Polyvinyl alcohol and amino acids as substitutes for bovine serum albumin in culture media for mouse preimplantation embryos

Human Reproduction Update 1997, Vol. 3, No. 2 pp. 125 135 European Society for Human Reproduction and Embryology Polyvinyl alcohol and amino acids as substitutes for bovine serum albumin in culture media for mouse preimplantation embryos John D.Biggers 1,3, Michael C.Summers 1,2 and Lynda K.McGinnis 1 1 Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA 2 Present address: Fertility Center of New England, 20 Pond Meadow Drive, Reading, MA 01867, USA TABLE OF CONTENTS Introduction 125 Materials and methods 126 Results 128 Discussion 133 Acknowledgements 135 References 135 The effect of replacing bovine serum albumin (BSA) in a simple defined medium (KSOM) with polyvinyl alcohol (PVA) and/or amino acids on the percentages of mouse zygotes that develop to at least the blastocyst stage and that hatch at least partially or completely is reported. Blastocysts could form when BSA was replaced with only PVA, but at a moderately reduced rate; however, partial hatching, and hence complete hatching, were severely impaired when BSA was replaced with only PVA. The substitution of BSA with amino acids alone resulted in a high rate of blastocyst formation and moderate impairment of hatching. The addition of PVA to BSA-free KSOM supplemented with amino acids had no extra effect. BSA had significant effects when added to BSA-free KSOM supplemented with amino acids. The BSA caused a significant increase in the rate of partial hatching, and may even have had a small effect on the rate of blastocyst formation. The results also showed that glucose, at a high concentration of 5.56 mm, does not inhibit the development of mouse zygotes to hatched blastocysts when cultured in KSOM supplemented with amino acids. Key words: amino acids/embryo culture/glucose/ polyvinyl alcohol Introduction The results reported here are concerned with whether polyvinyl alcohol (PVA) is an adequate replacement for bovine serum albumin (BSA) in media that support the development of mammalian pre-implantation embryos. These studies were motivated by the need to formulate chemically defined media for the cultivation of pre-implantation embryos that are serum-free or free of proteins with undefined functions. BSA has been incorporated in all media designed to support the development of mouse pre-implantation embryos in vitro (reviewed by Biggers, 1987, 1993; Bavister, 1995). The accepted need for macromolecules in these media stems from early studies which suggested that the development of 8-cell mouse embryos in vitro to blastocysts requires the incorporation of egg white (Hammond, 1949), and the subsequent observation that the essential components in egg white are non-dialysable (Whitten, 1956). The use of egg white was dropped when it was also shown that it could be replaced with crystalline BSA (Whitten, 1956). BSA may have nutritional functions, as a source of fixed nitrogen, in culture media that support pre-implantation development. One possible function is the provision of free amino acids as the result of hydrolysis of the protein. Historically the need to substitute a non-protein macromolecule for BSA first arose in the design of experiments to determine the requirement for exogenous amino acids in mouse pre-implantation development (Brinster, 1965). In a search for a synthetic substitute for BSA, Brinster (1965) found that polyvinylpyrrolidone (mol. wt 150 000; PVP 150 ), acacia, dextran and Ficoll were acceptable substitutes, but not methyl celluloses. However, he was unable to demonstrate a nutritional role for BSA in the development of the 2-cell mouse embryo to the blastocyst stage in vitro. He speculated that some of the beneficial effects of BSA and certain amino acids may be due to their action as chelating agents, regulators of oxidation reduction potential, cell surface protectors, or enzyme protectors. Later work drew attention to the fact that BSA is chemically a very variable product, whose composition depends on the degree to which different small molecules remain bound to the 1 To whom correspondence should be addressed

