BIOLOGY OF REPRODUCTION 58, 1316-1320 (1998)

Maturation In Vitro of Pig Oocytes in Protein-Free Culture Media: Fertilization and Subsequent Embryo Development In Vitro' Lalantha R. Abeydeera, Wei-hua Wang, Randall S. Prather, and Billy N. Day 2 Department of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri 6521 1 ABSTRACT In the present study, attempts were made to develop a protein-free (PF) in vitro maturation (IVM) system for pig oocytes and to examine subsequent embryo development after in vitro fertilization. In experiment 1, four IVM media were tested: 1) control: North Carolina State University (NCSU) 23+10% porcine follicular fluid; 2) PF-NCSU: NCSU 23+0.1% polyvinyl alcohol (PVA)+1 % amino acids; 3) PF-TCM: Tissue culture medium (TCM) 199+PVA; and 4) PF-WM: PF-Waymouth MB 752/1 medium (WM)+PVA. Oocytes were cultured in the respective media containing eCG and hCG (10 IU/ml each) for 20-22 h and then without hormonal supplements for an additional 2022 h. After culture, the degree of cumulus expansion and frequency of nuclear maturation were determined. Some oocytes were coincubated with frozen-thawed spermatozoa for 5-6 h in modified Tris-buffered medium containing caffeine and BSA. In experiment 2, oocytes were matured in control, PF-TCM, and PF-WM, fertilized in vitro, and cultured for 144 h in NSCU 23+BSA. Fewer (p < 0.01) oocytes reached metaphase IIstage in PF-NCSU (45% vs. 80-85%) than in the other media. Oocytes matured in control medium showed the most cumulus expansion, followed by those in PF-TCM and PF-WM; those in PFNCSU showed very slight expansion. A lower (p < 0.05) penetration rate was obtained for oocytes matured in PF-NCSU than in the control medium (59% vs. 81 %). In contrast to those in control (96%) and PF-TCM (93%), oocytes in PF-WM (65%) showed a lower male pronuclear formation. Compared to that in the control, a significantly lower (p < 0.05) cleavage rate was also observed for oocytes matured in PF-WM. Similar proportions of embryos developed to the blastocyst stage when oocytes were matured in control (22%) and PF-TCM (13%). These results indicate that pig oocytes can be successfully matured in a protein-free medium with subsequent development to the blastocyst stage. INTRODUCTION Successful methods for in vitro maturation/fertilization (IVM-IVF) [1-8] of porcine oocytes and culture [8-13] of porcine embryos have been developed. In general, most IVM media are usually supplemented with fetal calf serum (FCS) [1-4] or porcine follicular fluid (pFF) [5-8]. It is a well-known fact that growth factors and hormones, in addition to many other factors, are abundant in serum [14, 15] and follicular fluid [2, 14, 16]. Therefore, supplementation of IVM media with FCS or pFF undoubtedly conflicts with Accepted January 15, 1998. Received November 10, 1997. 'This work is supported as a part of the National Cooperative Program on Non-Human In Vitro Fertilization and Preimplantation Development and was funded by the National Institute of Child Health and Human Development through cooperative Agreement HD 34588 and is a contribution from the Missouri Agricultural Experimental station: Journal Series Number 12,701. 2Correspondence: B.N. Day, University of Missouri-Columbia, Department of Animal Sciences, 159 Animal Sciences Research Center, Columbia, MO 65211. FAX: (573) 884-7827; e-mail: [email protected]

