Human Reproduction, Vol. 15, No. 8, 1791-1801,
August 2000
© 2000 European Society of Human Reproduction and Embryology
IVF of mouse ova in a simplex optimized medium supplemented with amino acids
1 Fertility Center of New England, 20 Pond Meadow Drive, Suite 101, Reading, MA 01867 and 2 Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
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Abstract |
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The addition of amino acids to a modified simplex optimized medium (mKSOM) did not increase the percentage of blastocysts that develop from CF1 mouse ova fertilized in vitro. In contrast, the percentage of blastocysts that began to hatch and the number of cells in these blastocysts, particularly in the inner cell mass, was increased. The added amino acids also supported the development of a more organized extracellular matrix in the same blastocysts. The results suggest that zygotes produced in amino acid-supplemented mKSOM have a greater developmental potential, perhaps developing at a faster rate, than zygotes produced in mKSOM. This enhanced developmental potential may be caused by the alleviation of osmotic stress on the ova and zygotes by the amino acids that are osmolytes. The fertilization of human ova in vitro may benefit from the inclusion of free amino acids in the fertilizing medium. The availability of a medium that can be used to support both IVF and preimplantation development in the mouse is likely to benefit the recovery of mouse strains from cryopreserved spermatozoa.
Key words: amino acids/blastocyst/fetus/IVF/mouse
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Introduction |
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Three types of medium have been used to provide the environment in which IVF in the mouse has been successful. The first type of medium is a simple chemically defined medium which is no more than a classical physiological saline (Burton, 1975
) supplemented with a carbon source. Such a medium designed for the culture of mouse preimplantation embryos (Whitten and Biggers, 1968) was the first to be successfully employed to fertilize mouse ova in vitro (Whittingham, 1968; Mukherjee and Cohen, 1970). A modification of this medium (Whitten, 1971) was later used with comparable results (Hoppe and Pitts, 1973). Both of these media are derived from Krebs–Ringer's solution. An alternative medium (Whittingham, 1971), based on Tyrode's solution, has also been successfully employed to fertilize mouse ova in vitro (Frazer and Drury, 1975). The second type of medium is based on analyses of oviductal fluid. Mouse ova have been successfully fertilized in human tubal fluid (HTF) medium (Nakagata, 1996), which was designed for human IVF based on analyses of HTF (Quinn et al., 1985a,b). The third type of medium is a chemically defined medium containing many components in addition to the basic physiologcal saline, such as all 20 amino acids (AA). Minimum essential medium (MEM), based on Earle's balanced salt solution supplemented with the essential and non-essential AA required for the culture of certain human cell lines (Eagle, 1959), has also been used successfully to fertilize mouse ova in vitro (Ho et al., 1995).
Although all three types of media support IVF in the mouse they frequently do not permit development beyond the 2-cell stage, unless ova from F1 hybrids between two inbred lines are used, because of the so-called 2-cell block (review: Biggers, 1998). A medium with relatively few components, called KSOM (simplex optimized medium) (Lawitts and Biggers, 1993), has been shown to overcome the 2-cell block in many strains of mice, thus allowing complete development from the newly fertilized ovum to the blastocyst stage. In 1995 two papers reported results that independently extended the use of KSOM. The addition of essential and non-essential AA to KSOM, in the concentrations used by Eagle (1959) for the culture of some human cell lines, improved the development of preimplantation mouse embryos from the zygote to the blastocyst stage (Ho et al., 1995). Thus, after fertilizing mouse ova in vitro in MEM these investigators transferred the resulting zygotes to AA-supplemented KSOM for culture to the blastocyst stage. At the same time, it was shown that KSOM would support IVF in several strains of mice provided the glucose and bovine serum albumin (BSA) concentrations were raised from 0.2 to 5.56mmol/l and 1–4 mg/ml respectively (Summers et al., 1995). This medium was denoted mKSOM. Zygotes produced by IVF in mKSOM developed in high yield into blastocysts in KSOM, and then developed into newborns after transfer to the uterus of surrogate mothers. This result was also of particular interest because of the prevailing view that the development of the mouse embryos through the initial cleavage stages occurred in a concentration of glucose believed to be inhibitory (Barnett and Bavister, 1996).
