MOLECULAR REPRODUCTION AND DEVELOPMENT 30:226-231 (1991)
Reduction of Embryotoxicity by Protein in Embryo Culture Media LARRY P. FLOOD AND BARBARA SHIRLEY Department of Biological Sciences, University of Tulsa, Tulsa, Oklahoma
ABSTRACT Experiments tested the hypothesis that one role of protein in embryo culture media is protection of embryos against potentially embryotoxic substances in the media. Mouse embryos were cultured in modified Krebs-Ringer bicarbonate medium and in modified Tyrode’s medium, aliquots of which were supplemented with 4 mg/ml of the protein bovine serum albumin (BSA),while other aliquots were left protein free.The media were prepared using water samples that differed in purity, as reflected by differences in conductivity, with tap water being least pure (and considered to have the greatest potential for being embryotoxic) and water that had been purified by reverse osmosis, Milli-Q filtration, and triple distillation being most pure. Embryos were placed in the media while in the two-cell stage of development and their development was assessed after 24, 48, and 72 hr of culture. Rate of embryo development in BSA-supplemented media was greater than that in protein-free media only when the media were prepared with the least purified water samples. Because these water samples would have contained substances not contained in media prepared with purer water, or would have contained the substances in higher concentrations,the data supported the hypothesis that protein can protect embryos during culture by negating effects of embryotoxic substances in the media. Key Words: Bovine serum albumin, Embryo culture, Embryo development, Mouse embryos
INTRODUCTION Chemically defined media used for the culture of preimplantation mouse embryos typically contain the protein bovine serum albumin (BSA) (Whitten, 1956, 1957; Brinster, 1963,1965; Whitten and Biggers, 1968; Ackerman et al., 1984; Chatot et al., 1989). Yet, the contribution of protein, or amino acids, to development of preimplantation mammalian embryos has not been clearly defined. Although mouse embryos develop to blastocysts in media th at contain BSA, it has been variously reported that few embryos develop in medium that contains only a single amino acid (Brinster, 19651, that the presence of glycine alone supports development (Whitten, 1957; Bunim, 19601, and that embryos develop to blastocysts even in the absence of amino nitrogen (Cholewa and Whitten, 1970; Whitten, 1971; Caro and Trounson, 1984). The latter finding suggests t ha t amino acids are not required by mouse 0 1991 WILEY-LISS, INC.
embryos for nutritional purposes during the preimplantation stages of development but inconsistency in the requirement for protein andlor amino acids has caused the role of these substances in successful embryo culture to remain unclear. It has been postulated that protein, or amino acids, can play a role in chelation of embryotoxic components of culture media and some evidence supporting this notion has been obtained; after demonstrating that mouse embryos would develop in medium that contained only glycine as a source of fixed nitrogen, Bunim (1960) found that embryos would also develop in medium in which Versene (sodium edetate) was substituted for the glycine, i.e., in medium in which a chelating agent was substituted for a n amino acid. Similarly, addition of the chelating agent ethylenediamine tetraacetic acid (EDTA) to culture medium has been found to enhance embryo development (Abramczuk et al., 1977) and, furthermore, to permit embryo development in protein-free and amino acid-free media (Fissore et al., 1989; Mehta and Kiessling, 19901, suggesting that protein, amino acids, and EDTA may have similar functions in embryo culture media. If protein does indeed play a role in chelation, the presence of greater quantities of embryotoxic substances in culture media should be associated with a n increased need for protein, or some other chelator, in the culture medium and this study tested that association. A potential source of embryotoxic substances in culture media is the water from which the media are prepared, and the use of highly purified water samples for preparation of culture medium has been found to enhance embryo development in vitro (Fukuda et al., 1987). Although attention is given to water quality in laboratories in which embryos are cultured (Quinn et al., 1984; Condon-Mahony et al., 1985a; Fukuda et al., 1987; Han and Kiessling, 1988; Fissore et al., 1989; Mehta and Kiessling, 19901, the quality of the water used for culture medium preparation probably varies more from one laboratory to another than the quality of any other component of the chemically defined media used in embryo culture. This study tested the hypothesis that a principal
Received April 19, 1991; accepted July 9, 1991. Address reprint requests to Dr. Barbara Shirley, Department of Biological Sciences, University of Tulsa, Tulsa, OK 74104.
