Glycine transport by single human and mouse embryos

M.A. Hammer2,3, M. Kolajova2, M.-C. Léveillé1,3, P. Claman1,3 and J.M. Baltz1,2,3,4,5

1 Human In-Vitro Fertilization Program, Ottawa Hospital, 2 Loeb Research Institute, Departments of 3 Obstetrics and Gynecology (Division of Reproductive Medicine) and 4 Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario K1Y 4E9, Canada


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mouse zygotes and early cleavage-stage embryos have previously been shown to utilize glycine as an organic osmolyte, accumulating it to oppose any decrease in cell volume. Such glycine uptake in early cleavage-stage mouse embryos is via the glycine-specific Gly transporter. Mouse embryos also possess swelling-activated channels which function to release osmotically active glycine and other osmolytes when cell volume becomes too large. In this study it was found that human cleavage-stage embryos also transported glycine via a similarly saturable, sarcosine-inhibitable transporter, implying that the Gly transporter also mediates glycine transport in human embryos. Mouse zygotes have previously been shown to accumulate more intracellular glycine when cultured at increased osmolarities for 24 h. It was found in the current study that this ability was lost as preimplantation mouse embryo development proceeded, and that early cleavage-stage human embryos may also be capable of such osmosensitive accumulation of glycine. Finally, using spare human eggs which had failed to fertilize or cleave, the presence of swelling-activated currents resembling those in mouse zygotes was demonstrated. These data indicate that osmoregulation in early human embryos occurs via similar mechanisms as in the mouse.

Key words: egg/electrophysiology/embryo/glycine/transport


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mouse eggs and early cleavage-stage embryos, when freshly removed from the female genital tract, contain a high intracellular concentration of glycine relative to other {alpha}-amino acids (Schultz et al., 1981Go; Van Winkle and Dickinson, 1995Go). The source of this glycine is apparently follicular and oviductal fluids, where glycine has been shown to be one of the most abundant {alpha}-amino acids in a number of species (Nancarrow and Hill, 1994Go; Guérin et al., 1995Go). This high intracellular glycine concentration can be maintained in embryos during extended in-vitro culture only if exogenous glycine is included in the external medium (Van Winkle and Dickinson, 1995Go), indicating that transport of glycine into the egg or embryo is required. In eggs, zygotes and early cleavage-stage embryos of the mouse, it has been shown that uptake of glycine is mediated entirely by the Na+- and Cl-dependent Gly transporter (Hobbs and Kaye, 1985Go, 1986Go, 1990Go; Van Winkle et al., 1988Go). The reliance of this transport system on the inwardly directed Na+ and Cl gradients allows highly concentration-dependent uptake of glycine and the maintenance of steep concentration gradients across the cell membrane, permitting a high glycine content in eggs and early embryos, even when the extracellular concentration is much lower.

The function of intracellular glycine in eggs and embryos is unknown. A portion of glycine taken up by mouse embryos is metabolized, principally into serine and alanine, or incorporated into macromolecules such as proteins (Hobbs and Kaye, 1985Go), but the largest proportion remains as free glycine (Schultz et al., 1981Go; Dawson et al., 1998Go). Recently, glycine has been shown to function as an organic osmolyte in mouse embryos, providing intracellular osmotic support at the zygote and early cleavage stages (Van Winkle et al., 1990Go; Dawson and Baltz, 1997Go; Dawson et al., 1998Go). Presumably, osmotically active glycine is accumulated in the zygote and early cleavage-stage embryo via the Gly transporter. Glycine accumulation has been shown to be regulated by external osmolarity in mouse zygotes, with higher concentrations of glycine accumulated over 24 h in 310 or 340 mOsmol/l medium than in 250 mOsmol/l medium (Dawson et al., 1998Go). However, it is unknown if such osmosensitive glycine accumulation is a feature only of the zygote, or if it persists throughout preimplantation embryo development. Nor is it known whether embryos of other species, including humans, exhibit this phenomenon.

Although uptake of glycine via the Gly transporter can mediate accumulation of osmotically active glycine in the cytoplasm to protect against cell volume loss, it cannot perform the converse function of exporting glycine to alleviate cell swelling, since the Gly transporter is essentially irreversible in cells with normal, inwardly directed Na+ and Cl gradients. However, mouse embryos do possess a separate mechanism for releasing osmotically active cytoplasmic compounds upon swelling. This mechanism relies upon swelling-activated anion channels evident in zygotes and early cleavage-stage mouse embryos (Kolajova and Baltz, 1999Go; also unpublished results), which closely resemble the volume-sensitive channels found in a wide range of cells. These channels are highly permeable not only to anions such as Cl, but also to organic osmolytes including glycine (Strange et al., 1996Go; Okada, 1997Go). It has also been shown directly that early mouse embryos exhibit swelling-activated release of accumulated glycine and taurine (Dumoulin et al., 1997Go; Dawson et al., 1998Go), indicating that swelling-activated anion/osmolyte channels mediate organic osmolyte release from embryos. Thus, the mouse zygote appears capable of regulating its volume at least in part by transporting glycine into the cell via the Gly transporter, and releasing it via the swelling-activated anion/osmolyte channel. In this study, the osmoregulatory and glycine transport mechanisms in the mouse embryo were investigated further, and the early human embryo was examined as to whether it had similar mechanisms for glycine transport and possible osmoregulation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mouse embryos
Mouse embryos were obtained from female CF1 random-bred mice (Charles River, St. Hyacinth, PQ, Canada) which were superovulated by i.p. injections of 5 IU equine chorionic gonadotrophin (eCG) followed by 5 IU human chorionic gonadotrophin (HCG) 47.5 h later (eCG and HCG purchased from Sigma Chemical Co., St Louis, MO, USA) and mated with BDF males (Charles River). Zygotes were obtained on day 1 (i.e. the day following HCG administration), while later stages were obtained on subsequent days as specified. For zygotes, the cumulus mass was removed by brief exposure to hyaluronidase (0.3 mg/ml, 5–10 min; Sigma).

