Effects of Cumulus Cells on the Ability of Pig Oocytes to Utilize Cysteine or Cystine During Maturation In Vitro

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1 Journal of Reproduction and Development, Vol. 44, No. 2, 1998 Effects of Cumulus Cells on the Ability of Pig Oocytes to Utilize Cysteine or Cystine During Maturation In Vitro Ken SAWAI* ), Hiroaki FUNAHASHI and Koji NIWA Division of Animal Science and Technology, Faculty of Agriculture, Okayama University, Okayama , Japan * ) Present address: Hokkaido Prefectural Shintoku Animal Husbandry Experiment Station, Shintoku-cho, Kamikawa-gun, Hokkaido , Japan Abstract. The present study examined the effect of cysteine and cystine associated with cumulus cells in maturation medium on oocyte glutathione (GSH) concentration at the end of maturation culture (for 48 h) and male pronuclear (MPN) formation following in vitro fertilization of pig oocytes. When cumulus-enclosed oocytes were cultured in a serum-free medium containing 0.57 mm cysteine or cystine, their GSH concentration and incidence of MPN formation were higher than when they were cultured without cysteine and cystine. Supplementation with cysteine during the final 24 h of culture resulted in an increased GSH concentration of oocytes and a higher incidence of MPN formation, regardless of the presence of cumulus cells, than in those cultured without cysteine or cystine. The same high incidence of MPN formation was observed even when oocytes were denuded after 36 h of culture and exposed to cysteine for only 12 h. In contrast, when oocytes were denuded after 24 h of culture and exposed to cystine, neither GSH synthesis nor MPN formation was improved, but both parameters did improve when oocytes were not denuded. Exposure of cumulus-enclosed oocytes to cystine from 36 h of culture did not promote MPN formation. These results indicate that cumulus-free pig oocytes can synthesize GSH in the presence of cysteine and that the presence of cumulus cells is essential for maintaining a high concentration of GSH in oocytes in the presence of cystine. Key words: Pig, Cysteine, Cystine, Male pronuclear formation, Glutathione. (J. Reprod. Dev. 44: , 1998) I ntracellular glutathione (GSH) concentration of pig oocytes at the end of in vitro maturation is known to be positively correlated with male pronuclear (MPN) formation after in vitro fertilization [1 3]. Glutathione is a tripeptide that has intracellular functions in the reduction of the disulfide linkages of proteins and other molecules, amino acids transport and synthesis of proteins and DNA [4, 5]. Especially at the initiation of sperm decondensation in mature oocytes, GSH is believed to Accepted for publication: December 24, 1997 Correspondence: K. Niwa play an important role for reduction of penetrated sperm nuclear disulfide bonds [6 8]. Culture of cumulus-enclosed pig oocytes in the presence of a high concentration (0.57 mm) of cysteine in a maturation medium greatly increased oocyte GSH synthesis at the end of maturation culture and consequently improved MPN formation following sperm penetration [2, 9]. The GSH concentration of oocytes has been demonstrated to be highly correlated with that of the cumulus cell mass in the hamster [10] and pig [11]. However, since GSH synthesis in many kinds of cells is dependent on the availability of cysteine in the medium [12, 13],

2 162 SAWAI et al. it has been postulated that cumulus-free pig oocytes also may directly utilize cysteine for GSH synthesis. On the other hand, it is well known that cysteine in medium is oxidized under the usual culture conditions and forms cystine [14, 15]. Although mouse lymphocytes can not utilize cystine for the synthesis of GSH, when co-cultured with feeder cells, they can utilize cysteine formed from cystine by feeder cells [16]. Therefore, it is possible that cystine may not be utilized directly by oocytes but that it may be reduced to cysteine by cumulus cells and then affect oocytes for increasing GSH concentration. The present study was undertaken to explore these possibilities by culturing cumulus-enclosed or cumulus-free pig oocytes in medium supplemented with cysteine or cystine at various times after the start of culture for maturation. Materials and Methods Medium The basic medium used for the manipulation of oocytes and spermatozoa was TCM 199 with Earle s salts (Gibco, Grand Island, NY, USA) supplemented with 3.05 mm D-glucose, 0.91 mm sodium pyruvate, 75 µg/ml potassium penicillin G, 50 µg/ml streptomycin sulphate, and 1 mg/ml polyvinylalcohol (PVA). This medium was essentially the same as TCM-199B used by Wang et al. [17] except that calcium lactate was omitted and PVA was substituted for fetal calf serum (FCS) and therefore designated modified TCM-199B (mtcm- 199B, ph 