126 J.D.Biggers, M.C.Summers and L.K.McGinnis macromolecule during its preparation. Thus extensive literature arose describing the variable effects produced by different BSA preparations in pre-implantation embryo culture media (Kane, 1983; McKiernan and Bavister, 1992). Brinster (1965) favoured the use of PVP 150 as a substitute for BSA in his work on the need of 2-cell mouse embryos for a fixed nitrogen source in vitro. He emphasized the beneficial effect of this polymer in facilitating handling of the embryos, presumably by influencing the physico-chemical properties of the medium. Later, an alternative PVA was also found to be an acceptable substitute for BSA in media used for fertilizing hamster ova in vitro (Bavister, 1981). Since the publication of these results, the polyvinyl polymers PVP and PVA have been commonly used as substitutes for BSA in media for the culture of mammalian pre-implantation embryos, with PVA being the preferred substitute in recent times because of the reported toxicity of PVP (Ashwood-Smith and Warby, 1971). Nevertheless, results obtained with BSA-substituted media have not been consistent. In experiments using PVP 150 as a substitute for BSA, Brinster (1965) found that outbred 2-cell mouse embryos required a fixed nitrogen source to develop into blastocysts. This source could be in the form of BSA, an amino acid mixture simulating the composition of hydrolysed BSA (Brinster, 1965) or glutathionine (Brinster, 1968). In contrast, Cholewa and Whitten (1970), also using PVP as a substitute for BSA, were unable to show a need by 2-cell F 1 hybrid mouse embryos for a fixed nitrogen source. Moreover, outbred 8-cell mouse embryos did not require a fixed nitrogen source to develop into blastocysts (Brinster and Thomson, 1966). Recently, interest has been renewed on the need for amino acids in media used to culture mouse pre-implantation embryos (reviewed in Gardner, 1994; Bavister, 1995). Gardner and Lane (1994) and Lane and Gardner (1993) reported that the pre-implantation development of 1-cell F 1 hybrid mouse embryos was enhanced when a mixture of 20 amino acids was added to medium MTF (Gardner and Leese, 1990). Ho et al. (1995) have reported a similar enhancement using 1-cell embryos from C57BL/6J inbred mice when amino acids were added to KSOM (Lawitts and Biggers, 1993). The rate of development of blastocysts increased, as well as the rate of hatching and the blastocyst total cell number. The results of Gardner and Lane (1993) and Ho et al. (1995) are of particular interest because the addition of free amino acids enhanced development, even in the presence of BSA in the medium (4 mg/ml in MTF; 1 mg/ml in KSOM). We have therefore compared the effect of adding free amino acids to KSOM, in which the macromolecule is either BSA or PVA, on the development of outbred mouse zygotes to the hatching stage of blastocyst development. Our results also suggest that glucose does not inhibit the enhanced effects of amino acids and albumin on the development of the pre-implantation mouse embryos. These results are in contrast to the reported inhibitory effects of glucose when added to medium CZB (Chatot et al., 1989). Materials and methods Animals Outbred CF 1 female mice, 6-8 weeks old, initially from Charles River Laboratories (CRL) (Wilmington, MA, USA) and subsequently from Harlan Sprague Dawley (HSD) (Indianapolis, IN, USA) were used in this work. The source of the mice was changed when the strain from CRL became unavailable. Separate tests showed that the HSD mice were adequate substitutes. The females were mated to F 1 hybrid B6D2F 1 males, 2 11 months old, from CRL. Females were stimulated with 5 IU pregnant mare s serum gonadotrophin (Sigma Chemical Company, St Louis, MO, USA) and superovulated with 5 IU human chorionic gonadotrophin (HCG; Sigma) 48 h later. All females were killed 18 22 h post-hcg and any ova present recovered. Only those embryos showing two pronuclei were allocated to the experiments. Thus the embryos used were outbred prior to and after zygotic activation. Media Table I. Composition of medium designated as KSOM b (KSOM without bovine serum albumin) Component NaCl 95 KCl 2.5 KH 2 PO 4 0.35 MgSO 4 0.2 Lactate 10.0 Pyruvate 0.20 Glucose 0.20 NaHCO 3 25 CaCl 2 1.71 L-Glutamine 1.0 EDTA 0.01 Concentration (mm) The base medium used was KSOM minus the BSA, denoted KSOM b (Table I). It was supplemented with several macromolecules, which are denoted by superscripts. For example, KSOM b PVA indicates that the medium was supplemented with PVA. The base medium was also supplemented with a mixture of 19 amino acids (Table II), and is denoted KSOM b AA.

