| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
c Loeb Medical Research Institute,
d Human IVF Laboratory,
e Ottawa Civic Hospital, Departments of Obstetrics and Gynecology (Reproductive Biology Unit) and Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada K1Y 4E9
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We have recently reported that effective osmoprotectants for mouse embryo culture from the zygote stage are divided into two groups [6]. One group includes substrates of the Gly transporter, a Na+- and Cl--dependent transporter of glycine and its derivatives, which is found in cleavage-stage embryos [12–14], while the other includes substrates of the ß transporter, which is found throughout preimplantation development [15, 16]. Of the two groups, Gly substrates, including glycine, appeared to be the most effective osmoprotectants [6]. The osmoprotective effect of ß-amino acids is likely to be mediated by the ß transporter, which is an organic osmolyte transporter in other cells. Also, the rate of taurine (a model ß substrate) transport in early embryos is markedly stimulated by increased osmolarity [15]. However, how glycine might be accumulated as an osmolyte is not clear, since the Gly system has not, to our knowledge, been implicated in osmoregulation in other cell types. We have investigated whether glycine, a model Gly substrate present at high concentrations in oviductal fluid, is used by mouse zygotes and early cleavage-stage embryos as an osmolyte. Any organic compound that functions as an osmolyte must be accumulated to a higher intracellular concentration at higher external osmolarities. In addition, the availability of such osmolytes may confer improved ability to maintain cell volume when medium osmolarity is increased. Therefore, we have investigated whether glycine accumulation is regulated by external osmolarity. In addition, we have examined its transport as a function of osmolarity and its effect on cell volume regulation. Our results indicate that glycine has the properties of an organic osmolyte in the early preimplantation stages of the mouse embryo, and that the Gly transporter may function as an organic osmolyte transporter.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Components of the culture media used were obtained from Sigma (St. Louis, MO) and were of embryo culture grade (where available) or cell culture grade. The media used were based on KSOM embryo culture medium [17]. Polyvinyl alcohol (1 mg/ml) was used instead of BSA as the macromolecular component of the medium. For flushing zygotes from the oviduct, Hepes-KSOM was used, wherein all but 4 mM of the NaHCO3 in KSOM was replaced with an equimolar amount of Hepes free acid, and the medium adjusted to pH 7.4–7.5 using NaOH. Medium osmolarity was controlled independently of inorganic ion concentration using the trisaccharide D(+) raffinose, which is not transported or metabolized by mammalian cells. The media to which raffinose was added were either standard KSOM or Hepes-KSOM, whose osmolarities were about 250 mOsM, or KSOM in which the NaCl concentration had been reduced from 95 mM to 42 mM, whose osmolarity was about 150 mOsM. Raffinose was added to these to produce a range of osmolarities from 250 mOsm to 340 mOsM, or 150 mOsM to 340 mOsM, respectively. For experiments in which embryos were swelled, 180-mOsM medium was used in which the NaCl had been reduced to 58 mM. Osmolarities were determined using a Vapro model 5520 vapor pressure osmometer (Wescor, Logan, UT).
Embryos and Culture
Zygotes were obtained from female CF1 mice (Charles River Canada, St-Constant, PQ, Canada) that had been superovulated by i.p. injection of 5 IU eCG followed 47.5 h later by 5 IU hCG. Immediately after hCG, the females were caged with BDF1 males overnight. Female mice were killed by cervical dislocation approximately 24 h post-hCG, and zygotes were removed from excised oviducts by flushing with Hepes-KSOM containing 300 µg/ml hyaluronidase (to remove adherent cumulus). Zygotes were manipulated using flame-pulled, mouth-operated pasteur pipettes. After zygotes were washed, fertilization was confirmed by the presence of two pronuclei. Two-cell stage embryos were obtained similarly, but at approximately 48 h post-hCG, and hyaluronidase was omitted from the flushing medium. Zygotes were cultured according to standard techniques, in microdrop cultures under mineral oil (Sigma; embryo culture-tested) at 37°C in 5% CO2 and air [6, 17].