proper quality control and repeatability among laboratories. In order to eliminate such variability it is imperative to develop a more defined culture system for IVM. Development of a defined IVM system will help to advance understanding of the basic molecular mechanisms responsible for stimulating or inhibiting nuclear and/or cytoplasmic maturation of pig oocytes. Bovine oocytes matured in vitro under protein-free conditions can be successfully fertilized and can develop to the blastocyst stage in vitro [17, 18]. Similarly, porcine oocytes can also be matured in a protein-free medium with subsequent fertilization in vitro [19-21]. However, the developmental ability of IVM-IVF zygotes formed under these conditions is not known. In the present study, three different protein-free IVM media were tested for 1) the ability to induce nuclear maturation, 2) in vitro fertilizability after IVM, and 3) developmental competence of putative IVM-IVF zygotes to the blastocyst stage. MATERIALS AND METHODS Culture Media Unless otherwise stated, all chemicals used in this study were purchased from Sigma Chemical Company (St. Louis, MO). The control medium [7, 8] used for oocyte maturation was BSA-free North Carolina State University (NCSU) 23 medium [9] supplemented with 0.1 mg/ml cysteine and 10% (v:v) pFF The protein-free media consisted of 1) NCSU 23 medium supplemented with 0.1 mg/ml cysteine, 1% (v:v) minimum essential medium Eagle amino acids, and 1% (v:v) basal medium Eagle amino acids and 0.1% polyvinyl alcohol (PVA) (PF-NCSU); 2) tissue culture medium (TCM) 199 (Gibco, Grand Island, NY) supplemented with 0.1 mg/ml cysteine and 0.1% PVA (PF-TCM); and 3) Waymouth MB 752/1 medium containing 0.1% PVA (PFWM). All NM media were supplemented with 10 IU/ml eCG (Intervet America, Millsboro, DE) and 10 IU/ml hCG (Intervet). Porcine FF was collected from follicles 3-6 mm in diameter, centrifuged at 1500 x g for 30 min (4°C), filtered through 1.2-,um syringe filters, and stored at -20°C until use. The basic medium used for IVF was essentially the same as that used by Abeydeera and Day [8]. This medium, designated as modified Tris-buffered medium (mTBM), consists of 113.1 mM NaCl, 3 mM KCI, 7.5 mM CaC12.2H 0, 2 20 mM Tris (crystallized free base; Fisher Scientific, Fair Lawn, NJ), 11 mM glucose, 5 mM sodium pyruvate, 0.1% BSA (A 7888), and 1 mM caffeine. The embryo culture medium for embryo development was NCSU 23 containing 0.4% BSA (A 8022). Recovery and Culture of Oocytes Ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory in 0.9% 1316

MATURATION OF PIG OOCYTES IN PROTEIN-FREE MEDIA NaCl containing 75 jxg/ml potassium penicillin G and 50 pig/ml streptomycin sulfate maintained at 25-28°C. Oocytes were aspirated from medium-sized follicles (3-6 mm in diameter) with a 20-gauge needle fixed to a 10-ml disposable syringe. The cumulus-oocyte complexes surrounded by a compact cumulus mass and having evenly granulated cytoplasm were washed three times in the respective oocyte maturation media containing hormonal supplements. Then, 45-50 (to examine nuclear maturation) or 120 (60/500 Il) oocytes (to examine in vitro fertilization [IVF] and development) were transferred into a 35 x 10-mm 2 polystyrene culture dish (Becton Dickinson, Lincoln Park, NJ) containing a 500- 1l drop of the same culture medium, which had previously been covered with warm paraffin oil (light mineral oil; Fisher) and equilibrated in a 5% CO 2 incubator. After 20-22 h of maturation culture, oocytes were washed three times in the respective maturation media without hormonal supplements and transferred into 500-j1I drops of the same medium for an additional 20-22 h of culture. Each experiment (4 treatment groups) was repeated four times. Evaluation of Cumulus Expansion At the end of IVM culture, the degree of cumulus expansion was assessed subjectively under a stereomicroscope and classified into five categories: category 0, no expansion; category 1, very slight expansion observed only in the outermost layer; category 2, slight expansion observed in 2-3 layers from the periphery; category 3, moderate expansion, observed in about 50% of the cumulus cell layers; and category 4, full expansion except in the corona radiata layer. IVF After the completion of IVM culture in the four maturation media, cumulus cells were removed with 0.1% hyaluronidase in NCSU 23. Degenerated oocytes, constituting about - 3% of the total oocytes (n = 120) that underwent IVM in each treatment group, were discarded. As described previously [8], denuded oocytes (n = 105) were randomly selected from each treatment group and washed three times in IVF medium, and 35 oocytes were placed into each of three 50-Rl drops of the same medium, which had been covered with warm paraffin oil in a 35 x 10-mm 2 polystyrene culture dish. The dishes were kept in the incubator for about 30 min until spermatozoa were added for fertilization. A semen pellet was thawed and washed three times by centrifugation at 1900 x g for 4 min in Dulbecco's PBS (Gibco) supplemented with 0.1% BSA, 75 !xg/ml potassium penicillin G, and 50 jig/ml streptomycin sulfate (pH 7.2, 39°C). At the end of the washing procedure, the sperm pellet [8] was resuspended in IVF medium (same as above). After appropriate dilution, 50 1I of this sperm suspension was added to 50 l of the medium that contained oocytes to give a final sperm concentration of 5 X 105 cells/mi. Oocytes were coincubated with spermatozoa for 5-6 h at 39°C in an atmosphere of 5% CO 2 in air. Assessment of Meiotic Maturation and Fertilization Parameters After IVM culture or 12 h after IVF, oocytes were mounted and fixed for 48-72 h in 25% (v:v) acetic acid in ethanol at room temperature. They were then stained with 1% (w:v) orcein in 45% acetic acid (v:v) and examined under a phase-contrast microscope at X200 and x400 mag-