Polyvinyl alcohol and mouse embryo culture 127 Table II. Concentrations of amino acids added to KSOM and KSOM b to give KSOM AA and KSOM b AA respectively Amino acid Concentration (mm) Amino acid Concentration (mm) L-Alanine HCl 0.05 L-Leucine 0.2 L-Arginine HCl 0.3 L-Isoleucine 0.2 L-Asparagine H 2 O 0.05 L-Lysine HCl 0.2 L-Aspartic acid 0.05 L-Methionine 0.05 L-Cystine 0.05 L-Phenylalanine 0.1 L-Glutamic acid 0.05 L-Proline 0.05 Glycine 0.05 l-serine 0.05 L-Histidine HCl H 2 O 0.1 L-Threonine 0.2 L-Tyrosine 0.1 L-Tryptophan 0.025 L-Valine 0.2 All culture media were formulated from KSOM with 1.0 mg/ml BSA (Fraction V; Sigma, Cat. no. A9647, lot no. 15H0672) or 0.1 mg/ml PVA (10 kda; Sigma; P-8136). The KSOM used in these experiments was prepared as a 2 solution (complete KSOM without the CaCl 2, BSA or PVA), divided into aliquots in 50 ml culture tubes and frozen at 70 C for up to 3 months. Calcium chloride, BSA and PVA were prepared as 1 M stocks and either frozen (CaCl 2 and BSA) or stored at 4 C (PVA). At 1 day before embryo collection, one aliquot of 2 KSOM stock was thawed and supplemented with CaCl 2 and H 2 O. All chemicals used in the preparation of KSOM were from Sigma. Non-essential (NEAA) and essential (ESAA) amino acids (Eagle, 1959) were added in the concentrations used by Ho et al. (1995). The NEAA and ESAA stock amino acid solutions were purchased from Gibco BRL (Life Technologies Inc., Grand Island, NY, USA). Embryo culture Ova were flushed from the oviduct using a modification of the flushing medium described by Lawitts and Biggers (1993), called FHM, in which the BSA was replaced with 0.1 mg/ml PVA. The zygotes were then washed in 0.3 mg/ml hyaluronidase in the modified FHM to remove cumulus cells. At this step, the ova with two pronuclei were selected for culture. Embryos were cultured for 5 days (144 h post-hcg) in groups of 12 per 50 µl droplet of medium overlayered with embryo-tested light mineral oil (Sigma; M8410). The cultures were incubated at 37 C in modular incubator chambers (Billups-Rothenberg Inc., Del Mar, CA, USA), which were gassed with a mixture of 5% O 2, 6% CO 2 and 89% N 2 (Lawitts and Biggers, 1993). Culture plates (60 mm non-tissue culture-treated; Corning Inc., Corning, NY, USA) were prepared 1 day before embryo collection and equilibrated in the module overnight. Embryo evaluation Embryos were observed at 100 on a warmed microscope stage (35 C; Wild dissecting microscope) and graded for stage of development, including compaction, blastocoel formation and hatching, at 96, 120 and 144 h post-hcg. Cell counts Embryos were fixed 144 h post-hcg in 3% formaldehyde for 15 min at 37 C. After fixation, nuclei were stained with the fluorochrome, Hoechst 33258 (1 µg/ml) in Dulbecco s phosphate-buffered saline for 15 min at room temperature. Groups of one to four blastocysts were mounted onto glass slides and covered with a mounting medium (50% glycerol, 50% sodium azide and 1 µg/ml Hoechst 33258). Stained embryos were covered with a glass coverslip and sealed with clear nail polish. Nuclei were counted at 40 on an inverted Zeiss epifluorescence microscope with a 365 nm band pass excitation filter and a 420 nm long pass barrier filter. Statistical methods Experimental design Five experiments were performed using randomized block experimental designs. Experiments 1 and 2 were preliminary experiments, the results of which led to the design of the major experiments (nos. 3 5). In all experiments the experimental unit was the set of ova, containing two pronuclei, that was randomly allocated to each drop. In experiments 3, 4 and 5, each drop contained 12 zygotes. Each block in each experiment corresponded to a single replicate of the experiment. Within each replicate, the experimental units were allocated at random to the treatments being compared in each experiment. The number of experimental units allocated to each treatment within a replicate was the same, and ranged from one to four depending on the number of zygotes collected and the number of treatments. In experiments 1 and 2, the number of zygotes in a

