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Regular Article |
a Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
ABSTRACT
Development of outbred CF1 mouse zygotes in vitro was studied in a chemically defined, protein-free medium both with and without amino acids. The addition of amino acids to protein-free potassium simplex optimized medium (KSOM) had little effect on the proportion of embryos that developed at least to the zona-enclosed blastocyst stage. In contrast, amino acids stimulated very significantly, in a dilution-dependent way, the proportion of blastocysts that at least partially or completely hatched. Amino acids also stimulated cell proliferation in both the trophectoderm and inner cell mass (ICM) cells, at rates that favored proliferation of cells in the ICM; had no effect on the incidence of cell death (oncosis or apoptosis); and improved development of the basement membranes, which form on the blastocoelic surface of the trophectoderm and between the primitive endoderm and the primitive ectoderm. Thus, KSOM, supplemented with amino acids but containing no protein supplements, supports development of a newly fertilized ovum to the late blastocyst stage, in which its normal, three-dimensional structure is preserved and in which the ICM has been partitioned into the primitive ectoderm and primitive endoderm.
apoptosis, developmental biology, implantation/early development
INTRODUCTION
The morphological transition from the newly fertilized ovum to the blastocyst that is ready to gastrulate and implant involves both growth and pattern formation. These changes involve a series of cell divisions to form the morula, differentiation of the cells in the morula into two cell lineages (i.e., the trophoblast and inner cell mass [ICM] cells [1, 2]), the spatial orientation of these two lineages to form the blastocyst consisting of an ICM and a trophectoderm (TE), the pumping of fluid into the blastocoel [3, 4], and regional specification of the ICM into the primitive endoderm (i.e., hypoblast) and primitive ectoderm (PE; i.e., epiblast) by the formation of basement membrane-type extracellular matrix (ECM) [5].
Detailed analysis of these many changes during preimplantation development depends on the ability to study early mammalian development in vitro. Several media will support development of the mouse from the zygote to the blastocyst stage [6], but to our knowledge, relatively little attention has been given to how these media affect the ICM and TE lineages immediately after they are formed. The results presented here examine the effects of supplementing one of these media, potassium simplex optimized medium (KSOM), with amino acids (AA) on several of these developmental changes. Lawitts and Biggers [7] designed KSOM using the experimental strategy called sequential simplex optimization to overcome the two-cell block that occurs in many outbred and inbred strains of mice [8]. The medium was then fine-tuned based on analyses of the K+ and Na+ concentrations in single two-cell embryos by electron probe microanalysis [9]. An unexpected bonus of the medium was the finding that it supports development of a high yield of blastocysts from ova from the outbred CF1 strain, containing cells in greater numbers than have been reported using other media [10–12]. At approximately the same time, it was independently shown that supplementing KSOM with a mixture of AA "improved embryo development in terms of the rate of blastocyst development, blastocyst hatching, and the number of cells per blastocyst" [13]. The possibility that the effects of BSA could confound the actions of AA added to an embryo culture medium was recognized in the pioneering studies of Brinster [14] and Cholewa and Whitten [15] regarding the need of mouse preimplantation embryos for an exogenous fixed-nitrogen source. In both studies, BSA was replaced with polyvinylpyrrolidone. Many of the more recent studies regarding the effects of AA on the mouse preimplantation embryos have been done in media that included BSA [13, 16–18]. The need to eliminate the effects of BSA was confirmed when replacement of the BSA in KSOM with polyvinylalcohol (PVA) was found to partially depress the rate of blastocyst formation and to severely inhibit hatching [19]. The addition of AA to BSA-free KSOM was found to restore the rates of blastocyst formation and hatching. All the results reported in this paper, therefore, have been obtained using KSOM in which the BSA has been replaced with PVA (denoted as KSOMPVAAA). This work demonstrates that AA preferentially stimulates proliferation of cells in the ICM compared with the TE, has little demonstrable effect on cell death, and stimulates the organized appearance of ECM.
MATERIALS AND METHODS
Donors
CF1 female mice and 2- to 11-mo-old BDF male mice (Charles River Laboratories, Raleigh, NC) were used in this study. Animals were maintained in accordance with guidelines of the Committee on Care and Use of Laboratory Animal Resources, National Research Council. Females were superstimulated with 5 IU of pregnant mares serum gonadotropin (eCG) and superovulated with 5 IU of human chorionic gonadotropin (hCG) 48 h later. They were then mated to BDF males. Outbred one-cell BDF
x CF1
embryos were collected from the oviduct 22- to 26-h post-hCG, and those with two pronuclei were selected for culture. The medium used for oviduct flushing, embryo collection, and holding before culture was a modified flushing-holding medium (FHM) [7] in which the BSA was replaced with 1.0 mg ml-1 PVA.