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nification. The meiotic stage of oocytes was assessed according to Hunter and Polge [22]. Oocytes were considered penetrated when they had one or more swollen sperm heads and/or male pronuclei and their corresponding sperm tails. Culture of Embryos After sperm-oocyte coincubation (5-6 h), putative embryos were washed three times in embryo culture medium and transferred to a Nunc 4-well multidish (Nunc, Roskilde, Denmark) containing 500 Ril of the same medium per well and incubated at 39C in 5% CO 2 in air. At 48 and 144 h after insemination, cleavage rate and blastocyst formation, respectively, were evaluated under a stereomicroscope. Statistical Analysis Data were analyzed by ANOVA and Fisher's protected least significant difference test using the STATVIEW program (Abacus Concepts, Berkeley, CA). All percentage data were subjected to arcsine transformation before statistical analysis. Data are expressed as mean + SEM. A probability of p < 0.05 was considered statistically significant. RESULTS Effect of Various Protein-Free Media on Cumulus Expansion, Nuclear Maturation, and Fertilization Parameters Most cumulus-oocyte complexes showed cumulus expansion of category 4 after IVM in control medium. Oocytes matured in PF-TCM and PF-WM showed category 3 cumulus expansion, whereas those in PF-NCSU showed category 1 cumulus expansion. After 40-44 h of maturation culture, 80%, 86%, and 80% of oocytes reached metaphase II (M II) stage in control, PF-TCM, and PF-WM medium, respectively (Fig. 1). In contrast, significantly (p < 0.01) fewer oocytes (45%) that had matured in PF-NCSU medium reached M II stage, with a significantly higher (p < 0.01) proportion remaining at the germinal vesicle (GV) stage. For each IVM medium, a few oocytes ( 3%) showed clear signs of degeneration under the stereomicro0

v

100 80 U

0 0

60

0 4)

40

(1

20 0 Control (193)

PF-NCSU (174)

PF-TCM (185)

PF-WM (201)

In vitro maturation medium FIG. 1. Nuclear maturation of pig cumulus-oocyte complexes cultured in control and protein-free media. Values are presented as means SEM. The numbers in parentheses indicate the number of oocytes examined. Within a category, bars with different superscripts differ significantly, p < 0.01.

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ABEYDEERA ET AL.

TABLE 1. Effect of IVM medium on fertilization parameters of pig oocytes. Oocytes Maturation medium

Examined (no.)

Penetrated (%)

Polyspermic (%)

Control PF-NCSU PF-TCM PF-WM

139 133 136 133

81.4 1.4b 59.2 3.7 c 70.1 + 3. 6bc 70.9 ± 6 .8bc

42.8 3.6 57.7 + 11.6 58.2 ± 5.4 47.7 + 6.1

With male pronucleusa (%) 96.4 80.7 92.7 65.0 ±

2.1 b 9.4 bc 1.8b 11.5cd

Mean no. spermatozoa in penetrated oocyte 1.7 1.9 1.9 1.8

+ + ± +

0.1 0.2 0.1 0.1

a Percentage of the number of oocytes penetrated. b-d Within a column, values with different superscripts are significantly different (p < 0.05).

