Intracytoplasmic glutathione concentration and the role of β-mercaptoethanol in preimplantation development of bovine embryos

ELSEVIER

INTRACYTOPLASMIC GLUTATHIONE CONCENTRATION AND THE ROLE OF @MERCAPTOETHANOL IN PREIMPLANTATION DEVELOPMENT OF BOVINE EMBRYOS J.M. Lim’.‘, S.S. Lieu’,* and W. Hansel”2’a ‘Department

of Reproductive Biotechnology, Pennington Biomedical Research Center 2Department of Animal Science, Louisiana State University Baton Rouge, LA 70808, USA Received for publication: Accepted:

December 28, 1995 March I, 1996

ABSTRACT In vitro-matured/in vitro-fertilized bovine oocytes were cultured on cumulus cell layers in a serum-free medium (bovine embryo culture medium; BECM) supplemented with 3 mglml fatty acid-free BSA. The intracytoplasmic glutathione concentration of embryos was found to change significantly (PdO.008) during the preimplantation stages, beginning to increase at the 9- to 16-cell stage (20.7 PM/embryo) and reaching the highest (P~0.03) level at the hatched-blastocyst stage (36.7 PM/embryo). A significantly (PcO.06) lower concentration of glutathione was obtained at the 2- to 8-cell stage (7.1 PM/embryo) than at any other stage. When inseminated oocytes were cultured in BECM supplemented with different concentrations of 9-mercaptoethanol (2ME) to promote glutathione synthesis, higher (PcO.05) percentages of embryos developed to the 9- to 16-&l, morula and blastocyst stages at 96, 144 and 192 h post insemination, following the addition of 6.25 and 12.5 pM than after no supplementation with 2-ME. However, when 16-tell embryos were cultured in BECM supplemented with 6.25 and 12.5 pM of 2-ME, blastocyst formation was not significantly (PaO.9) increased. When the combined effects of 2-ME and/or cumulus cells were compared in a 2x2 factorial design, there was a significant (PcO.03) effect of 2-ME on the development of oocytes to blastocysts. The presence of cumulus cells significantly (PO.ll) interaction between 2-ME and cumulus cells. In conclusion, intracytoplasmic glutathione concentration of bovine embryos derived from in vitro-culture increases during preimplantation development. The glutathione synthesis promoter 2-ME exerts its embryotropic role on the development before the fourth cleavage, thus yielding an improvement in blastocyst formation. Key words:

bovine, embryo development, cells

glutathione, 8-mercaptoethanol.

cumulus

Acknowledgments This study was supported by a grant from the Gordon D. Cain Fund, and was approved for publication by the director of LSU Agricultural Center as manuscript number 96-l l-0006. We thank Dr. R.M. Blair, Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, LA, USA, for assistance with the glutathione assay. aCorrespondence and reprint requests.

Theriogenology 46:429439, 1996 0 1996 by Elsevier Science Inc.