Measurements of Glycine in Embryos
[3H]Glycine was obtained from Amersham ([2-3H]glycine, 10–30 Ci/mmol; Arlington Heights, IL) or New England Nuclear (30–60 Ci/mmol; Boston, MA) and added directly to the appropriate culture medium immediately before an experiment. Standard curves for each [3H]glycine batch were constructed by counting samples produced by serial dilutions of the [3H]glycine on our scintillation counter to allow conversion of counts per minute to glycine concentration, and the determination of the standard curve was repeated each week.
To determine the [3H]glycine content of embryos, ten embryos were removed from the [3H]glycine-containing medium, immediately washed several times in ice-cold Hepes-KSOM of the same osmolarity as the [3H]glycine-containing medium, and transferred to scintillation vials. This was repeated for subsequent groups of ten embryos until all of the embryos had been transferred to scintillation vials. Four milliliters of scintillation fluid (Scintiverse BD; Fisher Scientific, Pittsburgh, PA) was added to each vial. 3H was detected using a model 2200CA TriCarb liquid scintillation counter (Packard Instrument Co., Downer's Grove, IL), with each sample counted for 5 min. In addition to the embryo samples, backgrounds were measured by taking medium from the last wash drop and determining the residual counts, and this was subtracted from the counts obtained for the embryo samples. Background was always a small fraction of the counts obtained in embryo-containing samples. The total glycine content of embryos was calculated using the standard curves and expressed on a per-embryo basis.
Two types of measurements involving [3H]glycine were performed. The first was rate of glycine uptake. The rate of transport of glycine into zygotes was determined using 1 µM [3H]glycine. Even under conditions favoring the most rapid rate of transport (130 mM NaCl, 310 mOsM), uptake was linear for at least 1 h (data not shown). Uptake rates were calculated from the total amount of glycine in the embryos after a 45-min incubation with [3H]glycine, expressed as glycine uptake per min. The second measurement was amount of glycine accumulated during a 24-h incubation. Zygotes were cultured overnight, to the two-cell stage, in appropriate medium containing a mixture of [3H]glycine and glycine, at a fixed ratio of 1:1000. We found that a higher proportion of [3H]glycine, and hence a higher level of accumulated 3H, was toxic to the embryos, presumably because of radiation-induced damage. In calculating the total amount of glycine in embryos, we assumed that [3H]glycine and unlabeled glycine were indistinguishable for transport and metabolism.
Measurement of Embryo Size
Embryo dimensions were measured using an eyepiece reticle on an inverted Nikon Diaphot microscope with a 40x objective (Nikon Instruments, Garden City, NY). Measurements were made on embryos in microdrop cultures under oil in covered plastic culture dishes, which were removed from the incubator immediately before the measurement. For each zygote, two perpendicular cell diameters were measured. For each two-cell embryo, the major and minor diameters of each blastomere were measured. Zygotes were assumed to be spheres. Relative volumes were calculated using the average diameter if the two measurements were not identical. Relative volumes of two-cell embryos were calculated by assuming that each blastomere was a prolate ellipsoid, and by assuming that the third (unmeasured) axis was identical to the smaller of the two axes measured. The volumes of the two blastomeres were added to give a total volume. No attempt was made to account for deviations of zygotes or two-cell embryos from the assumed ideal geometries, and thus the volumes reported were used for comparisons only and were not assumed to reflect the true volumes.
