Theriogenology 59 (2003) 939949

Antioxidant requirements for bovine oocytes varies during in vitro maturation, fertilization and development
A.A. Ali, J.F. Bilodeau, M.A. Sirard*
Department of Animal Science, Centre de Recherche en Biologie de la Reproduction (CRBR), Laval University, Quebec, Canada G1K 7P4 Received 1 October 2001; accepted 10 June 2002

Abstract Antioxidants may be benecial additives to synthetic culture media because these well dened media lack serum or other macromolecules that serve as reactive oxygen species scavengers. In this study, three separate experiments were performed to determine the effects of antioxidants on the development of oocytes to the morula and blastocyst stage when added during in vitro maturation (IVM) of bovine oocytes, during in vitro fertilization (IVF), and during embryo culture for the rst 72 h of the development period. Bovine oocytes were matured, fertilized (under 20% O2), and embryos were cultured (under 7% O2) in dened conditioned medium in vitro with or without supplementation with the antioxidant cysteine, N-acetyl-L-cysteine (NAC), catalase and superoxide dismutase (SOD). Signicant improvements in the proportion of oocytes undergoing morula and blastocyst development (33.3% versus 20.3%, P < 0:05) were achieved when cysteine (0.6 mM) was added to the maturation medium as compared to control medium without antioxidant supplementation. However, the addition of NAC (0.6 mM), catalase (5 or 127 U/ml) or SOD (10 or 1000 U/ml) to the maturation medium did not improve the proportion of oocytes undergoing morula and blastocyst development. During the IVF period, addition of antioxidants (cysteine or NAC 0.6 mM, catalase 127 U/ml, SOD 100 U/ml) signicantly reduced the subsequent rate of bovine embryo development to the morula and blastocyst stage (P < 0:05). In a dened medium for embryo culture (7% O2), the addition of cysteine improved the development of bovine embryos while NAC, catalase and SOD had no positive effect on embryonic development. Our study showed that medium supplementation with cysteine during IVM and in vitro culture (IVC) improved the rate of bovine embryo development, in contrast to extracellular antioxidants like catalase and SOD that caused no improvement. # 2002 Elsevier Science Inc. All rights reserved.
Keywords: Bovine oocyte; Cysteine; N-Acetyl cysteine; Catalase; Superoxide dismutase

* Corresponding author. Tel.: 1-418-656-7359; fax: 1-418-656-3766. E-mail address: marc-andre.sirard@crbr.ulaval.ca (M.A. Sirard).

0093-691X/02/$ see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 1 1 2 5 - 1

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1. Introduction It is well known that oxygen concentration within the lumen of the female reproductive tract is about one-third (39%) that found under standard in vitro conditions [1]. The culture of embryos with a high oxygen tension in vitro (20%) may produce more free radicals [2] than embryo culture under 5% O2 or 7% O2 [3,4]. It appears that the balance between reactive oxygen species (ROS) production and scavenging is an important factor for the acquisition of fertilizing ability in vitro [5]. It seems likely that embryos cultured in vitro may be exposed to oxidative stress for which their defense mechanisms are insufcient to protect their delicate cellular structures. Detrimental effects of oxygenderived free radicals during in vitro culture (IVC) have been demonstrated in several species. It has been reported that ROS induce mitochondrial dysfunction, DNA, RNA and protein damage [6] as well as inhibiting spermoocyte fusion [7]. To protect oocytes and embryos from oxidative stress during IVC, various antioxidants can be added to culture media. For example, to modulate extracellular ROS media can be supplemented with extracellular enzymatic antioxidants such as superoxide dismutase or catalase. Superoxide dismutase (SOD) is the initial enzyme that induces the conversion of the superoxide anion (O2) to H2O2 which in turn is removed by catalase and glutathione peroxidase [8]. There are conicting reports in the literature regarding the effect of enzymes that neutralize ROS (e.g. catalase, SOD) on in vitro production of embryos. IVC media supplemented with SOD exerted a protective effect from oxidative stress on the development of mouse embryos fertilized in vitro and in vivo [9,10]. It has also been shown that supplementation of protein-free culture medium with superoxide dismutase increased the proportion of rabbit zygotes developing to the expanded blastocyst stage [11]. However, other authors have reported that the proportion of bovine oocytes developing to morula and blastocyst stages were not improved when in vitro maturation (IVM) and culture medium were supplemented with SOD [12,13]. In addition, the expression of embryonic SOD mRNA has been shown to be affected by culture conditions used [14]. It is also possible to add metabolizable antioxidants to the culture media, instead of enzymatic antioxidants. Glutathione (GSH) is a natural antioxidant present in both gametes but its level varies. An increase in the intracellular GSH concentration is shown as oocytes progress from the germinal vesicle to metaphase II stage, but GSH is lower in fertilized ova at the pronuclear stage when compared with mature oocytes [1518]. Moreover, GSH plays an important role in protecting the cell from oxidative damage [19], and in the formation of the male pronucleus in the hamster [16], mouse [15] and pig [20]. Thus, the levels of GSH found in oocytes at the end of maturation are considered as a good biochemical marker for oocyte viability [21]. The precursor of GSH in oocytes is cysteine [19], which may play an important role as an antioxidant supplement in IVC systems. During maturation of pig oocytes, cysteine is important for sperm nuclear decondensation [20], but little is known about other antioxidant requirements. During fertilization, when oocytes are denuded and exposed to high densities of live and dead sperm, the role of antioxidants has been shown to be positive for embryo survival but negative for fertilization rate [22]. During development, it is unclear if antioxidants are required at low oxygen concentration (7%), and if the protection must be extracellular or intracellular.

