In Vitro Fertilization of Bovine Oocytes in a Chemically Defined ...

BIOLOGY OF REPRODUCTION 50, 1231-1237 (1994)

In Vitro Fertilization of Bovine Oocytes in a Chemically Defined, Protein-Free Medium Varying the Bicarbonate Concentration1 PARVIZ TAJIK, WEI-HUA WANG, KIYOSHI OKUDA, and KOJI NIWA 2 Division of Animal Science and Technology, Faculty of Agriculture, Okayama University Okayama 700, Japan ABSTRACT Bovine cumulus-enclosed oocytes were matured in culture, freed from cumulus cells, and inseminated with frozen-thawed spermatozoa in a chemically defined protein-free medium containing 5 mM caffeine and 10 ljg/ml heparin. No penetration of oocytes was observed in the medium without polyvinylalcohol (PVA); but when the medium was supplemented with 0.1-5 mg/ ml PVA, penetration rates (9-16%) significantly increased. Sperm motility was also stimulated during incubation for 2 h in the presence of PVA. In the medium with 1 mg/ml PVA, a high penetration rate (24 of 62 = 39%) was observed at a sperm concentration of 10 x 106 cells/mi. When the bicarbonate concentration was changed in the fertilization medium containing I mg/ml PVA and 10 x 106 spermatozoa/ml, a high penetration rate (47 of 67 = 70%) and a high proportion (44 of 47 = 94%) of oocytes in which male and female pronuclei had developed were obtained at 46 mM NaHCO3. However, the penetration rate (58-95% ), the incidence of pronuclear formation (64-96% ), and the proportion of polyspermy (9-21% ) varied according to the animal (five different bulls). Spermatozoa obtained from two bulls started to penetrate oocytes 5 h after insemination in the presence of 46 mM NaHCO 3. This is the first report indicating that induction of capacitation of bull spermatozoa and penetration of oocytes matured in culture are possible in a chemically defined, protein-free medium.

INTRODUCTION Various chemicals such as caffeine [1, 2], heparin [3, 4], calcium ionophore A23187 [5], and caffeine plus heparin [6] or caffeine plus calcium ionophore A23187 [7] have been used to induce capacitation of bovine spermatozoa in vitro. However, BSA is always included in media used for in vitro fertilization in these experiments. Although proteinaceous macromolecules, especially BSA, may play an important role in inducing capacitation and the acrosome reaction of bovine spermatozoa [5], it has been shown that penetration in vitro of hamster [8], mouse [9], and bovine [10] oocytes is possible in medium without protein when cumulus-enclosed oocytes are inseminated. On the other hand, polyvinylalcohol (PVA) has been reported to decrease the spontaneous acrosome reaction in hamster [11] and mouse [9] spermatozoa. In cattle, it has been reported that PVA added to medium containing heparin can stimulate sperm capacitation but barely support the fertilization of cumulus-free oocytes compared to BSA [12]. Since both caffeine and heparin are known to induce capacitation of bovine spermatozoa, efficacy with respect to the penetration of oocytes may be dependent upon the role of cumulus cells and protein supplements. However, it is difficult to clarify which factor(s) can induce capacitation and affect the penetration of oocytes by sperm because many unknown factors are present in the cumulus mass and proteins. For a better understanding of penetration in vitro of bovine oocytes, it is Accepted January 26, 1994. Received November 29, 1993. 'This work was partly supported by a grant from the Ito Foundation, Tokyo, Japan. 2 Correspondence. FAX: (086) 254-0714.