7.4). Although this medium contains trace amount of cysteine (0.8 µm) and cystine (60 µm), these concentrations are reportedly too low to stimulate either GSH synthesis or MPN formation in pig oocytes [9, 17 19]. Preparation of oocytes Ovaries collected from maturing gilts at a local slaughterhouse were transported to the laboratory within h in 0.9% (w/v) NaCl solution containing 75 µg/ml potassium penicillin G and 50 µg/ml streptomycin sulfate at C. Cumulusenclosed oocytes were aspirated from antral follicles of 2 5 mm in diameter with an 18-G needle fixed to a 10-ml disposable syringe. Only oocytes with uniform ooplasm and enclosed by a compact cumulus cell mass were washed four times with maturation medium, mtcm-199b supplemented with 10 I.U./ml ecg and 10 I.U./ml hcg, and with or without 0.57 mm cysteine (Sigma Chemical Co., St Louis, MO, USA) or 0.57 mm cystine (Sigma). Ten cumulus-enclosed oocytes were transferred to 100 µl of the same medium and cultured for a total of 48 h at 39 C in an atmosphere of 5% CO 2 in air. Cumulus-enclosed oocytes after 48 h of culture (experiment 1) or oocytes freed from cumulus cells by repeated passage through a fine pipette at various times after the start of culture or at the end of culture (experiments 2 and 3) were washed twice with fertilization medium, mtcm- 199B (ph 7.8) containing 10 mm caffeine-sodium benzoate (Sigma), 2.92 mm calcium lactate, and 10% (v/v) FCS. Ten cumulus-enclosed or cumulus-free oocytes were placed into 50 µl of the same medium and kept in a CO 2 incubator (5% CO 2 in air at 39 C) for about 30 min until spermatozoa were added for fertilization. Maturation and fertilization media had been previously covered with warm paraffin oil in a polystyrene culture dish (No. 1008, Becton and Dickinson, Franklin Lakes, NJ, USA) and equilibrated in the same atmospheric condition for at least 3 h. Sperm preparation and in vitro fertilization Frozen ejaculated spermatozoa (three pellets with a 200-µl volume and containing about spermatozoa/pellet) were thawed in 2 ml fertilization medium without caffeine at 37 C for 1 min for each insemination. The medium had been previously equilibrated in an atmosphere of 5% CO 2 in air for at least 3 h. After thawing, 6 8 ml of the same medium was added and then spermatozoa were washed three times by centrifugation, each time at 550 g for 5 min. After washing, the sperm pellet was resuspended to give a concentration of spermatozoa/ml. A 50-µl aliquot of the sperm suspension was introduced into 50 µl of fertilization medium containing the cumulus-enclosed (experiment 1) or cumulus-free oocytes (experiments 2 and 3), thus producing a mixture with the final concentrations of spermatozoa/ml and 5 mm caffeine. Assessment for sperm penetration and oocyte nuclear maturation Fourteen hours after insemination, cumulus-enclosed oocytes were freed from cumulus cells as mentioned above. Oocytes were mounted, fixed

3 AVAILABILITY OF CYSTEINE OR CYSTINE BY PIG OOCYTES 163 for h in 25% (v/v) acetic acid in ethanol at room temperature, stained with 1% (w/v) orcein in 45% (v/v) acetic acid, and examined under a phase-contrast microscope at magnifications of 200 and 400. Oocytes were considered penetrated when they had one or more morphologically changed sperm nuclei and the corresponding sperm tails. Assay of GSH At the end of culture for maturation, oocytes were freed from cumulus cells by repeated passage through a fine pipette in PBS with 1 mg/l PVA (PBS-PVA), and all denuded oocytes were washed 3 times with the same medium. Groups of 30 oocytes in 10 µl of PBS-PVA were transferred to a 1.5-ml polypropylene microfuge tubes and frozen at 20 C. The frozen samples were thawed at room temperature, and 5 µl of 1.25 M H 3 PO 4 was added. The samples were refrozen for storage at 20 C until used for the assay of GSH. Blanks containing 5 µl of PBS-PVA without oocytes were treated similarly. Glutathione was determined by the enzymatic cycling assay of Tietze [20]. After thawing, the samples were diluted with 1.2 ml of H 2 O, and transferred to glass tubes. The following solutions were subsequently added to the tubes: 5 µl of 1.25 M H 3 PO 4, 1.2 ml of 0.2 M potassium phosphate in 10 mm EDTA buffer (ph 7.2), 100 µl of 10 µm 5,5-dithiobis-(2-nitrobenzonic acid) (DTNB; Sigma), 50 µl of 20 units/ml GSH reductase (Type III; Sigma), and 50 µl of 4 µm NADPH (Kohjin Co., Tokyo, Japan). After the addition of NADPH, the absorbance was immediately monitored at 412 nm with a spectrophotometer (Hitachi, Tokyo, Japan) and recorded at 0.5 and 5 min. Both the reagent blank and reduced GSH standards ( nm; Sigma) were also assayed under the same condition. The total amount of GSH measured was divided by the number of oocytes in the sample to obtain the total GSH content