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Using dened media for all three steps (IVM, in vitro fertilization (IVF) and IVC) and using metabolizable or extracellular enzymatic antioxidants, it would be possible to know when and which free radical protection is needed. Therefore, the present study was conducted to determine whether antioxidants (cysteine, NAC, catalase and SOD) at different concentrations might be benecial or detrimental to the development of oocytes to the morula and blastocyst stage when added during IVM of bovine oocytes in dened condition medium under 20% oxygen, during IVF under 20% oxygen, and during embryo culture for the rst 72 h in dened condition medium under low (7%) oxygen concentration. 2. Materials and methods 2.1. Oocytes recovery and selection Bovine ovaries were transported from the slaughterhouse to the laboratory in a 0.9% NaCl aqueous solution containing 100,000 IU/l penicillin, 100 mg/l streptomycin and 250 mg/l amphotericin B (SigmaAldrich, Oakville, Ont., Canada). Cumulus-oocyte complexes (COCs) were selected from 2 to 6 mm follicles and aspirated using a 18 g needle attached to a 10 ml syringe. Only oocytes with at least three layers of compact cumulus cells and homogenous cytoplasm were selected. These were then washed three times in Hepes buffered Tyrode's medium (TLH) supplemented with 0.3% bovine serum albumin (BSA fraction V; SigmaAldrich), 0.2 mM pyruvic acid (SigmaAldrich) and 50 mg/ml gentamicin (SigmaAldrich). 2.2. In vitro maturation COCs were incubated in groups of 10. Each 50 ml droplet of maturation medium consisted of modied synthetic oviduct uid (m-SOF) supplemented with MEM nonessential amino acids (Gibco, BRL, Burlington, Ont., Canada), MEM essential amino acids (Gibco), 1.5 mM glucose (SigmaAldrich) and 1 mM glutamine (SigmaAldrich). The SOF medium used in this study was based upon the original formulation [23] with subsequent modications [24]. The droplets were covered with mineral oil (Sigma Aldrich) and were pre-incubated under the maturation conditions for a minimum of 3 h (38.5 8C, 5% CO2 in air with 100% humidity) and then incubated for 2324 h after oocytes were added. 2.3. In vitro fertilization IVF took place in droplets (48 ml) composed of modied Tyrode lactate medium [25], supplemented with 0.6% BSA fatty acid free (SigmaAldrich), 0.2 mM pyruvic acid, 2 mg/ml heparin (SigmaAldrich), and 50 mg/ml gentamicin containing ve oocytes. Cumulus-oocyte complexes were previously washed twice for 5 min in TLH. Following the transfer of oocytes, 2 ml of PHE (2 mM penicillamine, 1 mM hypotaurine, 250 mM epinephrine; SigmaAldrich ) was added to each droplet. All experiments were carried out mination Articielle du Que bec; using frozen semen from the same bull (Centre d'Inse