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necessary to develop a chemically defined condition in which a high incidence of oocyte penetration occurs. Many other factors affect penetration of oocytes. Sperm concentration has been shown to be important for penetration of rat [13], pig [14], and human [15] oocytes. Bicarbonate has been reported to be essential for fertilization of mouse oocytes and suggested to be necessary for the Ca 2+ influx into spermatozoa to induce the acrosome reaction [16]. Bicarbonate also acts to increase the intracellular pH of caput and caudal bovine spermatozoa [17] and therefore is effective for maintaining sperm motility, which is one of the important factors supporting penetration of spermatozoa into oocytes [18]. The present study was performed to examine whether in vitro-matured bovine oocytes could be penetrated by spermatozoa in a chemically defined protein-free medium and to optimize conditions in the medium including the presence of PVA and the concentrations of PVA, spermatozoa, and bicarbonate. MATERIALS AND METHODS Media The medium used for maturation of oocytes was tissue culture medium (TCM) 199 with Earle's salts buffered with 25 mM Hepes and supplemented with 10% (v/v) heat-treated fetal calf serum (FCS; Gibco Labs., Life Technologies, Inc., Chagrin Falls, OH), 100 IU/ml penicillin G, and 100 $zg/ ml streptomycin sulfate (pH 7.4). The basic medium used for the treatment of spermatozoa and the fertilization of oocytes was essentially the same as that used by Brackett and Oliphant [19] for the fertilization of rabbit oocytes in vitro, except that BSA, phenol red, and penicillin G were

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TAJIK ET AL. TABLE 1. Effect of PVA on penetration in vitro of cumulus-free bovine oocytes matured in culture.' No. of oocytes penetrated PVA concentration (mg/ml)

No. of oocytes examined

Total (%)

Witt I swolli en sperm thead

With male and fem ale pronuclei

No. of polyspermic oocytes

0 5 5 9 7 5

0 0 0 0 0 0

0

62

0 ( 0 )b

0

0.1 0.5 1 3 5

65 66 63 64 62

6( 9)' 6(9) c 10(16)' c 8(13) 6(10)'

1 1 1 1 1

8

Experiments were repeated five times. bValues with different superscripts differ significantly (p < 0.05).

not added. This medium, designated BO medium, consisted of 112.0 mM NaCl, 4.02 mM KCI, 2.25 mM CaCI 2, 0.83 mM NaH 2 PO 4, 0.52 mM MgCl 2, 37.0 mM NaHCO 3, 13.9 mM glucose, and 1.25 mM sodium pyruvate (pH 7.8). The medium used for the culture of penetrated oocytes was a modified Tyrode's solution composed of 110.0 mM NaCl, 3.2 mM KCI, 2.0 mM CaC12, 0.5 mM MgC12, 0.35 mM NaH 2PO4, 25.0 mM NaHCO 3, 10.0 mM sodium lactate, 0.5 mM sodium pyruvate, 1 mg/ml PVA, and 1% (v/v) each of Basal Medium Eagle's amino acid solution (Gibco Labs., Gaithersburg, MD) and Minimal Essential Medium nonessential amino acid solution (Gibco Labs.). We have reported that 19-24% of bovine oocytes fertilized in an undefined condition and cultured in this chemically defined medium developed to the blastocyst stage by 192 h postinsemination [20]. Preparationof Oocytes Ovaries were removed from cows at a local slaughterhouse and were transported to the laboratory in 0.9% NaCI solution at 30-35°C within 2 h. Cumulus-oocyte complexes were aspirated from follicles 3-5 mm in diameter with a 24-gauge needle attached to a disposable syringe and were washed four times with maturation medium. Ten oocytes with compact cumulus cells were transferred into a 0.1-ml drop of the same medium under warm paraffin oil (no. 261-17, Nacalai Tesque, Inc., Kyoto, Japan) in a polystyrene culture dish (35 x 10 mm) that had been previously kept for about 2 h in a CO2 incubator; oocytes were cultured at 39°C in 5% CO2 in air. After culture for 22-24 h, the oocytes were freed from cumulus and corona cells by treatment with Dulbecco's PBS containing 0.1% hyaluronidase from bovine testes (Sigma Chemical Co., St. Louis, MO) for 10-20 min and by repeated passage through a fine pipette. Polyvinylpyrrolidone (10 mg/ml; PVP-40, Sigma) was added to PBS to prevent adhesion of denuded oocytes to the inner surface of the glass pipette during treatment. Only denuded oocytes with a polar body were washed twice and transferred into a 50-pl drop of BO medium containing 20 pg/ ml pig intestinal mucosal heparin (177 United States Pharmacopeia U/ml, Sigma) and 0-10 mg/ml (experiment 1) or 2 mg/ml (experiments 2-6) PVA under paraffin oil in a