Data Analysis
Plots were generated using SigmaPlot for Windows 1.0 (Jandel Scientific, San Rafael, CA). Data were expressed as the mean, and error bars represent the standard error of the mean (SEM). Where error bars do not appear associated with a symbol, they were smaller than the symbol. Since, as described above, groups of embryos were handled together (e.g., washing, uptake, culture) and then divided into groups of ten for counting, the values obtained for all of the sets of ten embryos from a single drop of [3H]glycine medium were averaged after measurement. Thus, for glycine determinations, the number of such replicates reported is the number of separate experiments done, not the number of groups of ten embryos counted. For volume measurements, each replicate was the averaged volume of five embryos handled together and measured simultaneously. The number of replicates is reported, with the mean and SEM for each set of replicates. Comparisons between means were made by ANOVA and the Tukey-Kramer Multiple Comparison test, or by Student's two-tailed t-test, as appropriate, using Instat (GraphPad, San Diego, CA). Regression analysis was performed with Excel (Microsoft, Redmond, WA).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To determine whether the amount of glycine accumulated by embryos is a function of osmolarity, zygotes were cultured in KSOM medium (250 mOsM, 95 mM NaCl) or KSOM with the osmolarity adjusted to 310 or 340 mOsM using raffinose. The media contained 0.1, 0.5, 1.0, or 2.0 mM total glycine (1:1000 [3H]glycine:glycine). When zygotes were cultured to the two-cell stage in the presence of 1 mM glycine (Fig. 1, inset; analyzed separately because this concentration was previously shown to be optimally beneficial to development [6]), significantly more glycine was accumulated at 310 and 340 mOsM than at 250 mOsM (p < 0.001 by ANOVA followed by Tukey-Kramer test). Accumulation reached a plateau with increasing glycine concentration in the medium (Fig. 1). Increasing the medium osmolarity from 250 to 310 mOsM resulted in a greater maximal accumulation of glycine. The maximal level of glycine accumulation did not change from 310 to 340 mOsM. However, maximal accumulation was reached at a lower concentration of total glycine in the medium at 340 mOsM, reaching a plateau by 0.5 mM total glycine at 340 mOsM vs. by 1.0 mM at 310 mOsM.
|
Most Glycine Accumulated during 24-h Culture Was Retained in a Releasable Form
To determine whether glycine accumulated by embryos cultured overnight is in an osmotically active, releasable form, or instead has been incorporated into proteins, used in nucleic acid synthesis, or otherwise metabolized, we exploited the swelling-activated permeability of embryos to small organic compounds (see Discussion); only small osmotically active compounds are released by cells upon swelling. After 24-h culture from the zygote stage in KSOM that contained 1.0 mM total glycine (as in Fig. 1, inset), the resulting two-cell embryos were swelled by exposing them to a hypotonic 180-mOsM medium containing no glycine. The amount of glycine remaining was determined as a function of time after transfer to hypotonic or isotonic KSOM. Upon swelling, embryos quickly released most of the accumulated glycine (Fig. 2, A and B). In contrast, only a very slow loss was evident in embryos that were transferred to isotonic glycine-free KSOM in which there was no swelling (Fig. 2, A and B). While most of the glycine was lost during swelling, a fraction was retained with no further decrease evident over at least 4 h. This nonreleasable fraction of glycine represented about 50% of the total glycine accumulated when embryos were cultured in 250 mOsM KSOM (Fig. 2A), but only 15% after culture at 310 mOsM (Fig. 2B).
|
The release of glycine was tightly correlated with embryo swelling. The dimensions of two-cell embryos treated identically to those used for efflux measurements (above, Fig. 2) were measured as a function of time after transfer to 180 mOsM KSOM (Fig. 3). The embryos swelled after transfer to 180 mOsM, with a greater volume increase occurring for embryos that had been cultured overnight at 310 mOsM (Fig. 3B) as compared to those that had been cultured overnight at 250 mOsM (Fig. 3A). Recovery from hypotonically induced swelling occurred with essentially the same time course as release of glycine from embryos subjected to identical hypotonic conditions (compare Figs. 2 and 3).
|
Rate of Glycine Uptake by Two-Cell Embryos Following 24-h Culture in 250, 310, or 340 mOsM KSOM Without Glycine
Zygotes were cultured for 24 h in the absence of glycine in either 250, 310, or 340 mOsM KSOM (95 mM NaCl). After culture they were transferred to identical media except with 1 µM [3H]glycine added, so that the rate of glycine uptake could be measured. Thus, any effect of culture osmolarity on glycine transport capacity of the resulting embryos would be revealed. Because these transfers were to isotonic media, no changes in cell volume occurred. No significant difference in glycine uptake rates was found among two-cell embryos after culture from zygotes at the different osmolarities (Fig. 4; p > 0.05 by ANOVA). Since the embryos cultured at 310 and 340 mOsM would have a severely compromised ability to develop in culture to the blastocyst stage in the absence of any osmolyte [6], this experiment was repeated with 1 mM glutamine present during culture so that developmental potential would be the same in each group. Again, no significant differences were found (data not shown).