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bec, Canada). Spermatozoa were thawed in a 35 8C water bath CIAQ, St-Hyacinthe, Que for 1 min and were then washed in a discontinuous Percoll gradient prepared by adding 2 ml of 90% Percoll under 2 ml of 45% Percoll in a 15 ml centrifuge tube (Falcon). The semen samples were added on top of the Percoll gradient and centrifuged at 700 g for 30 min at 26 8C. The pellet was removed and resuspended in 1 ml of modied Tyrode's medium (TALP) and centrifuged at 250 g for 5 min at 26 8C. After removal of the supernatant, spermatozoa were resuspended in IVF medium, counted in a hemocytometer chamber and 2 ml of sperm suspension (nal concentration 1 106 cells/ml) were added into each droplet. Incubation was carried out at 38.5 8C in 5% CO2 in air with saturated humidity for 1518 h. 2.4. In vitro culture In all experiments, embryo culture took place in m-SOF [24,26] under mineral oil in a humidied atmosphere of 5% CO2, 7% O2 at 38.5 8C. Between 15 and 18 h after insemination, presumptive zygotes were denuded of surrounding cumulus cells by repeated pipetting in phosphate buffered saline (PBS) and subsequently washed three times in PBS before being transferred to the culture droplets (50 ml) in groups of 2030 embryos. Cleavage was assessed after 72 h of culture and the number of embryos developing to the morula and blastocyst stage was assessed on Day 8. This study used a two-culture system. The rst system (SOFC1) medium contained 0.8% BSA, MEM nonessential amino acids, 1.5 mM glucose,1 mM glutamine and 10 mM EDTA (SigmaAldrich) for the rst 72 h. The medium was then replaced by the second system (SOFC2) containing 0.8% BSA, MEM nonessential amino acids, MEM essential amino acids, 1.5 mM glucose and 1 mM glutamine for the remaining 96 h of culture. To prevent toxic accumulation of ammonium as a result of amino acid degradation, SOFC2 medium was replaced after 72 h of culture [27]. 2.5. Experimental design Three separate experiments were performed to evaluate the effect of adding different concentrations of cysteine, NAC [20,28,29], catalase or SOD [22,30] on different phases of the in vitro production of bovine embryos from immature oocytes (Table 1). In Experiment 1, one of three different types of antioxidants cysteine (0.6 mM), NAC (0.6 mM), catalase (5 or 127 U/ml) or SOD (10 or 1000 U/ml) were added during IVM in dened condition medium containing low (1.5 mM) glucose concentration under 20% oxygen to reduce ROS production in the maturation medium. Oocytes cultured without antioxidant supplementation served as a control. In Experiment 2, after IVM of bovine oocytes in maturation medium control (antioxidant-free maturation medium) the effects of adding the antioxidants cysteine (0.1 or 0.6 mM), NAC (0.1 or 0.6 mM), catalase (5 or 127 U/ml) or SOD (10 or 100 U/ml) during the IVF period were compared to the control group (antioxidant-free fertilization medium) under high (20%) oxygen concentration. In Experiment 3, after IVM and IVF of oocytes in antioxidant-free medium, various concentrations of antioxidants (cysteine (0.6 mM), NAC (0.6 mM), catalase (5 or 127 U/ml)

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Table 1 The presence or absence of antioxidants and glucose during different phases of the in vitro production of bovine embryos from immature oocytes IVM IVF IVC SOFC1 Experiment 1 Experiment 2 Experiment 3 Glucose O2 1.5 mM 20% 20% 1.5 mM 7% SOFC2 1.5 mM 7%

() Medium supplementation with antioxidants; () medium without antioxidant supplementation.