0093-691X/96/$15.00 PII SO093691X(96)00165-&3

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430 INTRODUCTION

Bovine embryos derived from in vitro maturation (h/M) and in vitro fertilization (IVF) have been cultured in several systems utilizing defined media (14,15,24,26) and co-cultured with somatic cells (9,13,32,33) to enhance preimplantation development. In our in vitro culture (IVC) system based a serum-free medium and cumulus cell coculture (18) approximately 40 and 25% of inseminated oocytes developed to the 9- to 16-tell and blastocyst stages, respectively. Apparently, a considerable number of bovine oocytes cultured in vitro cease their development before the fourth cleavage. It is essential to elucidate the cause(s) of these early implantation stage embryo losses. Glutathione is one of the major factors sustaining various biological reactions occurring in the cytoplasm (3,16,19). Takahashi et al. (28) reported that the increase in intracytoplasmic glutathione concentration caused by the addition of 8-mercaptoethanol (2-ME) and cysteamine to the culture medium is beneficial for the development of 6- to 8-tell stage bovine embryos to the blastocyst stage. It was also reported (7) that a lower concentration of glutathione is found in mouse embryos derived from fertilization in vitro than in vivo, and that intracytoplasmic glutathione levels are quite different between the 2-ceII and the blastocyst stages embryos. If so, stimulation of the glutathione synthesis of in vitroderived embryos by 2-ME may promote the development of embryos to a specific stage that requires glutathione. The objective of this study was, therefore, to understand the fluctuations of intracytoplasmic glutathione concentration and the effect of addition of 2-ME to the culture medium during preimplantation development of in vitro-matured/in vitro-fertilized bovine embryos cultured on cumulus cell layers in a serum-free medium. In these experiments, we first measured intracytoplasmic glutathione concentrations of bovine embryos developed to various preimplantation stages. Then, we determined whether addition of 2-ME to the culture medium would enhance the development of either inseminated oocytes or embryos that had undergone the 4th cleavage (16-tell). Finally, we examined the combined effects of 2-ME and/or cumulus cells on embryo development, since 2-ME in the culture medium may act synergistically with co-cultured somatic cells. MATERIALS AND METHODS The basic medium assigned for maturation of oocytes was tissue culture medium (TCM)-199 with Earle’s salts buffered with 25 mM Hepes (No. 12340-014, Gibco BRL, Grand Island, NY) and supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL), 1 ug/ml estradiol-178 (NOBL Laboratories, Sioux City, IA), 3 pglml bovine FSH (NOBL Laboratories), 6 pg/ml bovine LH (NOBL Laboratories) and 25 pglml gentamycin (Sigma Chemical Co., St. Louis, MO). The basic medium used for treatment of spermatozoa and fertilization of oocytes was a modified Tyrode’s medium (22) supplemented with 0.25 mM sodium pyruvate (Gibco BRL), 6 mg/ml fatty acid-free bovine serum albumin (BSA; No. A-6003, Sigma Chemical), 15 pg/ml calcium heparin from porcine intestinal mucosa (183 USP unitslmg; H-8398, Sigma Chemical) and 25

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pg/ml gentamycin. The basic medium for culture of the embryos was a bovine embryo culture medium (BECM) consisting of 89 mM NaCI, 3.2 mM KCI, 2.0 mM CaC12.Hz0, 0.5 mM MgC12.6Hz0, 0.35 mM NaH2P04, 25 mM NaHC03, 0.5 mM sodium pyruvate, 10 mM sodium lactate, 1% Eagle basal medium amino acid solution (100x solution; Gibco BRL), 1% MEM nonessential amino acid solution (100x solution; Gibco BRL) and 3 mg/ml fatty acid-free BSA. This medium is basically the same as that described by Lim et al. (17) except that 1 mg/ml polyvinyl alcohol was replaced with 3 mglml fatty acidfree BSA. The osmolarity of BECM was within the range of 261 to 270 mOsm. All cumulus cell-enclosed oocytes used were collected in Madison, WI (BOMED, inc.) and shipped in 2-ml of maturation medium in a battery powered incubator via overnight express. Oocytes matured during transit and arrived within 24 h after exposure to maturation medium preequilibrated at 39°C in 5Oh COZ in air. At 22 to 24 h after exposure to maturation medium, cumulus cell-enclosed oocytes were washed 4 times in fertilization medium. Then ten to 12 oocytes were introduced into 5Oyl droplets of the same medium, which had previously been covered with warm mineral oil (No. M-8410, Sigma Chemical) in a 35 x 10 mm FalconTM plastic petridish (Becton and Dickinson, Lincoln Park, NJ), and placed in a COZ incubator until spermatozoa were added. One 0.5-ml straw of frozen semen collected from a Holstein bull was thawed in a 39 “C water bath and the semen were washed twice by centrifugation at 200 x g for a period of 6 min each after dilution with fertilization medium. The final sperm pellet was resuspended in the same medium to give a concentration of 2 to 3 x 10’ spermatozoa/ml. A 50-~1 sperm suspension was then introduced into the droplets containing the oocytes. The mixture was incubated at 39°C in 5% CO2 in air. Cumulus cell layers were prepared by the methods of Lim et al. (18). Briefly, cumulus cells used for co-culture were obtained by gentle pipetting of 280 to 300 oocytes matured for 22 to 24 h. After washing 3 times in TCM-199 with 10 OhFBS and antibiotics, cumulus cells were allotted to 20 wells of 4-well multidishes, each containing 0.5 ml of the same medium. The medium was changed every 4 d, and confluent cumulus cell monolayers IO to 14 d post seeding were provided for embryo culture. At 18 h post insemination, the inseminated oocytes were co-cultured on cumulus cell layers in a Nunc 4-well multidish (Nunc, Roskilde, Denmark). Each well of the multidish contained 17 to 20 oocytes in 0.8-ml BECM with various concentrations of 2-ME (No. M-7522, Sigma Chemical). According to the experimental design, oocytes 18 h post insemination were freed from cumulus cells by gently pipetting and cultured on the bottom of the culture dishes. The oocytes were dislodged from cumulus cells 48 to 60 h after insemination with a 27-gauge needle. Glutathione contents (oxidized plus reduced forms) of embryos were measured by an enzyme-linked assay as described by Tietze (31) with slight modification. Briefly, embryos were washed 3 times in Ca’* and Mg”-free PBS supplemented with 1 mg/ml

