IVF embryos in various concentrations of glucose

ELSEVIER

EFFECTS OF ANTIOXIDANTS ON THE DEVELOPMENT OF BOVINE IVM/IVF EMBRYOS IN VARIOUS CONCENTRATIONS OF GLUCOSE H. Iwata, la S. Akamatsu, 1 N. Minami 2 and M. Yamada 2 1

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Kobe City Horticulture Promoting Assecmtmn, Kobe 651-2204 Japan 2 Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan Received for publication: October 30, 1997 Accepted: May 20, 1998 ABSTRACT This study was undertaken to determine the effects of glucose, antioxidants and different oxygen tensions on the development of bovine embryos cultured in modified synthetic oviduct fluid (m-SOF) medium. In vitro matured (IVM) and fertilized (IVF) oocytes were incubated for 48 h. Embryos reaching at least the 4cell stage were selected for further culture under various conditions for 6 d. Supplementing the m-SOF media with 4.5 mM glucose resulted in a significantly lower (P<0.01) embryo developmental rate (21%; Day 8) than was obtained with 1.5 mM glucose (58 %; Day 8) or no glucose (53 %; Day 8). Antioxidants such as SOD, catalase and mannitol had no positive effect on embryo development in mSOF medium supplemented with 1.5 mM glucose. However, in m-SOF medium supplemented with 4.5 mM glucose, SOD and mannitol significantly (P<0.05) improved embryo development: SOD increased the developmental rate from 19 to 35 % (Day 8), while mannitol increased it from 13 to 30 % (Day 8). Low oxygen concentration improved embryo development significantly (P<0.05) in m-SOF medium supplemented with 4.5 mM glucose (low O2:31% vs high O2:14 %; Day 8) but not 0 mM glucose (low O2:58 % vs high O2:55 %; Day 8). Our data suggest that low concentration of glucose during culture of bovine embryos is beneficial, and that generation of free oxygen radicals is partly caused by a high concentration of glucose in the medium. © 1998 by ElsevierScience Inc.

Key words: antioxidants, bovine embryo, glucose, oxygen concentration,free oxygen radicals Acknowledgments Part of this work was supported by a grant from the Japan Society for the Promotion of Science (JSPS-RFTF 97L00905). e Correspondence and reprint requests: Kobe City Horticulture Promoting Association, 1557-1 Takawa Aza Seikaijiyama, Oshibedani-cho Nishi-ku, Kobe 651-22, Japan Theriogenology 50:365-375, 1998 © 1998 by Elsevier Science Inc.

0093-691X/98/$19.00 PII S0093-691X(98)00146-0

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INTRODUCTION Glucose is one of the major energy substrates for embryos cultured in vitro. In cattle embryos, glucose metabolism dramatically changes with embryo development (25). The glucose concentration of bovine oviductal fluid, in which most embryo development takes place, is about 0.05 to 0.2 mM (22), which is lower than that contained in calf selmm (5.56 raM). It has been reported that high glucose concentration inhibits early embryo development in vitro (26, 27, 30). Although it has been demonstrated that the inhibitory action of glucose is due to the Crabtreelike effect (28), the mechanism of this inhibition remains unclear. Recently, there have been several reports about the generation of free oxygen radicals by glucose autoxidation and phospho-ribosyltransferase (HPRT) inhibition (4, 7, 9). Moreover, we found that allopurinol, which is an inhibitor of xanthineoxidase (XOD), improves bovine embryo development in vitro (data not shown). Xanthine-oxidase and the hypoxanthine (HXT) system is one of the major sources of free oxygen radicals. It has been reported that glucose inhibits hypoxanthine metabolism by inhibition of HPRT. However, it is still unclear whether free oxygen radical generation is related to glucose. Almost all cells cultured in vitro are exposed to the risk of injury by free oxygen radicals. Some of the injury to in vitro embryos by fxee oxygen radicals may be due to the lack of scavenging systems such as SOD and to exposure to high oxygen concentration (31). Free oxygen radicals (Superoxide radical: O2", hydrogen peroxide: H202, hydroxyl radicals: • OH) generated in vitro react with cell proteins, lipids and DNA, resulting in inactivation of enzymes, lipid membrane peroxidation and DNA alterations (33). Thus, modification of the embryo culture environment, such as by reduction of atmospheric oxygen (1, 2, 5, 11, 18, 21, 31), or addition of substances to reduce free radical levels is required for the establishment of a suitable culture system (10, 11, 13, 15, 20, 29, 31). In these studies, however, there are contradictory results concerning the effects of antioxidant supplementation. For example, some studies that examined either rabbit embryos cultured in RD medium (10) or mouse embryos in BWW medium (20, 31) found that SOD improved embryo development to the blastocyst stage in vitro, while other studies using KSOM (11, 12) or SOF medium (13, 16) to culture bovine embryos found no evidence for improved development. In the present study, we investigated whether or not the inhibitory effect of glucose on embryo development is due to free radicals. We also examined the effects of antioxidants on the development of bovine embryos cultured in various concentration of glucose.