128 J.D.Biggers, M.C.Summers and L.K.McGinnis drop, and the number of drops allocated to a treatment, varied between replicates. Developmental responses In experiments 3, 4 and 5, the embryos were cultured for 144 h post-hcg. At 96, 120 and 144 h post-hcg the embryos were classified into one of a series of developmental states: pre-blastocyst, unhatched blastocyst, partially hatched blastocyst and completely hatched blastocyst. Thus statistically the observations are repeated measurements which, at each time, are a set of ordinal categorical responses (McCullagh and Nelder, 1989; Clogg and Shihadeh, 1994). For the statistical analysis, the observations were re-expressed as cumulative totals, i.e. the numbers of embryos developing to at least the blastocyst stage, at least the partially hatched blastocyst stage and at least the completely hatched blastocyst stage. Since the group size was constant throughout the data sets (n = 12), each of these cumulative sums was transformed before being used in analyses of variance (ANOVA) using the two-term inverse sine transformation proposed by Laubscher (1961): t 4 = n 1/2 sin 1 (x/n) 1/2 + (n + 1) 1/2 sin 1 [(x + 3/4)/(n + 3/2)] 1/2, where x = the number of responders (blastocysts) and n = the group size. The advantage of this transformation is that it is more stable for proportions <0.1 and >0.9, which occur frequently in our data. The theoretical variance of this distribution approaches 1 as n increases. Repeat measurement ANOVA Data from experiments 3, 4 and 5 were analysed by repeated measures ANOVA using the transformed data for each of the cumulative sums and the NCSS 6.0 Statistical Program (Jerry L.Hintze, Kaysville, UT, USA) [see Diggle et al. (1994) and Kshirsagar and Smith (1995) for the theory and assumptions of this analysis]. The statistical analysis is in two parts, each with separate error terms. One part analyses the effects of the experimental factors and their interactions [error (a)], while the other analyses the effect of time and the interactions between time and the experimental factors [error (b)]. The mean square for replicates was significant in several of the analyses. It is assumed that there were no large differences between the treatment effects within the replicates of each experiment. This assumption was checked graphically. Furthermore, the error mean squares used in the tests of significance include the variation attributable to the differential effects of the treatments between replicates. Data presentation The raw data from the experiments are not shown because of their extensive size. The data can be obtained from the first author (J.D.B.). Instead, the relevant information is summarized by the ANOVA of the transformed data which identify the significant effects. These significant effects are then illustrated graphically by plotting the relevant means of the transformed data after retransformation back to the percentage scale. This retransformation was performed using an iterative program written in QuickBasic. In all experiments, partial hatching and complete hatching had not started at 96 and 120 h post-hcg respectively. The data for these stages were therefore not included in the graphical and statistical analyses. Thus the number of repeat measurements for each response category, shown in Figures 1 3, diminishes from one category to the next. Other statistical procedures The preliminary results obtained in experiments 1 and 2 consisted of observations on the number of blastocysts formed (a single categorical variable) in drops that did not always contain the same number of embryos. The results were analysed after logistic transformation by a logistic regression analysis (Hosmer and Lemeshow, 1989) and the LogXact-Turbo Computer Program (Cytel Software Corporation, Cambridge, MA, USA). Experiment 2 also provided data on the number of cells that developed in the blastocysts formed. Data on cell counts were analysed by a two-way ANOVA using the NCSS 6.0 Statistical Program (Jerry L.Hintze). All effects with a probability value 0.05 were considered to be statistically significant. Results Preliminary observations (experiments 1 and 2) In experiment 1 the effects of adding either BSA (1.0 mg/ ml) or PVA (0.1 mg/ml) to KSOM b AA before or after freezing the medium were compared. Thus, there were four experimental treatments. Three replications were performed. Two of the replicates contained 10 embryos in each drop and the third replicate contained 11 embryos per drop. The embryos were cultured for 120 h post-hcg and the number of blastocysts recorded. A logistical regression analysis showed no significant replicate treatment interaction, so the data have been pooled over replicates. The results are summarized in Table III. Although there was a suggestion that the percentage of blastocysts that developed was less when PVA was substituted for BSA, and through freezing of the medium, the differences were not statistically significant. Analyses of the total cell number in blastocysts that developed under the four different conditions showed no statistically significant differences (Table III).

