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BIOLOGY OF REPRODUCTION 57, 1478-1483 (1997) Amino Acids in Maturation Medium and Presence of Cumulus Cells at Fertilization Promote Male Pronuclear Formation in Porcine Oocytes Matured and Penetrated In Vitro' Hak-Hyun Ka,3 5 Ken Sawai,s Wei-Hua Wang, 4'5 Kyung-Soon Im,6 and Koji Niwa2,s Division of Animal Science and Technology,s Faculty of Agriculture, Okayama University, Okayama 700, Japan Department of Animal Science and Technology,6 College of Agriculture and Life Sciences, Seoul National University, Suweon, 441-744, Korea ABSTRACT The present study was conducted to examine the ability of porcine oocytes to achieve male pronuclear (MPN) formation when they are matured and penetrated in vitro under various culture conditions. When cumulus-enclosed oocytes were cultured for 24-48 h in modified Whitten's medium (pH 7.4) supplemented with 10% porcine follicular fluid, 10 IU eCG/ml, and 10 IU hCG/ml (designated mWM-FG), nuclear maturation of oocytes reaching metaphase II was completed by 36 h after the start of culture. However, there were no differences in the proportions (94-95%) of oocytes penetrated in vitro by cryopreserved ejaculated spermatozoa or inthe rates (35-45%) of MPN formation between oocytes cultured for 36 and 48 h. When cumulus-enclosed oocytes were cultured for 36 h in mWM-FG supplemented with 2% (v:v) minimal essential medium (MEM) essential amino acids (EAA) with the addition of 0.1 mM glutamine and/or 1% (v:v) MEM nonessential amino acids (NEAA) and inseminated in vitro, 93-97% of oocytes were penetrated regardless of the presence of amino acids during maturation, but the rates of MPN formation were higher in the presence (7984%) than in the absence (51%) of any amino acids. The addition of EAA+ NEAA and/or 0.57 mM cysteine to mWM-FG also did not affect sperm penetration in vitro, while it promoted MPN formation (76-83%) in penetrated oocytes as compared with those matured in the absence of amino acids and cysteine (53%). When oocytes were freed from cumulus cells after culture in mWM-FG, sperm penetration rates were not different between cumulus-enclosed (100%) and cumulus-free (92%) oocytes, but the rate of MPN formation was higher in cumulusenclosed (53%) than in cumulus-free (28%) oocytes. When EAA+NEAA+cysteine was added to mWM-FG, MPN formation was not improved in cumulus-free oocytes but was much improved (78%) in cumulus-enclosed oocytes. These results indicate that MPN formation in porcine oocytes is promoted by the addition of amino acids and/or cysteine in simple maturation medium and by the presence of cumulus cells at fertilization in vitro. INTRODUCTION Male pronuclear (MPN) formation is generally considered a physiological measure of cytoplasmic maturation of Accepted August 5, 1997. Received May 27, 1997. 'This work was supported by Grant-in-Aid for Scientific Research B (no. 07456123) from the Ministry of Education, Science, Sports and Culture of Japan. 2Correspondence. FAX: (86) 254-0714; e-mail: [email protected] 3 Current address: Department of Animal Science and Technology, College of Agriculture and Life Sciences, Seoul National University, Suweon, 441-744, Korea. 4 Current address: Department of Animal Sciences, University of Missouri, Columbia, MO 65211. oocytes cultured in vitro [1, 2]. In the in vitro culture system for porcine oocytes, a low rate of MPN formation is one of the main problems along with a high incidence of polyspermy (for review see [3-5]). Improvements in MPN formation of porcine oocytes penetrated in vitro have been achieved by supplementing maturation medium with cysteine [6, 7], as a precursor of glutathione whose synthesis is known to be a prerequisite for sperm chromatin decondensation [8]; by supplementing with porcine follicular fluid (PFF) [9, 10]; by decreasing sodium chloride (NaCI) concentration in maturation medium [11]; and by coculturing oocytes with follicular shell [12, 13]. However, fully satisfactory culture systems for maturation and fertilization of porcine oocytes are yet to be established. Furthermore, there is little information about the factors affecting MPN formation with relation to the culture condition. Therefore, further studies of the effects of individual factors in media on maturation and fertilization