Human embryos
The human eggs and embryos used in these studies were excess to clinical requirements of the in-vitro fertilization (IVF) programme at the Ottawa Hospital and were to be discarded; they were released for research with patient consent. The human embryos available for these experiments were from patients who, for various reasons, had elected against cryopreservation. Therefore, the human embryos used for these experiments were those left over after the best two to four, as judged by cleavage stage and grade, had been selected for transfer. The experimental group was therefore somewhat biased towards lower-quality embryos, but consisted largely of embryos which would have been cryopreserved for subsequent transfer had the patients elected so to do. A standard ovarian stimulation protocol was used, with oocyte retrieval 36 h after triggering by i.m. injection of 10 000 IU HCG (HCG-Pregnyl®; Organon, Scarborough, Ontario, Canada), as described previously (Claman et al., 1996Go). Retrieved eggs were used for either standard IVF or intracytoplasmic sperm injection (ICSI) by standard procedures. The day of oocyte retrieval was designated as day 0. After IVF/ICSI, the eggs were placed into microdrop (20 µl) cultures of human tubal fluid (HTF) medium (Meditech 1st, Montreal, Quebec, Canada) with bovine serum albumin (BSA; Sigma) added, under paraffin oil at 37°C in 5% CO2/5% O2/90% N2. Pronuclear status was assessed 17–19 h post IVF/ICSI (on day 1). Embryo cleavage was assessed during culture, and embryo quality rated. Rating was from 1–5, based on the amount of fragmentation and regularity of the blastomeres, with grade 5 being of highest quality (Rattanachaiyanont et al., 1999Go). During the period of this study, most patients were scheduled for day 3 embryo transfers, while some were performed on day 2. Thus, embryos became available for this study on days 2 and 3 after oocyte retrieval. A few embryos were cultured for one additional day, and used on day 4. Preliminary analysis indicated no effects attributable to the day on which the embryo was obtained, so data were pooled. Those eggs which had undergone the IVF or ICSI procedure but had failed to cleave were here termed failed fertilized/failed cleaved eggs. They were left in culture in the same dishes (but separate drops) with fertilized, cleaving embryos until the day of embryo transfer, at which time they were released for research. Failed fertilized/failed cleaved eggs were not normally available before the day of embryo transfer, because clinical policy required that the dishes (which also contained cleaving embryos) should not be disturbed unnecessarily before embryo transfer; thus, no freshly obtained eggs were available for these studies.

Media
Mouse embryos were removed from oviducts using HEPES-KSOM medium (potassium supplemented simplex optimized medium; Lawitts and Biggers, 1993), modified by omitting glutamine and using polyvinyl alcohol (PVA) as the macromolecular component, as described previously (Dawson et al., 1998Go). A modification of G1 medium (Barnes et al., 1995Go), which had been developed for culture of human cleavage-stage embryos, was used for both mouse and human embryo experiments, under an atmosphere of 5% CO2 in air at 37°C. G1 medium was modified here by omitting all amino acids, except glycine or sarcosine which were added as specified, and by using PVA rather than albumin. Components of culture media were obtained commercially (Sigma), and were embryo culture grade or cell culture grade. A Vapro model 5520 vapour pressure osmometer (Wescor, Logan, UT, USA) was used to determine medium osmolality. As these were dilute solutions, the measured osmolality (osmoles/kg) was equivalent to osmolarity (osmoles/litre = Osmol/l), and thus osmolarity (with units of mOsmol/l) is referred to throughout. G1 (lacking amino acids) was ~ 250 mOsmol/l; raffinose (Sigma), a trisaccharide which is neither transported nor metabolized by mammalian cells, was added in one set of experiments to increase osmolarity to 310 mOsmol/l as described previously (Dawson et al., 1998Go).

Glycine measurements
[2-3H]Glycine (10–30 Ci/mmol; Amersham, Arlington Heights, IL, USA; alternatively, 30–60 Ci/mmol; New England Nuclear Boston, MA, USA) was added directly to the culture medium immediately before an experiment. Two types of protocols were used. Both protocols, and their validations, have been previously described (Dawson et al., 1998Go). The first was designed to measure the rate of uptake of glycine, which was done by incubating embryos for 45 min in modified G1 medium containing 1 or 2 µmol/l [3H]glycine (`45 min uptake'). The second was designed to measure accumulation of glycine and its metabolic products over a 24 h period (`24 h accumulation'), which was done by incubating embryos in modified G1 containing 1.0 or 0.1 mmol/l unlabelled glycine plus 1 µmol/l [3H]glycine. This reduced the specific activity of the glycine 1000-fold or 100-fold respectively, which has been shown to eliminate the toxic effects of large intracellular 3H concentrations while maintaining the total glycine concentration at values which supported maximal intracellular accumulation (Dawson et al., 1998Go). During the 45 min uptake or 24 h accumulation periods, embryos were kept in microdrops under mineral oil (embryo grade, Sigma) in an incubator which maintained 5% CO2 in air at 37°C.