Polyvinyl alcohol and mouse embryo culture 129 Table III. Results of experiment 1: the number of zygotes developing into blastocysts and the blastocyst cell number, after freezing and thawing of KSOM b AA containing either bovine serum albumin (BSA; 1 mg/ml) or polyvinyl alcohol (PVA; 0.1 mg/ml) Treatment No. of Blastocysts Blastocyst cell count a zygotes n % P b n Number ± SEM c BSA 31 28 90.3 18 88.30 ± 6.22 PVA 31 24 77.4 0.301 18 86.30 ± 6.22 BSA (frozen) 31 23 74.2 0.182 16 87.80 ± 6.59 PVA (frozen) 31 24 77.4 0.301 18 91.30 ± 6.22 KSOM = base medium; KSOM b = KSOM minus BSA; KSOM AA b = KSOM b supplemented with a mixture of 19 amino acids. a Probability that the treatment means are different = 0.953. b Fisher s exact test. c Calculated from the analysis of variance pooled error mean square: 695.72, df = 64. Table IV. Results of experiment 2: the number of zygotes developing into blastocysts and the blastocyst cell counts, when cultured in KSOM b AA containing various macromolecular supplements Macromolecular supplement No. of Blastocysts Blastocyst cell count zygotes n % P a n Number ± SEM b P BSA (control) 56 44 78.6 12 88.80 ± 6.69 None 56 42 75.0 0.823 11 55.9±6.99 0.001 Polyvinyl alcohol (0.1 mg/ml) 56 42 75.0 0.823 14 80.50 ± 6.20 0.364 Polyvinyl alcohol (1.0 mg/ml) 56 35 62.5 0.097 14 77.7 ± 6.20 0.226 Ficoll (1.0 mg/ml) 56 34 60.7 0.064 13 68.50 ± 6.43 0.033 KSOM b AA : see Table III. a Fisher s exact test. b Calculated from the analysis of variance pooled error mean square: 537.63, df = 54. In experiment 2 the effects of adding BSA, PVA or Ficoll to KSOM b AA were compared. The five treatments were KSOM AA containing 1.0 mg/ml BSA (positive control), KSOM b AA with no BSA (negative control), KSOM AA containing either 0.1 or 1.0 mg/ml PVA and KSOM AA containing 1.0 mg/ml Ficoll. Three replications were performed. The first replicate contained 12 embryos in a single drop, the second replicate contained 12 embryos in each of two drops, and the third replicate contained 10 embryos in each of two drops. The embryos were cultured for 120 h post- HCG and the number of blastocysts recorded. The statistical analyses of the separate replicates and examination of the replicate treatment interactions showed that the data were sufficiently homogeneous to be pooled (data not shown). The results of the pooled data are summarized in Table IV. After 120 h post-hcg in culture there were no significant differences in the percentage of embryos reaching the blastocyst stage, although the percentage was close to being significantly reduced in the group containing the higher concentration of PVA. The response to Ficoll was particularly variable, but overall the result was close to significance (P = 0.06). The total numbers of cells in the blastocysts that developed were significantly less in the group cultured without a macromolecular component (P = 0.001). In contrast, the total numbers of cells in the blastocyst were unaffected by any of the macromolecular additions, with the exception of Ficoll in which the total cell number was reduced (P = 0.03). Effects of an amino acid supplement to KSOM and KSOM b PVA In experiment 3 the effects of amino acid supplements to KSOM, and KSOM in which BSA was replaced with PVA, were examined. The experiment was a randomized block design with the treatments arranged in a 2 2 factorial array. The factors were BSA (1.0 mg/ml) versus PVA (0.1 mg/ml) and the presence or absence of the amino acids listed in Table II. The numbers of unhatched, partially hatched and completely hatched blastocysts were recorded at 120 and 144 h post-hcg for each of the four treatments. Five replications were performed. The results of adding the amino acids to KSOM or to KSOM b, at the concentrations shown in Table II, demonstrated clearly that there were large differences in the percentage of embryos that developed at the times of observation (120 and 144 h) at least to the zona-enclosed blastocyst, the partially and completely hatched blastocyst (Figure 1 and Table V).

130 J.D.Biggers, M.C.Summers and L.K.McGinnis Figure 1. The effects of bovine serum albumin (BSA) or polyvinyl alcohol (PVA) and the presence or absence of amino acids (AA) in KSOM b (base medium minus BSA) on the percentages of zygotes that develop to at least (A) zona-enclosed blastocysts, (B) partially hatched blastocysts and (C) completely hatched blastocysts at different times of culture after human chorionic gonadotrophin injection (experiment 3). Table V. Results of experiment 3: analysis of variance tables of the data shown in Figure 1 Variation df At least blastocysts At least partially hatched Completely hatched Mean square P Mean square P Mean square P Replicates 4 11.779 0.0113 19.983 <10 5 1.804 0.0812 Amino acids (A) 1 7.368 0.141 27.236 1.4e 4 30.141 <10 5 Macromolecules (M) 1 15.447 0.0347 33.245 <10 5 3.226 0.0525 AM 1 6.705 0.160 6.735 0.0479 13.938 1.2e 4 Error (a) 60 3.521 2.017 0.825 Time (T) 1 5.277 0.0208 83.068 <10 5 AT 1 0.366 0.535 5.880 0.0260 MT 1 0.308 0.569 0.208 0.699 AMT 1 0.668 0.402 3.477 0.0844 Error (b) 64 0.835 0.983 df = degrees of freedom. The ANOVA for the transformed proportions of embryos developing at least to the blastocyst stage demonstrated that all the interactions between the main effects (AM) and the main effects and time (AT, MT, AMT) were not significantly different. Thus the results can be summarized entirely in terms of the independent effects of the amino acids (A), the macromolecules (M) and time (T). The percentage of embryos that developed at least to the blastocyst stage increased significantly between 120 and 144 h of culture. The percentage increase was about the same in all four media, but amounted to only a few percentage points (~5.3%). Although the ANOVA showed that the addition of amino acids had no significant effect, while the effect of replacing BSA with PVA was only marginally significant, further statistical analyses demonstrated that significantly fewer blastocysts (~16.0%) developed in KSOM b PVA compared with the other three media (P = 0.0001). The ANOVA for the transformed proportions of embryos developing to at least the partially hatched blastocyst stage showed that the responses were complex. Although the main effects due to amino acids (A) and macromolecules (M) are both significant, one interaction (AT) was significant, two interactions (MT, AMT) were not significant and the remaining interaction (AM) was marginally significant. This pattern of significance was mainly due to the large effect of amino acids that increased the percentage of blastocysts which partially hatched between 120 and 144 h of culture in KSOM b PVA from 22.6 to 57.2% (Figure 1). The results, however, showed clearly that by 144 h of culture the addition of amino acids to KSOM b PVA enhanced the percentage that at least partially hatch. In contrast, the increase in partial hatching was only marginal when amino acids were added to KSOM.