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polyvinyl alcohol and were stored in Eppendorf microtubes (18 to 22 embryos/l 0 pl) at 80 “C. After thawing at room temperature, the samples were mixed with 0.8 ml of 0.2 M phosphate buffer (pH=7.1-7.2) supplemented with 10 mM EDTA (Sigma Chemical). The embryos were then broken by ultrasonication, and 0.59 ml of deionized distilled water was added to the suspension. immediately after mixing with 100 ul of 10 mM 5,5’dithiobis 2nitrobenzoic acid (Sigma Chemical), 50 ul (1 U/50 PI) of glutathione reductase (Sigma Chemical) and 50 ul of 4.3 mM NADPH (Sigma Chemical), the absorbance was monitored at 412 nm with a spectrophotometer (UV-16OU, Shimadzu Co., Osaka, Japan). The increase in absorbance observed between 30 set and 5 min after addition of NADPH to the standard and tested samples was measured. The amount of glutathione in each sample was determined by comparison with a standard curve prepared at the same time. After adjustment for dilution, the amount was divided by the number of embryos in the sample to obtain total glutathione content per embryo. Each experiment was replicated 4 times. The intracytoplasmic glutathione concentrations and the percentages of embryos developing to each stage (2- to 8cell, 9- to 16-tell, morula and blastocyst, at 48, 96, 144 and 192 h post insemination, respectively, were subjected to analysis of variance using the general linear model SAS program (27). The significances of the main effects of embryo stage (Experiment 1), 2ME concentration (Experiments 2 and 3) and the presence of 2-ME and cumulus cells in the culture system (Experiment 4) were tested by the least squares method. In Experiment 1, embryos developed to the l-cell (inseminated oocyte with two polar bodies), 2- to 8-&l, 9- to 16cell, morula, blastocyst and hatched blastocyst stages were collected at 18, 48, 96, 144, 192 and 216 h post insemination, respectively, and intracytoplasmic glutathione concentrations were measured. The effect of addition of 6 concentrations (0, 1.5625, 3.125, 6.25, 12.5 and 25 PM) of 2-ME to BECM on the development of inseminated oocytes to the blastocyst stage was examined in Experiment 2. The effect of addition of 3 concentrations (0, 6.25 and 12.5 PM) of 2-ME to BECM on the development of 16-tell embryos obtained 96 h post insemination was examined in Experiment 3. Combined effects of the presence of cumulus cells and/or the addition of 6.25 uM of 2-ME to BECM on the development of inseminated oocytes to the blastocyst stage were examined in a 2 x 2 factorial design in Experiment 4. RESULTS Experiment 1 As depicted in Figure 1, mean glutathione concentrations of embryos developed to the l-cell (n=82), 2- to 8-tell (n=75), 9- to 16-tell (n=71), morula (n=72), blastocyst (n=75) and hatched-blastocyst (n=75) stages were 20.5, 7.1, 20.7, 19.7, 20.7 and 36.7 PM/embryo, respectively. There was a significant (P
433

40

30

a

g 2 2 20 P

T

a

-

a

a

10

0 l-cell

2-8 cell

9-16 cell

Morula

Blastocyst (BL)