of porcine oocytes in vitro will be necessary. We have found that modified Whitten's medium containing PFF and gonadotropins could support nuclear maturation and penetration of porcine oocytes in vitro by frozen-thawed ejaculated spermatozoa, but that the rate of MPN formation was very low (unpublished results). Therefore, the present study was undertaken to determine whether various factors-including maturation time, the presence of amino acids in maturation medium, and the presence of cumulus cells during fertilization in vitro-affect MPN formation of penetrated porcine follicular oocytes. MATERIALS AND METHODS Media The basic medium used for the maturation of oocytes was BSA-free Whitten's medium [14] composed of 87.95 mM NaCl, 4.83 mM KC1, 1.18 mM KH2PO 4, 1.18 mM MgSO 4, 22.62 mM NaHCO 3, 5.56 mM glucose, 21.58 mM sodium lactate, 0.31 mM sodium pyruvate, 2.66 mM hemicalcium lactate, 80 fIg potassium penicillin G/ml, 50 jug streptomycin sulfate/ml, and 0.01 mg phenol red/ml and supplemented with 10% (v:v) PFF, 10 IU eCG/ml (Serotropin; Teikoku-Zoki Co., Tokyo, Japan) and 10 IU hCG/ml (Puberogen; Sankyo Co., Tokyo, Japan). This medium was designated mWM-FG. The PFF was withdrawn using an 18-gauge needle fixed to a 10-ml disposable syringe from superficial follicles (2-5 mm in diameter) on maturing porcine ovaries. The drawn PFF was centrifuged at 1500 x g for 15 min at room temperature to remove any cells or debris, and the supernatant was stored at -20°C until used [9]. The basic medium, designated TCM-199B (pH 7.8) and used for the treatment of spermatozoa and the fertilization in vitro of oocytes, was modified tissue culture medium 1478 1479 MALE PRONUCLEAR FORMATION IN PIG OOCYTES (TCM) 199 (with Earle's salts; Gibco Lab., Grand Island, NY) supplemented with 3.05 mM glucose, 0.91 mM sodium pyruvate, 75 jig potassium penicillin G/ml, and 50 jLg streptomycin sulfate/ml [15]. Preparation of Oocytes Ovaries were collected from maturing gilts at a local abattoir and transported to the laboratory within 1-1.5 h in 0.9% (w:v) NaCI containing 75 g potassium penicillin G/ml and 50 Ig streptomycin sulfate/ml at 37-39°C. Oocytes were aspirated from antral follicles (2-5 mm in diameter) with an 18-gauge needle fixed to a 10-ml disposable syringe and washed four times with mWM-FG with various supplements according to the experiment. Ten oocytes surrounded by compact cumulus were transferred to a 100-,ul drop of the same medium, which had previously been covered with warm paraffin oil in a polystyrene culture dish (35 x 10 mm; Becton Dickinson Labware, Lincoln Park, NJ) and equilibrated in an atmosphere of 5% CO 2 in air for a minimum of 3 h, and were cultured for 24-48 h (experiment 1) or 36 h (experiments 2-4) at 39°C under the same atmospheric conditions. For in vitro fertilization, cumulus-enclosed oocytes cultured for 36 h (experiments 1-4) or 48 h (experiment 1) were washed twice in TCM-199B supplemented with 10 mM caffeine-sodium benzoate (Sigma Chemical Co., St. Louis, MO), placed into 50 jil of the same medium, and kept in a CO2 incubator (5% CO2 in air at 39 0C) for 30 min until spermatozoa were added for fertilization. Sperm Preparation and In Vitro Fertilization Spermatozoa were prepared as described by Wang et al. [15]. Briefly, three 0.1-ml pellets of frozen ejaculated spermatozoa obtained from boars of proven fertility were thawed in 2 ml of TCM-199B at 37°C for 1 min. The medium had previously been equilibrated in an atmosphere of 5% CO 2 in air for a minimum of 3 h. After thawing, spermatozoa were washed three times in the same medium by centrifugation at 550 x g for 5 min at room temperature. The sperm pellet was then resuspended to give a sperm concentration of 2-3 X 107 cells/mil. A 50-pIl aliquot of final sperm suspension was introduced into 50 ptl of TCM199B containing 10 mM caffeine that included the oocytes, and the mixture was cultured at 39C in an atmosphere of 5% CO2 in air. The mixture had final concentrations of 11.5 x 107 spermatozoa/ml and 5 mM caffeine. Assessment of Nuclear Maturation After 24-48 h of maturation culture (experiment 1), the oocytes were freed from cumulus cells by treatment with Dulbecco's PBS (D-PBS; Sigma) containing 0.1% (w:v) hyaluronidase from bovine testis (Sigma), followed by repeated passage through a fine