Culture Media
All culture media were formulated from KSOM [7] in which BSA was replaced by 0.1 mg ml-1 PVA (Sigma Chemical Co., St. Louis, MO; catalog number P88136; average molecular weight, 10 000) (Table 1), denoted as KSOMPVA. Both the substitution of PVA with BSA and the freezing of KSOMPVA (CaCl2 supplemented after thawing) have been tested for their ability to support mouse embryo development from the zygote to the blastocyst stage [14]. The two procedures gave yields of blastocysts comparable to those obtained using freshly prepared KSOM containing BSA. The KSOMPVA was prepared from a 2x stock solution (complete KSOM without CaCl2 and PVA) and frozen in 50-ml culture tubes at -70°C for as long as 3 months. Stock solutions of CaCl2 (0.171 M stored at -20°C) and PVA (100 mg ml-1 stored at 4°C) were prepared. On the day before embryo collection, 50 ml of 2x KSOM stock solution was thawed and supplemented with 1 ml of thawed CaCl2 and 100 µl of PVA stock solutions, then made up to a final volume of 100 ml with distilled water. When KSOMPVAAA) (Table 2) was required, the AA were added from purchased stock solutions of Eagle mixtures of essential AA (Gibco BRL, Grand Island, NY; catalog number 11130-010) and nonessential AA (Gibco, catalog number 11140-019) [20] before making up the final volume with water. These AA stock solutions were kept at 4°C and should not be frozen. The volumes of the AA stock solutions added depended on the AA concentrations required. For example, one-half concentrations of AA were prepared by adding 1 ml of essential AA stock solution and 0.5 ml of nonessential AA stock solution to make 100 ml of KSOMPVAAA. All other chemicals, eCG, and hCG were from Sigma.
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Embryo Culture
Embryos were cultured in groups of 12 or 18 per 50-µl droplet of medium overlaid with embryo-tested mineral oil. Embryos were cultured in modular incubator chambers (Billups Rothenberg, Inc., Del Mar, CA), which were gassed with a mixture of 5% O2, 6% CO2, and 89% N2 [7]. The pH of the KSOMPVA and KSOMPVAAA was 7.3. Culture plates (60-mm suspension dish; Corning Inc., Corning, NY) were prepared 1 day before embryo collection and equilibrated in the module overnight. Embryos were cultured for 5 days (144-h post-hCG).
Embryo Evaluation
Embryos were observed at 40x magnification on a warmed microscopic stage at approximately 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. These times correspond to approximately 72-, 96-, and 120-h in culture.
Differential ICM and TE Cell Counts
After 144 h of culture, blastocysts were stained using a modification of a method originally described by Handyside and Hunter [21–23]. Blastocysts (3–5 at a time) were transferred from culture drops to acid Tyrode solution, under constant observation, for 5–15 sec until the zonae pellucidae were completely dissolved. The blastocysts were next removed to three rapid, successive washes in Minimal Essential Medium (MEM; Gibco) with 10% calf serum (CS; HyClone Laboratories, Inc., Logan, UT) and then for 30 min into rabbit antiserum to mouse red blood cells (Organon Teknika Corp., Durham, NC) diluted to 10% in MEM at 37°C. After 30 min, embryos were transferred through three successive, 5-min washes and then into MEM with 10% guinea pig complement (Gibco), 1 µg ml-1 bisbenzimide (Hoechst 33258), and 1 µg ml-1 propidium iodide (PI; Sigma) in MEM for 30 min at 37°C. This method was modified in two ways during our later work. The basic medium used for the staining of the blastocysts was changed from MEM (290 mOsm) to Hepes-buffered KSOM [7] in which BSA was replaced with PVA (FHMPVA; 260 mOsm). Almost all blastocysts remained intact after staining using FHMPVA. After lysis of the TE, the blastocysts were fixed in 4% formalin in FHMPVA with 1µg ml-1 PI and 1µg ml-1 Hoechst 33258 for 30 min and then stored in the staining solution at 4°C in the dark for 1–3 days. Each stained blastocyst was transferred to a clean glass slide, compressed under a glass coverslip, and viewed at 400x magnification on an inverted Zeiss microscope with epifluorescent attachments. The ICM cells fluoresce blue, and the TE cells stain red/orange. The numbers of nonmitotic, mitotic, and dead (i.e., degenerate and fragmented nuclei) cells were counted. The total number of surviving cells were the sum of the nonmitotic and mitotic cells.
Staining for Apoptosis
Zygotes were cultured in either KSOMPVA or KSOMPVAAA and graded for blastocoel expansion and zona hatching at 96-, 120-, and 144-h post-hCG. After grading at 144 h, half the blastocysts from each treatment were washed three times in FHMPVA (wash) and fixed in 4% paraformaldehyde (PFA) in wash overnight at 4°C. The remaining unfixed blastocysts were labeled for differential cell counts and then fixed in 4% formalin (in wash).