The results presented in this paper demonstrate that the addition of AA to mKSOM improves the development of zygotes produced by IVF in mKSOM. Our results confirm that this medium, containing 5.56 mmol/l glucose, does not inhibit early preimplantation development. More importantly, the results show that it is advantageous to use AA-supplemented mKSOM for the fertilization medium. Thus it is possible to fertilize mouse ova in vitro and cultivate them to the blastocyst stage in the same medium, thereby eliminating any stress that may occur as a result of suddenly transferring the zygotes from one environment to another. This result may be of practical importance for the reconstitution of mouse strains from cryopreserved sperm and in human IVF.
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Animals
Hybrid males (B6D2F1/CrlBR: Charles River Laboratories, Wilmington, MA, USA) were used as sperm donors. Unfertilized ova were obtained from outbred CF1 females (Harlan–Sprague–Dawley, Indianapolis, IN, USA). F1-hybrid females B6D2F1/J (C57BL/6J
xDBA/2J
) and B6CBAF1/J (C57BL/6JxCBA/J) (Jackson Laboratory, Bar Harbor, ME, USA) were used in a few experiments. Mice were maintained on a 14 h light:10 h dark cycle. Animals were killed by cervical dislocation.
Media
The following media were used: (i) KSOM (Lawitts and Biggers, 1993) (Table I); (ii) mKSOM, KSOM in which the D-glucose and BSA concentrations were raised to 5.56 mm and 4 mg/ml, respectively; (iii) FHM, a HEPES-buffered version of KSOM (Lawitts and Biggers, 1993); and (iv) mFHM, FHM containing 5.56 mmol/l glucose and 4 mg/ml BSA. If the AA shown in Table II were included in the medium, a superscript was added, e.g. KSOMAA, mKSOMAA. In some experiments, specifically identified in the text, the glucose concentration in KSOMAA was raised to 5.56 mmol/l, leaving the BSA concentration at 1 mg/ml.
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Media were prepared as 2x solutions (without the calcium chloride, BSA or glucose) and frozen at –70°C. Calcium chloride, BSA and glucose stocks were frozen separately at –20°C (Biggers et al., 1997
). All reagents were from Sigma Corporation (St Louis, MO, USA), unless otherwise stated. The BSA was Fraction V (Sigma, Cat. #A9647, lot #15H0672). Non-essential AA (NEAA) and essential AA (EAA) were added from premixed NEAA and EAA solutions (Gibco BRL, Grand Island, NY, USA) that were stored at 4°C. Water used for media preparation was 18 
M reverse-osmosis-filtered, twice demineralized-filtered, and then glass-distilled. Culture medium was prepared and filter-sterilized through a 0.2 µm filter (Millipore, Bedford, MA, USA) into a second sterile tube. The preparation of HEPES-buffered KSOM, denoted FHM, has been described elsewhere (Lawitts and Biggers, 1993; Summers et al., 1995).
Sperm preparation
Males of proven fertility were killed, and the cauda epididymis immediately removed to a 150 µl drop of modified Tyrode's medium under mineral oil (Sigma: embryo-tested, Cat. #M8410) (Summers et al., 1995). The epididymal contents were squeezed out and the residual caudal tissue was discarded. Capacitation was allowed to proceed for 1–2 h at 37°C in 5% CO2 in humidified air. Sperm concentrations were determined with a haemocytometer.
Oocyte collection
Female mice were superovulated by i.p. injection of 5 IU of pregnant mare's serum gonadotrophin (PMSG) (Sigma), followed by an i.p. injection of 5 IU of human chorionic gonadotrophin (HCG) (Sigma) 48 h later. Ova were collected between 13.5 and 14 h post-HCG administration by removing entire oviducts and placing them into 1ml drops of mFHM at 37°C (Summers et al., 1995). Cumuli were collected from an oviduct at the site of the cumulus bulge by tearing with a 25-gauge needle. Groups of 8–10 cumuli were then transferred through a second 1 ml drop of the same medium, then placed into 1 ml of the appropriate insemination medium under mineral oil.
