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Biology of Reproduction 66, 562-567 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Promoting Effect of ß-Mercaptoethanol on In Vitro Development under Oxidative Stress and Cystine Uptake of Bovine Embryos1

M. Takahashi2,a, T. Nagaib, N. Okamurac, H. Takahashid, and A. Okanod

a Department of Animal and Grassland Research, National Agricultural Center for Kyushu Okinawa Region, Kumamoto 861-1192, Japan b Developmental Biology Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan c Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan d Department of Animal Breeding and Reproduction, National Institute of Livestock and Grassland Science, Tsukuba Norin-kenkyu-danchi, Ibaraki 305-0901, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effects of ß-mercaptoethanol (ß-ME) on in vitro development under oxidative stress and cystine uptake of bovine embryos were investigated. Bovine 1-cell embryos obtained by in vitro fertilization were cultured in TCM-199 or synthetic oviductal fluid (SOF) in 20% O2 supplemented with ß-ME. Addition of ß-ME significantly (P < 0.01) promoted embryo development when cultured in both TCM-199 and SOF under high levels of O2, to almost the same rates when they were cultured in 5% O2. To investigate whether the growth-promoting effect of ß-ME was related to cystine uptake, which is an important amino acid for intracellular glutathione (GSH) synthesis, 1-cell, 8-cell, morula, and blastocyst stage embryos were incubated in cystine, cysteine-free TCM-199 containing radioisotope-labeled cystine supplemented with or without ß-ME. It was found that cystine uptake was consistently low in each embryo stage incubated without ß-ME. In contrast, addition of ß-ME significantly (P < 0.05 to 0.0001) promoted cystine uptake in each stage of embryo development. This increase of cystine uptake by ß-ME was significantly inhibited by supplementation of buthionine sulfoximine, a specific inhibitor of GSH biosynthesis (P < 0.0001). High-performance liquid chromatography (HPLC) analysis clearly revealed a decrease of cystine in culture medium after supplementation by ß-ME, thereby forming another peak. HPLC analysis also showed the incorporated cystine by supplementation of ß-ME was possibly metabolized for GSH synthesis in the embryos. These results indicate that ß-ME has a protective effect in embryo development against oxidative stress and that the effect of ß-ME is associated with the promotion of cystine uptake of low availability in embryos.

cystine uptake, early development, embryo, ß-mercaptoethanol


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is well known that in vitro development of bovine embryos is highly affected by culture conditions. Therefore, much effort has been made to develop effective culture methods that support embryo development in vitro. The detrimental effect of oxidative stress caused by a high oxygen concentration (95% air; approximately 20% O2 concentration) under culture conditions on the development of mammalian preimplantation embryos was recently studied. It was found that embryo development is promoted under a low O2 concentration as opposed to a high O2 concentration [1–5]. However, reduced embryo development in a high O2 concentration was restored by addition of free radical scavengers [6, 7]. Therefore, to protect embryos against oxidative stress seems to be one of the keys to improving development. We have demonstrated that development of bovine embryos was promoted by ß-mercaptoethanol (ß-ME), a low molecular weight thiol that is used as a reducing agent in culture medium [8]. Since that research was conducted, the beneficial effect of ß-ME has been widely investigated on maturation of bovine oocytes [9] and development of embryos in vitro [9–12] cultured in a 20% O2 concentration. However, the effect of ß-ME on in vitro development under conditions of oxidative stress such as a high O2 concentration, has not been elucidated precisely in comparison with development under conditions of low O2 concentration.

Further investigations have revealed that the effect of ß-ME on oocyte maturation and embryo development is correlated with biosynthesis of intracellular glutathione (GSH) [8, 9, 13], which is a tripeptide thiol that has many important roles in intracellular physiology and metabolism. One of the most important roles of GSH is to maintain the redox state in cells, protecting them against harmful effects caused by oxidative injuries. GSH synthesis is highly dependent on the availability of cysteine in the medium [14, 15]. Beneficial effects of cysteine in in vitro maturation of oocytes [9] and development of embryos [16] have been reported. However, cysteine in the medium is easily oxidized, forming cystine, a cysteine dimer, even under usual culture conditions [17, 18]. When cysteine is oxidized to cystine under usual culture conditions, some types of cells, such as lymphocytes, have difficulty utilizing cystine, resulting in a decrease in their proliferation and intracellular GSH concentration [14, 19]. Addition of thiols promotes the uptake of cystine in cells that have a poor ability to uptake cystine, thereby promoting their proliferation and GSH levels [14]. Although one report demonstrated the decrease of cystine in the culture medium by addition of ß-ME [11], the uptake and metabolism of the incorporated cystine in embryos by ß-ME has not been clearly elucidated in embryos. In the present study we investigated the protective effect of in vitro development under oxidative stress and enhancement of cystine uptake of bovine embryos by ß-ME.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In Vitro Maturation and Fertilization