Hatched BL

Figure 1. lntracytoplasmic glutathione concentration of bovine embryos developed to various preimplantation stages. Bar and error bars indicate mean values and standard errors, respectively. abCDifferent superscripts are significantly different (P
Experiment 2 There was a significant (PcO.05) effect of 2-ME concentration on development to the 9- to 16-&l, morula and blastocyst stages, but not on development to the 2- to 8cell stage. As shown in Table 1, higher (PcO.05) percentages of oocytes developed to the 9- to 16-tell (4652%) morula (3538%) and blastocyst (28-30%) stages after the addition of 6.25 and 12.5 pM than after the addition of 0 pM (36 Ohin 9- to 16-tell, 25% in morula and 17% in blastocyst) 2-ME.

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Table 1. Effect of the addition of 2-ME to BECM on the development of oocytes to the blastocyst stage No. (%)” of embryos developing to Concentrations No. of 2-8 cell 9-l 6cell Morula Blastoc st of 2-ME oocytes ! [1441b (PM) cultured [481b [961b 11921 0 77 52 (68) 28 (36)’ 19 (2qc 13 (17)c 1.5625 79 51 (65) 30 (38)cd 21 (27)’ 15 (19)* 3.125 79 55 (70) 36 (46)cd 28 (35)* 18 (23)* 6.25 76 56 (74) 39 (52)d 29 (38)d 23 (30)d 12.5 78 58 (74) 42 (54)d 34 Wd 22 (28)d 25 77 56 (73) 35 (45)cd 25 (32)cd 14 (la)* ‘Percentage of the number of embryos cultured. bNumbers in brackets indicate the time postinsemination. CdDifferent superscripts within the same column are significantly

different (PcO.05).

Experiment 3

Thirty-eight percent (144) of 381 oocytes developed to the 16-tell stage following co-culture on cumulus cell layers in BECM supplemented with 3 mglml fatty acid-free BSA. All 16-cell embryos were then pooled and randomly allotted to each treatment. In this case, there was no significant (PaO.9) effect of 2-ME concentration on development to either the morula or the blastocyst stage (Table 2).

Table 2.

Effect of the addition of 2-ME to BECM on the development of 16-tell embryos to the blastocyst stage No. (%)’ of embryos developing to Concentrations of No. of Blastocyst Morula 2-ME embryos cultured (clM) Wlb 0 48 35 (73) 6.25 48 35 (73) 12.5 48 35 (73) ‘Percentage of the number of embryos cultured. bNumbers in brackets indicate the time post-insemination.

WI 33 (69) 33 (69) 29 (60)

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Experiment 4 There were significant (PcO.03) effects of both cumulus cells and 2-ME at each developmental stage, except the 2- to 8-4~11 stage. As shown in Table 3, 2-ME significantly (PcO.03) affected the development of oocytes to the 9- to 16-tell, morula and blastocyst stages, and the presence of cumulus cells significantly (PO.ll) interaction between 2-ME and cumulus cells on development. More (PdO.04) 9- to 16-tell embryos were obtained after addition of 6.25 pM of 2-ME than after no supplementation, in both the presence (53 vs 34%) and the absence (51 vs 39%) of cumulus cells. When 2-ME was added to BECM, similar proportions of oocytes developed to the 9- to 16-tell stage in cumulus-free and cumulus-containing culture milieu. On the contrary, a’ higher (PcO.04) percentage of embryos developed to blastocysts in the presence (20 to 32%) than in the absence (7 to 12%) of cumulus cells, regardless of the presence or the absence of 2-ME in the culture medium.

Table 3.

Combined effects of cumulus cells and/or 2-ME on the development of oocytes to the blastocyst stage Treatments No. (%)” of embryos developing to No. of Blastocyst 2-8-cell 9-16-&l Morula Cumulus 2-ME oocytes [192]” [1441b cells (6.25 PM) cultured Wlb [481b Presence + 76 56 (74) 34 (4qc 40 (53)c 24 (32)’ 76 54 (71) 21 (28)d 15 (20)d 26 (34)d Absence

+

69 72

50 (72) 51 (71)

35 (51)G 28 (39)d

13 (19)de 9 (13)e

8 (12)e 5 ( 7)=

aPercentage of the number of embryos cultured. bNumbers in brackets indicate the time post-insemination. CdeDifferent superscripts within the same column are significantly

different (PcO.05).