Fertilization in vitro
Fertilization in vitro was carried out in 1 ml drops of either mKSOM or mKSOMAA under mineral oil. A preincubated, capacitated sperm suspension was gently added to the freshly ovulated ova to give a final motile sperm concentration of 1x106/ml. The combined sperm–oocyte suspension was incubated for 4 h. The ova were then washed through several changes of medium and finally incubated in 50 µl drops of medium under mineral oil. Fertilization was assessed by recording the number of 2-cell embryos 24 h after completion of fertilization in vitro.
Embryo culture
The ~95% of the ova exposed to spermatozoa that were not fragmented or degenerate were selected for culture. Groups of 12 of these putatively fertilized ova were cultured for 144 h post-HCG administration in a 50 µl droplet of medium of the indicated supplemented KSOM, overlayered with mineral oil. The cultures were incubated at 37°C in 5% CO2 in air in a humidified incubator. Culture plates (60 mm suspension culture dishes, Corning Inc., NY, USA) were prepared 1 day before embryo collection and equilibrated overnight. Groups of zygotes were washed through two drops of culture medium, then cultured in a third drop.
Embryo evaluation
Embryos were observed at x100 on a warmed microscope stage (35°C) of a Wild dissecting microscope. The numbers of zygotes that cleaved and developed by 96, 120 and 144 h post-HCG administration into zona-enclosed blastocyst, and partially and completely hatched, were counted.
Total cell counts
Groups of 8–10 blastocysts from each treatment were fixed [3% formalin in Dulbecco's phosphate-buffered saline (DPBS) at 37°C] for 15 min, then transferred into holding solution (DPBS, 2% sodium azide, 0.2% powdered milk, 2% goat serum. 2% BSA, 1 mol/l glycine and 0.01% Triton-X-100) with 1.0 µg/ml bisbenzimide (HO33258), and stored at 4°C in the dark (Ebert et al., 1985). Stained blastocysts were mounted on a glass slide under a coverslip using mounting medium (50% glycerol and 1 mg/ml HO33258 in DPBS containing 2% sodium azide). Nuclei were counted on a Zeiss Epifluorescence microscope with a 365-nm band pass excitation filter and a 420 nm long pass barrier filter.
Differential cell counts
Differential cell counts were determined by a modification of a method using bisbenzimide (Hoechst 33258) and propidium iodide (PI) (Handyside and Hunter, 1984; Papaioannou and Ebert, 1988). The zonae pellucidae were removed from blastocysts with acid Tyrode. After the zona had dissolved (~10 s), the blastocysts were placed into FHM + 10% fetal calf serum (wash A) on a rocker for 15 min at room temperature. They were then transferred into FHM containing 10% rabbit anti-mouse red blood cell antibody (ICN #844558), and placed on a rocker for 30 min. They were then washed three times in wash A and again placed on a rocker in wash A for 15 min at room temperature. The blastocysts were then transferred into a complement-stain solution (10% guinea-pig complement [Gibco (#191900032) + 1 µg/ml HO33258 + 1 µg/ml PI in FHM] and placed on a rocker in the dark for 30 min at room temperature. Labelled embryos were then washed three times in wash A before fixation in 4% formalin in FHM + PI + HO33258. They were then stored in the dark at 4°C for 1–3 days until the cells were counted. The nuclei in the trophectoderm (TE) fluoresced red-pink while the nuclei in the inner cell mass (ICM) fluoresced blue.