Bovine ovaries obtained at a slaughterhouse were transported to the laboratory in sterile saline at 37°C. Cumulus-oocyte complexes (COCs) were aspirated from the follicles (2–5 mm in diameter) with an 18-gauge needle attached to a 5-ml syringe.

COCs were washed twice in TCM-199 (Gibco BRL, Rockville, MD) containing 20 mM Hepes supplemented with 5% fetal calf serum (FCS). Twenty COCs were then placed in 100-µl drops of TCM-199 containing 5% FCS, 0.02 IU/ml FSH (Denka Co., Kawasaki, Japan), 1 µg/ml estradiol-17ß (Sigma Chemical Company, St. Louis, MO), and 10 µg/ml gentamycin (Sigma) in a 35-mm Petri dish (Nalge Nunc International, Rochester, NY). The drops were covered with mineral oil (Sigma) and COCs were then cultured for 20–21 h at 38.5°C under 5% CO2 and 95% air in a humidified atmosphere.

Sperm capacitation was carried out as described by Parrish et al. [20]. In brief, frozen-stored semen was thawed at 37°C. Semen was then suspended in 10 ml of BO solution [21] containing 10 mM caffeine (Sigma). After washing twice by centrifugation for 5 min at 800 x g, the concentration of spermatozoa was adjusted to 2 x 107/ml. The sperm suspension was then diluted 2-fold with BO solution containing 10 mg/ml BSA and 10 µg/ml heparin (Sigma). After 20–21 h of maturation, COCs were washed twice in BO solution and then placed into 0.5-ml drops of sperm suspension. In vitro fertilization was carried out for 5 h at 38.5°C in 5% CO2 and 95% air in a humidified atmosphere.

In Vitro Culture

After fertilization, cumulus cells surrounding fertilized oocytes were completely removed by pipetting. TCM-199 supplemented with 5% FCS (TCM-199) and synthetic oviductal fluid (SOF) [22] supplemented with an essential and nonessential amino acid solution (Sigma) and 5% FCS (SOFaa) was used for embryo culture. Twenty 1-cell embryos were placed in a 50-µl drop of TCM-199 or SOF supplemented with or without 50 µM ß-ME (Nakalai Inc., Kyoto, Japan) in a 35-mm Petri dish (Becton Dickinson Labware, Franklin Lakes, NJ). The concentration of ß-ME was determined according to the method detailed in our previous report [8]. Culture drops containing 1-cell embryos were covered with mineral oil (Sigma). Embryos in TCM-199 or SOF supplemented with or without ß-ME were cultured at 38.5°C in a high O2 concentration (5% CO2 and 95% air; 20% O2) in a humidified atmosphere. Embryos cultured in TCM-199 or SOF supplemented without ß-ME were also cultured in a low O2 concentration (5% CO2, 90% N2, and 5% O2) in a humidified atmosphere. The culture medium was changed every 2 days. After 8 days of culture from the day of in vitro fertilization, embryos that had developed to the blastocyst stage in each medium were observed using a dissecting microscope.

Effect of ß-ME on Cystine Uptake in Embryos

After in vitro fertilization, 1-cell, 8-cell, morula, and blastocyst stage embryos cultured in TCM-199 were collected. Embryos were then washed in cysteine-free and cystine-free TCM-199 (TCM-199 [-], Nissui Co., Japan) supplemented with 2 mg/ml polyvinyl pyrrolidone (PVP; average Mr 40 000, Sigma). Ten embryos at each stage were transferred to a 50-µl drop of TCM-199 (-) supplemented with PVP, 1 µCi/ml [35S]cystine (Amersham Pharmacia Biotech, Uppsala, Sweden), and with or without 100 µM ß-ME. Embryos were then incubated for 30 min at 39°C in 5% CO2 and 95% air in a humid atmosphere.