|
Rate of Glycine Uptake in Zygotes as a Function of Osmolarity
To determine whether medium osmolarity had an immediate effect on the rate of glycine transport by zygotes, the rate of glycine uptake into freshly obtained zygotes was measured in KSOM of various osmolarities, with 1 µM [3H]glycine in each. Two different series of media were used. One contained 42 mM NaCl, and thus osmolarities as low 150 mOsM could be obtained, with raffinose added for higher osmolarities at a constant NaCl concentration. The other contained 95 mM NaCl, as in normal KSOM, and thus osmolarities from 250 mOsM and higher could be obtained at a constant NaCl concentration. In separate experiments, we confirmed that the expected volume increases or decreases occurred, depending on osmolarity (data not shown). Glycine transport rates were measured immediately after transfer of zygotes from 250 mOsM Hepes-KSOM, exactly as if they were being placed into culture at various osmolarities. In the 42-mM NaCl media, there was an approximate 2-fold increase in the rate of glycine uptake as a function of osmolarity over the range of 150 to 340 mOsM (Fig. 5). Most or all of this increase occurred below 200 mOsM, with a significant difference between uptake at 150 mOsM and the other osmolarities (p < 0.05 by ANOVA followed by Tukey-Kramer test). No significant increase occurred over the range from 200 to 340 mOsM (p > 0.05 for a nonzero slope by regression analysis). Since these media contained only 42 mM NaCl, glycine transport was slow (see Discussion). Therefore, the effect of osmolarity in the range from 250 to 340 mOsM was also examined in medium containing 95 mM NaCl, which supports a much higher rate of glycine transport. In 95-mM NaCl media, a significant dependence of uptake rate on osmolarity was revealed, with transport at 340 mOsM occurring at about a 30% higher rate than at 250 mOsM (Fig. 5; p = 0.02 for a nonzero slope by regression analysis).
|
Competitive Inhibition Profile of Glycine Transport in Zygotes at 310 mOsM
We assessed the ability of several compounds, each present at 5 mM, to inhibit uptake of 1 µM [3H]glycine in zygotes (Fig. 6). To better reveal any significant Na+- and/or Cl--independent components, inhibition experiments were done in media containing 42 mM NaCl, a level at which glycine transport is fairly low (see Discussion), with osmolarity adjusted to 310 mOsM using raffinose (identical to the conditions used for measuring glycine uptake into zygotes, as in Fig. 5). Excess unlabeled glycine inhibited [3H]glycine uptake by about 95% (p < 0.001 vs. control, by ANOVA followed by Tukey-Kramer test), indicating that almost all of the measured uptake was attributable to specific saturable transport. A similar level of inhibition was seen with 5 mM sarcosine (p < 0.001). In contrast, glutamine, betaine, taurine, and ß-alanine had no significant effect. Proline partially inhibited glycine uptake (p < 0.01).
|
Relative Volume of Zygotes as a Function of Osmolarity and Glycine Concentration
To determine whether glycine had an effect on zygote volume, the volumes of freshly obtained zygotes transferred into 250, 310, or 340 mOsM KSOM (95 mM NaCl) in the presence or absence of 1.0 mM glycine were determined as a function of time after initial transfer to each medium. These are essentially the same conditions under which glycine uptake was measured as a function of osmolarity (Fig. 5). Zygotes placed into normal 250 mOsM KSOM had an increased volume at the earliest time at which measurements could be completed (10–15 min after transfer into medium). By 20 min, however, they had recovered to a volume of approximately 210 pL, which was not different in the presence or absence of glycine (Fig. 7A). When placed into 310 mOsM medium without glycine, zygotes decreased in volume from about 200 pL to about 180 pL, and remained there (Fig. 7B). However, in the presence of 1 mM glycine, such a decrease did not occur, and the zygotes retained their initial volumes (Fig. 7B). Thus, at 310 mOsM, zygotes had significantly larger volumes in the presence of glycine than in its absence (p values as shown, by t-test, comparing means ± glycine at each time independently). In contrast, when placed into 340-mOsM medium, zygotes had a greatly decreased volume. They then recovered only to about 180 pL regardless of whether glycine was present or not (Fig. 7C).