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MATERIALS AND METHODS In Vitro Maturation (IVM) Ovaries were collected from an abattoir and transported to the laboratory in physiological saline at approximately 32°C within 3 h. Cumulus-oocyte complexes (COCs) were aspirated from small antral follicles (3 to 5 mm in diameter) using a 20-g needle connected to a 10-mL syringe. The COCs were collected into mTyrode's solution (BO; 3) supplemented with 3 mg/mL BSA (Fraction-V, A-4503; Sigma Chemical Co., St. Louis, MO, USA). Only oocytes completely surrounded by unexpanded cumulus cells were selected and matured for 22 h in a 100-~L drop of maturation medium (10 oocytes/drop) under paraffin oil at 39°C in an atmosphere of 5 % CO2 in air with maximum humidity. The maturation medium was bicarbonate-buffered TCM-199 (Earl's salts; Gibco, Grand Island, NY, USA), supplemented with 10 % FCS (Gibco, 30K-0351), 5 mM taurine (Sigma, T-7146), 0.5 mM pyruvic acid (Sigma, P-2256), 100 U/mL penicillin and 100 ~tg/mL streptomycin. In Vitro Fertilization (IVF) The medium used for IVF medium was BO solution supplemented with 20 ~tg/mL heparin (Sigma, H-3393), 5 mM taurine, 3 mg/mL fatty acid-free BSA (Nacalai Chemicals Co., Tokyo, Japan, 012-79), 100 U/mL penicillin and 100 ~g/mL streptomycin. Frozen-thawed (37°C) semen from a Japanese Black bull was washed with 30 to 45 % discontinuous percoll (Pharmacia Co., Ltd., Uppsala, Sweden) gradient solution by centrifugation (500 x g) for 10 min (23). The washed semen was cultured for 2 h in BO solution under paraffin oil at a final concentration of 3 x l0 s sperm/mL. After preincubation, the semen was diluted at a concentration of 2.5 x 10~ sperm/mL with BO solution. Oocytes cultured for 22 h in maturation medium were washed with IVF medium and were transferred to 100-~L drops of sperm suspension for fertilization (10 oocytes/drop), and then cultured for 4 h. Incubation was carried out at 39°C in an atmosphere of 5 % CO2 in air with maximum humidity. In Vitro Culture and Experimental Design The medium used for in vitro culture (IVC) until 48 h after insemination was based on the composition reported by Takahashi and First (30) except that 1% FCS was used instead of BSA (m-SOF). Both essential and nonessential amino acids were added at the concentrations present in Eagle's minimal essential medium (MEM) and basal Eagle's medium, respectively, with the exception of glutamine

