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Biology of Reproduction 64, 1366-1374 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Differential Effect of Hexoses on Hamster Embryo Development in Culture1

Tenneille E. Ludwig2,a, Michelle Lane3,a, and Barry D. Bavister4,a

a Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706

ABSTRACT

The effects of glucose, fructose, and galactose on hamster embryo development in the absence of phosphate were studied in culture. One- and two-cell embryos were cultured to the blastocyst stage in HECM-9 medium without hexose or in medium with increasing concentrations of hexoses. Embryo development, cell number, and cell allocation were assessed in blastocysts. Blastocyst viability was determined by transfer to pseudopregnant recipients. Although 0.25 mM fructose increased mean cell number, low glucose concentrations had no stimulatory effect on development to blastocyst. Both galactose and 5.0 mM glucose were detrimental to embryos. Addition of 0.5 mM glucose increased implantation and fetal viability as compared with controls. Compared with 0.5 mM glucose, treatment with 0.25 mM fructose gave similar implantation and fetal viability, whereas 5.0 mM glucose tended to decrease implantation and significantly decreased fetal development. These data demonstrate that morphology is a poor indicator of embryo viability and that exposure of preimplantation embryos to glucose or fructose is important for embryo viability post-transfer. Although no difference in blastocyst viability was detected between embryos cultured with 0.25 mM fructose and those cultured with 0.5 mM glucose, increased cell numbers obtained with fructose suggest that fructose may be more appropriate than glucose for inclusion in culture medium.

developmental biology, implantation/early development, preimplantation embryo

INTRODUCTION

There is considerable evidence that inclusion of glucose in the culture medium at levels similar to those in plasma is inhibitory to embryo development in many species, including the hamster [1, 2], mouse [3–6], rat [7, 8], cow [9], sheep [10], and human [11, 12]. In the hamster, the combination of glucose and phosphate arrests development [1, 13], reduces respiration [14], and disrupts mitochondrial organization [15]. To prevent this inhibition and to promote embryo development, glucose and phosphate have frequently been omitted from embryo culture media [3, 5, 7, 12, 16].

Most of the previous work involving glucose has been performed in the presence of phosphate, and phosphate alone may be responsible for developmental inhibition previously attributed to both glucose and phosphate. Although phosphate alone appears not to be inhibitory to embryo development from the eight-cell stage [13], previous studies with embryos at earlier cleavage stages show that even in the absence of glucose, phosphate concentrations as low as 500 nM inhibit preimplantation embryo development in both hamsters and mice [1, 17–20]. Even with dramatic improvements in culture medium formulation, a recent report from our laboratory confirms that concentrations of phosphate as low as 2.5 µM alter ionic homeostasis and inhibit development of the embryo in culture [21]. Therefore, the complete removal of glucose from embryo culture medium may not be necessary to maintain developmental competence and in fact may be detrimental because it deprives the embryo of a physiological nutrient.

Although basal concentrations are species dependent (ranging between 0.5 and 3.0 mM), glucose is present in the female reproductive tract of all species examined, including the mouse [22], rabbit [23], sheep [24], pig [25], cow [26], and human [27]. A facilitated transport system for glucose is present in the mouse embryo from at least the two-cell stage [28–30], and the glucose transporter Glut 1 can be detected even in the unfertilized mouse oocyte [31]. Although the metabolism of glucose is low in the early stages of embryo development, glycolysis significantly increases at the blastocyst stage in hamsters [32], mice [33], and domestic animals [34–39]. In the mouse, glucose is the preferred energy substrate for the inner cell mass [40]. All of these data imply a physiological role for glucose in embryo development. Therefore, elimination of glucose from the culture medium results in an increasingly artificial environment for the developing embryo, the effects of which are unknown.

Like glucose, fructose and galactose are monosaccharide hexoses capable of entering the glycolytic pathway. Fructose is present in the reproductive tract of many species [41–46] and can be metabolized by preimplantation embryos [39]. In the presence of phosphate, replacement of glucose by fructose in culture medium alleviates the two-cell block and promotes blastocyst formation in outbred mouse strains [4]. For these reasons, we hypothesized that hexose sugars, in the absence of phosphate, can promote embryo development in vitro.

The aims of this study were to 1) examine the effect of glucose on embryo development and viability in the absence of exogenous phosphate and 2) determine whether fructose and/or galactose are practical alternatives to glucose for supporting embryo development. Elucidation of optimal energy sources for in vitro embryo culture will increase our understanding of embryo physiology and assist in the development of improved culture systems. These improvements will result in increased embryo competence, which in turn will advance the capabilities of developing technologies that rely on in vitro production of embryos.

MATERIALS AND METHODS

Culture Media

The base medium used in this study was a protein-free chemically defined medium (HECM-9) containing no phosphate [47]. The formulation is as follows: 113.8 mM NaCl, 3.0 mM KCl, 2.0 mM CaCl2·2H2O, 0.5 mM MgCl2·6H2O, 25.0 mM NaHCO3, 4.50 mM DL-sodium lactate, 0.01 mM asparagine, 0.01 mM aspartate, 0.01 mM cysteine, 0.01 mM glutamate, 0.20 mM glutamine, 0.01 mM glycine, 0.01 mM histidine, 0.01 mM lysine, 0.01 mM proline, 0.01 mM serine, 0.5 mM taurine, 3 µM pantothenate, and 0.1 mg/ml polyvinyl alcohol. Treatments were prepared by modifying the base medium to contain various concentrations of glucose, fructose, or galactose (0.0, 0.25, 0.5, 1.0, 2.0, or 5.0 mM). Media were prepared the day before use from stock solutions and stored at 4°C. The base medium used for staining procedures was a modified BM-3 medium [48] in which 20 mM NaHCO3 was replaced with 20 mM HEPES, pH 7.4 (H-BM-3). All salts, carbohydrates, amino acids, and vitamins were purchased from Sigma Chemical Co. (St. Louis, MO).