Effect of BSO on Cystine Uptake

Embryos that developed to the 8- to 16-cell stage after 2 days of culture were used for the experiment. After washing 3 times in TCM-199 (-), 10 embryos were then transferred to a 50-µl drop of TCM-199 (-) containing PVP, 1 µCi/ml [14C]cystine (NEN Life Science Products, Boston, MA), 100 µM ß-ME supplemented with or without 5 mM DL-buthionine-[S1R-sulfoximine (BSO), a specific inhibitor of GSH synthesis [23]. The concentration of BSO was determined according to the method detailed in our previous study in which the significant decrease of development was observed at the concentration [8]. Embryos were then incubated at 39°C for 10 min in 5% CO2 in air in a humid atmosphere.

After incubation in TCM-199 (-) containing radioisotope-labeled cystine, with or without ß-ME, embryos were then washed 6 times in Ca2+-free and Mg2+-free PBS containing 2 mg/ml PVP (PBS [-] plus PVP). Then, embryos suspended with 5 µl of PBS were transferred to a 0.5-ml microtube filled with 100 µl of 1 M NaOH. Five microliters of PBS used for the final embryo washing were also collected for background counting. Then the microtube containing embryos was boiled at 100°C for 5 min to lyse the embryos, followed by adding the same amount of 1 M HCl for neutralization. Then the solution was transferred to a plastic scintillation vial (Packard BioScience B.V., Groningen, The Netherlands). After the addition of a scintillation cocktail (Clear-sol I, Nakalai, Japan), the amount of radioactivity was counted with a scintillation counter (Packard Instrument Co., Meriden, CT). The radioactivity of incorporated cystine was calculated by subtracting the background count from the total count.

Statistical Analysis

Data were expressed as means ± SEM. Differences among the treatments in each experiment were analyzed by ANOVA using the Statview program (version 4.5J; Abacus Concepts Inc., Berkeley, CA). When ANOVA was less than 0.05, the data were then analyzed by the Fisher post-hoc test.

High-Performance Liquid Chromatography Analysis of Radioisotope-Labeled Cystine Incorporated in Embryos and Culture Medium With or Without ß-ME

Fifty embryos that had developed to the 8-cell stage were incubated for 30 min in a 50-µl drop of TCM-199 (-) containing PVP and 1 µCi/ml [14C]cystine. The incorporated [14C]cystine in embryos after 30 min of incubation with or without 50 µM ß-ME was analyzed by high-performance liquid chromatography (HPLC). After 30 min of incubation, embryos were washed 6 times in PBS (-) plus PVP. Then embryos with 5 µl of PBS were transferred to a 0.5-ml microtube filled with 100 µl of 10% (v/v) perchloric acid (Nakalai) diluted in distilled water. The tube was then frozen in the liquid nitrogen, followed by rapid thawing in hot water. After freezing and thawing the tube 3 times, the tube was sonicated for 30 min to rupture the embryos. The tube was centrifuged at 15 000 x g for 10 min, and the supernatant was used for HPLC analysis.

After a 30-min incubation of the medium containing [14C]cystine supplemented with or without ß-ME, 5 µl of medium was collected and transferred to a 0.5-ml microtube filled with 100 µl of 10% (v/v) perchloric acid diluted in distilled water. The tube was centrifuged at 15 000 x g for 10 min, and the supernatant was used for HPLC analysis. For detecting the peak of GSH, nonradiolabeled GSH (Sigma) was eluted as a standard.