To determine the 3H content of embryos, one (for most experiments) or 10 (for the initial experiment) embryos were removed from [3H]glycine-containing G1 medium, immediately washed several times in ice-cold HEPES-KSOM of the same osmolarity (adjusted where necessary with raffinose) as the [3H]glycine-containing G1 medium, and transferred to scintillation vials containing 4 ml each of scintillation fluid (Scintiverse BD, Fisher Scientific, Fairlawn, NJ, USA) and vortexed briefly. It had been observed previously that embryos lyse completely within minutes of transfer to scintillation fluid, even without vortexing; thus, no other means of cell lysis was required. [3H]glycine measurements were then made by liquid scintillation counting (model 2200CA TriCarb scintillation counter; Packard Instrument Co., Downer's Grove, IL, USA) as described previously (Dawson et al., 1998Go); raw data were obtained as counts per minute (c.p.m.). Background determinations were made on samples of the final wash medium, and these c.p.m. values were subtracted from the sample counts. The total 3H content of embryos was calculated using standard curves (Dawson et al., 1998Go), and expressed on a per embryo basis.

For the acute (45 min) uptake experiments, uptake by single embryos was proportional to external concentration for 1 and 2 µmol/l: preliminary comparisons showed that the total [3H]glycine taken up over 45 min was 1.74 ± 0.33-fold higher in 2 µmol/l than in 1 µmol/l external [3H]glycine (not different from the expected value of 2-fold). Thus, uptake into each embryo was reported as the rate of uptake of [3H]glycine in units of fmol/min; when 2 µmol/l glycine was used, data were normalized to the 1 µmol/l rate by dividing by 2. Because the signal was relatively low in 45 min uptake experiments with human embryos using 1 µmol/l external [3H]glycine (see below), most experiments with human embryos were performed using 2 µmol/l. Uptake was linear over several hours (Dawson et al., 1998; Van Winkle et al., 1988; also unpublished results), indicating that there was no saturation by 45 min.

Background c.p.m., obtained from the last wash drop, were the same for human or mouse embryos and for 45 min uptake or 24 h accumulation experiments, with means ranging from 15 to 19 c.p.m. in the four protocols. The mean background measured in a single mouse embryo was about 100–150 c.p.m., and in a single human embryo was 60–90 c.p.m., depending upon experimental protocol (except where transport was purposely inhibited, e.g. by sarcosine). The signal-to-background ratios for single embryo measurements in the various protocols were ~7–9 for mouse embryos, and 3–5 for human embryos; most 45 min uptake experiments with human embryos were carried out with 2 µmol/l external [3H]glycine, where signal-to-background ratios were about 5, as it was for 24 h uptake measurements.

Electrophysiological recordings
Whole-cell patch–clamp recordings were performed on human eggs which had failed to cleave following IVF or ICSI (failed fertilized/failed cleavage embryos). The recordings were made in identical manner to those described previously for mouse zygotes (Kolajova and Baltz, 1999Go). Briefly, gigaohm (G) level seals were achieved using patch microelectrodes filled with a high-K+, low-Cl solution where gluconate was the major anion (composition in mmol/l: 100 K+ gluconate, 10 KCl, 3.5 MgCl2, 1 Na2ATP, 1 EGTA, 10 HEPES, adjusted to pH 7.3 with NaOH). After achieving a G seal, the membrane within the pipette opening was ruptured by a brief suction pulse, to achieve whole-cell configuration. Currents and imposed voltages were monitored continuously with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA). A Digidata 1200B digitizer with p-CLAMP (v.6) software (Axon Instruments) was used on-line to sample current signals. Clampfit software (Axon Instruments) was used for analysis, and digitized current–voltage relationships obtained. Unless otherwise specified, voltages were corrected for the measured junction potential of about +10 mV. The failed fertilized embryos were continuously superfused, and external solution compositions changed by changing the superfusate source (Kolajova and Baltz, 1999Go). All electrophysiological recordings were carried out at room temperature, in HEPES-KSOM (Kolajova and Baltz, 1999Go).

Data analysis
Data are cited as mean ± SEM, except where noted. Data are presented graphically using SigmaPlot 5.0 for Windows (Statistical Package for Social Sciences, Chicago, IL, USA). Data were tabulated and F- and t-tests performed using Microsoft Excel 97. Bartlett's test for homogeneity of variances, and parametric and non-parametric one-way analysis of variance (ANOVA) with post-hoc tests (Tukey–Kramer or Dunn's respectively) were performed using Instat (GraphPad Software, San Diego, CA, USA). Two-way ANOVA was performed using the ViSta Visual Statistics System (Forrest W. Young, Psychometric Laboratory, University of North Carolina, Chapel Hill, NC, USA). Exact Pearson chi-square tests were performed with StatXact Turbo (Cytel, Cambridge, MA, USA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Measurement of [3H]glycine in single embryos
Due to the low numbers and variable developmental stages of spare human embryos which were available, it was necessary to measure the [3H]glycine content of single embryos. To validate single-embryo measurements under the conditions used, mouse zygotes were incubated with 1 µmol/l [3H]glycine for 45 min in KSOM, and then allocated randomly to scintillation vials either as single zygotes or groups of 10 zygotes. Measurements were made on 16 single zygotes, and nine groups of 10 zygotes (90 in total), obtained from five mice on five separate days.