Visualization of collagen IV by confocal microscopy
An immunofluorescence method (Palmieri et al., 1992, 1994) has been adapted for the detection of collagen type IV. Briefly, embryos were rinsed in 4 mg/ml of BSA/phosphate-buffered saline (PBS), and fixed in 3% paraformaldehyde in stabilization buffer (0.2 mol/l PIPES, 5 mmol/l MgCl2 and 2.5 mmol/l EGTA) for 30 min at 37°C. The fixed blastocysts were rinsed twice in BSA/PBS and incubated at room temperature for 45 min in 0.2% Triton BSA/PBS. Finally, the embryos were transferred to a blocking solution consisting of 3% fetal calf serum (FCS), 0.1% Tween-20, 0.02% Na-azide, and 0.4% powdered milk in PBS and incubated overnight at 4°C. Blastocysts were then treated with hyaluronidase (2 mg/ml) in BSA/PBS for 30 min at 37°C. After rinsing with BSA/PBS, 5–10 embryos were placed in 1/3 diluted blocking solution in PBS in 50 ml droplets, containing a 1:150 dilution of polyclonal rabbit anti-mouse collagen IV immunoglobulin (Ig)G (a1 and a2, 185 kDa and 170 kDa respectively) (Collaborative Biomedical Products, Bedford, MA, USA; lot no. 907304), and incubated for 1 h at 37°C. Embryos were rinsed three times in the blocking solution, and incubated for 1 h at room temperature in the same solution. The blastocysts were then incubated for 1h at 37°C in a secondary antibody, consisting of goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (FITC) (Boehringer Mannheim, Mannheim, Germany; lot no. 21014, 1:100 dilution in PBS) in BSA/PBS. After incubation, with the secondary antibody, the blastocysts were again rinsed in blocking solution, transferred through a glycerol series (2.5%, 5%, 10%, 20% and 50% glycerol in 3% FCS/PBS, and mounted on glass slides in glycerol with PI and 1,4-diazabicyclo [2,2,2]-octane. A cover slip was placed on the slide and sealed with clear nail polish. All appropriate control experiments to ensure antigen specific binding of the primary antibody control peptides have been described in detail elsewhere (Palmieri et al., 1994).
A Zeiss laser scan microscope 410 invert was used to examine the embryos. An argon laser excited the PI and FITC at a wavelength of 488 nm. A 590 nm longpass filter was used to detect fluorescent emission of the PI while a 510–525 nm bandpass filter was used to detect the FITC. The confocal settings were standardized using mouse blastocysts flushed from the uterus at 120 h post-HCG administration and labelled with the appropriate antibody. All embryos were viewed using the x40 objective at a zoom of two. Each embryo was scanned to find an optical section where (i) the ICM was best developed both in terms of cell number and organization, and (ii) the collagen IV IgG had demarcated the primitive endoderm (PE) and lined the inner aspect of the TE (Adamson and Acers, 1979; Leivo et al., 1980). Optical sections were recorded using Zeiss LSM software version 3.80. For image analysis, optical sections were retrieved using Adobe Photoshop version 3.0.
Embryo transfer
Pseudopregnant CD1 females were produced by mating CD1 females to vasectomized CD1 males. Blastocysts obtained after IVF in mKSOMAA and cultured in KSOMAA containing 5.56 mmol/l glucose were transferred into day 3 pseudopregnant females, six blastocysts chosen at random to each uterine horn. Fetuses were collected from pregnant mice on either day 14 or day 15 after IVF for gross examination and wet weight measurements. The embryo transfer data were the controls of a larger study that will be reported in detail elsewhere.
Biometrical considerations
Initial exploratory graphical analyses were done using the S-Plus 2000 package (MathSoft, Cambridge, MA, USA). Bivariate data are displayed using scatter plots after jittering the data to separate overlapping observations. Notched box plots, showing the confidence limits (P = 0.05) of the median, were used to test for the significance of the differences between medians. Two medians are significantly different if their confidence limits do not overlap (McGill et al., 1978).
Experimental design and the multivariate characteristics of the responses
All experiments used a randomized block experimental design, the experimental unit randomized being a set of 12 zygotes. Observations were made on the same embryos at three different times, so that the data are repeat (longitudinal) measurements (Diggle et al., 1994). At each time of observation the numbers of embryos that developed into three developmental stages, the zona-intact blastocyst, the partially hatched blastocyst and completely hatched blastocyst, were counted, so that the data are also ordinal categorical responses (Clogg and Shihadeh, 1994). Thus the multivariate observations are serially correlated both longitudinally and ordinally. The raw data have been re-expressed as the numbers of zygotes that at least developed into blastocysts and at least partially hatched by 120 and 144 h post-HCG administration. The responses were then expressed as the percentages of blastocysts that developed from zygotes and the percentages of these blastocysts that partially hatched.
