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a Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Removing serum and/or somatic cell coculture and replacing them with BSA as a protein source in a variety of culture media has resulted in good embryo development [8–11]. Another approach has been to include BSA or serum during only part of the embryo culture period ("two-step" culture systems), with the remaining period of culture being completely protein free [12, 13]. However, medium containing BSA is still poorly defined, and it contains proteins and other contaminants that may include energy substrates and growth factors [7]. In addition, the embryotrophic properties of BSA may vary greatly between suppliers, and even between lots [14]. This variation makes it difficult to obtain consistent results and to compare data between laboratories.
Relatively few attempts have been made to culture bovine embryos in completely defined, protein-free medium. Developmental results are generally lower than those reported for culture media supplemented with somatic cells, serum, or BSA [2, 15, 16]. An efficient system is necessary if differences in response to culture medium additives are to be determined. In addition, it is important to examine the viability of embryos cultured in a completely defined medium. Indicators of embryo viability such as metabolism, cell number, freezability, and especially pregnancy/calving results are critical if protein-free media are to be accepted for use in research laboratories and in the commercial bovine embryo transfer industry.
Metabolic activity of an embryo may have predictive value for evaluating viability after transfer under certain conditions. Glucose uptake and metabolism are related to embryo developmental competence, and probably reflect energy requirements of the embryo [2, 17, 18]. These requirements include synthetic precursors for protein synthesis, ATP, and energy demands for the activity of the Na+/K+ ATPase necessary for expansion of the blastocoele cavity [19]. Culture conditions are known to affect the ability of an embryo to metabolize glucose [20–22]. Embryos cultured in oviductal cell-conditioned medium had a higher rate of glucose metabolism but lower cell numbers and delayed development, suggesting that high rates of glucose metabolism may reflect less viable embryos [20].
The objective of this study was to relate metabolism to development supported by various culture media. We compared three semi-defined two-step culture systems: synthetic oviduct fluid (SOF), Gardner's G1/G2, and hamster embryo culture medium with 11 amino acids (HECM6) followed by tissue culture medium 199 with 10% bovine calf serum. These media were modified by reducing or removing protein and/or adding energy substrates. Viability of embryos was addressed by cell counts and measurement of embryo metabolism.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cumulus oocyte complexes (COC) were recovered from abattoir cow ovaries. Ovarian follicles measuring 2–10 mm were aspirated using vacuum suction (100 mmHg; 28 ml/min). COC were selected for a compact cumulus mass of at least 3 layers of cumulus cells and homogeneous cytoplasm. Selected COC were washed three times in modified basic medium-Hepes (mBM-Hepes, a modification of TL-Hepes-polyvinyl alcohol [TL-Hepes-PVA] [23] made by deleting glucose, phosphate, and lactate) and placed into 250 µl of maturation medium in 4-well plates (Nunc, Roskilde, Denmark), 25–50 oocytes per well. Oocyte maturation medium consisted of tissue culture medium 199 (TCM199; Gibco, Grand Island, NY) supplemented with 10% bovine calf serum (BCS; HyClone Laboratories, Logan, UT), 0.25 mM pyruvate (Sigma Chemical Co., St. Louis, MO), 0.01 U activity/ml each bLH and bFSH (from bovine pituitary; Sioux Biochemical, Sioux Center, IA), 50 ng/ml epidermal growth factor (Sigma), and 1 µg/ml estradiol (Sigma). Oocytes were matured for 24 h at 39°C in an atmosphere of 5% CO2 in air.
After maturation, all oocytes were washed three times in mBM-Hepes and transferred into 500 µl fertilization medium in a 4-well plate (25–50 oocytes per well). No selection was performed after maturation. Oil overlays were not used for in vitro maturation or fertilization. Fertilization medium consisted of a modified Tyrode's medium supplemented with 6 mg/ml fatty acid-free BSA (Sigma) and 0.25 mM pyruvate (Sigma) [24]. In addition, nonessential amino acids were included at 2 times the concentration present in Eagle's Minimal Essential medium (MEM) [25]. Frozen bull sperm (American Breeders Service, DeForest, WI) were thawed and prepared by a swim-up separation procedure. Oocytes were incubated with 1.0 x 106/ml sperm, 2 µg/ml heparin (Sigma), and PHE (penicillamine, 20 µM; hypotaurine, 10 µM; epinephrine 1 µM; [24, 26]) for 18–22 h at 39°C in an atmosphere of 5% CO2 in air.