or SOD (10 or 1000 U/ml)) were added to the SOFC1 under low oxygen (7%) and low glucose (1.5 mM) concentration for the rst 72 h of the 8-day development period (IVC). 2.6. Statistical analysis Data from three replicates were expressed as mean S:E:M. The data were analyzed by ANOVA using the STATVIEW program. All percentage data were subjected to arc sine transformation before statistical analysis. Fisher's least signicant difference (LSD) with a 5% signicance level (P < 0:05) was used to test the differences between treatments. 3. Results In the rst experiment, oocytes were cultured for 2324 h under low glucose concentration and high O2 tension (20%) in maturation medium containing various concentrations of antioxidants: cysteine 0.6 mM, NAC 0.6 mM, catalase 5 or 127 U/ml or SOD 10 or 1000 U/ml. The matured oocytes were then fertilized in vitro, transferred into development culture medium and examined after 72 h to assess cleavage rates. There were no signicant differences among treatments for the cleavage rates (Fig. 1). However, 8 days after transfer at 7% O2, a signicantly higher (P < 0:05) proportion of oocytes reached the morula and blastocyst stages in the presence of cysteine in maturation medium compared to control (antioxidant-free maturation medium) (33.3% versus 20.3%). The presence of NAC, catalase or SOD at low or high concentrations did not improve the proportions of oocytes reaching the morula and blastocyst stages. In the second experiment (Fig. 2), oocytes were matured in antioxidant-free maturation medium but the fertilization medium contained various concentrations of antioxidants: cysteine (0.1 or 0.6 mM), NAC (0.1 or 0.6 mM), catalase (5 or 127 U/ml) or SOD (10 or 100 U/ml). The presence of higher concentrations of cysteine, NAC or catalase signicantly decreased the cleavage rates observed after 72 h when compared to the control value (P < 0:05). The presence of low concentrations of cysteine, NAC, catalase or SOD during the IVF period did not improve the proportion of oocytes undergoing morula and blastocyst development. However, higher concentrations of these antioxidants signicantly reduced the percentage of oocytes developed to the morula and blastocyst stages (P < 0:05).

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Fig. 1. Effect on the developmental capacity of fertilized oocytes of cysteine, NAC, catalase and SOD added to the IVM medium. Pooled data from three replicates (mean S:E:M). (ac) Values with different superscripts are signicantly different (P < 0:05).

In the third experiment, oocytes were matured and fertilized in vitro in antioxidant-free medium. However, for the rst 72 h of the 8-day development period, the zygotes were cultured in medium containing various concentrations of antioxidants. There was no signicant difference among groups for cleavage rates, in contrast to what was observed for cysteine, NAC and catalase supplementation when added during IVF (Experiment 2). The presence of cysteine during the rst 72 h of the development period improved the number of embryos in the morula and blastocyst stages when compared to the control group in antioxidant-free medium (30.6% versus 22.2%, Fig. 3). However, the development of embryos to the morula and blastocyst stages was not improved by the supplementation of NAC, catalase or SOD to the medium used for IVC. 4. Discussion Our results showed cysteine supplementation during IVM or IVC improved the rate of bovine embryo development to the morula and blastocyst stags. However, the development

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Fig. 2. Effects of supplementation with cysteine, NAC, catalase and SOD during IVF on the development of in vitro inseminated bovine oocytes. Pooled data from three replicates (mean S:E:M). (ac) Values with different superscripts are signicantly different (P < 0:05).

of embryos to the morula and blastocyst stages was not improved by the supplementation of NAC, or the enzymatic antioxidants catalase and SOD, to media used for IVM, IVF or IVC. Our results indicated that the addition of 0.6 mM cysteine to the dened maturation medium containing low glucose concentration under high oxygen tension (Experiment 1) signicantly enhanced bovine embryo development to the morula and blastocyst stages (P < 0:05). This is in agreement with the results from de Matos et al. [28] who demonstrated that b-mercaptoethanol, cysteine and cystine added during IVM of bovine oocytes improved the rate of embryo development to the blastocyst stage. Recently, it has been reported that IVM medium supplemented with a high glucose concentration under 20% O2 impaired bovine oocyte developmental competence as a result of increased intracellular ROS, and decreased intracellular GSH. This occurs by a mechanism that impairs the expression of g-glutamylcysteine synthesis [31].

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Fig. 3. Effects of antioxidants on the development of IVMIVF bovine embryos in m-SOF. Pooled data from three replicates (mean S:E:M). (ac) Values with different superscripts are signicantly different (P < 0:05).