scope but were excluded from the data set. In comparison to that in control medium, a significantly (p < 0.05) lower penetration rate (81% vs. 59%) was observed for oocytes matured in PF-NCSU medium (Table 1). No difference in penetration rate was found between control, PF-TCM, and PF-WM or PF-NCSU, PF-TCM, and PF-WM. Between the four media, no difference was found in the rate of polyspermic fertilization (43-58%) or mean number of spermatozoa per oocyte (1.7-1.9). Compared to those in control (96%) and PF-TCM (93%), oocytes matured in PF-WM (65%) showed a significant (p < 0.05) reduction in male pronuclear formation after sperm penetration. However, no difference was found in rate of male pronuclear development between PF-NCSU, control, and PF-TCM or PFNCSU and PF-WM. Effect of Protein-Free IVM Medium on Embryo Development Compared to the cleavage rate for oocytes matured in control medium (51%), a significantly lower (p < 0.05) cleavage rate was obtained for oocytes matured in PF-WM (34%) following IVF (Fig. 2). No difference was found in cleavage rates for oocytes matured in control and PF-TCM (51% vs. 39%) or PF-TCM and PF-WM (39% vs. 34%). However, compared to that in PF-WM (2%), a higher (p < 0.05) proportion of putative IVM-IVF zygotes developed to the blastocyst stage when oocytes were matured in control (22%) and PF-TCM (13%), with no difference between them.

60

a

[] Cleavage (48 h) el Blastocysts (144

cFi ,T~

N 03

b

T

40

44 0 0, c) · a

h)

ab

O6

a 20

%)

./

b

0 Control (241)

PF-TCM

PF-WM

(242)

(243)

In vitro maturation medium FIG. 2. Cleavage and blastocyst formation rates after IVF of pig oocytes matured in control and protein-free media. The numbers in parentheses indicate the number of putative zygotes examined. Within a category, bars with different superscripts differ significantly, p < 0.05.

DISCUSSION The present study describes the successful production of porcine blastocysts through IVM-IVF techniques using protein-free culture conditions for oocyte maturation. Our main objective in this study was to develop a protein-free IVM system that could be used for the successful maturation of pig oocytes as measured by subsequent ability to develop to the blastocyst stage following IVE For this purpose, we selected three basic culture media (TCM 199, WM, and NCSU 23) that have been used to mature pig oocytes in vitro. These media were compared with the control medium, which will mature pig oocytes capable of developing to the blastocyst stage after IVF [8]. However, since TCM 199 and WM contain amino acids, NCSU 23 was supplemented with MEM essential (1%) and nonessential (1%) amino acids. Also, TCM 199 and NCSU 23 were supplemented with 0.1 mg/ml of cysteine, a concentration similar to that present in WM. The nuclear maturation rates obtained for oocytes matured in PF-TCM and PF-WM are in good agreement with the results of a similar study reported by Wang and Niwa [21]. Compared to that in PF-TCM and PF-WM, the increased cumulus expansion (category 3 vs. 4) observed for oocytes matured in control medium could be attributed to the presence of pFF Interestingly, in contrast to observations in pig oocytes, no cumulus expansion was observed in mouse [23] and hamster [24] oocytes matured with gonadotropins and in the presence of BSA and PVA, respectively. However, these oocytes did show cumulus expansion in the presence of gonadotropins and serum. In contrast, under similar protein-free culture conditions, cumulus expansion occurred in bovine oocytes [25]. These findings suggest that oocytes from domestic animals are capable of a certain degree of cumulus expansion in media that contain gonadotropins with no serum supplementation. In the mouse, cumulus expansion requires interoa-trypsin inhibitors, present in serum and follicular fluid, which stabilize the cumulus extracellular matrix through their direct binding with hyaluronic acid [26-28]. With respect to the present study, it would be of interest to know whether such an interaction occurred during cumulus expansion of pig oocytes matured in protein-free media. On the other hand, it is possible that some yet unknown factor(s) produced by the oocyte and/or cumulus cells bound with hyaluronic acid to stabilize the extracellular matrix, thus resulting in cumulus expansion. Despite the addition of amino acids, oocytes matured in PF-NCSU medium showed the minimal cumulus expansion (category 1). Interestingly, however, about half (45%) of the oocytes reached M II stage. Wang and Niwa [21] also obtained 33% of oocytes at M II stage with no cumulus expansion when pig oocytes were cultured for 48 h in serum-free TCM 199. Similar observations have also been