The TUNEL terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling procedures to label for apoptosis were based on those of Brison and Schultz [24]. Blastocysts fixed in PFA were washed three times, suspended in 50 µl of 50 mM Tris-HCl, allowed to settle to the bottom of the dish, transferred into DNase Rx mixture (10 µl of RQ1 DNase [Promega Corp., Madison, WI], 10x buffer [10 ml of 1 M Tris-HCl, 10 ml of 20 mM MgCl2, 20 mg of BSA], 5 µl of RQ1 DNase, 35 µl of H2O, and 50 µl of 50 mM Tris-HCl), and incubated in the dark at 37°C for 1 h. Blastocysts were then washed three times, placed into permeablizing solution (50 ml of FHMPVA and 50 µl of Triton-X100) for 40 min at room temperature, transferred into blocking solution (50 ml of FHMPVA, 50 µl of Triton-X100, and 500 mg of BSA) and incubated for 1 h at 37°C. Embryos were then washed three times and incubated in TUNEL Rx mixture (fluorescein-conjugated dUTP and TdT; Boehringer Mannheim, Indianapolis, IN) for 1 h at 37°C in the dark, washed three times, and incubated in RNase Rx mixture (500 µl of TE buffer and 1 µl of RNase A stock solution [100 µl of TE buffer and 5 mg of RNase A]) for 1 h at 37°C. They were subsequently washed three times and mounted on glass slides in mounting medium (2.5 ml of FHMPVA, 2.5 ml of glycerol, 2.5 mg of sodium azide, and PI and Hoechst 33258 (
10 µg/ml) and stored in the dark at 4°C. TUNEL-labeled blastocysts were examined at 400x magnification on an inverted Zeiss microscope with epifluorescent optics. The numbers of fluorescein isothiocyanate (FITC)-labeled apoptotic, normal, and mitotic cells were counted. The total numbers of cells reported includes the mitotic cells.
Confocal Microscopy
Whole-mount immunofluorescence
The whole-mount immunofluorescence procedure was a modification of that previously described by Palmieri et al. [25, 26]. Embryos were rinsed in 4 mg ml-1 of BSA/PBS and then fixed for 30 min at 37°C in 3% PFA in a stabilization buffer (0.1 M PIPES, 5.0 mM MgCl2·6H2O, and 2.5 mM EGTA). The embryos were rinsed two times in BSA/PBS and transferred to 0.2% Triton in BSA/PBS at room temperature for 45 min, then blocked overnight in 3% fetal calf serum, 0.1% Tween-20, 0.02% Na-azide, and 0.4% powdered milk. The following morning, the embryos were incubated in 2 mg ml-1 hyaluronidase in BSA/PBS for 30 min at 37°C [27, 28], rinsed twice in BSA/PBS, and transferred to a quench (0.05 M glycine, 34% blocking solution, and 0.013% Na-azide) in PBS for 15 min at room temperature. The embryos were then washed in diluted block (
x in PBS) and incubated in the primary antibody for 1 h at 37°C. Two different primary antibodies, a polyclonal rabbit antimouse collagen IV immunoglobulin (Ig) G (
1 and 2; Collaborative Biomedical Products, Bedford, MA; lot 907304) and a polyclonal rabbit antimouse laminin IgG (laminin 1; 1ß1
1; Collaborative Biomedical Products, lot 902384), were used at a dilution of 1:150 in PBS. Ten blastocysts were incubated in PBS/BSA without primary antibody (i.e., negative control). Embryos were rinsed three times and held in the blocking solution for 1 h at room temperature, then incubated in a secondary antibody for 1 h at 37°C. The secondary antibody was a goat antirabbit IgG conjugated to FITC (Boehringer Mannheim, Indianapolis, IN; lot 21014) at a 1:100 dilution in PBS. All blastocysts within a replicate were labeled at one time with a single set of primary and secondary antibodies. After incubation in the secondary antibody, the embryos were rinsed in the blocking solution and transferred through a glycerol series (2.5%, 5%, 10%, 20% and 50% glycerol in 3% CS/PBS). The embryos were mounted on glass slides in glycerol with PI (0.5 µg ml-1) and 1,4-diazabicyclo-[2,2,2]-octane (20 mg ml-1). A cover slip was placed on the slide and sealed with nail polish.
Control peptide To ensure the primary antibodies were binding specifically to the antigen to which they were raised, antibodies were preincubated (dilution, 1:150) with excess target peptide and possibly cross-reactive peptides [21]. The collagen IV IgG was incubated with mouse collagen type IV (Collaborative Biomedical Products, lot 907695) at concentrations of 0.5, 1, and 2 µM; human fibronectin at 1 µM (Collaborative Biomedical Products, lot 902795); and mouse laminin at 7.9 nM (Collaborative Biomedical Products, lot 905185). The collagen IV IgG and peptide solutions were used as the primary antibody in the whole-mount immunofluorescence procedure previously described. For the antimouse laminin IgG, the antibody was preincubated with mouse laminin at concentrations of 0, 4, 8, and 16 nM; mouse collagen type IV at 1 µM; and human fibronectin at 1 µM.
Fluorescence confocal microscopy A Zeiss Laser Scan Microscope 410 invert was used to examine the embryos. The confocal settings were standardized using mouse blastocysts flushed from the uterus 120-h post-HCG and labeled with the appropriate antibodies. All embryos were scanned through the z-axis, and optical sections were chosen for comparisons between the in vivo- and in vitro-produced embryos. Optical sections were recorded using Zeiss LSM software version 3.80.
Image analysis A complete set of serial sections from a blastocyst stained for collagen IV can be seen on the Internet (www.muritech.com).
