Fertilizability and developmental capacity of bovine oocytes cultured individually in a chemically defined maturation medium

ELSEVIER

FERTILIZABILITYAND DEVELOPMENTALCAPACITY OF BOVINE OOCYTES CULTURED INDIVIDUALLYIN A CHEMICALLYDEFINEDMATURATIONMEDIUM S. Mizushima and Y. Fukui' Laboratory of Animal Genetics and Reproduction, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan Received for publication: Hay 24, 2000 Accepted: November 13, 2000 ABSTRACT This study investigated effects of adding hypotaurine (HT), 13merocaptoethanol (I~-ME), or both into a chemically defined maturation medium (TCM-199 containing 0.1% polyvinyl alcohol: PVA) on maturation, fertilization and development of individually (single) cultured bovine oocytes. Mean GSH concentration in the oocytes cultured in the medium supplemented with either 13-ME(1.11 +0.05 nM) or HT plus 13-ME(0.97 _+ 0.03 nM) was significantly (P < 0.05) higher than that in the medium containing PVA alone (0.75 +_ 0.03 nM). Adding I~-ME showed a significantly (P < 0.05) higher rate of the second metaphase stage (93.6 ___ 3.3%) than in the medium containing PVA alone (single-control) (65.2 ___ 7.9%). Adding both HT and 13-MEshowed significantly (P < 0.05) higher rates (92.6 _+ Z.7%) of normal fertilization than did adding HT alone (63.5 +_ 4.6%). Also, adding both I-IT and 13-MEsignificantly (P < 0.05) lowered the polyspermy rate than did adding HT alone. Adding either p-ME or both HT and 13-MEshowed no significant difference in cleavage. Blastocyst development did not improve significantly adding either HT, 13-MEor both, although 13-MEalone or HT plus 13-MEtended to result in a higher rate of blastocysts (6.4 and 6.8%, respectively) than resulted without additives (1.6%). Our results show that adding 13-MEto a chemically defined maturation medium increased the intracellular GSH level of bovine oocytes cultured individually, and can improve the maturation rate leading to the blastocyst stage throughout in vitro production. © 2001 by ElsevierScience Inc.

Key words:individual culture, chemically defined medium, hypotaurine, ~-mercaptoethanol, IVM, bovine 'Correspondence and reprint requests: Y. Fukui, Fax: 81-155-49-5462; e-mail: [email protected]. Theriogenology 55:1431-1445, 2001 © 2001 Elsevier Science Inc.

OO93-691X/01/$-see front matter PIh S0093-691X(01)O0492-7

Theriogenology

1432 INTRODUCTION

Oocytes aspirated from the antral follicle of slaughtered or live animals are usually matured, fertilized and cultured in a group. Factors affecting oocyte maturation or embryonic development can not be determined by the group culture system. Several studies have cultured cattle (3, 6, 8, 14, 20-23, 30, 37), ovine (18), porcine (5), and mouse (26, 28, 38) oocytes or embryos individually. An individual in vitro production system is necessary for limited embryonic materials for IVC (such as an oocyte of valuable livestock or wildlife animals). An attempt must be made to pass each oocyte or embryo through each in vitro production step. This is especially true for oocyte collection by transvaginal oocyte recovery or embryo production with labor- intensive micro-manipulative techniques, such as nuclear transfer. Most culture media, however, were not chemically defined such as in granulosa or oviduct cell co-culture or other conditioned media, resulting in very low developmental capacity beyond the initial cleavage. Carolan et al. (8), Lim et al. (30) and Hangemann et al. (20, 21) achieved high blastocyst developmental rates (20 to 40 %) in a totally individual culture system. However, they supplemented fetal calf serum (FCS) or bovine serum albumin (BSA) in the maturation medium or used a co-culture system. In our previous study (16), we established a single oocyte culture system using a chemically semi-defined maturation medium containing BSA throughout in vi.tro production that was as effective as oocytes cultured in a group. However, polyvinyl alcohol (PVA) supplemented in the medium markedly decreased blastocyst development (7.1%) compared with BSA (15.6%). Either hypotaurine (HT) or taurine has been used in culture medium for IVF (16, 49) or IVC (4, 42) to protect against deleterious conditions in vitro, such as peroxidation of membrane lipids. Several other compounds, such as p-mercaptoethanol (p-ME), cysteine, cystine, and cysteamine also were used to increase glutathione (GSH) levels in bovine oocytes (1 O, 11 ) and in vitro- produced bovine embryos (7, 31,44, 48). The GSH plays an important role in protecting the cell from oxidative damage. We chose HT and p-ME in the present study, because the presence of either HT (10 mM) or p-ME (5 ~M) in a chemically defined maturation medium resulted in non-significant rates of blastocyst development (3.4 and 8.5% for HT and p-ME, respectively) compared with a single-culture medium containing FCS (8.8 and 7.9% for each control, respectively) in a preliminary study. Therefore, to establish a simple individual culture system of bovine oocytes using a completely defined, protein-free maturation medium, the additional effects of either HT, p-ME, or both in a maturation medium (TCM 199 + PVA) for IVM, were examined for intracellular glutathione