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and taurine, which were added separately at 1 and 5 m M , respectively.After insemination, oocytes were washed with m - S O F and transferredin 100-pL drops of m - S O F supplemented with 1% FCS (10 presumptive zygotes/drop) and then cultured for 48 h. At 48 h afterculture,cumulus cellswere removed from normally cleaved zygotes (>_- 4-cells)with a fine-drawn pasteur pipette, and then embryos were transferredto the culture medium (m-SOF supplemented with 5 % FCS) at 20 embryos/drop. The same number of embryos was randomly assigned to each experimental condition and was cultured for 144 h (Day 8 postinsemination).The number of embryos that reached the blastocyst stage at 6, 7 and 8 d after insemination was then compared. In Experiment 1, the effects of glucose supplementation (0, 1.5, 4.5 inM) on embryo development were examined. Incubation was performed at 39°C in an atmosphere of 5 % COs in air with maximum humidity. In all experiments, data were obtained from 4 replicates. To examine the effect of 02 concentration on the inhibitory action of glucose (Experiment 2), embryos were cultured under various concentrations of glucose (0, 1.5 or 4.5 raM) in low or high oxygen pressure (5 % C02 in air or 5 % CO2, 5 % 02, 90 % N2). In Experiment 3, we examined the effects of 3 types of antioxidants, 100 U/mL SOD (Nacalai), 100 U/mL catalase (Sigma) and 5 mM mannitol (Nacalai), on embryo development in the presence of 1.5 or 4.5 mM glucose under high oxygen pressure (20 %). Statistical Analysis The frequencies of development to the blastocyst stage were compared using Fisher's protected least significant difference (PLSD) test following an analysis of variance (ANOVA). All frequencies were subjected to an arcsine transformation before statistical analysis. A P value less than 0.05 was considered to be significant. ~S~TS In Experiment 1, supplementation of the culture medium with 4.5 mM glucose strongly inhibited embryo development to the blastocyst stage at all times postinsemination (Table 1). However, there were no significant differences between 1.5 mM and 0 mM supplementation. As shown in Table 2, when the embryos were cultured in m-SOF supplemented with 0 mM glucose, there was no significant difference in the development rates to the blastocyst stage between low and high oxygen pressure (0 mM glucose: 58 vs 55 % Day 8). On the other hand, there was a sJ~i~cant

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difference in the development rate to the blastocyst stage between low and high oxygen concentrations (31 vs 14 % Day 8) when the embryos were cultured in 4.5 mM glucose.

Table 1. Effect of glucose concentration on the development of embryos cultured in m-SOF containing 5 % FCS Glucose (raM)

No. of Trials

No. of Embryos ~

Day 6 b

No. (%) of blastocysts Day 7b

0

4

80

20(25) ¢

36(45) c

42(53) ¢

1.5 4.5

4 4

80 80

21(26) c 2(3) d

41(51) ¢ 12(15) 4

46(58) ¢ 17(21) d

Day 8 b

Normal cleaved embryo at the 4- to 6-cell stage 48 hours after insemination. b Days after insemination. c,d Different superscripts with in the same column differ significantly.

Table 2. Effect of oxygen concentration on the development of IVM/IVF bovine embryos in m - S O F medium with 2 different glucose concentrations Glucose (mM)

Oxygen Concentration

No. of No. of Trials Embryos a

No. (%) of blastocysts Day 6 b Day 7b Day 8 b

(%) 0

1.5

4.5

5

4

80

18(23) c

39(49) c

46(58) ~

20

4

80

18(23) ~

34(43) d

44(55) ~d

5

4

80

17(21) c

35(44) ca

47(59) c

20

4

80

13(16) d

28(35) e

41(51) d

5

4

80

3(4) e

18(23) f

25(31) e

4

80

O(O) f

7(9) g

11(14) f

20

a Normal cleaved embryo at the 4- to 6-cell stage 48 hours after insemination. b Days after insemination. c-g Different superscripts differ significantly within the same column.

Moreover, the percentage of embryos t h a t developed in m-SOF supplemented with 4.5 mM glucose was significantly lower in both low and high oxygen tensions

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t h a n in medium supplemented with 0 or 1.5 mM glucose (4.5 raM: 31% vs 1.5 raM: 59 % vs 0 raM: 58 % and 14 % vs 51% vs 55 %; Day 8). As shown in Table 3, addition of 100 U/mL SOD significantly improved embryo development with 4.5 mM glucose supplementation but not with 1.5 mM glucose. The effect of m a n n i t o l on embryo development was similar to that of SOD. The beneficial effect of m a n n i t o l was obsez~ced only in m-SOF supplemented with 4.5 mM glucose b u t not with 1.5 mM glucose. Catalase, however, had no significant effect on embryo development in either 4.5 or 1.5 mM glucose.