per oocyte. To calculate the mean cell volume, 100 oocytes were photographed by using microscope (Nikon, Tokyo, Japan) and the diameter of the ooplasm was measured (118.6 ± 0.7 µm); the calculated mean cell volume of porcine oocyte was pl. Then, the GSH concentration per oocyte was estimated according to Yoshida et al. [2]. Statistical analysis The percentages of penetration and pronuclear formation were subjected to an arc-sine transformation, and the transformed values were assigned for one-way or three way analysis of variance (ANOVA). When ANOVA revealed a significant treatment effect, the treatments were compared by Duncan s multiple range test. Concentrations of GSH were compared by Duncan s multiple range test. Experimental Deatails and Results Experiment 1 To examine the effects of cysteine or cystine present in medium throughout culture on GSH concentration of matured pig oocytes and MPN formation of penetrated oocytes in vitro, cumulusenclosed oocytes were cultured for 48 h in maturation medium supplemented with or without 0.57 mm cysteine or 0.57 mm cystine. Since preliminary investigations revealed that nuclear maturation was greatly inhibited when cumulusfree pig oocytes were cultured (data not shown), only cumulus-enclosed oocytes were used in this experiment. Oocytes used for assessment of GSH concentration were cultured separately from those used for in vitro fertilization. As shown in Fig. 1, the GSH concentration in oocytes matured in the presence of cysteine (7.6 ± 0.5 mm) or cystine (8.2 ± 0.5 mm) was significantly (P<0.01) higher than in control oocytes (3.0 ± 0.8 mm). Very high proportions (95 98%; Table 1) of oocytes were penetrated in vitro, regardless of whether cysteine or cystine had been present in the maturation medium. However, the incidence of MPN formation was significantly (P<0.01) higher in oocytes matured in the presence of cysteine (86%) or cystine (81%) than in control oocytes (38%). Experiment 2 In this experiment, the effects of cysteine or cystine, in the presence/absence of cumulus cells, during the latter half of culture on the final GSH concentration of matured oocytes and subsequent MPN formation of penetrated oocytes in vitro was examined. Cumulus-enclosed oocytes were first cultured for 24 h in medium without cysteine and cystine and then some oocytes were freed from cumulus cells. Both cumulus-enclosed and cumulus-free oocytes were transferred to newly prepared

4 164 SAWAI et al. medium supplemented or not with 0.57 mm cysteine or 0.57 mm cystine and cultured for a further 24 h. After a total of 48 h, cumulus-enclosed oocytes were denuded, and the denuded oocytes were fertilized in vitro. Oocytes used for assessment of GSH concentration were cultured separately from those used for in vitro fertilization. When cysteine was added to maturation medium 24 h after the start of culture, both the GSH concentration (8.2 ± ± 1.0 mm, Fig. 2) and the incidence of MPN formation (79 90%, Table 2) were similar in cumulus-enclosed and cumulusfree oocytes. These values were significantly (P<0.05) higher than those for control oocytes (3.5 ± ± 1.0 mm GSH and 22 25% MPN forma- Fig. 1. Glutathione concentration of cumulusenclosed pig oocytes which were cultured for 48 h in maturation medium with or without 0.57 mm cysteine or 0.57 mm cystine. Experiments were replicated 4 times using a group of 30 oocytes in each trial. The results represent the mean ± SEM. Values with different superscripts are different (P<0.01). Fig. 2. Effect of cysteine and cystine in the presence/ absence of cumulus cells, during the final 24 h of a 48 h maturation culture on the final glutathione content of pig oocytes. Cumulusenclosed oocytes were cultured in unsupplemented maturation medium for 24 h, then some oocytes were freed from cumulus cells. Cumulus-enclosed and cumulus-free oocytes were cultured in maturation medium with or without 0.57 mm cysteine or 0.57 mm cystine for a further 24 h. Experiments were replicated 4 times using a group of 30 oocytes in each trial. The results represent the mean ± SEM. Values with different superscripts are different (P<0.01). Table 1. Effects of cysteine (0.57 mm) or cystine (0.57 mm) during maturation on male pronuclear formation of cumulus-enclosed pig oocytes penetrated in vitro a No. of oocytes penetrated With swollen sperm head With male No. of at various stages and female oocytes Total Metaphase-II Female pronucleus pronuclei Supplement inseminated (%) (%) b (%) b (%) b 58 55(95) 0(0) 34(62) c 21(38) c Cysteine 62 59(95) 1(2) 7(12) d 51(86) d Cystine 58 57(98) 1(2) 10(18) d 46(81) d a Experiments were replicated four times. b Percentage of the total number of oocytes penetrated. c,d Values with different superscripts within each column are different (P<0.01).