pipette. The denuded oocytes were mounted, fixed for 48-72 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 phasecontrast microscope at magnifications of x200 or x400 for assessment of nuclear maturation. Oocytes at the stage of metaphase II (M-II1) were regarded as matured. TABLE 1. Composition of amino acids added to the medium. Amino acids mM EAA (2%, v:v)a L-Arginine HCI L-Cystine L-Glutamine L-Histidine HCIlH,O L-Isoleucine L-Leucine L-Lysine-HCI L-Methionine L-Phenylalanine L-Threonine L-Tryptophan L-Tyrosine L-Valine L-Glutamine 0.6 0.1 0.1 0.2 0.4 0.4 0.4 0.1 0.2 0.4 0.05 0.2 0.4 0.1 NEAA (1%, v:v)b L-Alanine L-AsparagineHO L-Aspartic acid L-Glutamic acid Glycine L-Proline L-Serine 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Minimal Essential Medium (MEM) essential amino acid solution (no. 320-1130; Gibco). b MEM nonessential amino acid solution (no. 320-1140; Gibco). a of sperm penetration as described above for the assessment of nuclear maturation. Oocytes were considered penetrated when they had one or more swollen sperm heads and/or male pronuclei and their corresponding sperm tails. Experimental Studies In experiment 1, to determine the optimal time for maturation of oocytes in mWM-FG, oocytes were cultured for periods ranging from 24 to 48 h. Furthermore, to determine the effect of maturation time on sperm penetration in vitro, cumulus-enclosed oocytes cultured for 36 and 48 h for maturation in mWM-FG were inseminated in vitro. In experiment 2, to study the effects of amino acids in maturation medium on sperm penetration in vitro, cumulus-enclosed oocytes were cultured for 36 h in mWM-FG supplemented with 2% (v:v) MEM essential amino acid solution (EAA; no. 320-1130; Gibco) with the addition of 0.1 mM glutamine (no. G-5763; Sigma) and/or 1% (v:v) MEM nonessential amino acid solution (NEAA; no. 320-1140; Gibco) and inseminated in vitro. The composition of amino acids added to the medium was exactly the same as that used by Miyoshi et al. [16] for the development of rat one-cell embryos as indicated in Table 1. In experiment 3, to examine the effects of amino acids and/or cysteine on sperm penetration in vitro, cumulus-enclosed oocytes were cultured for 36 h in mWM-FG supplemented with EAA+NEAA and/or 0.57 mM cysteine (Sigma) and inseminated in vitro. In experiment 4, to study the effects of the presence of cumulus cells during fertilization on sperm penetration in vitro, some of cumulus-enclosed oocytes that had been cultured for 36 h in mWM-FG with or without EAA+NEAA+cysteine were freed from cumulus cells. Then cumulus-enclosed and cumulus-free oocytes were inseminated in vitro. Assessment of Sperm Penetration Fourteen hours after insemination, oocytes were freed from cumulus cells by repeated passage through a fine pipette, mounted, fixed, stained, and examined for evidence Statistical Analysis The percentages of oocytes reaching M-II, penetration, MPN formation, and polyspermy were subjected to an arc- 1480 KA ET AL. (4 39 (42d 100- amino acids (Table 3). When the oocytes were matured in medium supplemented with amino acids (EAA and/or NEAA), the incidence (79-84%) of MPN formation was significantly (p < 0.01) greater than in the absence of amino acids (51%), while the incidence (97-99%) of polyspermy and the mean number (4.7-5.7) of spermatozoa in penetrated oocytes were not affected. 4(46) d 80c)q 0 (49 Experiment 3 U2 - CD V 20 (45) 20- (45) a 0- *y 24 - I I I 1 I 28 32 36 40 44 Maturation time (h) FIG. 1. Time course of nuclear maturation (metaphase II)of porcine oocytes cultured in mWM-FG containing 10% (v:v) PFF, 10 IU eCG/ml, and 10 IU hCG/ml. The numbers of oocytes examined at each time of culture are shown in parentheses. The values with different letters are significantly different (p < 0.01). Experiments were repeated three times. sine transformation, and the transformed values were analyzed using one-way (experiments 1 and 2) or two-way (experiments 3 and 4) ANOVA. When ANOVA revealed a significant effect, the treatments were compared by Duncan's Multiple Range test. Tukey's studentized range tests were applied to compare mean numbers of spermatozoa in penetrated oocytes. All analyses were carried out using GraphPad-PRISM (GraphPad Software, San Diego, CA). RESULTS Experiment 1 The proportion of oocytes completing