In our study, the addition of NAC, catalase or SOD during IVM in dened conditioned medium did not increase the percentage of embryos produced. The addition of higher concentrations of SOD signicantly reduced the number of blastocysts, suggesting that H2O2 generated by SOD is toxic during IVM. Conversely, the results from Blondin et al. [22] demonstrated that small amounts of superoxide anion (O2) can be benecial to oocytes during IVM. In addition, during IVM the oocyte itself may not be exposed to 20% O2 as a result of the active metabolism of the cumulus cell surrounding it. In our study, no benecial effect of extracellular antioxidants was seen, probably because the cumulus cells play a protective role that isolate the oocyte from the extracellular medium. However, de Matos et al. [28] found that cysteine is metabolized by the cells during IVM despite the presence of cumulus cells but NAC is not. The results of Experiment 2 demonstrated that the addition of various antioxidants at low concentrations to the IVF medium had no effect on the percentages of embryos produced, but higher concentrations of these antioxidants signicantly reduced the percentages of morula and blastocysts produced (P < 0:05). Previously, it has been reported that the addition of SOD during IVF signicantly reduced the percentage of embryos produced [13], which is in agreement with our ndings. These results show that ROS might play a positive role during IVF. It is possible that high SOD activity increases the amount of H2O2 in the media rather than O2 (Fig. 2), therefore, the detrimental effect of high concentration

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of SOD during the insemination interval could be attributed to increased H2O2 rather than reduced O2 in Experiment 2 [7,13]. The presence of higher concentrations of cysteine, NAC or catalase during the period of IVF signicantly reduced the cleavage rate and subsequent embryo development. A possible explanation for this may be that certain levels of ROS are required for IVF (Fig. 2). Our results and results from other groups suggest that small amounts of ROS, as discussed earlier, may be necessary for optimal fusion between the oocyte and spermatozoa [22], and sperm capacitation [32]. During development, it was unclear if supplementation with antioxidants is required at low oxygen concentration (7%) and if the protection must be intracellular or extracellular. Our study clearly demonstrated that the proportion of oocytes developing to the morula and blastocyst stages was not increased by the presence of NAC, catalase or SOD in dened culture medium containing low glucose concentrations under low oxygen tension (7%). A study reported by Nasr-Esfahani et al. [4] determined that in vitro culture medium supplemented with catalase did not affect endogenous H2O2 production. Moreover, Li et al. [33] showed that the addition of catalase to the culture medium had no effect on rabbit embryo development. These authors suggested that these results were due to the inability of catalase to cross the membrane. Moreover, m-SOF used in our study contains pyruvate, which has been reported to act as an antioxidant capable of reducing intracellular levels of hydrogen peroxidase in the embryo [34]. Many studies have reported the benecial effects of SOD on embryo development in vitro [33,35]. In these reports, the positive effects of SOD (more than 150 U/ml) are observed only when the embryos were cultured in medium containing high glucose concentrations (5.568.3 mM) under high O2 tension (20%), which is contrast with our conditions (low glucose concentration and low O2 tension). These results would support the hypothesis that generation of ROS is induced by high glucose or O2 concentrations. Therefore, a possible explanation for the benecial effect of low glucose concentration on embryo development compared to high glucose concentration, could be the reduced amount of ROS generated in vitro [33,35]. Interestingly, in our study the addition of cysteine to the development culture medium (Experiment 3) improved the development of bovine embryos. Caamano et al. [36] and Lim et al. [11] also reported benecial effects on bovine embryo development in vitro after supplementation of culture media with low molecular-weight thiols. Cysteine may be taken up by embryonic cells and can scavenge reactive oxygen species [11,18,36], leading to a decrease of the intracellular hydrogen peroxide level and better embryo development as shown in our study. Surprisingly, NAC did not improve the development of embryos despite its similarities to cysteine (Fig. 3). One hypothesis to explain this phenomenon would be that the benecial effect of NAC through its antioxidant properties is counteracted by its inhibitory effect on nuclear factor kappa B (NF-kB) transcription factor. NF-kB was shown to be critical for the development of mouse embryos from 1- to 4-cell stages [37]. We are currently investigating the exact role of NAC in the production of bovine embryos. In conclusion, this study shows that addition of cysteine, an amino acid that can be metabolized by cells during IVM and IVC in dened conditions containing low glucose concentration, improved the rate of bovine embryo development. However, NAC and

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extracellular enzymatic antioxidants like catalase and SOD did not improve the proportion of oocytes undergoing morula and blastocyst development. Acknowledgements This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). The authors thank Dr. Susan Novak for advice on English language usage in the manuscript. References
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