5 AVAILABILITY OF CYSTEINE OR CYSTINE BY PIG OOCYTES 165 tion). When cystine was added to maturation medium with cumulus-enclosed oocytes, the GSH concentration (7.2 ± 0.5 mm) and the incidence of MPN formation (90%) were comparable to values for oocytes matured with cysteine, but with cumulus-free oocytes, the values (2.4 ± 1.0 mm GSH and 16% MPN formation) were significantly (P<0.05) lower and similar to results obtained with control oocytes. Experiment 3 Our previous study [21] demonstrated that the presence of cysteine in the maturation medium during the last 12 h of a 48 h culture period can promote oocyte GSH synthesis and subsequent MPN formation after in vitro fertilization in cumulus-enclosed pig oocytes. In this next experiment, the effects of the presence of cysteine or cystine, along with cumulus cells during this period, on later MPN formation of oocytes were examined. The procedures were as described for experiment 2 except that treatment occurred at 36 h after the start of culture; oocyte GSH concentration was not determined. As shown in Table 3, when cysteine was added to maturation medium at 36 h after the start of culture, there was no significant difference in the incidence of MPN formation between cumulus-enclosed (92%) and cumulus-free oocytes (79%). These values were significantly (P<0.01) higher Table 2. Effects of cysteine (0.57 mm) or cystine (0.57 mm), in the presence/absence of cumulus cells, during the final 24 h of maturation culture on male pronuclear formation of pig oocytes penetrated in vitro a No. of oocytes penetrated With swollen sperm head With male Culture conditions No. of at various stages and female Cumulus oocytes Total Metaphase-II Female pronucleus pronuclei Cysteine Cystine cells inseminated (%) (%) b (%) b (%) b (79) 2 (5) 2 ( 5) c 38 (90) c (89) 1 (2) 9 (19) c 37 (79) c (94) 1 (2) 3 ( 6) c 46 (90) c (98) 1 (2) 45 (82) d 9 (16) d (89) 5 (10) 33 (67) d 11 (22) d (92) 0 (0) 35 (73) d 12 (25) d a Cumulus-enclosed oocytes were cultured for 24 h in medium without cysteine and cystine; conditions were then altered and culture continued for another 24 h (48 h total). Experiments were replicated four times. b Percentage of the total number of oocytes penetrated. c,d Values with different superscripts within each column differ significantly (P<0.05 at least). Table 3. Effects of cysteine (0.57 mm) or cystine (0.57 mm), in the presence/absence of cumulus cells during the final 12 h of maturation culture, on male pronuclear formation of pig oocytes penetrated in vitro a No. of oocytes penetrated With swollen sperm head With male Culture conditions No. of at various stages and female Cumulus oocytes Total Metaphase-II Female pronucleus pronuclei Cysteine Cystine cells inseminated (%) (%) b (%) b (%) b (88) 1 (2) 3 ( 6) c 46 (92) c (88) 3 (6) 8 (15) c 41 (79) c (84) 4 (8) 32 (63) d 15 (29) d (88) 1 (2) 40 (82) de 7 (14) de (85) 1 (2) 34 (77) de 9 (20) de (88) 2 (4) 44 (90) e 3 (6) e a Cumulus-enclosed oocytes were cultured for 36 h in medium without cysteine and cystine; conditions were then altered and culture continued for another 12 h (48 h total). Experiments were replicated four times. b Percentage of the total number of oocytes penetrated. c,d,e Values with different superscripts within each column differ significantly (P<0.01).