Animals and Embryo Collection

Embryos were collected from 3- to 6-mo-old cycling golden hamsters. Multiple ovulations were induced by an i.p. injection of 10–20 IU (indexed to body weight) eCG (Gestyl, Houston, TX) on the morning of the postestrus discharge (Day 1 of cycle). Females were mated to fertile males on the evening of Day 4. One-cell embryos were collected at 10 h post-egg activation (PEA, as previously described [49]). Two-cell embryos were collected at 32 h PEA. Embryos were flushed from the oviduct with warmed and equilibrated (10% CO2:5% O2:85% N2) HECM-9 medium (no hexose). All embryos were then washed twice in HECM-9 and once in the appropriate culture medium before being placed into culture.

Embryo Culture

All embryos were cultured in 35-µl drops of medium under mineral oil (Sigma) in groups of 8–12 embryos for either 72 h (to the blastocyst stage from collection at 10 h PEA one-cell stage) or 48 h (to the blastocyst stage from collection at 32 h PEA two-cell stage) at 37.5°C in a humidified atmosphere of 10% CO2:5% O2:85% N2. For all but the embryo transfer experiments, embryos from individual females were distributed among treatments so that each treatment group contained an equal number of embryos from any given female. Embryos from multiple females were pooled in each culture drop.

Morphology Assessment

Development to the eight-cell stage was assessed after 48 h of culture for embryos collected at 10 h PEA and after 24 h of culture for embryos collected at 32 h PEA. Morula and blastocyst development was assessed after 72 h of culture for embryos collected at 10 h PEA and after 48 h for embryos collected at 32 h PEA. Development to the morula and blastocyst stages was expressed as a single endpoint in addition to blastocyst development due to difficulties in accurately distinguishing morula from blastocyst stage hamster embryos. The hamster blastocyst in vitro undergoes repeated cycles of expansion and collapse [50], and following collapse a blastocyst is indistinguishable from a morula under low-power microscopy. Therefore, embryos identified as morula and blastocyst (morula/blastocyst) stages include all postcompaction embryos, and embryos identified as blastocysts include only those embryos exhibiting a distinct blastocoele cavity at the time of examination.

Differential Staining of Inner Cell Mass and Trophectoderm Cells

Embryo cell number and allocation of cells to the inner cell mass (ICM) and trophectoderm (TE) were determined by differentially staining the cell nuclei using a technique previously described [51]. Blastocysts were incubated in 0.5% pronase in H-BM-3 for 30 sec to dissolve the zona pellucida. Embryos were then washed in H-BM-3 and incubated in 0.25% picrylsulfonic acid (Sigma) for 10 min at 4°C before a further wash and 10 min of incubation in 0.1 mg/ml of anti-DNP BSA (ICN Technologies, Costa Mesa, CA) at 37°C. Following incubation with the antibody, embryos were again washed in H-BM-3 and incubated in a 1:5 dilution of guinea pig serum (ICN) containing 25 µg/ml of propidium iodide (Sigma) in H-BM-3 for 5 min. Embryos were subsequently placed in 25 µg/ml bisbenzimide (Hoechst 33258, Sigma) in ethanol overnight at 4°C. The following morning, differential staining of nuclei was determined using a fluorescence microscope.

Embryo Transfer

Viability of cultured embryos was assessed by transfer of blastocysts to pseudopregnant recipient females. Three experiments were performed, and each experiment compared two hexose treatments: 1) 0.0 mM glucose vs. 0.50 mM glucose; 2) 0.5 mM glucose vs. 5.0 mM glucose; 3) 0.5 mM glucose vs. 0.25 mM fructose. The hexose concentrations used were determined following the developmental and cell allocation experiments described previously (because galactose was detrimental to embryo development at the blastocyst stage, it was not investigated further). In each experiment, one-cell embryos from donor females were collected at 10 h PEA and cultured for 72 h to the morula/blastocyst stage. Exposure from the one-cell stage (10 h PEA) was selected because it is a more stringent test than exposure from the two-cell stage [52]. Embryos collected from a single female were distributed equally between the two treatments in each experiment, and embryos from individual females were not pooled during culture. For experiments 1 (0.0 mM glucose vs. 0.50 mM glucose) and 3 (0.5 mM glucose vs. 0.25 mM fructose), only embryos of good quality and exhibiting a blastocoele cavity occupying at least one quarter of their total volume were selected for possible transfer. For experiment 2 (0.5 mM glucose vs. 5.0 mM glucose), all blastocysts in each treatment group were selected for possible transfer regardless of quality and blastocoele cavity size. This modification of selection criteria for experiment 2 was necessary because of the difference in morphology of embryos cultured in 5.0 mM glucose (blastocysts obtained from culture with 5.0 mM glucose were overall generally smaller, with reduced blastocoele cavity area). Pseudopregnant recipient females were obtained by mating females to vasectomized males on Day 4 of the estrous cycle. Day 3 recipient females were asynchronous (-1 day) with respect to embryo donors to achieve successful pregnancy of in vitro cultured embryos [49]. For each experiment, eight randomly selected blastocysts from each treatment group were transferred into each recipient female (16 embryos/recipient). The hamster has a dual cervix and therefore an anatomical barrier to uterine migration of embryos. We placed embryos from one treatment into one uterine horn, and those from the other treatment into the contralateral horn, with the side (left vs. right) of embryo placement for each treatment alternating among recipients within each experiment. Within each recipient, the same number of embryos from a given donor female were transferred to left and right horns (i.e., both embryo culture treatments) to eliminate effects due to donor variation. Implantation sites, fetal development, and fetal weight were assessed on Day 14 of pregnancy relative to the recipient female (2 days before term).