nuclear maturation was significantly (p < 0.05) higher when they were cultured in mWM-FG for 36-48 h (90-98%) as compared to 24-32 h (0-43%; Fig. 1). There were no differences in the rates of nuclear maturation among oocytes cultured for 36-48 h. When oocytes cultured for 36 or 48 h were inseminated in vitro, very high penetration rates (94-95%) were obtained, and there were no differences in the proportions of MPN formation (35-45%) and polyspermy (8790%), or in the mean numbers (3.6-4.0) of spermatozoa in penetrated oocytes, between the two different culture periods (Table 2). Therefore, in all subsequent experiments, oocytes cultured for 36 h were used. Experiment 2 Very high penetration rates (93-97%) were obtained in oocytes matured both in the presence and in the absence of TABLE 2. When cysteine was added to the maturation medium without amino acids, the incidence (83%) of MPN formation was increased significantly (p < 0.05) as compared with the value (53%) in the absence of cysteine (Table 4). However, the beneficial effects of amino acids (EAA+NEAA) on MPN formation was not increased by supplementation with cysteine. Penetration rates (89100%), the incidence (85-98%) of polyspermy, and the mean number (4.3-6.4) of spermatozoa in penetrated oocytes were not affected by supplementation with amino acids and/or cysteine. Experiment 4 The proportions (92-100%) of oocytes penetrated were not different among different experimental groups, but the incidence of MPN formation was significantly (p < 0.01) higher in cumulus-enclosed (53-78%) than in cumulus-free (28%) oocytes matured in both the presence and absence of amino acids + cysteine (Table 5). In cumulus-enclosed oocytes, MPN formation was promoted when the oocytes were matured in the presence (78%) as compared to the absence (53%) of amino acids + cysteine (p < 0.05). The incidence (69-96%) of polyspermy and mean number (3.14.7) of spermatozoa in penetrated oocytes were not affected by the different conditions for maturation and fertilization in vitro. DISCUSSION The results of the present study indicate that 1) nuclear and cytoplasmic maturation of porcine oocytes can be completed by 36 h of culture in chemically simple medium (mWM-FG) containing 10% (v:v) PFF and gonadotropins (10 IU eCG/ml + 10 IU hCG/ml); 2) supplementation of the medium with amino acids (EAA and/or NEAA) or cysteine (0.57 mM) promotes MPN formation in penetrated oocytes; 3) the presence of cumulus cells at insemination also promotes MPN formation in penetrated oocytes; and 4) the efficacy of cumulus cells to promote MPN formation is higher when the oocytes are cultured for maturation in the presence as compared to the absence of amino acids and cysteine. Porcine oocytes usually require a long time for maturation, and various times in different culture conditions have been reported (36 h [15, 17, 18]; 40 h [11]; 44-46 h [1]; Effects of the time of maturation on sperm penetration in vitro of porcine oocytes.a Number of oocytes penetrated Time (h) of maturation 36 48 Number of oocytes examined 81 87 Total (%) With swollen sperm head (%)b With male pronucleus (%)b 77 (95) 82 (94) 42 (55) 53 (65) 35 (45) 29 (35) Number of polyspermic oocytes (%) b Mean number of spermatozoa in penetrated oocytes 69 (90) 71 (87) 4.0 3.6 a Cumulus-enclosed oocytes were cultured in modified Whitten's medium containing 10% (v:v) PFF, 10 IU eCG/ml, and 10 IU hCG/ml; experiments were repeated four times. b Percentage of oocytes penetrated. 1481 MALE PRONUCLEAR FORMATION IN PIG OOCYTES TABLE 3. Effects of addition of amino acids to the maturation medium (mWM-FG) on sperm penetration in vitro of porcine oocytes.a Number of oocytes penetrated Amino acidsb Number of oocytes inseminated None EAA NEAA EAA + NEAA 73 75 79 76 With swollen sperm head (%)c Total (%) 70 73 77 71 (96) (97) (97) (93) Number of With male pronucleus (%)' 34 (49)d 15 (21)e 12 (16)e 14 (20)e (51) d (79)e (84)e (80) e 36 58 65 57 Mean number of polyspermic oocytes spermatozoa in (%)c penetrated oocytes 68 72 75 69 (97) (99) (97) (97) 5.0 5.7 4.8 4.7 a Experiments were repeated four times. b EAA: minimal essential medium (MEM) essential amino acid solution (2%, v:v) + 0.1 mM glutamine; NEAA: MEM nonessential amino acid solution (1%, v:v). ' Percentage of oocytes penetrated. d. Values