General linear regression (McCullagh and Nelder, 1989)
All categorical responses described above have been analysed using general linear regression, assuming that the data had binomial errors and the link function was the logit transformation. The cell counts were also analysed by general linear regression, assuming that the errors arose from a multiplicative process with lognormally distributed errors where the link function was 1. All statistical analyses were summarized as analysis of deviance tables. The computations were done using S-Plus 2000 (MathSoft, Cambridge, MA, USA).
Fisher's exact significance test
This test for a 2x2 table was calculated using StatXact 4.0 (Cytel, Cambridge, MA, USA).
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Results |
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Development of embryos after the exposure of CF1 ova to B6D2F1 spermatozoa in vitro
Two experiments were done. In experiment 1, ova were exposed to spermatozoa in mKSOM for 4 h, and subsequently cultured in one of four media: KSOM, mKSOM, KSOMAA and mKSOMAA. Thus the treatments formed a 2x2 factorial array in which one factor was the medium and the other factor the presence or absence of AA. The experimental unit was a droplet containing 12 ova which had been exposed to spermatozoa. The four culture conditions were compared in a randomized block design consisting of three replicates. A smaller number of zygotes, produced by fertilization in vivo (controls), were also cultured simultaneously in the four media as controls on the media quality. In experiment 2, ova were randomly allotted to two treatments: fertilization in mKSOM and fertilization in mKSOMAA. The resulting zygotes, in groups of 12, were then cultured for 144 h post-HCG administration in KSOMAA containing 5.56 mmol/l glucose. Thus, the two treatments differed only in the 4 h exposure of the oocyte–cumulus complex during fertilization in vitro. Three replications were done. A control group of ova fertilized in vivo was included in each replicate.
Development to the 2-cell stage (experiment 1)
Table III summarizes the percentages of ova that cleaved to the 2-cell stage after 24 h culture in the four media. A high rate of IVF (94–97%), using cleavage to the 2-cell stage as a criterion, was obtained in all four media. The zygotes produced by fertilization in vivo cleaved to the 2-cell stage at a slightly lower rate (85–96%).
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Development to the 2-cell stage (experiment 2)
The number of 2-cell embryos that developed after 24 h is shown in Table IV
. Again very high rates of IVF, using cleavage to the 2-cell stage, were obtained in both mKSOM and mKSOMAA.
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Development to the blastocyst stage (experiment 1)
Figure 1
summarizes the percentages of ova fertilized in vitro that developed at least to the blastocyst stage and partially hatched by 120 and 144 h post-HCG administration, respectively.
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A high percentage of zygotes developed to at least the blastocyst stage, particularly if AA were in the embryo culture media (KSOMAA and mKSOMAA). Increasing the concentrations of glucose and BSA simultaneously in KSOMAA to give mKSOMAA had little effect. The analyses of deviance of the raw data are shown in Table V
. There was a very significant (P = 0.00008) replicate xAA interaction in the data obtained on blastocyst formation at 120 h post-HCG administration. This interaction was not significant, however, in the data on blastocyst formation by 144 h post-HCG administration. The change in the interaction was due to two low responses at 120 h post-HCG administration in the third replicate in media containing AA which caught up in development by 144 h post-HCG administration.
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A high percentage of the blastocysts partially hatched by 120 and 144 h post-HCG administration (up to ~80%), particularly if AA were in the embryo culture media (KSOMAA and mKSOMAA). Raising the concentrations of glucose and BSA simultaneously in KSOMAA to give mKSOMAA had little effect on partial hatching and was not significant. There are several interactions involving replicates in the data on partial hatching at 120 h post-HCG administration (Table V
). These interactions could not be attributed to any specific pattern of responses in the different replicates. Thus, there appeared to be no statistically significant effects on the percentage of partial hatching 120 h post-HCG administration. There was a very significant (P = 0.00033) replicate xAA interaction in the data obtained on partial hatching at 144 h post-HCG administration. This was due to a large variation in the percentages of zygotes partially hatching in the different replicates caused by the addition of AA to mKSOM.