Statistical Analysis

For culture experiments (including development and cell allocation experiments), data were subjected to least squares analysis of variance (ANOVA) using the general linear models procedure of the Statistical Analysis System (Cary, NC). Percentage data were arc sine transformed and weighted for the number of embryos in each experiment prior to analysis. The ANOVA was performed for a completely randomized design two-way treatment structure consisting of day and hexose concentration. All tests of hypotheses were performed using appropriate error terms according to the expectation of the mean squares. Differences were detected by least squares means.

Data for embryo transfer were analyzed using linear-logistic regression assuming a binomial distribution. The day of transfer was fitted as a variable. The log likelihood ratio statistic was used to detect between-treatment differences. The GLIM 4.0 statistical package (Numerical Algorithms Group, Oxford, U.K.) was used for statistical analysis of embryo transfer results.

RESULTS

Effect of Glucose on Development to the Eight-Cell Stage

Hamster one-cell embryos collected at 10 h PEA and cultured in the absence of glucose (HECM-9: control) had a mean (± SEM) cell number of 6.0 ± 0.2 after 48 h of culture. Addition of 0.25, 1.0, or 2.0 mM glucose significantly increased mean cell number at this time point when compared with control cultures. In contrast, culture with 0.5 or 5.0 mM glucose did not alter mean cell number as compared with controls (Table 1).


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  		TABLE 1. Effect of glucose concentration on development of hamster embryos cultured from the one-cell stage at 48 h of culture.*

Hamster two-cell embryos collected at 32 h PEA and cultured in control medium had a mean cell number of 6.9 ± 0.1 after 24 h of culture. Only treatment with 0.5 mM glucose significantly increased mean cell number at 24 h of culture (7.3 ± 0.1; P < 0.05). Treatment with 5 mM glucose significantly reduced mean cell number as compared with 0.5, 1.0, and 2.0 mM glucose (Table 2).


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  		TABLE 2. Effect of glucose concentration on development of hamster embryos cultured from the two-cell stage at 24 h of culture.*

Effect of Glucose on Development to Morula and Blastocyst Stages

Culture of 10 h PEA one-cell embryos with up to 2 mM glucose had no significant effect on either morula/blastocyst or blastocyst development as compared with controls (Fig. 1A). However, addition of 5.0 mM glucose significantly decreased morula/blastocyst development as compared with controls (P < 0.05). Additionally, 5.0 mM glucose decreased blastocyst development as compared with controls and with cultures with glucose concentrations of up to 1.0 mM (P < 0.02).



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  		FIG. 1. Effect of glucose concentration on development to morula and blastocyst stages in hamster embryos cultured from the one-cell (A) and two-cell (B) stages. Similarly shaded bars with different letters are significantly different within histograms (P < 0.05); 260 embryos/treatment, 14 replicates (A), and 260 embryos/treatment, 12 replicates (B)

Similarly, addition of glucose to culture medium had no effect on development of two-cell embryos to the morula/blastocyst stage (Fig. 1B). However, culture with 5.0 mM glucose inhibited development to the blastocyst stage (P < 0.01) as compared with controls. Additionally, culture with 5.0 mM glucose resulted in embryos that were morphologically different than those cultured in control medium or medium with 0.5 mM glucose.

Effect of Glucose on Cell Number and Allocation

Morulae and blastocysts derived from hamster one-cell embryos collected at 10 h PEA and cultured without glucose for 72 h had a total mean cell number of 19.4 ± 0.7. Of these cells, an average of 12.7 ± 0.5 were allocated to the TE and 6.9 ± 0.4 were allocated to the ICM. The presence of glucose from the one-cell stage had no significant effect on cell number or allocation as compared with controls (Fig. 2). However, compared to 0.5 mM glucose, culture with 5.0 mM glucose significantly reduced ICM and TE cell number as well as overall morula/blastocyst cell number (P < 0.05).



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  		FIG. 2. Effect of glucose concentration on morula and blastocyst mean cell numbers and allocation to the ICM or TE of embryos cultured from the one-cell (A) and two-cell (B) stages. Similarly shaded bars with different letters are significantly different within histograms (P < 0.05); 60–80 blastocysts stained/treatment, seven replicates (A), and 80–100 embryos/treatment, eight replicates (B)

Morulae and blastocysts derived from hamster two-cell embryos collected at 32 h PEA and cultured without glucose for 48 h had a total mean cell number of 27.2 ± 0.6. Of these cells, an average of 16.4 ± 0.4 were allocated to the TE and 10.6 ± 0.5 were allocated to the ICM (similar to cell counts for blastocysts produced in vivo, as previously reported by our laboratory [51]). Culture of two-cell embryos with glucose concentrations up to 1.0 mM did not significantly alter morula/blastocyst cell number or allocation (Fig. 2B). However, culture with glucose concentrations of 2.0 mM and above significantly decreased the mean total cell number and mean number of cells allocated to the ICM (P < 0.05). Additionally, culture with 5.0 mM glucose significantly reduced TE mean cell number (P < 0.05) as compared with concentrations up to 0.5 mM, as well as morula/blastocyst total and ICM mean cell number as compared with all treatments (P < 0.05).

Effect of Glucose on Implantation and Fetal Development after Transfer

Culture of one-cell embryos with 0.5 mM glucose to the blastocyst stage dramatically increased both implantation (P = 0.02) and fetal viability (P = 0.02) after transfer as compared with control (no glucose; Table 3). Fetal weight was not affected by addition of glucose to the culture medium.


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  		TABLE 3. Effect of glucose concentration on fetal development of hamster embryos cultured from the one-cell stage and transferred to pseudopregnant recipient females

Although implantation rates did not differ significantly between embryos cultured with 0.5 mM glucose and those cultured with 5.0 mM glucose, there is a trend (P = 0.07) toward decreased fetal development from blastocysts resulting from culture with high levels of glucose (Table 4). Additionally, culture with 5.0 mM glucose significantly reduced fetal development per implantation site (P < 0.01), indicating a higher level of fetal loss.