6 166 SAWAI et al. than those obtained when only cystine or neither was added to medium containing either cumulusenclosed or cumulus-free oocytes. Discussion Cysteine is an essential amino acid for many types of mammalian cells in culture [16]. Yoshida et al. [2] reported that oocyte GSH concentration and the incidence of MPN formation increased when cumulus-enclosed pig oocytes were matured in medium supplemented with cysteine during the entire culture period. The present study confirmed the stimulating effects of cysteine on oocyte GSH synthesis and MPN formation; however, both the intracellular GSH concentration of matured oocytes and the incidence of MPN formation after in vitro fertilization were still high even when oocytes were denuded of cumulus cells after 24 h of culture and then exposed to cysteine for the remaining 24 h of a total 48 h culture period. These data indicate that, in the absence of cumulus cells, pig oocytes can directly utilize cysteine to synthesize GSH and consequently increase the ability to form a male pronucleus. In our earlier study [21], oocyte GSH concentration and the incidence of MPN formation were reduced to a basal level when cysteine was removed after 36 h of a total 48 h culture of cumulus-enclosed pig oocytes, but in this study, the presence of cysteine for only the final 12 h culture was very effective for GSH synthesis and MPN formation by oocytes. Therefore, direct utilization of cysteine by oocytes during the final 12 h of culture appears to be sufficient for GSH synthesis associated with MPN formation following in vitro fertilization. When oocytes were denuded after 24 h of culture and then cultured for a further 24 h in the medium supplemented with cystine, both GSH and MPN values were significantly lower than those obtained with similar oocytes exposed to cysteine. Although about 90% of cysteine appears to be oxidized to cystine under usual culture conditions [15, 16], it has been reported that cysteine at a concentration as low as 0.08 mm stimulates GSH synthesis and MPN formation in cumulus-enclosed pig oocytes [2]. If cysteine is oxidized to cystine in medium within a few hours, then it would appear that cystine in medium may also be used for GSH synthesis and MPN formation in pig oocytes. The results of the present study demonstrated that the presence of a high concentration of cystine in the maturation medium during the final 24 h of culture was effective for promoting the ability of cumulus-enclosed oocytes to increase GSH concentration and to form male pronucleus, but that these effects disappeared when cumulus cells were removed indicating that cystine can not be utilized directly by oocytes for GSH synthesis. It has been reported that promoted growth of mouse lymphoma L1210 cells in co-culture with feeder cells is due to cysteine which is continuously provided by feeder cells and that the ability to form cysteine in medium by feeder cells is due to about 15 times greater capacity of feeder cells to take up cystine into the cells compared with L1210 cells [16]. It is unknown at present whether cumulus cells play a role in the same manner as feeder cells for utilizing cystine, but it may be postulated that cumulus cells have greater capacity to take up cystine compared with oocytes. Thus, cysteine in the medium and/or in cumulus cells may be incorporated into oocytes directly or through the gap junctions between the oocyte and cumulus cells, and utilized by oocytes for GSH synthesis. On the other hand, it is also possible that GSH synthesized in cumulus cells in the presence of cystine may be transported into oocytes through the gap junctions between the oocyte and cumulus cells. However, when cumulus-enclosed oocytes were only exposed to cystine during the last 12 h of culture, no increasing GSH concentration in oocytes could be detected. This may be due to decreased ability of cumulus cells to take up cystine and/or to form cysteine at this time of culture. The promotion of cystine uptake by chinese hamster ovary cells appears to be induced by exracellular thiols [22]. Exposure of cumulus-enclosed oocytes to cystine during the last 12 h of culture in experiment 3 might be insufficient to form adequate concentrations of cysteine and/or other thiols by cumulus cells. Alternatively, since intercellular coupling between the oocyte and cumulus cells is known to reach minimal level before completion of oocyte maturation [23], the loosened coupling may make it difficult to transport cysteine or GSH formed in cumulus cells into oocytes. However, our data do not allow us to draw any conclusions regarding the pricise mechanism of the involvement of cumulus cells in increasing concentration of GSH in oocytes cultured in the