Theriogenologx

1433

(GSH) concentration, nuclear maturity, fertilizability and developmental capacity of individually cultured bovine oocytes. MATERIALS AND METHODS Oocyte Collection and In Vitro Maturation (IVM) Ovaries from Holstein heifers and cows were collected at a local abattoir shortly after slaughter and were transported in physiological saline at 30 to 35t3 to the laboratory within 2 h. The ovaries then were washed in fresh saline. The contents of antral follicles (2 to 8 mm in diameter) were aspirated using an 18-gauge needle attached to a 5 mL disposable syringe. Cumulus-oocyte complexes (COOs) were washed in Tissue Culture Medium 199 (TCM199, Dainippon Pharmaceutical Co., Ltd. Australia) supplemented with 0.3% (w/v) BSA (fatty acid free, Fraction V; Sigma Chemical Co., St. Louis, MO, USA), 2 mM sodium bicarbonate and 10 mM HEPES (TCM199-HEPES). The COCs were cultured in microdrops (2 5 ~L) of a maturation medium supplemented with 25 mM NaHCO3and covered with mineral oil (Squibb & Sons Inc., Princeton, NJ, USA). The maturation medium was also supplemented with 0.0Z AU/mL of porcine pituitary FSH (Antrinr"; Denka Chemical Co., Ltd., kawasaki, Japan) and 1l~g/mL estradiol-17p (Sigma, USA). The oocytes were cultured for 24 h at 39t3 in an atmosphere of 5% COz in air and >95% humidity. The HT (Sigma, USA) and 13-ME(Sigma, USA) were weighed and were added to TCM199 supplemented with 0.1% PVA (Sigma, USA) to make up a chemically defined maturation medium. In all experiments, COOs with intact cumulus cells and evenly granulated ooplasm were selected and culture in1 ) group culture (5 to 6 oocytes/drop) in TCM199 supplemented with 10% (v/v) FCS (Life Technologies Oriental, Inc., Tokyo, JAPAN: heat-treated at 56t3 for 30 min); 2) individual culture (1 oocyte/drop) in TCM199 supplemented with 10% FCS; 3) individual culture in TCM199 supplemented with a) 0.1% PVA, b) 0.1% PVA and 10 mM HT, c) 0.1% PVA and 5 ~M I~-ME, or d) 0.1% PVA and 10 mM HT plus 5~M I~-ME.The concentrations of HT (10 mM) and p-ME (5 ~M) were chosen by our preliminary study showing that they resulted in similar rates of blastocyst development compared with a single-culture medium containing FCS. Sperm Preparation and In Vitro Fertilization (IVF) The media for IVF were prepared as described by Fukui (15). Each O.5-mL frozen straw of semen from three Holstein bulls was thawed at 37~2 in a water bath for 30 sec. An aliquot (0.2 mL) of thawed, pooled semen was placed under 1 mL of modified Tyrode's calcium-free medium (capacitation medium, pH 7.4) in conical tubes (Becton Dickinson