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  		TABLE 4. Effect of glucose concentration on fetal development of hamster embryos cultured from the one-cell stage and transferred to pseudopregnant recipient females

Effect of Galactose on Embryo Development and Cell Allocation

Culture with 5.0 mM galactose significantly reduced all parameters (P < 0.05; Tables 5 and 6). Culture with 0.5 mM galactose did result in increased morula/blastocyst development (P < 0.05) as compared with both controls (Table 5); however, that increase did not extend to blastocyst development (Table 5), mean cell number, or cell allocation (Table 6). Culture of embryos with all concentrations of galactose significantly decreased mean cell number at 72 h of culture (P < 0.05) as compared with embryos cultured without hexose (Table 6). High galactose concentrations (2.0 and 5.0 mM) decreased mean total and TE cell numbers (P < 0.05) as compared with both 0.0 and 0.5 mM glucose (controls; Table 6). Compared with culture with 0.5 mM glucose, culture of embryos with less than 5.0 mM galactose had no effect on mean cell number at 48 h (Table 5), blastocyst development, or ICM development. Culture with less than 2.0 mM galactose additionally did not affect total mean cell number, or TE cell number as compared with culture with 0.5 mM glucose (Table 6).


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  		TABLE 5. Effect of galactose concentration on development of hamster embryos cultured from the one-cell stage.*


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  		TABLE 6. Effect of galactose concentration on morula/blastocyst mean (± SEM) cell number and allocation in hamster embryos cultured from the one-cell stage.*

Effect of Fructose on Embryo Development and Cell Allocation

Culture of embryos with 0.25 mM fructose increased morula/blastocyst total and mean TE cell number as compared with controls (Fig. 3B). At 72 h of culture, fructose concentrations above 0.25 mM had no effect on blastocyst development (Fig. 3A), morula/blastocyst total, or ICM and TE mean cell numbers (Fig. 3B). Fructose had no effect on development of embryos cultured from the one-cell stage at 48 h of culture. Although glucose is inhibitory at high concentrations (Fig. 1, A and B), even the highest concentrations of fructose were not inhibitory as compared with 0.0 and 0.5 mM glucose treatments (Fig. 3, A and B).



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  		FIG. 3. Effect of fructose on development (A), and morula and blastocyst mean cell numbers and allocation to the ICM or TE (B) of embryos cultured from the one-cell stage. Similarly shaded bars with different letters are significantly different within histograms (P < 0.05); 160 embryos/treatment, eight replicates (A), and 40–60 blastocysts stained/treatment, five replicates (B)

Effect of Fructose on Implantation and Fetal Development after Transfer

A comparison was made of the viability of blastocysts cultured in either 0.5 mM glucose or 0.25 mM fructose. A total of 88 embryos per treatment from 11 individual females were transferred. No significant differences were noted following blastocyst transfer (data not shown). Embryos cultured in glucose and fructose exhibited similar percentages of implantation (62.5% and 60.2%, respectively), fetal development (51.1% and 53.4%), and fetuses per implantation (81.1% and 88.7%). Fetal weights were also similar (1.13 ± 0.03 g and 1.12 ± 0.03 g, respectively).

DISCUSSION

There has been considerable debate surrounding the inclusion of hexoses, particularly glucose, in embryo culture media. Several studies have shown that high concentrations (around 5.0 mM) of glucose are detrimental to embryo development in culture [1, 10, 12]. Paradoxically, glucose is the preferred energy substrate of somatic cells in culture and is a major precursor of DNA synthesis and phospholipid synthesis [53]. Glucose is certainly found in the female reproductive tract [22–27] and is the preferred energy substrate after compaction for many species of mammalian embryos [54], implying a physiological role for glucose in culture. When CZB medium is used, addition of glucose is necessary to support mouse blastocyst development [55], although exposure for as little as 1 min is enough to meet this glucose requirement [56]. In more optimized media containing amino acids, low concentrations of glucose are not inhibitory to embryo development [57, 58]. The study presented here reconciles these seemingly conflicting reports and demonstrates that the effect of glucose is biphasic and dose dependent. Furthermore, the results indicate that although galactose is detrimental to cleavage-stage embryos, preimplantation exposure to either glucose or fructose is important for maintenance of post-transfer viability, particularly appropriate implantation.

In this study, 5.0 mM glucose significantly inhibited development of one-cell and two-cell embryos to the morula and blastocyst stages (Fig. 1, A and B) and decreased mean cell number and cell allocation to the ICM (Fig. 2, A and B), supporting previous reports that high glucose levels are inhibitory to embryo development [1, 10, 12]. Low concentrations of glucose (below 2.0 mM), however, did not affect blastocyst formation or cell number at the morula/blastocyst stage, corroborating the previous reports that lower concentrations of glucose are not inhibitory in culture [57, 58]. These results should not be surprising, because concentrations of glucose in the female reproductive tract are considerably lower than those in plasma [22–27] and drop additionally following ovulation [25, 59], resulting in very low oviductal glucose concentrations in vivo at the time the cleavage stage embryo would be present. As in the study by Summers [57], our data show that although no inhibition was found, low concentrations of glucose have little benefit for preimplantation embryo development in culture. In fact, other than an increase in cell number at 24 h of culture in embryos cultured from the two-cell stage (Table 2), there was no observable benefit in ability to develop to the blastocyst stage gained by addition of 0.5 mM glucose in the culture medium. Why increases in cell number are seen when 0.5 mM glucose is administered from the two-cell stage but not from the one-cell stage remains unclear.