All samples were analyzed by reverse phase chromatographic analysis with an HPLC system (Beckman Coulter, Inc., Fullerton, CA) equipped with radioisotope detectors and a control system (System Gold program; Beckman). For HPLC analysis, a 5-µm Ultrasphere ODS column (250 x 4.6 mm, Beckman) was used. The solvent used for analysis was 0.1% (v/v) trifluoroacetic acid (TFA, Nacalai) in distilled water. In every chromatographic analysis, the flow rate was held constant at 1.0 ml/min.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of ß-ME on Development of Embryos under Oxidative Stress

Figure 1 shows the effect of ß-ME on in vitro development of bovine embryos cultured in TCM-199 in 20% O2. The rate of embryos that developed to the blastocyst stage was significantly (P < 0.01) lower when embryos were cultured in 20% O2 compared with 5% O2. However, the rate of development in 20% O2 was significantly (P < 0.05) increased by the addition of 50 µM ß-ME. A significantly lower rate of development was also observed when embryos were cultured in SOF in 20% O2 compared with 5% O2 (Fig. 2; P < 0.01), and this decrease in embryo development was significantly (P < 0.01) increased by the addition of ß-ME (Fig. 2).



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  		FIG. 1. Effect of ß-ME on in vitro development of bovine embryos cultured in TCM-199 in a 20% O2 concentration. Data show means ± SEM after 4 replications. n, The total number of embryos used for the experiment; a vs. b, significantly different (P < 0.01); b vs. c, significantly different (P < 0.05)



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  		FIG. 2. Effect of ß-ME on in vitro development of bovine embryos cultured in SOFaa in a 20% O2 concentration. Data show means ± SEM after 4 replications. n, The total number of embryos used for the experiment; a vs. b, significantly different (P < 0.01)

Effect of ß-ME on Cystine Uptake of Bovine Embryos

Figure 3 shows the effect of ß-ME on the uptake of [35S]cystine in bovine 1-cell, 8-cell, morula, and blastocyst stage embryos. Radioactivity of incorporated [35S]cystine was not significantly different among embryos at each stage when they were incubated without ß-ME. In contrast, once the culture medium was supplemented with ß-ME, the radioactivities were significantly increased in 1-cell (P < 0.05), 8-cell (P < 0.01), morula (P < 0.05), and blastocyst stage embryos (P < 0.001), respectively, compared with embryos cultured without ß-ME. A significant increase in the uptake of [35S]cystine was also observed in embryos at the blastocyst stage when they were incubated with ß-ME (P < 0.01) compared with other embryo stages.



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  		FIG. 3. Effect of ß-ME on [35S]cystine uptake of bovine embryos. Ten 1-cell, 8-cell, morula, and blastocyst stage embryos were incubated for 30 min in cystine, cysteine-free TCM-199 plus [35S]cystine with or without 100 µM ß-ME. After incubation with or without ß-ME, the radioactivity of incorporated [35S]cystine was measured with a scintillation counter. Data show means ± SEM. n, The number of replications; a vs. b, P < 0.05; a vs. c, P < 0.0001; b vs. c, P < 0.01

Effect of BSO on Cystine Uptake of Bovine Embryos

Radioactivity of incorporated [14C]cystine in embryos was significantly increased by supplementation of ß-ME compared with control embryos without ß-ME (P < 0.001, Fig. 4). However, the radioactivity was significantly decreased by supplementation of BSO despite the addition of ß-ME (P < 0.001).



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  		FIG. 4. Effect of BSO on [14C]cystine uptake of bovine embryos. Ten 8-cell stage embryos were incubated for 30 min in cystine, cysteine-free TCM-199 plus [14C]cystine containing 100 µM ß-mercaptoethanol with or without 5 mM BSO. After incubation with or without BSO, the radioactivity of incorporated [35S]cystine was measured with a scintillation counter. Data show means ± SEM. n, The number of replications; a vs. b, P < 0.0001; b vs. c, P < 0.0001; a vs. c, P < 0.01

HPLC Analysis

Figure 5 shows the reverse-phase HPLC chromatograms of [14C]cystine in TCM-199 after 30 min of incubation with or without ß-ME. A single peak of [14C]cystine was detected in the culture medium without ß-ME (Fig. 5A). In contrast, the peak of [14C]cysteine in the medium with ß-ME was drastically decreased, and a new peak was detected (Fig. 5B). After HPLC analysis of incorporated [14C]cystine in embryos incubated for 30 min with or without ß-ME, no peak of incorporated [14C]cystine was detected in embryos incubated without ß-ME (Fig. 6A). In contrast, several peaks of incorporated [14C]cystine were detected in the embryos incubated with ß-ME (Fig. 6B). After HPLC analysis of cold GSH as a standard, the elution time of the peak of GSH was almost the same as the highest peak of metabolite of incorporated radiolabeled cystine (Fig. 6B, arrow).