Scoring the confocal images Development of blastocysts was assessed using two scoring systems: cell organization of the TE, ICM, and primitive endoderm (PE); and development of the ECM associated with the TE, ICM, and PE. The cellular organizations of the TE, ICM, and PE were assessed separately as 0 (i.e., few or degenerating nuclei), 1 (i.e., disorganized cell assemblies), or 2 (i.e., normal cell assemblies). Development of the ECM was assessed as 0 (i.e., none or not obvious above background), 1 (i.e., diffuse or irregular, in which the labeled ECM appear on the outside surface of the TE or between the cells of the TE, ICM, and PE), 2 (i.e., patchy basement membranes, in which the ECM appear on the basolateral surface of individual TE cells but do not form a continuous layer), or 3 (i.e., normal continuous basement membrane, in which the ECM forms a continuous sheet covering at least two-thirds of the TE and clearly surrounding the ICM). All blastocysts within a replicate were scored blind on a single day by one observer, who was unaware of the treatments the individual blastocysts received.
Statistical Methods and Computer Packages
Exploratory graphical analyses All analyses were done using the S-Plus 4.5 package (MathSoft, Seattle, MA). Bivariate data were displayed using scatter plots after jittering the data to separate overlapping observations. Notched box plots [29] display the 10th, 25th, 50th (i.e., median), 75th, and 90th percentiles. Observations outside the 10th and the 90th percentiles were considered to be outliers. Notches on the boxplots are the median confidence limits. Two medians were considered to be significantly different if their confidence limits did not overlap.
Generalized linear models
General linear models [30–32] were introduced to extend the classical linear models of regression analysis, in which the random part of the model is assumed to be normal, to several one-parameter exponential distributions, and where the additivity of the effects of the explanatory variables are assumed to hold on some transformed scale defined by the link function. In this study, three types of distribution of errors and link functions were used according to the characteristics of the data being analyzed. All experiments analyzed were randomized block experiments having unequal numbers of cells within each treatment. Thus, the model used to analyze all the experiments was
where g(
) is the link function; b0 is the intercept; r is the replicate number; t is the treatment; b1, b2, and b3 are regression coefficients; and eij is the random error. For experiments in which the responses were quantal (i.e., blastocyst formation, partial and complete hatching), the data were assumed to have binomial errors and the link function to be the logit transformation. When the responses were cell counts, the errors were assumed to be formed by a multiplicative process, with log-normally distributed errors and the link function being 1. When the responses were counts of apoptotic cells and cells in mitosis, Poisson errors were assumed, and the link function was the logarithmic transformation. All computations were done with S-Plus 4.5.
Correlation coefficients Spearman rank correlations were computed using S-Plus 4.5 [32, 33].
Contingency tables The Fisher exact significance test for unordered r x c tables and the exact Kruskal-Wallis significance test for singly ordered r x c tables were calculated using StatXact 4.0 (Cytel, Cambridge, MA) [34].
RESULTS
Morphological Effects of KSOMPVAAA on Blastocyst Formation and Hatching
The effects of KSOMPVA supplemented with five different concentrations of AA on the development of mouse zygotes were compared with those of a control medium consisting of unsupplemented KSOMPVA. The composition of the medium containing the highest concentrations of amino acids is shown in Table 2; the concentrations of AA in the other four experimental media were 1:2, 1:4, 1:8, and 1:16 dilutions of this medium. The effects of the six media were compared in four replicates using a randomized block experimental design. The experimental unit randomized to the treatments consisted of 12 zygotes in the first replicate and 18 zygotes in the remaining replicates. One experimental unit was allotted at random to each treatment in the first, third, and fourth replicates. In the second replicate, each treatment was randomly allotted two experimental units. Observations were made on the same embryos at three different times (96-, 120-, and 144-h post-hCG) so that data were repeat (i.e., longitudinal) measurements [35]. At each time, the numbers of embryos that developed into three developmental stages (i.e., zona-intact blastocysts as well as partially and completely hatched blastocysts) were counted so that data within each time were ordinal categorical responses [36]. Thus, these data form a set of multivariate observations that are serially correlated, both ordinally in response and longitudinally in time. The statistical analyzes were done after re-expressing the data as cumulative sums (i.e., numbers of embryos developing at least to the zona-enclosed blastocyst stage and to at least the partially and the completely hatched blastocyst stages, respectively).
The total numbers of zygotes, summed over the four replicates that developed to at least the blastocyst, partially hatched, and completely hatched stages in the presence of different concentrations of AA, out of a total of 96 zygotes are shown in Table 3. That 67 of 96 (70%) of zygotes developed by 144-h post-hCG in the absence of AA, except glutamine, demonstrates that no absolute requirement exists for exogenous AA during blastocyst formation. In addition, 20 of 96 (21%) of these blastocysts began to hatch in the absence of AA. The exact Kruskal-Wallis tests of significance show that addition of AA to KSOMPVA enhanced significantly the development of zygotes by 144-h post-hCG. The analyses of deviance are summarized in Table 4. Although highly significant differences were found between replicates, the replicate x treatment interactions were not significant. Thus, the data were pooled to estimate the overall linear regressions of each logit response on log2 AA dilutions. The linear regressions (Table 4) were all highly significant, confirming that the effects of AA on blastocyst formation, partial hatching, and complete hatching are all concentration dependent. The fitted regressions are shown in Figure 1.