with different superscripts within each column differ significantly (p < 0.01). TABLE 4. oocytes.a Effects of addition of amino acids and/or cysteine (0.57 mM) to the maturation medium (mWM-FG) on sperm penetration in vitro of porcine Addition of EAA + NEAAb Cysteine _ + + + Number of oocytes inseminated Number of Mean number of With swollen sperm head (%)c polyspermic oocytes (%)c spermatozoa in penetrated oocytes Total (%) 44 48 46 45 + Number of oocytes penetrated 43 46 41 45 (98) (96), (89) (100) 20 8 10 10 With male pronucleus (%)c (47)d (17)e (24)0 (22)e 23 38 31 35 (53) d (83)e (76)e (78)0 41 42 39 44 (95) (91) (85) (98) 5.7 6.4 4.3 5.1 Experiments were repeated four times. EAA: minimal essential medium (MEM) essential amino acid solution (2%, v:v) + 0.1 mM glutamine; NEAA: MEM nonessential amino acid solution (1%, v:v). c Percentage of oocytes penetrated. d,eValues with different superscripts within each column differ significantly (p < 0.01). b 48 h [9, 12, 13]). The factors that regulate the timing of nuclear maturation in porcine oocytes in vitro have not been strictly identified. However, it has been reported that the proportion of porcine oocytes reaching M-II in vivo increased markedly 36 h after hCG injection [19]. This timing of oocyte maturation is comparable to that observed in vitro in complex medium, modified TCM-199 supplemented with fetal calf serum (FCS) [15, 18]. In the present study using simple medium supplemented with PFF and gonadotropins, most oocytes reached M-II also by 36 h of culture. These results may indicate that FCS, PFF, and gonadotropins contain the factors that regulate oocyte maturation. The maturation rate and/or the time for reaching M-II in the presence of EAA and/or NEAA were not examined in the present study. However, since in our previous study [15], a large proportion (73%) of porcine oocytes reaching M-II was not obtained until 36 h after culture in modified TCM- 199, which contains some amino acids, it is postulated that the addition of EAA and/or NEAA to mWH-FG may not alter the timing of meiotic progression of porcine oocytes. Although some degree of decondensation of the sperm nucleus can occur independently of oocyte maturation and activation [15], MPN formation is clearly dependent not only upon germinal vesicle breakdown but also on oocyte activation [20]. In the present study, oocytes had already been matured after 36 h of culture in mWM-FG and there were no differences in the rates of penetration in vitro and MPN formation between oocytes cultured for 36 h and 48 h. However, in a study involving hCG-regulated ovulation, Hunter [21] reported that aging in porcine oocytes was revealed within 6-8 h of ovulation and that oocytes penetrated by spermatozoa after these intervals showed an increased risk of polyspermic penetration and subsequent fragmentation. It has been also reported that as a result of TABLE 5. Effects of the presence of cumulus cells during fertilization on sperm penetration in vitro in porcine oocytes matured in the presence or absence of amino acids and cysteine (0.57 mM). Presence (+) or absence (-) of EAA + NEAA + cysteine during maturationb Number of oocytes penetrated Cumulus cells during fertilization + + + + Number of oocytes inseminated 50 43 45 46 Total (%) 46 43 43 45 (92) (100) (96) (98) With swollen sperm head (%)c 33 (72) d 20 (47)e 31 (72) d 10 (22 )f Mean number of With male pronucleus (%) 13 23 12 35 (28) d (53)e (28)d (78) f Number of polyspermic oocytes (%) 37 38 30 43 (80) (88) (69) (96) spermatozoa in penetrated oocytes 3.6 4.7 3.1 4.0 aExperiments were repeated four times. b EAA: minimal essential medium (MEM) essential amino acid solution (2%, v:v) + 0.1 mM glutamine; NEAA: MEM nonessential amino acid solution (1 %, v:v). Percentage of oocytes penetrated. d,-'fValues with different superscripts within each column differ significantly (d-e, d-f, p < 0.01; e-f, p < 0.05). 