Using the calibration curves, data in CPM were converted to [3H]glycine uptake after background subtraction. The mean (± SEM) rate of uptake of [3H]glycine measured using single embryos was 0.186 ± 0.012 fmol/zygote/min, compared with 0.157 ± 0.012 fmol/zygote/min using groups of 10. These means were not significantly different (P = 0.13 by t-test), and thus single embryos could be used for these measurements. The variability was slightly higher for single embryo measurements (SD = 0.049) compared with those using groups of 10 (SD = 0.037), but this difference was not significant (P = 0.22 by F-test). Similar results were obtained with 2-cell embryos (not shown). Thus, all subsequent [3H]glycine experiments with mouse embryos, and all experiments with human embryos, were performed using single embryos.

Glycine transport into mouse embryos
Mouse embryos were obtained as zygotes, 2-cell, 4-cell or 8-cell embryos by removing embryos from oviducts at the appropriate times. In each replicate, embryos were randomly allocated to drops of either G1 or G1 with the specified addition, and the rate of uptake of 1 or 2 µmol/l [3H]glycine into single embryos was measured. Glycine transport was saturable, as excess unlabelled glycine (5 mmol/l) almost completely abolished uptake of 3H-labelled glycine, as shown for zygotes (Figure 1AGo; P < 0.0001 by t-test). Glycine uptake was also almost completely abolished in the presence of 5 mmol/l sarcosine, as shown for zygotes and also for 2-cell, 4-cell and 8-cell embryos (Figure 1BGo), indicating that essentially all detectable glycine transport, under these conditions, was via a mechanism which could be competed by sarcosine. Analysis by two-way ANOVA (cell numberxsarcosine) showed that the inhibition by sarcosine was extremely significant (P < 0.0001). In addition, the rate of glycine uptake decreased after the 2-cell stage, so that the mean rate of uptake at the 8-cell stage was only about 50% that of zygotes and 2-cell embryos (Figure 1BGo); this decrease was also highly significant (P < 0.0001).



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Figure 1. Glycine uptake by mouse zygotes and cleavage-stage embryos. (A) Uptake (in fmol/embryo/min; mean ± SEM) of 1 µmol/l [3H]glycine by mouse zygotes (1c) in G1 medium or G1 + 5 mmol/l excess unlabelled glycine (n = 15 single embryos for each treatment, i.e. each bar, three replicates). (B) Uptake by mouse zygotes (1c), 2-cell (2c), 4-cell (4c) or 8-cell (8c) embryos in G1 (n = 13–18 single embryos per stage for each treatment, i.e. each bar, three replicates at each stage). Asterisks indicate significant inhibition by excess glycine or sarcosine (P < 0.0001), as indicated (tests described in text). In addition, the decrease in uptake at the 4- to 8-cell stages was significant (see text).

 
Glycine transport into human embryos
Human embryos which had cleaved to the 2-cell to 8-cell stages, as well as eggs which had failed to fertilize or cleave were used. Figure 2Go shows the rate of uptake of glycine into all single human embryos and failed fertilized/failed cleavage eggs in which such uptake was measured. The failed fertilized/failed cleavage eggs consistently imported glycine at a lower rate than fertilized, cleaved embryos. The mean uptake rates of cleavage-stage embryos appeared similar regardless of the number of cells, however. A Kruskal–Wallis non-parametric ANOVA (used since Bartlett's test indicated that the standard deviations differed significantly) was performed to determine if the median uptake rates differed when the data were grouped by cell number. The difference among rates of uptake differed significantly when grouped by cell number (P = 0.034); Dunn's Multiple Comparison test indicated that the failed fertilized/failed cleavage group accounted for the difference. These data were further analysed by testing whether cleaved embryos (2- to 8-cell, pooled) differed from the failed fertilized/failed cleavage group. This difference was highly significant (P < 10–6 by t-test, assuming unequal variances).



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Figure 2. Glycine uptake by human embryos and failed fertilized/failed cleaved eggs. Uptake of 1–2 µmol/l [3H]glycine by day 3–4 human embryos and failed fertilized/failed cleaved eggs. The dots each indicate the rate of uptake of a single embryo or egg, at the stage indicated at the bottom (FF = failed fertilized/failed cleaved eggs), obtained from 26 patients in total. The bars indicate the mean (± SEM) uptake for embryos grouped by cell number at time of measurement (n = 4–18 per bar; 66 total cleaved embryos). The uptake by failed fertilized/failed cleaved eggs was significantly less than that by other stages, as indicated by the asterisk (see text).

 
Most of the embryos (>70%) had quality ratings of 3. A plot of glycine uptake rate versus quality rating did not reveal any dependence (not shown), as confirmed by an ANOVA which indicated no significant difference between uptake rates when grouped by quality rating (P > 0.1).