|
Because volume measurements are somewhat subjective, we repeated the experiment as described, but in 320 mOsM KSOM and with the experiments performed by a different individual than with the first set of experiments. Again, we found that zygotes in the presence of 1 mM glycine maintained significantly larger volumes than in the absence of glycine (not shown).
Relative Volume of Two-Cell Embryos after 24-h Culture at 250 or 310 mOsM With or Without Glycine
Zygotes were cultured for 24 h to the two-cell stage in 250 or 310 mOsM KSOM, containing 0, 1, 10, or 20 mM glycine (for 10 and 20 mM glycine, osmolarity was kept constant by replacing NaCl with glycine or by adjusting raffinose concentration). The differences in volume among all groups (Fig. 8) were not significantly different by ANOVA (p > 0.05). The possibility that the mean volume of the group cultured without glycine was significantly less than the mean volume of those cultured in the presence of glycine at 310 mOsM (Fig. 8B) was tested by pooling all of the data for the glycine-containing groups (1, 10, and 20 mM) and comparing with 0 glycine using a one-tailed t-test. There was still, however, no significant difference (p = 0.07).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We have shown here that increased external osmolarity causes an increase in intracellular glycine accumulation by early mouse embryos cultured from zygotes to two-cell embryos (Figs. 1 and 2). We believe that this is a strong indication that glycine functions as an intracellular osmolyte in embryos. The increase in total accumulated glycine at higher osmolarity largely reflects increased intracellular concentration, since there was no significant difference between the volumes of two-cell embryos arising from culture in 240 vs. 310 mOsM (Fig. 8). Intracellular accumulation of glycine seems to account for the osmoprotective effect of adding glycine to culture media: half-maximal development to blastocysts [6] and half-maximal glycine accumulation both occurred at about 0.1 mM external glycine in 310 mOsM medium, and both glycine accumulation and blastocyst development [6] were maximal at 1 mM glycine.
Most of the glycine accumulated after overnight culture of zygotes to two-cell embryos in hypertonic medium seems to remain in a quickly diffusible, osmotically active form, since a large fraction of the glycine in such two-cell embryos is rapidly lost upon hypotonic swelling (Fig. 2). This indicates that it is probably free glycine, although we cannot rule out the possibility that some of the accumulated glycine has been metabolized into other small osmolytes such as alanine and serine [13].
Such an accumulation of a large amount of free glycine is not simply an artifact of our culture conditions, since freshly obtained two-cell embryos have been shown to contain approximately 10 pmol free glycine [18], comparable to the approximately 7 pmol that we found was accumulated in culture at 310 mOsM.
Many cells possess swelling-activated anion channels that are also permeable to organic osmolytes [19, 20], and such a mechanism probably mediates the release of glycine from swelled embryos. Three observations indicate that zygotes have such a mechanism. First, zygotes and two-cell embryos exhibit a swelling-activated permeability to organic compounds such as taurine, ß-alanine, and glycine [15, 21] (Figs. 2 and 3), which is blocked by Cl- channel blockers [15]. Second, Cl- channel blockers prevent volume recovery in hypotonically swelled zygotes [22]. Third, electrophysiological measurements indicate that Cl- channels are activated by swelling in zygotes (our unpublished results). Thus, the release of glycine upon swelling (Fig. 2) is due to the opening of these channels, and the cessation of efflux occurs either when all of the free glycine is lost, or when the channels close after cell volume is restored. We believe it is most likely that nearly all of the free glycine was released, as there was no further decrease even after 4 h in hypotonic, glycine-free medium (Fig. 2). This indicates that the remaining glycine is sequestered in the embryo, possibly having been used to synthesize proteins or nucleic acids, or otherwise metabolized. Total glycine is about 1.5-fold higher after culture at 310 mOsM than at 250 mOsM (Figs. 1 and 2). If the nonreleasable portion is excluded, then the amount of free glycine is shown to be 2.5-fold higher.