culture dish. The dishes were kept in a CO2 incubator (5% CO2 in air at 39°C) for about 30 min until spermatozoa were added for fertilization. Preparationof Sperm After two 0.5-ml straws of frozen bull semen were thawed in a water bath at 35-37 0C, spermatozoa were washed twice in BO medium supplemented with 10 mM caffeine-sodium benzoate (Sigma) by centrifugation at 833 x g, 10 min for each wash [1]. After washing, spermatozoa were resuspended in the same medium to give a sperm concentration of 2 x 106 (experiment 1), 2-40 x 106 (experiment 2), or 20 x 106 (experiments 3-6) cells/mi. In Vitro Fertilization and Examination of Oocytes A 50-ptl sample of the final sperm suspension containing 10 mM caffeine was added to medium that contained 20 Cg/ml heparin and various concentrations of PVA and included 10-15 cumulus-free oocytes. The mixture always gave final concentrations of 10 ptg/ml heparin and 5 mM caffeine. At 20-24 h after insemination, oocytes were mounted,, fixed for 48-96 h in 25% (v/v) acetic alcohol at room temperature, stained with 1% (v/v) orcein in 45% (v/v) acetic acid, and examined for evidence of sperm penetration as described previously [21]. In one case (experiment 6), 20 oocytes were cultured to study embryonic development. ExperimentalStudies Since there is no information about the effect of PVA concentration on in vitro penetration of cumulus-free bovine oocytes, this question was examined in experiment 1 using spermatozoa from one bull (P-52-18). For observation of sperm motility, a sperm suspension (100 p.l) was prepared in a culture dish by the same procedures as used for in vitro fertilization. At 0-8 h (at 1-h intervals) and at 20 h after the sperm suspension was made, sperm motility was estimated in a small drop of sperm suspension under a cover glass via a brightfield microscope (x 100) equipped with a microwarm plate (39°C; MP 10-DM, Kitazato Supply, Co., Shizuoka, Japan).

PENETRATION OF BOVINE OOCYTES IN A DEFINED MEDIUM

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FIG. 1. An oocyte penetrated 20 h after insemination in a chemically defined, protein-free medium supplemented with 1 mg/ml PVA, showing male (MP) and female (FP) pronuclei with a penetrating sperm midpiece (arrow) (a) and second polar body (PB) in a different focal plane (b). x304.

In experiment 2, the effect of sperm concentration on penetration of cumulus-free oocytes was examined in the presence of a fixed concentration (1 mg/ml) of PVA. The oocytes had been previously introduced into 50 pI of BO medium containing 20 RIg/ml heparin and 2 mg/ml PVA; then 50 Rli of sperm suspension (2-40 x 106 spermatozoa/ ml) from one bull (P-52-18) was added for insemination. In experiment 3, the effect of different concentrations of bicarbonate on penetration in vitro of oocytes was examined. Cumulus-free oocytes were introduced into 50 l of BO medium containing 20 jig/ml heparin, 2 mg/ml PVA, and concentrations of bicarbonate that were adjusted to 31, 37, 43, 49, 55, or 61 mM. The calculated osmolarity of the

60 o 50

- PVA + PVA

40

:.

30

C

20 10 0 0

5

10

15

medium was adjusted to 330 mOsm by changing the concentration of NaCl. Then 50 l1of sperm suspension (bull P-52-18) was added for insemination to give a final sperm concentration of 10 x 106 cells/mi. Thus, the final concentration of NaHCO 3 in the fertilization medium was 34, 37, 40, 43, 46, or 49 mM. The final concentrations of caffeine, heparin, and PVA were 5 mM, 10 g/ml, and 1 mg/ml, respectively. The pH of the medium at introduction of oocytes was 7.8 0.1. In experiment 4, the ability of spermatozoa from five different bulls to penetrate cumulus-free oocytes in vitro was compared. The final concentrations of NaHCO 3, PVA, and spermatozoa in fertilization medium were adjusted to 46 mM, 1 mg/ml, and 10 x 106 cells/ml, respectively. In experiment 5, to estimate the time necessary for inducing sperm capacitation under the same experimental conditions as in experiment 4, the time of sperm penetration into cumulus-free oocytes and of pronuclear formation was examined using spermatozoa obtained from two bulls, P-51-5 and B-36. In experiment 6, to examine whether penetrated oocytes could develop to the blastocyst stage in vitro, oocytes inseminated with spermatozoa from bull P-51-5, under the same experimental conditions as in experiments 4 and 5, were transferred into a chemically defined modified Tyrode's solution (0.5 ml) 8 h after insemination and cultured for 184 h at 390°C in 5% CO2 in air as reported previously [20].