1434

Theriogenology

Labware, Lincoln Park, NJ, USA) for a swim-up procedure (15, 39). The top O.8-mL of the medium then was collected after incubation for 1 h at 39T~. The pooled medium containing spermatozoa was washed twice (500 × g, 5 min) with capacitation medium. The final pellet of spermatozoa was resuspended in the capacitation medium to a concentration of 1 × 10 Gspermatozoa/mL. An equal volume of a 200 ~g/mL heparin (Sigma, USA) solution was added to the sperm suspension to yield spermatozoa and a heparin concentration of 12.5 × 10 Gspermatozoa/mL and 1O0 l~g/mL, respectively. The heparin-treated spermatozoa were incubated for 15 min at 39~, 5% COz in air and >95% humidity. After IVH the oocytes were washed three times with a modified Tyrode's medium (washing medium, pH 7.4) containing 2 mM CaCIz, 2 mM NaHCO3 and 10 mH HEPES.The COCs together with 1.5 i=L of washing medium were introduced into a fertilization drop (21.5 ~L, pH 7.8) of a modified Tyrode's medium under mineral oil. An aliquot (2 ~L) of the heparin-treated sperm suspension then was added to give a final concentration of 1 × 106 spermatozoa/mL. Gametes were co-incubated for 30 h at 39T~, 5% COz in air and >95% humidity. In Vitro Culture (IVC) The medium for IVC of oocytes was the synthetic oviduct fluid medium (SOFM) as described by Tervit et al. (45). It was modified by lowering the concentration of 8SA to 8 mg/mL. The SOFM was supplemented with 2% (v/v) MEM essential amino acids (Life Technologies, Inc., Grand Island, NY, USA) and 1% (v/v) MEM nonessential amino acids (Life Technologies, Inc., Grand Island, NY, USA). All media were passed through a 0.2 ~m membrane filter and were equilibrated overnight in an incubator at 39T~ in 5% COz in air and >95% humidity. After 30 h of fertilization, cumulus cells and residual spermatozoa were removed by repeated pipetting. Then, the oocytes were cultured (1 or 5 oocytes/drop) in microdrops (25 I~L) of SOFM at 39~ in an atmosphere of 5% COz, 7% Oz, 88% Nz and >95% humidity. Cleavage (>Z-cell and > 8-ceU) and embryonic development into blastocysts and hatched blastocysts were observed on Days 3, 8 and 10 (Day O=the day of insemination), respectively. Glutathione (GSH) Assay GSH levels were determined using the enzymatic cycling assay of Tietze (47). Oocytes were washed three times in PBS (Ca2÷, Mg2. free) containing 10% PVA and were stored in microtubes (30 oocytes/10 ~L). The oocytes then were frozen at -20% and were thawed twice. After freezing and thawing, 5 ~L ofl.25 M H3PO4was added and the oocytes were broken completely by ultrasonication. Samples were mixed with 1.2 mL of 0.2 M phosphate buffer containing 10 mM EDTA (Wako Pure Chemical Industries, LTD., Osaka, Japan) and 1.2 mL of distilled water. After rapid mixing with 100 I~L of 10 mM 5,5'-dithiobis 2-nitrobenzonic

Theriogenology

1435

acid (Wako, Japan), 50 ~L glutathione reductase (1 units/50 ~L; Wako, Japan), and 50 ~L of 4.3 mM NADPH (Wako, Japan), the increase in absorbance at 412 nm from 30 sec to 5 min was measured. A total GSH concentration (nM/30 oocytes/lO ~L) was calculated by reference to a standard curve prepared with authentic GSH. Oocyte Fixing and Staining At 24 h after IVM and 18 h after IVF, COCs were denuded from cumulus cells by repeated pipetting and were then mounted on glass slides. The oocytes were fixed in 25% (v/v) acetic acid in ethanol for 24 h, and in1% (w/v) orcein in 45% (v/v) acetic acid solution. To determine the nuclear stages, the proportion of oocytes at the second metaphase (M-II) was calculated. To determine the level of IVF, inseminated oocytes were examined for evidence of normal or polyspermic (more than two pronuclei and decondensed sperm heads) fertilization. Normal fertilization was defined as monospermic penetration with both male and female pronuclei. Oocytes penetrated with swollen sperm head(s), but without a male pronucleus were not recorded as normal fertilization. Experimental Design Experiment 1was designed to investigate the effects of additives (HT, 13-ME,and HT plus 13-ME)on intracellular GSH concentrations. Thirty oocytes cultured for IVM from each sample of the groups were pooled, and GSH concentrations were measured (3 replicates). Experiment 2 was designed to investigate effects of additives (HT, 13- ME, and HT plus p-ME) on the nuclear stages of bovine oocytes cultured for 24 h (5 replicates). Experiment 3 was designed to investigate the effects of additives (HT, p-ME, and HT plus 13-ME) on the fertilization of bovine oocytes at 18 h after IVF (4 replicates). Experiment 4 was designed to investigate the effects of additives (HT, 13-ME, and HT plus 13-ME) on cleavage and embryonic development to the blastocyst and hatched blastocyst stages after IVM and IVF (4 replicates). Statistical Analysis In Experiment 1, the GLM procedure of the SAS was used to assess the statistical difference among the me a n intracellular GSH concentrations in three replicates using six different IVM media with and P < 0.05 accepted as significant (43). In Experiments 2, 3 and 4, the mean proportions of the replicates for IVM, IVF and IVC were evaluated for analysis of variance by a general linear models (GLM) procedure using a Statistical Analysis System (SAS). Furthermore, significant differences were tested for the statistical difference among the means with P < 0.05 accepted as significant.