A series of measurements was performed, similar to those made with mouse embryos (above), to determine the characteristics of the glycine transport into cleaved human embryos. Here, 2- to 8-cell human embryos were allocated randomly to G1 or G1 with the specified addition, and the rate of uptake of 2 µmol/l [3H]glycine into single embryos was measured. Glycine uptake into human embryos was saturable, as excess unlabelled glycine (5 mmol/l) significantly decreased uptake of 3H-labelled glycine (Figure 3AGo; P < 0.0001 by t-test). Sarcosine (5 mmol/l) also significantly inhibited the inward transport of glycine, by a similar fraction (Figure 3BGo). A two-way ANOVA (cell numberxsarcosine) confirmed that there was no dependence on cell number (P = 0.50), but found a significant effect of sarcosine (P < 0.0001). As cell number did not exert a significant effect, data pooled by cell number are shown.



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Figure 3. Effect of excess glycine or sarcosine on glycine uptake by human embryos. (A) Uptake of [3H]glycine (2 µmol/l) by human cleaved embryos in G1 medium (n = 12) or G1 medium + 5 mmol/l excess unlabelled glycine (n = 13). Embryos are 2- to 8-cell, obtained from six patients total. (B) Uptake in G1 (n = 21) or G1 + 5 mmol/l sarcosine (n = 19). Embryos are 2- to 8-cell, obtained from 11 patients. Asterisks indicate significant differences from control (P < 0.0001, see text). The controls shown here are also incorporated into the overall control group shown in Figure 2Go. Bars indicate mean ± SEM.

 
Glycine transport into mouse eggs after in-vitro ageing
Because human failed fertilized/failed cleaved eggs, which had been maintained in culture for several days, exhibited a low rate of glycine uptake, it was investigated whether in-vitro ageing of ovulated mouse eggs would result in a similarly low glycine uptake. Mouse eggs were obtained ~17 h post HCG administration, and placed into culture. In each experiment, a randomly chosen subset of the eggs was removed, and glycine uptake measured immediately (`fresh eggs'). The remaining eggs were maintained in culture for 24 or 48 h, after which uptake was measured (`aged eggs'). Uptake of glycine by fresh eggs was 0.135 ± 0.027 fmol/zygote/min (n = 25 in five separate experiments); this was similar to, but slightly less than, that of zygotes (cf. Figure 1Go). Eggs from the same cohorts were cultured for 24 h or 48 h before glycine uptake was measured. At 24 h, the rate of glycine uptake in these eggs was almost twice that by fresh eggs (0.240 ± 0.044 fmol/zygote/min, n = 24 in five experiments), while at 48 h, the rate of uptake had reverted to a value only slightly higher than by fresh eggs (0.181 ± 0.063 fmol/zygote/min, n = 8 in two experiments). The differences among the groups were highly significant by Kruskal–Wallis non-parametric ANOVA (P < 0.0001); Dunn's test showed that this was due to a significant difference between fresh and 24 h eggs (P < 0.001). Longer culture times were not attempted, as most eggs had degenerated (fragmented) by 48 h; uptake rates by degenerated eggs were not included in the analysis, although they were found to take up glycine at approximately the same rate as intact eggs at 48 h (data not shown).

Osmosensitivity of glycine accumulation in mouse embryos
Mouse embryos obtained as in-vivo-produced zygotes, 2-cell embryos or 8-cell embryos were cultured for 24 h in the presence of 1 mmol/l glycine, with 1 µmol/l [3H]glycine added. The proportions of different stages arising after 24 h were not different between 250 and 310 mOsmol/l media for any group (Pearson's Exact chi-square test; P > 0.2). The total accumulated glycine in single embryos was then estimated from the accumulated 3H (Dawson et al., 1998Go). Zygotes cultured for 24 h to the 2-cell stage accumulated significantly more glycine when cultured in 310 mOsmol/l medium, than when cultured in 250 mOsmol/l medium (P < 0.0001 by t-test; Figure 4Go). Culturing 2-cell embryos for 24 h yielded a range of embryos from 4-cell to compacted (presumably 8-cell) embryos. For analysis, these were divided into the more slowly developing embryos (4-cell) and the more quickly developing embryos (6-cell, 8-cell, and compacted embryos). The 2-cell embryos which developed to the 4-cell stage had accumulated identical amounts of glycine, whether cultured at 250 or 310 mOsmol/l (P = 0.90; Figure 4Go). However, those which were faster-growing showed a slight stimulation of glycine accumulation by raised osmolarity (P = 0.002; Figure 4Go). When the >4-cell group was further divided into 6- and 8-cell and compacted groups (data not shown), accumulation within each group was still significantly greater at 310 versus 250 mOsmol/l (P = 0.03 and P = 0.002 respectively). The 8-cell embryos cultured to cavitating morulae and blastocysts after 24 h accumulated a low level of glycine which was not sensitive to osmolarity (P = 0.30; Figure 4Go).



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Figure 4. Osmosensitive glycine accumulation by mouse zygotes and cleavage-stage embryos. The total amount of 3H in mouse zygotes, 2-cell embryos or 8-cell embryos cultured in 1 mmol/l glycine (labelled with 1 µmol/l [3H]glycine) was measured after 24 h in culture at 250 or 310 mOsmol/l. Zygotes all reached the 2-cell stage (1c to 2c); 8-cell embryos which reached the cavitating morula or blastocyst stage were assayed (8c to B). 2-cell embryos were divided into those which divided to the 4-cell stage (2c to 4c) and those which reached the 6-cell, 8-cell or compacted stages (2c to >4c). P-values for differences between paired bars are shown (see text; ns = not significant at the P = 0.05 level). Bars indicate mean ± SEM.