Other hexoses affected embryo development to the blastocyst stage differently than did glucose. Galactose was detrimental to embryo development even at low levels. As little as 0.5 mM galactose in culture medium significantly decreased mean total and TE cell numbers (Table 6). The conversion of galactose to glucose (required for entry into the glycolytic pathway) requires specific enzyme activity [53]. One possible explanation for the inhibitory effect of galactose is that necessary enzymes are either absent or nonfunctional in cleavage-stage embryos, resulting in a buildup of intermediates that may be toxic to the embryo. In contrast, culture with low levels of fructose benefits embryos during the preimplantation stages. Although addition of fructose did not increase the percentage of morula/blastocyst (Fig. 3A), there was a significant increase in mean morula/blastocyst total cell numbers and TE cell numbers when embryos were cultured with 0.25 mM fructose (Fig. 3B).

Although low glucose concentrations resulted in little discernible benefit to embryos prior to transfer, culture with 0.5 mM glucose resulted in dramatically increased implantation rates and fetal development rates following transfer as compared with controls (Table 3). Culture of one-cell embryos to the blastocyst stage in medium containing 0.5 mM glucose elevated Day 14 percentage of fetal viability to rival that of in vivo produced transferred eight-cell embryos [60]. When compared with embryos cultured without exogenous glucose, the proportion of implanting embryos that go on to form viable fetuses (fetuses per implantation) was not different (Table 3). Because there was no difference in postimplantation viability (i.e., those embryos capable of appropriate implantation developed at similar rates), we conclude that the primary benefit of low glucose exposure occurs at implantation.

Preimplantation embryo exposure to 0.5 mM glucose resulted in a dramatic increase in fetal viability as compared with embryos cultured without hexose. Preimplantation embryo exposure to 0.25 mM fructose resulted in implantation rates similar to those of embryos cultured with 0.5 mM glucose. Collectively, these results clearly indicate that substantial benefit is derived from the inclusion of low concentrations of hexoses in culture media. Although exposure to 5.0 mM glucose reduced the percentage of blastocyst development, those embryos that did develop to the blastocyst stage did not have significantly different implantation rates when compared with embryos treated with 0.5 mM glucose (Table 4). However, although implantation rates were similar, there is a trend toward decreased percentage of fetal development and a significant decrease in viable fetuses per implantation site when embryos are exposed to 5.0 mM glucose. Although the effect of hexoses on implantation rate is not affected by concentration, postimplantation fetal viability decreases with high levels of glucose.

The beneficial effects of glucose and fructose may be linked to metabolism. Although the metabolic rate of the fertilized ovum is very low (close to that of bone), at the time of implantation the metabolic rate of the embryo is equal to that of brain tissue [61]. Energy-demanding biological processes, including protein synthesis, nucleic acid synthesis, and ion transport, increase the need for and cause an elevation in metabolic activity leading up to the blastocyst stage [61, 62]. Any alteration or compromise in normal metabolic processes results in the failure of the embryo to develop properly [14, 63, 64]. Alternatively, substrates capable of providing additional resources for the embryo to meet this high energy demand may increase the developmental potential of the embryo.

Glucose, fructose, and galactose are utilized by the glycolytic pathway. Increased glycolytic flux results in increased flux through the pentose phosphate pathway (PPP) [65]. Primary products of the PPP are ribose-5-phosphate and NADPH. Ribose-5-phosphate is an important component in the generation of ATP, NAD+, flavine adenine dinucleotide, and coenzyme A [53], resources important for energy storage and use in the cell. Additionally, ribose-5-phosphate is a precursor of 5-phosphoribosyl-1-pyrophosphate, which makes up the ribose phosphate portion of the purine and pyrimidine nucleotides. NADPH is utilized by the cell to convert oxidized glutathione to its reduced state. Reduced glutathione plays a key role in detoxification by reacting with hydrogen peroxide and organic peroxides, natural byproducts of oxidative phosphorylation that are harmful to the cell. Therefore, additional PPP activity increases the ability of the cell to protect itself from oxidative damage while providing necessary building blocks and energy resources for the embryo to maintain a rapid rate of development. Accordingly, increased viability seen in the presence of glucose could be due, at least in part, to the increased PPP activity, which would then result in a greater availability of ribose-5-phosphate and NADPH. Although fructose enters the glycolytic pathway below the flux-generating step for the PPP, it can be converted into ribose-5-phosphate without the production of NADPH. If the stimulatory effect of glucose is increased NADPH activity, then glucose and fructose may work together to further stimulate development.

Glucose and fructose may also benefit embryos by increasing the efficacy of protein synthesis. Both glucose and fructose are substrates for glutamine:fructose-6-phosphate amidotransferase (GFAT), the first and rate-limiting enzyme in the hexosamine pathway necessary for post-translational protein glycosylation [66]. Furthermore, there is evidence that increasing the availability of fructose-6-phosphate serves to regulate the activity of GFAT [67]. Clearly, the ability to efficiently produce appropriate glycosylated proteins is advantageous to embryo development, which may help explain the advantage conferred by glucose and fructose in culture medium.

Alternatively, the beneficial effect of glucose and fructose may be imparted only at the time of implantation and not prior to it. Although glucose alone is unable to support mouse embryo development prior to the eight-cell stage [68], it is able to support development after this stage [69] and is in fact the primary energy source postcompaction for mammalian embryos [54, 61]. This is not surprising, given that the implantation site is anoxic [54, 70] and therefore oxidative phosphorylation cannot be accomplished by the developing embryo until a maternal blood supply is established at the site of implantation and oxygen becomes available. Additional provision of glucose prior to implantation enables the embryo to increase glycogen stores, which are utilized during the period of anaerobic energy production occurring at implantation. In his 1974 review [61], Brinster noted that glycogen utilized near the time of implantation "could be a critical factor in the ability of the embryo to implant." Therefore, the additional glycogen resource available following preimplantation embryo development in hexose-supplemented media may explain the increased posttransfer viability seen in these embryos.