After incubation with sperm, all inseminated oocytes were placed into 1 ml mBM-Hepes and vortexed to remove cumulus cells, then washed an additional time in mBM-Hepes. No selection was performed after fertilization. Inseminated oocytes were then moved into 50-µl drops of culture medium under 10 ml of paraffin oil (Fisher Scientific, Pittsburgh, PA), 9–12 per drop. Embryo cultures were performed at 39°C in an atmosphere of 5% CO2:10% O2:85% N2. After 72 h of culture (96 h postinsemination; hpi), all inseminated oocytes were removed from culture drops, examined for cleavage, washed three times in mBM-Hepes, and placed into fresh preequilibrated culture drops. No embryos were removed from culture at this time. Embryo development was examined on Day 8 (192 hpi). At this time, morulae and blastocysts were evaluated for cell number and for glucose and pyruvate metabolism.
Culture Media
Experiment 1 Three semi-defined, two-step culture systems were compared for their ability to support bovine embryo development in vitro (Table 1). The first system was synthetic SOF [11, 27, 28] containing 8 mg/ml BSA (#81–001, lot 94, Pentex; Miles, Kankakee, IL), MEM essential and nonessential amino acids (ICN, Costa Mesa, CA), and 1 mM glutamine (Sigma), with 0.1 mM EDTA (Sigma) [29] for the first 72 h followed by SOF without EDTA with the addition of MEM vitamins (ICN) for the remaining 96 h of culture. SOF was adjusted to pH 8.0 with 0.1 M NaOH after addition of amino acids. The second system consisted of Gardner's G1/G2 culture media (G1/G2) [30, 31]. Gardner's G1 medium, used for the first 72 h of culture, contained 2 mg/ml BSA, MEM nonessential amino acids, and 1 mM glutamine, with 0.1 mM EDTA and 0.1 mM taurine (Sigma). Gardner's G2 medium, used for the remaining 96 h of culture, contained 2 mg/ml BSA (Miles), MEM nonessential amino acids, and 1 mM glutamine, with MEM essential amino acids. Both G1 and G2 media were adjusted to pH 7.6 with 0.1 M NaOH after addition of amino acids. Adjustments to pH were made to culture medium to stabilize bicarbonate during storage. The third culture system was one that is routinely used in our laboratory for the production of bovine embryos [13], HECM6 (chemically defined basic medium-3 containing PVA [32] supplemented with 11 amino acids [33]) for the initial 72 h of culture followed by TCM199 with 10% BCS (HyClone) for the remaining 96 h of culture (HECM6/199+BCS).
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Experiment 2 The effect of reducing or eliminating BSA in SOF medium on bovine embryo development in vitro was examined. Five variations in the BSA content of SOF medium were studied: SOF with 8 mg/ml BSA throughout culture as described for experiment 1 (SOF+8BSA); SOF with 2 mg/ml BSA throughout culture (SOF+2BSA); SOF with BSA completely replaced with 0.1 mg/ml PVA (Sigma) throughout culture (SOF+PVA); SOF with 2 mg/ml BSA during the first 72 h of culture with BSA replaced by PVA in the final 96 h of culture (SOF+2BSA/+PVA); and SOF with BSA replaced by PVA in the initial 72 h of culture with 2 mg/ml BSA in the final 96 h of culture (SOF+PVA/+2BSA). These media were compared to the control system, HECM6/199+BCS.
Experiment 3 Embryo development to the blastocyst stage in vitro, and the metabolic activity of the resulting embryos, were compared in three semi-defined two-step culture systems and one completely defined culture system. The culture media used were HECM6/199+BCS; SOF+8BSA; SOF with BSA replaced by PVA in the initial 72 h of culture with 8 mg/ml BSA in the final 96 h of culture (SOF+PVA/+8BSA); and HECM6, containing PVA, supplemented with 0.25 mM pyruvate and 2 mM glucose throughout culture (HECM6+P+G).