 
Osmosensitivity of glycine accumulation in human embryos
Human cleavage-stage embryos (4- or 8-cell, obtained on days 2–4 from 13 patients) were cultured for 24 h at 250 or 310 mOsmol/l in the presence of 0.1 mmol/l glycine with 1 µmol/l [3H]glycine added. After 24 h, the total glycine taken up was estimated from the 3H content, as described above for mouse embryos. There were sufficient 4-cell (n = 9 and 8 for 250 and 310 mOsmol/l respectively) and 6- and 8-cell (n = 5 and 4 respectively) embryos to perform statistical tests on these groupings. In both cases, the mean amount of glycine accumulated at 310 mOsmol/l was greater than at 250 mOsmol/l. For 4-cell embryos, the mean ± SEM accumulation was 0.32 ± 0.04 pmol/embryo (n = 9) at 250 mOsmol/l compared with 0.51 ± 0.06 (n = 8) at 310 mOsmol/l; for 6- to 8-cell embryos, the mean accumulation was 0.23 ± 0.05 pmol/embryo at 250 mOsmol/l (n = 5) compared with 0.38 ± 0.05 at 310 mOsmol/l (n = 4). The significance of these differences was low (P = 0.017 for 4-cell and 0.049 for 6- to 8-cell embryos; t-test). A few 5-cell embryos (n = 4 and 2 for 250 and 310 mOsmol/l respectively) were included in the assay, but have not been included in the above analysis; if these were included in the 4-cell group, the difference between 250 and 310 mOsmol/l became insignificant. Thus, there is a trend towards higher accumulation of glycine with increased osmolarity in these human embryos, though this effect had only borderline significance, at best, in this population of human embryos.

Swelling-activated currents in human failed-fertilized eggs
Six eggs which had failed to fertilize normally (two from IVF procedures, four from ICSI) were obtained from four patients. These became available for experiments on day 3 (i.e. the day of scheduled embryo transfer), and were used either the same day (n = 4), or the next day (n = 2). All were scored as having no pronuclei (PN) on day 1, except for one with 1 PN. Whole-cell currents in these eggs were measured as described in Materials and methods, using the whole-cell patch–clamp method. Whole-cell current was measured, as a function of applied voltage, in each egg. The initial measurements were done in medium whose osmolarity was 250 mOsmol/l. The external solution was then switched to 180 mOsmol/l medium, and current measured as the eggs swelled, continuing until the current reached steady state. After this, the external solution was again switched, to 180 mOsmol/l medium identical to the previous medium except that the anion channel-inhibitor DIDS (100 µmol/l) was added, and current again measured until it reached steady state.

Figure 5Go shows a typical set of current–voltage relationships obtained from one human egg. The current characteristics are nearly identical to those previously reported for mouse zygotes under the same conditions (Kolajova and Baltz, 1999Go): cell swelling activates an outwardly rectified current which is inhibited by DIDS in a voltage-dependent manner, with significant inhibition evident only at positive potentials. To analyse these data, the current at +60 mV was used as a measure of the magnitude of outwardly rectified current. The mean (± SEM) current was 2.8 ± 0.5 nA at 250 mOsmol/l medium, then 11.5 ± 1.5 nA after the cells had fully swelled in 180 mOsmol/l medium (n = 6). Introduction of DIDS decreased the mean current in the swelled cells to 3.6 ± 0.5 nA (n = 5; one seal was lost as DIDS was introduced). These data were analysed by ANOVA, followed by the Tukey–Kramer multiple comparison test. A preliminary repeated-measures ANOVA indicated that the between-subject variability was not significant, and that an ordinary ANOVA was more appropriate. The difference between the means was found to be significant (P < 0.0001); the Tukey–Kramer test indicated that the current in swelled eggs at 180 mOsmol/l was significantly higher (P < 0.001) than currents at either 250 mOsmol/l or with DIDS, which were not significantly different from each other (P > 0.05). There was no significant difference between measured reversal potentials (P = 0.40), which were –25 ± 1, –22 ± 1 and –24 ± 2 mV for 250, 180 and 180 mOsmol/l with DIDS respectively.



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Figure 5. Example of swelling-activated whole-cell current in a single human failed fertilized/failed cleavage egg. Voltage ramps were applied periodically (every 50 s) to measure whole-cell current as a function of transmembrane voltage. Each ramp consisted of a linear increase in imposed voltage from –60 to +80 mV in 2.5 s. This imposed voltage range corresponded to transmembrane voltages of –70 to +70 mV after correcting for junction potential. Between ramps, imposed holding potential was maintained at –20 mV. Shown here are three current-voltage plots obtained from such voltage ramps in one human egg, representative of data obtained from six separate eggs. The first plot was obtained in 250 mOsmol/l medium, the second was obtained after swelling in 180 mOsmol/l medium, and the third after application of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (100 µmol/l, plot shown is ~10 min after application) in 180 mOsmol/l medium.