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Effects of KSOMPVAAA on Cell Proliferation
A preliminary experiment was done to determine whether the addition of AA to KSOMPVA affected the proliferation of ICM and TE cells in blastocysts that developed from zygotes. Groups of 12 zygotes were cultured in either KSOMPVA or KSOMPVAAA containing AA at half the concentrations shown in Table 2. Half-concentrations of AA were used to repeat the conditions used by Ho et al. [13], who reported difficulties with the solubility of some AA when full-strength concentrations were used. Overall, the results suggested that proliferation of both TE and ICM cells was greater when embryos were cultured in KSOMPVAAA compared with KSOMPVA.
The preliminary experiment provided insufficient data, however, to determine whether addition of AA to KSOMPVA differentially stimulates proliferation of ICM and TE cells. Therefore, a larger experiment was done to examine the effect of adding AA to KSOMPVA on the numbers of ICM and TE cells 144-h post-hCG. Groups of 12 zygotes were cultured in either KSOMPVA or KSOMPVAAA containing AA at half the concentrations shown in Table 2, and the blastocysts that developed were randomly divided into two groups. One group was stained to primarily allow counts of ICM and TE cell numbers, and the second group was stained primarily to allow counts of apoptotic cell numbers. Cells not in mitosis and cells in the mitotic phase of the cell cycle could also be counted in both groups. Because blastocysts were allotted at random to the two groups, data from both parts of the experiment were used to examine the effect of adding exogenous AA to KSOMPVA on the numbers of ICM and TE cells (group1), the numbers of cells in the mitotic phase of cell division (groups 1 and 2), and the numbers of cells undergoing degeneration, fragmentation, and apoptosis (groups 1 and 2).
Numbers of developing cells in the ICM and TE Figure 2a shows bivariate plots of ICM and TE cell numbers in blastocysts that developed from zygotes cultured for 144-h post-hCG in either KSOMPVA (open circles) or KSOMPVAAA (closed circles). The graph also includes data from the preliminary experiment. The numbers of ICM and TE cells included both nonmitotic and mitotic cells. Although considerable overlap exists between the ICM and TE cell counts observed in blastocysts cultured in KSOMPVA and KSOMPVAAA, a clear tendency was found for the numbers of ICM and TE cells in the group cultured using KSOMPVAAA to be larger. In addition, the data suggest that the variance in the counts of both ICM and TE cells is proportional to the mean. The correlation between the numbers of ICM and TE cells in both sets of blastocysts was significantly positive (KSOMPVA: r = 0.311, n = 66, 0.01 < P < 0.001; KSOMPVAAA: r = 0.404, n = 78, P < 0.001).
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Figure 2b shows the marginal distributions of the cell counts in the ICM of blastocysts cultivated from zygotes in KSOMPVA and KSOMPVAAA. The cell counts varied over a wide range in both sets of data (KSOMPVA: minimum of 1, maximum of 34; KSOMPVAAA: minimum of 4, maximum of 62). Nevertheless, the notches of the two distributions, which indicate the approximate confidence limits for the medians of the two distributions, do not overlap, suggesting that addition of AA to KSOMPVA increases the number of cells that develop in the ICM. Partitioning the variance, however, shows that the large variation results from a significant replicate x AA interaction as well as from a significant difference between replicates (Table 5). The significant interaction can be explained by the increases in ICM cell counts caused by the added AA, which occurred in all replicates, varying widely. Despite this variability, the results show a significant stimulatory effect of adding AA to KSOMPVA on the numbers of cells that develop in the ICM. The median number of ICM cells in blastocysts produced at 144-h post-hCG in KSOMPVAAA is 20, and this number is almost identical to the number of ICM cells in mouse blastocysts, albeit of a different strain, developing in vivo [16].
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Figure 2c shows the marginal distributions of the cell counts in the TE of blastocysts cultivated from zygotes in KSOMPVA and KSOMPVAAA. The cell counts varied over a wide range in both sets of data (KSOMPVA: minimum of 24, maximum of 128; KSOMPVAAA: minimum of 27, maximum of 143). Nevertheless, the notches of the two distributions do not overlap, suggesting that addition of AA to KSOMPVA significantly increases the number of cells that develop in the TE. Partitioning the variance, however, showed that the large variation resulted from a significant replicate x AA interaction as well as a significant difference between replicates (Table 5). As with the ICM data, the significant interaction can be attributed to the increases in TE cell counts caused by the added AA, which occurred in all replicates, varying widely. Despite this variability, the results show a significant stimulatory effect of adding AA to KSOMPVA on the numbers of cells that develop in the TE. The median number of TE cells in blastocysts produced at 144-h post-hCG in KSOMPVAAA is 85, and this number is approximately 90% of the number of TE cells in blastocysts of a different strain developing in vivo [16].
