanne cedex and 3 Ecole Nationale Vétérinaire de Lyon, Unité biologie de la reproduction, CERREC, BP 83, 69280 Marcy l'étoile cedex France
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Abstract |
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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 transcription–polymerase chain reaction (RT–PCR). 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 3' end-specific primers for GPX and Mn-SOD, instead of the oligo(dT)12–18 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. antioxidant enzymes/free oxygen radicals/gene expression/oocyte maturation/preimplantation embryos
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Introduction |
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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 (Fe2+, Cu+), 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 that variations in maternal mRNA synthesis or accumulation during oocyte maturation may affect the in-vitro development of the embryo until zygotic gene activation (ZGA) (Pikó 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 -glutamylcysteine 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; Ménézo, 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 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 maturation. The mouse blastocyst content was also analysed as a control for post-genomic activation embryo development.
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Materials and methods |
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Collection of oocyte and embryo
Human oocytes were collected from an in-vitro fertilization (IVF) centre (IRH/Laboratoire Marcel Mérieux). 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 x200 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. Cumulus–oocyte 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.
Thermolysis of the oocytes and embryos
Single oocytes or blastocysts were placed in polymerase chain reaction (PCR) tubes in 2 µl of sterile diethylpyrocarbonate (DEPC)-treated water and overloaded with one drop of mineral oil. Before use, the oocytes underwent thermolysis for 1 min at 100°C in order to release nucleic acids (Kumazaki et al., 1994).
Reverse transcription (RT)
The reverse transcription reagents, RT buffer 1x, 10 mmol/l dithiothreitol (DTT), 0.5 mmol/l of each dNTP, 0.5 µg oligo(dT)12–18, 10 IU RNase inhibitor (Promega, Chrabonnières, France) and 200 IU superscript reverse transcriptase (Gibco-BRL, Cergy-Potoise, France) were mixed on ice in a total volume of 20 µl, and 18 µl of the RT mix was added to each single oocyte or blastocyst in tubes. RT was carried out at 42°C for 50 min followed by heating to 70°C for 15 min to inactivate the reaction and storage at 4°C. For each RT reaction, a positive control was performed on 1 µg 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 RT–PCR analyses for each enzyme were performed on single oocytes or embryos.
PCR analyses were carried out in 50 µl and contained cDNA (half of the RT product), 2 mmol/l MgCl2, 50 mmol/l KCl, 10 mmol/l Tris–HCl (pH 8.3), 0.2 mmol/l each of dNTP, 0.4 µmol/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 94°C, 35 amplification cycles were performed. Each cycle included denaturation at 94°C for 45 s, annealing at 56°C (GCS, GPX and Cu-Zn-SOD) or 55°C (Mn-SOD and catalase) for 1 min and extension at 72°C for 1 min. A final extension step of 5 min at 72°C was performed in order to complete the PCR reaction.
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To confirm the identity of the antioxidant enzyme transcripts, each RT–PCR reaction product cleaved with an appropriate restriction enzyme (Table I
). In order to determine the presence of a specific maturation polyadenylation, 3' end-specific primers of GPX, Mn-SOD, Cu-Zn-SOD and ß-actin were used for RT instead of an oligo(dT)12–18 primer (Johnson et al., 1997). Each RT reaction was split into two 10 µl aliquots in order to analyse the expression of antioxidant enzymes, in comparison with an internal control (ß-actin) (Runesson et al., 1996). The PCR reactions were prepared as described above in order to separate batches containing 0.1 µmol/l of 5'-specific end primers of the gene of interest and ß-actin respectively. The PCR conditions for antioxidant enzymes were used as described above. In order to eliminate the risk of genomic DNA contamination, RT–PCR was performed on mouse liver DNA using the same conditions as described above.
Gel electrophoresis
After amplification, 20% of the RT–PCR products was separated by agarose (2%) gel electrophoresis, stained by ethidium bromide and visualized under UV.
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Results |
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A summary of the results is given in Table II
. Gels corresponding to transcripts encoding for the antioxidant enzymes of individual immature human and mouse oocytes at the GV stage are shown in Figure 1. The size of amplicons were 346, 197, 246, 241 and 229 bp for GCS, GPX, Cu-Zn-SOD, Mn-SOD and catalase mRNA respectively.