The analysis of deviance of the logarithms of the total cell counts (Table VI) revealed that the effect of AA was highly significant (P = 0.043), whereas the effect of raising the concentrations of glucose and BSA was barely significant. There were no significant interactions. The data can, therefore, be summarized in terms of the distributions of the main effects as shown in box plots in Figure 2. The addition of AA to KSOM and mKSOM significantly (P < 0.05) increased the total number of cells in the blastocyst. Raising jointly the concentrations of glucose and BSA, however, slightly depressed the total cell count in the blastocyst.
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Table VII
shows the percentages of the control zygotes, recovered from naturally mated females, that developed at least to the blastocyst stage or at least partially hatched, and the total blastocyst cell counts. These values were very similar to the values obtained by producing zygotes by IVF (see Figure 1).
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Figure 3A,B
shows optical sections through two blastocysts that were fertilized in vivo, and flushed from the uterus 4 days later, stained for collagen IV to display the basement membrane-like extracellular matrix (ECM) that develops at this stage of development. Basement membrane develops beneath the cells of the mural TE, and it separates the primitive endoderm from the primitive ectoderm in the ICM. Fuller descriptions of these changes can be found elsewhere (Leivo et al., 1980; Thorsteinsdóttir, 1992; Biggers et al., 2000). Figure 3C shows a blastocyst, stained for collagen IV, that was fertilized in vitro in mKSOM and cultured in KSOM to the blastocyst stage. A basement membrane lining the mural TE was present, while the ECM in the ICM was poorly developed. Figure 3D shows a blastocyst, stained for collagen IV, that developed from an ovum fertilized in vitro in mKSOM and cultured in KSOMAA. A basement membrane had developed below the mural TE. The ICM was well-developed and ECM had developed between the primitive endoderm and the primitive ectoderm.
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Development to the blastocyst stage (experiment 2)
The percentages of zygotes that developed in KSOMAA containing 5.56 mmol/l glucose at least to the blastocyst stage are shown in Table VIII
. The analyses of deviance, shown in Table IX, demonstrate that there are highly significant differences between fertilizing in vivo and in vitro. The yields of blastocysts from ova fertilized in vivo were significantly greater (Fisher's exact test, P < 10–5) than the yield of blastocysts produced from zygotes produced in vitro. There was no statistically significant difference in the percentages of blastocysts that developed from zygotes produced by IVF in mKSOM and mKSOMAA.
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In contrast, the difference in the percentages of blastocysts that partially hatched from zygotes produced by IVF in mKSOM and mKSOMAA was just statistically significant (Fisher's exact test, P = 0.037). The yields of blastocysts that partially hatch was significantly less (Fisher's exact test, P < 10–5) than the yields that arose from zygotes produced in vivo.
Figure 4a is a scatter plot of the numbers of cells in the ICM and TE that developed by 144 h post-HCG administration in two of the replicates of experiment 2. There was a tendency to shift the counts upwards and to the right when mKSOMAA was used as the fertilization medium. This displacement is even more marked in blastocysts that developed from ova fertilized in vivo. Analyses of deviance (not given) of the log-transformed counts showed highly significant differences between the two replicates and the effects of the three modes of fertilization. Figure 4b,c shows the marginal distributions of the numbers of ICM and TE cells, in the form of box plots, for the three modes after adjusting for the replicate differences. The cell counts in the ICM in blastocysts produced from ova fertilized in vivo were significantly higher (P < 0.05) than in the blastocysts whose ova were fertilized in mKSOM but not significantly higher than those fertilized in mKSOMAA. The cell counts in the TE in blastocysts produced from ova fertilized in vivo were significantly higher (P < 0.05) than in the blastocysts whose ova were fertilized in mKSOM, but barely significantly higher (P < 0.05) than those fertilized in mKSOMAA. Figure 4d shows the distributions of the ratios of the numbers of ICM cells to the numbers of TE cells. The ratios for the different treatments are not significantly different.