Figure 2D shows the distributions of the ratio of the numbers of ICM and TE cells in blastocysts produced in KSOMPVA and KSOMPVAAA. The notches of the two distributions do not overlap, suggesting that addition of AA to KSOMPVA significantly increases the ratio of ICM to TE cells. Partitioning the variance, however, showed a highly significant effect of AA and no replicate x AA interaction (Table 5). Thus, these results confirm that the ICM:TE ratio is increased by the addition of exogenous AA to KSOMPVA.
Effect of KSOMPVAAA on cells in the mitotic phase Counts of mitotic figures were obtained using blastocysts cultured for 144-h post-hCG in either KSOMPVA or KSOMPVAAA and stained either by the TUNEL or the differential cell count method. The distributions of the counts are illustrated in Figure 3, a and b, and the analysis of deviance is shown in Table 6. No interactions were considered to be significant, so the main effects can be assumed to be independent and averaged over replicates. A highly significant difference was found in the numbers of mitoses detected by the two staining procedures. The counts obtained after staining with the TUNEL method are considered to be more reliable, because the blastocysts were fixed immediately after culture whereas those stained with the differential cell count method were not. The lack of fixation may account for the medians dropping from 1 to 0 in the two groups. Although adding exogenous AA to KSOMPVA has no significant effect on the mean numbers of mitoses in the TUNEL-stained group, the barplots suggest that slightly more mitoses occurred when AA was added to KSOMPVA. The numbers of blastocysts with zero mitoses was reduced, whereas the numbers of more than two mitoses slightly increased. This effect resulted in the interquartile range being changed from 0–2 to 1–2.
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Effect of KSOMPVAAA on apoptotic and dead cells The distributions of the numbers of apoptotic cells in blastocysts at 144-h post-hCG cultured in KSOMPVA and KSOMPVAAA are displayed in Figure 4, a and d, respectively. The analyses of deviance showed that the addition of AA to KSOMPVA had no effect on the numbers of apoptotic cells (Table 7). The median number of apoptotic cells was seven. Brison and Schultz [24, 37], using the TUNEL method, reported approximately three apoptotic cells per blastocyst cultured in KSOM supplemented with AA, but important differences exist between their studies and ours. First, we used outbred CF1 mice, whereas they used F1 hybrids. Second, they cultured from the two-cell stage, whereas we cultured from the zygote. Third, they terminated their experiment at 114-h post-hCG, whereas we terminated at 144-h post-hCG. The distributions of the numbers of dead cells in blastocysts at 144-h post-hCG cultured in KSOMPVA and KSOMPVAAA are displayed in Figure 4, b and e, respectively. The analyses of deviance showed that addition of AA to KSOMPVA had no significant effect on the total numbers of dead cells (Table 7). The median number of dead cells was five. Analyses of deviance also showed that the addition of AA to KSOMPVA had no effect on the numbers of dead cells in the ICM and TE (Table 7). The median numbers of dead cells in the ICM and TE were one and four, respectively. Figure 4, c and f, shows bivariate plots of the numbers of dead cells in the ICM and TE in each blastocyst produced in KSOMPVA or KSOMPVAAA.
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Bivariate plots showing the relation between numbers apoptotic cells and total numbers of live cells in blastocysts that developed from zygotes cultured for 144-h post-hCG in KSOMPVA and KSOMPVAAA are shown in Figure 5a. The nonparametric Spearman rank correlations (
) between numbers of apoptotic cells and total live cells produced after 144-h post-hCG in KSOMPVA and KSOMPVAAA were computed, both because the cell counts were not normally distributed and to avoid the effects of outlying observations. The results for KSOMPVA were = -0.0072 and P = 0.96; the results for KSOMPVAAA were = 0.0048 and P = 0.97. These results indicate no correlation between the numbers of apoptotic cells and total live cells. This conclusion is reflected in the almost horizontal linear regression lines shown in Figure 5a. In contrast, similar calculations suggest a significant negative correlation between numbers of dead cells and total live cells, particularly when embryos are cultured in KSOMPVA without AA. The results for KSOMPVA were = -0.403, P = 0.002; the results for KSOMPVAAA were = -0.223 and P = 0.07. The negative correlation coefficients are reflected in the downward slopes of the linear regressions shown in Figure 5b. The relation between cell death and total cell numbers of blastocysts produced by culture of zygotes in KSOM has been examined by Hardy [38], who plotting the dead cell index defined by Devreker and Hardy [39] as (number of dead cells/[total number of cells + number of dead cells])x 100. Their plots indicate that as the total cell number increases, the dead cell index falls, following a hyperbolic curve. This result is not inconsistent with our own, which follow a similar hyperbolic pattern when plotted similarly (plot not shown).
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Effects of Exogenous AA on the ECM of Blastocysts
Figure 6, A and B, shows optical sections obtained by the confocal microscope of two mouse blastocysts that developed in vivo for 120-h post-hCG. One was stained with a polyclonal antibody for collagen IV and the other with a polyclonal antibody for laminin. Both antibodies are not specific, because both have affinities for collagen IV, laminin, and nidogen (i.e., entactin), which are proteins specifically confined to basement membranes [40]. Thus, both these antibodies are useful reagents to demonstrate "basement membrane-type" ECM. A continuous basement membrane that lies adjacent to the basolateral surfaces of the mural trophoblast cells and faces the blastocoel was stained intensely by both antibodies. Staining also occurred in the continuous ECM between the primitive endoderm and the primitive ectoderm. A few endoderm cells were also seen on the blastocoel side of the basement membrane lining the mural TE. Occasionally, punctate staining of the ECM between the cells of the primitive ectoderm was observed, as well as of the ECM between the primitive ectoderm and the polar TE.