Although no difference in blastocyst viability was detected between embryos cultured with 0.25 mM fructose and those cultured with 0.5 mM glucose, increased cell numbers obtained with low concentrations of fructose (even lower than glucose concentrations shown to be beneficial to embryo viability; Fig. 3B), coupled with the fact that fructose does not cause developmental inhibition at high concentrations (as glucose does), demonstrate that fructose is a viable (and perhaps more appropriate) alternative to glucose in culture media.

Our results demonstrate that preimplantation embryo exposure to hexoses is not necessary to obtain adequate numbers of embryos developing to the blastocyst stage but is important for the continuing developmental competence of the blastocyst and its ability to undergo successful implantation. Although almost no benefit of hexose incorporation into culture medium was detectable at the blastocyst stage, dramatic increases in implantation rate were achieved when preimplantation embryos were exposed to low concentrations of glucose. Culture with low concentrations of fructose likewise resulted in exceptional fetal viability. The ability to obtain blastocysts has been generally accepted as the benchmark of a good culture system. This study clearly shows, however, that not all blastocysts are created equal and that by relying on assessment of preimplantation embryo development to evaluate culture systems we may ultimately compromise our ability to make improvements. Although expensive and time consuming in larger species, assessment of ultimate fetal viability should remain, whenever possible, the gold standard for comparison of culture systems.

ACKNOWLEDGMENTS

The authors thank Susan McKiernan, Dr. Randall Prather, and Dr. John Eppig for their critique of the manuscript.

FOOTNOTES

First decision: 2 May 2000.

1 This work was done as part of the National Cooperative Program on Non-Human In Vitro Fertilization and Preimplantation Embryo Development and was funded by the National Institute of Child Health and Human Development, NIH, through cooperative agreement HD-22023. Back

2 Correspondence: Tenneille Ludwig, Department of Animal Health and Biomedical Sciences, 1656 Linden Dr., University of Wisconsin-Madison, Madison, WI 53706. FAX: 608 262 7420; ludwig{at}ahabs.wisc.edu Back

3 Current address: Colorado Center for Reproductive Medicine, Englewood, CO 80110. Back

4 Current address: Department of Biological Sciences, University of New Orleans, New Orleans, LA 70122-3520. Back

Accepted: December 12, 2000.

Received: April 3, 2000.