The cytoplasmic glycine concentration is clearly high enough to be osmotically significant. Based on a 200-pL total volume for an embryo, the concentration of glycine accumulated after culture in 310 or 340 mOsM medium is 30 or 35 mM, using the values for free or total glycine, respectively. Thus, glycine appears to satisfy a basic requirement for an organic osmolyte: that free glycine is accumulated to a high level, and that the level of accumulation is a function of external osmolarity.
Effect of Osmolarity on Glycine Transport
Some cells have been shown to possess organic osmolyte transporters that are up-regulated in hypertonic media. This up-regulation is slow, requires mRNA synthesis, and results in an increase in the number of transporters without any change in their substrate affinity (i.e., Vmax increases while Km remains unchanged). Of known osmotically sensitive transporters, only the System A amino acid transporter carries glycine, but System A activity is absent from cleavage-stage preimplantation embryos [12, 23]. Nonetheless, it is possible that the increased glycine accumulation at higher osmolarity could involve a similar osmolarity-dependent increase in the number of Gly transporters in the embryo. To test this hypothesis, zygotes were cultured to the two-cell stage in 250, 310, or 340 mOsM medium in the absence of glycine. The resulting two-cell embryos were then exposed to 1 µM glycine, and the rate of glycine uptake was measured (Fig. 4). There was no difference in the uptake rates, regardless of the osmolarity at which they had been cultured. The simplest interpretation of this result is that Vmax was unchanged and hence no increase in the number of transporters had occurred as a result of increased osmolarity in culture.
Alternatively, it is possible that a hypertonically induced decrease in cell volume directly stimulates existing glycine transporters in zygotes and thus increases their activity. We therefore measured the rate of glycine uptake into zygotes immediately after transferring them to media of various osmolarities. Increasing the osmolarity from 150 to 340 mOsM (using raffinose addition to 42 mM NaCl KSOM) did indeed result in a significant increase in the rate of glycine transport (Fig. 5), with about a 2-fold increase in uptake rate over this range. However, all of the significant increase took place between 150 and 200 mOsM, below the range at which glycine accumulation was found to be stimulated during culture from the zygote to two-cell stage (Fig. 1). We also measured uptake in medium with 95 mM NaCl, whose osmolarity is 250 mOsM, and in the same medium with osmolarity increased using raffinose. The higher NaCl content in these media (95 mM) supports a greater rate of glycine transport by the Na+- and Cl--dependent Gly system and allows more precise measurements of any Gly activity increase. We found that there was a significant increase in glycine uptake as a function of osmolarity (Fig. 5). This could well have been due to stimulation of transport by decreased cell volumes, since the volumes of zygotes are significantly lower in 310- and 340-mOsM medium than in 250-mOsM medium in the absence of glycine. Since no glycine other than the 1 µM [3H]glycine used to measure transport rate was present in these short-term experiments, the zygotes presumably had the same volumes as zygotes in glycine-free media of the same osmolarities (Fig. 7). The stimulation was only about 30%, however, and it would seem unlikely that it plays a major role in controlling the amount of glycine accumulation.
Therefore, the question of how glycine accumulation is regulated by osmolarity or cell volume in the early embryo remains unanswered. It seems clear from our results that an increase in the rate of uptake does not play a major role in glycine accumulation, unlike the case for osmolyte accumulation in other cells, in which synthesis of new transporters leads to an eventual increase in transport rate. It also would appear different from the regulation of taurine transport in zygotes, in which uptake rate via the ß-amino acid transporter System ß is increased several-fold when osmolarity is increased [15]. This occurs quickly, and thus apparently without the need for mRNA and protein synthesis.