MATURATION OF PIG OOCYTES IN PROTEIN-FREE MEDIA reported for mouse [29] and cattle oocytes [30]. At present, the reason(s) for such a low M II rate and cumulus expansion for pig oocytes matured in PF-NCSU is not known. One possibility could be the absence of vitamins in PFNCSU as compared to PF-TCM and PF-WM. However, this is questionable, since the addition of vitamins did not improve the degree of cumulus expansion or rate of nuclear maturation of oocytes matured in PF-NCSU medium (unpublished results). However, this observation suggests that cumulus expansion and nuclear maturation are not necessarily totally dependent upon each other. IVF data provide evidence that, compared to those in the control medium, oocytes matured under protein-free conditions are equally penetrable, with the exception of oocytes matured in PF-NCSU medium. In contrast, mouse oocytes matured in a serum-free medium showed significant reduction in sperm penetration as compared to oocytes matured in the presence of serum [31]. This low penetration correlates well with zona hardening as evidenced by increased resistance to proteolytic digestion. Zona hardening is thought to be the result of the conversion of ZP2 to ZP2f catalyzed by a protease released during premature cortical granule exocytosis during IVM of mouse oocytes [32]. It has been shown that fetuin, a serum component, inhibits precocious hardening of the zona pellucida during spontaneous oocyte maturation in vitro [33]. In the present study, fertilization results suggest that pig oocytes matured in PFTCM and PF-WM do not exhibit zona hardening. A possible reason could be the absence of premature cortical granule exocytosis during IVM of pig oocytes. The low penetration rates obtained for oocytes matured in PF-NCSU medium are mainly attributable to the complete failure of penetration into GV-stage oocytes. According to Wang et al. [34], immature pig oocytes even at the GV stage can be penetrated with subsequent sperm nuclear decondensation. Different results could be attributed, at least in part, to the different IVF media (TCM 199 vs. mTBM) and/or macromolecular supplements (FCS vs. BSA) used in these studies. Careful examination of the nuclear maturation rate (45%) and fertilization rate (59%) for oocytes matured in PF-NCSU medium indicates that some oocytes may have been penetrated by sperm during the process of nuclear maturation. However, such evidence was not revealed by the examination of fixed oocytes 12 h after insemination. This observation suggests that some of those maturing oocytes, following sperm penetration, may have completed their nuclear maturation to M II and subsequently activated, forming male and female pronuclei. Nagai et al. [35] reported that pig oocytes inseminated at M I stage can form male and female pronuclei after sperm penetration. A similar phenomenon is also observed in bovine oocytes penetrated after GV breakdown [36]. Once penetrated, oocytes matured in control, PF-TCM, and PF-NCSU have an equal ability to transform a sperm nucleus into a male pronucleus. Surprisingly, oocytes matured in PF-WM showed a significantly lower ability to form male pronuclei when compared to those in control and PF-TCM media. According to Yoshida et al. [37, 38], oocytes matured in WM showed a higher incidence of male pronuclear formation following IVE However, in their study, both FCS (10%) and pFF (10%) were supplemented during IVM and may have been responsible for the improved male pronuclear formation. Intracellular glutathione (GSH) synthesis occurs throughout IVM of pig oocytes, and GSH concentration is an important cytoplasmic factor responsible for sperm nuclear decondensation and male

1319

pronuclear formation following sperm penetration [38]. In the present study, it is possible that oocytes matured in PFWM had a low intracellular GSH concentration compared to oocytes matured in control and PF-TCM medium. Although we did not examine it in this study, measurement of intracellular GSH concentration of oocytes matured in the media mentioned above may substantiate this hypothesis. Although the mathematical proportion was higher, no significant difference was observed in embryo development to the blastocyst stage of oocytes matured in control (22%) and PF-TCM (13%) following IVE There was no obvious difference in morphology or in total cell numbers (25-35 cells) in blastocysts derived from the two groups (data not shown). These results suggest that oocytes matured in PFTCM are similar to oocytes matured in control medium in terms of their developmental competence to form blastocysts in vitro. However, the viability of IVM-IVF-derived embryos was not confirmed by embryo transfer, the only unequivocal test. Only 2% of putative IVM-IVF embryos developed to the blastocyst stage when oocytes were matured in PF-WM. A similar low rate of blastocyst development was also reported for bovine oocytes matured in WM (3%) compared to TCM 199 (15%) under serum-free conditions and fertilized in vitro [39]. As hypothesized above, the extremely low embryo development after IVF of bovine [39] and pig oocytes (present study) matured in PF-WM could be related to a low intracellular GSH level. Oocyte GSH content after IVM is correlated with in vitro developmental ability of bovine [40] and pig (unpublished results) embryos to the blastocyst stage. In conclusion, the present study describes the successful IVM of pig oocytes in a protein-free culture medium and their subsequent embryonic development to the blastocyst stage after IVE ACKNOWLEDGMENTS The authors thank T.C. Cantley and R. Cabot for the transport of ovaries and B. Nichols for secretarial assistance with the preparation of this manuscript.