Determination of Blastocyst Cell Number
Two methods were used to count cell numbers of blastocysts. In the beginning of the first experiment, embryos were incubated for 30–60 sec in a 0.05% solution of Triton X (Sigma) in mBM-Hepes. Embryos were then washed and placed into a solution of 15 µg/ml propidium iodide in mBM-Hepes for 10 min. Embryos were removed from the propidium iodide solution and then mounted in a drop of glycerol on a siliconized slide and covered with a coverslip. At the end of the first experiment and in the entire second and third experiments, blastocysts were stained with Hoechst 33342 as described previously [34]. Briefly, embryos were placed into a 15-µl drop of 0.01% trypan blue solution on a siliconized slide for 30–60 sec. The trypan blue was removed from the slide, and 20 µl of 0.01 mg/ml Hoechst solution was placed over the embryo. The slide was incubated on a warming plate for 3–5 min. The solution was removed from the slide and 16 µl Permount placed over the embryo. A coverslip was then placed over the embryo. The total number of cells in each blastocyst in all experiments was counted using fluorescence microscopy.
Metabolic Measurements
A modification of the hanging drop technique [35, 36] was used to measure metabolism of D-[5-3H]glucose (specific activity 14.5 Ci/mmol; Amersham Life Science, Buckinghamshire, England) and pyruvic acid, sodium salt, [2-14C] (specific activity 6.25 mCi/mmol; American Radiolabeled Chemicals, St. Louis, MO). Labeled substrates were dried under nitrogen and taken up in BM3 medium [32], without lactate, supplemented with 0.5 mM unlabeled glucose (metabolic measurement medium). Metabolic measurement medium was used no more than 24 h after preparation, and was incubated at 37°C in 5% CO2 in air in a 50-µl drop under paraffin oil for 2–5 h before use. After assessment for development, morulae and blastocysts were removed from culture drops, washed in mBM3-Hepes, and placed into a 30-µl drop of preequilibrated metabolic measurement medium under oil, with individual treatments in separate drops. Each embryo was taken up in 2 µl of medium and placed in the cap of a 1.5-ml microcentrifuge tube (Fisher) together with 2 µl of preequilibrated metabolic measurement medium containing the radiolabeled substrates. The final concentrations of metabolic substrates present were 0.517 mM glucose and 0.5 mM pyruvate. The tube contained 1.5 ml of 25 mM bicarbonate solution preequilibrated with a 5% CO2:10% O2:85% N2 mixture. The cap was placed loosely over the tube, and the air space was gassed with the same mixture for 5 sec. The cap was then closed and the tube placed carefully into a 37°C incubator for 3 h. Five sham tubes and five total-count tubes, prepared in an identical manner but containing no embryo, were prepared for each replicate. Total-count tubes were shaken to mix the media before and after incubation.
At the end of the 3-h culture period, caps were carefully removed from the tubes, and 1 ml of bicarbonate solution was removed from each sample, sham, and total-count tube. The bicarbonate was placed into a glass scintillation vial containing 200 µl 0.1 M NaOH solution to convert the dissolved CO2 and bicarbonate into carbonate. Each sample tube was examined to verify the presence of 1 embryo, and the embryos were recovered into individual numbered wells of a 64-well plate (Falcon, Becton-Dickinson; Lincoln Park, NJ). Each embryo number corresponded to a scintillation vial. Embryos were then stained and counted for cell number. Scintillation vials were held at 4°C for 18–24 h, at which time 10 ml scintillation fluid (Readi-Safe; Beckman Instruments, Fullerton, CA; or Polyfluor; Packard Instrument Co., Meriden, CT) was added to each vial. The vials were held at room temperature in the dark for 24 h and then counted for 4 min in a liquid scintillation counter programmed for dual-label counting. The amount of glucose and pyruvate metabolized by each embryo was calculated as described by Tiffin et al. [37]. Recovery efficiency of each labeled substrate was examined using the technique described above with NaH14CO3 and 3H2O (American Radiolabeled Chemicals) and sampling every 30 min for 5 h, and was determined to be 100% at 3-h incubation.