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  		FIG. 5. HPLC chromatogram of [14C]cystine in culture medium after 30 min of incubation with or without ß-ME. A) [14C]Cystine in culture medium without ß-ME. B) [14C]Cystine in culture medium with 100 µM ß-ME. Note that the peak of [14C]cystine disappeared and a new peak appeared. The experiment was conducted 3 times



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  		FIG. 6. HPLC chromatogram of incorporated [14C]cystine in embryos with or without ß-ME. Fifty embryos were incubated for 30 min in TCM-199 containing [14C]cystine with or without 100 µM ß-ME. A) Control embryos incubated without ß-ME. B) Embryos incubated with ß-ME. Note that several peaks of [14C]cystine metabolites are observed. The experiment was conducted 3 times. The arrow in B shows the elution time of the GSH standard


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results obtained in the present study show that 1) addition of ß-ME improves the low rate of embryo development in 20% O2 to the same rates of the blastocyst stage in 5% O2, 2) the effect of ß-ME is to promote cystine uptake in embryos that have a low ability to uptake cystine, 3) the effect of ß-ME on cystine uptake was associated with the reaction of cystine with ß-ME to form the other compound, and 4) indicate that cystine uptake of embryos is probably correlated with glutathione metabolism.

In recent years, the detrimental effect of oxidative injury on the development of mammalian preimplantation embryos cultured in vitro has been studied [1–5]. Toxic effects of atmospheric O2 concentration on embryo development under ordinary culture conditions (5% CO2 and 95% air; approximately 20% O2) have been reported in mouse [1, 24, 25], hamster [2, 26], pig [5], sheep [3], and cattle [3, 4]. This reduction in development is likely due to oxidative stress, because the addition of free radical scavengers [6, 7] or reducing agents [8, 11, 12] to the culture medium improves the extent of development to the blastocyst stage under an atmospheric O2 concentration. In the present study, a high (20%) O2 concentration caused a significant decrease in development when embryos were cultured both in TCM-199 and SOFaa compared with embryos cultured under a low (5%) O2 concentration. In agreement with previous reports [3, 4], these results indicate that oxidative stress under a high O2 concentration is detrimental to embryo development.

Recent studies have demonstrated that ß-ME promotes in vitro development [8, 10–12] and DNA synthesis [27] of bovine embryos. Therefore, it could be suggested that addition of ß-ME promotes embryo development by promoting DNA synthesis. In addition, ß-ME promotes not only embryo development, but also intracellular glutathione synthesis [8, 13]. Considering that ß-ME is a thiol that acts as a reducing agent, the growth-promoting effect of ß-ME may have a correlation with keeping the redox state inside or outside of embryos that especially protect the embryos against oxidative stress.

The previous reports using somatic cells have revealed that the effect of ß-ME has a correlation with cystine uptake and GSH synthesis in the cells [28–30]. GSH is a tripeptide thiol synthesized by glutamic acid, cysteine, and glycine in the pathway of the {gamma}-glutamyl cycle [31]. GSH is known to have important roles for keeping intracellular redox state for protecting the cells against oxidative stress. In fact, oxidative stress caused serious DNA damage in bovine embryos and is associated with a low rate of embryo development [32]. Therefore, maintenance of the intracellular redox state by increasing the GSH level is an important factor for embryo development. In the process of GSH synthesis, cysteine is known to be a rate-limiting factor [31]. Furthermore, GSH synthesis is highly dependent on the availability of cysteine outside of the cells [14, 15, 28]. However, cysteine in the medium is easily oxidized even under usual culture conditions [17, 18], forming cystine, a dimer of cysteine. Once cysteine is changed to cystine, the growth of some cells such as lymphocytes is inhibited because of the low utility of cystine causing the depletion of GSH and growth inhibition [19]. Addition of thiols such as ß-ME or cysteamine promotes the uptake of cystine in cells that have a poor ability to uptake cystine, followed by enhanced GSH levels and cell growth [14, 29, 30].

The results obtained in the present study clearly showed that cystine uptake was low in all stages of bovine embryos when cultured in vitro, and that ß-ME dramatically promoted their cystine uptake. This low utility of cystine uptake without ß-ME was also observed in embryos cultured in the different culture media (unpublished data).