Polyvinyl alcohol and mouse embryo culture 131 Figure 2. The effects of bovine serum albumin (BSA) and/or polyvinyl alcohol (PVA) in KSOM b supplemented with a mixture of 19 amino acids (KSOM b AA ) on the percentages of zygotes that develop to at least (A) zona-enclosed blastocysts, (B) partially hatched blastocysts and (C) completely hatched blastocysts at different times of culture after human chorionic gonadotrophin injection (experiment 4). Table VI. Results of experiment 4: analysis of variance tables of the data shown in Figure 2 Variation df At least blastocysts df At least partially hatched df Completely hatched Mean square P Mean Square P Mean square P Replicates 2 23.071 4.6e 3 2 3.422 0.213 2 4.204 0.0260 BSA (B) 1 3.991 0.293 1 23.328 2.5e 3 1 2.439 0.130 PVA (P) 1 0.594 0.682 1 0.682 0.572 1 4.819 0.0370 BP 1 0.133 0.846 1 0.147 0.793 1 0.203 0.655 Error (a) 26 3.467 26 2.083 26 0.997 Time (T) 2 96.865 <10 5 1 48.040 <10 5 BT 2 1.114 0.440 1 0.633 0.530 PT 2 0.349 0.771 1 0.215 0.714 BPT 2 0.143 0.899 1 0.166 0.747 Error (b) 56 1.337 28 1.566 df = degrees of freedom. The effects of amino acids and macromolecules on the percentage of blastocysts that completely hatch by 144 h post-hcg reflected the observations on partial hatching. The ANOVA showed that, although the effect of macromolecules (M) on the percentage of blastocysts that completely hatched was not significant, the effect of amino acids (A) and the interaction between them (AM) were both highly significant. The addition of amino acids to KSOM resulted in a small effect on the percentage of blastocysts that completely hatched (from 12.7 to 17.2%). In contrast, the addition of amino acids to KSOM b PVA in which BSA was replaced with PVA had a much greater effect on the percentage of blastocysts that completely hatched (from 1.3 to 23.3%). Effects of PVA and BSA, separately and in combination, in KSOM b AA In experiment 4 the effects of the macromolecules BSA and PVA were compared in KSOM b AA in a randomized block design with the treatments arranged in a 2 2 factorial array. The factors were BSA (0.0 and 1.0 mg/ml) and PVA (0.0 and 0.1 mg/ml). The numbers of unhatched, partially hatched and completely hatched blastocysts were recorded at 96, 120 and 144 h post-hcg for each of the four treatments. Three replications were performed. The effects of adding BSA and/or PVA to KSOM b AA are summarized in Figure 2. The corresponding ANOVA are shown in Table VI. The results demonstrated that large

132 J.D.Biggers, M.C.Summers and L.K.McGinnis Figure 3. The effects of glucose in the presence and absence of bovine serum albumin (BSA) in KSOM b AA on the percentages of zygotes that develop to at least (A) zona-enclosed blastocysts, (B) partially hatched blastocysts and (C) completely hatched blastocysts at different times of culture after human chorionic gonadotrophin injection (experiment 5). differences occurred over time in the percentages of embryos that developed to at least the zona-enclosed blastocyst and the partially and completely hatched blastocyst stages. The ANOVA for the transformed proportions of embryos developing to at least the zona-enclosed blastocyst stage showed no significant main effects of BSA or PVA and their interaction. In contrast, there was a highly significant main effect of time in culture, but no significant interactions of BSA or PVA with time. The BSA and PVA increased the average percentage of zona-enclosed blastocysts over time by 4.6 and 1.0% respectively. However, these increases were not significant. Averaging over all treatments, 27.8, 73.0 and 76.3% of embryos developed to the zona-enclosed blastocyst stage by 96, 120 and 144 h respectively. The ANOVA for the transformed proportions of embryos developing to at least the partially hatched stage showed highly significant main effects of BSA and time, and no significant interactions. The addition of PVA had no significant effect. Pooling the non-significant effects of PVA with data on the effects of BSA, the percentage of embryos that at least partially hatched increased between 120 and 144 h from 21.0 to 50.4% when a macromolecule was absent or non-effective, and from 42.4 to 67.9% when BSA was present. The increase in percentage response produced by the addition of BSA was approximately the same at 120 and 144 h, and was 19.4%. The ANOVA for the transformed proportions of embryos that completely hatched showed that only the addition of PVA had a significant effect, but this was marginal. The