The major glycine transporter in mouse zygotes and two-cell embryos is the Na+- and Cl--dependent Gly transporter [12–14, 16]. We have confirmed that transport of glycine at 310 mOsM is almost entirely inhibited (about 95%) by either excess unlabeled glycine or sarcosine, the latter a specific substrate of the Gly transporter (Fig. 6). In contrast, 5 mM taurine or ß-alanine, substrates of the ß-amino acid transporter, had no effect. Betaine and glutamine are apparently low-affinity substrates of the Gly transporter [24, 25], and were unable to significantly inhibit transport of glycine, which has high affinity for the transporter. Proline partially inhibited glycine transport, in agreement with previous reports that it may be a system Gly substrate [12, 24]. Taken together, these results indicate that the glycine transport we have measured is overwhelmingly the result of Gly transporter activity. Thus, the Gly transporter may be a target of whatever regulatory mechanism controls glycine accumulation as a function of osmolarity. We are currently investigating the regulation of glycine accumulation in embryos.
Cell Volume
Organic osmolytes have been reported to confer protection from decreased cell volume during culture in other cell types, in addition to increased viability [26]. We have investigated whether the presence of glycine prevents a decrease in zygote volume when they are transferred into hypertonic medium. Upon transfer to normal KSOM, at 240 mOsM, zygotes initially had increased volumes from which they subsequently recovered (Fig. 7A), which is consistent with the idea that KSOM is hypotonic relative to oviductal fluid and that therefore zygotes swell upon transfer to KSOM. In support of this, we have preliminary results indicating that zygotes removed from the oviduct in undiluted oviductal fluid under oil are smaller than zygotes immediately after transfer to KSOM at 250 mOsM (unpublished). Recovery from this swelling was complete within 20 min (Fig. 7A), similar to the time-course of recovery we had previously found for swelling induced by exposure to 150-mOsM medium [22]. The volume of zygotes was not significantly different in the presence or absence of glycine.
In 340-mOsM medium (Fig. 7C), zygotes were initially smaller in volume than they were after 20 min. Zygotes then maintained a volume that was significantly less than the volume of zygotes at 240 mOsM, with no effect of glycine.
When zygotes were transferred into 310-mOsM medium, there was no immediate significant change in volume. However, zygotes in the absence of glycine exhibited a subsequent decrease in volume (Fig. 7B), so that by 60 min they were not very different in volume from zygotes in 340-mOsM medium (Fig. 7C). In contrast, zygotes transferred to 310-mOsM medium with glycine present did not exhibit this volume decrease, but instead showed a slight increase in volume over the course of the experiment. Their final volume at 310 mOsm was not significantly different from that of zygotes in 240-mOsM medium, indicating that glycine was effective at maintaining volume at this osmolarity.
We also examined whether there were differences in the volumes of two-cell embryos that had been cultured overnight from the zygote stage in 240- or 310-mOsM medium in glycine-free medium, and with various concentrations of glycine. Overall, the two-cell embryos resulting from such cultures did not differ significantly in their volumes among the various culture conditions (Fig. 8), although there was a trend toward lower volumes at 310 mOsM in the absence of glycine. Therefore, after overnight culture in glycine-free medium, two-cell embryos probably accumulate sufficient inorganic ions to nearly maintain their volumes, and this increased inorganic ion content may underlie the decreased development [2, 6] of such embryos. In the presence of glycine, however, the embryos preferentially accumulate glycine rather than inorganic ions for osmotic support, allowing improved development.