The addition of ß-ME in the radiolabeled cystine caused a significant decrease of cystine to form other components (Fig. 5B). Therefore, the newly appeared peak may be a key component for further incorporation and metabolism of cystine by ß-ME. Promotion of cystine uptake by ß-ME is reported to be due to a reaction of thiols with cystine to form mixed disulfides [15, 28, 29]. In particular, Ishii et al. [14] demonstrated that more than 40% of cystine in the medium is converted to mixed disulfide by the reaction of cysteine-ß-ME within 5 min. Also, the formation of a mixed disulfide by the reaction of cystine and ß-ME has been reported [29]. Considering these reports, the newly appeared peak after addition of ß-ME might be a mixed disulfide by reaction of cystine and ß-ME.

As shown in Figure 6B, several peaks of incorporated [14C]cystine have appeared in embryos cultured with ß-ME compared with embryos cultured without ß-ME (Fig. 6A). This result clearly demonstrates that the cystine that reacted with ß-ME is transported into embryos and metabolized. These data are compatible with the radioactivity of incorporated [35S]cystine in embryos cultured with or without ß-ME (Fig. 3).

After cystine is incorporated into the cells by ß-ME, about 40% is metabolized for GSH synthesis in lymphocytes [14, 29]. In the present study, the area of the main peak that had the same elution time with the GSH standard indicates that the incorporated cystine by ß-ME is metabolized for GSH synthesis. Because the promotion of cystine uptake with ß-ME was completely inhibited by BSO, the supply of cyst(e)ine may have a correlation with the metabolism of GSH synthesis in bovine embryos. The specific inhibitory effect of BSO on GSH synthesis and cystine uptake in the presence of a thiol is also reported in Chinese hamster ovary (CHO) cells [33]. We have already reported that the addition of BSO inhibits both development and intracellular GSH levels of bovine 6- to 8-cell embryos in the presence of ß-ME [8]. The same inhibitory effect of development is reported in mouse embryos [34]. Considering these results, the uptake of cystine that reacted with thiols is likely to be metabolized to GSH in the {gamma}-glutamyl cycle, and the block of the cycle at the step of {gamma}-glutamylcysteine synthetase with BSO may affect the supply of cystine with thiols (mixed disulfides) for GSH synthesis in bovine embryos.

With regard to the mechanism of cystine, cysteine, and mixed disulfides, several amino acid transport systems in the cells are reported. Cysteine, a main source of GSH synthesis, is mainly transported to the cells by the ASC system in a variety of cells [35]. In contrast, cystine is transported by a different system in fibroblasts and hepatic cells [36, 37], which is designated as system X. Some kinds of cells that have low cystine utility are reported to have weak cystine transport activity for system X [19, 28, 30, 33]. Therefore, the low utility of cystine in bovine embryos is probably the cause of the low activity of system X. It is interesting that the transporting system X is low in embryos, regardless of the undifferentiated state of cell development. However, the physiological activity of cystine in embryos is still unclear. It will be important to investigate the level of cystine uptake in in vivo embryos compared with in vitro embryos.

GSH has many important functions in cells or embryos, not only for protection from oxidative injuries, but also for DNA synthesis [27, 38], transcription [39], cell cycles [40], cytokine activity [41], and apoptosis [42]. Therefore, embryonic development is probably affected by the intracellular redox state correlated with GSH metabolism. For effective culture of bovine embryos, it is important to regulate intracellular GSH by controlling cystine and cysteine as a major source of GSH against the oxidative stress.


   FOOTNOTES
 
First decision: 1 June 2001.

1 This research was supported by the Ministry of Agriculture, Forestry, and Fisheries of Japan. Back

2 Correspondence: Masashi Takahashi, 2421 Suya, Nishigoshi, Kikuchi, Kumamoto 861-1192, Japan. FAX: 81 96 249 1002; masashi{at}affrc.go.jp Back

Accepted: October 4, 2001.

Received: May 14, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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H. Funahashi
Effect of beta-mercaptoethanol during in vitro fertilization procedures on sperm penetration into porcine oocytes and the early development in vitro
Reproduction, December 1, 2005; 130(6): 889 - 898.
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