DISCUSSION The results of this study clearly demonstrate that intracytoplasmic glutathione concentration changes during preimplantation development when IVMIVFderived bovine oocytes are co-cultured on cumulus cell layers in a serum-free medium containing BSA. Furthermore, activation of glutathione synthesis by treatment with 2ME is beneficial for the development of oocytes up to the third cleavage. However, morula compaction and blastocyst formation were not promoted when 16-tell embryos were treated with 2-ME.

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De novo synthesis of glutathione begins to increase (9- to 16cell stage) at the time of genomic activation of bovine embryos (29). Increase in glutathione synthesis may cause loss of embryotropic activity of 2-ME added at later preimplantation stages, as shown in Table 2. The reduced form of glutathione (GSH) is the major form present in embryo cytoplasm (7) and GSH augments the ability of the embryo to eliminate cytotoxic hydrogen peroxide at the time of genomic activation in mouse embryos (21). Furthermore, our data suggest that glutathione, in addition to its role as an antioxidant, may play an important intracellular role at specific stages in bovine embryo development. In the mouse, glutathione may be involved in various embryonic events, including cell proliferation and differentiation at later preimplantation stages, and may participate in energy-generating metabolism as a constituent of co-enzymes. It has been reported that the physiological action of glutathione involves promotion of male pronucleus formation in hamster (23) mouse (2) and pig (20,34,35) oocytes and preimplantation development in bovine (26) and pig (11) embryos. Glutathione also has an important role in the thermotolerance of preimplantation murine embryos (1). The fact that treatment of inseminated oocytes with 2-ME significantly enhances development before the 16-cell stage and not afterward implies that embryos can synthesize adequate amounts of glutathione after activation of the embryonic genome. Takahashi et al. (28) reported that treatment of bovine embryos with 2-ME significantly increases intracytoplasmic glutathione contents. In our preliminary experiment, we also found that higher glutathione levels were detected in 2- to 8-tell embryos treated with 2-ME than in untreated 2- to 8-tell embryos (26.1 vs 7.1 PM/embryo, respectively). In mouse embryos, blastocysts have the capacity to synthesize glutathione, but embryos developing from the 2-tell to the morula-stage have a limited ability to synthesize glutathione (8). Instead, early stage embryos accumulate large amounts of glutathione, which originates from the oocytes. The fluctuation of glutathione concentration in bovine embryos was partly consistent with that in mouse embryos, since a higher level of glutathione concentration in bovine embryos was found at the l-cell than the 2- to 8cell stage. However, the results of our study demonstrate that the biosynthesis of glutathione in the bovine embryo is quite different from that in the mouse. Perhaps, bovine embryos utilize mRNAs originating from both maternal and embryonic genomes for synthesizing glutathione. Our findings are supported by the report that preimplantation cow embryos derived from IVM/IVF express mRNAs for glutathione peroxidase and glutamylcysteine synthetase (12). It is not clear whether or not, compared with any other stage up to the hatched blastocyst, the significantly lower amount of glutathione found at the 2- to 8-cell stage represents a physiological level, since we did not examine in vivo-derived embryos. However, our results on the ability of 2-ME to promote development before the fourth cleavage indicate that low intracytoplasmic glutathione concentration may be one of the reasons for the 8-tell developmental block, which occurs in in vitro-cultured bovine embryos. This hypothesis is supported by the fact that development of l-cell pig embryos to the 8cell stage is significantly enhanced by addition of cysteamine, which also promotes glutathione production (1 I), and that GSH levels were lower in embryos