Theriogenology

1436 RESULTS

Experiment 1 Mean GSH concentrations in oocytes cultured in the FCS-containing media were not significantly different between the group (0.78 _+ 0.03 nM) and single culture (0.80 -+ 0.07 nM) systems. The mean GSH concentrations in oocytes cultured individually in the PVA medium supplemented with I~-ME (1.11 ___ 0.05 nM) or HT plus I~-ME (0.97 + 0.03 nM) were significantly (P<0.05) higher than those in the singleculture PVA medium without the additives (0.75 _+ 0.03 nM) (Table 1). The PVA medium supplemented with 13-MEalone showed the highest mean GSH concentration (1.11 nM), and it was significantly (P<0.05) higher than in the FCS-containing media for both group and single culture systems, and also higher than in the single-culture PVA medium supplemented with HT (0.83 +_ 0.04 nM). Experiment 2 The culture systems (group and single) did not affect the maturation rate, when FCS was added to the maturation medium. The proportion of matured (MI) oocytes in the FCS-containing media used for a group culture (82.7_+5.9%) and the PVA-medium supplemented with I~-ME (93.6_+3.3%) were significantly higher than that in the singleculture control medium (PVA only: 65.2_+7.9%) (Table 2).

Table 1. Intracelluar glutathione (GSH) concentration after 24 h of bovine oocytes cultured in maturation media supplemented with hypotaurine (HT) or 13-mercaptoethanol (t3-ME), or both in different culture systems Protein

Additives

Culture system

No. of GSH concentration replicates (nM/30 oocytes/10 I~L)

10 % FCS

Group

3

0.78

_+ 0.03 ,,b

10 % FCS

Single

3

0.80

_+ 0.07 ~,b

0.1% PVA

Single

3

0.75

+ 0.03 b

Single Single

3 3

0.83 1.11

+ 0.04 ,,b + 0.05 c

O.1% PVA 0.1% PVA

HT (1) I~-ME (2)

0.1% PVA (1) + (2) Single 3 0.97 _+ 0.03 a,c ~Values with different superscripts in a column are significantly different (P < 0.05)

Theriogenology

1437

Table 2. Effect of adding hypotaurine (HT) or 13-mercaptoethanol (13-ME) to in vitro maturation media on nuclear maturation of bovine oocytes cultured individually (single) or in a group

Protein

Additives

10 % FCS 10 % FCS

-

0.1% PVA

Culture system Group

No. of Total no. of replicates oocytes 5 52

Metaphase II (%+__SEM) 82.7 + 5.9 ~

Single

5

52

86.5 ±

4 . 3 ab

Single

5

46

65.2 ±

7.9 b

0.1%PVA

HT(1)

Single

5

47

74.5 ± 3.4 ab

0.1% PVA

13-ME(2)

Single

5

47

93.6 ± 3.3 a

Single

5

44

77.3 +11.2 ~b

0.1%PVA ( 1 ) + ( 2 )

a'DSignificant difference (P < 0.05). Experiment 3 The additives and culture systems did not affect the proportions of penetrated oocytes. Adding both HT and B-ME significantly (P < 0.05) increased the normal fertilization rate (92.6 ± 2.7%) more than adding HT alone (63.5 ± 4.5%), and also significantly (P < 0.05) lowered the polyspermy rate (7.4 ± 2.7%) than adding HT (36.5 ± 4.6%) (Table 3). Experiment 4 The proportion of cleaved oocytes to =>2-cell stage in the singleculture, PVA-containing medium (61.9 ± 7.3%) was significantly (P < 0.05) lower than in the FCS-containing medium used for a group culture (84.1 ± 3.9%). The proportions of >8-cell embryos in the FCScontaining medium in a group culture (44.9 ± 9.0%) was significantly higher than the other culture systems with different additives. The single-culture with FCS supplementation resulted in a nonsignificant rate of blastocyst development (16.5 ± 2.7%) in the group culture using FCS-containing medium (24.5 + 3.8%). Adding either B-ME or HT plush-ME in the single-culture, PVA-containing medium tended to improve the blastocyst rate (6.4 ± 1.2 and 6.8 ± 2.4%, respectively) compared with the PVA-medium without additives (1.6 ± 0.9%). The developmental rate to hatched blastocysts in the PVA-control medium (0.8 ± 0.7%) was significantly lower than the FCS-containing media in a group (14.7 ± 2.2%) and single culture (9.3 + 1.0%). Although the