Time of incubation (h) FIG. 2. Time course of changes in motility of bull spermatozoa incubated in a chemically defined, protein-free medium with or without 0.1-5.0 mg PVA/ml. Since sperm motility at each time after incubation was not different in relation to the different concentrations of PVA, the data obtained at each concentration of PVA were combined. All values are mean + SD (n = 5) for each time point.

StatisticalAnalysis In experiments 1, 2, 3, 4, and 5, the proportions of total oocytes penetrated, pronuclear oocytes, and polyspermic oocytes were subjected to an arc-sine transformation; the transformed values were assigned for one-way ANOVA. When

1234

TAJIK ET AL. TABLE 2. Effect of sperm concentration on penetration in vitro of cumulus-free bovine ooytes matured in culture.' No. of oocytes penetrated With swollen sperm head

With male and female pronuclei

No. of polyspermic oocytes

1 2(19 )b

1

11

0

16(27) c 24(39) 19(32) c

1 0 0

15 24 19

0 0 0


Total (%)

1

64

5 10 20

59 62 60

Sperm concentration (x 106 /ml)

b

'Fertilization medium contained 1 mg/ml PVA. Experiments were repeated five times. b'Values with different superscripts differ significantly (p < 0.05).

from values obtained at 5 and 20 x 106 cells/mi. As observed in experiment 1, no polyspermic penetration was obtained in this experiment.

ANOVA revealed a significant effect, values were compared by Duncan's Multiple Range test. RESULTS

Experiment 3: Effect of Concentration of Bicarbonate

Experiment 1: Effect of PVA None of the oocytes were penetrated when PVA was not included in the medium (Table 1).When the medium was supplemented with 0.1-5.0 mg/ml PVA, however, penetration rates significantly (p < 0.05) increased, although only up to 16% of oocytes were penetrated and most of them contained two pronuclei (Fig. 1). The highest observed rate, 16%, was obtained with 1 mg/ml PVA, but this value was not significantly different from values obtained with all other concentrations of PVA. No polyspermic penetration was seen in this experiment. When the sperm suspension was prepared in the medium with 0.1-5.0 mg/ml PVA, the proportion of motile spermatozoa increased until 2 h after insemination but gradually decreased with further incubation (Fig. 2). In contrast, sperm motility was completely lost within 2 h of incubation in the medium without PVA.

Oocyte penetration rates increased as the bicarbonate concentration increased (Table 3). The value (70%) at 46 mM NaHCO 3 was significantly (p < 0.05) higher than the values at 34-43 mM NaHCO 3 but not different from that at 49 mM NaHCO 3. Pronuclear formation was observed in a high proportion of oocytes penetrated. Comparatively higher proportions of polyspermy were observed at 40-46 mM than at 34-37 or 49 mM NaHCO3 , but there were no significant differences among these values. Experiment 4: Effect of Different Bulls

Experiment 2: Effect of Sperm Concentration As shown in Table 2, the proportion of oocytes penetrated increased significantly (p < 0.05) when sperm concentration increased from 1 (19%) to 10-20 (32-39%) x 106 cells/ml. A high penetration rate (39%) was obtained at 10 x 106 cells/ml, but this was not significantly different

The proportions (58-95%) of oocytes penetrated varied according to the bull, independently of the difference in sperm motility (Table 4). Spermatozoa from two bulls (P52-18 and P-173) had significantly (p < 0.05) less ability to penetrate cumulus-free oocytes than sperm from three others (P-51-5, B-36, and B-38). The proportion of oocytes penetrated and containing male and female pronuclei (6496%) also varied according to the bull (p < 0.05), but this variation was independent of the difference in penetration rates. However, the proportion of polyspermic oocytes (921%) was not significantly different among the five bulls.