1482 KA ET AL. aging the spindle of the second meiotic metaphase might break down, which results in the loss of chromosomes [22]. Therefore, in the present experimental conditions, it seems that oocytes cultured for 36 h are more suitable for use for in vitro fertilization than those cultured for 48 h. It has been reported that amino acids improve the development of mouse [23], rat [16], rabbit [24], hamster [25, 26], bovine [27], and ovine [28] embryos in vitro and also that they support rabbit [29] and hamster [30] oocyte maturation in vitro. The results of the present study show that the presence of EAA and NEAA, either alone or together in mWM-FG during maturation culture, is beneficial for MPN formation in porcine oocytes after penetration in vitro. The mechanism(s) by which amino acids during oocyte maturation improve MPN formation and the required amount or composition of amino acids are not known. However, it seems that amino acids in PFF may not be sufficient in their amounts or composition for porcine oocytes during maturation to acquire the ability to form male pronuclei after sperm penetration. In the present study, when mWM-FG was supplemented with 0.57 mM cysteine, MPN formation rate was increased significantly. This finding is consistent with the reports of Yoshida et al. [6] and Sawai et al. [7], who found that supplementing the maturation medium with cysteine as a substrate for glutathione synthesis is important for MPN formation in porcine oocytes after sperm penetration. However, when the medium was supplemented with amino acids (EAA+NEAA), additional beneficial effects of the addition of cysteine on MPN formation were not observed. These results might indicate that cysteine is not the only amino acid that can be utilized for glutathione synthesis or augment MPN formation and that other amino acids are also important for cytoplasmic maturation of oocytes, although it is unknown whether the effects of EAA, NEAA, and cysteine are brought about through the same mechanism or different mechanisms. Research in the pig and in other species showed that cumulus cells have a beneficial effect on maturation and fertilization of oocytes in vitro [1, 2, 31-33]. However, reports concerning the effect of cumulus cells during fertilization on MPN formation are very few. Recently it was reported that porcine oocytes surrounded by expanded cumulus cells exhibited higher frequency of MPN formation than did denuded oocytes [3, 15, 34]. Zhang et al. [35] also reported a significant decrease in fertilization rate when cumulus cells were removed from bovine oocytes 7 h after insemination. In agreement with these observations, the present study showed that the presence of cumulus cells during fertilization increased the incidence of MPN formation in porcine oocytes. Moreover, amino acids in maturation medium improved MPN formation only when cumulus cells were attached to the oocytes during fertilization in vitro. The fact that amino acids are transferred to the oocytes by the action of gap junctions between cumulus cells and an oocyte is well known [36-38], but the mechanisms by which amino acids and cumulus cells regulate MPN formation are unknown at present. Although no systematic assessment of the degree of cumulus expansion was done in the present study, full expansion of the cumulus mass was observed at 36 h of culture in mWM-FG, and no morphological changes in cumulus expansion were observed after the EAA and/or NEAA supplementation. However, one possibility is that amino acid supplementation alters the composition of extracellular matrix and subsequently changes the timing of sperm penetration, which influ- ences MPN formation. On the other hand, since intracellular uncoupling of porcine cumulus-oocyte complexes initiated during the first meiotic division is nearly completed at M-II [39], and since the ability of denuded oocytes to form male pronuclei is not increased when they are cocultured with cumulus-intact oocytes, Ball et al. [40] suggested that damage of oocytes by exposure to hyaluronidase and/or mechanical pipetting for the removal of cumulus cells may result in a low rate of MPN formation. Furthermore, Funahashi et al. [11] reported that decreasing the NaCl concentration of maturation medium can produce a high rate of MPN formation despite fertilization in vitro without cumulus cells. Since the requirement of the presence of cumulus cells could be reduced under conditions that are more favorable for oocyte maturation, additional studies will be necessary to elucidate the relationships between cumulus cells, culture conditions, and MPN formation. ACKNOWLEDGMENTS We thank Dr. A. 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