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Figure 6, C and D, show optical sections of two mouse blastocysts stained for either collagen IV or laminin that developed from zygotes cultivated in vitro for 144-h post-hCG in KSOMPVA. In both blastocysts, the basement membrane beneath the mural TE developed only in discrete areas beneath the body of each trophoblast cell. Confluence of these areas into a continuous sheet (i.e., the process by which the basement membrane finally forms) was not reached [41]. No continuous basement membrane was seen. Staining of the ECM occurred in both blastocysts between or on the relatively few cells of the ICM, but a clear separation of the primitive endoderm and primitive ectoderm was not observed.
Figure 6, E and F, shows optical sections of two mouse blastocysts that developed from zygotes in vitro for 144-h post-hCG in KSOMPVAAA containing half-strength AA stained for collagen IV or laminin. The basement membrane beneath the mural TE formed a continuous sheet and was more advanced than the blastocyst that developed in KSOMPVA. The primitive endoderm and primitive ectoderm were clearly delaminated by the formation of ECM, and some staining was seen of the marginal regions between the primitive ectoderm and the polar TE.
The cultured blastocysts shown in Figure 6, C through F, were selected to show specimens with similar morphology to "normal" blastocysts that developed in utero. Considerable variation exists, however, between blastocysts in the morphology and developmental stage reached when zygotes are cultured in KSOMPVA and KSOMPVAAA. An attempt to quantify these differences was made using two scoring systems, one assessing the cellular morphology and the other assessing the appearance of the extracellular matrix. Blastocysts were cultured in KSOMPVA and KSOMPVAAA for 144 h, and three replicates were done. The first two were stained for collagen IV, and the third was stained for laminin. The data on cell morphology were pooled over replicates for statistical analysis, because no significant differences were found between them. The distributions of the scores regarding the morphology of the TE for blastocysts produced in KSOMPVA and KSOMPVAAA are shown in Table 8. These distributions were not significantly different (P = 0.86), with 100% and 97.8% developing normally in the two media, respectively. In contrast, distributions regarding development of the ICM in the two media were very significantly different (P < 10-5). Only 35% developed well in blastocysts produced in KSOMPVA, compared with 79% in blastocysts produced in KSOMPVAAA. The contrast was even greater in development of the primitive endoderm (P < 10-5). Only 15% developed well in KSOMPVA, compared with 92% in KSOMPVAAA. The data obtained on the ECM, stained for either collagen IV or laminin, were also pooled, because both antibodies were nonspecific and merely detected ECM. The results of all three replicates were very similar. Distributions of the scores regarding development of the ECM associated with the mural TE were very significantly different (P = 0.0017; Fig. 7A). The basement membrane was completely developed in only 14% of blastocysts produced in KSOMPVA, compared with 46% of blastocysts produced in KSOMPVAAA. The distributions of the scores regarding development of the ECM associated with delamination of the primitive endoderm in KSOMPVA and KSOMPVAAA were also very significantly different (P < 10-5; Fig. 7B). The primitive endoderm was only completely delaminated in only 9% of the blastocysts produced in KSOMPVA, compared with 43% of the blastocysts produced in KSOMPVAAA.
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DISCUSSION
Effect of AA on Blastocyst Formation and Hatching
The results using CF1 x BDF1 zygotes summarized in Tables 3 and 4 and in Figure 1 demonstrate: 1) the blastocyst can develop 144-h post-hCG from approximately 70 percent of zygotes in KSOMPVA in which the only fixed-nitrogen source is glutamine, 2) addition of the 19 common AA shown in Table 3 to KSOMPVA increases the yield of blastocysts by a additional 20% (approximately) in a concentration-dependent manner, and 3) approximately 10% of zygotes are incapable of developing into a blastocyst when cultured in KSOMPVAAA. The number of nondevelopers is 10% less than that observed in previous experiments [19].
A parsimonious explanation for these results is that addition of AA to KSOMPVA accelerates the speed of development for all embryos in a concentration-dependent manner. Theoretically, let the time a zygote takes to develop into a recognizable blastocyst be t, and for the sake of argument, assume that the distribution of these times in a population of embryos is represented by the log-normal distribution (Fig. 8). (We exclude the 10% of nondevelopers.) The proportion of zygotes that develop by time T (e.g., 144-h post-hCG) is given by the area beneath the curve to the left of T. If the addition of some concentration of AA to the medium accelerates the blastocyst development times for all embryos, the distribution is displaced to the left (Fig. 8), with a consequent increase in the area under the curve to the left of T. The importance of this model is that it stresses the results can be explained in terms of a general effect of AA on all zygotes in the population. There is no need to postulate that something is unique regarding the ~20% of embryos that are specifically stimulated to develop into blastocysts by the addition of the AA. Evidence that addition of AA to KSOM increased the developmental rate was reported by Ho et al. [13]. Evidence also suggests that a subset of the 19 AA shown in Table 2 are sufficient to accelerate development. It has been reported that Eagle nonessential AA and glutamine accelerate the time of the first three cleavage divisions and increase the time of compaction in the mouse when added to modified mouse tubal fluid (mMTF) [18, 42]. The linear regression coefficients of the logit partial and complete hatching on AA log2 dilution are similar (Table 4). This result suggests that the increased hatching rates produced by addition of AA to KSOMPVA result from a general effect of accelerating development, as in the case of blastocyst formation.