TABLE 3. Effect of concentration of bicarbonate on penetration in vitro of cumulus-free bovine ooytes.

No. of

No. of oocytes penetrated Bicarbonate concentration (mM)


Total (%)

With swollen sperm head

a

With male and female pronuclei (%)b

polyspermic oocytes (%)b

34

67

12(18)'

1

11(92)

1( 9)

37 40 43 46

61 65 67 67

23 (3 8 )d de 26 (4 5 )de

2 2 4 3

21(91) 24(92) 27(87) 44(94)

1( 4) 3(12) 4(13) 8(17)

49

62

31 (4 6 )

4 7 (7 0 )'

3( 8) 34(92) 3 37(60) 'Medium contained 1 mg/ml PVA and final sperm concentration was 10 x 106 cells per milliliter. Experiments were repeated five times. bPercentage of the total number of oocytes penetrated. cdeValues with different superscripts differ significantly (p < 0.05).

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PENETRATION OF BOVINE OOCYTES IN A DEFINED MEDIUM TABLE 4. Penetration in vitro of cumulus-free bovine oocytes by frozen-thawed ejaculated spermatozoa obtained from 5 different bulls.'

Bull no.

Progressively motile spermatozoa at insemination %)


Total (%)

No. of oocytes penetrated With With male swollen and female sperm head pronuclei (%)b

P-52-18

20

76

53(70) c

P-51-5

30

63

57(90)

P-173 B-36 B-38

35

60

35(58) c

11

25

57

54(95)

2

30

61

56(92)

d

d

d

No. of polyspermic oocytes (%)b

4

49(92)d

9(17)

3

54(95)

7(12)

c

24(69) d' 52(96)

20

3( 9)

C

10(18)

36(64)'

12(21)

'Medium contained 1 mg/ml PVA and concentration of bicarbonate was adjusted to 46 mM. Final sperm concentration was 10 x 106 cells per milliliter. Experiments were repeated four times. bPercentage of the total number of oocytes penetrated. Cd'Values with different superscripts within each column differ significantly (p < 0.05 or higher).

Experiment 5: Time of Sperm Penetration As shown in Table 5, spermatozoa started to penetrate cumulus-free oocytes by 5 h after insemination. The first formation of male and female pronuclei was observed 2 h after the start of sperm penetration. Experiment 6 Development of Penetrated Oocytes in a Chemically Defined Medium In one trial, 4 (20%) of 20 oocytes cultured had developed to the blastocyst stage at 192 h after insemination. The appearance of these blastocysts was morphologically normal (Fig. 3). DISCUSSION The results of the present study indicate that a protein supplement in fertilization medium containing caffeine and heparin is not required for in vitro penetration of in vitromatured cumulus-free bovine oocytes when the medium contains PVA. According to Bavister [11], PVA can maintain motility of hamster spermatozoa but cannot support the fertilizing ability of spermatozoa in vitro. The inability of PVA to support fertilization of oocytes in vitro has also been

reported in mice [9] and cattle [12]. However, in the present study using a chemically defined medium, the penetration rate was substantially modified when concentrations of spermatozoa and bicarbonate were moderately adjusted. Therefore, these may be among the more important factors involved in inducing sperm capacitation and maintaining the fertilizing ability of spermatozoa in the chemically defined conditions used in the present study. In our previous study [10], very high proportions (9098%) of cumulus-intact bovine oocytes were penetrated in a medium with BSA containing 1.0-1.7 x 106 spermatozoa/ ml. However, in the present study, the penetration rate of cumulus-free oocytes was significantly increased at the sperm concentration of 10-20 (32-39%) compared to 1 (19%) x 106 cells/ml in the medium with PVA instead of BSA, although penetration was still much lower with PVA than with BSA. Why these higher sperm concentrations are necessary for maintaining the fertilizing ability of spermatozoa in the chemically defined condition is not known at present. Although we did not examine the acrosome reaction of spermatozoa in the present study, it has been suggested by others that the incidence of the spontaneous acrosome reaction in hamster [11] and mouse [9] spermatozoa decreases in a