Theriogenology

1438

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Theriogenology

1439

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Theriogenology

1440

adding of HT, 13-MEor both HT and I}-ME in the single culture PVA- medium tended to improve the mean rate of hatched blastocytsts (2.4 to 2.6%), there was no significant difference (Table 4). DISCUSSION In general, most IVM media are supplemented with FCS (12, 27, 33, 36, 50) or BSA (13). Growth factors and hormones, and many other unknown factors, are abundant in FCS (17, 40, 41 ) and BSA (2, 19, 24, 25). Therefore, IVM media supplemented with FCS or BSA undoubtedly conflict with proper quality control and repeatability among laboratories. To eliminate such variability, a more defined culture system for IVM must to be developed. The present study used a serum- and protein-free medium supplemented with PVA. In the control medium containing FCS, our single- culture system resulted in similar rates of maturation, normal fertilization and blastocyst development compared with the group culture, although the blastocyst rate tended to be lower in the single-culture system than in the group-culture system (16.5 and 24.5%, respectively). Using TCM199 supplemented with PVA adding p-ME, or both HT and I}-ME, the oocytes cultured individually were not significantly different in the rates of maturation, fertilization and cleavage compared with the singleculture medium containing FCS, but the rate of blastocyst development was still low in the chemically defined medium. The concentrations of HT (10 mM) and t3-ME (5 ~M) used in the present study were determined from a preliminary study. For a group culture during IVM or IVC, a higher I~-ME concentration (1 O0 to 500 I,M) was used (7, 1O, 44). Recently, a new IVC system (WOW method) for an individual bovine embryos has been established by Vajta et al. (48), and reported an encouraging result of blastocyst development compared with a group culture. In combination with such a new culture system, further investigation to determine an appropriate concentration of I}-ME for bovine oocytes cultured individually in a chemically defined maturation medium. Full maturation involves both nuclear and cytoplasmic maturation that confers on the oocyte capacity to support normal fertilization and early embryonic development. Observing nuclear meiosis can assess nuclear maturation, and GSH levels can assess cytoplasmic maturation. The concentration of intracellular GSH after IVM may be a valuable marker to assess the degree of cytoplasmic maturation in pig (1) and bovine (1 O, 11 ) oocytes. The GSH is a major non-protein sulfhydryl compound in mammalian cells (34). The concentration of intracellular GSH increases as cytoplasmic maturation is completed (1 O, 32, 34) and is capable of fully supporting bovine embryonic development. The results of the present study show that I}-ME is indicated for cytoplasmic maturation of bovine oocytes. It increases the intracellular GSH levels and maturation rate

Theriogenology

1441

compared with the single-cultured oocytes in the PVA medium without additives. However, the normal fertilizability and developmental capacity to the blastocyst stage did not improve significantly, although there was a tendency to improve blastocyst rates when 13-MEwas added in the medium. Low fertilizability and developmental competence results from deficiencies in cytoplasmic maturation at IVM (9, 29, 35, 46), especially for individually oocytes compared with oocytes cultured in a group (18, 28, 38), and our results support this hypothesis. However, some oocytes in this study appeared to have matured (M-II) normally at the nuclear level, but they did not mature completely at the cytoplasmic level. In this study, HT did not increase intracellular GSH concentration during oocyte maturation and a positive effect on embryonic development was not shown. The present results also showed no synergistic effect of HT and 13-MEon maturation, normal fertilization and blastocyst development. Oocytes cultured in the chemically defined IVM medium supplemented with B-ME increased their capacity to mature normally during IVM and then to prevent polyspermy in inseminated oocytes during IVF. Therefore, it appeared that adding ~-MIE during IVMI was more effective for an individual in vitro culture system than was adding HT. In conclusion, our results show that adding I~-MIEalone increases intracellular GSH concentration of bovine oocytes cultured individually during IVMt and increases in vitro maturation rates leading to the blastocyst stage throughout in vitro production system. However, the developmental capacity in this system is still low. Further research is needed to determine a more appropriate condition for an individual culture system using a chemically defined medium. REFERENCES

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Theriogenology

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