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factor and transforming growth factor-a on in vitro maturation of cumulus cell-enclosed bovine oocytes in a defined medium. J Reprod Fertil 1994; 100:439-446. 26. Chen L, Mao SJT, Larsen WJ. Identification of a factor in fetal bovine serum that stabilizes the cumulus extracellular matrix: a role for a member of the inter-ao-trypsin inhibitor family. J Biol Chem 1992; 267:12380-12386. 27. Chen L, Mao SJT, McLean LR, Powers RW, Larsen WJ. Proteins of the inter-ca-trypsin inhibitor family stabilize the cumulus extracellular matrix through their direct binding with hyaluronic acid. J Biol Chem 1994; 269:28282-28287. 28. Chen L, Zhang H, Powers RW, Russell PT, Larsen WJ. Covalent linkage between proteins of the inter-a-inhibitor family and hyaluronic acid is mediated by a factor produced by granulosa cells. J Biol Chem 1996; 271:19409-19414. 29. Downs SM. Specificity of epidermal growth factor action on maturation of the murine oocyte and cumulus oophorus in vitro. Biol Reprod 1989; 41:371-379. 30. Harper KM, Brackett BG. Bovine blastocyst development after in vitro maturation in a defined medium with epidermal growth factor and low concentrations of gonadotropins. Biol Reprod 1993; 48:409416. 31. Downs SM, Schroeder AC, Eppig JJ. Serum maintains the fertilizability of mouse oocytes matured in vitro by preventing hardening of the zona pellucida. Gamete Res 1986; 15:115-122. 32. Ducibella T, Kurasawa T, Rangarajan S, Kopf GS, Schultz RM. Precocious loss of cortical granules during oocyte meiotic maturation and correlation with an egg-induced modification of the zona pellucida. Dev Biol 1990; 137:46-55. 33. Schroeder AC, Schultz RM, Kopf GS, Taylor FR, Becker RB, Eppig JJ. Fetuin inhibits zona pellucida hardening and conversion of ZP2 to ZP2 f during spontaneous mouse oocyte maturation in vitro in the absence of serum. Biol Reprod 1990; 43:891-897. 34. Wang WH, Abeydeera LR, Okuda K, Niwa K. Penetration of porcine oocytes during maturation in vitro by cryopreserved, ejaculated spermatozoa. Biol Reprod 1994; 50:510-515. 35. Nagai T, Kikuchi K, Motlik J. In-vitro fertilization of pig follicular oocytes at metaphase-I. Theriogenology 1991; 35:248. 36. Abeydeera LR, Okuda K, Niwa K. Activation of bovine oocytes penetrated after germinal vesicle breakdown. Zygote 1994; 2:273-279. 37. Yoshida M, Ishigaki K, Pursel VG. Effect of maturation media on male pronucleus formation in pig oocytes matured in vitro. Mol Reprod Dev 1992; 31:68-71. 38. Yoshida M. Role of glutathione in the maturation and fertilization of pig oocytes in vitro. Mol Reprod Dev 1993; 35:76-81. 39. Rose TA, Bavister BD. Effect of oocyte maturation medium on in vitro development of in vitro fertilized bovine embryos. Mol Reprod Dev 1992; 31:72-77. 40. De Matos DG, Furnus CC, Moses DE Baldassarre H. Effect of cysteamine on glutathione level and developmental capacity of bovine oocyte matured in vitro. Mol Reprod Dev 1995; 42:432-436.