ROLE OF PROTEIN IN EMBRYO CULTURE MEDIA function of protein (BSA) in embryo culture media is the chelation of embryotoxic substances, one source of which may be the water from which the media are prepared, and that protein consequently enhances embryo development when embryotoxic substances are present in the medium but has little or no effect when reduction of toxicity is not required. To test this hypothesis, embryos were cultured in media prepared with and without BSA or other nitrogen-containing compounds and each type of medium to be tested was prepared from four grades of water that differed in purity. Tap water was the least pure of the water samples tested and was considered to have the greatest potential for contributing embryotoxic substances to the media. Other water samples were obtained after successive steps in purification of the tap water. Media prepared with and without BSA and prepared from each of the four grades of water were compared with regard to their capacities to support development of mouse embryos to specific developmental stages within specific culture periods. It was believed that a n association between water quality and the enhancement of embryo development by protein in the culture media, if demonstrable, could help explain why some investigators have reported beneficial effects from the inclusion of protein in embryo culture media, but others have found that embryos developed a t normal rates in media th at contained neither protein nor amino acids.
MATERIALS AND METHODS Water Samples Used in Media Preparation Four grades of water, designated TAP, RO, MQ, and 3X, were used in media preparation. TAP water was purified only by the city’s municipal water treatment plant, RO water was produced by further purification of the TAP water with reverse osmosis using a Milli-R04 Reagent Water System (Millipore Corporation, Bedford, MA), MQ water was produced by further purification of RO water with a Milli-Q Type 1 Reagent Grade System (Millipore), and 3X water was produced by subjecting MQ water to triple glass distillation. Mean electrical conductivities 2 SEM (micromhos) of the four grades of water were (1) TAP water: 193.75 2 1.83; (2) RO water: 36.42 2 1.25; (3) MQ water: 0.65 ? 0.04; and (4) 3X water: 1.04 t 0.01. Statistical comparisons of the electrical conductivities of the four grades of water indicated that TAP, RO, and MQ water samples differed significantly from one another in purity (P < 0.01) but that the 3X water was no purer than the RO water (P > 0.05).
The mKRB medium was the modified version of K r e b s Ringer bicarbonate described by Toyoda and Chang (19741, which in a protein-free form consisted of 94.6 mM NaC1, 4.78 mM KCI, 1.71 mM CaCl,, 1.19 mM KH,P04, 1.19 mM MgSO,, 25.07 mM NaHCO,, 21.58 mM Na lactate, 0.5 mM Na pyruvate, 5.56 mM glucose, 50 IU/ml of penicillin (Sigma Chemical Company, St. Louis, MO), and 50 pg/ml of streptomycin (Sigma). This medium was chosen because it has been widely used in the culture of mouse embryos (Ackerman and Swanson, 1983; Ackerman e t al., 1984; Condon-Mahony et al., 1985a,b; Shirley et al., 1985, 1987) and is known to support development to the blastocyst stage when it contains BSA. A protein-free version of mT6 medium (Quinn et al., 1982) was also prepared and consisted of 97.84 mM NaCI, 1.42 mM KCI, 1.78 mM CaCI,, 0.36 mM Na,HP04, 0.47 mM MgCl,, 25.0 mM NaHCO,, 24.90 mM Na lactate, 0.47 mM Na pyruvate, 5.56 mM glucose, 50 IU/ml of penicillin (Sigma), 50 pg/ml of streptomycin (Sigma), and 5 pg/ml of phenol red (Sigma). The mT6 medium was chosen for testing because it is a chemically defined medium that will support viability of both mouse and human gametes, although it is not the medium of choice for human embryo culture, and unlike some of the media used for the culture of human embryos, such as Ham’s F-10 medium (Ackerman et al., 1984; Quigley and Dandekar, 1984; Condon-Mahony et al., 1985a), it normally contains BSA but no amino acids and is used without the addition of blood serum. Thus, it, like the mKRB medium, could be prepared without a nitrogen source simply by excluding the BSA. An aliquot of each medium, mKRB and mT6, was supplemented with BSA (Fraction V, Sigma) in a concentration of 4 mg/ml and the remainder of each medium was used without the addition of BSA or any other source of nitrogen. The media with and without BSA were prepared using each of the four grades of water that had been subjected to different purification regimens. Each variant of the mKRB medium, protein-supplemented and protein-free, was adjusted to pH 7.4 and to a n osmolality of 280 ? 2 mOsm/kg and each variant of the mT6 medium was adjusted to a pH within the range of 7.3-7.45 and to a n osmolality of 260-280 mOsm/kg. Media were filter sterilized and transferred in 2-ml aliquots to flat-side Nunculon culture tubes, 110 x 16 mm (GIBCO, Grand Island, NY). The tubes were incubated, with their caps loosened, at 37°C in 5% COz in air, for 16-18 h r so th a t the media could equilibrate before embryos were placed in them for culture.