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Development of zygotes from F1 hybrid strains in KSOM, KSOMAA, mKSOM and mKSOMAA produced by IVF
Ova from two F1 hybrid stains of mice (B6CBAF1 and B6D2F1) were fertilized in vitro in mKSOM with males of the same strain. The F2 zygotes were then cultured to the blastocyst stage in either KSOM or mKSOM, with and without AA. The percentages of embryos reaching at least the zona-enclosed blastocyst stage, partially hatched, and completely hatched blastocyst stages at 120 h and 144 h were recorded. The results are summarized in Table X
. There was a high rate of development to the blastocyst stage in all treatment groups, whether or not AA were present in the media. In contrast, high rates of partial and complete hatching were observed only when AA were present in the media.
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Embryo transfer
Fifty-four blastocysts, produced by IVF in mKSOMAA and cultured in KSOMAA containing 5.56 mmol/l glucose, were transferred into nine pseudopregnant recipient mice. Forty-three (80%) of the embryos implanted, and 24 (44%) of them became fetuses. No gross morphological abnormalities were observed in any of these fetuses. The wet weights of the fetuses from three and nine of the mothers were measured on days 14 and 15 after fertilization. The distributions of these weights are shown as box plots in Figure 5
. The weights are within the normal range of mouse fetuses at this age (Rugh, 1968).
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Discussion |
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A very high yield of outbred CF1 2-cell mouse embryos was produced when fertilization in vitro was done in both mKSOM and mKSOMAA. (Tables III and IV
). The majority of these embryos were then capable of developing in vitro into blastocysts in either KSOM and mKSOM, particularly if AA were added to the medium (Figure 1a). The presence of AA in the media favoured a high rate of partial hatching, as high as 80% of the blastocysts that developed. Blastocysts produced in media containing AA also contained a larger number of cells (Figures 2 and 4), had larger ICM and more organized ECM (Figure 3), than blastocysts that developed in media without AA. Blastocysts produced in mKSOMAA were also capable of developing into normal fetuses after transfer into the uterus of surrogate mothers. None of the 24 fetuses produced showed any sign of gross morphological abnormality, such as exencephaly which has been attributed to the damaging effects of ammonium produced by the decomposition of glutamine in modified mouse tubal fluid medium (mMTF) (Lane and Gardner, 1994). A high rate of development into hatched blastocysts was also observed when ova from two F1 hybrid strains were fertilized in vitro and cultured in mKSOMAA (Table X).
The enhanced effect of adding AA to the fertilizing medium, followed by culture in mKSOMAA, suggests that zygotes produced have a greater developmental potential, perhaps developing at a faster rate, than zygotes produced in an AA-free medium. These results may be due to the effect of the AA on the unfertilized ovum and zygote, rather than on the spermatozoon, since it has been shown that adding non-essential AA (Eagle, 1959) and glutamine to the medium used to collect CF1 zygotes fertilized in vivo also enhances development to the blastocyst stage (Gardner and Lane, 1996, 1999). Further work is needed to show whether the AA influence cellular functions in spermatozoa.
The mouse unfertilized ovum contains a significant free amino acid pool, consisting mainly of taurine, glycine, alanine, glutamate and aspartate (Schultz et al., 1981). The functions that have been proposed for the intracellular AA in preimplantation embryos fall into three categories (Edwards et al., 1998): (i) homeostatic (osmolytes, pH regulators), (ii) protective (chelators of heavy metals), (iii) metabolic (substrates, precursors for protein sysnthesis, regulators of metabolic function). Reliable analyses of the concentrations of AA in the mouse oviduct where fertilization occurs are not available (Guérin et al., 1995). These and other investigators, however, have reported significant concentrations of free AA, particularly glycine, in the oviduct of other species. A reasonable conjecture is that significant AA are present in the ampulla of the mouse oviduct where fertilization occurs. When an unfertilized mouse ovum is placed in mKSOM, it is exposed to sudden changes in its environment. These changes include: (i) a drop in osmolality from 290 to 300 mOs/kg in the oviduct (Collins and Baltz, 1999) to about 270 mOs/kg in mKSOM, (ii) an increased exposure to weak acids, particularly lactic acid, whose concentration is increased from 4.79 mmol/l in the ampulla of the oviduct (Gardner and Leese, 1990) to 10 mmol/l in mKSOM, and (iii) the total