7 AVAILABILITY OF CYSTEINE OR CYSTINE BY PIG OOCYTES 167 presence of cystine. In conclusion, the results of the present study suggest that, while cysteine can be directly incorporated by pig oocytes in vitro and utilized for GSH synthesis, cumulus cells play an important role in allowing pig oocytes to increase GSH concentration, important for MPN formation after in vitro fertilization. Furthermore, the critical period during which cysteine is required is the final 12 h of the 48 h culture period. With further experimentation, it may be possible to define the critical period even more precisely. Acknowledgments We thank Professor L. R. Fraser, King s College London, for critical reading of the manuscript. This work was supported by Grant-in-Aid for Scientific Research B (number ) from the Ministry of Education, Science, Sport and Culture of Japan. K. Sawai was a recipient of the Japan Society for Promotion of Science (JSPS) Fellowship (number 5501). References 1. Yoshida M. Role of glutathione in the maturation and fertilization of pig oocytes in vitro. Mol Reprod Dev 1993; 35: Yoshida M, Ishigaki K, Nagai T, Chikyu M, Pursel VG. Glutathione concentration during maturation and after fertilization in pig oocytes: relevance to the ability of oocytes to form male pronucleus. Biol Reprod 1993; 49: Funahashi H, Cantley TC, Stumpf TT, Terlouw SL, Day BN. Use of low salt culture medium for in vitro maturation of porcine oocytes is associated with elevated oocyte glutathione levels and enhanced male pronuclear formation after in vitro fertilization. Biol Reprod 1994; 51: Kosower NS, Kosower EM. The glutathione status of cells. Int Rev Cytol 1978; 54: Meister A, Anderson ME. Glutathione. Ann Rev Biochem 1983; 52: Griffith OW, Meister A. Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J Biol Chem 1979; 254: Perreault SD, Wolff RA, Zirkin BR. The role of disulfide bond reduction during mammalian sperm nuclear decondensation in vivo. Dev Biol 1984; 101: Calvin HI, Grosshans K, Blake EJ. Estimation and manipulation of glutathione levels in prepuberal mouse ovaries and ova: Relevance to sperm nucleus transformation in the fertilized egg. Gamete Res 1986; 14: Yoshida M, Ishigaki K, Pursel VG. Effect of maturation media on male pronucleus formation in pig oocytes matured in vitro. Mol Reprod Dev 1992; 31: Zuelke KA, Perreault SD. Hamster oocyte and cumulus cell glutathione concentrations increase rapidly during in vivo meiotic maturation. Biol Reprod 1994; 50 (Suppl. 1): 144 (abstr). 11. Funahashi H, Day BN. Effects of cumulus cells on glutathione content of porcine oocyte during in vitro maturation. J Anim Sci 1995; 73 (Suppl. 1): 90 (abstr). 12. Tateishi N, Higashi T, Shinya S, Naruse A, Sakamoto Y. Studies on the regulation of glutathione level in rat liver. J Biochem 1974; 75: Meister A, Tate SS. Glutathione and related gamma-glutamyl compounds: biosynthesis and utilization. Ann Rev Biochem 1976; 45: Toohey JI. Sulhydryl dependence in primary explant hematopoietic cells. Inhibition of growth in vitro with vitamin B12 compounds. Proc Natl Acad Sci USA 1975; 72: Mohindru A, Fisher JM, Rabinovitz M. Endogenous copper is cytotoxic to lymphoma in primary culture which requires thiols for growth. Experimentia 1985; 41: Ishii T, Hishinuma I, Bannai S, Sugita Y. Mechanism of growth promotion of mouse lymphoma L1210 cells in vitro by feeder layer or 2-mercaptoethanol. J Cell Physiol 1981; 107: Wang WH, Niwa K, Okuda K. In-vitro penetration of pig oocytes matured in culture by frozenthawed ejaculated spermatozoa. J Reprod Fertil 1991; 93: Funahashi H, Day BN. Effects of different serum supplements in maturation medium on meiotic and cytoplasmic maturation of pig oocytes. Theriogenology 1993; 39: Funahashi H, Cantley TC, Stumpf TT, Terlouw SL, Day BN. In vitro development of in vitro matured porcine oocytes following chemical activation or in vitro fertilization. Biol Reprod 1994; 50: Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: application to mammalian

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