Media Preparation Two types of chemically defined media, modified Krebs-Ringer bicarbonate (mKRB) medium (Toyoda and Chang, 1974), and modified Tyrode’s (mT6) medium (Quinn et al., 1982), were selected for testing the relationship between water quality, BSA content of the culture medium, and development of embryos in vitro.
Embryo Collection and Culture Hybrid female mice (C57BL female x CBA male), seven to nine weeks of age, were obtained from the Jackson Laboratory (Bar Harbor, ME) or obtained from Phillips University, Enid, OK, where they were colony bred. The mice were provided Rodent Laboratory Chow 5001 (Ralston Purina Company, St. Louis, MO) and
L.P. FLOOD AND B. SHIRLEY
water ad libitum and were maintained on a light-dark cycle with lights on a t 5:OO AM and off at 7:OO PM. Animals were allowed to acclimatize for at least 2 weeks before being used for experiments. The hybrid females were superovulated with i.p. injections of 5 IU of pregnant mares' serum gonadotropin (Sigma) in 0.1 ml of distilled water, given at 4:OO PM, followed by i.p. injections of 5 IU of hCG (Sigma) in 0.1 ml of distilled water 48 hr later. Immediately after being given the hCG injections, the females were placed individually in the cages of proven male breeders of the CD-1 strain (colony bred using stock from Charles River Breeding Laboratories, Wilmington, MA). The morning after being paired with a male, each female was checked for evidence of mating, the presence of a vaginal plug or redness and distention of the vaginal orifice suggestive of the recent presence of a vaginal plug. Females that showed evidence of mating were sacrificed by cervical dislocation the following day, 42-46 h r after hCG had been injected. Their oviducts and segments of their uterine horns were removed, under sterile conditions, and placed in culture dishes that contained 2 ml of mKRB, or mT6, medium. Oviducts were cut free from the uterine tissue and embryos were flushed from them into the surrounding medium. Embryos were transferred to culture tubes in which culture medium had been allowed to equilibrate to culture conditions since the previous day. Embryos, 5-10 per tube, were cultured for 72 hr, and the development of each embryo was assessed after 24, 48, and 72 h r of culture. Embryos were expected to reach a t least the
four-cell stage within 24 h r of culture, a t least the morula stage within 48 hr, and the blastocyst stage within 72 h r (Saito e t al., 1984a).
Statistical Analysis of Data Chi square tests were used to determine significant differences between numbers of embryos that reached expected stages of development within 24,48, and 72 h r of culture in media with and without BSA and prepared from water samples with different degrees of purity. RESULTS Embryo Development in Modified Krebs-Ringer Bicarbonate Medium Significantly smaller numbers of embryos developed to expected stages within 24,48, and 72 h r of culture in BSA-free medium prepared from TAP water than in BSA-supplemented medium prepared from water of the same grade (Table 1).The mKRB medium prepared from the somewhat purer RO water supported embryo development quite well for 48 hr, regardless of whether it contained BSA, but a significantly smaller number of embryos reached the blastocyst stage within 72 h r of culture in BSA-free than in BSA-supplemented medium prepared from RO water. In mKRB medium prepared from the more highly purified samples of water (MQ and 3x1, a lack of BSA had no significant effect on numbers of embryos that reached the expected stages of development within 24, 48, or 72 h r of culture (Table 1).
TABLE 1. Development of Mouse Embryos in Modified Krebs-Ringer Bicarbonate (mKRB) Medium Prepared With a n d Without Bovine Serum Albumin (BSA) a n d Prepared From W a t e r Samples T h a t Differed in Purity
Water in mediuma TAP
Embryo development to expected stages within specific periods" (No. of embryos a t expected stagekotal number cultured) 48 hr 72 hr 24 hr -BSA +BSA -BSA fBSA -BSA fBSA 57/66'sC (86%) 95/105' (90%) 109/113' (96%) 109/116' (94%)
57/58' 39/66C,D 57/58' 31/66C,D 52/58' (98%) (59%) (98%) (47%) (90%) RO 109/113' 93/105d 107/113' 87/105d!E 105/113' (96%) (89%) (95%) (83%) (93%) 98/104' 109/113d 981104' 106/113e 113/124' MQ (94%) (96%) (94%) (94%) (91%) 3x 120/124' 109/116d 119/124' 106/116e 113/124' (97%) (94%) (96%) (91%) (91%) Sum of TAP, RO, MQ, 3X RO, MQ, 3X MQ, 3X 370/400E 384/399 311/334 3241341 2121229 211/228 (93%) (96%) (93%) (95%) (93%) (93%) aWater samples listed in order of increasing purity. "Embryos were expected to reach the 4-cell stage of development in 24 hr, to reach the morula stage in 48 hr, and to reach the blastocyst stage in 72 hr of culture.
