Osmolarity-Dependent Glycine Accumulation Indicates a Role for Glycine as an Organic Osmolyte in Early Preimplantation Mouse Embryos

Citation

Kerri M. Dawson, Jennifer L. Collins, and Jay M. Baltz. "Osmolarity-Dependent Glycine Accumulation Indicates a Role for Glycine as an Organic Osmolyte in Early Preimplantation Mouse Embryos". Biology of Reproduction (1998), Volume 59, 225-232

Summary

In this paper, Dawson et al. tested the osmoregulatory effects of glycine on mouse zygotes. In mice, oviductal fluid has a high osmolarity above 340 mOsM and contains a high concentration of glycine. However, mouse zygotes don’t develop in vitro at osmolarities higher than 300 mOsM unless some organic molecules are present in the media. Dawson et al. hypothesized that glycine transport is affected by changes in osmolarity and plays a role in regulating cell volume. They extracted the embryos with Hepes-KSOM media and cultured them in pH 7.4-7.5 KSOM media. The media osmolarity was modulated using different concentrations of D(+) raffinose, a trisaccharide that is not transported or metabolized. The osmolarities used included 150, 180, 250, 310, or 340 mOsM with 42mM or 95 mM NaCl for the experiments. Glycine content in embryos was measured using [³H]glycine and scintillation and quantification as done using a standard curve. Cell volume was measured by measuring the major and minor axes using an eyepiece at 40x on an inverted microscope.

Culturing zygotes at 250, 310, or 340 mOsM with different glycine concentrations showed that at 310 and 340 mOsM the cells accumulated more glycine than at 250 mOsM over 24 hours (figure 1). Moreover, when these zygotes were transferred to hypotonic media, they swelled and released glycine (figure 2). In order to see if there was an effect of varying osmolarity and changing cell volume on glycine uptake, 2 cell embryos were cultured in 250, 310, and 340 mOsm media without glycine to equilibrate and then transferred to the same osmolarity media with glycine. No significant differences were found in glycine uptake rates. Experiments were then done on zygotes to measure the initial effects of osmolarity. Zygotes were cultured in media from 150 mOsM to 350 mOsM. Zygotes in 150 mOsM showed the lowest rate of glycine uptake, and there was a significant jump in uptake rate between 150 and 200 mOsM (figure 5). To increase transport rates, more NaCl was added, and showed a greater difference in transport rates from 200 to 340 mOsM. Cells were then tested to see if there were compounds that inhibited uptake of [_­³H]glycine. It was found that high concentrations of glycine and sarcosine greatly inhibited [³H]glycine uptake with proline mildly inhibited uptake. To see whether glycine affected zygote volume, zygotes were put into 250, 310 or 340 mOsM media wither with or without glycine. Results showed that cells initially swelled in 250 mOsM media, but then returned to normal volume both with and without glycine. Cells in 310 and 340 mOsM media shrank in no glycine culture but with glycine present, the cells didn’t change volume significantly (figure 7). The experiment was then done with a time length of 24 hrs in 250 and 310 mOsM and varying glycine concentrations, but the results showed that there was no statistical difference in cell volumes.

From these results, Dawson et al. concluded that glycine is used by mouse zygotes to balance osmolarites inside and outside the cell due to the accumulation of free glycine inside cells at high osmolarities. They could not draw any conclusions on the mechanism of regulation of increased glycine uptake, because the results showed no difference in uptake rate at varying osmolarities. Additionally, increased rates of uptake also did not greatly affect the total accumulation of glycine in cells. However, the results do show that glycine presence helps zygotes maintain cell volume in hypertonic solutions.

Critique:

This paper showed good experimental design that accounts for many different factors that may affect glycine transport in glycine. Additionally, the conclusions Dawson et al. draw are do not overly generalize or assume things not shown in the results – they admit that some of their data is inconclusive. However, one control they could have included was the negative control - culturing zygotes in isotonic media and measuring glycine uptake. Among the osmolarities that they tested, 250 mOsM was hypotonic while 310 mOsM was hypertonic and they did not test any osmolarities in between. Also, I believe that some of their conclusions may not be fully supported. They claim that after hypotonic swelling, all of the free glycine was released since they couldn’t detect further decrease even after 4 hours. This could also be because the cell has already reached its original volume and equilibrated with the media, so the glycine channels closed to prevent further release of glycine. The paper’s results make it seem like glycine is the most important osmoregulator for mouse zygotes, but this may or may not be true and the paper does not explore this idea. On a different note, the method of calculating volumes was not very precise. Though the paper notes this and tries to account for it by having multiple people perform the test, it doesn’t change the fact that the true volumes of the zygotes were not obtained. This could lead to false data and erroneous conclusions could have been drawn. Next, in the discussion, 240 mOsM culture is mentioned, but it is not mentioned anywhere else in the paper, making me believe that it is either a typo, or they forgot to list the data in the methods and results sections. Also, when the paper mentions decreased or improved development of the 2 celled embryos in the results section “Rate of Glycine Uptake by Two-Cell Embryos Following…” , it does not mention what that means and then uses it as a justification to add 1mM glutamine, an additional variable that may affect results. Finally, in the methods section, the paper mentions manipulation zygotes by pipetting by mouth, a very unsafe lab procedure.