Table 3. Effect of antioxidants on the development of IVM-IVF bovine embryo in m-SOF medium with two different glucose concentrations Glucose (raM)

Antioxidants Concentration

1.5

SOD(100 U/mL) +

4.5

1.5

4.5

1.5

4.5

+ Catalase(100 U/mL) +

+ Mannitol(5 mM) +

+

No. of No. of Trials Embryos"

No.(%) of blastocysts Day 65 Day 7b Day 85

4

80

20(25) c

33(41) c

43(54) c

4

80

17(21) c

31(39) c

42(53) c

4

80

9(11) d

20(25) d

28(35) d

4

80

4(5) e

10(13) e

15(19) e

4

80

13(16) c

30(38) °

41(51) c

4

80

15(19) ~

29(36) c

40(50) ¢

4

80

3(4) d

7(9) d

11(14) d

4

80

4(5) d

8(10) d

12(15) d

4

80

20(25) ~

37(46) ~

45(56) c

4

S0

19(24) ~

35(44) c

43(54) c

4

80

0(0) d

19(24) d

24(30) d

4

80

0(0) d

8(10) e

10(13) e

a Normal cleaved embryo at the 4- to 6-cell stage 48 hours after insemination b Days after insemination. c~ Different superscripts within the same column of each antioxidant t r e a t m e n t differ significantly.

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DISCUSSION Glucose metabolism is reported to change dramatically throughout embryo development (24). At the early stages of development, glucose metabolism is low compared with that at the morula or blastocyst stage. Moreover, glucose concentration in oviductal fluid is reported to be 0.05 to 0.2 mM (22). In our present study, as shown in Table 1, there was no significant difference in the percentages of blastocysts that developed (Day 8) with 0 and 1.5 mM glucose supplementation. This may be due to the presence of 5 % glucose in FCS (0.28 mM), however, a high concentration of glucose (4.5 mM) inhibits embryo development from the 4-ceU to the blastocyst stage. This inhibitory effect of glucose on embryo development is similar to that previously reported (30). In one study (32) a high glucose concentration increased glycolytic activity. High glycolytic activity is detrimental to the early embryo due to a Crabtree-like effect (28); however, it remains unclear how glucose inhibits embryo development. Recently, it was suggested that glucose induces cell injury through free radicals generated by autoxidation (4, 7) or hypoxanthine accumulation (9). More free oxygen radicals are generated under in vitro culture conditions than under in vivo conditions due to suboptimal oxygen pressure and suboptimal scavenging systems. It is reported that the oxygen pressure in the rabbit oviduct at the early stages of embryo development is about 5 % (14). It has also been reported that high oxygen concentration may lead to developmental arrest by formation of oxygen radicals in mouse embryos (21). Moreover, a high concentration of SOD has been found in mouse oviductal fluid, while taurine has been found in bovine oviductal fluid (12). Thus, it is inferred that low oxygen pressure and the addition of antioxidants to the culture medium may reduce free radical levels in the culture medium. There have been some reports that taurine acts as an antioxidant (8, 12); however, in our preliminary experiments, neither the effects of antioxidants (SOD, catalase and mannitol) nor of oxygen tension on the development of bovine embryos was affected by taurine supplementation (data not shown). The mechanism of the action of taurine may be different from that of the antioxidants used in our study. There have been numerous reports about the beneficial effects of both low oxygen concentration in the mouse (5, 21, 31),sheep (1, 2) and cow (11, 18) and antioxidant supplementation (10, 20, 31) on embryo development in vitro. In Experiment 2, it was demonstrated that the inhibitory action of glucose on embryo development occurs at a concentration of 4.5 mM, and this inhibitory effect is improved when the oxygen pressure is decreased (Table 1). However, no positive