Statistical Analysis
Data were collected in 6 replicates over days for experiments 1 and 2, and in 7 replicates over days for experiment 3. Metabolic data were collected from 5 of the 7 replicates in experiment 3. For evaluation of differences between development of in vitro-produced bovine embryos from all selected oocytes to the morula and/or blastocyst stages and frequency of hatching, data were arcsin transformed and analyzed using a randomized block design ANOVA, blocked for replicate. Treatment differences were determined using a Bonferroni (all-pairwise) multiple comparison test. Cell numbers and metabolic measurements were also analyzed using a randomized block design ANOVA blocked for replicate, with treatment differences determined using a Bonferroni (all-pairwise) multiple comparison test. The p value used to determine significance in all tests was 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There were no differences in development to morula and blastocyst of embryos cultured in SOF, G1/G2, or HECM6/199+BCS (Table 2). However, significantly more embryos developed to the blastocyst stage in HECM6/199+BCS than in G1/G2. The percentage of cleaved embryos and mean cell number of blastocysts resulting from these three culture systems did not differ (Table 2). The method of staining embryos was changed in the middle of the first experiment owing to difficulties in staining serum-derived embryos with propidium iodide. Embryos grown in serum-free conditions (SOF, G1/G2) stained very clearly, but embryos cultured with serum (HECM6/199+BCS) appeared smudged and fuzzy, and cell numbers were often difficult to count. This may have been due to the presence of lipid inclusions in the serum-derived embryos. Staining with Hoechst 33342 produced clear, brightly stained nuclei with all culture systems. There was also no difference in embryo development at 72 h of culture between SOF, G1/G2, and HECM6/199+BCS media (8 cells or more: 56%, 57%, 66%; 12–16 cells or more: 34%, 25%, 28%, respectively). There were differences in the percentage of embryos reaching the hatching blastocyst stage as a percentage of the total number of inseminated oocytes between HECM6/199+BCS (35%), SOF (10%), and G1/G2 (7%). There were no differences in morphological grade of morulae and blastocysts between the treatments (data not shown).
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Experiment 2: The Effect of Reducing or Eliminating BSA in SOF Medium on Bovine Embryo Development In Vitro
The results of experiment 2 are presented in Table 3. Development to morula and/or blastocyst was significantly decreased by replacing BSA with PVA in SOF culture medium when compared to HECM6/199+BCS or SOF+8BSA. Removing BSA from the final 96 h of culture was more detrimental than removing BSA from the initial 72 h of culture when compared to SOF+8BSA. Removing BSA from either the initial 72 h or the final 96 h of culture had no effect on development in comparison to reduced concentrations of BSA present throughout culture. There was no significant difference in embryo cleavage or blastocyst cell number among the treatments. In this experiment, there was no difference in the number of blastocysts that were able to hatch from the zona pellucida as a percentage of total inseminated oocytes between HECM6/199+BCS (34%) and SOF+8BSA (25%).
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Experiment 3: Development and Metabolism of Bovine Embryos after Culture in Semi-Defined and Completely Defined Culture Systems
Development to morula and/or blastocyst of embryos cultured in the completely defined medium HECM6+P+G was significantly reduced in comparison to that in control and standard SOF media (Table 4). However, morula and blastocyst development (21%) was attained. Development of embryos cultured in completely defined medium was not significantly reduced when compared to that in culture in SOF with BSA present in only the final 96 h of culture. Development of embryos cultured in SOF with BSA present in the late stages of culture was not significantly reduced. Culture in either SOF treatment resulted in blastocysts that had significantly higher mean cell numbers than blastocysts cultured in control medium (Table 4). Cell numbers of embryos cultured in completely defined medium were not reduced compared to control values. However, there were significant differences in embryo hatching as a percentage of inseminated oocytes (HECM6/199+BCS = 27%, SOF+8BSA = 36%, SOF+PVA/+8BSA = 19%, HECM6+P+G = 3%). Across all experiments, the hatching frequency (data not shown) for HECM6/199+BCS (76 of 243, 31%) was not significantly different from that for SOF+8BSA (52 of 225, 23%). Embryos grown in the presence of serum appeared dark and granular compared to embryos grown in the presence of BSA or PVA (Fig. 1).