REFERENCES

  1. Schini SA, Bavister BD. Two-cell block to development of cultured hamster embryos is caused by phosphate and glucose. Biol Reprod 1988; 39:1183–1192[Abstract]
  2. Barnett DK, Bavister BD. What is the relationship between the metabolism of preimplantation embryos and their developmental competence? Mol Reprod Dev 1996; 43:105–133[CrossRef][Medline]
  3. Chatot CL, Ziomek CA, Bavister BD, Lewis JL, Torres I. An improved culture medium supports development of random-bred one-cell mouse embryos in vitro. J Reprod Fertil 1989; 86:679–688[Abstract/Free Full Text]
  4. Menezo Y, Katchadourian C. Implication de l'activitie glucose 6 phospahte isomerase (EC 5.3.1.9) dans l'arret de la segmentation de l'oeuf de souris au stade 2 cellules in vitro. CR Acad Sci Paris Ser III 1990; 310:297–301
  5. Lawitts JA, Biggers JD. Overcoming the 2-cell block by modifying standard components in a mouse culture medium. Biol Reprod 1991; 45:245–251[Abstract]
  6. Scott L, Whittingham DG. Influence of genetic background and media components on the development of mouse embryos in vitro. Mol Reprod Dev 1996; 43:336–346[CrossRef][Medline]
  7. Kishi J, Noda Y, Narimoto K, Umaoka Y, Mori T. Block to development in cultured rat one-cell embryos is overcome using medium HECM-1. Hum Reprod 1991; 6:1445–1448[Abstract/Free Full Text]
  8. Miyoshi K, Funahashi H, Okuda K, Niwa K. Development of rat one-cell embryos in a chemically defined medium: effects of glucose, phosphate and osmolarity. J Reprod Fertil 1994; 100:21–26[Abstract/Free Full Text]
  9. Kim JH, Niwa K, Lim JM, Okuda K. Effects of phosphate, energy substrates and amino acids on development of in vitro-matured, in vitro-fertilized bovine oocytes in a chemically defined, protein-free culture medium. Biol Reprod 1993; 48:1320–1325[Abstract]
  10. Thompson JG, Simpson AC, Pugh PA, Tervit HR. Requirement for glucose during in vitro culture of sheep preimplantaion embryos. Mol Reprod Dev 1992; 31:253–257[CrossRef][Medline]
  11. Conaghan J, Handyside AH, Winston RM, Leese HL. Effects of pyruvate and glucose on the development of human preimplantation embryos in vitro. J Reprod Fertil 1993; 99:87–95[Abstract/Free Full Text]
  12. Quinn P. Enhanced results in mouse and human embryo culture using a modified human tubal fluid medium lacking glucose and phosphate. J Assist Reprod Genet 1995; 12:97–105[CrossRef][Medline]
  13. Seshagiri PB, Bavister BD. Glucose inhibits development of hamster 8-cell embryos in vitro. Biol Reprod 1989; 40:599–606[Abstract]
  14. Seshagiri PB, Bavister BD. Glucose and phosphate inhibit respiration and oxidative metabolism in cultured hamster eight-cell embryos: evidence for the "Crabtree effect." Mol Reprod Dev 1991; 30:105–111[CrossRef][Medline]
  15. Barnett DK, Clayton MK, Kimura J, Bavister BD. Glucose and phosphate toxicity in hamster preimplantation embryos involves disruption of cellular organization, including distribution of active mitochondria. Mol Reprod Dev 1997; 48:227–237[CrossRef][Medline]
  16. Schini SA, Bavister BD. Development of golden hamster embryos through the two-cell block in chemically defined medium. J Exp Zool 1988; 245:111–115[CrossRef][Medline]
  17. Meyer M, Seshagiri PB. Analysis of phosphate-inhibition of development of hamster 2-cell embryos in vitro. Biol Reprod 1990; 42(suppl 1):56
  18. Monis H, Bavister BD. Analysis of the inhibitory effect of inorganic phosphate on development of four-cell hamster embryos in vitro. J Exp Zool 1990; 256:75–83[CrossRef][Medline]
  19. Monis H, Bavister BD. Development of four-cell hamster embryos to the blastocyst stage in vitro and its regulation by components of the culture milieu. Reprod Fertil Dev 1990; 2:1–9[CrossRef][Medline]
  20. Lawitts JA, Biggers JD. Optimization of mouse embryo culture media using simplex methods. J Reprod Fertil 1991; 91:543–556[Abstract/Free Full Text]
  21. Lane M, Ludwig TE, Bavister BD. Phosphate induced developmental arrest of hamster two-cell embryos is associated with disrupted ionic homeostasis. Mol Reprod Dev 1999; 54:410–417[CrossRef][Medline]
  22. Gardner DK, Leese HJ. Concentrations of nutrients in mouse oviduct fluid and their effects on embryo development and metabolism in vitro. J Reprod Fertil 1990; 88:361–368[Abstract/Free Full Text]
  23. Leese H. The formation and function of oviduct fluid. J Reprod Fertil 1988; 82:843–856[CrossRef][Medline]
  24. Wales RG. The uterus of the ewe. II. Chemical analysis of the uterine fluid collected by cannulation. Aust J Biol Sci 1973; 26:947–959[Medline]
  25. Nichol R, Hunter RHF, Gardner DK, Leese HJ, Cooke GM. Concentrations of energy substrates in oviductal fluid and blood plasma of pigs during the peri-ovulatory period. J Reprod Fertil 1992; 96:699–707[Abstract/Free Full Text]
  26. Carlson D, Black DL, Howe GR. Oviduct secretion in the cow. J Reprod Fertil 1970; 22:549–552[Abstract/Free Full Text]
  27. Lippes J, Enders RG, Pragay DA, Bartholomew WR. The collection and analysis of human fallopian tubal fluid. Contraception 1972; 5:85–103[CrossRef][Medline]
  28. Gardner DK, Leese HJ. The role of glucose and pyruvate transport in regulating nutrient utilization by preimplantation mouse embryos. Development 1988; 104:423–429[Abstract/Free Full Text]
  29. Morita Y, Tsutsumi O, Oka Y, Taketani Y. Glucose transporter GLUT1 mRNA expression in the ontogeny of glucose incorporation in mouse preimplantation embryos. Biochem Biophys Res Commun 1994; 199:1525–1531[CrossRef][Medline]
  30. Pantaleon M, Kaye PL. IGF-I and insulin regulate glucose transport in mouse blastocysts via IGF-I receptor. Mol Reprod Dev 1997; 44:71–76
  31. Aghayan M, Rao LV, Smith RM, Jarett L, Charron MJ, Thorens B, Heyner S. Developmental expression and cellular localization of glucose transporter molecules during mouse preimplantation development. Development 1992; 115:305–312[Abstract]
  32. Barnett DK, Reiger D, Bavister BD. Changes in the metabolism of glucose and glutamine during development of the hamster embryo. Theriogenology 1993; 39:185[CrossRef]
  33. Lane M, Gardner DK. Amino acids and vitamins prevent culture-induced metabolic perturbations and associated loss of viability of mouse blastocysts. Hum Reprod 1998; 13:991–997[Abstract/Free Full Text]
  34. Flood MR, Weibold JL. Glucose metabolism by preimplantation pig embryos. J Reprod Fertil 1988; 72:9–13