BP interaction was not significant. Averaging over all groups, the percentage of blastocysts that completely hatched was 29.6%. Effects of glucose and BSA in KSOM AA In experiment 5 the effects of glucose in the absence and presence of BSA in KSOM AA were compared in a randomized block design with the treatments arranged in a 2 2 factorial array. The factors were BSA (0.0 and 1 mg/ml) and glucose (0.20 and 5.56 mm). The numbers of unhatched, partially hatched and completely hatched blastocysts were recorded at 96, 120 and 144 h post-hcg of the four treatments. Three replications were performed. The effects of adding glucose and BSA to amino acid-supplemented KSOM are summarized in Figure 3. The corresponding ANOVA are shown in Table VII. A comparison of the results summarized in the three panels in Figure 3 demonstrated that large differences occurred in the percentages of embryos that developed over time to at least the zona-enclosed blastocyst and the partially and completely hatched blastocyst stages. The ANOVA for the transformed proportions of embryos developing to at least the zona-enclosed blastocyst stage showed significant main effects of BSA and time. The main effect of glucose and all corresponding interactions were not significant. Furthermore, there was a highly significant effect of time in culture, but no significant interactions with time. The BSA and glucose increased the average percentage of zona-enclosed blastocysts over time by 10.7 and 0.4% respectively. These responses were reflected in the percentage of embryos that partially hatched. Glucose had no effect, and BSA significantly increased the percentages of partially hatched blastocysts at both time points by 15.4%. The glucose and BSA had no effect at 144 h on the percentages of blastocysts that had completely hatched. The percentage hatching was 23.5% when data were pooled from the four groups.

Polyvinyl alcohol and mouse embryo culture 133 Table VII. Results of experiment 5: analysis of variance tables of the data shown in Figure 3 Variation df At least blastocysts df At least partially hatched df Completely hatched Mean square P Mean square P Mean square P Replicates 2 32.264 <10 5 2 21.923 <10 5 2 27.208 <10 5 BSA (B) 1 26.337 1.1e 3 1 28.045 5.3e 5 1 0.364 0.526 Glucose (G) 1 0.00821 0.951 1 0.0193 0.907 1 0.0111 0.912 BG 1 3.826 0.188 1 0.0687 0.825 1 2.406 0.108 Error (a) 42 2.135 42 1.384 42 0.892 Time (T) 2 270.129 <10 5 1 105.313 <10 5 BT 2 0.307 0.785 1 0.0269 0.857 GT 2 1.347 0.349 1 1.019 0.270 BGT 2 1.262 0.373 1 0.250 0.583 Error (b) 88 1.264 44 0.816 df = degrees of freedom. Discussion Three morphological responses, which form a developmental series, have been used to assess the development of the zygotes after explantation: the proportions that developed into zona-enclosed blastocysts, partially hatched blastocysts and completely hatched blastocysts respectively. The results summarized in Figures 2A and 3A provide rough indications of the cumulative distributions of the times that embryos take to cavitate, measured from the time of HCG injection. The graphs seem to reach a maximum value between 75 and 85%, suggesting that there may be a 15 25% incidence of zygotes that may be unable to develop into blastocysts under any conditions. The results of experiment 3 show that outbred CF 1 zygotes develop into zona-enclosed blastocysts in KSOMb PVA (Figure 1 and Table IV). Under these conditions the only fixed nitrogen source is glutamine. This result differs from that obtained by Brinster (1965), who reported some cleavage of 2-cell embryos, but no blastocyst formation occurred when glutamine was added to another medium in which PVP replaced BSA. Whether this discrepancy is due to differences in the composition of the two media is unknown. The results obtained in experiment 3 also show that the yield of blastocysts cultured in KSOM b PVA is significantly less than that obtained using KSOM where BSA is the macromolecule. Thus BSA provides something over and above the other constituents of KSOM that enhances the incidence of blastocyst formation. Furthermore, the addition of amino acids, at the concentrations shown in Table II, to KSOM b PVA also increased the yield of blastocysts to that observed using KSOM. The BSA and amino acids may have three functions: to provide amino acids for metabolism, to chelating contaminating toxic divalent metals, and to act as scavengers of free radicals. Similar and more spectacular effects were observed when partially hatched