C-eValuesin same column but with different superscripts (lower case) differ significantly (P< 0.05); data from groups not significantly different are pooled and compared at the bottom of the table. C-EValuesignificantly different from that in the column to the right: C (P< 0.02); D (P< 0.001); E (P< 0.05).
ROLE OF PROTEIN IN EMBRYO CULTURE MEDIA Embryo Development in Modified Tyrode's Medium Results obtained with mT6 medium were similar to those obtained with mKRB medium. When the mT6 medium was prepared from TAP water, significantly smaller numbers of embryos developed to the expected stages within 24, 48, and 72 hr of culture in BSA-free than in BSA supplemented medium (Table 2). However, lack of BSA in the medium had no deleterious effect on rate of embryo development when the medium was prepared from any grade of water that was more highly purified than the TAP water. When mT6 medium was prepared from RO, MQ, or 3X water samples, more than 90% of the embryos reached the expected stages of development within 24, 48, or 72 hr of culture regardless of whether the medium contained BSA. Furthermore, the pooled data obtained with mT6 medium prepared from these three grades of water revealed that significantly greater numbers of embryos were a t expected stages of development in BSA-free than in BSA-supplemented medium after 24 and after 72 hr of culture (Table 2). DISCUSSION Data supported the hypothesis that a function of protein in embryo culture medium is protection of embryos from the effects of potentially embryotoxic substances in the medium and that the usefulness of the protein is reduced or eliminated when the medium is prepared from water that has been purified to the extent that it contributes few foreign substances to the medium. When media were prepared from TAP water, the least pure of the water samples used, supplemen-
tation of the media with BSA caused a significant enhancement of embryo development but when media were prepared from water samples purer than TAP water, in most cases BSA caused no significant improvement in embryo development over that seen in BSA-free media (Tables 1, 2) and the presence of protein was associated with a reduction in rate of' embryo development in some cases (Table 2). Although embryo development was inhibited in protein-free mKRB and protein-free mT6 media prepared from TAP water in this study, mouse embryos have been cultured successfully in protein-free Ham's F-10 medium constituted with tap water (Silverman et al., 1987). However, Ham's F-10 medium (Ham, 1963) contains 20 amino acids in different concentrations and one or more of these may have reduced embryotoxicity of substances in the medium as amino acids have been found to be effective chelators (Lindenbaum, 1973).It is important to consider as well that the tap water used by Silverman et al. (1987) for preparation of Ham's F-10 medium may have contained fewer embryotoxic substances than the TAP water used in the present study, thereby reducing the need for chelation, and that this may be yet another instance in which variability in water quality between laboratories makes difficult a comparison of experimental results obtained at different sites. Since the inclusion of protein in embryo culture media was seen to benefit embryo development in some cases but not others, the results in toto paralleled the previous conflicting reports regarding the benefit derived from supplementing culture media with proteins o r amino acids. However, in some of the previous studies the protein-free and amino acid-free media that
TABLE 2 . Development of Mouse Embryos in Modified Tyrode's (mT6) Medium Prepared With and Without Bovine Serum Albumin (BSA) and Prepared From Water Samples That Differed in Purity Embryo development to expected stages within specific periodsb (No. of embryos at expected stage/total number cultured) 24 hr 48 hr 72 hr Water in mediuma -BSA +BSA -BSA +BSA -BSA fBSA 92/94d 0/96'*' 91/94d 17/96'.' 92/94d 32/96'.' TAP (97%) (18%) (98%) (33%j (0%) (98%) 89/98d 94/98d 88/92d 89/92d 94/98d RO 91/92 ~. (96%) (96%) (91%) (97%) (96%) (99%) 82/8gd 95/100d 82/8gd 97/100d 99/100d 84/8gd MQ (92%) (95%) (92%) (97%) (94%) (99%) 79/82d 112/114d 77/82d 113/114d 80/82d 114/114d 3x (96%) (98%) (94%) (99%) (98%) (100%) Sum of RO, MQ, 3X 295/306E 2481269 255/269 299/306 304/306D 258/269 (98%) (96%) (92%) (96%) (99%) (95%) ~
aWater samples listed in order of increasing purity. bEmbryos were expected to reach the 4-cell stage of development in 24 hr, to reach the morula stage in 48 hr, and to reach the blastocyst stage in 72 hr of culture. C,dValuesin same column but with different superscripts (lower case) differ significantly (P< 0.001); data from groups not significantly different are pooled and compared at the bottom of the table. C,D,EValue significantlydifferent from that in the column to the right C (P