  35. Thompson JG, Simpson AC, Pugh PA, Wright RW, Tervit HR. Glucose utilization by sheep embryos derived in vivo and in vitro. Reprod Fertil Dev 1991; 3:571–576[CrossRef][Medline]
  36. Rieger D, Luskutoff NM, Betteridge KJ. Developmentally related changes in the metabolism of glucose and glutamine by cattle embryos produced and co-cultured in vitro. J Reprod Fertil 1992; 95:585–595[Abstract/Free Full Text]
  37. Rieger D, Luskutoff NM, Betteridge KJ. Developmentally related changes in the uptake and metabolism of glucose, glutamine and pyruvate by cattle embryos produced in vitro. Reprod Fertil Dev 1992; 4:547–557[CrossRef][Medline]
  38. Gardner DK, Lane M, Batt P. Uptake and metabolism of pyruvate and glucose by individual sheep preattachment embryos developed in vivo. Mol Reprod Fertil 1993; 36:313–319
  39. Guyader-Joly C, Kahtchadourian C, Menezo Y. Comparative glucose and fructose incorporation and conversion by in vitro produced bovine embryos. Zygote 1996; 4:85–91[Medline]
  40. Hewitson LC, Leese HJ. Energy metabolism of the trophectoderm and inner cell mass of the mouse blastocyst. J Exp Zool 1993; 267:337–343[CrossRef][Medline]
  41. Gregorie AT, Gibbon R. Glucosyl oligosacchrides of the rabbit genital tract: effects of ovarian hormone administration. Int J Fertil 1965; 10:151–157
  42. Haynes NB, Lamming GE. Carbohydrate content of sow uterine flushings. J Reprod Fertil 1967; 14:335–337[Abstract/Free Full Text]
  43. Douglas CP, Garrow JS, Pugh EW. Investigation into the sugar content of endometrial secretion. J Obstet Gynaecol Br Commonw 1970; 77:891–894[Medline]
  44. Suga T, Masaki J. Studies on the secretions of the cow. 6. Sugar and polyol constituents in the luminal fluid of the bovine uterus. Jpn J Anim Reprod 1973; 18:143–147
  45. Aitken RJ. Uterine secretion of fructose in the roe deer. J Reprod Fertil 1976; 46:439–440[Abstract/Free Full Text]
  46. Greven H. The distribution of monosaccharides and hexosamines in the oviduct of Salamandra salamandra (L.) (Amphibia, Urodela). Comp Biochem Physiol B 1984; 79:229–232[CrossRef][Medline]
  47. McKiernan SH, Bavister BD. Culture of one-cell hamster embryos with water soluble vitamins: pantothenate stimulates blastocyst production. Hum Reprod 2000; 15:157–164[Abstract/Free Full Text]
  48. McKiernan SH, Bavister BD, Tasca RJ. Energy substrate requirements for in-vitro development of hamster 1- and 2-cell embryos to the blastocyst stage. Hum Reprod 1991; 6:64–75[Abstract/Free Full Text]
  49. Barnett DK, Bavister BD. Hypotaurine requirement for in vitro development of golden hamster one-cell embryos into morulae and blastocysts, and production of term offspring from in vitro-fertilized ova. Biol Reprod 1992; 47:297–304[Abstract]
  50. Gonzales DS, Bavister BD. Zona pellucida escape by hamster blastocysts in vitro is delayed and morphologically different compared with zona escape in vivo. Biol Reprod 1995; 52:470–480[Abstract]
  51. Lane M, Boatman DE, Albrecht RM, Bavister BD. Intracellular divalent cation homeostasis and developmental competence in the hamster preimplantation embryo. Mol Reprod Dev 1998; 50:443–450[CrossRef][Medline]
  52. Davidson A, Vermesh M, Lobo RA, Paulson RJ. Mouse embryo culture as quality control for human in vitro fertilization: the one-cell versus the two-cell model. Fertil Steril 1988; 49:516–521[Medline]
  53. Stryer L. Biochemistry. New York: WH Freeman and Co.; 1995
  54. Leese HJ. Metabolism of the preimplantation mammalian embryo. Oxf Rev Reprod Biol 1991; 13:35–72[Medline]
  55. Chatot CL, Lewis JL, Torres I, Ziomek CA. Development of 1-cell embryos from different strains of mice in CZB medium. Biol Reprod 1990; 42:432–440[Abstract]
  56. Chatot CL, Lewis-Williams J, Torres I, Ziomek CA. One-minute exposure of 4-cell mouse embryos to glucose overcomes morula block in CZB medium. Mol Reprod Dev 1994; 37:407–412[CrossRef][Medline]
  57. Summers MC, Bhatnagar PR, Lawitts JA, Biggers JD. Fertilization in vitro of mouse ova from inbred and outbred strains: complete preimplantation embryo development in glucose-supplemented KSOM. Biol Reprod 1995; 53:431–437[Abstract]
  58. Gardner DK, Lane M. Alleviation of the "2-cell block" and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA and physical parameters. Hum Reprod 1996; 11:2703–2712[Abstract/Free Full Text]
  59. Gardner DK, Lane M, Calderon I, Leeton J. Environment of the preimplantation human embryo in vivo: metabolite analysis of oviduct and uterine fluids and metabolism of cumulus cells [see comments]. Fertil Steril 1996; 65:349–353[Medline]
  60. Hoppe RW, Bavister BD. Evaluation of the fluorescein diacetate (FDA) vital dye viability test with hamster and bovine embryos. Anim Reprod Sci 1984; 6:323–335[CrossRef]
  61. Brinster RL. Embryo development. J Anim Sci 1974; 38:1003–1012
  62. Wales RG. Maturation of the mammalian embryo: biochemical aspects. Biol Reprod 1975; 12:66–81[CrossRef][Medline]
  63. Brison DR, Leese HJ. Blastocoel cavity formation by preimplantation rat embryos in the presence of cyanide and other inhibitors of oxidative phosphorylation. J Reprod Fertil 1994; 101:305–309[Abstract/Free Full Text]
  64. Houghton FD, Thompson JG, Kennedy CJ, Leese HJ. Oxygen consumption and energy metabolism of the early mouse embryo. Mol Reprod Dev 1996; 44:476–485[CrossRef][Medline]
  65. Katz J, Wood HG. The use of C14O2 yields from glucose-1 and-6-C14 for the evaluation of the pathways of glucose metabolism. J Biol Chem 1963; 238:517–523[Free Full Text]
  66. Traxinger RR, Marshall S. Coordinated regulation of glutamine:fructose-6-phosphate amidotransferase activity by insulin, glucose and glutamine: role of hexosamine biosynthesis in enzyme regulation. J Biol Chem 1991; 266:10148–10154[Abstract/Free Full Text]
  67. Marshall S, Bacote V, Traxinger RR. Complete inhibition of glucose-induced desensitization of the glucose transport system by inhibitors of mRNA synthesis: evidence for rapid turnover of glutamine:fructose-6-phosphate amidotransferase. J Biol Chem 1991; 266:10155–10161[Abstract/Free Full Text]
  68. Brinster RL. Studies on the development of mouse embryos in vitro. II. The effect of energy source. J Exp Zool 1965; 158:59–68[CrossRef][Medline]
  69. Whitten WK. Culture of tubal ova. Nature 1957; 179:1081–1082
  70. Rogers PA, Gannon BJ. The vascular and microvascular anatomy of the rat uterus during the oestrous cycle. Aust J Exp Biol Med Sci 1981; 59:667–679[Medline]



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