TABLE 5. Time of penetration in vitro of cumulus-free bovine ooytes by spermatozoa. 8 Time of examination (h after insemination)

No. of oocytes penetrated With With male swollen and female sperm head pronuclei (%)b


Total (%)

1 2 3 4 5 6

57 50 52 53 55 69

0( 0)c 0( 0)c 0( 0) 0( 0) 15(27)d 37(54)e

0 0 0 0 15 37

7

51

4 0(78)

36

8 22

58 50

4 8 (8 3 )f 45(90)'

f

27 5

0( 0) 0( 0) 0( o) 0( 0) 0( 0) 0( 0)C 4 (1 0)d

21(44) e 40 (89 )f

No. of polyspermic oocytes (%)b

0( 0) 0( 0) 0( 0) 0( 0) 0( 0)c 1( 3)cd 5(13)d 1( 2)cd 5(11)d

'Spermatozoa from 2 bulls, P-51-5 and B-36, were used and experiment was repeated twice for each bull. Since similar results were obtained for each bull, the data were combined. Experimental conditions were completely the same as in Table 4. bPercentage of the total number of oocytes penetrated. cdfValues with different superscripts within each column differ significantly (p < 0.05 or higher).

1236

TAJIK ET AL.

FIG. 3. A blastocyst obtained 184 h after culture in a chemically defined culture medium for bovine oocytes that were matured in vitro, inseminated with spermatozoa (10 x 106 cells/ml) in a chemically defined medium containing 5 mM caffeine, 10 g/ml heparin, 1 mg/ml PVA, and 46 mM NaHCO3, and transferred into the culture medium 8 h after insemination. x280.

medium with PVA. Therefore, it seems that the number of acrosome-reacted spermatozoa increases under a higher sperm concentration by which the penetration rate increases in the chemically defined condition. Bicarbonate has been found to play an important role in hyperactivation of mouse [22], guinea pig [23], and hamster [24] spermatozoa. Hyne [25] reported that the range of extracellular pH needed for guinea pig spermatozoa to continue undergoing the acrosome reaction is narrow and assumed a role for bicarbonate as a medium-buffering component. However, in a constant pH condition, the concentration of bicarbonate may be critical for supporting the acrosome reaction [22]. Therefore, it is postulated that bicarbonate has a role or roles other than as a pH-buffering molecule in maintaining the penetrability of spermatozoa. In the present study, little change of extracellular pH (7.77.9) was observed in the presence of 34-49 mM bicarbonate. Nevertheless, the proportion of oocytes penetrated increased as the concentration of bicarbonate increased. This may indicate that a role for bicarbonate as a pH-buffering factor is not important, but that the increased intracellular pH due to a higher concentration of bicarbonate can stimulate the acrosome reaction and the penetrability of bull spermatozoa. Since we did not measure the intracellular pH of bull spermatozoa in the present study, further experiments are necessary to clarify the role(s) of bicarbonate in fertilization of bovine oocytes. The variations observed in the incidence of polyspermy among different concentrations of NaHCO3 and among an-

imals were not statistically significant and were independent of the difference in penetration rates. It has also been reported in cumulus-free and cumulus-intact bovine oocytes that there is no relation between penetration rate and polyspermy in medium with BSA [10]. At present, the factors that affect polyspermy are not clearly defined. Xu and Greve [26] have reported that it takes fresh or frozen-thawed ejaculated bovine spermatozoa, treated with heparin, 6 h to start penetrating oocytes. Although Parrish et al. [12] have reported that glycolyzable monosaccharides delay capacitation of bovine spermatozoa, the data obtained in our laboratory have shown that when bull spermatozoa are treated with caffeine and heparin in a medium containing BSA and glucose, they start to penetrate cumulusintact and cumulus-free bovine oocytes 3 and 1 h after insemination, respectively [27]. In the present study, it took 5 h for frozen-thawed bovine spermatozoa to start penetrating the cumulus-free oocytes in a protein-free medium containing caffeine, heparin, and glucose. Therefore, it is postulated that protein supplement facilitates capacitation of bovine spermatozoa and/or penetration of oocytes. Since a portion of oocytes penetrated under the present experimental condition could develop to the blastocyst stage in a chemically defined medium, it seems that cumulus-free oocytes penetrated in medium without protein have the capacity for early development. In conclusion, this is the first report of the observation that in vitro-matured cumulus-free bovine oocytes can be penetrated in a chemically defined, protein-free medium. This system might be useful for analyzing the factors necessary for capacitation and the acrosome reaction of bull spermatozoa and for fertilization of bovine oocytes. ACKNOWLEDGMENT We thank Dr. Lynn R. Fraser, King's College London, UK, for valuable advice on preparing this manuscript.