Conclusion
Glycine functions as an organic osmolyte in mouse zygotes and two-cell embryos, and zygotes accumulate it at a much higher level when osmolarity is increased during overnight culture. Furthermore, zygotes can maintain their volumes against moderately increased osmolarity in the presence of glycine, but not in its absence. Presumably, the Gly transporter, which is the major transport pathway for glycine in early embryos, functions in the accumulation of this osmolyte. However, the rate of transport does not increase very substantially when osmolarity is increased over the relevant range, nor is it increased after overnight culture at higher osmolarity. Thus, the regulation of glycine accumulation by osmolarity or cell volume remains to be elucidated.
|
FOOTNOTES |
|---|
2 Correspondence: Jay M. Baltz, Loeb Medical Research Institute, 725 Parkdale Ave., Ottawa, ON, Canada K1Y 4E9. FAX: (613) 761–5327; jay{at}civich.ottawa.on.ca
Accepted: March 10, 1998.
Received: December 8, 1997.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  T. Hadi, M.-A. Hammer, C. Algire, T. Richards, and J. M. Baltz Similar Effects of Osmolarity, Glucose, and Phosphate on Cleavage past the 2-Cell Stage in Mouse Embryos from Outbred and F1 Hybrid Females Biol Reprod, January 1, 2005; 72(1): 179 - 187. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. M Orsi and H. J Leese Ammonium exposure and pyruvate affect the amino acid metabolism of bovine blastocysts in vitro Reproduction, January 1, 2004; 127(1): 131 - 140. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Steeves, M.-A. Hammer, G. B. Walker, D. Rae, N. A. Stewart, and J. M. Baltz The glycine neurotransmitter transporter GLYT1 is an organic osmolyte transporter regulating cell volume in cleavage-stage embryos PNAS, November 25, 2003; 100(24): 13982 - 13987. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Sonoda, F. Okamoto, H. Kajiya, Y. Inoue, K. Honjo, Y. Sumii, T. Kawarabayashi, and K. Okabe Amino Acid-Permeable Anion Channels in Early Mouse Embryos and Their Possible Effects on Cleavage Biol Reprod, March 1, 2003; 68(3): 947 - 953. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Vallee, F. Guay, D. Beaudry, J. Matte, R. Blouin, J.-P. Laforest, M. Lessard, and M.-F. Palin Effects of Breed, Parity, and Folic Acid Supplement on the Expression of Folate Metabolism Genes in Endometrial and Embryonic Tissues from Sows in Early Pregnancy Biol Reprod, October 1, 2002; 67(4): 1259 - 1267. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. D. Houghton1, J. A. Hawkhead, P. G. Humpherson, J. E. Hogg, A. H. Balen, A. J. Rutherford, and H. J. Leese Non-invasive amino acid turnover predicts human embryo developmental capacity Hum. Reprod., April 1, 2002; 17(4): 999 - 1005. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. G. Chung, M. R.W. Mann, M. S. Bartolomei, and K. E. Latham Nuclear-Cytoplasmic "Tug of War" During Cloning: Effects of Somatic Cell Nuclei on Culture Medium Preferences of Preimplantation Cloned Mouse Embryos Biol Reprod, April 1, 2002; 66(4): 1178 - 1184. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kolajova, M.-A. Hammer, J. L. Collins, and J. M. Baltz Developmentally regulated cell cycle dependence of swelling-activated anion channel activity in the mouse embryo Development, September 15, 2001; 128(18): 3427 - 3434. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. J. Van Winkle Amino Acid Transport Regulation and Early Embryo Development Biol Reprod, January 1, 2001; 64(1): 1 - 12. [Abstract] [Full Text]
|
|
|
|
|
|
M.A. Hammer, M. Kolajova, M.-C. Leveille, P. Claman, and J.M. Baltz Glycine transport by single human and mouse embryos Hum. Reprod., February 1, 2000; 15(2): 419 - 426. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Collins and J. M. Baltz Estimates of Mouse Oviductal Fluid Tonicity Based on Osmotic Responses of Embryos Biol Reprod, May 1, 1999; 60(5): 1188 - 1193. [Abstract] [Full Text]
|
|
|
|
|
|
M. Kolajova and J. M. Baltz Volume-Regulated Anion and Organic Osmolyte Channels in Mouse Zygotes Biol Reprod, April 1, 1999; 60(4): 964 - 972. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