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effect of low oxygen tension was observed when embryos were cultured in medium supplemented with 0 mM glucose. It is inferred that embryo development improved by reduction of oxygen pressure represents the percentage of embryos injured by oxygen radicals. In Experiment 3, both SOD (100 U/mL) and mannitol (5 mM) improved embryo development when the embryos were cultured in m-SOF containing 4.5 mM glucose, but no positive effect of antioxidant was observed when embryos were cultured in medium supplemented with 1.5 mM glucose (Table 3). From these results, it is demonstrated that a low glucose concentration produces optimal culture conditions for embryo development, and it is inferred that high glucose concentration induces free radical generation. The superoxide anion (O~), hydrogen peroxide (I-I202) and hydroxyl radical (" OH), are major oxidative species. It is known that SOD catalyzes the dismutation of 05", and catalase rapidly reduces hydrogen peroxide (H202) to H20 and molecular 02. Mannitol is a sugar alcohol and is also reported to be one of the scavengers of hydroxyl radicals ( . O H ) in vitro (9). Several reports have examined the effects of SOD on the development of embryos (10, 11, 13, 20, 31). Li et al. (10) reported that addition of 100 to 500 U/mL SOD improved rabbit embryo development in RD medium which contained 8.3 mM glucose under high oxygen tension (20 %). Umaoka et al. (31) and Noda et al. (20) reported that the addition of more than 150 U/mL SOD improved mouse embryo development in BWW medium which contained 5.56 mM glucose under high oxygen tension (20 %). Lovoni et al. (13), however, reported that the addition of 1500 U/mL SOD did not improve bovine embryo development in SOF medium which contained 1.5 mM glucose under low oxygen tension. Liu and Foote (11,12) also reported that KSOM medium supplemented with 0 to 1200 U/mL SOD and containing 0.1 mM glucose did not improve bovine embryo development under either low or high oxygen tension. Matsuyama et al. (16) reported that neither 0 to 750 ~g/mL SOD nor 5 to 50 t~g/mL catalase, or combinations of these antioxidants, could improve bovine embryo development in SOF medium which contained 1.5 mM glucose under either low or high oxygen tension. From these results, it is suggested that the positive effects of SOD on embryo development are observed only when rabbit or mouse embryos were cultured in medium cont,lning high glucose concentrations. However, in our present study, SOD improved bovine embryo development in medium supplemented with 4.5 mM glucose but not with 1.5 mM glucose under high oxygen tension (20 %). These results suggest that O~" generation is partly dependent on the glucose concentration. Moreover, in our experiments, mannitol, like SOD, had a positive effect on embryo development only in medium

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supplemented with 4.5 mM glucose. Furthermore, addition of mannitol to TCM199 medium which contains 5.56 mM glucose improved embryo development to the blastocyst stage (data not shown). These data suggest that .OH generation also occurs in media containing a high concentration of glucose and that m~nnltol scavenges free radicals. The effect of catalase, however, was different from that of SOD or m~nnitol. Nasr-Esfahani et al. (19) reported that addition of catalase to culture medium did not affect endogenous H~O2 production, although microinjection of catalase into 2cell mouse embryos inhibited endogenous H202 production. Moreover, Li et al. (10) reported that addition of catalase to the culture medium had no effect on rabbit embryo development, and suggested that this was due to the inability of catalase to cross the membrane. Although it has been reported that SOD can not cross the cell membrane (17) and that mannitol may also be unable to cross the membrane (6), our data suggest that these antioxidants affect embryo development. Contradictory results on antioxidants may be due to differences between the experimental species (cow, rabbit, mice etc), and between developmental stages when antioxidants are added to the culture media (zygote to blastocyst or 4-cell to blastocyst stage). However, the reasons for the contradictory results with respect to the role of scavengers remain to be explained. Recently, it was reported that oxidase systems such as the XOD-HXT system are a major source of free oxygen radicals in vivo, and glucose inhibits HXT metabolism (9). In our preliminary experiment, it is suggested that allopurinol, which is an inhibitor of XOD, improved embryo development when the culture medium contained glucose (data not shown). This indicates that generation of free radicals by the XOD-HXT system also occurs in bovine embryo culture systems. However, it is still not understood how glucose induces the generation of the radicals. In conclusion, the present study suggests that free oxygen radical generation is induced by high glucose concentration and that low glucose concentration is beneficial for embryo development. REFERENCES 1. Bernardi ML, Flechon JE, Delouis C. Influence of culture system and oxygen tension on the development of ovine zygotes matured and fertilized in vitro. J Reprod Fertil 1996; 106: 161-167. 2. Betterbed B, Wright RW. Development of one-cell ovine embryos in two culture media under two gas atmospheres. Theriogenology 1985; 23: 547-553.

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