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PENETRATION OF BOVINE OOCYIES IN A DEFINED MEDIUM 10. Tajik P, Niwa K, Murase T. Effect of different protein supplements in fertilization medium on in vitro penetration of cumulus-intact and cumulus-free bovine oocytes matured in culture. Theriogenology 1993; 40:949-958. 11. Bavister BD. Substitution of a synthetic polymer for protein in a mammalian gamete culture system. J Exp Zool 1981; 217:45-51. 12. Parrish 1, Susko-Parrish J, First NL. Capacitation of bovine sperm by heparin: inhibitory effect of glucose and role of intracellular pH. Biol Reprod 1989; 41:683-

699. 13. Niw/ K, Chang C. Effect of sperm concentration on the capacitation of rat spermatozoa. J Exp Zool 1974; 189:353-356. 14. Nagai T, Niwa K, Iritani A Effect of sperm concentration during preincubation in a defined medium on fertilization in vitro of pig follicular oocytes. J Reprod Fertil 1984; 70:271-275. 15. Wolf DP, Byrd W, Dandekar P, Quigley MM. Sperm concentration and the fertilization of human eggs in vitro. Biol Reprod 1984; 31:837-848. 16. Lee MA, Storey BT. Bicarbonate is essential for fertilization of mouse eggs: mouse sperm require it to undergo the acrosome reaction. Biol Reprod 1986; 34:349356. 17. Vijayaraghavan S, Critchlow LM, Hoskins DD. Evidence for a role for cellular alkalinization in the cyclic adenosine 3',5'-monophosphate-mediated initiation of motility in bovine caput spermatozoa. Biol Reprod 1985; 32:489-500. 18. Fraser LR, Ahuja KK Metabolic and surface events in fertilization. Gamete Res 1988; 20:491-519. 19. Brackett BG, Oliphant G. Capacitation of rabbit spermatozoa in vitro. Biol Reprod 1975; 12:260-274.

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20. Kim JH, Niwa K, Lim JM, Okuda K. Effects of phosphate, energy substrates, and amino acids on development of in vitro-matured, in vitro-fertilized bovine oocytes in a chemically defined, protein-free culture medium. Biol Reprod 1993; 48:1320-1325. 21. Ohgoda O, Niwa K, Yuhara M, Takahashi S, Kanoya K Variations in penetration rates in vitro of bovine follicular oocytes do not reflect conception rates after artificial insemination using frozen semen from different bulls. Theriogenology 1988; 29:1375-1381. 22. Neill JM, Olds-Clarke P. A computer-assisted assay for mouse sperm hyperactivation demonstrates that bicarbonate but not bovine serum albumin is required. Gamete Res 1987; 18:121-140. 23. Bhattacharyya A, Yanagimachi R Synthetic organic pH buffers can support fertilization of guinea pig eggs, but not as efficiently as bicarbonate buffer. Gamete Res 1988; 19:123-129. 24. Boatman DE, Robbins RS. Bicarbonate: carbon-dioxide regulation of sperm capacitation, hyperactivated motility, and acrosome reaction. Biol Reprod 1991; 44:806-813. 25. Hyne RV. Bicarbonate- and caldum-dependent induction of rapid guinea pig sperm acrosome reaction by monovalent ionophores. Biol Reprod 1984; 31:312-323. 26. Xu KP, Greve T. A detailed analysis of early events during in-vitro fertilization of bovine follicular oocytes. J Reprod Fertil 1988; 82:127-134. 27. Park CK, Ohgoda O, Niwa K. Penetration of bovine follicular oocytes by frozenthawed spermatozoa in the presence of caffeine and heparin. J Reprod Fertil 1989; 86:577-582.