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The oxidation of pyruvate per embryo did not differ in morulae and blastocysts cultured in any of the treatment media (Table 5). The oxidation of pyruvate per cell was lower in embryos cultured in SOF without BSA in the initial stages of culture than in standard SOF medium. Utilization of glucose per embryo was lower in embryos that developed in completely defined medium than in those cultured in control medium (Table 5). When normalized to a per cell basis, glycolytic rate was also significantly higher in embryos cultured in control medium than in medium without serum or BSA. Both glycolysis and oxidation rates per embryo increased as embryos progressed from the morula through the hatched blastocyst stages (Fig. 2A). However, when glycolysis and oxidation rates are expressed on a per cell basis, there is no significant increase as cells progress from the morula through the hatched blastocyst stages (Fig. 2B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study delineates the important interval for inclusion of BSA in culture medium as a component of the in vitro system employed. Embryos cultured without BSA in the final stages of culture developed at a lower frequency and had reduced blastocyst cell numbers (Table 3), while embryos cultured without BSA in the initial stages of culture developed as well as embryos cultured in the continuous presence of BSA (standard SOF). This is true regardless of the BSA concentration used in the second step (8 mg/ml versus 2 mg/ml). This indicates that the metabolic requirements of early cleavage stage embryos can be met in the absence of protein, while requirements for embryos at the 12- to 16-cell stage and later are lacking in protein-free medium. Whether BSA contributes a factor to help in blastocoele formation and expansion, or whether BSA is providing the embryo with a metabolic substrate lacking in the culture medium, is unknown. Alternatively, albumin itself could be an important macromolecule in embryo development.
The metabolic activity of preimplantation-stage bovine embryos can be evaluated by measuring substrate utilization. It appears that bovine embryos utilize glutamine and pyruvate in the early cleavage stages, with glycolysis becoming the predominant pathway after the morula stage [19, 35]. Thompson et al. [41] demonstrated that the major metabolic fate of glucose in postcompaction in vitro-produced bovine embryos is lactate production. Pyruvate metabolism increases after morula formation, indicating that oxidative metabolism does play a role in blastocyst formation, probably as a source of energy [19]. In agreement with Rieger et al. [19], the results presented here showed an increase in oxidative metabolism from the morula through the blastocyst stage. However, on a per cell basis, oxidative metabolism remained constant. These findings support the hypothesis that oxidative metabolism does play an important role in blastocyst formation, expansion, and hatching and may provide the cell with enough ATP to complete these tasks. Glucose metabolism via the Krebs cycle does not play a large metabolic role at any stage [35].
The present study and others [42] demonstrate that culture medium can affect metabolism of the resulting embryos, so it is unclear how much of what is currently known about embryo metabolism may be an artifact of the culture conditions. Concentrations of glucose above physiological levels in the culture medium may artificially drive glycolysis to abnormally high levels, depending on stage of preimplantation development [20, 43]. Simply because an embryo metabolizes a substrate does not mean that this is a normal pathway in vivo, particularly when substrates are used at nonphysiological concentrations. There is also a question as to the reliability of using metabolism of an embryo to provide information on its viability and developmental competence. Rieger et al. [20] showed that embryos that are delayed in reaching the blastocyst stage and that have fewer cell numbers, traits associated with reduced viability, displayed increased rates of glycolysis. The best way to evaluate embryo metabolism may be to compare metabolic pathways of in vitro-produced embryos to in vivo-derived embryos of the same stage within the same experiment. In the present study, embryos grown in completely defined culture medium had rates of glycolysis closely resembling those reported for in vivo embryos [44], whereas embryos cultured in medium containing serum had significantly higher glycolytic rates. It is difficult, however, to compare results between these studies, as different techniques and substrate concentrations were used. An increased rate of glycolysis at the blastocyst stage may be an indication of inadequate culture conditions and may reflect reduced embryo viability, as is the case in mice [45]. However, this remains to be demonstrated in cattle. In this study, embryos cultured in the presence of serum had very good development to the blastocyst stage, with cell numbers equal to or lower than those cultured in BSA, but had elevated glycolytic rates. Whether elevated glycolysis does indeed indicate reduced viability in the cattle cannot be definitively known until direct comparisons to in vivo-produced embryos and the appropriate embryo transfer experiments are conducted.
In conclusion, during the first 3 days of preimplantation development in vitro there is little requirement for protein, but thereafter, protein stimulates development to the blastocyst stage. The mechanism for this stimulation is undetermined. Embryos grown in the presence of serum had elevated glycolytic rates and appeared dark and granular. Removing serum from the culture medium altogether and replacing it with BSA in only the final stages of culture resulted in development equal to that with serum. Although development was reduced when neither serum nor BSA present, this defined culture system can be used for the specific purpose of examining substrate requirements.
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FOOTNOTES |
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2 Correspondence and current address: Rebecca L. Krisher, Department of Animal Sciences, 1151 Lilly Hall, Purdue University, West Lafayette, IN 47907–1151. FAX: 765 494 9346; rkrisher{at}purdue.edu
Accepted: January 15, 1999.
Received: March 9, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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