 

    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
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The rate of glycine uptake measured in mouse embryos was highest at the zygote and 2-cell stages, and decreased at the 4- and 8-cell stages, similar to the pattern described previously (Hobbs and Kaye, 1985Go; Van Winkle et al., 1988Go). Under the conditions used in this study, essentially all glycine uptake by cleavage-stage mouse embryos was saturable and sarcosine-sensitive, consistent with the previous finding that Gly activity is responsible for virtually all of the glycine transport by mouse zygotes and cleavage-stage embryos (Hobbs and Kaye, 1985Go; Van Winkle et al., 1988Go). It appears from the data presented here that, as in the mouse, glycine uptake by human embryos is likely mediated by the Gly transporter. Uptake was saturable, decreasing by ~75% in the presence of excess unlabelled glycine (Figure 3AGo), and almost all of this saturable uptake was sarcosine-sensitive (Figure 3BGo). Although sarcosine sensitivity is generally taken to be a hallmark of transport by the Gly system, based on these limited experiments in human embryos, the existence of another heretofore unidentified sarcosine-sensitive transporter mediating some or all of this transport in human embryos cannot be ruled out. However, based on the current data and comparison with previous extensive experiments with mouse embryos (Van Winkle et al., 1988Go), it is proposed that the sarcosine-sensitive transport in human embryos is also likely due to Gly activity.

The human embryos used in these experiments were obtained mainly on day 3, when normal in-vitro embryo development is to at least the 4- to 8-cell stage. Human embryos at these stages took up glycine at ~50% of the rate of uptake of 4- to 8-cell mouse embryos (compare Figures 1 and 2GoGo). Less advanced embryos (2- to 3-cell) transported glycine at approximately the same rate as later cleavage-stage embryos, but unlike mouse embryos at the zygote and 2-cell stages, which show higher uptake rates. The human and mouse experiments, however, are not directly comparable, as the mouse embryos were obtained on the appropriate day of development for each stage, while the human 2- to 3-cell embryos on day 3 post-oocyte retrieval were clearly delayed or arrested. Thus, the absence of a higher rate of glycine uptake in these earlier cleavage stages could be due either to a different developmental pattern of glycine transport in humans, or more likely to their delayed/arrested development. This is an inevitable complication of using human embryos which are excess to clinical requirements, and thus further studies with fresh, stage- appropriate human, or at least primate, embryos are required for resolution.

In contrast to cleaved human embryos, human eggs which failed to fertilize or cleave exhibited a significantly lower rate of glycine uptake (Figure 2Go). Their uptake rate did not differ much from the sarcosine- and excess glycine-insensitive portion of glycine uptake by cleaved embryos, indicating that failed fertilized/failed cleavage eggs do not specifically transport glycine at a significant rate, and thus lack significant Gly activity. This could arise in two different ways: either these eggs were never capable of glycine transport, or they lost this ability when kept in culture for an extended time, as degenerative processes proceeded. The human eggs available for this study had failed to fertilize or cleave, and were necessarily kept in culture for an extended period, due to clinical policy (see Materials and methods). As fresh, normal human eggs were not available for study, mouse eggs were used to investigate the effect of in-vitro ageing on glycine transport by unfertilized eggs. Freshly obtained unfertilized, ovulated mouse eggs transported glycine at approximately the same rate as zygotes and 2-cell embryos, in agreement with previously published data (Hobbs and Kaye, 1985Go; Van Winkle et al., 1988Go). It was found that the rate of glycine uptake by unfertilized mouse eggs increased markedly after 24 h in culture, rather than decreasing, as the unfertilized eggs were aged in vitro, and maintained at least the same rate of uptake as fresh eggs even after 48 h. A similar increase was reported in leucine uptake by unfertilized mouse eggs aged for 24 h (Carroll and Longo, 1981Go), and therefore increased amino acid uptake by aged unfertilized eggs may be a general phenomenon. Thus, it is possible that those human eggs which failed to fertilize or cleave were already deficient in glycine uptake even before ageing, rather than this decrease being due to ageing in vitro. This needs to be further investigated, as it would be intriguing if a subpopulation of eggs could be identified on the basis of glycine uptake ability which correlated with their subsequent ability to become fertilized and cleave.

We have replicated our previous finding (Dawson et al., 1998Go) that the amount of glycine accumulated by mouse zygotes cultured for 24 h to the 2-cell stage increases when external osmolarity is increased, except that the measurements here were made with single embryos rather than groups of 10. The amounts of glycine accumulation at 250 and 310 mOsmol/l were essentially identical to those reported previously. Furthermore, these experiments were extended to mouse 2-cell and 8-cell embryos cultured for 24 h. In contrast to zygotes, there was little effect of raised osmolarity on glycine accumulation by subsequent stages (Figure 4Go). Embryos cultured from the 2-cell stage were divided into two groups: those that cleaved to the 4-cell stage (which were slowed in their development), and those which cleaved to >4-cell stage (6-cell, 8-cell and compacted embryos). The most rapidly developing embryos showed a slight, but very significant, stimulation of glycine accumulation by increased osmolarity, while the more slowly cleaving embryos did not. Glycine accumulation by embryos cultured from the 8-cell stage was unaffected by increased osmolarity. Thus, osmosensitive accumulation of glycine, presumably for use as an organic osmolyte (Van Winkle et al., 1990Go; Dawson and Baltz, 1997Go), appears to be a feature of the earliest stages of embryo development, but is subsequently lost. It is interesting to note that the ability to accumulate glycine in response to increased osmolarity parallels the activity of the Gly transporter, offering support for the hypothesis that transport via the Gly transporter mediates such osmosensitive accumulation.