removal of AA. The ovum swells when placed in KSOM (Sequin and Baltz, 1997; Kolajova and Baltz, 1999; Hammer et al., 2000) and becomes highly permeable to AA (J.M.Baltz, Loeb Medical Research Institute, Ottawa Civic Hospital, Ontario, Canada; personal communication). The ovum will thus lose the protective effect of AA against a sudden intracellular acid load (Edwards et al., 1998). Further, the absence of taurine and glycine in the culture medium deprives the ovum of two of the organic osmolytes needed to maintain its normal cell volume (Van Winkle et al., 1994; Kolajova and Baltz, 1999). These effects are examples of processes that may occur in the so-called shock phase which occurs when tissues are placed in culture (Biggers, 1963). If the stresses are not severe, the tissues can adapt to the new conditions and enter a stable phase. It is clear that when a mouse ovum is fertilized in vitro in mKSOM, or in similar media, it experiences a shock phase to which it is able to adapt, thus allowing the zygote to develop to the blastocyst stage. The addition of AA to the fertilizing medium ameliorates these stresses, possibly allowing more rapid development. The possibility that the addition of AA to media would reduce the shock experienced by the unfertilized ova and spermatozoa during IVF in the human should be considered. Most media used for this purpose do not contain free AA. Gardner and Lane (1999) suggest that AA should be included in all media used for the culture of the human preimplantation embryo. The results of the current study suggest that AA should also be included in the fertilizing medium.
The observations in this study show that a single medium developed specifically for the mouse (mKSOMAA) can be used to support fertilization of mouse ova in vitro in high yield, and that the zygotes produced can be cultured in the same medium to the blastocyst stage. This capability is significant in the design of a protocol for recovering mouse strains stored by cryopreserving their spermatozoa, and after thawing using the spermatozoa for IVF (Nakagata, 1996; Sztein et al., 1997; Marschall et al., 1999; Thornton et al., 1999). At present the media that are being used for fertilization have been developed for other purposes, and a switch is made after fertilization to a medium of different composition to support further development. One medium that has been used for IVF in the mouse (Sztein et al., 1997; Thornton et al., 1999) is a modification of a medium developed for the culture of human cell lines (Eagle, 1959). After fertilization the ova with two pronuclei are transferred to fresh MEM and cultured to the 2-cell stage after which they are transferred to the oviduct of surrogate mothers. Alternately the embryos are transferred to KSOM supplemented with AA (Ho et al., 1995) and cultured to the blastocyst stage after which they may be transferred to the uterus of foster mothers. Another medium that has been used for IVF in the mouse (Marschall et al., 1999) is HTF which was developed for the culture of human preimplantation embryos (Quinn et al., 1985a,b). After fertilization the ova with two pronuclei are transferred to KSOM supplemented with AA (Ho et al., 1995) for culture to the 2-cell stage after which they are transferred to the oviduct of surrogate mothers. Replacement of these procedures with a single medium developed specifically for IVF in the mouse and culture through the preimplantation stages should be considered. Under these conditions the stresses the gametes and preimplantation embryos experience are likely to be minimized.
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This work has been supported as part of the National Cooperative Program on Non-Human In Vitro Fertilization and Preimplantation Development and was funded by the National Institute of Child Health and Human Development, National Institute of Health (NIH), through cooperative agreement HD21988. Animals used in this study were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and those prepared by the Committee on Care and Use of Laboratory Animal Resources, National Research Council [DHHS publication No. (NIH) 85–23, revised 1985]. We thank Dr. Jay Baltz for valuable discussions and Dr. Betsey S. Williams for helpful criticism of the manuscript. Cell and Molecular Technology, Inc. markets KSOM and KSOMAA and gives Harvard Medical School a small amount of its profits. The Company does not control the research that we do.
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3 Present address: Beth Israel Deaconess Hospital, Boston, MA 02115, USA
4 Present address: Department of Biology, University of Michigan, Ann Arbor, MI 48109, USA
5 To whom correspondence should be addressed at: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA. E-mail: john_biggers{at}hms.harvard.edu.
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