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Effect of AA on Proliferation of Total Cells in the Blastocyst
The data summarized in Table 5 and Figure 2 demonstrate that addition of AA to KSOMPVA significantly increases the numbers of cells observed in a blastocyst 144-h post-hCG. The number of cells observed is the resultant of the number of cells produced by multiplication and the number of cells destroyed by cell death, either by oncosis or apoptosis [43–45]. Cell death has been recognized as a mechanism for eliminating redundant cells in normal preimplantation embryos since the pioneering work of El-Shershaby and Hinchliffe [38, 46]. The maximum incidence of cell death occurs approximately 97-h postcoitum, and it largely is confined to the ICM [23]. Application of the TUNEL reaction has shown this natural cell death to result from apoptosis [24, 38]. The effects of adding AA to KSOMPVA on cell death in blastocysts produced from zygotes in vitro over 144 h have been examined using two stains: the TUNEL reaction, which detects apoptosis; and fluorochromes such as PI, which labels nuclear fragments. The results are summarized in Figure 4 and 5 and in Table 7. We found no statistically significant evidence that addition of AA to KSOMPVA influences the incidence of cell death as detected by either method. Addition of AA to KSOMPVA does not affect the numbers of apoptotic cells in blastocysts cultured from the zygote, but the percentage of apoptotic cells in blastocysts produced in KSOMPVA (10%) is less than the percentage of apoptotic cells in blastocysts produced in KSOMPVAAA (6%). The difference results from the increase in total cells in the blastocysts produced by the added AA.
Interpretation of results from the two staining methods for cell death requires some care, because the cells these two methods detect may partially overlap. In addition, apoptosis does not occur until after the morula stage, whereas cell death can occur at the earliest stages of culture because of adverse culture conditions. The significant negative correlation between the numbers of dead cells and total live cells may well result from cell loss early during development creating a geometric loss of cells at the blastocyst stage because of the loss of daughter cells. As a result, blastocysts containing larger cell numbers would be associated with a low incidence of death during the early stages. In contrast, the loss of cells soon after the morula stage because of apoptosis would only have an additive effect, because their daughter cells would be few in number.
Effect of AA on Pattern Formation in the Blastocyst
Differentiation of the morula involves not only the formation of two populations of cells (i.e., the ICM and TE) but also their location in different regions of the blastocyst. The results summarized in Figure 2d and Table 5 show that addition of AA to KSOMPVA increases the number of cells in both the ICM and TE. Furthermore, the ratio of ICM to TE cells is significantly greater among embryos cultured in KSOMPVAAA. Thus, the added AA significantly favors the division of cells that will occupy the ICM. The numbers of cells observed in the ICM and TE and the rates of division in each region, however, are determined, in part, by the partition of cells into the two cell lineages, which normally occurs at the 16- to 32-cell stage [47, 48]. Whether the added AA affect the stage at which this partition of cells occurs is unknown. Our results differ from those of Lane and Gardner [49], who found that adding AA to medium mMTF, which contains 4 mg ml-1 BSA, increased only the number of ICM cells and not the number of TE cells.
The results displayed in Figures 6 and 7 show that addition of AA to KSOMPVA affects the deposition of collagen IV and laminin. The display of these ECM proteins is particularly useful for demonstrating delamination of the primitive endoderm from the ectoderm [5, 28, 49–52]. The results we obtained by the study of serial optical sections using confocal microscopy demonstrate that blastocysts cultured from the zygote in KSOMPVAAA retain their three-dimensional form, and that the primitive ectoderm and primitive endoderm become separated by the deposition of a basement membrane-type ECM similar to that which occurs in blastocysts that develop in utero.
ACKNOWLEDGMENTS
We wish to thank Dr. Susan Palmieri for helpful advice on confocal microscopy. We are also indebted to Drs. John Eppig, Richard Tasca, and Betsey Williams for helpful criticisms of the manuscript.
FOOTNOTES
First decision: 22 October 1999.
1 Supported as part of the National Cooperative Program on Non-Human In Vitro Fertilization and Preimplantation Development and funded by the National Institute of Child Health and Human Development, NIH, through cooperative agreement HD21988. 
2 Correspondence: John D. Biggers, Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115. FAX: 617 432 2229; john_biggers{at}hms.harvard.edu
3 Current address: Department of Biology, University of Michigan, Ann Arbor, MI 48109.
Accepted: February 29, 2000.
Received: July 22, 1999.
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