Glycine accumulation by the human embryos available for this study was slightly stimulated by increased osmolarity, with the greatest and most significant increase seen when culturing 4-cell embryos. Thus, it appears that human embryos, like those of the mouse, are stimulated to accumulate glycine as an organic osmolyte by increased external osmolarity. Although normal human zygotes could not be obtained, it is predicted that the largest osmosensitive accumulation would be found at that stage, as in the mouse. This remains to be investigated with human or primate zygotes, however.

Although in short-term uptake experiments, the metabolism of glycine would not be a major factor, in these 24 h accumulation experiments this could be a significant factor. As we were measuring the total amount of 3H (rather than directly measuring glycine) in embryos, any compound to which the 3H radiolabel had been transferred would have been detected along with [3H]glycine. This is true, however, only of relatively impermeant compounds which are retained within the embryo; highly permeant compounds such as water would rapidly be lost during the washes. In contrast, any metabolite of glycine which functions as an osmolyte would be retained within the embryo, and would be functionally equivalent to glycine itself. Because of the limited material available, we did not attempt to determine (e.g. by direct amino acid analysis) if significant conversion of glycine to other such osmolytes occurred during the 24 h period, but instead measured total 3H osmolyte content and reported it as the equivalent glycine concentration. In contrast, any metabolism of glycine to fixed cellular components (e.g. protein or nucleic acids through purine synthesis) would remove it from the pool of osmolytes. To exclude this latter possibility in mice, we confirmed that most of the 3H accumulated after 24 h was osmotically active, and releasable through swelling-activated osmolyte channels upon hypotonic swelling of the embryo (Dawson et al., 1998Go), ruling out the incorporation of 3H into fixed components of the cell such as proteins for the majority of accumulated glycine. Thus, we cannot exclude that glycine was converted to small, osmotically active substances, such as serine and/or alanine. However, our conclusion would be unchanged: that these substances are accumulated for osmotic support. We could not confirm that [3H]glycine was in the form of small osmolytes in human embryos by showing hypotonic release of accumulated 3H, due to the extremely limited amount of material available. However, by analogy with the mouse embryos, the majority is expected to be osmotically active.

In mouse embryos, osmotically active glycine is released via swelling-activated anion channels which are detectable both by measuring the increase in glycine permeability upon cell swelling (Van Winkle et al., 1994; Dawson et al., 1998; D.G.Séguin, K.M.Dawson, M.A.Hammer and J.M.Baltz, unpublished observations), and by measuring the current increase upon swelling (Kolajova and Baltz, 1999; also unpublished results). In this study, DIDS-inhibitable, swelling-activated currents were detected in each of six human failed fertilized or failed cleavage eggs. The currents in unswelled human eggs (mean 2.8 nA at +60 mV) were approximately the same as those found in mouse zygotes (mean 2.4 nA; Kolajova and Baltz, 1999), while the currents in swelled human failed fertilized or failed cleavage eggs (mean 11.5 nA) were approximately twice those in mouse zygotes (6.6 nA; Kolajova and Baltz, 1999), possibly indicating a greater number of swelling-activated channels in human eggs (and perhaps reflecting their greater surface area). DIDS inhibited this current with the same characteristic voltage dependence (inhibition at positive voltages) as seen in mouse zygotes (Kolajova and Baltz, 1999Go) and other cells (Okada, 1997Go). This swelling-activated pathway has been shown to be responsible for the release of intracellular glycine when mouse embryos are swelled, allowing them to regulate against detrimental volume increases (Dawson et al., 1998Go; Kolajova and Baltz, 1999Go). Thus, this mechanism forms part of the total volume-regulatory mechanism of the embryo, which consists at least in part of glycine accumulation by the Gly transporter and release via swelling-activated channels. As human embryos exhibit both pathways, it is proposed that early cleavage-stage human embryos resemble mouse embryos in their use of glycine for osmoregulation depending on these two opposing mechanisms.

Further work is required to determine the characteristics of glycine transport in normal human eggs and zygotes. In addition, the relationship—if any—between the rate of glycine uptake by eggs or early embryos and subsequent developmental potential, remains to be determined. However, it is clear from these data that human embryos possess the mechanisms needed to transport and utilize glycine, including the glycine-specific Gly transporter, supporting the inclusion of glycine in human embryo culture media.


    Acknowledgments
 
The authors thank the staff of the Human IVF Laboratory at the Ottawa Hospital, Dianne Hoppe, Carole Lawrence, Peggy Philion, Michelle Schonfeldt and Alena Spacek, for their generous help. This work was supported by the Division of Reproductive Medicine, Department of Obstetrics and Gynecology, University of Ottawa. Some of the work with mouse embryos was partially supported by the Medical Research Council, Canada (Operating Grant MT12040 to J.M.B.).


    Notes
 
5 To whom correspondence should be addressed at: Loeb Research Institute, 725 Parkdale Avenue, Ottawa, ON K1Y 4E9, Canada

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    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on July 26, 1999; accepted on November 10, 1999.