blastocysts developed. These results do not establish whether the outbred CF 1 pre-implantation mouse embryo needs a fixed nitrogen source. However, they do show that the provision of amino acids or BSA can cause more embryos to reach the zona-enclosed blastocyst stage by 120 h after the administration of HCG. The results summarized in Figure 2 demonstrate that CF 1 zygotes will develop into blastocysts in high yield (74%) by 144 h in KSOM b AA without any macromolecule. Furthermore, 48% of the embryos partially hatched and 21% completely hatched. Our results on the development to the blastocyst stage agree with the observations of Fissore et al. (1989), who showed that BSA could be omitted from the medium if EDTA was present. Our results also demonstrate that the addition of BSA and/or PVA to KSOM b AA did not significantly affect the dispersion of the cumulative distributions of cavitation times of zygotes developing into blastocysts. In contrast, the addition of BSA to KSOM b AA resulted in a shift in location, as shown by a very significantly higher percentage of partial hatching, while the addition of PVA in the absence and presence of BSA had no effect. These results (Figure 3B) confirm the strong effect of BSA in promoting partial hatching in KSOM b AA, and are in agreement with the results of Wright et al. (1978) who reported that the incidence of hatching of 2-cell mouse embryos in vitro is dependent on the concentration of BSA to which the embryos are exposed. The results shown in Figure 3A also suggest that the addition of BSA to KSO b AA increased the yield of zona-enclosed blastocysts, an effect not statistically confirmed for the data shown in Figures 1A and 2A. Thus the possibility that the stimulatory effects of BSA begin to exert themselves before cavitation should not be ignored.

134 J.D.Biggers, M.C.Summers and L.K.McGinnis The overall conclusion from this work is that PVA does not substitute completely for BSA as the macromolecular constituent of KSOM or KSOM AA for the culture of mouse zygotes to the blastocyst stage. The effect of PVA on the rate of development to the zona-enclosed blastocyst is only slightly less than with BSA; in contrast, the rate of partial hatching is significantly less. The distributions of the development times, however, are merely displaced because within experiments 3 5 all the time response curves are parallel (no main effect time interactions) with one exception. Thus, from an experimental point of view, the substitution of BSA with PVA will merely lower the responses overall, and not lead to serious complications caused by the differential effects of the two macromolecules. The reason why the particular batch of BSA we used to supplement KSOM b AA enhances development, particularly hatching, is unknown. There are at least three possibilities: (i) the BSA contributes additional amino acids that supplement those included in KSOM b AA; (ii) the BSA provides unknown bound non-amino acid molecules which stimulate development; or (iii) the BSA provides additional chelating functions in addition to those provided by EDTA and amino acids already in KSOM b AA. We should also recognize that the findings may not be universally true because of the variability between different batches of BSA. McKiernan and Bavister (1992) found that some batches of BSA stimulated the development of 2-cell hamster embryos in vitro, while others inhibited development. The results obtained in experiment 5 also show that the addition of 5.56 mm glucose to KSOM b AA or KSOM AA has no effect on the rate of development of outbred CF 1 zygotes to the partially hatching blastocyst stage. These results further confirmed that glucose has no inhibitory effect on the pre-implantation development of mice in vitro under the conditions provided by the KSOM family of media (Lawitts and Biggers, 1992; Summers et al., 1995). Members of this family all contain phosphate (0.35 mm KH 2 PO 4 ). Thus our results differ significantly from those reported for the mouse by Chatot et al. (1989), and for the hamster and other species where glucose and phosphate both inhibit early cleavage (Barnett and Bavister, 1996). The reason for these major differences has yet to be resolved. Acknowledgements We thank Michael Raffin and Bret Nelson for their technical assistance. We also thank Drs Barry Bavister, John Eppig, Robert Foote, Richard Tasca and Betsey Williams for critical comments on the manuscript. 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