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Transcripts encoding for GCS, GPX, Cu-Zn-SOD, Mn-SOD were observed in all mouse GV oocytes analysed. On the other hand, the transcripts for GPX and Mn-SOD were never detected at the GV stage in human.
The expression of transcripts encoding antioxidant enzymes of non-fertilized human and mouse oocytes at the MII stage is shown in Figure 2. Transcripts for GCS, GPX, Cu-Zn-SOD, and Mn-SOD were detected in human and mouse MII oocytes. To confirm the identity of the amplicons, each RT–PCR product was digested with restriction enzymes (Table I). Figure 3 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. The predicted digestion product sizes were 260 and 82 for GCS, 167 and 30 bp for GPX and 156 and 85 bp for Mn-SOD.
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In both species, catalase transcripts were never observed whatever the maturation stage. The liver control was positive for all the enzymes tested. Figure 4
summarizes the expression patterns of the antioxidant enzyme transcripts within mouse blastocysts. Transcripts encoding GCS, GPX, Cu-Zn-SOD, Mn-SOD, catalase and ß-actin were detected.
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In human, GPX and Mn-SOD transcripts were not detected at GV stage but only at the MII stage of oocyte maturation (Figures 1 and 2
). Nevertheless, using 3' end-specific primers of GPX, Mn-SOD and ß-actin instead of the oligo(dT)18 primer, for RT reaction, transcripts of these antioxidant enzymes were detected in human oocytes at the GV stage (Figure 5).
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Discussion |
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In mammals, oxidative stress interferes severely with in-vitro embryo development retardation and arrest. The series of antioxidant enzymes studied protects gametes and embryos against FOR damage during maturation. The resulting impaired cellular function affects further embryo development (Beckman and Ames, 1997
; Lopes et al., 1998). The presence of four out of five enzyme transcripts investigated at the MII stage in human and mouse oocytes suggests that these defence mechanisms are conserved and are important for the final steps of maturation and for early preimplantation embryo development. As these enzymes are polyadenylated at the MII 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 Fenton's 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 (Guérin and Ménézo, 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 3' end-specific primers instead of an oligo(dT)12–18 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 (Pikó and Clegg, 1982). Final modifications of mRNA polyadenylation, in order to regulate further expression/translation, have been already 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 alteration 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.
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Acknowledgments |
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This research was supported by grants from ARCEFAR and AKZO-Organon. We also thank Dr Heddi Abdelaziz and Chaqué Khatchadourian for supplying the restriction enzymes.
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Notes |
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4 To whom correspondence should be addressed at: INSA Biologie 406, Unité biologie du développement préimplantatoire, 20 Avenue A.Einstein, 69621 Villeurbanne cedex France
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Submitted on February 22, 1999; accepted on May 13, 1999.
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R. E. Spindler and D. E. Wildt Quality and Age of Companion Felid Embryos Modulate Enhanced Development by Group Culture Biol Reprod, January 1, 2002; 66(1): 167 - 173. [Abstract] [Full Text]
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S. Dinara, K. Sengoku, K. Tamate, M. Horikawa, and M. Ishikawa Effects of supplementation with free radical scavengers on the survival and fertilization rates of mouse cryopreserved oocytes Hum. Reprod., September 1, 2001; 16(9): 1976 - 1981. [Abstract] [Full Text] [PDF]
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E. T. Mystkowska, A. Niemierko, A. Komar, and W. Sawicki Embryotoxicity of magainin-2-amide and its enhancement by cyclodextrin, albumin, hydrogen peroxide and acidification Hum. Reprod., July 1, 2001; 16(7): 1457 - 1463. [Abstract] [Full Text] [PDF]
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Y. Hu, I. Betzendahl, R. Cortvrindt, J. Smitz, and U. Eichenlaub-Ritter Effects of low O2 and ageing on spindles and chromosomes in mouse oocytes from pre-antral follicle culture Hum. Reprod., April 1, 2001; 16(4): 737 - 748. [Abstract] [Full Text] [PDF]
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