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cultured in vitro than in embryos developed in vivo (7). Conversely, the finding that there is no difference in the developmental capacity of oocytes cultured either with cumulus cell layers or in 2-ME-containing medium to the 9 to 16-tell stage, suggests that glutathione and other defined medium ingredients are capable of fully supporting development to this stage. Activation of glutathione synthesis by treatment with 2-ME is not beneficial, however, to the formation of blastocysts from 16cell embryos (Table 2). Furthermore, small numbers of 9- to 16cell embryos developed to the blastocyst stage even though they were cultured in 2-ME-supplemented medium under cumulus cell-free conditions (Table 3). In contrast to the development of oocytes up to the third cleavage, blastocyst formation of embryos that have completed the fourth cleavage are not stimulated by addition of 2-ME to the culture medium. Additionally, a number of factors such as growth factors, inorganic salts, glycoproteins and macromolecules may affect morula and blastocyst formation. The optimal range (6.25 to 12.5 pM) of 2-ME concentrations (Table 1) was different from that (50 PM) reported by Takahashi et al. (28). This may be related to different compositions of the culture media (TCM-199 supplemented with 10% FBS vs BECM supplemented with 3 mg/ml BSA) and to the presence of cumulus cells. We also used a serum-free BECM, which has less NaCl than TCM-199. The NaCl concentration in culture medium affects intracellular concentration of glutathione in pig oocytes (5). There are 2 major actions of somatic cells to support embryo development: one is to secrete various mitogens including growth factors (30) and macromolecules (6), and the other is to remove cytotoxic products such as reactive oxygen radicals (4,10) and carbohydrate (18) from the culture medium. We assume that one of the beneficial effects of co-culture on cumulus cell layers is the production of antioxidants such as glutathione. This suggestion is supported by the finding that co-cultured reproductive tissue cells contain several kinds of mRNA for synthesis of antioxidants (12). However, the effect of 2-ME on embryo development is independent of the action of cumulus cell glutathione secretion, since there was no interaction between the two (Table 3). Furthermore, it is questionable whether most cells can take up the intact form of glutathione from the medium (25). REFERENCES I. Arechiga CF, Ear-y AD, Hansen PJ. Evidence that glutathione is involved in thermotolerance of preimplantation murine embryos. Biol Reprod 1995; 52:12961301. 2. Calvin HI, Grosshans K, Blake EJ. Estimation and manipulation of glutathione levels in prepubertal mouse ovaries and ova: relevance to sperm nucleus transformation in the fertilized egg. Gamete Res 1986; 14:265-275. 3. Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev 1979; 59:527-605.

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Theriogenology

4. Flood LP, Shirley 8. Reduction of embryo toxicity by protein in embryo culture media. Mol Reprod Dev 1991; 30:228-231. 5. 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; 51:633639. 6. Gandolfi F, Brevini TAL, Richardson L, Brown CR, Moor RM. Characterization of proteins secreted by sheep oviduct epithelial cells and their function in embryonic development. Development 1989; 106:203-213. 7. Gardiner CS, Reed DJ. Status of glutathione during oxidant-induced oxidative stress in the preimplantation mouse embryo. Biol Reprod 1994; 51: 1307-l 314. 8. Gardiner CS, Reed DJ. Synthesis of glutathione in the preimplantation mouse embryo. Archi Biochem Biophys 1995; 318:30-36. 9. Goto K, Kajihara Y, Kosaka S, Koba M, Nakanishi Y, Ogawa K Pregnancies after w-culture of cumulus cells with bovine embryos derived from in-vitro fertilization of in vitro-matured follicular oocytes. J Reprod Fertil 1988; 83:753-758. 10. Goto Y, Noda Y, Mori T, Nakao M. Increased generation of reactive oxygen species in embryos cultured in vitro. Free Radical Biol Med 1993; 15:69-75. 11. Grupen CG, Nagashima H, Nottle MB. Cysteamine enhances in vitro development of porcine oocytes matured and fertilized in vitro. Biol Reprod 1995; 53: 173-178. 12. Harvey MB, Arcellana-Panlilio MY, Zhang X, Schultz GA, Watson AJ. Expression of gene encoding antioxidant enzymes in preimplantation mouse and cow embryos and primary bovine oviduct cultures employed for embryo coculture. Biol Reprod 1995; 53:532-540. 13. Heyman Y, Menezo Y, Chesne P, Camous S, Gardier V. In vitro cleavage of bovine and ovine early embryos: improved development using coculture with trophoblastic vesicles. Theriogenology 1987; 27:59-68. 14.Keskintepe L, Burnley CA, Brackett BG. Production of viable bovine blastocysts in defined in vitro conditions. Biol Reprod 1995; 52: 141 O-l 417. 15.Kim JH, Niwa K, Lim JM, Okuda K Effects of phosphate, energy substrates and amino acids on development of in vitro-matured and in vitro-fertilized bovine oocytes in a chemically defined, protein-free culture medium. Biol Reprod 1993; 48:1320-l 325. 16. Kosower NS. The glutathione status of cells. Int Rev Cytol 1978; 54:109-160. 17.Lim JM, Okitsu 0, Okuda K, Niwa K Effects of fetal calf serum in culture medium on the development of bovine oocytes matured and fertilized in vitro. Theriogenology 1994; 41:1091-1098. 18. Lim JM, Rocha A, Hansel W. A serum-free medium for use in a cumulus-cell coculture system for bovine embryos derived from in vitro-maturation and in vitrofertilization. Theriogenology 1996; 45: 1081-l 089. 19. Meister A, Anderson ME. Glutathione. Ann Rev Biochem 1983; 52:1 l-60. 20.Naito K, Toyoda Y. Effects of microinjection of glutathione on male pronucleus formation in porcine oocytes matured in vitro. J Reprod Dev 1992; 38:173-178.

Theriogenology

439

21. Nasr-esfahani MH, Aitken JR, Johnson MH. Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developed in vitro or in vivo. Development 1990; 109501-507. 22. Parrish JJ, Susko-Parrish JL, Winter WA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod 1988; 38: 1171-l 180. 23. Perreault SD, Barbee RR, Slott VL. Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in maturing hamster oocytes. Dev Biol 1988; 125: 181-l 86. 24. Pinyopummintr T, Bavister BD. In vitro-matured/in vitro-fertilized bovine oocytes can develop into morulaelblastocysts in chemically defined, protein-free culture media Biol Reprod 1991; 45736-742. 25Reed DJ. Mechanisms of chemically induced cell injury and cellular protection mechanisms. In: Hodgson E, Levi PE (eds), Introduction to Biochemical Toxicology. Norwalk, CT: Appleton & Lange; 1994; 265295. 26.Rosenkrans CF Jr, Zeng GQ, McNamara GT, Schoff PK, First NL. Development of bovine embryos in vitro as affected by energy substrates. Biol Reprod 1993; 49:459-462. 27.SAS. SAS User’s Guide, Statistics, Cary, NC: Statistical Analysis System Institute Inc; 1992. 28.Takahashi M, Nagai T, Hamano S, Kuwayama M, Okamura N, Okano A. Effect of thiol compounds on in vitro development and intracellular glutathione content of bovine embryos. Biol Reprod 1993; 49:228-232. 29.Telford NA, Watson AJ, Schultz GA. Transition from maternal to embryonic control in early mammalian development: a comparison of several species. Mol Reprod Dev 1990; 26:90-l 00. BO.Thibodeaux JK, Del Vecchio RP, Hansel W. The role of platelet derived growth factor (PDGF) in development of early bovine embryos. J Reprod Fertil 1993; 98:6166. 31.Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Application to mammalian blood and other tissues. Analy Biochem 1969; 27:502-522. 32.Voekle SA, Amborsk GF, Hill KG, Godke RA. Use of uterine cell monolayer culture system for micromanipulated bovine embryos. Theriogenology 1985; 24:271-281. 33.Xu KP, Yadav BR, Rorie RW, Plante L, Betteridge KJ, King WA. Development and viability of bovine embryos derived from oocytes matured and fertilized in vitro and w-cultured with bovine oviductal epithelial cells. J Reprod Fertil 1992; 94:33-43. 34.Yoshida M. Role of glutathione in the maturation and fertilization of pig oocytes in vitro. Mol Reprod Dev 1993; 35:76-81. 35.Yoshida M, lshigaki K, Nagai T, Chikyu M, Purse1 VG. Glutathione concentration during maturation and after fertilization in pig oocytes: relevance to the ability of oocytes to form male pronucleus. Biol Reprod 1993; 49:89-94.